Transcription-replication interactions reveal bacterial genome regulation.

Organisms determine the transcription rates of thousands of genes through a few modes of regulation that recur across the genome1. In bacteria, the relationship between the regulatory architecture of a gene and its expression is well understood for individual model gene circuits2,3. However, a broader perspective of these dynamics at the genome scale is lacking, in part because bacterial transcriptomics has hitherto captured only a static snapshot of expression averaged across millions of cells4. As a result, the full diversity of gene expression dynamics and their relation to regulatory architecture remains unknown. Here we present a novel genome-wide classification of regulatory modes based on the transcriptional response of each gene to its own replication, which we term the transcription-replication interaction profile (TRIP). Analysing single-bacterium RNA-sequencing data, we found that the response to the universal perturbation of chromosomal replication integrates biological regulatory factors with biophysical molecular events on the chromosome to reveal the local regulatory context of a gene. Whereas the TRIPs of many genes conform to a gene dosage-dependent pattern, others diverge in distinct ways, and this is shaped by factors such as intra-operon position and repression state. By revealing the underlying mechanistic drivers of gene expression heterogeneity, this work provides a quantitative, biophysical framework for modelling replication-dependent expression dynamics.

Bacterial contact induces polar plug disintegration to mediate whipworm egg hatching.

The bacterial microbiota promotes the life cycle of the intestine-dwelling whipworm Trichuris by mediating hatching of parasite eggs ingested by the mammalian host. Despite the enormous disease burden associated with Trichuris colonization, the mechanisms underlying this transkingdom interaction have been obscure. Here, we used a multiscale microscopy approach to define the structural events associated with bacteria-mediated hatching of eggs for the murine model parasite Trichuris muris. Through the combination of scanning electron microscopy (SEM) and serial block face SEM (SBFSEM), we visualized the outer surface morphology of the shell and generated 3D structures of the egg and larva during the hatching process. These images revealed that exposure to hatching-inducing bacteria catalyzed asymmetric degradation of the polar plugs prior to exit by the larva. Unrelated bacteria induced similar loss of electron density and dissolution of the structural integrity of the plugs. Egg hatching was most efficient when high densities of bacteria were bound to the poles. Consistent with the ability of taxonomically distant bacteria to induce hatching, additional results suggest chitinase released from larva within the eggs degrade the plugs from the inside instead of enzymes produced by bacteria in the external environment. These findings define at ultrastructure resolution the evolutionary adaptation of a parasite for the microbe-rich environment of the mammalian gut.

ACE2-containing defensosomes serve as decoys to inhibit SARS-CoV-2 infection.

Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19). Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchoalveolar lavage fluid (BALF) from critically ill COVID-19 patients was associated with reduced intensive care unit (ICU) and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.

Staphylococcus aureus senses human neutrophils via PerR to coordinate the expression of the toxin LukAB.

Staphylococcus aureus is a gram-positive pathogen that poses a major health concern, in part due to its large array of virulence factors that allow infection and evasion of the immune system. One of these virulence factors is the bicomponent pore-forming leukocidin LukAB. The regulation of lukAB expression is not completely understood, especially in the presence of immune cells such as human polymorphonuclear neutrophils (hPMNs). Here, we screened for transcriptional regulators of lukAB during the infection of primary hPMNs. We uncovered that PerR, a peroxide sensor, is vital for hPMN-mediated induction of lukAB and that PerR upregulates cytotoxicity during the infection of hPMNs. Exposure of S. aureus to hydrogen peroxide (H2O2) alone also results in increased lukAB promoter activity, a phenotype dependent on PerR. Collectively, our data suggest that S. aureus uses PerR to sense the H2O2 produced by hPMNs to stimulate the expression of lukAB, allowing the bacteria to withstand these critical innate immune cells.IMPORTANCEStaphylococcus aureus utilizes a diverse set of virulence factors, such as leukocidins, to subvert human neutrophils, but how these toxins are regulated is incompletely defined. Here, we identified the peroxide-sensitive repressor, PerR, as a required protein involved in the induction of lukAB in the presence of primary human neutrophils, a phenotype directly linked to the ability of PerR to sense H2O2. Thus, we show that S. aureus coordinates sensing and resistance to oxidative stress with toxin production to promote pathogen survival.

Priorities and Progress in Gram-positive Bacterial Infection Research by the Antibacterial Resistance Leadership Group: A Narrative Review.

The Antibacterial Resistance Leadership Group (ARLG) has prioritized infections caused by gram-positive bacteria as one of its core areas of emphasis. The ARLG Gram-positive Committee has focused on studies responding to 3 main identified research priorities: (1) investigation of strategies or therapies for infections predominantly caused by gram-positive bacteria, (2) evaluation of the efficacy of novel agents for infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci, and (3) optimization of dosing and duration of antimicrobial agents for gram-positive infections. Herein, we summarize ARLG accomplishments in gram-positive bacterial infection research, including studies aiming to (1) inform optimal vancomycin dosing, (2) determine the role of dalbavancin in MRSA bloodstream infection, (3) characterize enterococcal bloodstream infections, (4) demonstrate the benefits of short-course therapy for pediatric community-acquired pneumonia, (5) develop quality of life measures for use in clinical trials, and (6) advance understanding of the microbiome. Future studies will incorporate innovative methodologies with a focus on interventional clinical trials that have the potential to change clinical practice for difficult-to-treat infections, such as MRSA bloodstream infections.

The purine biosynthesis regulator PurR moonlights as a virulence regulator in Staphylococcus aureus.

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR's canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.

Sequential evolution of virulence and resistance during clonal spread of community-acquired methicillin-resistant Staphylococcus aureus.

The past two decades have witnessed an alarming expansion of staphylococcal disease caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The factors underlying the epidemic expansion of CA-MRSA lineages such as USA300, the predominant CA-MRSA clone in the United States, are largely unknown. Previously described virulence and antimicrobial resistance genes that promote the dissemination of CA-MRSA are carried by mobile genetic elements, including phages and plasmids. Here, we used high-resolution genomics and experimental infections to characterize the evolution of a USA300 variant plaguing a patient population at increased risk of infection to understand the mechanisms underlying the emergence of genetic elements that facilitate clonal spread of the pathogen. Genetic analyses provided conclusive evidence that fitness (manifest as emergence of a dominant clone) changed coincidently with the stepwise emergence of (i) a unique prophage and mutation of the regulator of the pyrimidine nucleotide biosynthetic operon that promoted abscess formation and colonization, respectively, thereby priming the clone for success; and (ii) a unique plasmid that conferred resistance to two topical microbiocides, mupirocin and chlorhexidine, frequently used for decolonization and infection prevention. The resistance plasmid evolved through successive incorporation of DNA elements from non-S. aureus spp. into an indigenous cryptic plasmid, suggesting a mechanism for interspecies genetic exchange that promotes antimicrobial resistance. Collectively, the data suggest that clonal spread in a vulnerable population resulted from extensive clinical intervention and intense selection pressure toward a pathogen lifestyle that involved the evolution of consequential mutations and mobile genetic elements.

Unbiased Identification of Immunogenic Staphylococcus aureus Leukotoxin B-Cell Epitopes.

Unbiased identification of individual immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as few as one major pathogen antigen. Using the bicomponent pore-forming leukocidin (Luk) exotoxins of the major pathogen Staphylococcus aureus as a prototype, we randomly fragmented and separately ligated the hemolysin gamma A (HlgA) and LukS genes into a custom-built phage display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk monoclonal antibody (MAb) recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino-acid sequence that is highly conserved in four Luk toxin subunits and is ubiquitous in representation within S. aureus clinical isolates. The isolated 11-amino-acid peptide probe was predicted to retain the native three-dimensional (3D) conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk MAb, and, using mutated peptides, we showed that a particular amino acid side chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment- and phage display-based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much-needed S. aureus-protective subunit vaccine.

Using Quantitative Spectrometry to Understand the Influence of Genetics and Nutritional Perturbations On the Virulence Potential of Staphylococcus aureus.

Staphylococcus aureus (Sa) is the leading cause of a variety of bacterial infections ranging from superficial skin infections to invasive and life threatening diseases such as septic bacteremia, necrotizing pneumonia, and endocarditis. The success of Sa as a human pathogen is contributed to its ability to adapt to different environments by changing expression, production, or secretion of virulence factors. Although Sa immune evasion is well-studied, the regulation of virulence factors under different nutrient and growth conditions is still not well understood. Here, we used label-free quantitative mass spectrometry to quantify and compare the Sa exoproteins (i.e. exoproteomes) of master regulator mutants or established reference strains. Different environmental conditions were addressed by growing the bacteria in rich or minimal media at different phases of growth. We observed clear differences in the composition of the exoproteomes depending on the genetic background or growth conditions. The relative abundance of cytotoxins determined in our study correlated well with differences in cytotoxicity measured by lysis of human neutrophils. Our findings demonstrate that label-free quantitative mass spectrometry is a versatile tool for predicting the virulence of bacterial strains and highlights the importance of the experimental design for in vitro studies. Furthermore, the results indicate that label-free proteomics can be used to cluster isolates into groups with similar virulence properties, highlighting the power of label-free quantitative mass spectrometry to distinguish Sa strains.

Increased Staphylococcus aureus Nasal Carriage Rates in Rheumatoid Arthritis Patients on Biologic Therapy.

BACKGROUND: Rheumatoid arthritis patients are at increased risk for periprosthetic joint infection after arthroplasty. The reason is multifactorial. Nasal colonization with Staphylococcus aureus is a modifiable risk factor; carriage rates in RA patients are unknown. The goal of this study is to determine the S aureus nasal carriage rates of RA patients on biologics, RA patients on traditional disease-modifying anti-rheumatic drugs (DMARDs), and osteoarthritis. METHODS: Consecutive patients with RA on biologics (±DMARDs), RA on non-biologic DMARDs, or OA were prospectively enrolled from April 2017 to May 2018. One hundred twenty-three patients were determined necessary per group to show a difference in carriage rates. Patients underwent a nasal swab and answered questions to identify additional risk factors. S aureus positive swabs were further categorized using spa typing. Logistic regression evaluated the association with S aureus colonization between the groups after controlling for known risk factors. RESULTS: RA patients on biologics, 70% of whom were on DMARDs, had statistically significant increase in S aureus colonization (37%) compared to RA on DMARDs alone (24%), or OA (20%) (P = .01 overall). After controlling for glucocorticoids, antibiotic use, recent hospitalization, and diabetes, RA on biologics had a significant increased risk of S aureus nasal colonization (Odds ratio 1.80, 95% confidence interval 1.00-3.22, P = .047). CONCLUSION: S aureus colonization risk was increased for RA on biologics compared to RA not on biologics and OA. Nasal S aureus carriage increases the risk of surgical site infection; this modifiable risk factor should be addressed prior to total joint arthroplasty for this higher risk patient group.

Genome Plasticity of agr-Defective Staphylococcus aureus during Clinical Infection.

Therapy for bacteremia caused by Staphylococcus aureus is often ineffective, even when treatment conditions are optimal according to experimental protocols. Adapted subclones, such as those bearing mutations that attenuate agr-mediated virulence activation, are associated with persistent infection and patient mortality. To identify additional alterations in agr-defective mutants, we sequenced and assembled the complete genomes of clone pairs from colonizing and infected sites of several patients in whom S. aureus demonstrated a within-host loss of agr function. We report that events associated with agr inactivation result in agr-defective blood and nares strain pairs that are enriched in mutations compared to pairs from wild-type controls. The random distribution of mutations between colonizing and infecting strains from the same patient, and between strains from different patients, suggests that much of the genetic complexity of agr-defective strains results from prolonged infection or therapy-induced stress. However, in one of the agr-defective infecting strains, multiple genetic changes resulted in increased virulence in a murine model of bloodstream infection, bypassing the mutation of agr and raising the possibility that some changes were selected. Expression profiling correlated the elevated virulence of this agr-defective mutant to restored expression of the agr-regulated ESAT6-like type VII secretion system, a known virulence factor. Thus, additional mutations outside the agr locus can contribute to diversification and adaptation during infection by S. aureus agr mutants associated with poor patient outcomes.

Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E (pafE) Mutant.

Mycobacterium tuberculosis uses a proteasome to degrade proteins by both ATP-dependent and -independent pathways. While much has been learned about ATP-dependent degradation, relatively little is understood about the ATP-independent pathway, which is controlled by Mycobacterium tuberculosisproteasome accessory factor E (PafE). Recently, we found that a Mycobacterium tuberculosispafE mutant has slowed growth in vitro and is sensitive to killing by heat stress. However, we did not know if these phenotypes were caused by an inability to degrade the PafE-proteasome substrate HspR (heat shock protein repressor), an inability to degrade any damaged or misfolded proteins, or a defect in another protein quality control pathway. To address this question, we characterized pafE suppressor mutants that grew similarly to pafE+ bacteria under normal culture conditions. All but one suppressor mutant analyzed contained mutations that inactivated HspR function, demonstrating that the slowed growth and heat shock sensitivity of a pafE mutant were caused primarily by the inability of the proteasome to degrade HspR.IMPORTANCEMycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required for virulence. We recently discovered a proteasome cofactor, PafE, which is required for the normal growth, heat shock resistance, and full virulence of M. tuberculosis In this study, we demonstrate that PafE influences this phenotype primarily by promoting the expression of protein chaperone genes that are necessary for surviving proteotoxic stress.

Staphylococcus aureus Strain Newman D2C Contains Mutations in Major Regulatory Pathways That Cripple Its Pathogenesis.

Staphylococcus aureus is a major human pathogen that imposes a great burden on the health care system. In the development of antistaphylococcal modalities intended to reduce the burden of staphylococcal disease, it is imperative to select appropriate models of S. aureus strains when assessing the efficacy of novel agents. Here, using whole-genome sequencing, we reveal that the commonly used strain Newman D2C from the American Type Culture Collection (ATCC) contains mutations that render the strain essentially avirulent. Importantly, Newman D2C is often inaccurately referred to as simply "Newman" in many publications, leading investigators to believe it is the well-described pathogenic strain Newman. This study reveals that Newman D2C carries a stop mutation in the open reading frame of the virulence gene regulator, agrA In addition, Newman D2C carries a single-nucleotide polymorphism (SNP) in the global virulence regulator gene saeR that results in loss of protein function. This loss of function is highlighted by complementation studies, where the saeR allele from Newman D2C is incapable of restoring functionality to an saeR-null mutant. Additional functional assessment was achieved through the use of biochemical assays for protein secretion, ex vivo intoxications of human immune cells, and in vivo infections. Altogether, our study highlights the importance of judiciously screening for genetic changes in model S. aureus strains when assessing pathogenesis or the efficacy of novel agents. Moreover, we have identified a novel SNP in the virulence regulator gene saeR that directly affects the ability of the protein product to activate S. aureus virulence pathways.IMPORTANCEStaphylococcus aureus is a human pathogen that imposes an enormous burden on health care systems worldwide. This bacterium is capable of evoking a multitude of disease states that can range from self-limiting skin infections to life-threatening bacteremia. To combat these infections, numerous investigations are under way to develop therapeutics capable of thwarting the deadly effects of the bacterium. To generate successful treatments, it is of paramount importance that investigators use suitable models for examining the efficacy of the drugs under study. Here, we demonstrate that a strain of S. aureus commonly used for drug efficacy studies is severely mutated and displays markedly reduced pathogenicity. As such, the organism is an inappropriate model for disease studies.

Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection.

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.

The cutaneous microbiome in outpatients presenting with acute skin abscesses.

BACKGROUND: Previous studies have demonstrated an association between antibiotic use and the development of skin abscesses. We tested the hypothesis that alterations in the composition of the cutaneous microbiota may predispose individuals to skin abscesses. METHODS: We studied 25 patients with skin abscesses and 25 age-matched controls, who each completed a questionnaire. Skin swab samples were obtained for DNA analysis from 4 sites around the abscess site (hereafter, "peri-abscess specimens") and from similar sites on the patient's contralateral side and on healthy control subjects. DNA was extracted and analyzed by quantitative polymerase chain reaction (qPCR) and high-throughput sequencing. The purulent abscess drainage was sent for culture. RESULTS: Fifteen patients with abscess were infected with Staphylococcus aureus. Use of nuc qPCR to quantitate S. aureus revealed a significantly greater frequency of positive results for peri-abscess and contralateral skin samples, compared with control skin specimens. Analysis of community structure showed greater heterogeneity in the control samples than in the peri-abscess and contralateral samples. Metagenomic analysis detected significantly more predicted genes related to metabolic activity in the peri-abscess specimens than in the control samples. CONCLUSIONS: The peri-abscess microbiome was similar to the contralateral microbiome, but both microbiomes differed from that for control patients. Host characteristics affecting microbial populations might be important determinants of abscess risk.

Cytotoxic Virulence Predicts Mortality in Nosocomial Pneumonia Due to Methicillin-Resistant Staphylococcus aureus.

The current study identified bacterial factors that may improve management of methicillin-resistant Staphylococcus aureus (MRSA) nosocomial pneumonia. Isolates were obtained from 386 patients enrolled in a randomized, controlled study of antibiotic efficacy. Isolates were screened for production of virulence factors and for vancomycin susceptibility. After adjustment for host factors such as severity of illness and treatment modality, cytotoxic activity was strongly and inversely associated with mortality; however, it had no effect on clinical cure. Isolates having low cytotoxicity, which were derived largely from healthcare-associated clones, exhibited a greater prevalence of vancomycin heteroresistance, and they were recovered more often from patients who were older and frailer. Additionally, a clone with low cytotoxic activity was associated with death and poor clinical improvement. Clone specificity and attenuated virulence appear to be associated with outcome. To our knowledge, these are the first correlations between MRSA virulence and mortality in nosocomial pneumonia.

Gastrointestinal dissemination and transmission of Staphylococcus aureus following bacteremia.

Mutations that alter virulence and antibiotic susceptibility arise and persist during Staphylococcus aureus bacteremia. However, an experimental system demonstrating transmission following bacteremia has been lacking, and thus implications of within-host adaptation for between-host transmission are unknown. We report that S. aureus disseminates to the gastrointestinal tract of mice following intravenous injection and readily transmits to cohoused naive mice. Both intestinal dissemination and transmission were linked to the production of virulence factors based on gene deletion studies of the sae and agr two-component systems. Furthermore, antimicrobial selection for antibiotic-resistant S. aureus displaced susceptible S. aureus from the intestine of infected hosts, which led to the preferential transmission and dominance of antibiotic-resistant bacteria among cohoused untreated mice. These findings establish an animal model to investigate gastrointestinal dissemination and transmission of S. aureus and suggest that adaptation during the course of systemic infection has implications beyond the level of a single host.

Evolution of hypervirulence by a MRSA clone through acquisition of a transposable element.

Staphylococcus aureus has evolved as a pathogen that causes a range of diseases in humans. There are two dominant modes of evolution thought to explain most of the virulence differences between strains. First, virulence genes may be acquired from other organisms. Second, mutations may cause changes in the regulation and expression of genes. Here we describe an evolutionary event in which transposition of an IS element has a direct impact on virulence gene regulation resulting in hypervirulence. Whole-genome analysis of a methicillin-resistant S. aureus (MRSA) strain USA500 revealed acquisition of a transposable element (IS256) that is absent from close relatives of this strain. Of the multiple copies of IS256 found in the USA500 genome, one was inserted in the promoter sequence of repressor of toxins (Rot), a master transcriptional regulator responsible for the expression of virulence factors in S. aureus. We show that insertion into the rot promoter by IS256 results in the derepression of cytotoxin expression and increased virulence. Taken together, this work provides new insight into evolutionary strategies by which S. aureus is able to modify its virulence properties and demonstrates a novel mechanism by which horizontal gene transfer directly impacts virulence through altering toxin regulation.

An rpsL-based allelic exchange vector for Staphylococcus aureus.

Staphylococcus aureus is one of the most successful bacterial pathogens, harboring a vast repertoire of virulence factors in its arsenal. As such, the genetic manipulation of S. aureus chromosomal DNA is an important tool for the study of genes involved in virulence and survival in the host. Previously reported allelic exchange vectors for S. aureus are shuttle vectors that can be propagated in Escherichia coli, so that standard genetic manipulations can be carried out. Most of the vectors currently in use carry the temperature-sensitive replicon (pE194ts) that was originally developed for use in Bacillus subtilis. Here we show that in S. aureus, the thermosensitivity of a pE194ts vector is incomplete at standard non-permissive temperatures (42 °C), and replication of the plasmid is impaired but not abolished. We report rpsL-based counterselection vectors, with an improved temperature-sensitive replicon (pT181 repC3) that is completely blocked for replication in S. aureus at non-permissive and standard growth temperature (37 °C). We also describe a set of temperature-sensitive vectors that can be cured at standard growth temperature. These vectors provide highly effective tools for rapidly generating allelic replacement mutations and curing expression plasmids, and expand the genetic tool set available for the study of S. aureus.

Microbiota and metabolic adaptation shape Staphylococcus aureus virulence and antimicrobial resistance during intestinal colonization.

Depletion of microbiota increases susceptibility to gastrointestinal colonization and subsequent infection by opportunistic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). How the absence of gut microbiota impacts the evolution of MRSA is unknown. The present report used germ-free mice to investigate the evolutionary dynamics of MRSA in the absence of gut microbiota. Through genomic analyses and competition assays, we found that MRSA adapts to the microbiota-free gut through sequential genetic mutations and structural changes that enhance fitness. Initially, these adaptations increase carbohydrate transport; subsequently, evolutionary pathways largely diverge to enhance either arginine metabolism or cell wall biosynthesis. Increased fitness in arginine pathway mutants depended on arginine catabolic genes, especially nos and arcC, which promote microaerobic respiration and ATP generation, respectively. Thus, arginine adaptation likely improves redox balance and energy production in the oxygen-limited gut environment. Findings were supported by human gut metagenomic analyses, which suggest the influence of arginine metabolism on colonization. Surprisingly, these adaptive genetic changes often reduced MRSA's antimicrobial resistance and virulence. Furthermore, resistance mutation, typically associated with decreased virulence, also reduced colonization fitness, indicating evolutionary trade-offs among these traits. The presence of normal microbiota inhibited these adaptations, preserving MRSA's wild-type characteristics that effectively balance virulence, resistance, and colonization fitness. The results highlight the protective role of gut microbiota in preserving a balance of key MRSA traits for long-term ecological success in commensal populations, underscoring the potential consequences on MRSA's survival and fitness during and after host hospitalization and antimicrobial treatment.