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, 93 (4), 664-81

Evolution of Hypervirulence by a MRSA Clone Through Acquisition of a Transposable Element

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Evolution of Hypervirulence by a MRSA Clone Through Acquisition of a Transposable Element

Meredith A Benson et al. Mol Microbiol.

Abstract

Staphylococcus aureus has evolved as a pathogen that causes a range of diseases in humans. There are two dominant modes of evolution thought to explain most of the virulence differences between strains. First, virulence genes may be acquired from other organisms. Second, mutations may cause changes in the regulation and expression of genes. Here we describe an evolutionary event in which transposition of an IS element has a direct impact on virulence gene regulation resulting in hypervirulence. Whole-genome analysis of a methicillin-resistant S. aureus (MRSA) strain USA500 revealed acquisition of a transposable element (IS256) that is absent from close relatives of this strain. Of the multiple copies of IS256 found in the USA500 genome, one was inserted in the promoter sequence of repressor of toxins (Rot), a master transcriptional regulator responsible for the expression of virulence factors in S. aureus. We show that insertion into the rot promoter by IS256 results in the derepression of cytotoxin expression and increased virulence. Taken together, this work provides new insight into evolutionary strategies by which S. aureus is able to modify its virulence properties and demonstrates a novel mechanism by which horizontal gene transfer directly impacts virulence through altering toxin regulation.

Figures

Fig. 1
Fig. 1. Genome sequence analysis of USA500 strain 2395
(A) Circular map of the genome of USA500 strain 2395 (Red) and USA300 strain FPR3757 (Black) compared to that of USA300 strain TCH1516. Width of red and black band is proportional to percent nucleotide identity. Numbered lines show locations of IS256 elements. Green arcs denote key areas of difference between strains. (B) Circular map of pUSA500. Key elements of the plasmid are denoted. (C) Phylogram and virulence gene distribution in close relatives of USA500. Tree is based on single nucleotide polymorphisms (SNPs) determined by whole genome analysis, branch length is proportional to inferred substitutions per site. Numbers on branches are bootstrap percentages showing support for each node. Heat map of gene presence/absence shows proportion of nucleotide identity over the full-length gene using either the gene from FPR3757 genome (Query USA300) or the 2395 genome (Query USA500).
Fig. 2
Fig. 2. IS256 alters the production of virulence factors in USA500 clinical isolates
(A) USA300, USA500, and Newman wild type strains were transformed with reporter plasmid pDB59 containing the rnaIII promoter driving expression of yfp. YFP fluorescence was measured at indicated time points. Values represent the average of three independent experiments ± S.D. (B) USA500 clinical isolates were transformed with pDB59. YFP fluorescence was measured after 10 hours of growth and normalized to the OD600. Values represent the average of two independent experiments each performed with two colonies of each strain ± S.D. (C) Time course comparing expression of PrnaIII-yfp in IS256 + versus IS256 − USA500 clinical isolates. Values represent the average of two independent experiments each performed with two colonies of each strain ± S.E.M. (D) Exoprotein comparison of supernatants from USA500 clinical isolates grown to stationary phase. Total exoprotein profiles were examined by coomassie blue staining (CB). LukA and LukD abundance was analyzed by immunoblot (IB), with specific antibodies. (E and F) Cytotoxicity profile of USA500 clinical isolates. Primary human neutrophils from three independent donors were assayed using culture filtrates from two independent colonies of each strain. (A, C, and F). Asterisk (*) denotes statistical significance determined by Student’s t test (P≤0.05).
Fig. 3
Fig. 3. Influence of IS256 insertion on the rot promoter
(A) PCR amplification of the rot promoter from strain Newman, USA300, and USA500 and schematic of the rot promoter locus in USA500. (B) USA500 was transformed with reporter plasmids containing the rot promoter of Newman/USA300 or USA500 controlling gfp expression, and GFP fluorescence was monitored at the indicated time points. (C) Transcript levels of rot from USA300 and USA500 as measured by qRT-PCR. (D) Immunoblot analysis of Rot levels in Newman, USA300 and USA500 grown to early stationary phase. Bottom graph represents quantification of immunoblots. (B–D) Values represent the average of three independent experiments ± S.D. Asterisk (*) denotes statistical significance determined by Student’s t test (P≤0.05).
Fig. 4
Fig. 4. Rot directly regulates the expression of cytotoxins
(A) USA300 wildtype and an isogenic rot mutant were transformed with reporter plasmids containing the hlgCB, lukED, hla, and lukAB promoters driving gfp expression. GFP fluorescence was monitored at the indicated time points. (B) EMSA analysis of Rot binding to cytotoxin promoters. 40 fmol of biotinylated promoter DNA was incubated with Rot alone, or in the presence of 30x molar excess of non-biotinylated probe DNA, or non-specific DNA. Arrow indicates non-shifted DNA, bracket indicates shifted DNA. (C) Transcript abundance of hlgC and lukE in USA300 and USA500 as measured by qRT-PCR. (D) Protein abundance of HlgC and LukD in USA300 and USA500. (A, C–D) Values represent the average of three independent experiments ± S.D. Asterisk (*) denotes statistical significance determined by Student’s t test (P≤0.05).
Fig. 5
Fig. 5. The effect of IS256 on cytotoxin production in USA500
(A) PCR amplification of the rot promoter from wildtype USA500, the isogenic ΔIS256 mutant, USA300 and Newman. (B) rot transcript abundance in USA500 and the isogenic ΔIS256 mutant as measured by qRT-PCR. (C) Immunoblot analysis of Rot in strains from Panel A grown to early stationary phase. (D) lukA, hla, lukE, hlgC, hlgA, and spa transcript levels in the strains from Panel A as measured by qRT-PCR. (E) Immunoblot analysis of cytotoxin in stationary phase supernatants collected from strains used in Panel A. (B–E) Values represent the average of three independent experiments ± S.D. Asterisk (*) denotes statistical significance determined by Student’s t test (P≤0.05).
Fig. 6
Fig. 6. The contribution of altered rot expression to USA500 virulence
(A) Log10CFU of bacteria recovered from the spleen of mice retro-orbitally infected with 1x107 CFU of wildtype USA500 and the isogenic ΔIS256 mutant. Each symbol represents an individual mouse (n=20). Asterisk (*) denotes statistical significance determined by Student’s t test (P=0.0335). (B) Percent recovered bacteria of indicated strains after 3hr infection of primary human neutrophils compared to input. (C) Increasing amounts of stationary phase culture filtrates from indicated strains were incubated with sheep red blood cells for 45 minutes and lysis determined by measuring Ab 405. (B–C) Values represent the average of three independent experiments ± S.D. Asterisk (*) denotes statistical significance determined by Student’s t test (P≤0.05).

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