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.

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.

Analysis of Murein Hydrolases and Proteases Through Zymography.

Zymography has been used to analyze enzymatic activity and processing of enzymes for many years. We have used bacterial cells copolymerized into the acrylamide gel to analyze specific activity of murein hydrolases of interest. In addition, this method has been widely used to examine and distinguish protease activities using different substrates. This chapter provides instruction for zymography of both extracellular murein hydrolases and proteases produced by Staphylococcus aureus.

Measuring Staphylococcal Promoter Activities Using a Codon-Optimized ß-Galactosidase Reporter.

The lacZ gene and corresponding ß-galactosidase enzyme has been a mainstay for bacterial reporter systems for decades. We have used this versatile reporter to analyze expression profiles from strains grown both on solid media and from broth culture. The standard broth protocol can also be adapted for a 96-well plate to allow high-throughput screening of promoter reporter constructs under a variety of conditions. Furthermore, codon-optimization of the E. coli lacZ gene has greatly improved activity levels of ß-galactosidase in S. aureus, facilitating improved sensitivity for screening assays, detection of low-activity promoters, and use of small sample volumes. In this chapter, details are provided for both standard and high-throughput quantitative assays that we have routinely used for S. aureus transcriptional profiling.