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Staphylococcus aureus is an important human pathogen causing a wide range of disease presentations. It harbors a vast array of virulence factors and drug-resistance determinants. All of these factors are coordinately regulated by a hand full of key transcriptional regulators. The regulation and expression of these factors are tightly intertwined with the metabolic state of the cell. Furthermore, alterations in central metabolism are also key to the ability of S. aureus to resist clearance by the host innate immune response, including nitric oxide (NO·) production. Given the fact that central metabolism directly influences virulence, drug resistance and immune tolerance in S. aureus, a better understanding of the metabolic capabilities of this pathogen is critical. This work highlights some of the major findings within the last five years surrounding S. aureus central metabolism, both organic and inorganic. These are also put in the context of the unique NO·-resistance associated with this pathogen as well as their contributions to virulence. The more we understand the intersection between central metabolism and virulence capabilities in S. aureus, the better the chances of developing novel therapeutics so desperately needed to treat this pathogen.

The high antibiotic tolerance of Staphylococcus aureus biofilms is associated with challenges for treating periprosthetic joint infection. The toxin–antitoxin system, YefM–YoeB, is thought to be a regulator for antibiotic tolerance, but its physiological role is unknown. The objective of this study was to determine the biofilm and antibiotic susceptibility phenotypes associated with S. aureus yoeB homologs. We hypothesized the toxin–antitoxin yoeB homologs contribute to biofilm formation and antibiotic susceptibility. Disruption of yoeB1 and yoeB2 resulted in decreased biofilm formation in comparison to Newman and JE2 wild-type (WT) S. aureus strains. In comparison to yoeB mutants, both Newman and JE2 WT strains had higher polysaccharide intercellular adhesin (PIA) production. Treatment with sodium metaperiodate increased biofilm formation in Newman WT, indicating biofilm formation may be increased under conditions of oxidative stress. DNase I treatment decreased biofilm formation in Newman WT but not in the absence of yoeB1 or yoeB2. Additionally, WT strains had a higher extracellular DNA (eDNA) content in comparison to yoeB mutants but no differences in biofilm protein content. Moreover, loss of yoeB1 and yoeB2 decreased biofilm survival in both Newman and JE2 strains. Finally, in a neutropenic mouse abscess model, deletion of yoeB1 and yoeB2 resulted in reduced bacterial burden. In conclusion, our data suggest that yoeB1 and yoeB2 are associated with S. aureus planktonic growth, extracellular dependent biofilm formation, antibiotic tolerance, and virulence.

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 can complicate preceding viral infections, including influenza virus. A bacterial infection combined with a preceeding viral infection, known as super-infection, leads to worse outcomes compared to single infection. Most of the super-infection literature focuses on the changes in immune responses to bacteria between homeostatic and virally infected lungs. However, it is unclear how much of an influence bacterial virulence factors have in super-infection. Staphylococcal species express a broad range of cell wall-anchored proteins (CWAs) that have roles in host adhesion, nutrient acquisition, and immune evasion. We screened the importance of these CWAs using mutants lacking individual CWAs in vivo in both bacterial pneumonia and influenza super-infection. In bacterial pneumonia, lacking individual CWAs led to varying decreases in bacterial burden, lung damage, and immune infiltration into the lung. However, the presence of a preceding influenza infection partially abrogated the requirement for CWAs. In the screen, we found that the uncharacterized CWA S. aureus surface protein D (SasD) induced changes in both inflammatory and homeostatic lung markers. We further characterized a SasD mutant (sasD A50.1) in the context of pneumonia. Mice infected with sasD A50.1 had decreased bacterial burden, inflammatory responses, and mortalty compared to wildtype S. aureus. Mice also had reduced levels of IL-1β compared with wildtype, likely derived from macrophages. Reductions in IL-1β transcript levels as well as increased macrophage viability implicate altered macrophage cell death pathways. These data identify a novel virulence factor for S. aureus that influences inflammatory signaling within the lung.ImportanceStaphylococcus aureus is a common commensal bacteria that can cause severe infections, such as pneumonia. In the lung, viral infections increase the risk of staphylococcal pneumonia, leading to combined infections known as super-infections. The most common virus associated with S. aureus pneumonia is influenza, and super-infections lead to worse patient outcomes compared to either infection alone. While there is much known about how the immune system differs between healthy and virally infected lungs, the role of bacterial virulence factors in super-infection is less understood. The significance of our research is identifying new bacterial virulence factors that play a role in the initiation of infection and lung injury, which could lead to future therapies to prevent pulmonary single or super-infection with S. aureus.

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 airways of people 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 sufficient to select mutants with problematic clinical phenotypes including increased biofilm and altered quorum sensing. IMPORTANCE Pseudomonas 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 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.

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.

Diving behaviour of ‘surfacers' such as sea snakes, cetaceans and turtles is complex and multi-dimensional, thus may be better captured by multi-sensor biologging data. However, analysing these large multi-faceted datasets remains challenging, though a high priority. We used high-resolution multi-sensor biologging data to provide the first detailed description of the environmental influences on flatback turtle ( Natator depressus ) diving behaviour, during its foraging life-history stage. We developed an analytical method to investigate seasonal, diel and tidal effects on diving behaviour for 24 adult flatback turtles tagged with biologgers. We extracted 16 dive variables associated with three-dimensional and kinematic characteristics for 4128 dives. K -means and hierarchical cluster analyses failed to identify distinct dive types. Instead, principal component analysis objectively condensed the dive variables, removing collinearity and highlighting the main features of diving behaviour. Generalized additive mixed models of the main principal components identified significant seasonal, diel and tidal effects on flatback turtle diving behaviour. Flatback turtles altered their diving behaviour in response to extreme tidal and water temperature ranges, displaying thermoregulation and predator avoidance strategies while likely optimizing foraging in this challenging environment. This study demonstrates an alternative statistical technique for objectively interpreting diving behaviour from multivariate collinear data derived from biologgers.

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.

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.….

Elevated blood/tissue glucose is a hallmark feature of advanced diabetes, and people with diabetes are prone to more frequent and invasive infections with Staphylococcus aureus. Phagocytes must markedly increase glucose consumption during infection to generate and oxidative burst and kill invading bacteria. Similarly, glucose is essential for S. aureus survival in an infection and competition with the host, for this limited resource is reminiscent of nutritional immunity. Here, we show that infiltrating phagocytes do not express their high-efficiency glucose transporters in modeled diabetic infections, resulting in a diminished respiratory burst and increased glucose availability for S. aureus We show that excess glucose in these hyperglycemic abscesses significantly enhances S. aureus virulence potential, resulting in worse infection outcomes. Last, we show that two glucose transporters recently acquired by S. aureus are essential for excess virulence factor production and the concomitant increase in disease severity in hyperglycemic infections.

The high antibiotic tolerance of Staphylococcus aureus biofilms is associated with challenges for treating periprosthetic joint infection. The toxin–antitoxin system, YefM–YoeB, is thought to be a regulator for antibiotic tolerance, but its physiological role is unknown. The objective of this study was to determine the biofilm and antibiotic susceptibility phenotypes associated with S. aureus yoeB homologs. We hypothesized the toxin–antitoxin yoeB homologs contribute to biofilm formation and antibiotic susceptibility. Disruption of yoeB1 and yoeB2 resulted in decreased biofilm formation in comparison to Newman and JE2 wild-type (WT) S. aureus strains. In comparison to yoeB mutants, both Newman and JE2 WT strains had higher polysaccharide intercellular adhesin (PIA) production. Treatment with sodium metaperiodate increased biofilm formation in Newman WT, indicating biofilm formation may be increased under conditions of oxidative stress. DNase I treatment decreased biofilm formation in Newman WT but not in the absence of yoeB1 or yoeB2. Additionally, WT strains had a higher extracellular DNA (eDNA) content in comparison to yoeB mutants but no differences in biofilm protein content. Moreover, loss of yoeB1 and yoeB2 decreased biofilm survival in both Newman and JE2 strains. Finally, in a neutropenic mouse abscess model, deletion of yoeB1 and yoeB2 resulted in reduced bacterial burden. In conclusion, our data suggest that yoeB1 and yoeB2 are associated with S. aureus planktonic growth, extracellular dependent biofilm formation, antibiotic tolerance, and virulence.

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.

Nutritional immunity defects explain the severity of S. aureus skin infections in patients with diabetes., Elevated blood/tissue glucose is a hallmark feature of advanced diabetes, and people with diabetes are prone to more frequent and invasive infections with Staphylococcus aureus. Phagocytes must markedly increase glucose consumption during infection to generate and oxidative burst and kill invading bacteria. Similarly, glucose is essential for S. aureus survival in an infection and competition with the host, for this limited resource is reminiscent of nutritional immunity. Here, we show that infiltrating phagocytes do not express their high-efficiency glucose transporters in modeled diabetic infections, resulting in a diminished respiratory burst and increased glucose availability for S. aureus. We show that excess glucose in these hyperglycemic abscesses significantly enhances S. aureus virulence potential, resulting in worse infection outcomes. Last, we show that two glucose transporters recently acquired by S. aureus are essential for excess virulence factor production and the concomitant increase in disease severity in hyperglycemic infections.

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.

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 must rapidly adapt to a variety of carbon and nitrogen sources during invasion of a host. Within a staphylococcal abscess, preferred carbon sources such as glucose are limiting, suggesting that S. aureus survives through the catabolism of secondary carbon sources. S. aureus encodes pathways to catabolize multiple amino acids, including those that generate pyruvate, 2-oxoglutarate, and oxaloacetate. To assess amino acid catabolism, S. aureus JE2 and mutants were grown in complete defined medium containing 18 amino acids but lacking glucose (CDM). A mutation in the gudB gene, coding for glutamate dehydrogenase, which generates 2-oxoglutarate from glutamate, significantly reduced growth in CDM, suggesting that glutamate and those amino acids generating glutamate, particularly proline, serve as the major carbon source in this medium. Nuclear magnetic resonance (NMR) studies confirmed this supposition. Furthermore, a mutation in the ackA gene, coding for acetate kinase, also abrogated growth of JE2 in CDM, suggesting that ATP production from pyruvate-producing amino acids is also critical for growth. In addition, although a functional respiratory chain was absolutely required for growth, the oxygen consumption rate and intracellular ATP concentration were significantly lower during growth in CDM than during growth in glucose-containing media. Finally, transcriptional analyses demonstrated that expression levels of genes coding for the enzymes that synthesize glutamate from proline, arginine, and histidine are repressed by CcpA and carbon catabolite repression. These data show that pathways important for glutamate catabolism or ATP generation via Pta/AckA are important for growth in niches where glucose is not abundant, such as abscesses within skin and soft tissue infections., IMPORTANCE S. aureus is a significant cause of both morbidity and mortality worldwide. This bacterium causes infections in a wide variety of organ systems, the most common being skin and soft tissue. Within a staphylococcal abscess, levels of glucose, a preferred carbon source, are limited due to the host immune response. Therefore, S. aureus must utilize other available carbon sources such as amino acids or peptides to proliferate. Our results show that glutamate and amino acids that serve as substrates for glutamate synthesis, particularly proline, function as major carbon sources during growth, whereas other amino acids that generate pyruvate are important for ATP synthesis via substrate-level phosphorylation in the Pta-AckA pathway. Our data support a model whereby certain amino acid catabolic pathways, and acquisition of those particular amino acids, are crucial for growth in niches where glucose is not abundant.

A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK is not only critical for bacterial survival which would make it a target for development of novel antibiotics, but it is reported to be one of the most highly connected 'hub proteins' in MRSA, and thus should be very sensitive to mutations and making it difficult for the bacteria to develop resistance. From the co-crystal structure of cis-3-4-dihydrohamacanthin B (4) bound to S. aureus PK we were able to identify the pharmacophore needed for activity. Consequently, we prepared simple direct linked bis-indoles such as 10b that have similar anti-MRSA activity as compound 4. Structure-activity relationship (SAR) studies were carried out on 10b and led us to discover more potent compounds such as 10c, 10d, 10k and 10 m with enzyme inhibiting activities in the low nanomolar range that effectively inhibited the bacteria growth in culture with minimum inhibitory concentrations (MIC) for MRSA as low as 0.5 μg/ml. Some potent PK inhibitors, such as 10b, exhibited attenuated antibacterial activity and were found to be substrates for an efflux mechanism in S. aureus. Studies comparing a wild type S. aureus with a construct (S. aureus LAC Δpyk::Erm(R)) that lacks PK activity confirmed that bactericidal activity of 10d was PK-dependant.

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⋅.