Purpose: The purpose of this study was to analyze associations between the oral health literacy of refugees and two oral health outcomes: dental care utilization and oral health self-efficacy.Methods: A convenience sample of refugees in the greater Los Angeles area attending English as a second language (ESL) classes sponsored by two refugee assistance organizations was used for this cross-sectional, correlational study. Participants responded to a questionnaire using items from the Health Literacy in Dentistry (HeLD) scale, in addition to items concerning dental care utilization and oral health self-efficacy. Descriptive statistics, chi-square and Fisher's Exact tests were used to analyze results.Results: Sixty-two refugees volunteered to participate (n=62). A majority of the respondents were female from Iraq or Syria, and selected the item “with little difficulty” for all oral health literacy tasks. In regards to dental care utilization, more than half of the respondents were considered high utilizers (63%, n=34) meaning they had visited a dental office within the last year; while a little more than one-third (37%, n=20), were low utilizers, indicating they had either never been to a dental office or it had been more than one year since they had dental treatment. Statistical analysis showed associations between oral health literacy and dental care utilization. However, few associations between oral health literacy and oral health self-efficacy were identified (p=0.0045).Conclusions: Results support the provision of easily obtainable and understandable oral health information to increase oral health literacy and dental care utilization among refugee populations. Future research is needed to examine the oral health literacy among refugees resettling in the United States.

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (^–/–) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.­

The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K₂ subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R^–/–) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by ²H/¹³C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.

Cellular membranes are not homogenous mixtures of proteins; rather, they are segregated into microdomains on the basis of preferential association between specific lipids and proteins. These microdomains, called lipid rafts, are well known for their role in receptor signaling on the plasma membrane (PM) and are essential to such cellular functions as signal transduction and spatial organization of the PM. A number of disease states, including atherosclerosis and other cardiovascular disorders, may be caused by dysfunctional maintenance of lipid rafts. Lipid rafts do not occur only in the PM but also have been found in intracellular membranes and extracellular vesicles (EVs). Here, we focus on discussing newly discovered functions of lipid rafts and microdomains in intracellular membranes, including lipid and protein trafficking from the ER, Golgi bodies, and endosomes to the PM, and we examine lipid raft involvement in the production and composition of EVs. Because lipid rafts are small and transient, visualization remains challenging. Future work with advanced techniques will continue to expand our knowledge about the roles of lipid rafts in cellular functioning.

Lipid rafts are highly ordered regions of the plasma membrane that are enriched in cholesterol and sphingolipids and play important roles in many cells. In hematopoietic stem and progenitor cells (HSPCs), lipid rafts house receptors critical for normal hematopoiesis. Lipid rafts also can bind and sequester kinases that induce negative feedback pathways to limit proliferative cytokine receptor cycling back to the cell membrane. Modulation of lipid rafts occurs through an array of mechanisms, with optimal cholesterol efflux one of the major regulators. As such, cholesterol homeostasis also regulates hematopoiesis. Increased lipid raft content, which occurs in response to changes in cholesterol efflux in the membrane, can result in prolonged receptor occupancy in the cell membrane and enhanced signaling. In addition, certain diseases, like diabetes, may contribute to lipid raft formation and affect cholesterol retention in rafts. In this review, we explore the role of lipid raft-related mechanisms in hematopoiesis and CVD (specifically, atherosclerosis) and discuss how defective cholesterol efflux pathways in HSPCs contribute to expansion of lipid rafts, thereby promoting myelopoiesis and thrombopoiesis. We also discuss the utility of cholesterol acceptors in contributing to lipid raft regulation and disruption, and highlight the potential to manipulate these pathways for therapeutic gain in CVD as well as other disorders with aberrant hematopoiesis.

Activation of microglia and astrocytes secondary to inflammatory processes contributes to the development and perpetuation of pain with a neuropathic phenotype. This pain state presents as a chronic debilitating condition and affects a large population of patients with conditions like rheumatoid arthritis and diabetes, or after surgery, trauma, or chemotherapy. Here, we review the regulation of lipid rafts in glial cells and the role they play as a key component of neuroinflammatory sensitization of central pain signaling pathways. In this context, we introduce the concept of an inflammaraft (i-raft), enlarged lipid rafts harboring activated receptors and adaptor molecules and serving as an organizing platform to initiate inflammatory signaling and the cellular response. Characteristics of the inflammaraft include increased relative abundance of lipid rafts in inflammatory cells, increased content of cholesterol per raft, and increased levels of inflammatory receptors, such as toll-like receptor (TLR)4, adaptor molecules, ion channels, and enzymes in lipid rafts. This inflammaraft motif serves an important role in the membrane assembly of protein complexes, for example, TLR4 dimerization. Operating within this framework, we demonstrate the involvement of inflammatory receptors, redox molecules, and ion channels in the inflammaraft formation and the regulation of cholesterol and sphingolipid metabolism in the inflammaraft maintenance and disruption. Strategies for targeting inflammarafts, without affecting the integrity of lipid rafts in noninflammatory cells, may lead to developing novel therapies for neuropathic pain states and other neuroinflammatory conditions.

Lipid rafts are small, dynamic membrane areas characterized by the clustering of selected membrane lipids as the result of the spontaneous separation of glycolipids, sphingolipids, and cholesterol in a liquid-ordered phase. The exact dynamics underlying phase separation of membrane lipids in the complex biological membranes are still not fully understood. Nevertheless, alterations in the membrane lipid composition affect the lateral organization of molecules belonging to lipid rafts. Neural lipid rafts are found in brain cells, including neurons, astrocytes, and microglia, and are characterized by a high enrichment of specific lipids depending on the cell type. These lipid rafts seem to organize and determine the function of multiprotein complexes involved in several aspects of signal transduction, thus regulating the homeostasis of the brain. The progressive decline of brain performance along with physiological aging is at least in part associated with alterations in the composition and structure of neural lipid rafts. In addition, neurodegenerative conditions, such as lysosomal storage disorders, multiple sclerosis, and Parkinson’s, Huntington’s, and Alzheimer’s diseases, are frequently characterized by dysregulated lipid metabolism, which in turn affects the structure of lipid rafts. Several events underlying the pathogenesis of these diseases appear to depend on the altered composition of lipid rafts. Thus, the structure and function of lipid rafts play a central role in the pathogenesis of many common neurodegenerative diseases.

Cholesterol/sphingolipid-rich membrane domains, known as lipid rafts or membrane rafts, play a critical role in the compartmentalization of signaling pathways. Physical segregation of proteins in lipid rafts may modulate the accessibility of proteins to regulatory or effector molecules. Thus, lipid rafts serve as sorting platforms and hubs for signal transduction proteins. Cancer cells contain higher levels of intracellular cholesterol and lipid rafts than their normal non-tumorigenic counterparts. Many signal transduction processes involved in cancer development (insulin-like growth factor system and phosphatidylinositol 3-kinase-AKT) and metastasis [cluster of differentiation (CD)44] are dependent on or modulated by lipid rafts. Additional proteins playing an important role in several malignant cancers (e.g., transmembrane glycoprotein mucin 1) are also being detected in association with lipid rafts, suggesting a major role of lipid rafts in tumor progression. Conversely, lipid rafts also serve as scaffolds for the recruitment and clustering of Fas/CD95 death receptors and downstream signaling molecules leading to cell death-promoting raft platforms. The partition of death receptors and downstream signaling molecules in aggregated lipid rafts has led to the formation of the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER, which leads to apoptosis amplification and can be pharmacologically modulated. These death-promoting rafts can be viewed as a linchpin from which apoptotic signals are launched. In this review, we discuss the involvement of lipid rafts in major signaling processes in cancer cells, including cell survival, cell death, and metastasis, and we consider the potential of lipid raft modulation as a promising target in cancer therapy.

Lipid rafts, solid regions of the plasma membrane enriched in cholesterol and glycosphingolipids, are essential parts of a cell. Functionally, lipid rafts present a platform that facilitates interaction of cells with the outside world. However, the unique properties of lipid rafts required to fulfill this function at the same time make them susceptible to exploitation by pathogens. Many steps of pathogen interaction with host cells, and sometimes all steps within the entire lifecycle of various pathogens, rely on host lipid rafts. Such steps as binding of pathogens to the host cells, invasion of intracellular parasites into the cell, the intracellular dwelling of parasites, microbial assembly and exit from the host cell, and microbe transfer from one cell to another all involve lipid rafts. Interaction also includes modification of lipid rafts in host cells, inflicted by pathogens from both inside and outside the cell, through contact or remotely, to advance pathogen replication, to utilize cellular resources, and/or to mitigate immune response. Here, we provide a systematic overview of how and why pathogens interact with and exploit host lipid rafts, as well as the consequences of this interaction for the host, locally and systemically, and for the microbe. We also raise the possibility of modulation of lipid rafts as a therapeutic approach against a variety of infectious agents.

Lipid rafts are organized plasma membrane microdomains, which provide a distinct level of regulation of cellular metabolism and response to extracellular stimuli, affecting a diverse range of physiologic and pathologic processes. This Thematic Review Series focuses on Biology of Lipid Rafts rather than on their composition or structure. The aim is to provide an overview of ideas on how lipid rafts are involved in regulation of different pathways and how they interact with other layers of metabolic regulation. Articles in the series will review the involvement of lipid rafts in regulation of hematopoiesis, production of extracellular vesicles, host interaction with infection, and the development and progression of cancer, neuroinflammation, and neurodegeneration, as well as the current outlook on therapeutic targeting of lipid rafts.

ABSTRACT

The luminous marine Gram-negative bacterium Vibrio (Aliivibrio) fischeri is the natural light organ symbiont of several squid species, including the Hawaiian bobtail squid, Euprymna scolopes, and the Japanese bobtail squid, Euprymna morsei. Work with E. scolopes has shown how the bacteria establish their niche in the light organ of the newly hatched host. Two types of V. fischeri strains have been distinguished based upon their behavior in cocolonization competition assays in juvenile E. scolopes, i.e., (i) niche-sharing or (ii) niche-dominant behavior. This study aimed to determine whether these behaviors are observed with other V. fischeri strains or whether they are specific to those isolated from E. scolopes light organs. Cocolonization competition assays between V. fischeri strains isolated from the congeneric squid E. morsei or from other marine animals revealed the same sharing or dominant behaviors. In addition, whole-genome sequencing of these strains showed that the dominant behavior is polyphyletic and not associated with the presence or absence of a single gene or genes. Comparative genomics of 44 squid light organ isolates from around the globe led to the identification of symbiosis-specific candidates in the genomes of these strains. Colonization assays using genetic derivatives with deletions of these candidates established the importance of two such genes in colonization. This study has allowed us to expand the concept of distinct colonization behaviors to strains isolated from a number of squid and fish hosts.

IMPORTANCE There is an increasing recognition of the importance of strain differences in the ecology of a symbiotic bacterial species and, in particular, how these differences underlie crucial interactions with their host. Nevertheless, little is known about the genetic bases for these differences, how they manifest themselves in specific behaviors, and their distribution among symbionts of different host species. In this study, we sequenced the genomes of Vibrio fischeri isolated from the tissues of squids and fishes and applied comparative genomics approaches to look for patterns between symbiont lineages and host colonization behavior. In addition, we identified the only two genes that were exclusively present in all V. fischeri strains isolated from the light organs of sepiolid squid species. Mutational studies of these genes indicated that they both played a role in colonization of the squid light organ, emphasizing the value of applying a comparative genomics approach in the study of symbioses.

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) establishes lifelong infections in humans, a process that relies on its ability to thwart innate and adaptive immune defenses of the host. Recently, we reported that HIV-1 infection results in a dramatic reduction of the cellular peroxisome pool. Peroxisomes are metabolic organelles that also function as signaling platforms in the innate immune response. Here, we show that the HIV-1 accessory protein Vpu is necessary and sufficient for the depletion of cellular peroxisomes during infection. Vpu induces the expression of four microRNAs that target mRNAs encoding proteins required for peroxisome formation and metabolic function. The ability of Vpu to downregulate peroxisomes was found to be dependent upon the Wnt/β-catenin signaling pathway. Given the importance of peroxisomes in innate immune signaling and central nervous system function, the roles of Vpu in dampening antiviral signaling appear to be more diverse than previously realized. Finally, our findings highlight a potential role for Wnt/β-catenin signaling in peroxisome homeostasis through modulating the production of biogenesis factors.

IMPORTANCE People living with HIV can experience accelerated aging and the development of neurological disorders. Recently, we reported that HIV-1 infection results in a dramatic loss of peroxisomes in macrophages and brain tissue. This is significant because (i) peroxisomes are important for the innate immune response and (ii) loss of peroxisome function is associated with cellular aging and neurodegeneration. Accordingly, understanding how HIV-1 infection causes peroxisome depletion may provide clues regarding how the virus establishes persistent infections and, potentially, the development of neurological disorders. Here, we show that the accessory protein Vpu is necessary and sufficient for the induction of microRNAs that target peroxisome biogenesis factors. The ability of Vpu to downregulate peroxisome formation depends on the Wnt/β-catenin pathway. Thus, in addition to revealing a novel mechanism by which HIV-1 uses intracellular signaling pathways to target antiviral signaling platforms (peroxisomes), we have uncovered a previously unknown link between the Wnt/β-catenin pathway and peroxisome homeostasis.

ABSTRACT

Arthritogenic alphaviruses such as Ross River and Chikungunya viruses cause debilitating muscle and joint pain and pose significant challenges in the light of recent outbreaks. How host immune responses are orchestrated after alphaviral infections and lead to musculoskeletal inflammation remains poorly understood. Here, we show that myositis induced by Ross River virus (RRV) infection is driven by CD11b^hi Ly6C^hi inflammatory monocytes and followed by the establishment of a CD11b^hi Ly6C^lo CX₃CR1⁺ macrophage population in the muscle upon recovery. Selective modulation of CD11b^hi Ly6C^hi monocyte migration to infected muscle using immune-modifying microparticles (IMP) reduced disease score, tissue damage, and inflammation and promoted the accumulation of CX₃CR1⁺ macrophages, enhancing recovery and resolution. Here, we detail the role of immune pathology, describing a poorly characterized muscle macrophage subset as part of the dynamics of alphavirus-induced myositis and tissue recovery and identify IMP as an effective immunomodulatory approach. Given the lack of specific treatments available for alphavirus-induced pathologies, this study highlights a therapeutic potential for simple immune modulation by IMP in infected individuals in the event of large alphavirus outbreaks.

IMPORTANCE Arthritogenic alphaviruses cause debilitating inflammatory disease, and current therapies are restricted to palliative approaches. Here, we show that following monocyte-driven muscle inflammation, tissue recovery is associated with the accumulation of CX₃CR1⁺ macrophages in the muscle. Modulating inflammatory monocyte infiltration using immune-modifying microparticles (IMP) reduced tissue damage and inflammation and enhanced the formation of tissue repair-associated CX₃CR1⁺ macrophages in the muscle. This shows that modulating key effectors of viral inflammation using microparticles can alter the outcome of disease by facilitating the accumulation of macrophage subsets associated with tissue repair.

ABSTRACT

Synthesis and cleavage of the cell wall polymer peptidoglycan (PG) are carefully orchestrated processes and are essential for the growth and survival of bacteria. Yet, the function and importance of many enzymes that act on PG in Mycobacterium tuberculosis remain to be elucidated. We demonstrate that the activity of the N-acetylmuramyl-l-alanine amidase Ami1 is dispensable for cell division in M. tuberculosis in vitro yet contributes to the bacterium’s ability to persist during chronic infection in mice. Furthermore, the d,l-endopeptidase RipA, a predicted essential enzyme, is dispensable for the viability of M. tuberculosis but required for efficient cell division in vitro and in vivo. Depletion of RipA sensitizes M. tuberculosis to rifampin and to cell envelope-targeting antibiotics. Ami1 helps sustain residual cell division in cells lacking RipA, but the partial redundancy provided by Ami1 is not sufficient during infection, as depletion of RipA prevents M. tuberculosis from replicating in macrophages and leads to dramatic killing of the bacteria in mice. Notably, RipA is essential for persistence of M. tuberculosis in mice, suggesting that cell division is required during chronic mouse infection. Despite the multiplicity of enzymes acting on PG with redundant functions, we have identified two PG hydrolases that are important for M. tuberculosis to replicate and persist in the host.

IMPORTANCE Tuberculosis (TB) is a major global heath burden, with 1.6 million people succumbing to the disease every year. The search for new drugs to improve the current chemotherapeutic regimen is crucial to reducing this global health burden. The cell wall polymer peptidoglycan (PG) has emerged as a very successful drug target in bacterial pathogens, as many currently used antibiotics target the synthesis of this macromolecule. However, the multitude of genes encoding PG-synthesizing and PG-modifying enzymes with apparent redundant functions has hindered the identification of novel drug targets in PG synthesis in Mycobacterium tuberculosis. Here, we demonstrate that two PG-cleaving enzymes are important for virulence of M. tuberculosis. In particular, the d,l-endopeptidase RipA represents a potentially attractive drug target, as its depletion results in the clearance of M. tuberculosis from the host and renders the bacteria hypersusceptible to rifampin, a frontline TB drug, and to several cell wall-targeting antibiotics.

ABSTRACT

The synergy between Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) interferes with therapy and facilitates the pathogenesis of both human pathogens. Fundamental mechanisms by which M. tuberculosis exacerbates HIV-1 infection are not clear. Here, we show that exosomes secreted by macrophages infected with M. tuberculosis, including drug-resistant clinical strains, reactivated HIV-1 by inducing oxidative stress. Mechanistically, M. tuberculosis-specific exosomes realigned mitochondrial and nonmitochondrial oxygen consumption rates (OCR) and modulated the expression of host genes mediating oxidative stress response, inflammation, and HIV-1 transactivation. Proteomics analyses revealed the enrichment of several host factors (e.g., HIF-1α, galectins, and Hsp90) known to promote HIV-1 reactivation in M. tuberculosis-specific exosomes. Treatment with a known antioxidant—N-acetyl cysteine (NAC)—or with inhibitors of host factors—galectins and Hsp90—attenuated HIV-1 reactivation by M. tuberculosis-specific exosomes. Our findings uncover new paradigms for understanding the redox and bioenergetics bases of HIV-M. tuberculosis coinfection, which will enable the design of effective therapeutic strategies.

IMPORTANCE Globally, individuals coinfected with the AIDS virus (HIV-1) and with M. tuberculosis (causative agent of tuberculosis [TB]) pose major obstacles in the clinical management of both diseases. At the heart of this issue is the apparent synergy between the two human pathogens. On the one hand, mechanisms induced by HIV-1 for reactivation of TB in AIDS patients are well characterized. On the other hand, while clinical findings clearly identified TB as a risk factor for HIV-1 reactivation and associated mortality, basic mechanisms by which M. tuberculosis exacerbates HIV-1 replication and infection remain poorly characterized. The significance of our research is in identifying the role of fundamental mechanisms such as redox and energy metabolism in catalyzing HIV-M. tuberculosis synergy. The quantification of redox and respiratory parameters affected by M. tuberculosis in stimulating HIV-1 will greatly enhance our understanding of HIV-M. tuberculosis coinfection, leading to a wider impact on the biomedical research community and creating new translational opportunities.

ABSTRACT

Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage-infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra- and interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy.

IMPORTANCE We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets.

ABSTRACT

Bacterial flagella are rotating nanomachines required for motility. Flagellar gene expression and protein secretion are coordinated for efficient flagellar biogenesis. Polar flagellates, unlike peritrichous bacteria, commonly order flagellar rod and hook gene transcription as a separate step after production of the MS ring, C ring, and flagellar type III secretion system (fT3SS) core proteins that form a competent fT3SS. Conserved regulatory mechanisms in diverse polar flagellates to create this polar flagellar transcriptional program have not been thoroughly assimilated. Using in silico and genetic analyses and our previous findings in Campylobacter jejuni as a foundation, we observed a large subset of Gram-negative bacteria with the FlhF/FlhG regulatory system for polar flagellation to possess flagellum-associated two-component signal transduction systems (TCSs). We present data supporting a general theme in polar flagellates whereby MS ring, rotor, and fT3SS proteins contribute to a regulatory checkpoint during polar flagellar biogenesis. We demonstrate that Vibrio cholerae and Pseudomonas aeruginosa require the formation of this regulatory checkpoint for the TCSs to directly activate subsequent rod and hook gene transcription, which are hallmarks of the polar flagellar transcriptional program. By reprogramming transcription in V. cholerae to more closely follow the peritrichous flagellar transcriptional program, we discovered a link between the polar flagellar transcription program and the activity of FlhF/FlhG flagellar biogenesis regulators in which the transcriptional program allows polar flagellates to continue to produce flagella for motility when FlhF or FlhG activity may be altered. Our findings integrate flagellar transcriptional and biogenesis regulatory processes involved in polar flagellation in many species.

IMPORTANCE Relative to peritrichous bacteria, polar flagellates possess regulatory systems that order flagellar gene transcription differently and produce flagella in specific numbers only at poles. How transcriptional and flagellar biogenesis regulatory systems are interlinked to promote the correct synthesis of polar flagella in diverse species has largely been unexplored. We found evidence for many Gram-negative polar flagellates encoding two-component signal transduction systems with activity linked to the formation of flagellar type III secretion systems to enable production of flagellar rod and hook proteins at a discrete, subsequent stage during flagellar assembly. This polar flagellar transcriptional program assists, in some manner, the FlhF/FlhG flagellar biogenesis regulatory system, which forms specific flagellation patterns in polar flagellates in maintaining flagellation and motility when activity of FlhF or FlhG might be altered. Our work provides insight into the multiple regulatory processes required for polar flagellation.

ABSTRACT

In the absence of a vaccine, multidrug-resistant Neisseria gonorrhoeae has emerged as a major human health threat, and new approaches to treat gonorrhea are urgently needed. N. gonorrhoeae pili are posttranslationally modified by a glycan that terminates in a galactose. The terminal galactose is critical for initial contact with the human cervical mucosa via an interaction with the I-domain of complement receptor 3 (CR3). We have now identified the I-domain galactose-binding epitope and characterized its galactose-specific lectin activity. Using surface plasmon resonance and cellular infection assays, we found that a peptide mimic of this galactose-binding region competitively inhibited the N. gonorrhoeae-CR3 interaction. A compound library was screened for potential drugs that could similarly prohibit the N. gonorrhoeae-CR3 interaction and be repurposed as novel host-targeted therapeutics for multidrug-resistant gonococcal infections in women. Two drugs, methyldopa and carbamazepine, prevented and cured cervical cell infection by multidrug-resistant gonococci by blocking the gonococcal-CR3 I-domain interaction.

IMPORTANCE Novel therapies that avert the problem of Neisseria gonorrhoeae with acquired antibiotic resistance are urgently needed. Gonococcal infection of the human cervix is initiated by an interaction between a galactose modification made to its surface appendages, pili, and the I-domain region of (host) complement receptor 3 (CR3). By targeting this crucial gonococcal–I-domain interaction, it may be possible to prevent cervical infection in females. To this end, we identified the I-domain galactose-binding epitope of CR3 and characterized its galactose lectin activity. Moreover, we identified two drugs, carbamazepine and methyldopa, as effective host-targeted therapies for gonorrhea treatment. At doses below those currently used for their respective existing indications, both carbamazepine and methyldopa were more effective than ceftriaxone in curing cervical infection ex vivo. This host-targeted approach would not be subject to N. gonorrhoeae drug resistance mechanisms. Thus, our data suggest a long-term solution to the growing problem of multidrug-resistant N. gonorrhoeae infections.

ABSTRACT

Transporters belonging to the chromosomally encoded resistance-nodulation-division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria. However, the cotransfer of large gene clusters encoding RND-type pumps from the chromosome to a plasmid appears infrequent, and no plasmid-mediated RND efflux pump gene cluster has yet been found to confer resistance to tigecycline. Here, we identified a novel RND efflux pump gene cluster, designated tmexCD1-toprJ1, on plasmids from five pandrug-resistant Klebsiella pneumoniae isolates of animal origin. TMexCD1-TOprJ1 increased (by 4- to 32-fold) the MICs of tetracyclines (including tigecycline and eravacycline), quinolones, cephalosporins, and aminoglycosides for K. pneumoniae, Escherichia coli, and Salmonella. TMexCD1-TOprJ1 is closely related (64.5% to 77.8% amino acid identity) to the MexCD-OprJ efflux pump encoded on the chromosome of Pseudomonas aeruginosa. In an IncFIA plasmid, pHNAH8I, the tmexCD1-toprJ1 gene cluster lies adjacent to two genes encoding site-specific integrases, which may have been responsible for its acquisition. Expression of TMexCD1-TOprJ1 in E. coli resulted in increased tigecycline efflux and in K. pneumoniae negated the efficacy of tigecycline in an in vivo infection model. Expression of TMexCD1-TOprJ1 reduced the growth of E. coli and Salmonella but not K. pneumoniae. tmexCD1-toprJ1-positive Enterobacteriaceae isolates were rare in humans (0.08%) but more common in chicken fecal (14.3%) and retail meat (3.4%) samples. Plasmid-borne tmexCD1-toprJ1-like gene clusters were identified in sequences in GenBank from Enterobacteriaceae and Pseudomonas strains from multiple continents. The possibility of further global dissemination of the tmexCD1-toprJ1 gene cluster and its analogues in Enterobacteriaceae via plasmids may be an important consideration for public health planning.

IMPORTANCE In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae, the most problematic pathogens in human clinical settings—especially carbapenem-resistant K. pneumoniae. Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a "One Health" perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this.

ABSTRACT

Host-associated microbial communities are shaped by extrinsic and intrinsic factors to the holobiont organism. Environmental factors and microbe-microbe interactions act simultaneously on the microbial community structure, making the microbiome dynamics challenging to predict. The coral microbiome is essential to the health of coral reefs and sensitive to environmental changes. Here, we develop a dynamic model to determine the microbial community structure associated with the surface mucus layer (SML) of corals using temperature as an extrinsic factor and microbial network as an intrinsic factor. The model was validated by comparing the predicted relative abundances of microbial taxa to the relative abundances of microbial taxa from the sample data. The SML microbiome from Pseudodiploria strigosa was collected across reef zones in Bermuda, where inner and outer reefs are exposed to distinct thermal profiles. A shotgun metagenomics approach was used to describe the taxonomic composition and the microbial network of the coral SML microbiome. By simulating the annual temperature fluctuations at each reef zone, the model output is statistically identical to the observed data. The model was further applied to six scenarios that combined different profiles of temperature and microbial network to investigate the influence of each of these two factors on the model accuracy. The SML microbiome was best predicted by model scenarios with the temperature profile that was closest to the local thermal environment, regardless of the microbial network profile. Our model shows that the SML microbiome of P. strigosa in Bermuda is primarily structured by seasonal fluctuations in temperature at a reef scale, while the microbial network is a secondary driver.

IMPORTANCE Coral microbiome dysbiosis (i.e., shifts in the microbial community structure or complete loss of microbial symbionts) caused by environmental changes is a key player in the decline of coral health worldwide. Multiple factors in the water column and the surrounding biological community influence the dynamics of the coral microbiome. However, by including only temperature as an external factor, our model proved to be successful in describing the microbial community associated with the surface mucus layer (SML) of the coral P. strigosa. The dynamic model developed and validated in this study is a potential tool to predict the coral microbiome under different temperature conditions.

ABSTRACT

Horizontal gene transfer (HGT) promotes the spread of genes within bacterial communities. Among the HGT mechanisms, natural transformation stands out as being encoded by the bacterial core genome. Natural transformation is often viewed as a way to acquire new genes and to generate genetic mixing within bacterial populations. Another recently proposed function is the curing of bacterial genomes of their infectious parasitic mobile genetic elements (MGEs). Here, we propose that these seemingly opposing theoretical points of view can be unified. Although costly for bacterial cells, MGEs can carry functions that are at points in time beneficial to bacteria under stressful conditions (e.g., antibiotic resistance genes). Using computational modeling, we show that, in stochastic environments, an intermediate transformation rate maximizes bacterial fitness by allowing the reversible integration of MGEs carrying resistance genes, although these MGEs are costly for host cell replication. Based on this dual function (MGE acquisition and removal), transformation would be a key mechanism for stabilizing the bacterial genome in the long term, and this would explain its striking conservation.

IMPORTANCE Natural transformation is the acquisition, controlled by bacteria, of extracellular DNA and is one of the most common mechanisms of horizontal gene transfer, promoting the spread of resistance genes. However, its evolutionary function remains elusive, and two main roles have been proposed: (i) the new gene acquisition and genetic mixing within bacterial populations and (ii) the removal of infectious parasitic mobile genetic elements (MGEs). While the first one promotes genetic diversification, the other one promotes the removal of foreign DNA and thus genome stability, making these two functions apparently antagonistic. Using a computational model, we show that intermediate transformation rates, commonly observed in bacteria, allow the acquisition then removal of MGEs. The transient acquisition of costly MGEs with resistance genes maximizes bacterial fitness in environments with stochastic stress exposure. Thus, transformation would ensure both a strong dynamic of the bacterial genome in the short term and its long-term stabilization.

ABSTRACT

Population-level analyses are rapidly becoming inadequate to answer many of biomedical science and microbial ecology’s most pressing questions. The role of microbial populations within ecosystems and the evolutionary selective pressure on individuals depend fundamentally on the metabolic activity of single cells. Yet, many existing single-cell technologies provide only indirect evidence of metabolic specialization because they rely on correlations between transcription and phenotype established at the level of the population to infer activity. In this study, we take a top-down approach using isotope labels and secondary ion mass spectrometry to track the uptake of carbon and nitrogen atoms from different sources into biomass and directly observe dynamic changes in anabolic specialization at the level of single cells. We investigate the classic microbiological phenomenon of diauxic growth at the single-cell level in the model methylotroph Methylobacterium extorquens. In nature, this organism inhabits the phyllosphere, where it experiences diurnal changes in the available carbon substrates, necessitating an overhaul of central carbon metabolism. We show that the population exhibits a unimodal response to the changing availability of viable substrates, a conclusion that supports the canonical model but has thus far been supported by only indirect evidence. We anticipate that the ability to monitor the dynamics of anabolism in individual cells directly will have important applications across the fields of ecology, medicine, and biogeochemistry, especially where regulation downstream of transcription has the potential to manifest as heterogeneity that would be undetectable with other existing single-cell approaches.

IMPORTANCE Understanding how genetic information is realized as the behavior of individual cells is a long-term goal of biology but represents a significant technological challenge. In clonal microbial populations, variation in gene regulation is often interpreted as metabolic heterogeneity. This follows the central dogma of biology, in which information flows from DNA to RNA to protein and ultimately manifests as activity. At present, DNA and RNA can be characterized in single cells, but the abundance and activity of proteins cannot. Inferences about metabolic activity usually therefore rely on the assumption that transcription reflects activity. By tracking the atoms from which they build their biomass, we make direct observations of growth rate and substrate specialization in individual cells throughout a period of growth in a changing environment. This approach allows the flow of information from DNA to be constrained from the distal end of the regulatory cascade and will become an essential tool in the rapidly advancing field of single-cell metabolism.

ABSTRACT

Human genetics influence a range of pathological and clinical phenotypes in respiratory infections; however, the contributions of disease modifiers remain underappreciated. We exploited the Collaborative Cross (CC) mouse genetic-reference population to map genetic modifiers that affect the severity of Pseudomonas aeruginosa lung infection. Screening for P. aeruginosa respiratory infection in a cohort of 39 CC lines exhibits distinct disease phenotypes ranging from complete resistance to lethal disease. Based on major changes in the survival times, a quantitative-trait locus (QTL) was mapped on murine chromosome 3 to the genomic interval of Mb 110.4 to 120.5. Within this locus, composed of 31 protein-coding genes, two candidate genes, namely, dihydropyrimidine dehydrogenase (Dpyd) and sphingosine-1-phosphate receptor 1 (S1pr1), were identified according to the level of genome-wide significance and disease gene prioritization. Functional validation of the S1pr1 gene by pharmacological targeting in C57BL/6NCrl mice confirmed its relevance in P. aeruginosa pathophysiology. However, in a cohort of Canadian patients with cystic fibrosis (CF) disease, regional genetic-association analysis of the syntenic human locus on chromosome 1 (Mb 97.0 to 105.0) identified two single-nucleotide polymorphisms (rs10875080 and rs11582736) annotated to the Dpyd gene that were significantly associated with age at first P. aeruginosa infection. Thus, there is evidence that both genes might be implicated in this disease. Our results demonstrate that the discovery of murine modifier loci may generate information that is relevant to human disease progression.

IMPORTANCE Respiratory infection caused by P. aeruginosa is one of the most critical health burdens worldwide. People affected by P. aeruginosa infection include patients with a weakened immune system, such as those with cystic fibrosis (CF) genetic disease or non-CF bronchiectasis. Disease outcomes range from fatal pneumonia to chronic life-threatening infection and inflammation leading to the progressive deterioration of pulmonary function. The development of these respiratory infections is mediated by multiple causes. However, the genetic factors underlying infection susceptibility are poorly known and difficult to predict. Our study employed novel approaches and improved mouse disease models to identify genetic modifiers that affect the severity of P. aeruginosa lung infection. We identified candidate genes to enhance our understanding of P. aeruginosa infection in humans and provide a proof of concept that could be exploited for other human pathologies mediated by bacterial infection.

ABSTRACT

One of the primary functions of the mucosal barrier, found lining epithelial cells, is to serve as a first-line of defense against microbial pathogens. The major structural components of mucus are heavily glycosylated proteins called mucins. Mucins are key components of the innate immune system as they aid in the clearance of pathogens and can decrease pathogen virulence. It has also been recently reported that individual mucins and derived glycans can attenuate the virulence of the human pathogen Pseudomonas aeruginosa. Here, we show data indicating that mucins not only play a role in host defense but that they can also be subverted by P. aeruginosa to cause disease. We found that the mucin MUL-1 and mucin-derived monosaccharides N-acetyl-galactosamine and N-acetylglucosamine are required for P. aeruginosa killing of Caenorhabditis elegans. We also found that the defective adhesion of P. aeruginosa to human lung alveolar epithelial cells, deficient in the mucin MUC1, can be reversed by the addition of individual monosaccharides. The monosaccharides identified in this study are found in a wide range of organisms where they act as host factors required for bacterial pathogenesis. While mucins in C. elegans lack sialic acid caps, which makes their monosaccharides readily available, they are capped in other species. Pathogens such as P. aeruginosa that lack sialidases may rely on enzymes from other bacteria to utilize mucin-derived monosaccharides.

IMPORTANCE One of the first lines of defense present at mucosal epithelial tissues is mucus, which is a highly viscous material formed by mucin glycoproteins. Mucins serve various functions, but importantly they aid in the clearance of pathogens and debris from epithelial barriers and serve as innate immune factors. In this study, we describe a requirement of host monosaccharides, likely derived from host mucins, for the ability of Pseudomonas aeruginosa to colonize the intestine and ultimately cause death in Caenorhabditis elegans. We also demonstrate that monosaccharides alter the ability of bacteria to bind to both Caenorhabditis elegans intestinal cells and human lung alveolar epithelial cells, suggesting that there are conserved mechanisms underlying host-pathogen interactions in a range of organisms. By gaining a better understanding of pathogen-mucin interactions, we can develop better approaches to protect against pathogen infection.

ABSTRACT

Anti-galactose-α-1,3-galactose (anti-α-Gal) antibody is naturally expressed at a high level in humans. It constitutes about 1% of immunoglobulins found in human blood. Here, we designed a live attenuated influenza virus vaccine that can generate α-Gal epitopes in infected cells in order to facilitate opsonization of infected cells, thereby enhancing vaccine-induced immune responses. In the presence of normal human sera, cells infected with this mutant can enhance phagocytosis of human macrophages and cytotoxicity of NK cells in vitro. Using a knockout mouse strain that allows expression of anti-α-Gal antibody in vivo, we showed that this strategy can increase vaccine immunogenicity and the breadth of protection. This vaccine can induce 100% protection against a lethal heterosubtypic group 1 (H5) or group 2 (mouse-adapted H3) influenza virus challenge in the mouse model. In contrast, its heterosubtypic protective effect in wild-type or knockout mice that do not have anti-α-Gal antibody expression is only partial, demonstrating that the enhanced vaccine-induced protection requires anti-α-Gal antibody upon vaccination. Anti-α-Gal-expressing knockout mice immunized with this vaccine produce robust humoral and cell-mediated responses upon a lethal virus challenge. This vaccine can stimulate CD11b^lo/– pulmonary dendritic cells, which are known to be crucial for clearance of influenza virus. Our approach provides a novel strategy for developing next-generation influenza virus vaccines.

IMPORTANCE Influenza A viruses have multiple HA subtypes that are antigenically diverse. Classical influenza virus vaccines are subtype specific, and they cannot induce satisfactory heterosubtypic immunity against multiple influenza virus subtypes. Here, we developed a live attenuated H1N1 influenza virus vaccine that allows the expression of α-Gal epitopes by infected cells. Anti-α-Gal antibody is naturally produced by humans. In the presence of this antibody, human cells infected with this experimental vaccine virus can enhance several antibody-mediated immune responses in vitro. Importantly, mice expressing anti-α-Gal antibody in vivo can be fully protected by this H1N1 vaccine against a lethal H5 or H3 virus challenge. Our work demonstrates a new strategy for using a single influenza virus strain to induce broadly cross-reactive immune responses against different influenza virus subtypes.

ABSTRACT

The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa. We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD⁺ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period.

IMPORTANCE The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a further challenge to the development of therapeutics. Here, we identify that NADH activity has an important role in the induction of virulence in the pathogen P. aeruginosa. This finding, coupled with the ubiquity of NADH in bacterial pathogens, opens up the possibility of targeting enzymes that process NADH as a potential broad antivirulence approach.

ABSTRACT

The cell cycle is a critical component of cellular proliferation, differentiation, and response to stress, yet its role in the regulation of intracellular symbioses is not well understood. To explore host-symbiont cell cycle coordination in a marine symbiosis, we employed a model for coral-dinoflagellate associations: the tropical sea anemone Aiptasia (Exaiptasia pallida) and its native microalgal photosymbionts (Breviolum minutum and Breviolum psygmophilum). Using fluorescent labeling and spatial point-pattern image analyses to characterize cell population distributions in both partners, we developed protocols that are tailored to the three-dimensional cellular landscape of a symbiotic sea anemone tentacle. Introducing cultured symbiont cells to symbiont-free adult hosts increased overall host cell proliferation rates. The acceleration occurred predominantly in the symbiont-containing gastrodermis near clusters of symbionts but was also observed in symbiont-free epidermal tissue layers, indicating that the presence of symbionts contributes to elevated proliferation rates in the entire host during colonization. Symbiont cell cycle progression differed between cultured algae and those residing within hosts; the endosymbiotic state resulted in increased S-phase but decreased G₂/M-phase symbiont populations. These phenotypes and the deceleration of cell cycle progression varied with symbiont identity and host nutritional status. These results demonstrate that host and symbiont cells have substantial and species-specific effects on the proliferation rates of their mutualistic partners. This is the first empirical evidence to support species-specific regulation of the symbiont cell cycle within a single cnidarian-dinoflagellate association; similar regulatory mechanisms likely govern interpartner coordination in other coral-algal symbioses and shape their ecophysiological responses to a changing climate.

IMPORTANCE Biomass regulation is critical to the overall health of cnidarian-dinoflagellate symbioses. Despite the central role of the cell cycle in the growth and proliferation of cnidarian host cells and dinoflagellate symbionts, there are few studies that have examined the potential for host-symbiont coregulation. This study provides evidence for the acceleration of host cell proliferation when in local proximity to clusters of symbionts within cnidarian tentacles. The findings suggest that symbionts augment the cell cycle of not only their enveloping host cells but also neighboring cells in the epidermis and gastrodermis. This provides a possible mechanism for rapid colonization of cnidarian tissues. In addition, the cell cycles of symbionts differed depending on nutritional regime, symbiotic state, and species identity. The responses of cell cycle profiles to these different factors implicate a role for species-specific regulation of symbiont cell cycles within host cnidarian tissues.

ABSTRACT

The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3' untranslated regions (UTRs). The umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3' UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant’s vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana, p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3' UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor.

IMPORTANCE Nonsense-mediated decay (NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3' untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.

ABSTRACT

During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. Here, we identified a novel cis-acting sequence (5'-GUA AAG CUA CGG GAC GGU-3'), intronic splicing enhancer 3 (ISE3), which lies 72 nucleotides upstream of the second splice acceptor (A2-2) site of the second intron that defines the exon of the mRNA encoding the 11-kDa viral nonstructural protein. RNA binding motif protein 45 (RBM45) specifically binds to ISE3 with high affinity (equilibrium dissociation constant [K_D] = 33 nM) mediated by its RNA recognition domain and 2-homo-oligomer assembly domain (RRM2-HOA). Knockdown of RBM45 expression or ectopic overexpression of RRM2-HOA in human erythroid progenitor cells (EPCs) expanded ex vivo significantly decreased the level of viral mRNA spliced at the A2-2 acceptor but not that of the mRNA spliced at A2-1 that encodes VP2. Moreover, silent mutations of ISE3 in an infectious DNA of B19V significantly reduced 11-kDa expression. Notably, RBM45 also specifically interacts in vitro with ISE2, which shares the octanucleotide (GGGACGGU) with ISE3. Taken together, our results suggest that RBM45, through binding to both ISE2 and ISE3, is an essential host factor for maturation of 11-kDa-encoding mRNA.

IMPORTANCE Human parvovirus B19 (B19V) is a human pathogen that causes severe hematological disorders in immunocompromised individuals. B19V infection has a remarkable tropism with respect to human erythroid progenitor cells (EPCs) in human bone marrow and fetal liver. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. Our studies revealed that a cellular RNA binding protein, RBM45, binds to two intron splicing enhancers and is essential for the maturation of the small nonstructural protein 11-kDa-encoding mRNA. The 11-kDa protein plays an important role not only in B19V infection-induced apoptosis but also in viral DNA replication. Thus, the identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders.

ABSTRACT

Each year, >180,000 infants become infected via mother-to-child transmission (MTCT) of HIV despite the availability of effective maternal antiretroviral treatments, underlining the need for a maternal HIV vaccine. We characterized 224 maternal HIV envelope (Env)-specific IgG monoclonal antibodies (MAbs) from seven nontransmitting and transmitting HIV-infected U.S. and Malawian mothers and examined their neutralization activities against nontransmitted autologous circulating viruses and infant-transmitted founder (infant-T/F) viruses. Only a small subset of maternal viruses, 3 of 72 (4%), were weakly neutralized by maternal linear V3 epitope-specific IgG MAbs, whereas 6 out of 6 (100%) infant-T/F viruses were neutralization resistant to these V3-specific IgG MAbs. We also show that maternal-plasma broadly neutralizing antibody (bNAb) responses targeting the V3 glycan supersite in a transmitting woman may have selected for an N332 V3 glycan neutralization-resistant infant-T/F virus. These data have important implications for bNAb-eliciting vaccines and passively administered bNAbs in the setting of MTCT.

IMPORTANCE Efforts to eliminate MTCT of HIV with antiretroviral therapy (ART) have met little success, with >180,000 infant infections each year worldwide. It is therefore likely that additional immunologic strategies that can synergize with ART will be required to eliminate MTCT of HIV. To this end, understanding the role of maternal HIV Env-specific IgG antibodies in the setting of MTCT is crucial. In this study, we found that maternal-plasma broadly neutralizing antibody (bNAb) responses can select for T/F viruses that initiate infection in infants. We propose that clinical trials testing the efficacy of single bNAb specificities should not include HIV-infected pregnant women, as a single bNAb might select for neutralization-resistant infant-T/F viruses.

ABSTRACT

Gram-negative bacteria are intrinsically resistant to many antibiotics due to their outer membrane barrier. Although the outer membrane has been studied for decades, there is much to uncover about the biology and permeability of this complex structure. Investigating synthetic genetic interactions can reveal a great deal of information about genetic function and pathway interconnectivity. Here, we performed synthetic genetic arrays (SGAs) in Escherichia coli by crossing a subset of gene deletion strains implicated in outer membrane permeability with nonessential gene and small RNA (sRNA) deletion collections. Some 155,400 double-deletion strains were grown on rich microbiological medium with and without subinhibitory concentrations of two antibiotics excluded by the outer membrane, vancomycin and rifampin, to probe both genetic interactions and permeability. The genetic interactions of interest were synthetic sick or lethal (SSL) gene deletions that were detrimental to the cell in combination but had a negligible impact on viability individually. On average, there were ~30, ~36, and ~40 SSL interactions per gene under no-drug, rifampin, and vancomycin conditions, respectively; however, many of these involved frequent interactors. Our data sets have been compiled into an interactive database called the Outer Membrane Interaction (OMI) Explorer, where genetic interactions can be searched, visualized across the genome, compared between conditions, and enriched for gene ontology (GO) terms. A set of SSL interactions revealed connectivity and permeability links between enterobacterial common antigen (ECA) and lipopolysaccharide (LPS) of the outer membrane. This data set provides a novel platform to generate hypotheses about outer membrane biology and permeability.

IMPORTANCE Gram-negative bacteria are a major concern for public health, particularly due to the rise of antibiotic resistance. It is important to understand the biology and permeability of the outer membrane of these bacteria in order to increase the efficacy of antibiotics that have difficulty penetrating this structure. Here, we studied the genetic interactions of a subset of outer membrane-related gene deletions in the model Gram-negative bacterium E. coli. We systematically combined these mutants with 3,985 nonessential gene and small RNA deletion mutations in the genome. We examined the viability of these double-deletion strains and probed their permeability characteristics using two antibiotics that have difficulty crossing the outer membrane barrier. An understanding of the genetic basis for outer membrane integrity can assist in the development of new antibiotics with favorable permeability properties and the discovery of compounds capable of increasing outer membrane permeability to enhance the activity of existing antibiotics.

ABSTRACT

Microbes affect vertebrates on timescales from daily to evolutionary, and the cumulative effect of these interactions is immense. However, how microbiomes compare across (host) species is poorly understood, as most studies focus on relatively few species. A recent mBio article by S. J. Song, J. G. Sanders, F. Delsuc, J. Metcalf, et al. (mBio 11:e02901-19, 2019, https://doi.org/10.1128/mBio.02901-19) expands our collective understanding of the vertebrate microbiome by analyzing ~900 species. They demonstrate that patterns within mammals contrast with those within birds. Their results suggest many hypotheses about the role of host ecology and evolution on microbiome variation. Bats, the only volant mammals, appear to contradict many of the general mammal microbiome trends, in some ways resembling birds. What role has powered flight, and the evolution thereof, played in microbiome structure and function? Comparative methods, mechanistic hypotheses, and theory will elucidate this exciting question (and others) that we can ask using Song, Sanders et al.’s data and results.

ABSTRACT

Merkel cell polyomavirus (MCPyV) is the only polyomavirus known to be associated with tumorigenesis in humans. Similarly to other polyomaviruses, MCPyV expresses a large tumor antigen (LT-Ag) that, together with a small tumor antigen (sT-Ag), contributes to cellular transformation and that is of critical importance for the initiation of the viral DNA replication. Understanding the cellular protein network regulated by MCPyV early proteins will significantly contribute to our understanding of the natural MCPyV life cycle as well as of the mechanisms by which the virus contributes to cellular transformation. We here describe KRAB-associated protein 1 (Kap1), a chromatin remodeling factor involved in cotranscriptional regulation, as a novel protein interaction partner of MCPyV T antigens sT and LT. Kap1 knockout results in a significant increase in the level of viral DNA replication that is highly suggestive of Kap1 being an important host restriction factor during MCPyV infection. Differently from other DNA viruses, MCPyV gene expression is unaffected in the absence of Kap1 and Kap1 does not associate with the viral genome. Instead, we show that in primary normal human dermal fibroblast (nHDF) cells, MCPyV DNA replication, but not T antigen expression alone, induces ataxia telangiectasia mutated (ATM) kinase-dependent Kap1 S824 phosphorylation, a mechanism that typically facilitates repair of double-strand breaks in heterochromatin by arresting the cells in G₂. We show that MCPyV-induced inhibition of cell proliferation is mainly conferred by residues within the origin binding domain and thereby by viral DNA replication. Our data suggest that phosphorylation of Kap1 and subsequent Kap1-dependent G₂ arrest/senescence represent host defense mechanisms against MCPyV replication in nHDF cells.

IMPORTANCE We here describe Kap1 as a restriction factor in MCPyV infection. We report a novel, indirect mechanism by which Kap1 affects MCPyV replication. In contrast with from other DNA viruses, Kap1 does not associate with the viral genome in MCPyV infection and has no impact on viral gene expression. In MCPyV-infected nHDF cells, Kap1 phosphorylation (pKap1 S824) accumulates because of genomic stress mainly induced by viral DNA replication. In contrast, ectopic expression of LT or LT MCPyV mutants, previously shown to be important for induction of genotoxic stress, does not result in a similar extent of pKap1 accumulation. We show that cells actively replicating MCPyV accumulate pKap1 (in a manner dependent on the presence of ATM) and display a senescence phenotype reflected by G₂ arrest. These results are supported by transcriptome analyses showing that LT antigen, in a manner dependent on the presence of Kap1, induces expression of secreted factors, which is known as the senescence-associated secretory phenotype (SASP).

ABSTRACT

Although the pathogen recognition receptor pathways that activate cell-intrinsic antiviral responses are well delineated, less is known about how the host regulates this response to prevent sustained signaling and possible immune-mediated damage. Using a genome-wide CRISPR-Cas9 screening approach to identify host factors that modulate interferon-stimulated gene (ISG) expression, we identified the DNA binding protein Barrier-to-autointegration factor 1 (Banf1), a previously described inhibitor of retrovirus integration, as a modulator of basal cell-intrinsic immunity. Ablation of Banf1 by gene editing resulted in chromatin activation near host defense genes with associated increased expression of ISGs, including Oas2, Rsad2 (viperin), Ifit1, and ISG15. The phenotype in Banf1-deficient cells occurred through a cGAS-, STING-, and IRF3-dependent signaling axis, was associated with reduced infection of RNA and DNA viruses, and was reversed in Banf1 complemented cells. Confocal microscopy and biochemical studies revealed that a loss of Banf1 expression resulted in higher level of cytosolic double-stranded DNA at baseline. Our study identifies an undescribed role for Banf1 in regulating the levels of cytoplasmic DNA and cGAS-dependent ISG homeostasis and suggests possible therapeutic directions for promoting or inhibiting cell-intrinsic innate immune responses.

IMPORTANCE Although the interferon (IFN) signaling pathway is a key host mechanism to restrict infection of a diverse range of viral pathogens, its unrestrained activity either at baseline or in the context of an immune response can result in host cell damage and injury. Here, we used a genome-wide CRISPR-Cas9 screen and identified the DNA binding protein Barrier-to-autointegration factor 1 (Banf1) as a modulator of basal cell-intrinsic immunity. A loss of Banf1 expression resulted in higher level of cytosolic double-stranded DNA at baseline, which triggered IFN-stimulated gene expression via a cGAS-STING-IRF3 axis that did not require type I IFN or STAT1 signaling. Our experiments define a regulatory network in which Banf1 limits basal inflammation by preventing self DNA accumulation in the cytosol.

ABSTRACT

Recent global advocacy efforts have highlighted the importance of development of a vaccine against group A Streptococcus (GAS). Combo5 is a non-M protein-based vaccine that provides protection against GAS skin infection in mice and reduces the severity of pharyngitis in nonhuman primates. However, Combo5 with the addition of aluminum hydroxide (alum) as an adjuvant failed to protect against invasive GAS infection of mice. Here, we show that formulation of Combo5 with adjuvants containing saponin QS21 significantly improves protective efficacy, even though all 7 adjuvants tested generated high antigen-specific IgG antibody titers, including alum. Detailed characterization of Combo5 formulated with SMQ adjuvant, a squalene-in-water emulsion containing a TLR4 agonist and QS21, showed significant differences from the results obtained with alum in IgG subclasses generated following immunization, with an absence of GAS opsonizing antibodies. SMQ, but not alum, generated strong interleukin-6 (IL-6), gamma interferon (IFN-), and tumor necrosis alpha (TNF-α) responses. This work highlights the importance of adjuvant selection for non-M protein-based GAS vaccines to optimize immune responses and protective efficacy.

IMPORTANCE Availability of a group A Streptococcus vaccine remains an unmet public health need. Here, we tested different adjuvant formulations to improve the protective efficacy of non-M protein vaccine Combo5 in an invasive disease model. We show that novel adjuvants can dramatically shape the type of immune response developed following immunization with Combo5 and significantly improve protection. In addition, protection afforded by Combo5 is not mediated by opsonizing antibodies, believed to be the main correlate of protection against GAS infections. Overall, this report highlights the importance of adjuvant selection in raising protective immune responses against GAS invasive infection. Adjuvants that can provide a more balanced Th1/Th2-type response may be required to optimize protection of GAS vaccines, particularly those based on non-M protein antigens.

ABSTRACT

OleA, a member of the thiolase superfamily, is known to catalyze the Claisen condensation of long-chain acyl coenzyme A (acyl-CoA) substrates, initiating metabolic pathways in bacteria for the production of membrane lipids and β-lactone natural products. OleA homologs are found in diverse bacterial phyla, but to date, only one homodimeric OleA has been successfully purified to homogeneity and characterized in vitro. A major impediment for the identification of new OleA enzymes has been protein instability and time-consuming in vitro assays. Here, we developed a bioinformatic pipeline to identify OleA homologs and a new rapid assay to screen OleA enzyme activity in vivo and map their taxonomic diversity. The screen is based on the discovery that OleA displayed surprisingly high rates of p-nitrophenyl ester hydrolysis, an activity not shared by other thiolases, including FabH. The high rates allowed activity to be determined in vitro and with heterologously expressed OleA in vivo via the release of the yellow p-nitrophenol product. Seventy-four putative oleA genes identified in the genomes of diverse bacteria were heterologously expressed in Escherichia coli, and 25 showed activity with p-nitrophenyl esters. The OleA proteins tested were encoded in variable genomic contexts from seven different phyla and are predicted to function in distinct membrane lipid and β-lactone natural product metabolic pathways. This study highlights the diversity of unstudied OleA proteins and presents a rapid method for their identification and characterization.

IMPORTANCE Microbially produced β-lactones are found in antibiotic, antitumor, and antiobesity drugs. Long-chain olefinic membrane hydrocarbons have potential utility as fuels and specialty chemicals. The metabolic pathway to both end products share bacterial enzymes denoted as OleA, OleC, and OleD that transform acyl-CoA cellular intermediates into β-lactones. Bacteria producing membrane hydrocarbons via the Ole pathway additionally express a β-lactone decarboxylase, OleB. Both β-lactone and olefin biosynthesis pathways are initiated by OleA enzymes that define the overall structure of the final product. There is currently very limited information on OleA enzymes apart from the single representative from Xanthomonas campestris. In this study, bioinformatic analysis identified hundreds of new, putative OleA proteins, 74 proteins were screened via a rapid whole-cell method, leading to the identification of 25 stably expressed OleA proteins representing seven bacteria phyla.

ABSTRACT

Iron is a vital mineral for almost all living organisms and has a pivotal role in central metabolism. Despite its great abundance on earth, the accessibility for microorganisms is often limited, because poorly soluble ferric iron (Fe³⁺) is the predominant oxidation state in an aerobic environment. Hence, the reduction of Fe³⁺ is of essential importance to meet the cellular demand of ferrous iron (Fe²⁺) but might become detrimental as excessive amounts of intracellular Fe²⁺ tend to undergo the cytotoxic Fenton reaction in the presence of hydrogen peroxide. We demonstrate that the complex formation rate of Fe³⁺ and phenolic compounds like protocatechuic acid was increased by 46% in the presence of HCO₃^– and thus accelerated the subsequent redox reaction, yielding reduced Fe²⁺. Consequently, elevated CO₂/HCO₃^– levels increased the intracellular Fe²⁺ availability, which resulted in at least 50% higher biomass-specific fluorescence of a DtxR-based Corynebacterium glutamicum reporter strain, and stimulated growth. Since the increased Fe²⁺ availability was attributed to the interaction of HCO₃^– and chemical iron reduction, the abiotic effect postulated in this study is of general relevance in geochemical and biological environments.

IMPORTANCE In an oxygenic environment, poorly soluble Fe³⁺ must be reduced to meet the cellular Fe²⁺ demand. This study demonstrates that elevated CO₂/HCO₃^– levels accelerate chemical Fe³⁺ reduction through phenolic compounds, thus increasing intracellular Fe²⁺ availability. A number of biological environments are characterized by the presence of phenolic compounds and elevated HCO₃^– levels and include soil habitats and the human body. Fe²⁺ availability is of particular interest in the latter, as it controls the infectiousness of pathogens. Since the effect postulated here is abiotic, it generally affects the Fe²⁺ distribution in nature.

ABSTRACT

Frequent and excessive use of antibiotics primes patients to Clostridioides difficile infection (CDI), which leads to fatal pseudomembranous colitis, with limited treatment options. In earlier reports, we used a drug repurposing strategy and identified amoxapine (an antidepressant), doxapram (a breathing stimulant), and trifluoperazine (an antipsychotic), which provided significant protection to mice against lethal infections with several pathogens, including C. difficile. However, the mechanisms of action of these drugs were not known. Here, we provide evidence that all three drugs offered protection against experimental CDI by reducing bacterial burden and toxin levels, although the drugs were neither bacteriostatic nor bactericidal in nature and had minimal impact on the composition of the microbiota. Drug-mediated protection was dependent on the presence of the microbiota, implicating its role in evoking host defenses that promoted protective immunity. By utilizing transcriptome sequencing (RNA-seq), we identified that each drug increased expression of several innate immune response-related genes, including those involved in the recruitment of neutrophils, the production of interleukin 33 (IL-33), and the IL-22 signaling pathway. The RNA-seq data on selected genes were confirmed by quantitative real-time PCR (qRT-PCR) and protein assays. Focusing on amoxapine, which had the best anti-CDI outcome, we demonstrated that neutralization of IL-33 or depletion of neutrophils resulted in loss of drug efficacy. Overall, our lead drugs promote disease alleviation and survival in the murine model through activation of IL-33 and by clearing the pathogen through host defense mechanisms that critically include an early influx of neutrophils.

IMPORTANCE Clostridioides difficile is a spore-forming anaerobic bacterium and the leading cause of antibiotic-associated colitis. With few therapeutic options and high rates of disease recurrence, the need to develop new treatment options is urgent. Prior studies utilizing a repurposing approach identified three nonantibiotic Food and Drug Administration-approved drugs, amoxapine, doxapram, and trifluoperazine, with efficacy against a broad range of human pathogens; however, the protective mechanisms remained unknown. Here, we identified mechanisms leading to drug efficacy in a murine model of lethal C. difficile infection (CDI), advancing our understanding of the role of these drugs in infectious disease pathogenesis that center on host immune responses to C. difficile. Overall, these studies highlight the crucial involvement of innate immune responses, as well as the importance of immunomodulation as a potential therapeutic option to combat CDI.

ABSTRACT

The ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain chronic infection of its host for prolonged periods. Despite this, little is known regarding whether and how T. gondii bradyzoites, a quasi-dormant life stage residing within intracellular cysts, manipulate the host cell to maintain persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro. Because MYR1 is known to be involved in the translocation of parasite-derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show, by immunofluorescence assays, that most effectors accumulate in the host nucleus at early but not late time points after infection, during the tachyzoite-to-bradyzoite transition and when parasites further along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon gamma signaling, which was previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with bradyzoites and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications regarding how this highly successful parasite maintains persistent infection of its host.

IMPORTANCE Toxoplasma bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life stage successfully establishes and maintains persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different time points after bradyzoite differentiation and found that they accumulated largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediated its function after prolonged infection with bradyzoites, and we provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.

ABSTRACT

Bacteria harbor viruses called bacteriophages that, like all viruses, co-opt the host cellular machinery to replicate. Although this relationship is at first glance parasitic, there are social interactions among and between bacteriophages and their bacterial hosts. These social interactions can take on many forms, including cooperation, altruism, and cheating. Such behaviors among individuals in groups of bacteria have been well described. However, the social nature of some interactions between phages or phages and bacteria is only now becoming clear. We are just beginning to understand how bacteriophages affect the sociobiology of bacteria, and we know even less about social interactions within bacteriophage populations. In this review, we discuss recent developments in our understanding of bacteriophage sociobiology, including how selective pressures influence the outcomes of social interactions between populations of bacteria and bacteriophages. We also explore how tripartite social interactions between bacteria, bacteriophages, and an animal host affect host-microbe interactions. Finally, we argue that understanding the sociobiology of bacteriophages will have implications for the therapeutic use of bacteriophages to treat bacterial infections.

ABSTRACT

Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile. Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms.

IMPORTANCE Clostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficile in vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.

ABSTRACT

Fungal infections are a major contributor to infectious disease-related deaths worldwide. Recently, global emergence of the fungal pathogen Candida auris has caused considerable concern because most C. auris isolates are resistant to fluconazole, the most commonly administered antifungal, and some isolates are resistant to drugs from all three major antifungal classes. To identify novel agents with bioactivity against C. auris, we screened 2,454 compounds from a diversity-oriented synthesis collection. Of the five hits identified, most shared a common rocaglate core structure and displayed fungicidal activity against C. auris. These rocaglate hits inhibited translation in C. auris but not in its pathogenic relative Candida albicans. Species specificity was contingent on variation at a single amino acid residue in Tif1, a fungal member of the eukaryotic initiation factor 4A (eIF4A) family of translation initiation factors known to be targeted by rocaglates. Rocaglate-mediated inhibition of translation in C. auris activated a cell death program characterized by loss of mitochondrial membrane potential, increased caspase-like activity, and disrupted vacuolar homeostasis. In a rocaglate-sensitized C. albicans mutant engineered to express translation initiation factor 1 (Tif1) with the variant amino acid that we had identified in C. auris, translation was inhibited but no programmed cell death phenotypes were observed. This surprising finding suggests divergence between these related fungal pathogens in their pathways of cellular responses to translation inhibition. From a therapeutic perspective, the chemical biology that we have uncovered reveals species-specific vulnerability in C. auris and identifies a promising target for development of new, mechanistically distinct antifungals in the battle against this emerging pathogen.

IMPORTANCE Emergence of the fungal pathogen Candida auris has ignited intrigue and alarm within the medical community and the public at large. This pathogen is unusually resistant to antifungals, threatening to overwhelm current management options. By screening a library of structurally diverse molecules, we found that C. auris is surprisingly sensitive to translation inhibition by a class of compounds known as rocaglates (also known as flavaglines). Despite the high level of conservation across fungi in their protein synthesis machinery, these compounds inhibited translation initiation and activated a cell death program in C. auris but not in its relative Candida albicans. Our findings highlight a surprising divergence across the cell death programs operating in Candida species and underscore the need to understand the specific biology of a pathogen in attempting to develop more-effective treatments against it.

ABSTRACT

The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as a virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as a cross-linker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of β1-integrins on the basolateral plasma membrane of epithelial cells and causes rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive β1-integrins and also of complete α3β1-integrin–laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and causes the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by cross-linking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild-type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results, we suggest that LecB has a role in disseminating bacteria along the cell-basement membrane interface.

IMPORTANCE Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes of nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interactions is not well understood. The significance of our research is in identifying cellular mechanisms of how LecB facilitates P. aeruginosa infection. We introduce LecB as a new member of the list of bacterial molecules that bind integrins and show that P. aeruginosa can move forward underneath attached epithelial cells by loosening cell-basement membrane attachment in a LecB-dependent manner.

ABSTRACT

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria.

IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa. In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.

ABSTRACT

The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp⁰] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase.

IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.