What We Do
Molecular Pathogenesis Program in Staphylococcus aureus
1 Regulation of virulence factors by global regulatory elements in S. aureus including the SarA protein family and two component regulatory systems.
To adapt to the ever changing microenvironments, S. aureus uses an array of two component signal transduction systems and proteins within the SarA protein family to create an intricate regulatory network to respond to environmental stimuli (23, 24). Sequence alignment and structural data reveal that the SarA protein family can be divided into three subfamilies: 1) single domain proteins; 2) double domain proteins and 3) MarR homologs. Structural studies divulge that SarA, SarR, SarS, MgrA and possibly all members of the SarA protein family are winged helix proteins with variations. Mutagenesis studies disclose that the winged helix motifs within this protein family are important for DNA binding and function (97). Our goal here is to decipher the molecular mechanism of activation and/or repression of target genes by evaluating the transcription requirement of specific promotion, using in vitro transcription, transcriptional fusion, Northern blots and protein-DNA interaction studies.
Legend. Regulation of virulence determinants in S. aureus by the SarA protein family adapted from the figure in Crossley and Archer. Normally, the synthesis of cell surface adhesins such as fibronectin binding protein A (FnbA) during the exponential phase coincides with the expression of SarA and SaeRS. In transition from exponential to postexponential phase, the synthesis of cell wall proteins is disrupted and the production of extracellular toxins such as -toxin would begin. This transition corresponds to the maximal expression of SarA and the ensuing activation of agr. SarA expression is repressed by SarA and SarR. SigB, a stress-induced transcription factor, also activates one of the sarA promoter (the P3 promoter). On the other hand, agr is controlled by SarA, a quorum sensing autoinducing peptide, other TCRS, SarR, MgrA, SarX and SarU. MgrA, in turn, is controlled by SarZ. Activation of agr would lead to up-regulation of another TCRS system called SaeRS and down-regulation of a SarA protein homolog called Rot. This will eventually lead to repression of two gene products called SarT and subsequently SarS. SarT is an activator of SarS, which is a repressor of alpha toxin production and an activator of protein A synthesis, thus explaining the elevated production of -toxin and repression of protein A upon agr activation. Activation of agr would also result in the amplification of the original signal by activating SarU, which is a positive regulator of agr.
2 Characterization of the toxin-antitoxin (TA) family of S. aureus and their contribution to stress response.
TA systems are prevalent in prokaryotes including S. aureus. Most studies of TA systems are conducted in E. coli, but the S. aureus counterparts can differ from E. coli in important ways. There are three TA systems in S. aureus. We have initially focused our studies on MazEF. The MazEF system is unique in that these genes lie directly upstream of the sigB operon which encompasses rsbUVW-sigB, but lacks the sensory input for environmental stress (RsbRST) that is typically found in B. subtilis. We have found that the mazEF promoter can drive a 3.6 kb transcript that encompasses mazEF and rsbUVW-sigB and contributes to ~50% of the sigB activity. In distinct to other well described systems of MazEF, SigB can repress the mazEF promoter, thus forming a negative feedback loop to down-regulate sigB activity to dampen the enhanced stress response (110). Another feature of the system in S. aureus is the MazF toxin cleaves at VUUVí where V is A, C or G but not U (96). This contrasts with the ACA cleavage site of MazF of E. coli. Ectopic expression of the MazF toxin from S. aureus led to a rapid decrease in colony-formation unit (CFU) count, but most cells remained viable as determined by differential Syto 9 and propridium iodide staining after MazFsa induction. This finding suggested that the toxin MazFsa induced cell stasis rather than cell death. We also showed that MazF of S. aureus selectively cleaves cellular mRNAs in vivo, avoiding ìimportantî transcripts such as recA, gyrB and sarA mRNAs in MazF-induced cells while these three mRNAs can be cleaved in vitro. Northwestern blotting showed that both sarA and recA mRNAs bind strongly to a putative RNA-binding protein (109). These data suggest that S. aureus likely undergoes stasis by protecting selective mRNA with RNA-binding proteins upon expression of MazFsa in vivo. Preliminary identification of the mRNA binding protein is in progress. We speculate that inactivation of this mRNA binding protein of S. aureus may lead to more uniform cleavage of mRNA and hence a rapid progression of cell death.
Legend. Proposed model for mazEF regulation in S. aureus. Transcription initiated from PmazE combined with a weak rho-independent terminator nadownstream of mazF creates both the 0.7 kb mazEF transcript and a 3.7 kb mazEFrsbUVWsigB transcript. This latter transcript is required for full σB activity, and contributes significantly to rsbU-related transcripts, as the activity of the PA promoter is weak. Transcription from PmazE is stimulated by environmental and antibiotic stress, which may be mediated by the direct binding of SarA at the -35 site of the promoter. Transcription of the 0.7 and 3.7 kb mRNAs is also negatively regulated by sigB, likely through an intermediary. It is possible that this establishes a negative feedback loop wherein sigB represses PmazE. The mazEF translational products ultimately produce a toxin-antitoxin system in which the antitoxin MazE binds and inactivates MazF in a higher order stoichiometry. Following proteolytic degradation of MazE by ATP-dependent proteases, MazF of S. aureus degrades mRNA at the VUUV’ sequence in susceptible mRNA.
3 The fate of S. aureus within non-phagocytic cells and the role of quorum sensing in bacterial survival.
Cystic fibrosis patients are highly susceptible to S. aureus infections. To delineate this susceptibility, we have evaluated the interaction of S. aureus with a cultured cystic fibrosis cell line CFT-1. Our lab has shown that S. aureus can invade and replicate within CFT-1 (85), and that these internalized bacteria escape from the endocytic vesicle into the host cell cytoplasm following internalization (102). The accessory gene regulator, agr, in S. aureus has been shown to control the expression of a large number of secreted toxins involved in virulence by virtue of a four-component signal transduction system that encodes a quorum sensing peptide synthesis scheme and a two-component regulatory system that senses the cognate peptide. We showed that an agr mutant of S. aureus strain RN6390 was unable to escape from the endocytic vesicle after invasion of the CFT-1 cells, using markers of vesicular trafficking (LAMP-1 and 2, LysoTracker and Vacuolar-ATPase). Further, expression of a specific agr regulated toxin, alpha-hemolysin, under an inducible promoter in an agr mutant of S. aureus restored the phagosome-escaping phenotype of an agr mutant, thus demonstrating that the expression of agr is required for vesicular escape and that biologically active alpha-hemolysin alone within the endosome is sufficient for S. aureus escape from the endocytic vesicle into the host cytosol (102). Although the above studies suggest that quorum sensing within the confine of the endosome may be required for expression of the agr-regulated factors to promote endosomal escape, it is also possible that other factors such as the dimensions and diffusional characteristics of the endosome could influence quorum sensing (QS) and induction of genetic reprogramming. In collaboration with Jeff Brinker at Sandia National Lab, we described a physical system that mimics isolation of a bacterium, such as within an endosome or phagosome during infection (112), and maintains cell viability under conditions of complete chemical and physical isolation. Importantly, we showed that quorum sensing and genetic reprogramming can occur in a single isolated organism within a confined environment. Accordingly, quorum sensing allows S. aureus to sense confinement and to activate virulence and metabolic pathways needed for survival (112). Interestingly, we found that quorum-sensing bacteria have significantly greater viability over non-QS bacteria. Future studies will focus on the role of factors besides the autoinducing peptide in promoting the agr expression system via the quorum sensing pathway.
Legend. Quorum sensing and regulation of gene expression in individual (and groups of) S. aureus encapsulated within an isolated, nanostructured host matrix. (A) Schematic of physical system; (B) Confocal florescence images of ALC1743 cells 1 or 8 hours post-encapsulation; (C) Confocal images of ALC1740 cells 1 or 8 hours post-encapsulation. The large lower magnification frame in B and C is the merged red, green, and blue channels and locates the cell (stained red with SYTO 64) within the matrix (blue channel). The higher magnification images are the corresponding red, green (green fluorescent protein) and merged (red+green+blue) images, where yellow corresponds to co-localization of the cell and GFP. After 8 hours individual cells strongly express GFP (B,C, lower panels); (D) Time-course images for ALC1743 cells in groups (upper) and single cells,1, 4, or 8 hours post-encapsulation. For all images, fluorescence emission fingerprinting and linear unmixing were used to subtract green matrix autofluorescence (shown as blue) from red SYTO 64 and GFP.
4 Regulation of ABC transporters by two component regulatory systems and their impacts on sensitivity of S. aureus to cationic antimicrobial peptides.
Current treatment for serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA) relies heavily upon the glycopeptide antibiotic vancomycin. Unfortunately, this practice has led to an intermediate resistance phenotype that is particularly difficult to treat. Previously, whole-genome array studies listed numerous genes to be over-expressed in vancomycin intermediate sensitive strains including graRS (SA0614/0615), encoding a two-component regulatory system, as well as the adjacent vraFG (SA0616/0617), encoding an ATP-binding cassette (ABC) transporter; but the exact contribution of these genes to increased vancomycin resistance has not been defined. In a published report (91), we showed that isogenic strains with mutations in genes encoding the GraRS TCRS and the VraFG ABC-transporter are hypersensitive to vancomycin, polymyxin B and selective antimicrobial peptides including RP-1 and hNP-1. Mutations of graRS and vraFG also led to increased autolytic rates and a more negative net surface charge, which may explain, in part, to their increased sensitivity to cationic antimicrobial peptides (91,94). Taken together, these data reveal an important genetic mediator to the VISA phenotype and may hold clues to the selective pressures on staphylococci upon exposure to selective cationic peptide antibiotics. Further characterization of GraRS and VraFG and other related loci and their role in innate vancomycin resistance is in progress.
5 The molecular basis of antibiotic resistance in hospital-acquired and community-acquired methicillin resistant S. aureus.
Recent cases of infections caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strains in healthy individuals have raised concerns worldwide. CA-MRSA strains differ from hospital-acquired MRSAs by virtue of their genomic background and increased virulence in animal models. We found that in two common CA-MRSA isolates, USA300 and MW2 (USA400), a loss of penicillin-binding protein 4 (PBP4) is sufficient to cause a 16-fold reduction in oxacillin and nafcillin resistance, thus demonstrating that mecA, encoding PBP2A, is not the sole determinant of methicillin resistance in CA-MRSA (103). Loss of PBP4 was also found to severely affect the transcription of PBP2 in cells after challenge with oxacillin, thus leading to a significant decrease in peptidoglycan cross-linking. Autolysis, which is commonly associated with the killing mechanism of penicillin and -lactams, does not play a role in the reduced resistance phenotype associated with loss of PBP4. We also showed that cefoxitin, a semisynthetic -lactam that binds irreversibly to PBP4, is synergistic with oxacillin in killing CA-MRSA strains, including clinical CA-MRSA isolates. Characterization of genes that confer enhanced sensitivity of pbp4 mutants of CA-MRSA strains to cefoxitin is in progress. Our studies also infer that a combination of cefoxitin and synthetic penicillins may be an effective therapy for CA-MRSA infections.