ABSTRACT

Many HIV prevention strategies are currently under consideration where it is highly informative to know the study participants’ times of infection. These can be estimated using viral sequence data sampled early in infection. However, there are several scenarios that, if not addressed, can skew timing estimates. These include multiple transmitted/founder (TF) viruses, APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like)-mediated mutational enrichment, and recombination. Here, we suggest a pipeline to identify these problems and resolve the biases that they introduce. We then compare two modeling strategies to obtain timing estimates from sequence data. The first, Poisson Fitter (PF), is based on a Poisson model of random accumulation of mutations relative to the TF virus (or viruses) that established the infection. The second uses a coalescence-based phylogenetic strategy as implemented in BEAST. The comparison is based on timing predictions using plasma viral RNA (cDNA) sequence data from 28 simian-human immunodeficiency virus (SHIV)-infected animals for which the exact day of infection is known. In this particular setting, based on nucleotide sequences from samples obtained in early infection, the Poisson method yielded more accurate, more precise, and unbiased estimates for the time of infection than did the explored implementations of BEAST.

IMPORTANCE The inference of the time of infection is a critical parameter in testing the efficacy of clinical interventions in protecting against HIV-1 infection. For example, in clinical trials evaluating the efficacy of passively delivered antibodies (Abs) for preventing infections, accurate time of infection data are essential for discerning levels of the Abs required to confer protection, given the natural Ab decay rate in the human body. In such trials, genetic sequences from early in the infection are regularly sampled from study participants, generally prior to immune selection, when the viral population is still expanding and genetic diversity is low. In this particular setting of early viral growth, the Poisson method is superior to the alternative approach based on coalescent methods. This approach can also be applied in human vaccine trials, where accurate estimates of infection times help ascertain if vaccine-elicited immune protection wanes over time.

ABSTRACT

Bacillus anthracis is a spore-forming bacterium that causes devastating infections and has been used as a bioterror agent. This pathogen can survive hostile environments through the signaling activity of two-component systems, which couple environmental sensing with transcriptional activation to initiate a coordinated response to stress. In this work, we describe the identification of a two-component system, EdsRS, which mediates the B. anthracis response to the antimicrobial compound targocil. Targocil is a cell envelope-targeting compound that is toxic to B. anthracis at high concentrations. Exposure to targocil causes damage to the cellular barrier and activates EdsRS to induce expression of a previously uncharacterized cardiolipin synthase, which we have named ClsT. Both EdsRS and ClsT are required for protection against targocil-dependent damage. Induction of clsT by EdsRS during targocil treatment results in an increase in cardiolipin levels, which protects B. anthracis from envelope damage. Together, these results reveal that a two-component system signaling response to an envelope-targeting antimicrobial induces production of a phospholipid associated with stabilization of the membrane. Cardiolipin is then used to repair envelope damage and promote B. anthracis viability.

IMPORTANCE Compromising the integrity of the bacterial cell barrier is a common action of antimicrobials. Targocil is an antimicrobial that is active against the bacterial envelope. We hypothesized that Bacillus anthracis, a potential weapon of bioterror, senses and responds to targocil to alleviate targocil-dependent cell damage. Here, we show that targocil treatment increases the permeability of the cellular envelope and is particularly toxic to B. anthracis spores during outgrowth. In vegetative cells, two-component system signaling through EdsRS is activated by targocil. This results in an increase in the production of cardiolipin via a cardiolipin synthase, ClsT, which restores the loss of barrier function, thereby reducing the effectiveness of targocil. By elucidating the B. anthracis response to targocil, we have uncovered an intrinsic mechanism that this pathogen employs to resist toxicity and have revealed therapeutic targets that are important for bacterial defense against structural damage.

ABSTRACT

Intracellular bacterial pathogens remodel cellular functions during their infectious cycle via the coordinated actions of effector molecules delivered through dedicated secretion systems. While the function of many individual effectors is known, how they interact to promote pathogenesis is rarely understood. The zoonotic bacterium Brucella abortus, the causative agent of brucellosis, delivers effector proteins via its VirB type IV secretion system (T4SS) which mediate biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV). Here, we show that T4SS effectors BspB and RicA display epistatic interactions in Brucella replication. Defects in rBCV biogenesis and Brucella replication caused by deletion of bspB were dependent on the host GTPase Rab2a and suppressed by the deletion of ricA, indicating a role of Rab2-binding effector RicA in these phenotypic defects. Rab2a requirements for rBCV biogenesis and Brucella intracellular replication were abolished upon deletion of both bspB and ricA, demonstrating that the functional interaction of these effectors engages Rab2-dependent transport in the Brucella intracellular cycle. Expression of RicA impaired host secretion and caused Golgi fragmentation. While BspB-mediated changes in ER-to-Golgi transport were independent of RicA and Rab2a, BspB-driven alterations in Golgi vesicular traffic also involved RicA and Rab2a, defining BspB and RicA’s functional interplay at the Golgi interface. Altogether, these findings support a model where RicA modulation of Rab2a functions impairs Brucella replication but is compensated by BspB-mediated remodeling of Golgi apparatus-associated vesicular transport, revealing an epistatic interaction between these T4SS effectors.

IMPORTANCE Bacterial pathogens with an intracellular lifestyle modulate many host cellular processes to promote their infectious cycle. They do so by delivering effector proteins into host cells via dedicated secretion systems that target specific host functions. While the roles of many individual effectors are known, how their modes of action are coordinated is rarely understood. Here, we show that the zoonotic bacterium Brucella abortus delivers the BspB effector that mitigates the negative effect on bacterial replication that the RicA effector exerts via modulation of the host small GTPase Rab2. These findings provide an example of functional integration between bacterial effectors that promotes proliferation of pathogens.

ABSTRACT

The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of adcAII in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 adcAII mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated adcAII mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the adcAII mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated adcAII strains. However, transformation of adcAII with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated adcAII strains contained the same single nucleotide polymorphism in the capsule locus gene cps2E, which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of adcAII. Further investigation will be needed to characterize how mutation of adcAII affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype.

IMPORTANCE The Streptococcus pneumoniae capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of adcAII, a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of adcAII resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased S. pneumoniae virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the adcAII strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to affect capsule thickness. Our data provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identify an unexpected link between capsule thickness and adcAII, further investigation of which could further characterize mechanisms of capsule regulation.

ABSTRACT

Hepatitis C virus (HCV) infects millions of people worldwide, causing chronic liver disease that can lead to cirrhosis, hepatocellular carcinoma, and liver transplant. In the last several years, the advent of direct-acting antivirals, including NS3/4A protease inhibitors (PIs), has remarkably improved treatment outcomes of HCV-infected patients. However, selection of resistance-associated substitutions and polymorphisms among genotypes can lead to drug resistance and in some cases treatment failure. A proactive strategy to combat resistance is to constrain PIs within evolutionarily conserved regions in the protease active site. Designing PIs using the substrate envelope is a rational strategy to decrease the susceptibility to resistance by using the constraints of substrate recognition. We successfully designed two series of HCV NS3/4A PIs to leverage unexploited areas in the substrate envelope to improve potency, specifically against resistance-associated substitutions at D168. Our design strategy achieved better resistance profiles over both the FDA-approved NS3/4A PI grazoprevir and the parent compound against the clinically relevant D168A substitution. Crystallographic structural analysis and inhibition assays confirmed that optimally filling the substrate envelope is critical to improve inhibitor potency while avoiding resistance. Specifically, inhibitors that enhanced hydrophobic packing in the S4 pocket and avoided an energetically frustrated pocket performed the best. Thus, the HCV substrate envelope proved to be a powerful tool to design robust PIs, offering a strategy that can be translated to other targets for rational design of inhibitors with improved potency and resistance profiles.

IMPORTANCE Despite significant progress, hepatitis C virus (HCV) continues to be a major health problem with millions of people infected worldwide and thousands dying annually due to resulting complications. Recent antiviral combinations can achieve >95% cure, but late diagnosis, low access to treatment, and treatment failure due to drug resistance continue to be roadblocks against eradication of the virus. We report the rational design of two series of HCV NS3/4A protease inhibitors with improved resistance profiles by exploiting evolutionarily constrained regions of the active site using the substrate envelope model. Optimally filling the S4 pocket is critical to avoid resistance and improve potency. Our results provide drug design strategies to avoid resistance that are applicable to other quickly evolving viral drug targets.

ABSTRACT

Coinfections shape immunity and influence the development of inflammatory diseases, resulting in detrimental or beneficial outcome. Coinfections with concurrent Plasmodium species can alter malaria clinical evolution, and malaria infection itself can modulate autoimmune reactions. Yet, the underlying mechanisms remain ill defined. Here, we demonstrate that the protective effects of some rodent malaria strains on T cell-mediated inflammatory pathologies are due to an RNA virus cohosted in malaria-parasitized blood. We show that live and extracts of blood parasitized by Plasmodium berghei K173 or Plasmodium yoelii 17X YM, protect against P. berghei ANKA-induced experimental cerebral malaria (ECM) and myelin oligodendrocyte glycoprotein (MOG)/complete Freund’s adjuvant (CFA)-induced experimental autoimmune encephalomyelitis (EAE), and that protection is associated with a strong type I interferon (IFN-I) signature. We detected the presence of the RNA virus lactate dehydrogenase-elevating virus (LDV) in the protective Plasmodium stabilates and we established that LDV infection alone was necessary and sufficient to recapitulate the protective effects on ECM and EAE. In ECM, protection resulted from an IFN-I-mediated reduction in the abundance of splenic conventional dendritic cell and impairment of their ability to produce interleukin (IL)-12p70, leading to a decrease in pathogenic CD4⁺ Th1 responses. In EAE, LDV infection induced IFN-I-mediated abrogation of IL-23, thereby preventing the differentiation of granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing encephalitogenic CD4⁺ T cells. Our work identifies a virus cohosted in several Plasmodium stabilates across the community and deciphers its major consequences on the host immune system. More generally, our data emphasize the importance of considering contemporaneous infections for the understanding of malaria-associated and autoimmune diseases.

IMPORTANCE Any infection modifies the host immune status, potentially ameliorating or aggravating the pathophysiology of a simultaneous inflammatory condition. In the course of investigating how malaria infection modulates the severity of contemporaneous inflammatory diseases, we identified a nonpathogenic mouse virus in stabilates of two widely used rodent parasite lines: Plasmodium berghei K173 and Plasmodium yoelii 17X YM. We established that the protective effects of these Plasmodium lines on cerebral malaria and multiple sclerosis are exclusively due to this virus. The virus induces a massive type I interferon (IFN-I) response and causes quantitative and qualitative defects in the ability of dendritic cells to promote pathogenic T cell responses. Beyond revealing a possible confounding factor in rodent malaria models, our work uncovers some bases by which a seemingly innocuous viral (co)infection profoundly changes the immunopathophysiology of inflammatory diseases.

ABSTRACT

Microbial pathogens exploit host nutrients to proliferate and cause disease. Intracellular pathogens, particularly those exclusively living in the phagosome such as Histoplasma capsulatum, must adapt and acquire nutrients within the nutrient-limited phagosomal environment. In this study, we investigated which host nutrients could be utilized by Histoplasma as carbon sources to proliferate within macrophages. Histoplasma yeasts can grow on hexoses and amino acids but not fatty acids as the carbon source in vitro. Transcriptional analysis and metabolism profiling showed that Histoplasma yeasts downregulate glycolysis and fatty acid utilization but upregulate gluconeogenesis within macrophages. Depletion of glycolysis or fatty acid utilization pathways does not prevent Histoplasma growth within macrophages or impair virulence in vivo. However, loss of function in Pck1, the enzyme catalyzing the first committed step of gluconeogenesis, impairs Histoplasma growth within macrophages and severely attenuates virulence in vivo, indicating that Histoplasma yeasts rely on catabolism of gluconeogenic substrates (e.g., amino acids) to proliferate within macrophages.

IMPORTANCE Histoplasma is a primary human fungal pathogen that survives and proliferates within host immune cells, particularly within the macrophage phagosome compartment. The phagosome compartment is a nutrient-limited environment, requiring Histoplasma yeasts to be able to assimilate available carbon sources within the phagosome to meet their nutritional needs. In this study, we showed that Histoplasma yeasts do not utilize fatty acids or hexoses for growth within macrophages. Instead, Histoplasma yeasts consume gluconeogenic substrates to proliferate in macrophages. These findings reveal the phagosome composition from a nutrient standpoint and highlight essential metabolic pathways that are required for a phagosomal pathogen to proliferate in this intracellular environment.

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans and remains endemic in the Middle East since first being identified in 2012. There are currently no approved vaccines or therapies available for MERS-CoV. In this study, we evaluated parainfluenza virus 5 (PIV5)-based vaccine expressing the MERS-CoV envelope spike protein (PIV5/MERS-S) in a human DPP4 knockin C57BL/6 congenic mouse model (hDPP4 KI). Following a single-dose intranasal immunization, PIV5-MERS-S induced neutralizing antibody and robust T cell responses in hDPP4 KI mice. A single intranasal administration of 10⁴ PFU PIV5-MERS-S provided complete protection against a lethal challenge with mouse-adapted MERS-CoV (MERS_MA6.1.2) and improved virus clearance in the lung. In comparison, single-dose intramuscular immunization with 10⁶ PFU UV-inactivated MERS_MA6.1.2 mixed with Imject alum provided protection to only 25% of immunized mice. Intriguingly, an influx of eosinophils was observed only in the lungs of mice immunized with inactivated MERS-CoV, suggestive of a hypersensitivity-type response. Overall, our study indicated that PIV5-MERS-S is a promising effective vaccine candidate against MERS-CoV infection.

IMPORTANCE MERS-CoV causes lethal infection in humans, and there is no vaccine. Our work demonstrates that PIV5 is a promising vector for developing a MERS vaccine. Furthermore, success of PIV5-based MERS vaccine can be employed to develop a vaccine for emerging CoVs such as SARS-CoV-2, which causes COVID-19.

ABSTRACT

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often rewired such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this rewired background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations as PAO1, and in E90, RhlR plays a significant role in mediating cytotoxicity in a three-dimensional lung epithelium cell model. These data illuminate a rewired LasR-independent RhlR regulon in chronic infection isolates and suggest further investigation of RhlR as a possible target for therapeutic development in chronic infections.

IMPORTANCE Pseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Many isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR in chronic P. aeruginosa infections.

ABSTRACT

Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944–3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790–6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome.

IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

ABSTRACT

Aquifers, which are essential underground freshwater reservoirs worldwide, are understudied ecosystems that harbor diverse forms of microbial life. This study investigated the abundance and composition of prokaryotic and viral communities in the outflow of five springs across northern Florida, USA, as a proxy of microbial communities found in one of the most productive aquifers in the world, the Floridan aquifer. The average abundances of virus-like particles and prokaryotic cells were slightly lower than those reported from other groundwater systems, ranging from 9.6 x 10³ ml^–1 to 1.1 x 10⁵ ml^–1 and 2.2 x 10³ ml^–1 to 3.4 x 10⁴ ml^–1, respectively. Despite all of the springs being fed by the Floridan aquifer, sequencing of 16S rRNA genes and viral metagenomes (viromes) revealed unique communities in each spring, suggesting that groundwater microbial communities are influenced by land usage in recharge zones. The prokaryotic communities were dominated by Bacteria, and though the most abundant phyla (Proteobacteria, Cyanobacteria, and Bacteroidetes) were found in relatively high abundance across springs, variation was seen at finer taxonomic resolution. The viral sequences were most similar to those described from other aquatic environments. Sequencing resulted in the completion of 58 novel viral genomes representing members of the order Caudovirales as well as prokaryotic and eukaryotic single-stranded DNA (ssDNA) viruses. Sequences similar to those of ssDNA viruses were detected at all spring sites and dominated the identifiable sequences at one spring site, showing that these small viruses merit further investigation in groundwater systems.

IMPORTANCE Aquifer systems may hold up to 40% of the total microbial biomass on Earth. However, little is known about the composition of microbial communities within these critical freshwater ecosystems. Here, we took advantage of Florida’s first-magnitude springs (the highest spring classification based on water discharge), each discharging at least 246 million liters of water each day from the Floridan aquifer system (FAS), to investigate prokaryotic and viral communities from the aquifer. The FAS serves as a major source of potable water in the Southeastern United States, providing water for large cities and citizens in three states. Unfortunately, the health of the FAS and its associated springs has declined in the past few decades due to nutrient loading, increased urbanization and agricultural activity in aquifer recharge zones, and saltwater intrusion. This is the first study to describe the prokaryotic and viral communities in Florida’s first-magnitude springs, providing a baseline against which to compare future ecosystem change.

ABSTRACT

The multifunctional nature of viral proteins is essentially driven by posttranslational modifications (PTMs) and is key for the successful outcome of infection. For influenza A viruses (IAVs), a composite pattern of PTMs regulates the activity of viral proteins. However, almost none are known that target the PB2 replication protein, except for inducing its degradation. We show here that PB2 undergoes a nonproteolytic ubiquitination during infection. We identified E3 ubiquitin ligases catalyzing this ubiquitination as two multicomponent RING-E3 ligases based on cullin 4 (CRL4s), which are both contributing to the levels of ubiquitinated forms of PB2 in infected cells. The CRL4 E3 ligase activity is required for the normal progression of the viral cycle and for maximal virion production, indicating that the CRL4s mediate a ubiquitin signaling that promotes infection. The CRL4s are recruiting PB2 through an unconventional bimodal interaction with both the DDB1 adaptor and DCAF substrate receptors. While able to bind to PB2 when engaged in the viral polymerase complex, the CRL4 factors do not alter transcription and replication of the viral segments during infection. CRL4 ligases catalyze different patterns of lysine ubiquitination on PB2. Recombinant viruses mutated in the targeted lysines showed attenuated viral production, suggesting that CRL4-mediated ubiquitination of PB2 contributes to IAV infection. We identified K29-linked ubiquitin chains as main components of the nonproteolytic PB2 ubiquitination mediated by the CRL4s, providing the first example of the role of this atypical ubiquitin linkage in the regulation of a viral infection.

IMPORTANCE Successful infection by influenza A virus, a pathogen of major public health importance, involves fine regulation of the multiple functions of the viral proteins, which often relies on post-translational modifications (PTMs). The PB2 protein of influenza A viruses is essential for viral replication and a key determinant of host range. While PTMs of PB2 inducing its degradation have been identified, here we show that PB2 undergoes a regulating PTM signaling detected during infection, based on an atypical K29-linked ubiquitination and mediated by two multicomponent E3 ubiquitin ligases. Recombinant viruses impaired for CRL4-mediated ubiquitination are attenuated, indicating that ubiquitination of PB2 is necessary for an optimal influenza A virus infection. The CRL4 E3 ligases are required for normal viral cycle progression and for maximal virion production. Consequently, they represent potential candidate host factors for antiviral targets.

ABSTRACT

Selectable markers are indispensable for genetic engineering, yet their number and variety are limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs of interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, stress resistance, or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes, including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use by inserting three genes encoding fluorescent proteins into a single strain for simultaneous multicolor localization microscopy. In addition to A. fumigatus, we validated the applicability of this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum. Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering.

IMPORTANCE This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Aspergillus fumigatus. Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.

ABSTRACT

Obesity and associated metabolic disorders are worldwide public health issues. The gut microbiota plays a key role in the pathophysiology of diet-induced obesity. Glycerol monolaurate (GML) is a widely consumed food emulsifier with antibacterial properties. Here, we explore the anti-obesity effect of GML (1,600 mg/kg of body weight) in high-fat diet (HFD)-fed mice. HFD-fed mice were treated with 1,600 mg/kg GML. Integrated microbiome, metabolome, and transcriptome analyses were used to systematically investigate the metabolic effects of GML, and antibiotic treatment was used to assess the effects of GML on the gut microbiota. Our data indicated that GML significantly reduced body weight and visceral fat deposition, improved hyperlipidemia and hepatic lipid metabolism, and ameliorated glucose homeostasis and inflammation in HFD-fed mice. Importantly, GML modulated HFD-induced gut microbiota dysbiosis and selectively increased the abundance of Bifidobacterium pseudolongum. Antibiotic treatment abolished all the GML-mediated metabolic improvements. A multiomics (microbiome, metabolome, and transcriptome) association study showed that GML significantly modulated glycerophospholipid metabolism, and the abundance of Bifidobacterium pseudolongum strongly correlated with the metabolites and genes that participated in glycerophospholipid metabolism. Our results indicated that GML may be provided for obesity prevention by targeting the gut microbiota and regulating glycerophospholipid metabolism.

ABSTRACT

The human gut microbiota (HGM) has far-reaching impacts on human health and nutrition, which are fueled primarily by the metabolism of otherwise indigestible complex carbohydrates commonly known as dietary fiber. However, the molecular basis of the ability of individual taxa of the HGM to address specific dietary glycan structures remains largely unclear. In particular, the utilization of β(1,3)-glucans, which are widespread in the human diet as yeast, seaweed, and plant cell walls, had not previously been resolved. Through a systems-based approach, here we show that the symbiont Bacteroides uniformis deploys a single, exemplar polysaccharide utilization locus (PUL) to access yeast β(1,3)-glucan, brown seaweed β(1,3)-glucan (laminarin), and cereal mixed-linkage β(1,3)/β(1,4)-glucan. Combined biochemical, enzymatic, and structural analysis of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) illuminates a concerted molecular system by which B. uniformis recognizes and saccharifies these distinct β-glucans. Strikingly, the functional characterization of homologous β(1,3)-glucan utilization loci (1,3GUL) in other Bacteroides further demonstrated that the ability of individual taxa to utilize β(1,3)-glucan variants and/or β(1,3)/β(1,4)-glucans arises combinatorially from the individual specificities of SGBPs and GHs at the cell surface, which feed corresponding signals to periplasmic hybrid two-component sensors (HTCSs) via TonB-dependent transporters (TBDTs). These data reveal the importance of cooperativity in the adaptive evolution of GH and SGBP cohorts to address individual polysaccharide structures. We anticipate that this fine-grained knowledge of PUL function will inform metabolic network analysis and proactive manipulation of the HGM. Indeed, a survey of 2,441 public human metagenomes revealed the international, yet individual-specific, distribution of each 1,3GUL.

IMPORTANCE Bacteroidetes are a dominant phylum of the human gut microbiota (HGM) that target otherwise indigestible dietary fiber with an arsenal of polysaccharide utilization loci (PULs), each of which is dedicated to the utilization of a specific complex carbohydrate. Here, we provide novel insight into this paradigm through functional characterization of homologous PULs from three autochthonous Bacteroides species, which target the family of dietary β(1,3)-glucans. Through detailed biochemical and protein structural analysis, we observed an unexpected diversity in the substrate specificity of PUL glycosidases and glycan-binding proteins with regard to β(1,3)-glucan linkage and branching patterns. In combination, these individual enzyme and protein specificities support taxon-specific growth on individual β(1,3)-glucans. This detailed metabolic insight, together with a comprehensive survey of individual 1,3GULs across human populations, further expands the fundamental roadmap of the HGM, with potential application to the future development of microbial intervention therapies.

ABSTRACT

Bacteria that encounter antibiotics can efficiently change their physiology to develop resistance. This intrinsic antibiotic resistance is mediated by multiple pathways, including a regulatory system(s) that activates specific genes. In some Streptomyces and Mycobacterium spp., the WblC/WhiB7 transcription factor is required for intrinsic resistance to translation-targeting antibiotics. Wide conservation of WblC/WhiB7 within Actinobacteria indicates a critical role of WblC/WhiB7 in developing resistance to such antibiotics. Here, we identified 312 WblC target genes in Streptomyces coelicolor, a model antibiotic-producing bacterium, using a combined analysis of RNA sequencing and chromatin immunoprecipitation sequencing. Interestingly, WblC controls many genes involved in translation, in addition to previously identified antibiotic resistance genes. Moreover, WblC promotes translation rate during antibiotic stress by altering the ribosome-associated protein composition. Our genome-wide analyses highlight a previously unappreciated antibiotic resistance mechanism that modifies ribosome composition and maintains the translation rate in the presence of sub-MIC levels of antibiotics.

IMPORTANCE The emergence of antibiotic-resistant bacteria is one of the top threats in human health. Therefore, we need to understand how bacteria acquire resistance to antibiotics and continue growth even in the presence of antibiotics. Streptomyces coelicolor, an antibiotic-producing soil bacterium, intrinsically develops resistance to translation-targeting antibiotics. Intrinsic resistance is controlled by the WblC/WhiB7 transcription factor that is highly conserved within Actinobacteria, including Mycobacterium tuberculosis. Here, identification of the WblC/WhiB7 regulon revealed that WblC/WhiB7 controls ribosome maintenance genes and promotes translation in the presence of antibiotics by altering the composition of ribosome-associated proteins. Also, the WblC-mediated ribosomal alteration is indeed required for resistance to translation-targeting antibiotics. This suggests that inactivation of the WblC/WhiB7 regulon could be a potential target to treat antibiotic-resistant mycobacteria.

ABSTRACT

The translocation of effectors into the host cell through type 3 secretion systems (T3SS) is a sophisticated strategy employed by pathogenic bacteria to subvert host responses and facilitate colonization. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) utilize the Tir and EspFu (also known as TccP) effectors to remodel the host cytoskeleton, culminating in the formation of attaching and effacing (AE) lesions on enterocytes. While some EPEC strains require tyrosine phosphorylation of Tir and recruitment of the host Nck to trigger actin polymerization, EHEC and certain EPEC strains, whose Tir is not phosphorylated, rely on the effector EspFu for efficient actin remodeling. Here, we investigated the role played by Tir-Nck and Tir-EspFu actin polymerization pathways during the infection of epithelial cells, as well as the host transcriptional response to the AE lesion formation induced by EPEC. We found that EspFu-mediated actin assembly promotes bacterial attachment and epithelial colonization more efficiently than Tir-Nck. Moreover, we showed that both actin polymerization mechanisms can activate inflammatory pathways and reverse the anti-inflammatory response induced by EPEC in epithelial cells. However, this activity is remarkably more evident in infections with EspFu-expressing EPEC strains. This study demonstrates the complex interactions between effector-mediated actin remodeling and inflammation. Different strains carry different combinations of these two effectors, highlighting the plasticity of pathogenic E. coli enteric infections.

IMPORTANCE EPEC is among the leading causes of diarrheal disease worldwide. The colonization of the gut mucosa by EPEC results in actin pedestal formation at the site of bacterial attachment. These pedestals are referred to as attaching and effacing (AE) lesions. Here, we exploit the different molecular mechanisms used by EPEC to induce AE lesions on epithelial cells, showing that the effector EspFu is associated with increased bacterial attachment and enhanced epithelial colonization compared to the Tir-Nck pathway. Moreover, we also showed that actin pedestal formation can counterbalance the anti-inflammatory activity induced by EPEC, especially when driven by EspFu. Collectively, our findings provide new insights into virulence mechanisms employed by EPEC to colonize epithelial cells, as well as the host response to this enteric pathogen.

ABSTRACT

In Gram-negative bacteria, the permeability of the outer membrane governs rates of antibiotic uptake and thus the efficacy of antimicrobial treatment. Hydrophilic drugs like β-lactam antibiotics depend on diffusion through pore-forming outer membrane proteins to reach their intracellular targets. In this study, we investigated the distribution of porin genes in more than 2,700 Klebsiella isolates and found a widespread loss of OmpK35 functionality, particularly in those strains isolated from clinical environments. Using a defined set of outer-membrane-remodeled mutants, the major porin OmpK35 was shown to be largely responsible for β-lactam permeation. Sequence similarity network analysis characterized the porin protein subfamilies and led to discovery of a new porin family member, OmpK38. Structure-based comparisons of OmpK35, OmpK36, OmpK37, OmpK38, and PhoE showed near-identical pore frameworks but defining differences in the sequence characteristics of the extracellular loops. Antibiotic sensitivity profiles of isogenic Klebsiella pneumoniae strains, each expressing a different porin as its dominant pore, revealed striking differences in the antibiotic permeability characteristics of each channel in a physiological context. Since K. pneumoniae is a nosocomial pathogen with high rates of antimicrobial resistance and concurrent mortality, these experiments elucidate the role of porins in conferring specific drug-resistant phenotypes in a global context, informing future research to combat antimicrobial resistance in K. pneumoniae.

IMPORTANCE Klebsiella pneumoniae is a pathogen of humans with high rates of mortality and a recognized global rise in incidence of carbapenem-resistant K. pneumoniae (CRKP). The outer membrane of K. pneumoniae forms a permeability barrier that modulates the ability of antibiotics to reach their intracellular target. OmpK35, OmpK36, OmpK37, OmpK38, PhoE, and OmpK26 are porins in the outer membrane of K. pneumoniae, demonstrated here to have a causative relationship to drug resistance phenotypes in a physiological context. The data highlight that currently trialed combination treatments with a carbapenem and β-lactamase inhibitors could be effective on porin-deficient K. pneumoniae. Together with structural data, the results reveal the role of outer membrane proteome remodeling in antimicrobial resistance of K. pneumoniae and point to the role of extracellular loops, not channel parameters, in drug permeation. This significant finding warrants care in the development of phage therapies for K. pneumoniae infections, given the way porin expression will be modulated to confer phage-resistant—and collateral drug-resistant—phenotypes in K. pneumoniae.

ABSTRACT

Lipopolysaccharide (LPS) is an essential glycolipid present in the outer membrane (OM) of many Gram-negative bacteria. Balanced biosynthesis of LPS is critical for cell viability; too little LPS weakens the OM, while too much LPS is lethal. In Escherichia coli, this balance is maintained by the YciM/FtsH protease complex, which adjusts LPS levels by degrading the LPS biosynthesis enzyme LpxC. Here, we provide evidence that activity of the YciM/FtsH protease complex is inhibited by the essential protein YejM. Using strains in which LpxC activity is reduced, we show that yciM is epistatic to yejM, demonstrating that YejM acts upstream of YciM to prevent toxic overproduction of LPS. Previous studies have shown that this toxicity can be suppressed by deleting lpp, which codes for a highly abundant OM lipoprotein. It was assumed that deletion of lpp restores lipid balance by increasing the number of acyl chains available for glycerophospholipid biosynthesis. We show that this is not the case. Rather, our data suggest that preventing attachment of lpp to the peptidoglycan sacculus allows excess LPS to be shed in vesicles. We propose that this loss of OM material allows continued transport of LPS to the OM, thus preventing lethal accumulation of LPS within the inner membrane. Overall, our data justify the commitment of three essential inner membrane proteins to avoid toxic over- or underproduction of LPS.

IMPORTANCE Gram-negative bacteria are encapsulated by an outer membrane (OM) that is impermeable to large and hydrophobic molecules. As such, these bacteria are intrinsically resistant to several clinically relevant antibiotics. To better understand how the OM is established or maintained, we sought to clarify the function of the essential protein YejM in Escherichia coli. Here, we show that YejM inhibits activity of the YciM/FtsH protease complex, which regulates synthesis of the essential OM glycolipid lipopolysaccharide (LPS). Our data suggest that disrupting proper communication between LPS synthesis and transport to the OM leads to accumulation of LPS within the inner membrane (IM). The lethality associated with this event can be suppressed by increasing OM vesiculation. Our research has identified a completely novel signaling pathway that we propose coordinates LPS synthesis and transport.

ABSTRACT

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatus.

IMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

ABSTRACT

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.

IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

ABSTRACT

Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureus.

IMPORTANCE The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time. Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus. Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections.

ABSTRACT

Staphylococcus aureus can colonize the human host and cause a variety of superficial and invasive infections. The success of S. aureus as a pathogen derives from its ability to modulate its virulence through the release, sensing of and response to cyclic signaling peptides. Here we provide, for the first time, evidence that S. aureus processes and secretes small linear peptides through a specialized pathway that converts a lipoprotein leader into an extracellular peptide signal. We have identified and confirmed the machinery for each step and demonstrate that the putative membrane metalloprotease Eep and the EcsAB transporter are required to complete the processing and secretion of the peptides. In addition, we have identified several linear peptides, including the interspecies signaling molecule staph-cAM373, that are dependent on this processing and secretion pathway. These findings are particularly important because multiple Gram-positive bacteria rely on small linear peptides to control bacterial gene expression and virulence.

IMPORTANCE Here, we provide evidence indicating that S. aureus secretes small linear peptides into the environment via a novel processing and secretion pathway. The discovery of a specialized pathway for the production of small linear peptides and the identification of these peptides leads to several important questions regarding their role in S. aureus biology, most interestingly, their potential to act as signaling molecules. The observations in this study provide a foundation for further in-depth studies into the biological activity of small linear peptides in S. aureus.

ABSTRACT

Reversible repression of HIV-1 5' long terminal repeat (5'-LTR)-mediated transcription represents the main mechanism for HIV-1 to maintain latency. Identification of host factors that modulate LTR activity and viral latency may help develop new antiretroviral therapies. The heterogeneous nuclear ribonucleoproteins (hnRNPs) are known to regulate gene expression and possess multiple physiological functions. hnRNP family members have recently been identified as the sensors for viral nucleic acids to induce antiviral responses, highlighting the crucial roles of hnRNPs in regulating viral infection. A member of the hnRNP family, X-linked RNA-binding motif protein (RBMX), has been identified in this study as a novel HIV-1 restriction factor that modulates HIV-1 5'-LTR-driven transcription of viral genome in CD4⁺ T cells. Mechanistically, RBMX binds to HIV-1 proviral DNA at the LTR downstream region and maintains the repressive trimethylation of histone H3 lysine 9 (H3K9me3), leading to a blockage of the recruitment of the positive transcription factor phosphorylated RNA polymerase II (RNA pol II) and consequential impediment of transcription elongation. This RBMX-mediated modulation of HIV-1 transcription maintains viral latency by inhibiting viral reactivation from an integrated proviral DNA. Our findings provide a new understanding of how host factors modulate HIV-1 infection and latency and suggest a potential new target for the development of HIV-1 therapies.

IMPORTANCE HIV-1 latency featuring silence of transcription from HIV-1 proviral DNA represents a major obstacle for HIV-1 eradication. Reversible repression of HIV-1 5'-LTR-mediated transcription represents the main mechanism for HIV-1 to maintain latency. The 5'-LTR-driven HIV gene transcription can be modulated by multiple host factors and mechanisms. The hnRNPs are known to regulate gene expression. A member of the hnRNP family, RBMX, has been identified in this study as a novel HIV-1 restriction factor that modulates HIV-1 5'-LTR-driven transcription of viral genome in CD4⁺ T cells and maintains viral latency. These findings provide a new understanding of how host factors modulate HIV-1 infection and latency and suggest a potential new target for the development of HIV-1 therapies.

ABSTRACT

Channelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. "Bacteriorhodopsin-like" cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, the sequences of BCCR match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here, we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption, Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function.

IMPORTANCE Cation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-visible (UV-vis) spectroscopy and photoelectrophysiology reveal that the desensitization is caused by rapid accumulation of a nonconductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

ABSTRACT

A large portion of biological iron is found in the form of an iron-protoporphyrin IX complex, or heme. In the human host environment, which is exceptionally poor in free iron, heme iron, particularly from hemoglobin, constitutes a major source of iron for invading microbial pathogens. Several fungi were shown to utilize free heme, and Candida albicans, a major opportunistic pathogen, is able both to capture free heme and to extract heme from hemoglobin using a network of extracellular hemophores. Human serum albumin (HSA) is the most abundant host heme-scavenging protein. Tight binding of heme by HSA restricts its toxic chemical reactivity and could diminish its availability as an iron source for pathogenic microbes. We found, however, that rather than inhibiting heme utilization, HSA greatly increases availability of heme as an iron source for C. albicans and other fungi. In contrast, hemopexin, a low-abundance but high-affinity heme-scavenging serum protein, does inhibit heme utilization by C. albicans. However, inhibition by hemopexin is mitigated in the presence of HSA. Utilization of albumin-bound heme requires the same hemophore cascade as that which mediates hemoglobin-iron utilization. Accordingly, we found that the C. albicans hemophores are able to extract heme bound to HSA in vitro. Since many common drugs are known to bind to HSA, we tested whether they could interfere with heme-iron utilization. We show that utilization of albumin-bound heme by C. albicans can be inhibited by the anti-inflammatory drugs naproxen and salicylic acid.

IMPORTANCE Heme constitutes a major iron source for microorganisms and particularly for pathogenic microbes; to overcome the iron scarcity in the animal host, many pathogenic bacteria and fungi have developed systems to extract and take up heme from host proteins such as hemoglobin. Microbial heme uptake mechanisms are usually studied using growth media containing free heme or hemoglobin as a sole iron source. However, the animal host contains heme-scavenging proteins that could prevent this uptake. In the human host in particular, the most abundant serum heme-binding protein is albumin. Surprisingly, however, we found that in the case of fungi of the Candida species family, albumin promoted rather than prevented heme utilization. Albumin thus constitutes a human-specific factor that can affect heme-iron utilization and could serve as target for preventing heme-iron utilization by fungal pathogens. As a proof of principle, we identify two drugs that can inhibit albumin-stimulated heme utilization.

ABSTRACT

Simian immunodeficiency virus (SIV)-infected nonhuman primates can serve as a relevant model for AIDS neuropathogenesis. Current SIV-induced encephalitis (SIVE)/neurological complications of AIDS (neuroAIDS) models are generally associated with rapid progression to neuroAIDS, which does not reflect the tempo of neuroAIDS progression in humans. Recently, we isolated a neuropathogenic clone, SIVsm804E-CL757 (CL757), obtained from an SIV-infected rhesus macaque (RM). CL757 causes a more protracted progression to disease, inducing SIVE in 50% of inoculated animals, with high cerebral spinal fluid viral loads, multinucleated giant cells (MNGCs), and perivascular lymphocytic cuffing in the central nervous system (CNS). This latter finding is reminiscent of human immunodeficiency virus (HIV) encephalitis in humans but not generally observed in rapid progressor animals with neuroAIDS. Here, we studied which subsets of cells within the CNS were targeted by CL757 in animals with neurological symptoms of SIVE. Immunohistochemistry of brain sections demonstrated infiltration of CD4⁺ T cells (CD4) and macrophages (Ms) to the site of MNGCs. Moreover, an increase in mononuclear cells isolated from the brain tissues of RMs with SIVE correlated with increased cerebrospinal fluid (CSF) viral load. Subset analysis showed a specific increase in brain CD4⁺ memory T cells (Br-mCD4), brain-Ms (Br-Ms), and brain B cells (Br-B cells). Both Br-mCD4s and Br-Ms harbored replication-competent viral DNA, as demonstrated by virus isolation by coculture. However, only in animals exhibiting SIVE/neuroAIDS was virus isolated from Br-Ms. These findings support the use of CL757 to study the pathogenesis of AIDS viruses in the central nervous system and indicate a previously unanticipated role of CD4s cells as a potential reservoir in the brain.

IMPORTANCE While the use of combination antiretroviral therapy effectively suppresses systemic viral replication in the body, neurocognitive disorders as a result of HIV infection of the central nervous system (CNS) remain a clinical problem. Therefore, the use of nonhuman primate models is necessary to study mechanisms of neuropathogenesis. The neurotropic, molecular clone SIVsm804E-CL757 (CL757) results in neuroAIDS in 50% of infected rhesus macaques approximately 1 year postinfection. Using CL757-infected macaques, we investigate disease progression by examining subsets of cells within the CNS that were targeted by CL757 and could potentially serve as viral reservoirs. By isolating mononuclear cells from the brains of SIV-infected rhesus macaques with and without encephalitis, we show that immune cells invade the neuroparenchyma and increase in number in the CNS in animals with SIV-induced encephalitis (SIVE). Of these cells, both brain macrophages and brain memory CD4⁺ T cells harbor replication-competent SIV DNA; however, only brain CD4⁺ T cells harbored SIV DNA in animals without SIVE. These findings support use of CL757 as an important model to investigate disease progression in the CNS and as a model to study virus reservoirs in the CNS.

ABSTRACT

Cold seeps and hydrothermal vents deliver large amounts of methane and other gaseous alkanes into marine surface sediments. Consortia of archaea and partner bacteria thrive on the oxidation of these alkanes and its coupling to sulfate reduction. The inherently slow growth of the involved organisms and the lack of pure cultures have impeded the understanding of the molecular mechanisms of archaeal alkane degradation. Here, using hydrothermal sediments of the Guaymas Basin (Gulf of California) and ethane as the substrate, we cultured microbial consortia of a novel anaerobic ethane oxidizer, "Candidatus Ethanoperedens thermophilum" (GoM-Arc1 clade), and its partner bacterium "Candidatus Desulfofervidus auxilii," previously known from methane-oxidizing consortia. The sulfate reduction activity of the culture doubled within one week, indicating a much faster growth than in any other alkane-oxidizing archaea described before. The dominance of a single archaeal phylotype in this culture allowed retrieval of a closed genome of "Ca. Ethanoperedens," a sister genus of the recently reported ethane oxidizer "Candidatus Argoarchaeum." The metagenome-assembled genome of "Ca. Ethanoperedens" encoded a complete methanogenesis pathway including a methyl-coenzyme M reductase (MCR) that is highly divergent from those of methanogens and methanotrophs. Combined substrate and metabolite analysis showed ethane as the sole growth substrate and production of ethyl-coenzyme M as the activation product. Stable isotope probing demonstrated that the enzymatic mechanism of ethane oxidation in "Ca. Ethanoperedens" is fully reversible; thus, its enzymatic machinery has potential for the biotechnological development of microbial ethane production from carbon dioxide.

IMPORTANCE In the seabed, gaseous alkanes are oxidized by syntrophic microbial consortia that thereby reduce fluxes of these compounds into the water column. Because of the immense quantities of seabed alkane fluxes, these consortia are key catalysts of the global carbon cycle. Due to their obligate syntrophic lifestyle, the physiology of alkane-degrading archaea remains poorly understood. We have now cultivated a thermophilic, relatively fast-growing ethane oxidizer in partnership with a sulfate-reducing bacterium known to aid in methane oxidation and have retrieved the first complete genome of a short-chain alkane-degrading archaeon. This will greatly enhance the understanding of nonmethane alkane activation by noncanonical methyl-coenzyme M reductase enzymes and provide insights into additional metabolic steps and the mechanisms underlying syntrophic partnerships. Ultimately, this knowledge could lead to the biotechnological development of alkanogenic microorganisms to support the carbon neutrality of industrial processes.

ABSTRACT

Symbiotic microorganisms can have a profound impact on the host physiology and behavior, and novel relationships between symbionts and their hosts are continually discovered. A colony of social ants consists of various castes that exhibit distinct lifestyles and is, thus, a unique model for investigating how symbionts may be involved in host eusociality. Yet our knowledge of social ant-symbiont dynamics has remained rudimentary. Through 16S rRNA gene deep sequencing of the carpenter ant Camponotus japonicus symbiont community across various castes, we here report caste-dependent diversity of commensal gut microbiota and lineage divergence of "Candidatus Blochmannia," an obligate endosymbiont. While most prevalent gut-associated bacterial populations are found across all castes (Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria), we also discovered uncultured populations that are found only in males (belonging to Corynebacteriales, Alkanindiges, and Burkholderia). Most of those populations are not detected in laboratory-maintained queens and workers, suggesting that they are facultative gut symbionts introduced via environmental acquisition. Further inspection of "Ca. Blochmannia" endosymbionts reveals that two populations are dominant in all individuals across all castes but that males preferentially contain two different sublineages that are diversified from others. Clearly, each caste has distinct symbiont communities, suggesting an overlooked biological aspect of host-symbiont interaction in social insects.

IMPORTANCE Social animals, such as primates and some insects, have been shown to exchange symbiotic microbes among individuals through sharing diet or habitats, resulting in increased consistency of microbiota among social partners. The ant is a representative of social insects exhibiting various castes within a colony; queens, males, and nonreproductive females (so-called workers) show distinct morphologies, physiologies, and behaviors but tightly interact with each other in the nest. However, how this social context affects their gut microbiota has remained unclear. In this study, we deeply sequenced the gut symbiont community across various castes of the carpenter ant Camponotus japonicus. We report caste-dependent diversity of commensal gut microbial community and lineage divergence of the mutualistic endosymbiont "Candidatus Blochmannia." This report sheds light on the hidden diversity in microbial populations and community structure associated with guts of males in social ants.

ABSTRACT

The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of Ehrlichia surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C) directly binds mammalian DNase X, a glycosylphosphatidylinositol-anchored cell surface receptor and that binding is required to induce bacterial entry and simultaneously to block the generation of reactive oxygen species (ROS) by host monocytes and macrophages. However, how the EtpE-C–DNase X complex mediates the ROS blockade was unknown. A mammalian transmembrane glycoprotein CD147 (basigin) binds to the EtpE-DNase X complex and is required for Ehrlichia entry and infection of host cells. Here, we found that bone marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced Ehrlichia-induced or EtpE-C-induced blockade of ROS generation in response to phorbol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the Ehrlichia-mediated inhibition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to Ehrlichia infection. Moreover, in human monocytes, anti-CD147 partially abrogated EtpE-C-induced blockade of ROS generation. Both Ehrlichia and EtpE-C could block activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor by phorbol myristate acetate. Vav1 suppression by Ehrlichia was CD147 dependent. E. chaffeensis is the first example of pathogens that block Rac1 activation to colonize macrophages. Furthermore, Ehrlichia uses EtpE to hijack the unique host DNase X-CD147-Vav1 signaling to block Rac1 activation.

IMPORTANCE Ehrlichia chaffeensis is an obligatory intracellular bacterium with the capability of causing an emerging infectious disease called human monocytic ehrlichiosis. E. chaffeensis preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of antimicrobial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering bacteria. As Ehrlichia isolated from host cells are readily killed upon exposure to ROS, Ehrlichia must have evolved a unique mechanism to safely enter phagocytes. We discovered that binding of the Ehrlichia surface invasin to the host cell surface receptor not only triggers Ehrlichia entry but also blocks ROS generation by the host cells by mobilizing a novel intracellular signaling pathway. Knowledge of the mechanisms by which ROS production is inhibited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies.

ABSTRACT

Candida auris has emerged globally as a multidrug-resistant yeast that can spread via nosocomial transmission. An initial phylogenetic study of isolates from Japan, India, Pakistan, South Africa, and Venezuela revealed four populations (clades I, II, III, and IV) corresponding to these geographic regions. Since this description, C. auris has been reported in more than 30 additional countries. To trace this global emergence, we compared the genomes of 304 C. auris isolates from 19 countries on six continents. We found that four predominant clades persist across wide geographic locations. We observed phylogeographic mixing in most clades; clade IV, with isolates mainly from South America, demonstrated the strongest phylogeographic substructure. C. auris isolates from two clades with opposite mating types were detected contemporaneously in a single health care facility in Kenya. We estimated a Bayesian molecular clock phylogeny and dated the origin of each clade within the last 360 years; outbreak-causing clusters from clades I, III, and IV originated 36 to 38 years ago. We observed high rates of antifungal resistance in clade I, including four isolates resistant to all three major classes of antifungals. Mutations that contribute to resistance varied between the clades, with Y132F in ERG11 as the most widespread mutation associated with azole resistance and S639P in FKS1 for echinocandin resistance. Copy number variants in ERG11 predominantly appeared in clade III and were associated with fluconazole resistance. These results provide a global context for the phylogeography, population structure, and mechanisms associated with antifungal resistance in C. auris.

IMPORTANCE In less than a decade, C. auris has emerged in health care settings worldwide; this species is capable of colonizing skin and causing outbreaks of invasive candidiasis. In contrast to other Candida species, C. auris is unique in its ability to spread via nosocomial transmission and its high rates of drug resistance. As part of the public health response, whole-genome sequencing has played a major role in characterizing transmission dynamics and detecting new C. auris introductions. Through a global collaboration, we assessed genome evolution of isolates of C. auris from 19 countries. Here, we described estimated timing of the expansion of each C. auris clade and of fluconazole resistance, characterized discrete phylogeographic population structure of each clade, and compared genome data to sensitivity measurements to describe how antifungal resistance mechanisms vary across the population. These efforts are critical for a sustained, robust public health response that effectively utilizes molecular epidemiology.

ABSTRACT

Humans encode proteins, called restriction factors, that inhibit replication of viruses such as HIV-1. The members of one family of antiviral proteins, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; shortened here to A3), act by deaminating cytidines to uridines during the reverse transcription reaction of HIV-1. The A3 locus encodes seven genes, named A3A to A3H. These genes have either one or two cytidine deaminase domains, and several of these A3s potently restrict HIV-1. A3C, which has only a single cytidine deaminase domain, however, inhibits HIV-1 only very weakly. We tested novel double domain protein combinations by genetically linking two A3C genes to make a synthetic tandem domain protein. This protein created a "super restriction factor" that had more potent antiviral activity than the native A3C protein, which correlated with increased packaging into virions. Furthermore, disabling one of the active sites of the synthetic tandem domain protein resulted in an even greater increase in the antiviral activity—recapitulating a similar evolution seen in A3F and A3G (double domain A3s that use only a single catalytically active deaminase domain). These A3C tandem domain proteins do not have an increase in mutational activity but instead inhibit formation of reverse transcription products, which correlates with their ability to form large higher-order complexes in cells. Finally, the A3C-A3C super restriction factor largely escaped antagonism by the HIV-1 viral protein Vif.

IMPORTANCE As a part of the innate immune system, humans encode proteins that inhibit viruses such as HIV-1. These broadly acting antiviral proteins do not protect humans from viral infections because viruses encode proteins that antagonize the host antiviral proteins to evade the innate immune system. One such example of a host antiviral protein is APOBEC3C (A3C), which weakly inhibits HIV-1. Here, we show that we can improve the antiviral activity of A3C by duplicating the DNA sequence to create a synthetic tandem domain and, furthermore, that the proteins thus generated are relatively resistant to the viral antagonist Vif. Together, these data give insights about how nature has evolved a defense against viral pathogens such as HIV.

ABSTRACT

Urinary tract infections (UTI) affect half of all women at least once during their lifetime. The rise in the numbers of extended-spectrum beta-lactamase-producing strains and the potential for carbapenem resistance within uropathogenic Escherichia coli (UPEC), the most common causative agent of UTI, create an urgent need for vaccine development. Intranasal immunization of mice with UPEC outer membrane iron receptors FyuA, Hma, IreA, and IutA, conjugated to cholera toxin, provides protection in the bladder or kidneys under conditions of challenge with UPEC strain CFT073 or strain 536. On the basis of these data, we sought to optimize the vaccination route (intramuscular, intranasal, or subcutaneous) in combination with adjuvants suitable for human use, including aluminum hydroxide gel (alum), monophosphoryl lipid A (MPLA), unmethylated CpG synthetic oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (polyIC), and mutated heat-labile E. coli enterotoxin (dmLT). Mice intranasally vaccinated with dmLT-IutA and dmLT-Hma displayed significant reductions in bladder colonization (86-fold and 32-fold, respectively), with 40% to 42% of mice having no detectable CFU. Intranasal vaccination of mice with CpG-IutA and polyIC-IutA significantly reduced kidney colonization (131-fold) and urine CFU (22-fold), respectively. dmLT generated the most consistently robust antibody response in intranasally immunized mice, while MPLA and alum produced greater concentrations of antigen-specific serum IgG with intramuscular immunization. On the basis of these results, we conclude that intranasal administration of Hma or IutA formulated with dmLT adjuvant provides the greatest protection from UPEC UTI. This report advances our progress toward a vaccine against uncomplicated UTI, which will significantly improve the quality of life for women burdened by recurrent UTI and enable better antibiotic stewardship.

IMPORTANCE Urinary tract infections (UTI) are among the most common bacterial infection in humans, affecting half of all women at least once during their lifetimes. The rise in antibiotic resistance and health care costs emphasizes the need to develop a vaccine against the most common UTI pathogen, Escherichia coli. Vaccinating mice intranasally with a detoxified heat-labile enterotoxin and two surface-exposed receptors, Hma or IutA, significantly reduced bacterial burden in the bladder. This work highlights progress in the development of a UTI vaccine formulated with adjuvants suitable for human use and antigens that encode outer membrane iron receptors required for infection in the iron-limited urinary tract.

ABSTRACT

Ever since the discovery of the first rare earth element (REE)-dependent enzyme, the physiological role of lanthanides has become an emerging field of research due to the environmental implications and biotechnological opportunities. In Pseudomonas putida KT2440, the two pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) PedE and PedH are inversely regulated in response to REE availability. This transcriptional switch is orchestrated by a complex regulatory network that includes the PedR2/PedS2 two-component system and is important for efficient growth on several alcoholic volatiles. To study whether cellular responses beyond the REE switch exist, the differential proteomic responses that occur during growth on various model carbon sources were analyzed. Apart from the Ca²⁺-dependent enzyme PedE, the differential abundances of most identified proteins were conditional. During growth on glycerol—and concomitant with the proteomic changes—lanthanum (La³⁺) availability affected different growth parameters, including the onset of logarithmic growth and final optical densities. Studies with mutant strains revealed a novel metabolic route for glycerol utilization, initiated by PedE and/or PedH activity. Upon oxidation to glycerate via glyceraldehyde, phosphorylation by the glycerate kinase GarK most likely yields glycerate-2-phosphate, which is eventually channeled into the central metabolism of the cell. This new route functions in parallel with the main degradation pathway encoded by the glpFKRD operon and provides a growth advantage to the cells by allowing an earlier onset of growth with glycerol as the sole source of carbon and energy.

IMPORTANCE The biological role of REEs has long been underestimated, and research has mainly focused on methanotrophic and methylotrophic bacteria. We have recently demonstrated that P. putida, a plant growth-promoting bacterium that thrives in the rhizosphere of various food crops, possesses a REE-dependent alcohol dehydrogenase (PedH), but knowledge about REE-specific effects on physiological traits in nonmethylotrophic bacteria is still scarce. This study demonstrates that the cellular response of P. putida to lanthanum (La³⁺) is mostly substrate specific and that La³⁺ availability highly affects the growth of cells on glycerol. Further, a novel route for glycerol metabolism is identified, which is initiated by PedE and/or PedH activity and provides a growth advantage to this biotechnologically relevant organism by allowing a faster onset of growth. Overall, these findings demonstrate that lanthanides can affect physiological traits in nonmethylotrophic bacteria and might influence their competitiveness in various environmental niches.

ABSTRACT

DNA phosphorothioate (PT) modification, in which the nonbridging oxygen in the sugar-phosphate backbone is substituted by sulfur, is catalyzed by DndABCDE or SspABCD in a double-stranded or single-stranded manner, respectively. In Dnd and Ssp systems, mobilization of sulfur in PT formation starts with the activation of the sulfur atom of cysteine catalyzed by the DndA and SspA cysteine desulfurases, respectively. Despite playing the same biochemical role, SspA cannot be functionally replaced by DndA, indicating its unique physiological properties. In this study, we solved the crystal structure of Vibrio cyclitrophicus SspA in complex with its natural substrate, cysteine, and cofactor, pyridoxal phosphate (PLP), at a resolution of 1.80 Å. Our solved structure revealed the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor, suggesting a common binding mode shared by cysteine desulfurases. In addition, although the distance between the catalytic Cys314 and the substrate cysteine is 8.9 Å, which is too far for direct interaction, our structural modeling and biochemical analysis revealed a conformational change in the active site region toward the cysteine substrate to move them close to each other to facilitate the nucleophilic attack. Finally, the pulldown analysis showed that SspA could form a complex with SspD, an ATP pyrophosphatase, suggesting that SspD might potentially accept the activated sulfur atom directly from SspA, providing further insights into the biochemical pathway of Ssp-mediated PT modification.

IMPORTANCE Apart from its roles in Fe-S cluster assembly, tRNA thiolation, and sulfur-containing cofactor biosynthesis, cysteine desulfurase serves as a sulfur donor in the DNA PT modification, in which a sulfur atom substitutes a nonbridging oxygen in the DNA phosphodiester backbone. The initial sulfur mobilization from l-cysteine is catalyzed by the SspA cysteine desulfurase in the SspABCD-mediated DNA PT modification system. By determining the crystal structure of SspA, the study presents the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor. To overcome the long distance (8.9 Å) between the catalytic Cys314 and the cysteine substrate, a conformational change occurs to bring Cys314 to the vicinity of the substrate, allowing for nucleophilic attack.

ABSTRACT

Cell division requires proper spatial coordination with the chromosome, which undergoes dynamic changes during chromosome replication and segregation. FtsZ is a bacterial cytoskeletal protein that assembles into the Z-ring, providing a platform to build the cell division apparatus. In the model bacterium Caulobacter crescentus, the cellular localization of the Z-ring is controlled during the cell cycle in a chromosome replication-coupled manner. Although dynamic localization of the Z-ring at midcell is driven primarily by the replication origin-associated FtsZ inhibitor MipZ, the mechanism ensuring accurate positioning of the Z-ring remains unclear. In this study, we showed that the Z-ring colocalizes with the replication terminus region, located opposite the origin, throughout most of the C. crescentus cell cycle. Spatial organization of the two is mediated by ZapT, a previously uncharacterized protein that interacts with the terminus region and associates with ZapA and ZauP, both of which are part of the incipient division apparatus. While the Z-ring and the terminus region coincided with the presence of ZapT, colocalization of the two was perturbed in cells lacking zapT, which is accompanied by delayed midcellular positioning of the Z-ring. Moreover, cells overexpressing ZapT showed compromised positioning of the Z-ring and MipZ. These findings underscore the important role of ZapT in controlling cell division processes. We propose that ZapT acts as a molecular bridge that physically links the terminus region to the Z-ring, thereby ensuring accurate site selection for the Z-ring. Because ZapT is conserved in proteobacteria, these findings may define a general mechanism coordinating cell division with chromosome organization.

IMPORTANCE Growing bacteria require careful tuning of cell division processes with dynamic organization of replicating chromosomes. In enteric bacteria, ZapA associates with the cytoskeletal Z-ring and establishes a physical linkage to the chromosomal replication terminus through its interaction with ZapB-MatP-DNA complexes. However, because ZapB and MatP are found only in enteric bacteria, it remains unclear how the Z-ring and the terminus are coordinated in the vast majority of bacteria. Here, we provide evidence that a novel conserved protein, termed ZapT, mediates colocalization of the Z-ring with the terminus in Caulobacter crescentus, a model organism that is phylogenetically distant from enteric bacteria. Given that ZapT facilitates cell division processes in C. crescentus, this study highlights the universal importance of the physical linkage between the Z-ring and the terminus in maintaining cell integrity.

ABSTRACT

Theory, simulation, and experimental evolution demonstrate that diversified CRISPR-Cas immunity to lytic viruses can lead to stochastic virus extinction due to a limited number of susceptible hosts available to each potential new protospacer escape mutation. Under such conditions, theory predicts that to evade extinction, viruses evolve toward decreased virulence and promote vertical transmission and persistence in infected hosts. To better understand the evolution of host-virus interactions in microbial populations with active CRISPR-Cas immunity, we studied the interaction between CRISPR-immune Sulfolobus islandicus cells and immune-deficient strains that are infected by the chronic virus SSV9. We demonstrate that Sulfolobus islandicus cells infected with SSV9, and with other related SSVs, kill uninfected, immune strains through an antagonistic mechanism that is a protein and is independent of infectious virus. Cells that are infected with SSV9 are protected from killing and persist in the population. We hypothesize that this infection acts as a form of mutualism between the host and the virus by removing competitors in the population and ensuring continued vertical transmission of the virus within populations with diversified CRISPR-Cas immunity.

IMPORTANCE Multiple studies, especially those focusing on the role of lytic viruses in key model systems, have shown the importance of viruses in shaping microbial populations. However, it has become increasingly clear that viruses with a long host-virus interaction, such as those with a chronic lifestyle, can be important drivers of evolution and have large impacts on host ecology. In this work, we describe one such interaction with the acidic crenarchaeon Sulfolobus islandicus and its chronic virus Sulfolobus spindle-shaped virus 9. Our work expands the view in which this symbiosis between host and virus evolved, describing a killing phenotype which we hypothesize has evolved in part due to the high prevalence and diversity of CRISPR-Cas immunity seen in natural populations. We explore the implications of this phenotype in population dynamics and host ecology, as well as the implications of mutualism between this virus-host pair.

ABSTRACT

All enterococci produce a complex polysaccharide called the enterococcal polysaccharide antigen (EPA). This polymer is required for normal cell growth and division and for resistance to cephalosporins and plays a critical role in host-pathogen interaction. The EPA contributes to host colonization and is essential for virulence, conferring resistance to phagocytosis during the infection. Recent studies revealed that the "decorations" of the EPA polymer, encoded by genetic loci that are variable between isolates, underpin the biological activity of this surface polysaccharide. In this work, we investigated the structure of the EPA polymer produced by the high-risk enterococcal clonal complex Enterococcus faecalis V583. We analyzed purified EPA from the wild-type strain and a mutant lacking decorations and elucidated the structure of the EPA backbone and decorations. We showed that the rhamnan backbone of EPA is composed of a hexasaccharide repeat unit of C2- and C3-linked rhamnan chains, partially substituted in the C3 position by α-glucose (α-Glc) and in the C2 position by β-N-acetylglucosamine (β-GlcNAc). The so-called "EPA decorations" consist of phosphopolysaccharide chains corresponding to teichoic acids covalently bound to the rhamnan backbone. The elucidation of the complete EPA structure allowed us to propose a biosynthetic pathway, a first essential step toward the design of antimicrobials targeting the synthesis of this virulence factor.

IMPORTANCE Enterococci are opportunistic pathogens responsible for hospital- and community-acquired infections. All enterococci produce a surface polysaccharide called EPA (enterococcal polysaccharide antigen) required for biofilm formation, antibiotic resistance, and pathogenesis. Despite the critical role of EPA in cell growth and division and as a major virulence factor, no information is available on its structure. Here, we report the complete structure of the EPA polymer produced by the model strain E. faecalis V583. We describe the structure of the EPA backbone, made of a rhamnan hexasaccharide substituted by Glc and GlcNAc residues, and show that teichoic acids are covalently bound to this rhamnan chain, forming the so-called "EPA decorations" essential for host colonization and pathogenesis. This report represents a key step in efforts to identify the structural properties of EPA that are essential for its biological activity and to identify novel targets to develop preventive and therapeutic approaches against enterococci.

ABSTRACT

The capacity of Candida albicans to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes ALS3 and ECE1, which are involved in pathogenicity processes such as invasion, iron acquisition, and host cell damage. The global repressor Tup1 and its cofactor Nrg1 are considered to be the main antagonists of hyphal development in C. albicans. However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of ALS3 and ECE1. In contrast to NRG1, overexpression of TUP1 was found to inhibit neither filamentous growth nor transcription of ALS3 and ECE1. In addition, we identified the transcription factor Ahr1 as being required for full expression of both genes. A hyperactive version of Ahr1 bound directly to the promoters of ALS3 and ECE1 and induced their transcription even in the absence of environmental stimuli. This regulation worked even in the absence of the crucial hyphal growth regulators Cph1 and Efg1 but was dependent on the presence of Tup1. Overall, our results show that Ahr1 and Tup1 are key contributors in the complex regulation of virulence-associated genes in the different C. albicans morphologies.

IMPORTANCE Candida albicans is a major human fungal pathogen and the leading cause of systemic Candida infections. In recent years, Als3 and Ece1 were identified as important factors for fungal virulence. Transcription of both corresponding genes is closely associated with hyphal growth. Here, we describe how Tup1, normally a global repressor of gene expression as well as of filamentation, and the transcription factor Ahr1 contribute to full expression of ALS3 and ECE1 in C. albicans hyphae. Both regulators are required for high mRNA amounts of the two genes to ensure functional relevant protein synthesis and localization. These observations identified a new aspect of regulation in the complex transcriptional control of virulence-associated genes in C. albicans.

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