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Staphylococcus aureus is a common organism in orthopaedic infections, but little is known about the genetic diversity of strains during an infectious process. Using periprosthetic joint infection (PJI) as a model, a prospective study was designed to quantify genetic variation among S. aureus strains both among and within patients. Whole genome sequencing and multilocus sequence typing (MLST) was performed to genotype these two populations at high resolution. In nasal cultures, 78% of strains were of clonal complexes CC5, CC8, and CC30. In PJI cultures, only 63% could be classified in these common clonal complexes. The PJI cultures had a larger proportion of atypical strains, and these atypical strains were associated with poor host status and compromised immune conditions. Mutations in genes involved in fibronectin binding (ebh, fnbA, clfA, clfB) systematically distinguished later PJI isolates from the first PJI isolate from each patient. Repeated mutations in S. aureus genes associated with extracellular matrix binding were identified suggesting an adaptive, parallel evolution in S. aureus during the development of PJI.

Staphylococcus aureus is the most pathogenic member of the Staphylococcaceae. While it acquired an arsenal of canonical virulence determinants that mediate pathogenicity, it has also metabolically adapted to thrive at sites of inflammation. Notably, it has evolved to grow in the presence of nitric oxide (NO·). To this end, we note that the Rex regulon, composed of genes encoding dehydrogenases, metabolite transporters, and regulators, is much larger in S. aureus than other Staphylococcus species. Here, we demonstrate that this expanded Rex regulon is necessary and sufficient for NO· resistance. Preventing its expression results in NO· sensitivity, and the closely related species, Staphylococcus simiae, also possesses an expanded Rex regulon and exhibits NO· resistance. We hypothesize that the expanded Rex regulon initially evolved to provide efficient anaerobic metabolism but that S. aureus has co-opted this feature to thrive at sites of inflammation where respiration is limited. One distinguishing feature of the Rex regulon in S. aureus is that it contains the srrAB two-component system. Here, we show that Rex blocks the ability of SrrA to auto-induce the operon, thereby preventing maximal SrrAB expression. This results in NO·-responsive srrAB expression in S. aureus but not in other staphylococci. Consequently, higher expression of cytochromes and NO· detoxification are also observed in S. aureus alone, allowing for continued respiration at NO· concentrations beyond that of S. simiae. We therefore contend that the intersection of the Rex and SrrAB regulons represents an evolutionary event that allowed S. aureus to metabolically adapt to host inflammatory radicals during infection. IMPORTANCE Pathogens must evolve virulence potential to improve transmission to new hosts as well as evolve metabolically to thrive within their current host. Staphylococcus aureus has achieved both of these, and here, we show that one such metabolic adaptation was the expansion of the Rex regulon. First, it affords S. aureus with efficient respiration-independent growth critical to surviving the inflammatory environment replete with respiration-inhibiting immune radicals. Second, it includes the srrAB operon encoding a two-component system critical to maximizing respiratory capacity in the face of host nitric oxide (NO·), a potent respiratory inhibitor. This second facet is only apparent in S. aureus and not in other closely related species. Thus, evolutionarily, it must have occurred relatively recently. The intertwining of the Rex and SrrAB regulons represents an important evolutionary event that affords S. aureus the metabolic flexibility required to thrive within inflamed tissue and cause disease.

As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall’s correlation=0.896–0.897) although the latter remain superior owing to their accessibility and high sequencing depth.

The evolution of bacterial populations during infections can be influenced by various factors including available nutrients, the immune system, and competing microbes, rendering it difficult to identify the specific forces that select on evolved traits. The genomes of Pseudomonas aeruginosa isolated from the airway of patients with cystic fibrosis (CF), for example, have revealed commonly mutated genes, but which phenotypes led to their prevalence is often uncertain. Here, we focus on effects of nutritional components of the CF airway on genetic adaptations by P. aeruginosa grown in either well-mixed (planktonic) or biofilm-associated conditions. After only 80 generations of experimental evolution in a simple medium with glucose, lactate, and amino acids, all planktonic populations diversified into lineages with mutated genes common to CF infections: morA, encoding a regulator of biofilm formation, or lasR, encoding a quorum sensing regulator that modulates the expression of virulence factors. Although mutated quorum sensing is often thought to be selected in vivo due to altered virulence phenotypes or social cheating, isolates with lasR mutations demonstrated increased fitness when grown alone and outcompeted the ancestral PA14 strain. Nonsynonymous SNPs in morA increased fitness in a nutrient concentration-dependent manner during planktonic growth and surprisingly also increased biofilm production. Populations propagated in biofilm conditions also acquired mutations in loci associated with chronic infections, including lasR and cyclic-di-GMP regulators roeA and wspF. These findings demonstrate that nutrient conditions and biofilm selection are alone sufficient to select mutants with problematic clinical phenotypes including increased biofilm and altered quorum sensing.ImportancePseudomonas aeruginosa produces dangerous chronic infections that are known for their rapid diversification and recalcitrance to treatment. We performed evolution experiments to identify adaptations selected by two specific aspects of the CF respiratory environment: nutrient levels and surface attachment. Propagation of P. aeruginosa in nutrients present within the CF airway was alone sufficient to drive diversification into subpopulations with identical mutations in regulators of biofilm and quorum sensing to those arising during infection. Thus, the adaptation of opportunistic pathogens to nutrients found in the host may select mutants with phenotypes that complicate treatment and clearance of infection.

Coronavirus disease 2019 (COVID-19) has dramatically altered our lives in 2020, a vivid reminder that infectious disease continues to threaten society.….

The human skin is a major barrier for host defense against many human pathogens, with several pathogens directly targeting the skin for replication and disease. The skin is also the primary route of infection for a myriad of vector-borne diseases; thus cutaneous immune cells play a major role in modulating transmission for such infectious diseases. Several human pathogens that target the skin as a major route of infection are unable to infect traditional rodent models or recapitulate the pathogenesis in humans. It is well established that differences exist in human skin and immune cell biology compared to rodent models. Therefore, rodent (mouse and rat) models that incorporate human skin and immune cells would addressed the above discussed technical gap, and enable in vivo mechanistic studies of human host-skin pathogen interactions, and support the development of novel therapeutics. Here, we introduce the novel human Skin and Immune System (hSIS)-humanized NOD-scid IL2Rgnull (NSG) mouse and Sprague-Dawley-Rag2tm2hera Il2rgtm1hera (SRG) rat models, co-engrafted with full-thickness human fetal skin, autologous fetal lymphoid tissues, and fetal liver-derived hematopoietic stem cells. hSIS-humanized rodents support the development of adult-like, full-thickness human skin and human lymphoid tissues, and support human immune cell development. Furthermore, the engrafted human skin supports Methicillin-resistant Staphylococcus aureus infection, demonstrating the utility of these humanized rodent models in studying human disease.

Staphylococcus aureus is a common organism in periprosthetic joint infection (PJI). Little is known about S. aureus genetic diversity in PJI as compared to nasal carriage. We hypothesized PJI S. aureus strains would be associated with increased virulence as compared to those from nasal carriage. Whole genome sequencing and multilocus sequence typing (MLST) was performed to genotype these two populations at high resolution. MLST revealed a variety of genotypes in both populations but many belonged to the most common clonal complexes. In nasal cultures, 69% of strains were of clonal complexes CC5, CC8, and CC30. In PJI cultures, only 51% could be classified in these common clonal complexes. Remaining strains were atypical, and these atypical strains in PJI were associated with poor host status and compromised immune conditions. Mutations in genes involved in fibronectin binding (ebh, fnbA, clfA, clfB) systematically distinguished later PJI isolates from the first PJI isolate from each patient. S. aureus isolated from nasal carriage and PJI specimens differ significantly, with the latter being more diverse. Strains associated with lower pathogenicity tended to be found in immunocompromised patients, suggesting the host immune system plays an important role in preventing PJI. Repeated mutations in S. aureus genes associated with extracellular matrix binding were identified suggesting an adaptive, parallel evolution in S. aureus during the development of PJI.

150 years after Louis Pasteur9s germ theory ushered in the “golden age of microbiology” (1), infectious disease research is taking center stage again.…

Bacterial pneumonia is a leading cause of death late after burn injury due to the severe immune dysfunction that follows this traumatic injury. The Mechanistic/Mammalian Target of Rapamycin (mTOR) pathway drives many effector functions of innate immune cells required for bacterial clearance. Studies have demonstrated alterations in multiple cellular processes in patients and animal models following burn injury in which mTOR is a central component. Goals of this study were to (1) investigate the importance of mTOR signaling in antimicrobial activity by neutrophils and (2) therapeutically target mTOR to promote normalization of the immune response. We utilized a murine model of 20% total body surface area burn and the mTOR-specific inhibitor rapamycin. Burn injury led to innate immune hyperresponsiveness in the lung including recruitment of neutrophils with greater ex vivo oxidative activity compared with neutrophils from sham-injured mice. Elevated oxidative function correlated with improved clearance of Pseudomonas aeruginosa, despite down-regulated expression of the bacterial-sensing TLR molecules. Rapamycin administration reversed the burn injury-induced lung innate immune hyperresponsiveness and inhibited enhanced bacterial clearance in burn mice compared with untreated burn mice, resulting in significantly higher mortality. Neutrophil ex vivo oxidative burst was decreased by rapamycin treatment. These data indicate that (1) neutrophil function within the lung is more important than recruitment for bacterial clearance following burn injury and (2) mTOR inhibition significantly impacts innate immune hyperresponsiveness, including neutrophil effector function, allowing normalization of the immune response late after burn injury.

Human skin is commonly colonized and infected by Staphylococcus aureus. Exactly how these organisms are sensed by keratinocytes has not been clearly delineated. Using a combination of metabolic and transcriptomic methodologies, we found that S. aureus infection is sensed as a metabolic stress by the hypoxic keratinocytes. This induces HIF1a signaling, which promotes IL-1 β production and stimulates aerobic glycolysis to meet the metabolic requirements of infection. We demonstrate that staphylococci capable of glycolysis, including WT and agr mutants, readily induce HIF1a responses. In contrast, Δpyk glycolytic mutants fail to compete with keratinocytes for their metabolic needs. Suppression of glycolysis using 2-DG blocked keratinocyte production of IL-1 β in vitro and significantly exacerbated the S. aureus cutaneous infection in a murine model. Our data suggest that S. aureus impose a metabolic stress on keratinocytes that initiates signaling necessary to promote both glycolysis and the proinflammatory response to infection.

Infection with Staphylococcus aureus does not induce long-lived protective immunity for reasons that are not completely understood. Human and murine vaccine studies support a role for Abs in protecting against recurring infections, but S. aureus modulates the B cell response through expression of staphylococcus protein A (SpA), a surface protein that drives polyclonal B cell expansion and induces cell death in the absence of costimulation. In this murine study, we show that SpA altered the fate of plasmablasts and plasma cells (PCs) by enhancing the short-lived extrafollicular response and reducing the pool of bone marrow (BM)-resident long-lived PCs. The absence of long-lived PCs was associated with a rapid decline in Ag-specific class-switched Ab. In contrast, when previously inoculated mice were challenged with an isogenic SpA-deficient S. aureus mutant, cells proliferated in the BM survival niches and sustained long-term Ab titers. The effects of SpA on PC fate were limited to the secondary response, because Ab levels and the formation of B cell memory occurred normally during the primary response in mice inoculated with wild-type or SpA-deficient S. aureus mutant. Thus, failure to establish long-term protective Ab titers against S. aureus was not a consequence of diminished formation of B cell memory; instead, SpA reduced the proliferative capacity of PCs that entered the BM, diminishing the number of cells in the long-lived pool.

Surgical infections are one of the most common types of infections encountered in a hospital. Staphylococcus aureus is the most common pathogen associated with this infection. These infections are resilient and difficult to eradicate, as the bacteria form biofilm, a community of bacteria held together by an extracellular matrix. Compared to bacteria that are planktonic, bacteria in a biofilm are more resistant to antibiotics. The mechanism behind how bacteria develop this resistance and establish a chronic infection is unknown. We demonstrate that mazEF, a toxin-antitoxin gene, inhibits biofilm formation and promotes biofilm antibiotic tolerance which allows S. aureus to transition from an acute to chronic infection that cannot be eradicated with antibiotics but is less virulent. This gene not only makes the bacteria more tolerant to antibiotics but makes the bacteria more tolerant to the host., Staphylococcus aureus is the major organism responsible for surgical implant infections. Antimicrobial treatment of these infections often fails, leading to expensive surgical intervention and increased risk of mortality to the patient. The challenge in treating these infections is associated with the high tolerance of S. aureus biofilm to antibiotics. MazEF, a toxin-antitoxin system, is thought to be an important regulator of this phenotype, but its physiological function in S. aureus is controversial. Here, we examined the role of MazEF in developing chronic infections by comparing growth and antibiotic tolerance phenotypes in three S. aureus strains to their corresponding strains with disruption of mazF expression. Strains lacking mazF production showed increased biofilm growth and decreased biofilm antibiotic tolerance. Deletion of icaADBC in the mazF::Tn background suppressed the growth phenotype observed with mazF-disrupted strains, suggesting the phenotype was ica dependent. We confirmed these phenotypes in our murine animal model. Loss of mazF resulted in increased bacterial burden and decreased survival rate of mice compared to its wild-type strain demonstrating that loss of the mazF gene caused an increase in S. aureus virulence. Although lack of mazF gene expression increased S. aureus virulence, it was more susceptible to antibiotics in vivo. Combined, the ability of mazF to inhibit biofilm formation and promote biofilm antibiotic tolerance plays a critical role in transitioning from an acute to chronic infection that is difficult to eradicate with antibiotics alone.

Staphylococcus aureusis the major organism responsible for surgical implant infections. Antimicrobial treatment of these infections often fails leading to expensive surgical intervention and increased risk of mortality to the patient. The challenge in treating these infections is associated with the high tolerance ofS. aureusbiofilm to antibiotics. MazEF, a toxin-antitoxin system, is thought to be an important regulator of this phenotype, but its physiological function inS. aureusis controversial. Here, we examined the role of MazEF in developing chronic infections by comparing growth and antibiotic tolerance phenotypes in threeS. aureusstrains to their corresponding strains with disruption ofmazFexpression. Strains lackingmazFproduction showed increased biofilm growth, and decreased biofilm antibiotic tolerance. Deletion oficaADBCin themazF::tn background suppressed the growth phenotype observed withmazF-disrupted strains, suggesting the phenotype wasica-dependent. We confirmed these phenotypes in our murine animal model. Loss ofmazFresulted in increased bacterial burden and decreased survival rate compared to its wild-type strain demonstrating that loss of themazFgene caused an increase inS. aureusvirulence. Although lack ofmazFgene expression increasedS. aureusvirulence, it was more susceptible to antibioticsin vivo. Combined, the ability ofmazFto inhibit biofilm formation and promote biofilm antibiotic tolerance plays a critical role in transitioning from an acute to chronic infection that is difficult to eradicate with antibiotics alone.ImportanceSurgical infections are one of the most common types of infections obtained in a hospital.Staphylococcus aureusis the most common pathogen associated with this infection. These infections are resilient and difficult to eradicate as the bacteria form a biofilm, a community of bacteria held together by an extracellular matrix. Compared to bacteria floating in liquid, bacteria in a biofilm are more resistant to antibiotics. The mechanism behind how bacteria develop this resistance and establish a chronic infection is unknown. We demonstrate thatmazEF, a toxin-antitoxin gene, inhibits biofilm formation and promotes biofilm antibiotic tolerance which allowsS. aureusto transition from an acute to chronic infection that cannot be eradicated with antibiotics but is less virulent. This gene not only makes the bacteria more tolerant to antibiotics but makes the bacteria more tolerant to the host.

Staphylococcus aureus is clearly the most pathogenic member of the Staphylococcaceae . This is in large part due to the acquisition of an impressive arsenal of virulence factors that are coordinately regulated by a series of dedicated transcription factors. What is becoming more and more appreciated in the field is the influence of the metabolic state of S. aureus on the activity of these virulence regulators and their roles in modulating metabolic gene expression. Here I highlight recent advances in S. aureus metabolism as it pertains to virulence. Specifically, mechanisms of nutrient acquisition are outlined including carbohydrate and non-carbohydrate carbon/energy sources as well as micronutrient (Fe, Mn, Zn and S) acquisition. Additionally, energy producing strategies (respiration versus fermentation) are discussed and put in the context of pathogenesis. Finally, transcriptional regulators that coordinate metabolic gene expression are outlined, particularly those that affect the activities of major virulence factor regulators. This chapter essentially connects many recent observations that link the metabolism of S. aureus to its overall pathogenesis and hints that the mere presence of a plethora of virulence factors may not entirely explain the extraordinary pathogenic potential of S. aureus .

The ability of Staphylococcus aureus to resist host innate immunity augments the severity and pervasiveness of its pathogenesis. Nitric oxide (NO˙) is an innate immune radical that is critical for the efficient clearance of a wide range of microbial pathogens. Exposure of microbes to NO˙ typically results in growth inhibition and induction of stress regulons. S. aureus , however, induces a metabolic state in response to NO˙ that allows for continued replication and precludes stress regulon induction. The regulatory factors mediating this distinctive response remain largely undefined. Here, we employ a targeted transposon screen and transcriptomics to identify and characterize five regulons essential for NO˙ resistance in S. aureus : three virulence regulons not formerly associated with NO˙ resistance, SarA, CodY, and Rot, as well as two regulons with established roles, Fur and SrrAB. We provide new insights into the contributions of Fur and SrrAB during NO˙ stress and show that the S. aureus Δ sarA mutant, the most sensitive of the newly identified mutants, exhibits metabolic dysfunction and widespread transcriptional dysregulation following NO˙ exposure. Altogether, our results broadly characterize the regulatory requirements for NO˙ resistance in S. aureus and suggest an intriguing overlap between the regulation of NO˙ resistance and virulence in this well-adapted human pathogen. IMPORTANCE The prolific human pathogen Staphylococcus aureus is uniquely capable of resisting the antimicrobial radical nitric oxide (NO˙), a crucial component of the innate immune response. However, a complete understanding of how S. aureus regulates an effective response to NO˙ is lacking. Here, we implicate three central virulence regulators, SarA, CodY, and Rot, as major players in the S. aureus NO˙ response. Additionally, we elaborate on the contribution of two regulators, SrrAB and Fur, already known to play a crucial role in S. aureus NO˙ resistance. Our study sheds light on a unique facet of S. aureus pathogenicity and demonstrates that the transcriptional response of S. aureus to NO˙ is highly pleiotropic and intrinsically tied to metabolism and virulence regulation.

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., 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.

Staphylococcus aureus is a versatile bacterial pathogen that can cause significant disease burden and mortality. Like other pathogens, S. aureus must adapt to its environment to produce virulence factors to survive the immune responses evoked by infection. Despite the importance of environmental signals for S. aureus pathogenicity, only a limited number of these signals have been investigated in detail for their ability to modulate virulence. Here we show that pyruvate, a central metabolite, causes alterations in the overall metabolic flux of S. aureus and enhances its pathogenicity. We demonstrate that pyruvate induces the production of virulence factors such as the pore-forming leucocidins and that this induction results in increased virulence of community-acquired methicillin-resistant S. aureus (CA-MRSA) clone USA300. Specifically, we show that an efficient “pyruvate response” requires the activation of S. aureus master regulators AgrAC and SaeRS as well as the ArlRS two-component system. Altogether, our report further establishes a strong relationship between metabolism and virulence and identifies pyruvate as a novel regulatory signal for the coordination of the S. aureus virulon through intricate regulatory networks., IMPORTANCE Delineation of the influence of host-derived small molecules on the makeup of human pathogens is a growing field in understanding host-pathogen interactions. S. aureus is a prominent pathogen that colonizes up to one-third of the human population and can cause serious infections that result in mortality in ~15% of cases. Here, we show that pyruvate, a key nutrient and central metabolite, causes global changes to the metabolic flux of S. aureus and activates regulatory networks that allow significant increases in the production of leucocidins. These and other virulence factors are critical for S. aureus to infect diverse host niches, initiate infections, and effectively subvert host immune responses. Understanding how environmental signals, particularly ones that are essential to and prominent in the human host, affect virulence will allow us to better understand pathogenicity and consider more-targeted approaches to tackling the current S. aureus epidemic.

Skin/soft tissue infections (SSTIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) represent serious healthcare burdens worldwide. The host initially controls these infections with a pro-inflammatory infiltrate. However, once established, MRSA viability remains constant. To clear established MRSA SSTIs, the host must transition into the post-inflammatory resolution phase marked by infiltration of alternatively activated macrophages. Here we show that the host nuclear receptor, peroxisome proliferation activator receptor γ (PPARγ), is essential for this transition and MRSA clearance. Chemical PPARγ inhibition or genetic ablation of PPARγ in myeloid cells results in an extended inflammatory phase and exacerbated MRSA SSTIs. Conversely, treating mice with PPARγ agonists hastens the onset of the resolution phase and improves MRSA clearance in a myeloid-dependent fashion. The resolving fibrotic abscess lacks abundant glucose and oxygen but is replete with antimicrobial peptides, which together contribute to MRSA clearance. Thus, PPARγ agonists may serve as viable treatment options for complicated MRSA SSTIs.

Staphylococcus aureus is a significant infectious threat to global public health. Acquisition or synthesis of heme is required for S. aureus to capture energy through respiration, but an excess of this critical cofactor is toxic to bacteria. S. aureus employs the heme sensor system (HssRS) to overcome heme toxicity; however, the mechanism of heme sensing is not defined. Here, we describe the identification of a small molecule activator of HssRS that induces endogenous heme biosynthesis by perturbing central metabolism. This molecule is toxic to fermenting S. aureus , including clinically relevant small colony variants. The utility of targeting fermenting bacteria is exemplified by the fact that this compound prevents the emergence of antibiotic resistance, enhances phagocyte killing, and reduces S. aureus pathogenesis. Not only is this small molecule a powerful tool for studying bacterial heme biosynthesis and central metabolism; it also establishes targeting of fermentation as a viable antibacterial strategy.

Pantothenate, commonly referred to as vitamin B(5), is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ΔpanG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ΔpanG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.

Acquisition of numerous virulence determinants affords Staphylococcus aureus greater pathogenicity than other skin-colonizing staphylococci in humans. Additionally, the metabolic adaptation of S. aureus to nonrespiratory conditions encountered during infection (e.g., hypoxia, nitric oxide, iron chelation) has been implicated as contributing to S. aureus virulence. Specifically, S. aureus has been shown to ferment glycolytic substrates in nonrespiratory environments encountered within the host. Here, we show that S. aureus has acquired unique carbohydrate transporters that facilitate the maximal uptake of host sugars and serve to support nonrespiratory growth in inflamed tissue. The carbohydrate substrates of 11 S. aureus transporters were identified, and at least four of their genes encode S. aureus glucose transporters (glcA, glcB, glcC, and glcU). Moreover, two transporter genes (glcA and glcC) are unique to S. aureus and contribute disproportionately to the nonrespiratory growth of S. aureus on glucose. Targeted inactivation of sugar transporters reduced glucose uptake and attenuated S. aureus in a murine model of skin and soft tissue infections. These data expand the evidence for metabolic adaptation of S. aureus to invasive infection and demonstrate the specific requirement for the fermentation of glucose over all other available carbohydrates. Ultimately, acquisition of foreign genes allows S. aureus to adopt a metabolic strategy resembling that of infiltrating host immune cells: high glycolytic flux coupled to lactate excretion., IMPORTANCE The bacterial pathogen Staphylococcus aureus causes a wide range of human infections that are costly and difficult to treat. S. aureus differs from closely related commensal staphylococci in its ability to flourish following the invasion of deeper tissue from the skin surface. There, S. aureus primarily uses glucose to grow under respiration-limiting conditions imposed by the immune system. It was previously unclear how S. aureus thrives in this environment when other Staphylococcus species cannot. Our results provide evidence that S. aureus has acquired an expanded repertoire of carbohydrate transporters. In particular, four glucose transporters contribute to efficient S. aureus growth during infection. Thus, S. aureus has evolved to maximize its glucose uptake abilities for enhanced glycolytic flux during tissue invasion. This dependence on glucose acquisition for S. aureus virulence may also explain links between serious infectious complications associated with diabetic patients exhibiting elevated blood glucose levels.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to worldwide health. Historically, MRSA clones have strictly been associated with hospital settings, and most hospital-associated MRSA (HA-MRSA) disease resulted from a limited number of virulent clones. Recently, MRSA has spread into the community causing disease in otherwise healthy people with no discernible contact with healthcare environments. These community-associated MRSA clones (CA-MRSA) are phylogenetically distinct from traditional HA-MRSA clones, and CA-MRSA strains seem to exhibit hypervirulence and more efficient host : host transmission. Consequently, CA-MRSA clones belonging to the USA300 lineage have become dominant sources of MRSA infections in North America. The rise of this successful USA300 lineage represents an important step in the evolution of emerging pathogens and a great deal of effort has been exerted to understand how these clones evolved. Here, we review much of the recent literature aimed at illuminating the source of USA300 success and broadly categorize these findings into three main categories: newly acquired virulence genes, altered expression of common virulence determinants and alterations in protein sequence that increase fitness. We argue that none of these evolutionary events alone account for the success of USA300, but rather their combination may be responsible for the rise and spread of CA-MRSA.

Polyamines, including spermine (Spm) and spermidine (Spd), are aliphatic cations that are reportedly synthesized by all living organisms. They exert pleiotropic effects on cells and are required for efficient nucleic acid and protein synthesis. Here, we report that the human pathogen Staphylococcus aureus lacks identifiable polyamine biosynthetic genes, and consequently produces no Spm/Spd or their precursor compounds putrescine and agmatine. Moreover, while supplementing defined medium with polyamines generally enhances bacterial growth, Spm and Spd exert bactericidal effects on S. aureus at physiological concentrations. Small colony variants specifically lacking menaquinone biosynthesis arose after prolonged Spm exposure and exhibited reduced polyamine sensitivity. However, other respiratory-defective mutants were no less susceptible to Spm implying menaquinone itself rather than general respiration is required for full Spm toxicity. Polyamine hypersensitivity distinguishes S. aureus from other bacteria and is exhibited by all tested strains save those belonging to the USA-300 group of community-associated methicillin-resistant S. aureus (CA-MRSA). We identified one gene within the USA-300-specific arginine catabolic mobile element (ACME) encoding a Spm/Spd N-acetyltransferase that is necessary and sufficient for polyamine resistance. S. aureus encounters significant polyamine levels during infection; however, the acquisition of ACME encoded speG allows USA-300 clones to circumvent polyamine hypersensitivity, a peculiar trait of S. aureus.

SummaryHost nitric oxide (NO⋅) production is important for controlling intracellular bacterial pathogens, including Salmonella enterica serovar Typhimurium, but the underlying mechanisms are incompletely understood. S. Typhmurium 14028s is prototrophic for all amino acids but cannot synthesize methionine (M) or lysine (K) during nitrosative stress. Here, we show that NO⋅-induced MK auxotrophy results from reduced succinyl-CoA availability as a consequence of NO⋅ targeting of lipoamide-dependent lipoamide dehydrogenase (LpdA) activity. LpdA is an essential component of the pyruvate and α-ketoglutarate dehydrogenase complexes. Additional effects of NO⋅ on gene regulation prevent compensatory pathways of succinyl-CoA production. Microarray analysis indicates that over 50% of the transcriptional response of S. Typhimurium to nitrosative stress is attributable to LpdA inhibition. Bacterial methionine transport is essential for virulence in NO⋅-producing mice, demonstrating that NO⋅-induced MK auxotrophy occurs in vivo. These observations underscore the importance of metabolic targets for antimicrobial actions of NO⋅.

Ferritins are ubiquitous iron (Fe) storage proteins that play a fundamental role in cellular Fe homeostasis. The enteric pathogen Salmonella enterica serovar Typhimurium possesses four ferritins: bacterioferritin, ferritin A, ferritin B and Dps. The haem-containing bacterioferritin (Bfr) accounts for the majority of stored Fe, followed by ferritin A (FtnA). Inactivation of bfr elevates the intracellular free Fe concentration and enhances susceptibility to H2O2 stress. The DNA-binding Dps protein provides protection from oxidative damage without affecting the steady-state intracellular free Fe concentration. FtnB appears to be particularly important for the repair of oxidatively damaged Fe-sulphur clusters of aconitase and, in contrast to Bfr and FtnA, is required for Salmonella virulence in mice. Moreover, ftnB and dps are repressed by the Fe-responsive regulator Fur and induced under conditions of Fe limitation, whereas bfr and ftnA are maximally expressed when Fe is abundant. The absence of a conserved ferroxidase domain and the potentiation of oxidative stress by FtnB in some strains lacking Dps suggest that FtnB serves as a facile cellular reservoir of Fe2+.

Staphylococcus aureus is a prolific human pathogen capable of causing severe invasive disease with a myriad of presentations. The ability of S. aureus to cause infection is strongly linked with its capacity to overcome the effects of innate immunity, whether by directly killing immune cells or expressing factors that diminish the impact of immune effectors. One such scenario is the induction of lactic acid fermentation by S. aureus in response to host nitric oxide (NO·). This fermentative activity allows S. aureus to balance redox during NO·-induced respiration inhibition. However, little is known about the metabolic substrates and pathways that support this activity. Here, we identify glycolytic hexose catabolism as being essential for S. aureus growth in the presence of high levels of NO·. We determine that glycolysis supports S. aureus NO· resistance by allowing for ATP and precursor metabolite production in a redox-balanced and respiration-independent manner. We further demonstrate that glycolysis is required for NO· resistance during phagocytosis and that increased levels of extracellular glucose limit the effectiveness of phagocytic killing by enhancing NO· resistance. Finally, we demonstrate that S. aureus glycolysis is essential for virulence in both sepsis and skin/soft tissue models of infection in a time frame consistent with the induction of innate immunity and host NO· production., IMPORTANCE Staphylococcus aureus is a leading human bacterial pathogen capable of causing a wide variety of diseases that, as a result of antibiotic resistance, are very difficult to treat. The frequency of S. aureus tissue invasion suggests that this bacterium has evolved to resist innate immunity and grow using the nutrients present in otherwise sterile host tissue. We have identified glycolysis as an essential component of S. aureus virulence and attribute its importance to promoting nitric oxide resistance and growth under low oxygen conditions. Our data suggest that diabetics, a patient population characterized by excess serum glucose, may be more susceptible to S. aureus as a result of increased glucose availability. Furthermore, the essential nature of S. aureus glycolysis indicates that a newly developed glycolysis inhibitor may be a highly effective treatment for S. aureus infections.

Neisseria meningitidis has evolved the ability to control the expression-state of numerous genes by phase variation. It has been proposed that the process aids this human pathogen in coping with the diversity of microenvironments and host immune systems. Therefore, increased frequencies of phase variation may augment the organism's adaptability and virulence. In this study, we found that DNA derived from various neisserial co-colonizers of the human nasopharynx increased N. meningitidis switching frequencies, indicating that heterologous neisserial DNA modulates phase variation in a transformation-dependent manner. In order to determine whether the effect of heterologous DNA was specific to the Hb receptor, HmbR, we constructed a Universal Rates of Switching cassette (UROS). With this cassette, we demonstrated that heterologous DNA positively affects phase variation throughout the meningococcal genome, as UROS phase variation frequencies were also increased in the presence of neisserial DNA. Overexpressing components of the neisserial mismatch repair system partially alleviated DNA-induced changes in phase variation frequencies, thus implicating mismatch repair titration as a cause of these transformation-dependent increases in switching. The DNA-dependent effect on phase variation was transient and may serve as a mechanism for meningococcal genetic variability that avoids the fitness costs encountered by global mutators.

For bacterial species that are not known to be naturally competent, such as Staphylococcus aureus and Staphylococcus epidermidis, electroporation is an efficient method for introducing genetic material into the cell. The technique utilizes electrical pulses to transiently permeabilize bacterial cell membranes, which allows for the passage of plasmid DNA across the membranes. Here, we describe methods for preparing electrocompetent S. aureus and S. epidermidis cells and outline a procedure for electroporation of the prepared competent cells.

Neisseria meningitidis controls the expression of several genes involved in host adaptation by a process known as phase variation. The phase variation frequency of haemoglobin (Hb) receptors among clinical isolates of serogroups A, B and C differed drastically, ranging from approximately 10(-6) to 10(-2) cfu-1. Frequencies of phase variation are a genetic trait of a particular strain, as two unlinked Hb receptors, hpuAB and hmbR, phase varied with similar frequencies within a given isolate. Based on these frequencies, six Neisserial clinical isolates could be grouped into three distinct classes; slow, medium and fast. An increase in phase variation frequency was accompanied by high rates of spontaneous mutation to rifampicin and nalidixic acid resistance in one medium and one fast strain. The remaining three medium strains displayed elevated levels of phase variation without increases in overall mutability, as they possessed low rates of spontaneous mutation to drug resistance. The mismatch repair system of N. meningitidis was found to play an important role in determining the overall mutability of the clinical isolates. Inactivation of mismatch repair in any strain, regardless of its original phenotype, increased mutability to a level seen in the fast strain. Insertional inactivation of mutS and mutL in the slow strain led to 500- and 250-fold increases in hmbR switching frequency respectively. Concurrently, the frequency of spontaneous point mutations of mutS and mutL mutants from the slow strain was increased 20- to 30-fold to the level seen in the high strain. The status of Dam methylation did not correlate with either the phase variation frequency of Hb receptors or the general mutability of Neisserial strains. Analysis of an expanded set of isolates identified defects in mismatch repair as the genetic basis for strains displaying both the fast Hb switching and high mutation rate phenotypes. In conclusion, elevated frequencies of phase variation were accompanied by increased overall mutability in some N. meningitidis isolates including strains shown to be mismatch repair defective. Other isolates have evolved mechanisms that seem to affect only the switching frequency of phase-variable genes without an accompanied increased accumulation of spontaneous mutations.

Staphylococcus aureus is an important bacterial pathogen in the hospital and community settings, especially Staphylococcus aureus clones that exhibit methicillin-resistance (MRSA). Many strains of S. aureus are utilized in the laboratory, underscoring the genetic differences inherent in clinical isolates. S. aureus grows quickly at 37°C with aeration in rich media (e.g., BHI) and exhibits a preference for glycolytic carbon sources. Furthermore, S. aureus has a gold pigmentation, exhibits β-hemolysis, and is catalase and coagulase positive. The four basic laboratory protocols presented in this unit describe how to culture S. aureus on liquid and solid media, how to identify S. aureus strains as methicillin resistant, and how to generate a freezer stock of S. aureus for long-term storage.

Lactate Dehydrogenase 1 (Ldh1) is a key enzyme involved in Staphylococcus aureus NO·-resistance. Full ldh1-induction requires the presence of glucose, and mutants lacking the Carbon-Catabolite Protein (CcpA) exhibit decreased ldh1 transcription and diminished Ldh1 activity. The redox-regulator Rex represses ldh1 directly by binding to Rex-sites within the ldh1 promoter (P(ldh1)). In the absence of Rex, neither glucose nor CcpA affect ldh1 expression implying that glucose/CcpA-mediated activation requires Rex activity. Rex-mediated repression of ldh1 depends on cellular redox status and is maximal when NADH levels are low. However, compared to WT cells, the ΔccpA mutant exhibited impaired redox balance with relatively high NADH levels, yet ldh1 was still poorly expressed. Furthermore, CcpA did not drastically alter Rex transcript levels, nor did glucose or CcpA affect the expression of other Rex-regulated genes indicating that the glucose/CcpA effect is specific for P(ldh1). A putative catabolite response element (CRE) is located ∼30 bp upstream of the promoter-distal Rex-binding site in P(ldh1). However, CcpA had no affinity for P(ldh1) in vitro and a genomic mutation of CRE upstream of P(ldh1) in S. aureus had no affect on Ldh1 expression in vivo. In contrast to WT, ΔccpA S. aureus preferentially consumes non-glycolytic carbon sources. However when grown in defined medium with glucose as the primary carbon source, ΔccpA mutants express high levels of Ldh1 compared to growth in media devoid of glucose. Thus, the actual consumption of glucose stimulates Ldh1 expression rather than direct CcpA interaction at P(ldh1).

Staphylococcus aureus is an important human pathogen commonly infecting nearly every host tissue. The ability of S. aureus to resist innate immunity is critical to its success as a pathogen, including its propensity to grow in the presence of host nitric oxide (NO·). Upon exogenous NO· exposure, S. aureus immediately excretes copious amounts of L-lactate to maintain redox balance. However, after prolonged NO·-exposure, S. aureus reassimilates L-lactate specifically and in this work, we identify the enzyme responsible for this L-lactate consumption as a L-lactate-quinone oxidoreductase (Lqo, SACOL2623). Originally annotated as Mqo2 and thought to oxidize malate, we show that this enzyme exhibits no affinity for malate but reacts specifically with L-lactate (KM = ~330 µM). In addition to its requirement for reassimilation of L-lactate during NO·-stress, Lqo is also critical to respiratory growth on L-lactate as a sole carbon source. Moreover, ∆lqo mutants exhibit attenuation in a murine model of sepsis, particularly in their ability to cause myocarditis. Interestingly, this cardiac-specific attenuation is completely abrogated in mice unable to synthesize inflammatory NO· (iNOS-/-). We demonstrate that S. aureus NO·-resistance is highly dependent on the availability of a glycolytic carbon sources. However, S. aureus can utilize the combination of peptides and L-lactate as carbon sources during NO·-stress in an Lqo-dependent fashion. Murine cardiac tissue has markedly high levels of L-lactate in comparison to renal or hepatic tissue consistent with the NO·-dependent requirement for Lqo in S. aureus myocarditis. Thus, Lqo provides S. aureus with yet another means of replicating in the presence of host NO·.

Exotoxins, including the hemolysins known as the alpha (alpha) and beta (beta) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

Microbes are subjected to selective pressures during chronic infections of host tissues. Pseudomonas aeruginosa isolates with inactivating mutations in the transcriptional regulator LasR are frequently selected within the airways of people with cystic fibrosis (CF), and infection with these isolates has been associated with poorer lung function outcomes. The mechanisms underlying selection for lasR mutation are unknown but have been postulated to involve the abundance of specific nutrients within CF airway secretions. We characterized lasR mutant P. aeruginosa strains and isolates to identify conditions found in CF airways that select for growth of lasR mutants. Relative to wild-type P. aeruginosa, lasR mutants exhibited a dramatic metabolic shift, including decreased oxygen consumption and increased nitrate utilization, that is predicted to confer increased fitness within the nutrient conditions known to occur in CF airways. This metabolic shift exhibited by lasR mutants conferred resistance to two antibiotics used frequently in CF care, tobramycin and ciprofloxacin, even under oxygen-dependent growth conditions, yet selection for these mutants in vitro did not require preceding antibiotic exposure. The selection for loss of LasR function in vivo, and the associated adverse clinical impact, could be due to increased bacterial growth in the oxygen-poor and nitrate-rich CF airway, and from the resulting resistance to therapeutic antibiotics. The metabolic similarities among diverse chronic infection-adapted bacteria suggest a common mode of adaptation and antibiotic resistance during chronic infection that is primarily driven by bacterial metabolic shifts in response to nutrient availability within host tissues., Author Summary Chronic infections are distinguished from many other infections in that they are difficult to eradicate with antibiotics. Thus, the microbes that cause chronic infections persist within host tissues for long periods despite our best treatment efforts. During the course of these chronic infections, the causative microbes often change genetically. For example, a bacterium that commonly infects the lungs of people with the genetic disease cystic fibrosis (CF) undergoes several known changes that affect the growth of this pathogen. However, the causes and clinical impact of the changes undergone by this and other chronically infecting microbes are unclear. We show that a common, early mutation found in bacteria isolated from chronically infected CF airways renders these bacteria better able to grow in the nutrients found in CF lung secretions. Interestingly, these same changes also confer resistance to several antibiotics used commonly to treat CF patients. Many of the characteristics conferred by this mutation are exhibited by other microbes found in chronic infections, suggesting that adaptation of these microbes to host tissue nutrient environments may be a common mechanism of antibiotic resistance in chronic infections.

Intracellular pathogens must withstand nitric oxide (NO·) generated by host phagocytes. Salmonella enterica serovar Typhimurium interferes with intracellular trafficking of inducible nitric oxide synthase (iNOS) and possesses multiple systems to detoxify NO·. Consequently, the level of NO· stress encountered by S. Typhimurium during infection in vivo has been unknown. The Base Excision Repair (BER) system recognizes and repairs damaged DNA bases including cytosine and guanine residues modified by reactive nitrogen species. Apurinic/apyrimidinic (AP) sites generated by BER glycosylases require subsequent processing by AP endonucleases. S. Typhimurium xth nfo mutants lacking AP endonuclease activity exhibit increased NO· sensitivity resulting from chromosomal fragmentation at unprocessed AP sites. BER mutant strains were thus used to probe the nature and extent of nitrosative damage sustained by intracellular bacteria during infection. Here we show that an xth nfo S. Typhimurium mutant is attenuated for virulence in C3H/HeN mice, and virulence can be completely restored by the iNOS inhibitor L-NIL. Inactivation of the ung or fpg glycosylase genes partially restores virulence to xth nfo mutant S. Typhimurium, demonstrating that NO· fluxes in vivo are sufficient to modify cytosine and guanine bases, respectively. Mutants lacking ung or fpg exhibit NO·–dependent hypermutability during infection, underscoring the importance of BER in protecting Salmonella from the genotoxic effects of host NO·. These observations demonstrate that host-derived NO· damages Salmonella DNA in vivo, and the BER system is required to maintain bacterial genomic integrity., Author Summary The host innate immune system represents the first line of defense against invading microorganisms. An integral part of this response involves the production of nitric oxide (NO·), a potent antimicrobial effector. Bacterial pathogens have evolved a variety of systems to counteract the inhibitory effects of host NO·. Here we describe the role of a complex DNA repair pathway in the bacterium Salmonella Typhimurium that minimizes the mutagenic nature of host NO·. We have determined the mutation rate of bacteria during a model infection. Our results reveal that the Salmonella Base Excision Repair system (BER), comprised of DNA glycosylases and AP endonucleases, is able to eliminate excess mutations that accumulate in NO·–exposed cells during their interaction with the host. Thus, during Salmonella infections, the BER system protects the bacterium against potentially detrimental DNA damage arising from NO·–exposure. This provides genomic stability to a pathogenic microorganism that has evolved to survive within the genotoxic intracellular environment of host phagocytes.

Growth of Moraxella catarrhalis in a biofilm resulted in marked upregulation of two open reading frames (ORFs), aniA and norB , predicted to encode a nitrite reductase and a nitric oxide reductase, respectively (W. Wang, L. Reitzer, D. A. Rasko, M. M. Pearson, R. J. Blick, C. Laurence, and E. J. Hansen, Infect. Immun. 75:4959-4971, 2007). An ORF designated nsrR , which was located between aniA and norB , was shown to encode a predicted transcriptional regulator. Inactivation of nsrR resulted in increased expression of aniA and norB in three different M. catarrhalis strains, as measured by both DNA microarray analysis and quantitative reverse transcriptase PCR. Provision of a wild-type nsrR gene in trans in an nsrR mutant resulted in decreased expression of the AniA protein. DNA microarray analysis revealed that two other ORFs (MC ORF 683 and MC ORF 1550) were also consistently upregulated in an nsrR mutant. Consumption of both nitrite and nitric oxide occurred more rapidly with cells of an nsrR mutant than with wild-type cells. However, growth of nsrR mutants was completely inhibited by a low level of sodium nitrite. This inhibition of growth by nitrite was significantly reversed by introduction of an aniA mutation into the nsrR mutant and was completely reversed by the presence of a wild-type nsrR gene in trans . NsrR regulation of the expression of aniA was sensitive to nitrite, whereas NsrR regulation of norB was sensitive to nitric oxide.

Staphylococcus aureus is one of the most successful human pathogens, colonizing 2 billion individuals worldwide and causing invasive infections even in immunocompetent hosts. S. aureus can evade multiple components of host innate immunity, including the antimicrobial radical nitric oxide (NO ⚫ ) produced by activated phagocytes. We show that S. aureus is capable of metabolically adapting to nitrosative stress by expressing an NO ⚫ -inducible l -lactate dehydrogenase ( ldh 1, SACOL0222) divergently transcribed from the NO ⚫ -detoxifying flavohemoglobin ( hmp ). l -Lactate production allows S. aureus to maintain redox homeostasis during nitrosative stress and is essential for virulence. NO ⚫ -inducible lactate dehydrogenase activity and NO ⚫ resistance distinguish S. aureus from the closely related commensal species S. epidermidis and S. saprophyticus .

Staphylococcus aureus is a highly virulent human pathogen with an extensive array of strategies to subvert the innate immune response. An important aspect of innate immunity is the production of the nitrogen monoxide radical (Nitric Oxide, NO.). Here we describe an adaptive response to nitrosative stress that allows S. aureus to replicate at high concentrations of NO.. Microarray analysis revealed 84 staphylococcal genes with significantly altered expression following NO. exposure. Of these, 30 are involved with iron-homeostasis, potentially under the control of the Fur regulator. Another seven induced genes are involved in hypoxic/fermentative metabolism, including the flavohaemoprotein, Hmp. The SrrAB two-component system has been shown to regulate the expression of many of the NO.-induced metabolic genes. Indeed, inactivation of hmp, srrAB and fur resulted in heightened NO. sensitivity. Hmp was responsible for c. 90% of measurable staphylococcal NO. consumption and therefore critical for efficient NO. detoxification. While SrrAB was required for maximal hmp expression, srrAB mutants still exhibited significant NO. scavenging and NO.-dependent induction of hmp. Yet S. aureus lacking SrrAB were more sensitive to nitrosative stress than hmp mutants, indicating that the contribution of SrrAB to NO. resistance extends beyond the regulation of hmp expression. Both Hmp and SrrAB were required for full virulence in a murine sepsis model, however, only the attenuation of the hmp mutant was restored by the abrogation of host NO. production. Thus, the S. aureus Hmp protein has evolved to serve as an iNOS-dependent virulence determinant.

Many so-called pathogenic bacteria such as Neisseria meningitidis, Haemophilus influenzae, Staphylococcus aureus and Streptococcus pneumoniae are far more likely to colonize and maintain populations in healthy individuals asymptomatically than to cause disease. Disease is a dead-end for these bacteria: virulence shortens the window of time during which transmission to new hosts can occur and the subpopulations of bacteria actually responsible for disease, like those in the blood or cerebral spinal fluid, are rarely transmitted to new hosts. Hence, the virulence factors underlying their occasional pathogenicity must evolve in response to selection for something other than making their hosts sick. What are those selective pressures? We address this general question of the evolution of virulence in the context of phase shifting in N. meningitidis, a mutational process that turns specific genes on and off, and, in particular, contingency loci that code for virulence determinants such as pili, lipopolysaccharides, capsular polysaccharides and outer membrane proteins. We use mathematical models of the epidemiology and the within-host infection dynamics of N. meningitidis to make the case that rapid phase shifting evolves as an adaptation for colonization of diverse hosts and that the virulence of this bacterium is an inadvertent consequence of short-sighted within-host evolution, which is exasperated by the increased mutation rates associated with phase shifting. We present evidence for and suggest experimental and retrospective tests of these hypotheses.

Serogroup A Neisseria meningitidis has repeatedly caused widespread epidemics of meningitis and septicemia throughout the 20th century. Recently, in a limited collection of strains, epidemic serogroup A isolates were found to have elevated mutation rates that was caused by defects in mismatch repair pathways. To ascertain the role of these mutators in the epidemic spread of this serogroup, the prevalence of hypermutability in a collection of 95 serogroup A N. meningitidis invasive isolates was determined. Overall mutability in Neisseriae can be described by measuring both missense mutation rates as well as phase variation frequencies of “contingency loci.” Fifty-seven percent of serogroup A isolates possessed elevated mutability, which could be divided into two classes: intermediate and high level. Eleven of 20 high-level mutators, with phase variation rates >100-fold higher than wild-type isolates, were defective in mismatch repair. Ten of the 34 intermediate mutators possessing >10-fold increases in phase variation rates could be partially complemented by a wild-type mutL allele. A high prevalence of mutators in epidemic isolates indicates that hypermutability may play a major role in the transmission of this pathogen. The added diversity derived from increased phase variation rates may allow fixation of mutator alleles more frequently during epidemic spread.

Nitric oxide (NO) is emerging as an important regulator of bacterial stress resistance, biofilm development, and virulence. One potential source of endogenous NO production in the pathogen Staphylococcus aureus is its NO-synthase (saNOS) enzyme, encoded by the nos gene. Although a role for saNOS in oxidative stress resistance, antibiotic resistance, and virulence has been recently-described, insights into the regulation of nos expression and saNOS enzyme activity remain elusive. To this end, transcriptional analysis of the nos gene in S. aureus strain UAMS-1 was performed, which revealed that nos expression increases during low-oxygen growth and is growth-phase dependent. Furthermore, nos is co-transcribed with a downstream gene, designated pdt, which encodes a prephenate dehydratase (PDT) enzyme involved in phenylalanine biosynthesis. Deletion of pdt significantly impaired the ability of UAMS-1 to grow in chemically-defined media lacking phenylalanine, confirming the function of this enzyme. Bioinformatics analysis revealed that the operon organization of nos-pdt appears to be unique to the staphylococci. As described for other S. aureus nos mutants, inactivation of nos in UAMS-1 conferred sensitivity to oxidative stress, while deletion of pdt did not affect this phenotype. The nos mutant also displayed reduced virulence in a murine sepsis infection model, and increased carotenoid pigmentation when cultured on agar plates, both previously-undescribed nos mutant phenotypes. Utilizing the fluorescent stain 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM) diacetate, decreased levels of intracellular NO/reactive nitrogen species (RNS) were detected in the nos mutant on agar plates. These results reinforce the important role of saNOS in S. aureus physiology and virulence, and have identified an in vitro growth condition under which saNOS activity appears to be upregulated. However, the significance of the operon organization of nos-pdt and potential relationship between these two enzymes remains to be elucidated.

Heme compounds are an important source of iron for neisseriae. We have identified a neisserial gene, hemO , that is essential for heme, hemoglobin (Hb), and haptoglobin-Hb utilization. The hemO gene is located 178 bp upstream of the hmbR Hb receptor gene in Neisseria meningitidis isolates. The product of the hemO gene is homologous to enzymes that degrade heme; 21% of its amino acid residues are identical, and 44% are similar, to those of the human heme oxygenase-1. DNA sequences homologous to hemO were ubiquitous in commensal and pathogenic neisseriae. HemO genetic knockout strains of Neisseria gonorrhoeae and N. meningitidis were unable to use any heme source, while the assimilation of transferrin-iron and iron-citrate complexes was unaffected. A phenotypic characterization of a conditional hemO mutant, constructed by inserting an isopropyl-β- d -thiogalactopyranoside (IPTG)-regulated promoter upstream of the ribosomal binding site of hemO , confirmed the indispensability of the HemO protein in heme utilization. The expression of HemO also protected N. meningitidis cells against heme toxicity. hemO mutants were still able to transport heme into the cell, since both heme and Hb could complement an N. meningitidis hemA hemO double mutant for growth. The expression of the HmbR receptor was reduced significantly by the inactivation of the hemO gene, suggesting that hemO and hmbR are transcriptionally linked. The expression of the unlinked Hb receptor, HpuAB, was not altered. Comparison of the polypeptide patterns of the wild type and the hemO mutant led to detection of six protein spots with an altered expression pattern, suggesting a more general role of HemO in the regulation of gene expression in Neisseriae.

The gram-negative diplococcus Neisseria meningitidis is a major cause of bacterial meningitis and is a significant public health problem throughout the world (7, 38). The incidence of meningococcal disease in developing countries ranges between 10 and 25 per 100,000, with periodic epidemics having attack rates of up to 1,000 per 100,000 (6, 12, 45). Approximately 2,600 cases of meningococcal disease occur annually in the United States, predominantly in children less than 2 years old (7). Since the success of the current vaccine against Haemophilus influenzae type b, N. meningitidis is the leading cause of meningitis in children in the United States (7). Despite its relatively low incidence, several facts about meningococcal disease make it a potential public health problem of major importance. Mortality from meningococcal meningitis remains, despite optimal antibiotic and supportive therapy, about 5% in children and 10 to 15% in adults (33). There have been recent reports from Europe, Canada, South Africa, and the United States of N. meningitidis isolates that show moderate resistance to penicillin (48, 51). The occurrence of a new, more virulent strain of N. meningitidis serogroup C associated with an increase in both incidence and mortality of meningococcal disease in Canada is a reminder that the evolution of N. meningitidis virulence is ongoing (49). The best preventive measure against meningococcal disease is a polyvalent polysaccharide vaccine. The vaccine is not protective against serogroup B meningococcus, however, nor can it protect children under 2 years of age (7, 14). Among new vaccine targets against N. meningitidis being explored are iron-regulated outer membrane proteins (2, 24). These proteins are attractive vaccine candidates due to their important role in virulence and surface accessibility (2, 24, 39). N. meningitidis expresses several outer membrane receptors that enable the bacterium to use human transferrin, lactoferrin, hemoglobin (Hb), heme, and haptoglobin-hemoglobin complexes (Hpt-Hb) as sources of iron (11, 13, 22, 23, 36, 42). Heme and Hb are the most abundant sources of iron in the body (29). Recently, two neisserial Hb-binding outer membrane receptors have been identified. HmbR is an iron-regulated, 89.5-kDa protein that binds Hb, extracts the heme from it, and transports the heme into the periplasm (42–44). The hmbR mutant of N. meningitidis was attenuated in an infant rat infection model, confirming the importance of Hb acquisition in meningococcal virulence (42). A second Hb-binding protein is the HpuAB bipartite receptor involved in use of Hb and Hpt-Hb complexes in N. meningitidis and Neisseria gonorrhoeae (8, 22, 23). In addition to Hb, the HpuAB receptor binds Hpt-Hb complexes and apo-Hpt (22). The HpuAB bipartite receptor is most likely the main Hb receptor of gonococci, since the hmbR gene contains a premature stop codon in all gonococcal strains tested (8, 43). Recently, the expression of the hpuAB receptor genes in N. gonorrhoeae was shown to undergo phase variation (9). A limited analysis of meningococcal clinical isolates revealed functional redundancy in their Hb utilization systems (43). In this communication, we show that this functional redundancy is a common phenomenon in clinical isolates of N. meningitidis. Moreover, we show that the expression of hmbR undergoes phase variation due to the slipped-strand mispairing (SSM) of a poly(G) tract beginning at position +1164 in the hmbR coding sequence. The rate of switching between the off and on phases of hmbR differs more than 1,000-fold between different clinical isolates. These results establish a rational explanation for the existence of functional redundancy in Hb utilization systems and support a novel hypothesis correlating the rates of phase variation and virulence.

SummaryThe USA300 community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) lineage causes the majority of skin and soft tissue infections (SSTIs) and is highly associated with the carriage of the arginine catabolic mobile element (ACME). However, the contribution of ACME to USA300’s success in SSTIs is not completely understood. We show that the constitutive ACME-encoded arginine-deiminase system (Arc) allows USA300 to thrive in acidic environments that mimic human skin. Consequently, the ACME-Arc system drives excessive production of host polyamines, compounds uniquely toxic to S. aureus. To mitigate this, ACME also encodes SpeG, a polyamine-resistance enzyme that is essential for combating excess host polyamines in a murine SSTI model. Inhibiting host polyamine production not only restored ΔspeG persistence within infected wounds but also severely altered the host healing process, implying that polyamines play an integral role in coordinating the wound-healing response. Together, these data underscore the functional modularity of ACME and its contribution to the success of USA300 CA-MRSA.