The contribution of DNA repair pathways to Staphylococcus aureus fitness and fidelity during nitric oxide stress.

Staphylococcus aureus is a major human pathogen that causes a variety of illnesses, ranging from minor skin and soft tissue infections to more severe systemic infections. Although the primary host immune response can typically clear bacterial infections, S. aureus is uniquely resistant to inflammation. For instance, our laboratory has determined that S. aureus is highly resistant to nitric oxide (NO⋅), an important component of the innate immune response that plays a role in both immunomodulatory and antibacterial processes. Additionally, NO⋅ and its derivatives can cause damage to S. aureus DNA, more specifically, deamination and/or oxidation of DNA bases; however, regulation and repair mechanisms of DNA in S. aureus are understudied. Thus, we hypothesize that several DNA repair mechanisms may account for the replication fidelity of S. aureus and may contribute to fitness in the presence of NO⋅. Here, we show the role of several DNA repair mechanisms in S. aureus. More specifically, we found that recombinational repair genes recJ, recG, and polA may play a role in the repair of NO⋅-induced replication fork collapses. We also show the role of the base excision repair pathway protein, MutY, in reducing NO⋅-mediated mutagenesis. Overall, our results suggest that NO⋅ leads to DNA damage, which subsequently induces the activity of several DNA repair pathways, contributing to the replication fidelity and fitness of S. aureus.IMPORTANCEPathogenic bacteria must evolve various mechanisms in order to evade the host immune response that they are infecting. One aspect of the primary host immune response to an infection is the production of an inflammatory effector component, nitric oxide (NO⋅). Staphylococcus aureus has uniquely evolved a diverse array of strategies to circumvent the inhibitory activity of nitric oxide. One such mechanism by which S. aureus has evolved allows the pathogen to survive and maintain its genomic integrity in this environment. For instance, here, our results suggest that S. aureus employs several DNA repair pathways to ensure replicative fitness and fidelity under NO⋅ stress. Thus, our study presents evidence of an additional strategy that allows S. aureus to evade the cytotoxic effects of host NO⋅.

Specialized phosphate transport is essential for Staphylococcus aureus nitric oxide resistance.

Staphylococcus aureus is a major human pathogen capable of causing a variety of diseases ranging from skin and soft tissue infections to systemic presentations such as sepsis, endocarditis, and osteomyelitis. For S. aureus to persist as a pathogen in these environments, it must be able to resist the host immune response, including the production of reactive oxygen and nitrogen species (e.g., nitric oxide, NO·). Extensive work from our lab has shown that S. aureus is highly resistant to NO·, especially in the presence of glucose. RNA-seq performed on S. aureus exposed to NO· in the presence and absence of glucose showed a new system important for NO· resistance-phosphate transport. The phosphate transport systems pstSCAB and nptA are both upregulated upon NO·-exposure, particularly in the presence of glucose. Both are key for phosphate transport at an alkaline pH, which the cytosol of S. aureus becomes under NO· stress. Accordingly, the ΔpstSΔnptA mutant is attenuated under NO stress in vitro as well as in macrophage and murine infection models. This work defines a new role in infection for two phosphate transporters in S. aureus and provides insight into the complex system that is NO· resistance in S. aureus.IMPORTANCEStaphylococcus aureus is a bacterial pathogen capable of causing a wide variety of disease in humans. S. aureus is unique in its ability to resist the host immune response, including the antibacterial compound known as nitric oxide (NO·). We used an RNA-sequencing approach to better understand the impact of NO· on S. aureus in different environments. We discovered that inorganic phosphate transport is induced by the presence of NO·. Phosphate is important for the generation of energy from glucose, a carbon source favored by S. aureus. We show that the absence of these phosphate transporters causes lowered energy levels in S. aureus. We find that these phosphate transporters are essential for S. aureus to grow in the presence of NO· and to cause infection. Our work here contributes significantly to our understanding of S. aureus NO· resistance and provides a new context in which S. aureus phosphate transporters are essential.

Changing careers: Skin pathogen evolves to infect the bloodstream.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) rose to clinical dominance decades ago and predominantly manifested as skin and soft-tissue infections (SSTIs). These clones were distinct from those causing hospital acquired (HA-MRSA) infections. Dyzenhaus et al. describe the evolutionary changes necessary for CA-MRSA clones to cause bloodstream infections (BSIs).

Recent developments in our understanding of the physiology and nitric oxide-resistance of Staphylococcus aureus.

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.

Mechanisms Behind the Indirect Impact of Metabolic Regulators on Virulence Factor Production in Staphylococcus aureus.

Staphylococcus aureus is a human skin pathogen capable of causing invasive infections in many tissues in the human body. The host of virulence factors, such as toxins and proteases, available to S. aureus contribute to its diverse disease presentations. The majority of these virulence factors are under the control of the Agr quorum sensing system. The interaction between the Agr system and some well-established metabolic regulators has long been noted, but no mechanism has been provided as to these indirect interactions. In this study, we examine the connection between Agr and CcpA, a regulator of central carbon metabolism with a known positive impact on Agr function. We further investigated the interaction of Agr and CodY, a regulator of amino acid metabolism and a member of the stringent response with a known negative impact on Agr function. We show that though there are alterations in intracellular amino acid levels in each of these mutants that are consistent with their effect on Agr, there does not seem to be a direct impact on the translation of the Agr system itself that contributes to the altered expression observed in these mutants. Given the changes in cellular metabolism in a ΔccpA mutant, we find reduced levels of intracellular ATP even in the presence of glucose. This reduction in ATP, combined with the reduced affinity of the AgrC sensor kinase for ATP, explains the reduction in Agr activity long observed in ΔccpA strains. IMPORTANCE The human pathogen Staphylococcus aureus produces a great number of virulence factors that contribute to the pathogen's ability to cause dangerous, invasive infections. Understanding the full scope of the regulation of these virulence factors can provide us with new information about how to target virulence factor production. For years, researchers in the field have observed an impact of metabolic regulators on virulence factor production with no mechanistic explanation. Here, we describe the role of two of these regulators, CcpA and CodY, in virulence factor expression and provide evidence of indirect mechanisms contributing to the control of the Agr system and virulence factor production by these two metabolic regulators. Our study sheds light on the interplay between metabolism and virulence in S. aureus and provides an explanation as to how these concepts are linked.

The Nutritional Environment Is Sufficient To Select Coexisting Biofilm and Quorum Sensing Mutants of Pseudomonas aeruginosa.

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.

The Intersection of the Staphylococcus aureus Rex and SrrAB Regulons: an Example of Metabolic Evolution That Maximizes Resistance to Immune Radicals.

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.

Lack of nutritional immunity in diabetic skin infections promotes Staphylococcus aureus virulence.

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.