Molecular basis for the activation of the Fatty Acid Kinase complex of Staphylococcus aureus.

Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. The complex, consisting of the fatty acid kinase, FakA, and an acyl carrier protein, FakB, is known to impact virulence and disease outcomes. However, FAK's structure and enzymatic mechanism remain poorly understood. Here, we used a combination of modeling, biochemical, and cell-based approaches to establish critical details of FAK activity. Solved structures of the apo and ligand-bound FakA kinase domain captured the protein state through ATP hydrolysis. Additionally, targeted mutagenesis of an understudied FakA Middle domain identified critical residues within a metal-binding pocket that contribute to FakA dimer stability and protein function. Regarding the complex, we demonstrated nanomolar affinity between FakA and FakB and generated computational models of the complex's quaternary structure. Together, these data provide critical insight into the structure and function of the FAK complex which is essential for understanding its mechanism.

Factors impacting the regulation of nos gene expression in Staphylococcus aureus.

Staphylococcus aureus nitric oxide synthase (saNOS) contributes to oxidative stress resistance, antibiotic tolerance, virulence, and modulation of aerobic and nitrate-based cellular respiration. Despite its involvement in these essential processes, the genetic regulation of nos expression has not been well characterized. 5' rapid amplification of cDNA ends on nos RNA isolated from S. aureus UAMS-1 (USA200 strain) and AH1263 (USA300 strain) revealed that the nos transcriptional start site mapped to an adenine nucleotide in the predicted Shine-Dalgarno site located 11 bp upstream of the nos ATG start codon, suggesting that the nos transcript may have a leaderless organization or may be subject to processing. The SrrAB two-component system (TCS) was previously identified as a positive regulator of nos RNA levels, and experiments using a ß-galactosidase reporter plasmid confirmed that SrrAB is a positive regulator of nos promoter activity. In addition, the quorum-sensing system Agr was identified as a negative regulator of low-oxygen nos expression in UAMS-1, with activity epistatic to SrrAB. Involvement of Agr was strain dependent, as nos expression remained unchanged in an AH1263 agr mutant, which has higher Agr activity compared to UAMS-1. Furthermore, nos promoter activity and RNA levels were significantly stronger in AH1263 relative to UAMS-1 during late-exponential low-oxygen growth, when nos expression is maximal. Global regulators Rex and MgrA were also implicated as negative regulators of low-oxygen nos promoter activity in UAMS-1. Collectively, these results provide new insight into factors that control nos expression.IMPORTANCEBacterial nitric oxide synthase (bNOS) has recently emerged in several species as a key player in resistance to stresses commonly encountered during infection. Although Staphylococcus aureus (sa)NOS has been suggested to be a promising drug target in S. aureus, an obstacle to this in practice is the existence of mammalian NOS, whose oxygenase domain is like bacterial NOS. Increased understanding of the nos regulatory network in S. aureus could allow targeting of saNOS through its regulators, bypassing the issue of also inhibiting mammalian NOS. Furthermore, the observed strain-dependent differences in S. aureus nos regulation presented in this study reinforce the importance of studying bacterial NOS regulation and function at both the strain and species levels.

Kinetic Characterization of the Immune Response to Methicillin-Resistant Staphylococcus aureus Subcutaneous Skin Infection.

Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTIs). Studies examining the immune response to S. aureus have been conducted, yet our understanding of the kinetic response to S. aureus subcutaneous skin infection remains incomplete. In this study, we used C57BL/6J mice and USA300 S. aureus to examine the host-pathogen interface from 8 h postinfection to 15 days postinfection (dpi), with the following outcomes measured: lesion size, bacterial titers, local cytokine and chemokine levels, phenotype of the responding leukocytes, and histopathology and Gram staining of skin tissue. Lesions were largest at 1 dpi, with peak necrotic tissue areas at 3 dpi, and were largely resolved by 15 dpi. During early infection, bacterial titers were high, neutrophils were the most abundant immune cell type, there was a decrease in most leukocyte populations found in uninfected skin, and many different cytokines were produced. Histopathological analysis demonstrated swift and extensive keratinocyte death and robust and persistent neutrophil infiltration. Gram staining revealed subdermal S. aureus colonization and, later, limited migration into upper skin layers. Interleukin-17A/F (IL-17A/F) was detected only starting at 5 dpi and coincided with an immediate decrease in bacterial numbers in the following days. After 9 days, neutrophils were no longer the most abundant immune cell type present as most other leukocyte subsets returned, and surface wounds resolved coincident with declining bacterial titers. Collectively, these data illustrate a dynamic immune response to S. aureus skin infection and suggest a key role for precisely timed IL-17 production for infection clearance and healthy tissue formation.

Fatty acids can inhibit Staphylococcus aureus SaeS activity at the membrane independent of alterations in respiration.

In Staphylococcus aureus, the two-component system SaeRS is responsible for regulating various virulence factors essential for the success of this pathogen. SaeRS can be stimulated by neutrophil-derived products but has also recently been shown to be inactivated by the presence of free fatty acids. A mechanism for how fatty acids negatively impacts SaeRS has not been described. We found that unsaturated fatty acids, as well as fatty acids not commonly found in Staphylococcal membranes, prevent the activation of SaeRS at a lower concentration than their saturated counterparts. These fatty acids can negatively impact SaeRS without altering the respiratory capacity of the bacterium. To uncover a potential mechanism for how fatty acids impact SaeRS function/activity, we utilized a naturally occurring point mutation found in S. aureus as well as chimeric SaeS proteins. Using these tools, we identified that the native transmembrane domains of SaeS dictate the transcriptional response to fatty acids in S. aureus. Our data support a model where free fatty acids alter the activity of the two-component system SaeRS directly through the sensor kinase SaeS and is dependent on the transmembrane domains of the protein.

Quantitative Hemolysis Assays.

Many strains of Staphylococcus aureus produce a variety of cytolysins that target many different cell types to both fight the immune system and acquire nutrients. This includes hemolysins which destroy erythrocytes and are well studied virulence factors. Traditionally, hemolysin activity is measured on blood agar plates due to the simplicity of the assay. While this is telling, it cannot encapsulate the full story because S. aureus is known to behave differently in broth and on agar. Furthermore, plate-based assays are primarily semiquantitative and often a more accurate determination of hemolytic potential is needed to discern differences between strains. Here, we describe a method to quantify hemolysin activity from broth or similarly grown cells.

Bone Marrow-Derived Macrophage Infection Assay.

The use of cultured mammalian cells, whether immortalized cell lines or primary cells, is a well-known technique used as a substitute or prescreen for in vivo virulence potential of bacterial pathogens. This technique is also a way to examine host-pathogen interactions in a less complex environment compared to that found in whole animals. To this end, macrophage infection assays have become a key technique for studying the molecular mechanisms by which bacteria interact with the host. Herein, this chapter describes both how to produce macrophages from mouse bone marrow and the subsequent infection assays.

Staphylococcus aureus Fatty Acid Kinase FakA Modulates Pathogenesis during Skin Infection via Proteases.

Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro, and that the ΔfakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla ΔfakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the ΔfakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the ΔfakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the ΔfakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in ΔfakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δaur and ΔsspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the ΔfakA mutant during skin infection.

Contribution of YjbIH to Virulence Factor Expression and Host Colonization in Staphylococcus aureus.

To persist within the host and cause disease, Staphylococcus aureus relies on its ability to precisely fine-tune virulence factor expression in response to rapidly changing environments. During an unbiased transposon mutant screen, we observed that disruption of a two-gene operon, yjbIH, resulted in decreased levels of pigmentation and aureolysin (Aur) activity relative to the wild-type strain. Further analyses revealed that YjbH, a predicted thioredoxin-like oxidoreductase, is predominantly responsible for the observed yjbIH mutant phenotypes, though a minor role exists for the putative truncated hemoglobin YjbI. These differences were due to significantly decreased expression of crtOPQMN and aur Previous studies found that YjbH targets the disulfide- and oxidative stress-responsive regulator Spx for degradation by ClpXP. The absence of yjbH or yjbI resulted in altered sensitivities to nitrosative and oxidative stress and iron deprivation. Additionally, aconitase activity was altered in the yjbH and yjbI mutant strains. Decreased levels of pigmentation and aureolysin (Aur) activity in the yjbH mutant were found to be Spx dependent. Lastly, we used a murine sepsis model to determine the effect of the yjbIH deletion on pathogenesis and found that the mutant was better able to colonize the kidneys and spleens during an acute infection than the wild-type strain. These studies identified changes in pigmentation and protease activity in response to YjbIH and are the first to have shown a role for these proteins during infection.

Interplay of Nitric Oxide Synthase (NOS) and SrrAB in Modulation of Staphylococcus aureus Metabolism and Virulence.

Staphylococcus aureus nitric oxide synthase (saNOS) is a major contributor to virulence, stress resistance, and physiology, yet the specific mechanism(s) by which saNOS intersects with other known regulatory circuits is largely unknown. The SrrAB two-component system, which modulates gene expression in response to the reduced state of respiratory menaquinones, is a positive regulator of nos expression. Several SrrAB-regulated genes were also previously shown to be induced in an aerobically respiring nos mutant, suggesting a potential interplay between saNOS and SrrAB. Therefore, a combination of genetic, molecular, and physiological approaches was employed to characterize a nos srrAB mutant, which had significant reductions in the maximum specific growth rate and oxygen consumption when cultured under conditions promoting aerobic respiration. The nos srrAB mutant secreted elevated lactate levels, correlating with the increased transcription of lactate dehydrogenases. Expression of nitrate and nitrite reductase genes was also significantly enhanced in the nos srrAB double mutant, and its aerobic growth defect could be partially rescued with supplementation with nitrate, nitrite, or ammonia. Furthermore, elevated ornithine and citrulline levels and highly upregulated expression of arginine deiminase genes were observed in the double mutant. These data suggest that a dual deficiency in saNOS and SrrAB limits S. aureus to fermentative metabolism, with a reliance on nitrate assimilation and the urea cycle to help fuel energy production. The nos, srrAB, and nos srrAB mutants showed comparable defects in endothelial intracellular survival, whereas the srrAB and nos srrAB mutants were highly attenuated during murine sepsis, suggesting that SrrAB-mediated metabolic versatility is dominant in vivo.

Inactivation of the exogenous fatty acid utilization pathway leads to increased resistance to unsaturated fatty acids in Staphylococcus aureus.

The human pathogen Staphylococcus aureus produces saturated fatty acids, but can incorporate both exogenous saturated and unsaturated fatty acids into its lipid membrane. S. aureus encounters unsaturated fatty acids in the host skin where they serve as an innate immune defence due to their toxicity. Previously, we identified a fatty acid kinase in S. aureus that is necessary for the utilization of exogenous fatty acids. The goal of this study was to determine the effects of fatty acids on mutants deficient in the exogenous fatty acid utilization machinery. We have demonstrated that mutants lacking a functional fatty acid kinase (fakA) or both fatty acid carrier proteins (fakB1 fakB2) are more resistant to unsaturated fatty acids. Previous studies suggested a role for ammonia-producing enzymes in resistance to unsaturated fatty acids, but these enzymes do not contribute to the resistance of the fakA mutant, despite increased urease transcription and protein activity in the mutant. Additionally, while pigment is altered in mutants unable to use exogenous fatty acids, staphyloxanthin does not contribute to fatty acid resistance of an fakA mutant. Because exposure to unsaturated fatty acids probably initiates a stress response, we investigated the role of the alternative sigma factor σB and determined if it is necessary for the fatty acid resistance observed in the fakA mutant. Collectively, this study demonstrates that the inability to incorporate unsaturated fatty acids leads to increased resistance to those fatty acids, and that resistance requires a σB stress response.