Differential Effects of Glutamine Inhibition Strategies on Antitumor CD8 T Cells.

Activated T cells undergo metabolic reprogramming to meet anabolic, differentiation, and functional demands. Glutamine supports many processes in activated T cells, and inhibition of glutamine metabolism alters T cell function in autoimmune disease and cancer. Multiple glutamine-targeting molecules are under investigation, yet the precise mechanisms of glutamine-dependent CD8 T cell differentiation remain unclear. We show that distinct strategies of glutamine inhibition by glutaminase-specific inhibition with small molecule CB-839, pan-glutamine inhibition with 6-diazo-5-oxo-l-norleucine (DON), or by glutamine-depleted conditions (No Q) produce distinct metabolic differentiation trajectories in murine CD8 T cells. T cell activation with CB-839 treatment had a milder effect than did DON or No Q treatment. A key difference was that CB-839-treated cells compensated with increased glycolytic metabolism, whereas DON and No Q-treated cells increased oxidative metabolism. However, all glutamine treatment strategies elevated CD8 T cell dependence on glucose metabolism, and No Q treatment caused adaptation toward reduced glutamine dependence. DON treatment reduced histone modifications and numbers of persisting cells in adoptive transfer studies, but those T cells that remained could expand normally upon secondary Ag encounter. In contrast, No Q-treated cells persisted well yet demonstrated decreased secondary expansion. Consistent with reduced persistence, CD8 T cells activated in the presence of DON had reduced ability to control tumor growth and reduced tumor infiltration in adoptive cell therapy. Overall, each approach to inhibit glutamine metabolism confers distinct effects on CD8 T cells and highlights that targeting the same pathway in different ways can elicit opposing metabolic and functional outcomes.

Pathogen elimination by probiotic Bacillus via signalling interference.

Probiotic nutrition is frequently claimed to improve human health. In particular, live probiotic bacteria obtained with food are thought to reduce intestinal colonization by pathogens, and thus to reduce susceptibility to infection. However, the mechanisms that underlie these effects remain poorly understood. Here we report that the consumption of probiotic Bacillus bacteria comprehensively abolished colonization by the dangerous pathogen Staphylococcus aureus in a rural Thai population. We show that a widespread class of Bacillus lipopeptides, the fengycins, eliminates S. aureus by inhibiting S. aureus quorum sensing-a process through which bacteria respond to their population density by altering gene regulation. Our study presents a detailed molecular mechanism that underlines the importance of probiotic nutrition in reducing infectious disease. We also provide evidence that supports the biological significance of probiotic bacterial interference in humans, and show that such interference can be achieved by blocking a pathogen's signalling system. Furthermore, our findings suggest a probiotic-based method for S. aureus decolonization and new ways to fight S. aureus infections.

Contribution of Staphylococcal Enterotoxin B to Staphylococcus aureus Systemic Infection.

Staphylococcal enterotoxin B (SEB), which is produced by the major human pathogen, Staphylococcus aureus, represents a powerful superantigenic toxin and is considered a bioweapon. However, the contribution of SEB to S. aureus pathogenesis has never been directly demonstrated with genetically defined mutants in clinically relevant strains. Many isolates of the predominant Asian community-associated methicillin-resistant S. aureus lineage sequence type (ST) 59 harbor seb, implying a significant role of SEB in the observed hypervirulence of this lineage. We created an isogenic seb mutant in a representative ST59 isolate and assessed its virulence potential in mouse infection models. We detected a significant contribution of seb to systemic ST59 infection that was associated with a cytokine storm. Our results directly demonstrate that seb contributes to S. aureus pathogenesis, suggesting the value of including SEB as a target in multipronged antistaphylococcal drug development strategies. Furthermore, they indicate that seb contributes to fatal exacerbation of community-associated methicillin-resistant S. aureus infection.

Influenza vaccine community outreach: Leveraging an interprofessional healthcare student workforce to immunize marginalized populations.

Vulnerable populations such as the uninsured, unemployed, and unhoused face significant morbidity and mortality from influenza but are less likely to receive the annual vaccine and have limited access to medical care. We describe an interprofessional, student-run vaccine outreach program (VOP) in Davidson County, Tennessee that lowers barriers to vaccination through free vaccination events in nontraditional community locations. We provide this framework as a model to expand novel, seasonal, or outbreak-oriented vaccine outreach to resource-poor populations. Demographic data were collected from the patients who received an influenza vaccine between 2015 and 2019 through an optional survey to determine whether these events were reaching unhoused, uninsured, and/or unemployed individuals. Of 1,803 patients, 1,733 (96.1%) completed at least one field of the demographic form. Overall, 481 (27.8%) were individuals without homes or living in temporary housing and 673 (38.8%) were unemployed. Most patients, 1,109 (64.0%), did not have health insurance at any point during the prior two years. With the addition of a nurse practitioner student to VOP leadership, the 2018-2019 VOP reached the most unhoused or temporarily-housed (228, 32.3%), unemployed (313, 18.5%), and disabled (60, 8.5%) patients. The VOP can be adapted to meet community needs, funding, and volunteer interest. The VOP model may be applicable to a SARS-CoV-2 vaccine, especially since the economic impact of COVID-19 has increased unemployment rates and housing instability. Healthcare students serve as an eager, underutilized resource who can be leveraged to disseminate vaccines to individuals with limited access to care.

Toxin Mediates Sepsis Caused by Methicillin-Resistant Staphylococcus epidermidis.

Bacterial sepsis is a major killer in hospitalized patients. Coagulase-negative staphylococci (CNS) with the leading species Staphylococcus epidermidis are the most frequent causes of nosocomial sepsis, with most infectious isolates being methicillin-resistant. However, which bacterial factors underlie the pathogenesis of CNS sepsis is unknown. While it has been commonly believed that invariant structures on the surface of CNS trigger sepsis by causing an over-reaction of the immune system, we show here that sepsis caused by methicillin-resistant S. epidermidis is to a large extent mediated by the methicillin resistance island-encoded peptide toxin, PSM-mec. PSM-mec contributed to bacterial survival in whole human blood and resistance to neutrophil-mediated killing, and caused significantly increased mortality and cytokine expression in a mouse sepsis model. Furthermore, we show that the PSM-mec peptide itself, rather than the regulatory RNA in which its gene is embedded, is responsible for the observed virulence phenotype. This finding is of particular importance given the contrasting roles of the psm-mec locus that have been reported in S. aureus strains, inasmuch as our findings suggest that the psm-mec locus may exert effects in the background of S. aureus strains that differ from its original role in the CNS environment due to originally "unintended" interferences. Notably, while toxins have never been clearly implied in CNS infections, our tissue culture and mouse infection model data indicate that an important type of infection caused by the predominant CNS species is mediated to a large extent by a toxin. These findings suggest that CNS infections may be amenable to virulence-targeted drug development approaches.

Antimicrobial Peptide Resistance Mechanism Contributes to Staphylococcus aureus Infection.

Antimicrobial peptides (AMPs) constitute an important part of innate host defense. Possibly limiting the therapeutic potential of AMPs is the fact that bacteria have developed AMP resistance mechanisms during their co-evolution with humans. However, there is no direct evidence that AMP resistance per se is important during an infection. Here we show that the Staphylococcus aureus Pmt ABC transporter defends the bacteria from killing by important human AMPs and elimination by human neutrophils. By showing that Pmt contributes to virulence during skin infection in an AMP-dependent manner, we provide evidence that AMP resistance plays a key role in bacterial infection.

Functional overlap of inborn errors of immunity and metabolism genes defines T cell metabolic vulnerabilities.

Inborn errors of metabolism (IEMs) and immunity (IEIs) are Mendelian diseases in which complex phenotypes and patient rarity have limited clinical understanding. Whereas few genes have been annotated as contributing to both IEMs and IEIs, immunometabolic demands suggested greater functional overlap. Here, CRISPR screens tested IEM genes for immunologic roles and IEI genes for metabolic effects and found considerable previously unappreciated crossover. Analysis of IEMs showed that N-linked glycosylation and the hexosamine pathway enzyme Gfpt1 are critical for T cell expansion and function. Further, T helper (TH1) cells synthesized uridine diphosphate N-acetylglucosamine more rapidly and were more impaired by Gfpt1 deficiency than TH17 cells. Screening IEI genes found that Bcl11b promotes the CD4 T cell mitochondrial activity and Mcl1 expression necessary to prevent metabolic stress. Thus, a high degree of functional overlap exists between IEM and IEI genes, and immunometabolic mechanisms may underlie a previously underappreciated intersection of these disorders.

Subset-specific mitochondrial stress and DNA damage shape T cell responses to fever and inflammation.

Heat is a cardinal feature of inflammation, yet its impacts on immune cells remain uncertain. We show that moderate-grade fever temperatures (39°C) increased murine CD4 T cell metabolism, proliferation, and inflammatory effector activity while decreasing regulatory T cell suppressive capacity. However, heat-exposed T helper 1 (TH1) cells selectively developed mitochondrial stress and DNA damage that activated Trp53 and stimulator of interferon genes pathways. Although many TH1 cells subjected to such temperatures died, surviving TH1 cells exhibited increased mitochondrial mass and enhanced activity. Electron transport chain complex 1 (ETC1) was rapidly impaired under fever-range temperatures, a phenomenon that was specifically detrimental to TH1 cells. TH1 cells with elevated DNA damage and ETC1 signatures were also detected in human chronic inflammation. Thus, fever-relevant temperatures disrupt ETC1 to selectively drive apoptosis or adaptation of TH1 cells to maintain genomic integrity and enhance effector functions.

Functional Overlap of Inborn Errors of Immunity and Metabolism Genes Define T Cell Immunometabolic Vulnerabilities.

Inborn Errors of Metabolism (IEM) and Immunity (IEI) are Mendelian diseases in which complex phenotypes and patient rarity can limit clinical annotations. Few genes are assigned to both IEM and IEI, but immunometabolic demands suggest functional overlap is underestimated. We applied CRISPR screens to test IEM genes for immunologic roles and IEI genes for metabolic effects and found considerable crossover. Analysis of IEM showed N-linked glycosylation and the de novo hexosamine synthesis enzyme, Gfpt1 , are critical for T cell expansion and function. Interestingly, Gfpt1 -deficient T H 1 cells were more affected than T H 17 cells, which had increased Nagk for salvage UDP-GlcNAc synthesis. Screening IEI genes showed the transcription factor Bcl11b promotes CD4 + T cell mitochondrial activity and Mcl1 expression necessary to prevent metabolic stress. These data illustrate a high degree of functional overlap of IEM and IEI genes and point to potential immunometabolic mechanisms for a previously unappreciated set of these disorders. HIGHLIGHTS: Inborn errors of immunity and metabolism have greater overlap than previously known Gfpt1 deficiency causes an IEM but also selectively regulates T cell subset fate Loss of Bcl11b causes a T cell deficiency IEI but also harms mitochondrial function Many IEM may have immune defects and IEI may be driven by metabolic mechanisms.

Protein kinase C delta regulates mononuclear phagocytes and hinders response to immunotherapy in cancer.

Mononuclear phagocytes (MPs) play a crucial role in tissue homeostasis; however, MPs also contribute to tumor progression and resistance to immune checkpoint blockade (ICB). Targeting MPs could be an effective strategy to enhance ICB efficacy. We report that protein kinase C delta (PKCδ), a serine/threonine kinase, is abundantly expressed by MPs in human and mouse tumors. PKCδ-/- mice displayed reduced tumor progression compared to wild types, with increased response to anti-PD-1. Tumors from PKCδ-/- mice demonstrated TH1-skewed immune response including increased antigen presentation and T cell activation. Depletion of MPs in vivo altered tumor growth in control but not PKCδ-/- mice. Coinjection of PKCδ-/- M2-like macrophages with cancer cells into wild-type mice markedly delayed tumor growth and significantly increased intratumoral T cell activation compared to PKCδ+/+ controls. PKCδ deficiency reprogrammed MPs by activating type I and type II interferon signaling. Thus, PKCδ might be targeted to reprogram MPs to augment ICB efficacy.

Microenvironmental influences on T cell immunity in cancer and inflammation.

T cell metabolism is dynamic and highly regulated. While the intrinsic metabolic programs of T cell subsets are integral to their distinct differentiation and functional patterns, the ability of cells to acquire nutrients and cope with hostile microenvironments can limit these pathways. T cells must function in a wide variety of tissue settings, and how T cells interpret these signals to maintain an appropriate metabolic program for their demands or if metabolic mechanisms of immune suppression restrain immunity is an area of growing importance. Both in inflamed and cancer tissues, a wide range of changes in physical conditions and nutrient availability are now acknowledged to shape immunity. These include fever and increased temperatures, depletion of critical micro and macro-nutrients, and accumulation of inhibitory waste products. Here we review several of these factors and how the tissue microenvironment both shapes and constrains immunity.

Commensal Staphylococcus epidermidis contributes to skin barrier homeostasis by generating protective ceramides.

Previously either regarded as insignificant or feared as potential sources of infection, the bacteria living on our skin are increasingly recognized for their role in benefitting human health. Skin commensals modulate mucosal immune defenses and directly interfere with pathogens; however, their contribution to the skin's physical integrity is less understood. Here, we show that the abundant skin commensal Staphylococcus epidermidis contributes to skin barrier integrity. S. epidermidis secretes a sphingomyelinase that acquires essential nutrients for the bacteria and assists the host in producing ceramides, the main constituent of the epithelial barrier that averts skin dehydration and aging. In mouse models, S. epidermidis significantly increases skin ceramide levels and prevents water loss of damaged skin in a fashion entirely dependent on its sphingomyelinase. Our findings reveal a symbiotic mechanism that demonstrates an important role of the skin microbiota in the maintenance of the skin's protective barrier.

COVID-19 vaccination program at a student-run free clinic: A descriptive study.

People historically excluded from receiving medical care in the United States, in addition to being at greater risk for SARS-CoV-2 infection, have had slower vaccine uptake due to structural barriers to availability. We present one student-run free clinic's SARS-CoV-2 vaccination program from January 15 to August 1, 2021, in Nashville, Tennessee. We tracked SARS-CoV-2 vaccine primary series completion among 273 free clinic patients with the help of medical student volunteers, who scheduled appointments and answered vaccine-related questions. We worked with our academic medical center partner to host a single-dose vaccination event at our clinic. We compared vaccine series completion in our clinic to adult vaccine completion in Davidson County, Tennessee on August 1, 2021. Of the 273 free clinic participants, 144 identified as Spanish-speaking (52.7%) and 172 (63%) had at least one qualifying comorbidity per the December 30, 2020, Tennessee COVID-19 Vaccination Plan. As such, 183 (67%) were characterized as vaccine eligible in Phase 1a2, 1b, or 1c. On August 1, 2021, 63.1% of free clinic patients had completed their primary SARS-CoV-2 vaccination series compared with 58.9% of adults in Davidson County, Tennessee (RD 4.2%, 95% CI: -1.5% to 9.9%). Spanish-speaking free clinic patients were most likely to have completed their vaccination series. We describe a framework for a patient-centered vaccination effort to reach individuals traditionally missed by large vaccination campaigns. We highlight structural hurdles experienced by vulnerable populations, including language barriers, lack of technology or reliable internet access, inflexible working schedules, lack of transportation, and vaccine misinformation.

Rapid pathogen-specific recruitment of immune effector cells in the skin by secreted toxins.

Swift recruitment of phagocytic leucocytes is critical in preventing infection when bacteria breach through the protective layers of the skin. According to canonical models, this occurs via an indirect process that is initiated by contact of bacteria with resident skin cells and which is independent of the pathogenic potential of the invader. Here we describe a more rapid mechanism of leucocyte recruitment to the site of intrusion of the important skin pathogen Staphylococcus aureus that is based on direct recognition of specific bacterial toxins, the phenol-soluble modulins (PSMs), by circulating leucocytes. We used a combination of intravital imaging, ear infection and skin abscess models, and in vitro gene expression studies to demonstrate that this early recruitment was dependent on the transcription factor EGR1 and contributed to the prevention of infection. Our findings refine the classical notion of the non-specific and resident cell-dependent character of the innate immune response to bacterial infection by demonstrating a pathogen-specific high-alert mechanism involving direct recruitment of immune effector cells by secreted bacterial products.

Role of Phenol-Soluble Modulins in Staphylococcus epidermidis Biofilm Formation and Infection of Indwelling Medical Devices.

Phenol-soluble modulins (PSMs) are amphipathic, alpha-helical peptides that are secreted by staphylococci in high amounts in a quorum-sensing-controlled fashion. Studies performed predominantly in Staphylococcus aureus showed that PSMs structure biofilms, which results in reduced biofilm mass, while it has also been reported that S. aureus PSMs stabilize biofilms due to amyloid formation. We here analyzed the roles of PSMs in in vitro and in vivo biofilms of Staphylococcus epidermidis, the leading cause of indwelling device-associated biofilm infection. We produced isogenic deletion mutants for every S. epidermidis psm locus and a sequential deletion mutant in which production of all PSMs was abolished. In vitro analysis substantiated the role of all PSMs in biofilm structuring. PSM-dependent biofilm expansion was not observed, in accordance with our finding that no S. epidermidis PSM produced amyloids. In a mouse model of indwelling device-associated infection, the total psm deletion mutant had a significant defect in dissemination. Notably, the total psm mutant produced a significantly more substantial biofilm on the implanted catheter than the wild-type strain. Our study, which for the first time directly quantified the impact of PSMs on biofilm expansion on an implanted device, shows that the in vivo biofilm infection phenotype in S. epidermidis is in accordance with the PSM biofilm structuring and detachment model, which has important implications for the potential therapeutic application of quorum-sensing blockers.

Basis of Virulence in Enterotoxin-Mediated Staphylococcal Food Poisoning.

The Staphylococcus aureus enterotoxins are a superfamily of secreted virulence factors that share structural and functional similarities and possess potent superantigenic activity causing disruptions in adaptive immunity. The enterotoxins can be separated into two groups; the classical (SEA-SEE) and the newer (SEG-SElY and counting) enterotoxin groups. Many members from both these groups contribute to the pathogenesis of several serious human diseases, including toxic shock syndrome, pneumonia, and sepsis-related infections. Additionally, many members demonstrate emetic activity and are frequently responsible for food poisoning outbreaks. Due to their robust tolerance to denaturing, the enterotoxins retain activity in food contaminated previously with S. aureus. The genes encoding the enterotoxins are found mostly on a variety of different mobile genetic elements. Therefore, the presence of enterotoxins can vary widely among different S. aureus isolates. Additionally, the enterotoxins are regulated by multiple, and often overlapping, regulatory pathways, which are influenced by environmental factors. In this review, we also will focus on the newer enterotoxins (SEG-SElY), which matter for the role of S. aureus as an enteropathogen, and summarize our current knowledge on their prevalence in recent food poisoning outbreaks. Finally, we will review the current literature regarding the key elements that govern the complex regulation of enterotoxins, the molecular mechanisms underlying their enterotoxigenic, superantigenic, and immunomodulatory functions, and discuss how these activities may collectively contribute to the overall manifestation of staphylococcal food poisoning.