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).

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.

Contribution of the nos-pdt operon to virulence phenotypes in methicillin-sensitive Staphylococcus aureus.

Nitric oxide (NO) is emerging as an important regulator of bacterial stress resistance, biofilm development, and virulence. One potential source of endogenous NO production in the pathogen Staphylococcus aureus is its NO-synthase (saNOS) enzyme, encoded by the nos gene. Although a role for saNOS in oxidative stress resistance, antibiotic resistance, and virulence has been recently-described, insights into the regulation of nos expression and saNOS enzyme activity remain elusive. To this end, transcriptional analysis of the nos gene in S. aureus strain UAMS-1 was performed, which revealed that nos expression increases during low-oxygen growth and is growth-phase dependent. Furthermore, nos is co-transcribed with a downstream gene, designated pdt, which encodes a prephenate dehydratase (PDT) enzyme involved in phenylalanine biosynthesis. Deletion of pdt significantly impaired the ability of UAMS-1 to grow in chemically-defined media lacking phenylalanine, confirming the function of this enzyme. Bioinformatics analysis revealed that the operon organization of nos-pdt appears to be unique to the staphylococci. As described for other S. aureus nos mutants, inactivation of nos in UAMS-1 conferred sensitivity to oxidative stress, while deletion of pdt did not affect this phenotype. The nos mutant also displayed reduced virulence in a murine sepsis infection model, and increased carotenoid pigmentation when cultured on agar plates, both previously-undescribed nos mutant phenotypes. Utilizing the fluorescent stain 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM) diacetate, decreased levels of intracellular NO/reactive nitrogen species (RNS) were detected in the nos mutant on agar plates. These results reinforce the important role of saNOS in S. aureus physiology and virulence, and have identified an in vitro growth condition under which saNOS activity appears to be upregulated. However, the significance of the operon organization of nos-pdt and potential relationship between these two enzymes remains to be elucidated.

The lone S41 family C-terminal processing protease in Staphylococcus aureus is localized to the cell wall and contributes to virulence.

Staphylococcus aureus is a versatile pathogen of humans and a continued public health concern due to the rise and spread of multidrug-resistant strains. As part of an ongoing investigation into the pathogenic mechanisms of this organism we previously demonstrated that an intracellular N-terminal processing protease is required for S. aureus virulence. Following on from this, here we examine the role of CtpA, the lone C-terminal processing protease of S. aureus. CtpA, a member of the S41 family, is a serine protease whose homologues in Gram-negative bacteria have been implicated in a range of biological functions, including pathogenesis. We demonstrate that S. aureus CtpA is localized to the bacterial cell wall and expression of the ctpA gene is maximal upon exposure to conditions encountered during infection. Disruption of the ctpA gene leads to decreased heat tolerance and increased sensitivity when exposed to components of the host immune system. Finally we demonstrate that the ctpA(-) mutant strain is attenuated for virulence in a murine model of infection. Our results represent the first characterization of a C-terminal processing protease in a pathogenic Gram-positive bacterium and show that it plays a critical role during infection.

Extracellular proteases are key mediators of Staphylococcus aureus virulence via the global modulation of virulence-determinant stability.

Staphylococcus aureus is a highly virulent and successful pathogen that causes a diverse array of diseases. Recently, an increase of severe infections in healthy subjects has been observed, caused by community-associated methicillin-resistant S. aureus (CA-MRSA). The reason for enhanced CA-MRSA virulence is unclear; however, work suggests that it results from hypersecretion of agr-regulated toxins, including secreted proteases. In this study, we explore the contribution of exo-proteases to CA-MRSA pathogenesis using a mutant lacking all 10 enzymes. We show that they are required for growth in peptide-rich environments, serum, in the presence of antimicrobial peptides (AMPs), and in human blood. We also reveal that extracellular proteases are important for resisting phagocytosis by human leukocytes. Using murine infection models, we reveal contrasting roles for the proteases in morbidity and mortality. Upon exo-protease deletion, we observed decreases in abscess formation, and impairment during organ invasion. In contrast, we observed hypervirulence of the protease-null strain in the context of mortality. This dichotomy is explained by proteomic analyses, which demonstrates exo-proteases to be key mediators of virulence-determinant stability. Specifically, increased abundance of both secreted (e.g. α-toxin, Psms, LukAB, LukE, PVL, Sbi, γ-hemolysin) and surface-associated (e.g. ClfA+B, FnbA+B, IsdA, Spa) proteins was observed upon protease deletion. Collectively, our findings provide a unique insight into the progression of CA-MRSA infections, and the role of secreted proteolytic enzymes.

Identification of an intracellular M17 family leucine aminopeptidase that is required for virulence in Staphylococcus aureus.

Staphylococcus aureus is a highly virulent bacterial pathogen capable of causing a variety of ailments throughout the human body. It is a major public health concern due to the continued emergence of highly pathogenic methicillin resistant strains (MRSA) both within hospitals and in the community. Virulence in S. aureus is mediated by an array of secreted and cell wall associated virulence factors, including toxins, hemolysins and proteases. In this work we identify a leucine aminopeptidase (LAP, pepZ) that strongly impacts the pathogenic abilities of S. aureus. Disruption of the pepZ gene in either Newman or USA300 resulted in a dramatic attenuation of virulence in both localized and systemic models of infection. LAP is required for survival inside human macrophages and gene expression analysis shows that pepZ expression is highest in the intracellular environment. We examine the cellular location of LAP and demonstrate that it is localized to the bacterial cytosol. These results identify for the first time an intracellular leucine aminopeptidase that influences disease causation in a Gram-positive bacterium.

The impact of CodY on virulence determinant production in community-associated methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is a leading human pathogen of both hospital and community-associated diseases worldwide. This organism causes a wealth of infections within the human host as a result of the vast arsenal of toxins encoded within its genome. Previous transcriptomic studies have shown that toxin production in S. aureus can be strongly impacted by the negative regulator CodY. CodY acts by directly, and indirectly (via Agr), repressing toxin production during times of plentiful nutrition. In this study, we use iTRAQ-based proteomics for the first time to study virulence determinant production in S. aureus, so as to correlate transcriptional observations with actual changes in protein synthesis. Using a codY mutant in the epidemic CA-MRSA clone USA300 we demonstrate that deletion of this transcription factor results in a major upregulation of toxin synthesis in both post-exponential and stationary growth. Specifically, we observe hyper-production of secreted proteases, leukocidins and hemolysins in both growth phases in the USA300 codY mutant. Our findings demonstrate the power of mass spectrometry-based quantitative proteomics for studying toxin production in S. aureus, and the importance of CodY to this central process in disease causation and infection.

NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus.

Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.