Ellen Murphy

Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci.

The advent of transcription profiling technologies has provided researchers with an unprecedented ability to study biological processes. Accordingly, a custom-made Affymetrix GeneChip, constituting >86% of the Staphylococcus aureus genome, was used to identify open reading frames that are regulated by agr and/or SarA, the two best-studied regulators of the organisms virulence response. RNA extracted from wild-type cells and agr, sarA, and agr sarA mutant cells in the early-, mid-, and late-log and stationary phases of growth was analyzed. Open reading frames with transcription patterns expected of genes either up- or downregulated in an agr- and/or SarA-dependent manner were identified. Oligonucleotide microarray and Northern blot analyses confirmed that the transcription of several known virulence genes, including hla (alpha-toxin) and spa (protein A), is regulated by each effector and provided insights about the regulatory cascades involved in both alpha-hemolysin and protein A expression. Several putative virulence factors were also identified as regulated by agr and/or SarA. In addition, genes that are involved in several biological processes but which are difficult to reconcile as playing a direct role in the organisms pathogenesis also appeared to be regulated by each effector, suggesting that products of both the agr and the sarA locus are more-global transcription regulators than previously realized.

Global Gene Expression in Staphylococcus aureus Biofilms

We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K. E. Beenken, J. S. Blevins, and M. S. Smeltzer, Infect. Immun. 71:4206-4211, 2003). In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo. Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo. In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S. aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential- and stationary-phase planktonic cultures. Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S. aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476). The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture. Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions. Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions. A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions.

Microarray-Based Analysis of the Staphylococcus aureus σB Regulon

Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by σB activity. While σB was found to positively control 198 genes by a factor of ≥2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of σB. Gene products that were found to be influenced by σB are putatively involved in all manner of cellular processes, including cell envelope biosynthesis and turnover, intermediary metabolism, and signaling pathways. Most of the genes and/or operons identified as upregulated by σB were preceded by a nucleotide sequence that resembled the σB consensus promoter sequence of Bacillus subtilis. A conspicuous number of virulence-associated genes were identified as regulated by σB activity, with many adhesins upregulated and prominently represented in this group, while transcription of various exoproteins and toxins were repressed. The data presented here suggest that the σB of S. aureus controls a large regulon and is an important modulator of virulence gene expression that is likely to act conversely to RNAIII, the effector molecule of the agr locus. We propose that this alternative transcription factor may be of importance for the invading pathogen to fine-tune its virulence factor production in response to changing host environments.

Global Regulation of Staphylococcus aureus Genes by Rot

Staphylococcus aureus produces a wide array of cell surface and extracellular proteins involved in virulence. Expression of these virulence factors is tightly controlled by numerous regulatory loci, including agr, sar, sigB, sae, and arl, as well as by a number of proteins with homology to SarA. Rot (repressor of toxins), a SarA homologue, was previously identified in a library of transposon-induced mutants created in an agr-negative strain by screening for restored protease and alpha-toxin. To date, all of the SarA homologues have been shown to act as global regulators of virulence genes. Therefore, we investigated the extent of transcriptional regulation of staphylococcal genes by Rot. We compared the transcriptional profile of a rot agr double mutant to that of its agr parental strain by using custom-made Affymetrix GeneChips. Our findings indicate that Rot is not only a repressor but a global regulator with both positive and negative effects on the expression of S. aureus genes. Our data also indicate that Rot and agr have opposing effects on select target genes. These results provide further insight into the role of Rot in the regulatory cascade of S. aureus virulence gene expression.

Overexpression of Genes of the Cell Wall Stimulon in Clinical Isolates of Staphylococcus aureus Exhibiting Vancomycin-Intermediate- S. aureus-Type Resistance to Vancomycin

Custom-designed gene chips (Affymetrix) were used to determine genetic relatedness and gene expression profiles in Staphylococcus aureus isolates with increasing MICs of vancomycin that were recovered over a period of several weeks from the blood and heart valve of a patient undergoing extensive vancomycin therapy. The isolates were found to be isogenic as determined by the GeneChip based genotyping approach and thus represented a unique opportunity to study changes in gene expression that may contribute to the vancomycin resistance phenotype. No differences in gene expression were detected between the parent strain, JH1, and JH15, isolated from the nares of a patient contact. Few expression changes were observed between blood and heart valve isolates with identical vancomycin MICs. A large number of genes had altered expression in the late stage JH9 isolate (MIC = 8 microg/ml) compared to JH1 (MIC = 1 microg/ml). Most genes with altered expression were involved in housekeeping functions or cell wall biosynthesis and regulation. The sortase-encoding genes, srtA and srtB, as well as several surface protein-encoding genes were downregulated in JH9. Two hypothetical protein-encoding genes, SAS016 and SA2343, were dramatically overexpressed in JH9. Interestingly, 27 of the genes with altered expression in JH9 grown in drug-free medium were found to be also overexpressed when the parental strain JH1 was briefly exposed to inhibitory concentrations of vancomycin, and more than half (17 of 27) of the genes with altered expression belonged to determinants that were proposed to form part of a general cell wall stress stimulon (S. Utaida et al., Microbiology 149:2719-2732, 2003).

Penicillinase plasmids of Staphylococcus aureus: Restriction-deletion maps

Abstract The derivation of physical-genetic maps of two Staphylococcus aureus penicillinase plasmids—pI258 [28.2 kilobases (kb)] and pII147 (32.6 kb)—is described. The maps are based on data obtained by recombination, deletion, restriction endonuclease digestion, molecular cloning, and insertional inactivation. Evidence is presented for the existence of at least three separate operons transcribed in different directions. Data are presented to show that these plasmids are closely related physically as well as genetically to several others with which they can recombine. Physical mapping studies of one recombinant have helped to pinpoint structural differences between the two parental plasmids.

Transcription Profiling of the mgrA Regulon in Staphylococcus aureus

MgrA has been shown to affect multiple Staphylococcus aureus genes involved in virulence and antibiotic resistance. To comprehensively identify the target genes regulated by mgrA, we employed a microarray method to analyze the transcription profiles of S. aureus Newman, its isogeneic mgrA mutant, and an MgrA-overproducing derivative. We compared genes that were differentially expressed at exponential or early stationary growth phases. Our results showed that MgrA affected an impressive number of genes, 175 of which were positively regulated and 180 of which were negatively regulated in an mgrA-specific manner. The target genes included all functional categories. The microarray results were validated by real-time reverse transcription-PCR quantitation of a set of selected genes from different functional categories. Our data also indicate that mgrA regulates virulence factors in a fashion analogous to that of the accessory gene regulatory locus (agr). Accordingly, exoproteins are upregulated and surface proteins are downregulated by the regulator, suggesting that mgrA may function in concert with agr. The fact that a large number of genes are regulated by mgrA implies that MgrA is a major global regulator in S. aureus.

A novel MATE family efflux pump contributes to the reduced susceptibility of laboratory-derived Staphylococcus aureus mutants to tigecycline

ABSTRACT Tigecycline, an expanded-broad-spectrum glycylcycline antibiotic is not affected by the classical tetracycline resistance determinants found in Staphylococcus aureus. The in vitro selection of mutants with reduced susceptibility to tigecycline was evaluated for two methicillin-resistant S. aureus strains by serial passage in increasing concentrations of tigecycline. Both strains showed a stepwise elevation in tigecycline MIC over a period of 16 days, resulting in an increase in tigecycline MIC of 16- and 32-fold for N315 and Mu3, respectively. Transcriptional profiling revealed that both mutants exhibited over 100-fold increased expression of a gene cluster, mepRAB (multidrug export protein), encoding a MarR-like transcriptional regulator (mepR), a novel MATE family efflux pump (mepA), and a hypothetical protein of unknown function (mepB). Sequencing of the mepR gene in the mutant strains identified changes that presumably inactivated the MepR protein, which suggested that MepR functions as a repressor of mepA. Overexpression of mepA in a wild-type background caused a decrease in susceptibility to tigecycline and other substrates for MATE-type efflux pumps, although it was not sufficient to confer high-level resistance to tigecycline. Complementation of the mepR defect by overexpressing a wild-type mepR gene reduced mepA transcription and lowered the tigecycline MIC in the mutants. Transcription of tet(M) also increased by over 40-fold in the Mu3 mutant. This was attributed to a deletion in the promoter region of the gene that removed a stem-loop responsible for transcriptional attenuation. However, overexpression of the tet(M) transcript in a tigecycline-susceptible strain was not enough to significantly increase the MIC of tigecycline. These results suggest that the overexpression of mepA but not tet(M) may contribute to decreased susceptibility of tigecycline in S. aureus.

Effect of Mild Acid on Gene Expression in Staphylococcus aureus

During staphylococcal growth in glucose-supplemented medium, the pH of a culture starting near neutrality typically decreases by about 2 units due to the fermentation of glucose. Many species can comfortably tolerate the resulting mildly acidic conditions (pH, approximately 5.5) by mounting a cellular response, which serves to defend the intracellular pH and, in principle, to modify gene expression for optimal performance in a mildly acidic infection site. In this report, we show that changes in staphylococcal gene expression formerly thought to represent a glucose effect are largely the result of declining pH. We examine the cellular response to mild acid by microarray analysis and define the affected gene set as the mild acid stimulon. Many of the genes encoding extracellular virulence factors are affected, as are genes involved in regulation of virulence factor gene expression, transport of sugars and peptides, intermediary metabolism, and pH homeostasis. Key results are verified by gene fusion and Northern blot hybridization analyses. The results point to, but do not define, possible regulatory pathways by which the organism senses and responds to a pH stimulus.

Proteomic Analysis and Identification of Streptococcus pyogenes Surface-Associated Proteins

Streptococcus pyogenes is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of S. pyogenes. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in S. pyogenes. Several proteins were expressed in Escherichia coli, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzyme-linked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by S. pyogenes.