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

Abstract

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 g/ml) compared to JH1 (MIC ‫؍‬ 1 g/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). Staphylococcus aureus strains with decreased susceptibility to vancomycin (so-called vancomycin-intermediate S. aureus[VISA] strains) have been identified in clinical specimens in several countries during the past decade (10,11,25). Although several abnormal physiological properties have been described in VISA isolates, the mechanisms of resistance and whether or not it involves acquisition of genes has remained unclear. A major problem in mechanistic studies has been the lack of availability of isogenic vancomycin-susceptible strains that could be considered the parental strains of the VISA isolates. Even when a VISA isolate shared a common multilocus sequence type with a fully sequenced vancomycin-susceptible strain, the clinical origins and times of isolation were far apart, excluding the possibility of attributing genetic and phenotypic differences to the antibiotic susceptibility phenotype.A recently described series of methicillin-resistant S. aureus (MRSA) isolates (JH1 through JH15) were recovered from a single patient during extensive chemotherapy with vancomycin (24). Bacterial isolates recovered at various times during therapy showed increasing vancomycin MICs which in isolates JH9 and JH14 eventually reached 8 g/ml, a value typical of many clinical VISA isolates. The first isolate chronologically, JH1, and all of the subsequent isolates sha...

Diversity of Penicillin-binding Proteins

“…This is in agreement with the current knowledge that PBPs, besides their direct role in peptidoglycan synthesis, may interact with other proteins that could be involved in regulation of peptidoglycan biosynthesis or its degradation (68). Interestingly, fmtA expression has been reported to increase in the presence of different cell wall-active antibiotics (24,(32)(33)(34)(35). These observations support our proposal that FmtA may directly compensate for peptidoglycan biosynthesis and indirectly prevent cell wall degradation.…”

“…This proposal is in agreement with observations that inactivation of fmtA reduces methicillin resistance and peptidoglycan cross-linking (24). It is also consistent with the recent transcriptomics studies that have identified fmtA as an active participant of the S. aureus response to cell wall-active antibiotics (24,(32)(33)(34)(35).…”

“…Several reports have inferred that FmtA is involved with peptidoglycan biosynthesis (24,(32)(33)(34)(35), but the function of FmtA is not known. We show that FmtA ⌬27 is capable of binding to peptidoglycan in vitro with high binding affinity.…”

“…A few of these additional factors, notably the proteins encoded by the fem genes, have been characterized functionally (17,30,31). Among the identified methicillin resistance factors, fmtA in particular has been reported to be part of the cell wall stimulon (32)(33)(34)(35), which is a group of genes that is commonly induced upon treatment with cell wallactive antibiotics, such as ␤-lactams. Furthermore, inactivation of fmtA has been shown to affect peptidoglycan structure (24), suggesting that this gene is an active participant in peptidoglycan biosynthesis under antibiotic-induced cell wall stress conditions.…”

Gene expression profiling ofLeishmania (Leishmania) donovani: overcoming technical variation and exploiting biological variation

“…Gene expression profiling is becoming an important tool to study pathogenesis of infectious diseases as it allows molecular characterization of the cellular responses of host and pathogen (Chaussabel et al 2003 ;Hromatka et al 2005 ;McAleese et al 2006). This type of study has become feasible since the introduction of 2 high-throughput expression profiling techniques : (i) microarrays, used for comparative analysis of thousands of genes in distinct RNA populations (Schena et al 1995), and (ii) quantitative real-time PCR (Q-PCR), used for rapid simultaneous analysis of a specific set of genes in large sample collections (Heid et al 1996 ;Vandesompele et al 2002).…”