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Transcriptomic and functional analysis of an autolysis-deficient, teicoplanin-resistant derivative of methicillin-resistant Staphylococcus aureus.

ABSTRACT: The molecular basis of glycopeptide-intermediate S. aureus (GISA) isolates is not well defined though frequently involves phenotypes such as thickened cell walls and decreased autolysis. We have exploited an isogenic pair of teicoplanin-susceptible (strain MRGR3) and teicoplanin-resistant (strain 14-4) methicillin-resistant S. aureus strains for detailed transcriptomic profiling and analysis of altered autolytic properties. Strain 14-4 displayed markedly deficient Triton X-100-triggered autolysis compared to its teicoplanin-susceptible parent, although microarray analysis paradoxically did not reveal significant reductions in expression levels of major autolytic genes atl, lytM, and lytN, except for sle1, which showed a slight decrease. The most important paradox was a more-than-twofold increase in expression of the cidABC operon in 14-4 compared to MRGR3, which was correlated with decreased expression of autolysis negative regulators lytSR and lrgAB. In contrast, the autolysis-deficient phenotype of 14-4 was correlated with both increased expression of negative autolysis regulators (arlRS, mgrA, and sarA) and decreased expression of positive regulators (agr RNAII and RNAIII). Quantitative bacteriolytic assays and zymographic analysis of concentrated culture supernatants showed a striking reduction in Atl-derived, extracellular bacteriolytic hydrolase activities in 14-4 compared to MRGR3. This observed difference was independent of the source of cell wall substrate (MRGR3 or 14-4) used for analysis. Collectively, our results suggest that altered autolytic properties in 14-4 are apparently not driven by significant changes in the transcription of key autolytic effectors. Instead, our analysis points to alternate regulatory mechanisms that impact autolysis effectors which may include changes in posttranscriptional processing or export.

SUBMITTER: Renzoni A

PROVIDER: S-EPMC1563528 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

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Transcriptomic and functional analysis of an autolysis-deficient, teicoplanin-resistant derivative of methicillin-resistant Staphylococcus aureus.

Renzoni Adriana A Barras Christine C François Patrice P Charbonnier Yvan Y Huggler Elzbieta E Garzoni Christian C Kelley William L WL Majcherczyk Paul P Schrenzel Jacques J Lew Daniel P DP Vaudaux Pierre P

Antimicrobial agents and chemotherapy 20060901 9

The molecular basis of glycopeptide-intermediate S. aureus (GISA) isolates is not well defined though frequently involves phenotypes such as thickened cell walls and decreased autolysis. We have exploited an isogenic pair of teicoplanin-susceptible (strain MRGR3) and teicoplanin-resistant (strain 14-4) methicillin-resistant S. aureus strains for detailed transcriptomic profiling and analysis of altered autolytic properties. Strain 14-4 displayed markedly deficient Triton X-100-triggered autolysi ...[more]

PMID: 16940101

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Project description:Methicillin resistant Staphylococcus aureus (MRSA) infection is becoming refractory to existing antibiotic therapy owing to the inherent ability of S. aureus to develop rapid resistance and is considered a major threat to public health. We found that a natural isolate of Bacillus pumilus from the Columbia River Estuary produces a strong anti-MRSA compound, amicoumacin A. As amicoumacin A has been reported to exhibit anti-microbial, anti-inflammatory, and anti-ulcer activities, we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in the expression of genes involved in several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which has been shown to be induced in response to a collapse of membrane potential. In order to gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by serial passage method was carried out. Single point mutations resulting in codon substitutions were uncovered in several distinct genes: ksgA, RNA dimethyltranferase; fusA, elongation factor G; dnaG, primase, ; lacD, tagatose 1,6-bisphosphate aldolase, ; and SACOL0611, encoding a putative glycosyl transferase gene. Based on these results, a candidate approach was undertaken to recreate the same amino acid substitution individually in FusA and KsgA, each of which resulted in two-fold resistance towards amicoumacin A. The fusA gene is known as the site for fusidic acid- resistant mutations; however the codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance occur in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability.

Project description:BackgroundStaphylococcus aureus is a human pathogen responsible for substantial morbidity and mortality through its ability to cause a number of human infections including bacteremia, pneumonia and soft tissue infections. Of great concern is the emergence and dissemination of methicillin-resistant Staphylococcus aureus strains (MRSA) that are resistant to nearly all β-lactams. The emergence of the USA300 MRSA genetic background among community associated S. aureus infections (CA-MRSA) in the USA was followed by the disappearance of USA400 CA-MRSA isolates.ResultsTo gain a greater understanding of the potential fitness advantages and virulence capacity of S. aureus USA300 clones, we performed whole genome sequencing of 15 USA300 and 4 USA400 clinical isolates. A comparison of representative genomes of the USA300 and USA400 pulsotypes indicates a number of differences in mobile genome elements. We examined the in vitro gene expression profiles by microarray hybridization and the in vivo transcriptomes during lung infection in mice of a USA300 and a USA400 MRSA strain by performing complete genome qRT-PCR analysis. The unique presence and increased expression of 6 exotoxins in USA300 (12- to 600-fold) compared to USA400 may contribute to the increased virulence of USA300 clones. Importantly, we also observed the up-regulation of prophage genes in USA300 (compared with USA400) during mouse lung infection (including genes encoded by both prophages ΦSa2usa and ΦSa3usa), suggesting that these prophages may play an important role in vivo by contributing to the elevated virulence characteristic of the USA300 clone.ConclusionsWe observed differences in the genetic content of USA300 and USA400 strains, as well as significant differences of in vitro and in vivo gene expression of mobile elements in a lung pneumonia model. This is the first study to document the global transcription differences between USA300 and USA400 strains during both in vitro and in vivo growth.

Dataset Information

Transcriptomic and functional analysis of an autolysis-deficient, teicoplanin-resistant derivative of methicillin-resistant Staphylococcus aureus.

Publications

Transcriptomic and functional analysis of an autolysis-deficient, teicoplanin-resistant derivative of methicillin-resistant Staphylococcus aureus.

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