The spirochete Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect against LD infection. In this study, we demonstrated that immunization with CspZ-YA, a CspZ mutant protein with no FH-binding activity, protected mice from infection by several spirochete genotypes introduced via tick feeding. We found that the sera from CspZ-YA-vaccinated mice more efficiently eliminated spirochetes and blocked CspZ FH-binding activity than sera from CspZ-immunized mice. We also found that vaccination with CspZ, but not CspZ-YA, triggered the production of anti-FH antibodies, justifying CspZ-YA as an LD vaccine candidate. The mechanistic and efficacy information derived from this study provides insights into the development of a CspZ-based LD vaccine.

LuxS/AI-2 is an important quorum sensing system which affects the growth, biofilm formation, virulence, and metabolism of bacteria. LuxS is encoded by the luxS gene, but how this gene is associated with a diverse array of physiological activities in Edwardsiella piscicida (E. piscicida) is not known. Here, we constructed an luxS gene mutant strain, the luxS strain, to identify how LuxS/AI-2 affects pathogenicity. The results showed that LuxS was not found in the luxS gene mutant strain, and this gene deletion decreased E. piscicida growth compared to that of the wild-type strain. Meanwhile, the wild-type strain significantly increased penetration and motility in mucin compared to levels with the luxS strain. The 50% lethal dose (LD₅₀) of the E. piscicida luxS strain for zebrafish was significantly higher than that of the wild-type strain, which suggested that the luxS gene deletion could attenuate the strain’s virulence. The AI-2 activities of EIB202 were 56-fold higher than those in the luxS strain, suggesting that the luxS gene promotes AI-2 production. Transcriptome results demonstrated that between cells infected with the luxS strain and those infected with the wild-type strain 46 genes were significantly differentially regulated, which included 34 upregulated genes and 12 downregulated genes. Among these genes, the largest number were closely related to cell immunity and signaling systems. In addition, the biofilm formation ability of EIB202 was significantly higher than that of the luxS strain. The supernatant of EIB202 increased the biofilm formation ability of the luxS strain, which suggested that the luxS gene and its product LuxS enhanced biofilm formation in E. piscicida. All results indicate that the LuxS/AI-2 quorum sensing system in E. piscicida promotes its pathogenicity through increasing a diverse array of physiological activities.

Endothelial activation and microvascular dysfunction are key pathogenic processes in severe malaria. We evaluated the early role of these processes in experimentally induced Plasmodium falciparum and P. vivax infection. Participants were enrolled in induced blood-stage malaria clinical trials. Plasma osteoprotegerin, angiopoietin-2, and von Willebrand Factor (vWF) levels were measured as biomarkers of endothelial activation. Microvascular function was assessed using peripheral arterial tonometry and near-infrared spectroscopy, and the endothelial glycocalyx was assessed by sublingual videomicroscopy and measurement of biomarkers of degradation. Forty-five healthy, malaria-naive participants were recruited from 5 studies. Osteoprotegerin and vWF levels increased in participants following inoculation with P. vivax (n = 16) or P. falciparum (n = 15), with the angiopoietin-2 level also increasing in participants following inoculation with P. falciparum. For both species, the most pronounced increase was seen in osteoprotegerin. This was particularly marked in participants inoculated with P. vivax, where the osteoprotegerin level correlated with the levels of parasitemia and the malaria clinical score. There were no changes in measures of endothelial glycocalyx or microvascular function. Plasma biomarkers of endothelial activation increased in early P. falciparum and P. vivax infection and preceded changes in the endothelial glycocalyx or microvascular function. The more pronounced increase in osteoprotegerin suggests that this biomarker may play a role in disease pathogenesis.

Localized skin lesions are characteristic of cutaneous leishmaniasis (CL); however, Leishmania (Viannia) species, which are responsible for most CL cases in the Americas, can spread systemically, sometimes resulting in mucosal disease. Detection of Leishmania has been documented in healthy mucosal tissues (conjunctiva, tonsils, and nasal mucosa) and healthy skin of CL patients and in individuals with asymptomatic infection in areas of endemicity of L. (V.) panamensis and L. (V.) braziliensis transmission. However, the conditions and mechanisms that favor parasite persistence in healthy mucosal tissues are unknown. In this descriptive study, we compared the cell populations of the nasal mucosa (NM) of healthy donors and patients with active CL and explored the immune gene expression signatures related to molecular detection of Leishmania in this tissue in the absence of clinical signs or symptoms of mucosal disease. The cellular composition and gene expression profiles of NM samples from active CL patients were similar to those of healthy volunteers, with a predominance of epithelial over immune cells, and within the CD45⁺ cell population, a higher frequency of CD66b⁺ followed by CD14⁺ and CD3⁺ cells. In CL patients with molecular evidence of Leishmania persistence in the NM, genes characteristic of an anti-inflammatory and tissue repair responses (IL4R, IL5RA, POSTN, and SATB1) were overexpressed relative to NM samples from CL patients in which Leishmania was not detected. Here, we report the first immunological description of subclinically infected NM tissues of CL patients and provide evidence of a local anti-inflammatory environment favoring parasite persistence in the NM.

Glaesserella (Haemophilus) parasuis is a commensal bacterium of the upper respiratory tract in pigs and also the causative agent of Glässer’s disease, which causes significant morbidity and mortality in pigs worldwide. Isolates are characterized into 15 serovars by their capsular polysaccharide, which has shown a correlation with isolate pathogenicity. To investigate the role the capsule plays in G. parasuis virulence and host interaction, a capsule mutant of the serovar 5 strain HS069 was generated (HS069cap) through allelic exchange following natural transformation. HS069cap was unable to cause signs of systemic disease during a pig challenge study and had increased sensitivity to complement killing and phagocytosis by alveolar macrophages. Compared with the parent strain, HS069cap produced more robust biofilm and adhered equivalently to 3D4/31 cells; however, it was unable to persistently colonize the nasal cavity of inoculated pigs, with all pigs clearing HS069cap by 5 days postchallenge. Our results indicate the importance of the capsular polysaccharide to G. parasuis virulence as well as nasal colonization in pigs.

Efficient delivery of antigenic cargo to trigger protective immune responses is critical to the success of vaccination. Genetically engineered microorganisms, including virus, bacteria, and protozoa, can be modified to carry and deliver heterologous antigens to the host immune system. The biological vectors can induce a broad range of immune responses and enhance heterologous antigen-specific immunological outcomes. The protozoan genus Eimeria is widespread in domestic animals, causing serious coccidiosis. Eimeria parasites with strong immunogenicity are potent coccidiosis vaccine candidates and offer a valuable model of live vaccines against infectious diseases in animals. Eimeria parasites can also function as a vaccine vector. Herein, we review recent advances in design and application of recombinant Eimeria as a vaccine vector, which has been a topic of ongoing research in our laboratory. By recapitulating the establishment of an Eimeria transfection platform and its application, it will help lay the foundation for the future development of effective parasite-based vaccine delivery vectors and beyond.

Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. Staphylococcus aureus is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the S. aureus fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that S. aureus FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment of device-associated infections.

Legionella pneumophila, the etiological agent of Legionnaires’ disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

Nutrient acquisition is a central challenge for all organisms. For the fungal pathogen Candida albicans, utilization of amino acids has been shown to be critical for survival, immune evasion, and escape, while the importance of catabolism of host-derived proteins and peptides in vivo is less well understood. Stp1 and Stp2 are paralogous transcription factors (TFs) regulated by the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing system and have been proposed to have distinct, if uncertain, roles in protein and amino acid utilization. We show here that Stp1 is required for proper utilization of peptides but has no effect on amino acid catabolism. In contrast, Stp2 is critical for utilization of both carbon sources. Commensurate with this observation, we found that Stp1 controls a very limited set of genes, while Stp2 has a much more extensive regulon that is partly dependent on the Ssy1 amino acid sensor (amino acid uptake and catabolism) and partly Ssy1 independent (genes associated with filamentous growth, including the regulators UME6 and SFL2). The ssy1/ and stp2/ mutants showed reduced fitness in a gastrointestinal (GI) colonization model, yet induced greater damage to epithelial cells and macrophages in a manner that was highly dependent on the growth status of the fungal cells. Surprisingly, the stp1/ mutant was better able to colonize the gut but the mutation had no effect on host cell damage. Thus, proper protein and amino acid utilization are both required for normal host interaction and are controlled by an interrelated network that includes Stp1 and Stp2.

Staphylococcus aureus is a noted human and animal pathogen. Despite decades of research on this important bacterium, there are still many unanswered questions regarding the pathogenic mechanisms it uses to infect the mammalian host. This can be attributed to it possessing a plethora of virulence factors and complex virulence factor and metabolic regulation. PurR, the purine biosynthesis regulator, was recently also shown to regulate virulence factors in S. aureus, and mutations in purR result in derepression of fibronectin binding proteins (FnBPs) and extracellular toxins, required for a so-called hypervirulent phenotype. Here, we show that hypervirulent strains containing purR mutations can be attenuated with the addition of purine biosynthesis mutations, implicating the necessity for de novo purine biosynthesis in this phenotype and indicating that S. aureus in the mammalian host experiences purine limitation. Using cell culture, we showed that while purR mutants are not altered in epithelial cell binding, compared to that of wild-type (WT) S. aureus, purR mutants have enhanced invasion of these nonprofessional phagocytes, consistent with the requirement of FnBPs for invasion of these cells. This correlates with purR mutants having increased transcription of fnb genes, resulting in higher levels of surface-exposed FnBPs to promote invasion. These data provide important contributions to our understanding of how the pathogenesis of S. aureus is affected by sensing of purine levels during infection of the mammalian host.

Peptidoglycan, the sugar-amino acid polymer that composes the bacterial cell wall, requires a significant expenditure of energy to synthesize and is highly immunogenic. To minimize the loss of an energetically expensive metabolite and avoid host detection, bacteria often recycle their peptidoglycan, transporting its components back into the cytoplasm, where they can be used for subsequent rounds of new synthesis. The peptidoglycan-recycling substrate binding protein (SBP) MppA, which is responsible for recycling peptidoglycan fragments in Escherichia coli, has not been annotated for most intracellular pathogens. One such pathogen, Chlamydia trachomatis, has a limited capacity to synthesize amino acids de novo and therefore must obtain oligopeptides from its host cell for growth. Bioinformatics analysis suggests that the putative C. trachomatis oligopeptide transporter OppABCDF (OppABCDF_Ct) encodes multiple SBPs (OppA1_Ct, OppA2_Ct, and OppA3_Ct). Intracellular pathogens often encode multiple SBPs, while only one, OppA, is encoded in the E. coli opp operon. We hypothesized that the putative OppABCDF transporter of C. trachomatis functions in both oligopeptide transport and peptidoglycan recycling. We coexpressed the putative SBP genes (oppA1_Ct, oppA2_Ct, oppA3_Ct) along with oppBCDF_Ct in an E. coli mutant lacking the Opp transporter and determined that all three chlamydial OppA subunits supported oligopeptide transport. We also demonstrated the in vivo functionality of the chlamydial Opp transporter in C. trachomatis. Importantly, we found that one chlamydial SBP, OppA3_Ct, possessed dual substrate recognition properties and is capable of transporting peptidoglycan fragments (tri-diaminopimelic acid) in E. coli and in C. trachomatis. These findings suggest that Chlamydia evolved an oligopeptide transporter to facilitate the acquisition of oligopeptides for growth while simultaneously reducing the accumulation of immunostimulatory peptidoglycan fragments in the host cell cytosol. The latter property reflects bacterial pathoadaptation that dampens the host innate immune response to Chlamydia infection.

A Yersinia pestis mutant synthesizing an adjuvant form of lipid A (monophosphoryl lipid A, MPLA) displayed increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, we constructed an Asd-based balanced-lethal host-vector system that oversynthesized the LcrV antigen of Y. pestis, raised the amounts of LcrV enclosed in OMVs by the type II secretion system, and eliminated harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. Vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge with 8 x 10⁵ CFU (80,000 50% lethal dose [LD₅₀]) and intranasal challenge with 5 x 10³ CFU (50 LD₅₀) of virulent Y. pestis. This protection was significantly superior to that resulting from vaccination with LcrV/alhydrogel or rF1-V/alhydrogel. At week 4 postimmunization, the OMV-immunized mice showed more robust titers of antibodies against LcrV, Y. pestis whole-cell lysate (YPL), and F1 antigen and more balanced IgG1:IgG2a/IgG2b-derived Th1 and Th2 responses than LcrV-immunized mice. Moreover, potent adaptive and innate immune responses were stimulated in the OMV-immunized mice. Our findings demonstrate that self-adjuvanting Y. pestis OMVs provide a novel plague vaccine candidate and that the rational design of OMVs could serve as a robust approach for vaccine development.

Antimicrobial peptides play an important role in host defense against Vibrio cholerae. Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS, regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carR^r and carR^s alleles, respectively, and replaced the carR^s allele of a sensitive strain with the carR^r allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarR^s protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR-regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca²⁺ but increased with a rise in pH. The downregulation of almEFG in CarR^s strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains.

Brucella spp. are facultative intracellular bacteria notorious for their ability to induce a chronic, and often lifelong, infection known as brucellosis. To date, no licensed vaccine exists for prevention of human disease, and mechanisms underlying chronic illness and immune evasion remain elusive. We and others have observed that B cell-deficient mice challenged with Brucella display reduced bacterial burden following infection, but the underlying mechanism has not been clearly defined. Here, we show that at 1 month postinfection, B cell deficiency alone enhanced resistance to splenic infection ~100-fold; however, combined B and T cell deficiency did not impact bacterial burden, indicating that B cells only enhance susceptibility to infection when T cells are present. Therefore, we investigated whether B cells inhibit T cell-mediated protection against Brucella. Using B and T cell-deficient Rag1^–/– animals as recipients, we demonstrate that adoptive transfer of CD4⁺ T cells alone confers marked protection against Brucella melitensis that is abrogated by cotransfer of B cells. Interestingly, depletion of CD4⁺ T cells from B cell-deficient, but not wild-type, mice enhanced susceptibility to infection, further confirming that CD4⁺ T cell-mediated immunity against Brucella is inhibited by B cells. In addition, we found that the ability of B cells to suppress CD4⁺ T cell-mediated immunity and modulate CD4⁺ T cell effector responses during infection was major histocompatibility complex class II (MHCII)-dependent. Collectively, these findings indicate that B cells modulate CD4⁺ T cell function through an MHCII-dependent mechanism which enhances susceptibility to Brucella infection.

Gamma interferon (IFN-)-induced innate immune responses play important roles in the inhibition of Toxoplasma gondii infection. It has been reported that IFN- stimulates non-acidification-dependent growth restriction of T. gondii in HeLa cells, but the mechanism remains unclear. Here, we found that -aminobutyric acid (GABA) receptor-associated protein-like 2 (GABARAPL2) plays a critical role in parasite restriction in IFN--treated HeLa cells. GABARAPL2 is recruited to membrane structures surrounding parasitophorous vacuoles (PV). Autophagy adaptors are required for the proper localization and function of GABARAPL2 in the IFN- -induced immune response. These findings provide further understanding of a noncanonical autophagy pathway responsible for IFN--dependent inhibition of T. gondii growth in human HeLa cells and demonstrate the critical role of GABARAPL2 in this response.

Mycoplasma gallisepticum is the primary etiological agent of chronic respiratory disease in chickens. Live attenuated vaccines are most commonly used in the field to control the disease, but current vaccines have some limitations. Vaxsafe MG (strain ts-304) is a new vaccine candidate that is efficacious at a lower dose than the current commercial vaccine strain ts-11, from which it is derived. In this study, the transcriptional profiles of the trachea of unvaccinated chickens and chickens vaccinated with strain ts-304 were compared 2 weeks after challenge with M. gallisepticum strain Ap3AS during the chronic stage of infection. After challenge, genes, gene ontologies, pathways, and protein classes involved in inflammation, cytokine production and signaling, and cell proliferation were upregulated, while those involved in formation and motor movement of cilia, formation of intercellular junctional complexes, and formation of the cytoskeleton were downregulated in the unvaccinated birds compared to the vaccinated birds, reflecting immune dysregulation and the pathological changes induced in the trachea by infection with M. gallisepticum. Vaccination appears to protect the structural and functional integrity of the tracheal mucosa 2 weeks after infection with M. gallisepticum.

Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis (P. gingivalis), a keystone pathogen for the development of periodontitis. The bacterial cell wall component peptidoglycan uses bacterial outer membrane vesicles to be taken up by cells. Internalized peptidoglycan triggers cytosolic receptors to induce PD-L1 expression in a myeloid differentiation primary response 88 (Myd88)-independent and receptor-interacting serine/threonine-protein kinase 2 (RIP2)-dependent fashion. Interference with the kinase activity of RIP2 or mitogen-activated protein (MAP) kinases interferes with inducible PD-L1 expression.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa. However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa. However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.

The intracellular lifestyle of bacteria is widely acknowledged to be an important mechanism in chronic and recurring infection. Among the Staphylococcus genus, only Staphylococcus aureus and Staphylococcus pseudintermedius have been clearly identified as intracellular in nonprofessional phagocytic cells (NPPCs), for which the mechanism is mainly fibronectin-binding dependent. Here, we used bioinformatics tools to search for possible new fibronectin-binding proteins (FnBP-like) in other Staphylococcus species. We found a protein in Staphylococcus delphini called Staphylococcus delphini surface protein Y (SdsY). This protein shares 68% identity with the Staphylococcus pseudintermedius surface protein D (SpsD), 36% identity with S. aureus FnBPA, and 39% identity with S. aureus FnBPB. The SdsY protein possesses the typical structure of FnBP-like proteins, including an N-terminal signal sequence, an A domain, a characteristic repeated pattern, and an LPXTG cell wall anchor motif. The level of adhesion to immobilized fibronectin was significantly higher in all S. delphini strains tested than in the fibronectin-binding-deficient S. aureus DU5883 strain. By using a model of human osteoblast infection, the level of internalization of all strains tested was significantly higher than with the invasive-incompetent S. aureus DU5883. These findings were confirmed by phenotype restoration after transformation of DU5883 by a plasmid expression vector encoding the SdsY repeats. Additionally, using fibronectin-depleted serum and murine osteoblast cell lines deficient for the β₁ integrin, the involvement of fibronectin and β₁ integrin was demonstrated in S. delphini internalization. The present study demonstrates that additional staphylococcal species are able to invade NPPCs and proposes a method to identify FnBP-like proteins.

This article is meant as a personal reflection on my unexpected retirement, and includes memories and thoughts about my career with the Indian Health Service near the border with Mexico, serving the Tohono O’odham tribe.

Background:

Patient identification is an important step for advance care planning (ACP) discussions.

There are approximately 1 million transgender and gender-diverse adults in the United States. Despite increased awareness and acceptance, they frequently encounter medical settings that are not welcoming and/or health care providers who are not knowledgeable about their health needs. Use of correct terminology, following best practices for name and pronoun use, and knowledge of gender-affirming interventions can create office environments that are welcoming to transgender clients. Health disparities faced by transgender patients that impact access to care include higher rates of mental health issues, substance use disorders, violence, and poverty. Transgender women are at greater risk for HIV acquisition and are less likely to achieve viral suppression compared with cisgender (nontransgender) individuals. Medical providers can facilitate HIV prevention efforts by offering pre- and postexposure prophylaxis to transgender patients at risk for HIV infection. Improving health outcomes requires attention to cultural competency and an understanding of lived experiences and priorities of transgender people.

Cervical spondylotic myelopathy (CSM) is a neurologic condition that develops insidiously over time as degenerative changes of the spine result in compression of the cord and nearby structures. It is the most common form of spinal cord injury in adults; yet, its diagnosis is often delayed. The purpose of this article is to review the pathophysiology, natural history, diagnosis, and management of CSM with a focus on the recommended timeline for physicians suspecting CSM to refer patients to a spine surgeon. Various processes underlie spondylotic changes of the canal and are separated into static and dynamic factors. Not all patients with evidence of cord compression will present with symptoms, and the progression of disease varies by patient. The hallmark symptoms of CSM include decreased hand dexterity and gait instability as well as sensory and motor dysfunction. magnetic resonance imaging is the imaging modality of choice in patients with suspected CSM, but computed tomography myelography may be used in patients with contraindications. Patients with mild CSM may be treated surgically or nonoperatively, whereas those with moderate-severe disease are treated operatively. Due to the long-term disability that may result from a delay in diagnosis and management, prompt referral to a spine surgeon is recommended for any patient suspected of having CSM. This review provides information and guidelines for practitioners to develop an actionable awareness of CSM.

Inhaled corticosteroid (ICS)–based therapy is often used for patients with chronic obstructive pulmonary disease (COPD). However, this approach is under scrutiny because of ICS overuse in patients for whom it is not recommended and because of concerns about adverse events, particularly pneumonia, with long-term ICS use. Evidence suggests ICS may be beneficial in specific patients, namely, those with high blood eosinophil counts (eg, ≥300 cells/µL) or who are at a high risk of exacerbations. According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2020 ABCD assessment tool, these patients belong in group D. For these patients, recommended initial treatment includes ICS in combination with long-acting β2-agonists (LABAs) when blood eosinophil counts are ≥300 cells/µL or LABA + long-acting muscarinic antagonist (LAMA) when patients are highly symptomatic, that is, with greater dyspnea and/or exercise limitation. Follow-up treatments for patients with persistent dyspnea and/or exacerbations may include LABA + ICS, LABA + LAMA, or LABA + LAMA + ICS, with use of ICS being guided by blood eosinophil counts. In this review, differences in the inflammatory mechanism underlying COPD and asthma and the role of ICS treatment in COPD are summarized. Furthermore, findings from recent clinical trials where use of ICS-based dual or triple therapy in COPD was compared with LABA + LAMA therapy and trials in which ICS withdrawal was evaluated in patients with COPD are reviewed. Finally, a step-by-step guide for ICS withdrawal in patients who are unlikely to benefit from this treatment is proposed. A video of the author discussing the overall takeaway of the review article could be downloaded from the link provided: https://www.youtube.com/watch?v=Uq7Sr5jqPDI.

Background:

Thyroid disorders are among the most commonly treated conditions by the United States health care system. The number of patients reporting thyroid hormone use has increased in recent years, but it is unknown if there have been differential increases in the number of treated individuals within different demographic groups. Previous research has also not evaluated how expenditures for different thyroid hormone medications have changed in recent years.

Background:

Direct-to-consumer prescription drug advertising is prevalent and affects patient care. Previous research that examined its effect on the patient-provider relationship predates many changes in the advertising and medical landscape that have occurred in the last decade, such as the rise in online promotion and the push for value-based medicine.

Background:

Despite widespread recognition that adherence to clinical preventive guidelines improves patient outcomes, clinicians struggle to implement guideline changes in a timely manner. Multiple factors influence guideline adoption and effective implementation. However, few studies evaluate their collective and inter-related effects. This qualitative study provides a comprehensive picture of the interplay between multiple factors on uptake of new or changed preventive guidelines.

Background:

Prediabetes is increasing in prevalence and is associated with risk of developing diabetes, heart disease, stroke, and retinopathy. Clinicians have limited tools to facilitate prediabetes discussions within primary care visits.

Purpose:

Clinical evidence shows minimal benefit to vitamin D screening and subsequent treatment in the general population. This study aims to assess the effectiveness of 2 light-touch interventions on reducing vitamin D test orders.

Background:

In 2015, Vyvanse (lisdexamfetamine) became the first Food and Drug Administration (FDA)-approved treatment for binge-eating disorder (BED), a condition first recognized by the DSM–V in 2013. Because pharmaceutical companies use continuing medical education (CME) to help sell drugs, we explored possible bias in CME modules on BED.

Background:

Facilitation is an effective approach for helping practices implement sustainable evidence-based practice improvements. Few studies examine the facilitation infrastructure and support needed for large-scale dissemination and implementation initiatives.

Background and Objectives:

The goal of this study was to decrease admission and readmission rate for the 2296 Medicaid patients in our clinic. Our focus was to eliminate patient identified barriers to care that led to decreased quality of care. The identified barriers for our clinic included distance to care, poor same-day access, communication, and fragmented care. A team-based, collaborative approach using members from all aspects of patient care.

Introduction:

Understanding patients’ perspectives about their diabetes and what causes those perspectives to shift is critical to building a treatment strategy with the patient and facilitating patient self-management behavior. Key "turning points" can provide crucial opportunities to enact a change in perspective. The goal of this study is to identify "turning points" that have significance to diabetes-related health.

Purpose:

To identify specific actions and characteristics of health care providers (HCPs) in the United States and Canada that influenced patients with type 2 diabetes who were initially reluctant to begin insulin.

Purpose:

Excess weight gain during pregnancy is at epidemic proportions, and pregnancy complications are also on the rise. We sought to determine whether better weight gain counseling of expectant mothers will improve obstetric outcomes.

Access to services related to reproductive and sexual health is critical to the health of women but has been threatened in recent years. Family physicians are trained to provide a range of women’s health care services and are an essential part of the health care workforce in rural and underserved areas, where access to these services may be limited.

Evaluations of health care–based screening programs for social risks often report that a relatively small proportion of patients screening positive for social risk factors are interested in receiving assistance from their health care teams to address them. The relatively low number of patients who desire assistance is relevant to the growing number of initiatives in US health care settings designed to collect data on and address patients’ social risks. We highlight multiple studies that have found differences between positive risks screens and desire for assistance. We explore possible explanations for those differences—focusing on the fallibility of screening tools as well as patient preferences, priorities, and lived experiences—and the potential implications for health equity.

JABFM seeks to widely disseminate its peer-reviewed publications, increasing article visibility for the purpose of advancing scientific knowledge. We describe the journal’s approach to dissemination and recommend a number of strategies for authors to implement, including press releases and social media. Providing the article’s digital object identifier (DOI) is most useful, compared with links that can break, or attaching the article PDF, which will depress reader metrics. All JABFM articles are freely accessible online worldwide.

Family physician researchers continue to provide assistance to improve family medicine care. Commentaries on social determinants of health lead off this issue. Next, we have several papers on successful interventions by clinicians and/or patients to improve diabetes control, and then other provide information on other practice interventions that make a difference in overall care. Drug advertising continues to mislead. There is costly and nonproductive overuse of specific types of care. Herein is also a Scoping Review of possible indices for determining timely initiation of advance care planning. The issue’s clinical reviews on use of transgender care, cervical myelopathy, and inhaled steroids for chronic obstructive pulmonary disease are pertinent, thorough, and timely.