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.

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.

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.

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.

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System.

In previous studies, we identified the fatty acid kinase virulence factor regulator B (VfrB) as a potent regulator of α-hemolysin and other virulence factors in Staphylococcus aureus In this study, we demonstrated that VfrB is a positive activator of the SaeRS two-component regulatory system. Analysis of vfrB, saeR, and saeS mutant strains revealed that VfrB functions in the same pathway as SaeRS. At the transcriptional level, the promoter activities of SaeRS class I (coa) and class II (hla) target genes were downregulated during the exponential growth phase in the vfrB mutant, compared to the wild-type strain. In addition, saePQRS expression was decreased in the vfrB mutant strain, demonstrating a need for this protein in the autoregulation of SaeRS. The requirement for VfrB-mediated activation was circumvented when SaeS was constitutively active due to an SaeS (L18P) substitution. Furthermore, activation of SaeS via human neutrophil peptide 1 (HNP-1) overcame the dependence on VfrB for transcription from class I Sae promoters. Consistent with the role of VfrB in fatty acid metabolism, hla expression was decreased in the vfrB mutant with the addition of exogenous myristic acid. Lastly, we determined that aspartic acid residues D38 and D40, which are predicted to be key to VfrB enzymatic activity, were required for VfrB-mediated α-hemolysin production. Collectively, this study implicates VfrB as a novel accessory protein needed for the activation of SaeRS in S. aureusIMPORTANCE The SaeRS two-component system is a key regulator of virulence determinant production in Staphylococcus aureus Although the regulon of this two-component system is well characterized, the activation mechanisms, including the specific signaling molecules, remain elusive. Elucidating the complex regulatory circuit of SaeRS regulation is important for understanding how the system contributes to disease causation by this pathogen. To this end, we have identified the fatty acid kinase VfrB as a positive regulatory modulator of SaeRS-mediated transcription of virulence factors in S. aureus In addition to describing a new regulatory aspect of SaeRS, this study establishes a link between fatty acid kinase activity and virulence factor regulation.

Generation of a Stable Plasmid for In Vitro and In Vivo Studies of Staphylococcus Species.

A major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as during in vivo models of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid in Staphylococcus aureus USA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools for Staphylococcus species. LAC-p01 was genetically manipulated into an Escherichia coli-S. aureus shuttle vector that remained stable for at least 100 generations without antibiotic selection. The double- and single-stranded (dso and sso) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LAC-p01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use in S. aureus USA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable in Staphylococcus epidermidis Moreover, pKK22 was maintained for 7 days postinoculation during a murine model of S. aureus systemic infection and successfully complemented an hla mutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during both in vitro and in vivo experiments are powerful new tools for studies of StaphylococcusIMPORTANCE Plasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation during in vivo studies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from an S. aureus USA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generations in vitro and in vivo without antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies of Staphylococcus species.

Confirming Sterility of an Autoclaved Infected Femoral Component for Use in an Articulated Antibiotic Knee Spacer: A Pilot Study.

Antibiotic spacer designs have proven effective at eradicating infection during a two-stage revision arthroplasty. Temporary reuse of the steam-sterilized femoral component and a new all poly tibia component has been described as an effective articulating antibiotic spacer, but sterility concerns persist. Six explanted cobalt chrome femurs from patients with grossly infected TKA's and six stock femurs inoculated with different bacterial species were confirmed to be bacteria-free after autoclaving under a standard gravity-displacement cycle. The effect of steam sterilization on cobalt chrome fragments contaminated with MRSA biofilm was analyzed microscopically to quantify remaining biofilm. The autoclave significantly reduced the biofilm burden on the cobalt chrome fragments. This study confirmed sterility of the femur after a standard gravity-displacement cycle (132°C, 27 PSIG, 10 minutes).

Understanding Staphylococcal Nomenclature.

Bacteria are often grouped by a variety of properties, including biochemical activity, appearance, and more recently, nucleic acid sequence differences. In the case of human pathogens, significant work goes into "typing" strains to understand relatedness. This is especially true when trying to understand the epidemiology of these organisms. In attempts to group Staphylococci, a variety of methods and nomenclatures have been employed, which can often serve as a point of confusion to those entering the field. Therefore, the intent of this chapter is to give a brief overview of some common methods and associated nomenclature used to type Staphylococci, with S. aureus as an example.

The membrane protein PrsS mimics σS in protecting Staphylococcus aureus against cell wall-targeting antibiotics and DNA-damaging agents.

Staphylococcus aureus possesses a lone extracytoplasmic function (ECF) sigma factor, σ(S). In Bacillus subtilis, the ECF sigma factor, σ(W), is activated through a proteolytic cascade that begins with cleavage of the RsiW anti-sigma factor by a site-1 protease (S1P), PrsW. We have identified a PrsW homologue in S. aureus (termed PrsS) and explored its role in σ(S) regulation. Herein, we demonstrate that although a cognate σ(S) anti-sigma factor currently remains elusive, prsS phenocopies sigS in a wealth of regards. Specifically, prsS expression mimics the upregulation observed for sigS in response to DNA-damaging agents, cell wall-targeting antibiotics and during ex vivo growth in human serum and murine macrophages. prsS mutants also display the same sensitivities of sigS mutants to the DNA-damaging agents methyl methane sulfonate (MMS) and hydrogen peroxide, and the cell wall-targeting antibiotics ampicillin, bacitracin and penicillin-G. These phenotypes appear to be explained by alterations in abundance of proteins involved in drug resistance (Pbp2a, FemB, HmrA) and the response to DNA damage (BmrA, Hpt, Tag). Our findings seem to be mediated by putative proteolytic activity of PrsS, as site-directed mutagenesis of predicted catalytic residues fails to rescue the sensitivity of the mutant to H2O2 and MMS. Finally, a role for PrsS in S. aureus virulence was identified using human and murine models of infection. Collectively, our data indicate that PrsS and σ(S) function in a similar manner, and perhaps mediate virulence and resistance to DNA damage and cell wall-targeting antibiotics, via a common pathway.

The disruption of prenylation leads to pleiotropic rearrangements in cellular behavior in Staphylococcus aureus.

Prenylation is the addition of prenyl groups to peptide chains or metabolites via the condensation of geranyl- or isopentenyl-diphosphate moieties by geranyltranstransferases. Although this process is extensively studied in eukaryotes, little is known about the influence of prenylation in prokaryotic species. To explore the role of this modification in bacteria, we generated a mutation in the geranyltranstransferase (IspA) of Staphylococcus aureus. Quite strikingly, the ispA mutant completely lacked pigment and exhibited a previously undescribed small colony variant-like phenotype. Further pleiotropic defects in cellular behavior were noted, including impaired growth, decreased ATP production, increased sensitivity to oxidative stress, increased resistance to aminoglycosides and cationic antimicrobial peptides, and decreased resistance to cell wall-targeting antibiotics. These latter effects appear to result from differences in envelope composition as ispA mutants have highly diffuse cell walls (particularly at the septum), marked alterations in fatty acid composition and increased membrane fluidity. Taken together, these data present an important characterization of prokaryotic prenylation and demonstrate that this process is central to a wealth of pathways involved in mediating cellular homeostasis in S. aureus.

Investigating the genetic regulation of the ECF sigma factor σS in Staphylococcus aureus.

BACKGROUND: We previously identified an ECF sigma factor, σS, that is important in the stress and virulence response of Staphylococcus aureus. Transcriptional profiling of sigS revealed that it is differentially expressed in many laboratory and clinical isolates, suggesting the existence of regulatory networks that modulates its expression. RESULTS: To identify regulators of sigS, we performed a pull down assay using S. aureus lysates and the sigS promoter. Through this we identified CymR as a negative effector of sigS expression. Electrophoretic mobility shift assays (EMSAs) revealed that CymR directly binds to the sigS promoter and negatively effects transcription. To more globally explore genetic regulation of sigS, a Tn551 transposon screen was performed, and identified insertions in genes that are involved in amino acid biosynthesis, DNA replication, recombination and repair pathways, and transcriptional regulators. In efforts to identify gain of function mutations, methyl nitro-nitrosoguanidine mutagenesis was performed on a sigS-lacZ reporter fusion strain. From this a number of clones displaying sigS upregulation were subject to whole genome sequencing, leading to the identification of the lactose phosphotransferase repressor, lacR, and the membrane histidine kinase, kdpD, as central regulators of sigS expression. Again using EMSAs we determined that LacR is an indirect regulator of sigS expression, while the response regulator, KdpE, directly binds to the promoter region of sigS. CONCLUSIONS: Collectively, our work suggests a complex regulatory network exists in S. aureus that modulates expression of the ECF sigma factor, σS.

The extracytoplasmic function sigma factor σS protects against both intracellular and extracytoplasmic stresses in Staphylococcus aureus.

Previously we identified a novel component of the Staphylococcus aureus regulatory network, an extracytoplasmic function σ-factor, σ(S), involved in stress response and disease causation. Here we present additional characterization of σ(S), demonstrating a role for it in protection against DNA damage, cell wall disruption, and interaction with components of the innate immune system. Promoter mapping reveals the existence of three unique sigS start sites, one of which appears to be subject to autoregulation. Transcriptional profiling revealed that sigS expression remains low in a number of S. aureus wild types but is upregulated in the highly mutated strain RN4220. Further analysis demonstrates that sigS expression is inducible upon exposure to a variety of chemical stressors that elicit DNA damage, including methyl methanesulfonate and ciprofloxacin, as well as those that disrupt cell wall stability, such as ampicillin and oxacillin. Significantly, expression of sigS is highly induced during growth in serum and upon phagocytosis by RAW 264.7 murine macrophage-like cells. Phenotypically, σ(S) mutants display sensitivity to a broad range of DNA-damaging agents and cell wall-targeting antibiotics. Furthermore, the survivability of σ(S) mutants is strongly impacted during challenge by components of the innate immune system. Collectively, our data suggest that σ(S) likely serves dual functions within the S. aureus cell, protecting against both cytoplasmic and extracytoplasmic stresses. This further argues for its important, and perhaps novel, role in the S. aureus stress and virulence responses.

The impact of fatty acids on the antibacterial properties of N-thiolated ß-lactams.

Bacterial fatty acid synthesis (FAS) is a potentially important, albeit controversial, target for antimicrobial therapy. Recent studies have suggested that the addition of exogenous fatty acids (FAs) to growth media can circumvent the effects of FAS-targeting compounds on bacterial growth. Consequently, such agents may have limited in vivo applicability for the treatment of human disease, as free FAs are abundant within the body. Our group has previously developed N-thiolated ß-lactams and found they function by interfering with FAS in select pathogenic bacteria, including MRSA. To determine if the FAS targeting activity of N-thiolated ß-lactams can be abrogated by exogenous fatty acids, we performed MIC determinations for MRSA strains cultured with the fatty acids oleic acid and Tween 80. We find that, whilst the activity of the known FAS inhibitor triclosan is severely compromised by the addition of both oleic acid and Tween 80, exogenous FAs do not mitigate the antibacterial activity of N-thiolated ß-lactams towards MRSA. Consequently, we propose that N-thiolated ß-lactams are unique amongst FAS-inhibiting antimicrobials, as their effects are unimpeded by exogenous FAs.