Naturally occurring single amino acid replacements in a regulatory protein alter streptococcal gene expression and virulence in mice
Ronan K. Carroll, … , Anthony R. Flores, James M. Musser
Ronan K. Carroll, … , Anthony R. Flores, James M. Musser
Published April 1, 2011
Citation Information: J Clin Invest. 2011;121(5):1956-1968. https://doi.org/10.1172/JCI45169.
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Naturally occurring single amino acid replacements in a regulatory protein alter streptococcal gene expression and virulence in mice

Abstract

Infection with different strains of the same species of bacteria often results in vastly different clinical outcomes. Despite extensive investigation, the genetic basis of microbial strain-specific virulence remains poorly understood. Recent whole-genome sequencing has revealed that SNPs are the most prevalent form of genetic diversity among different strains of the same species of bacteria. For invasive serotype M3 group A streptococci (GAS) strains, the gene encoding regulator of proteinase B (RopB) has the highest frequency of SNPs. Here, we have determined that ropB polymorphisms alter RopB function and modulate GAS host-pathogen interactions. Sequencing of ropB in 171 invasive serotype M3 GAS strains identified 19 distinct ropB alleles. Inactivation of the ropB gene in strains producing distinct RopB variants had dramatically divergent effects on GAS global gene expression. Additionally, generation of isoallelic GAS strains differing only by a single amino acid in RopB confirmed that variant proteins affected transcript levels of the gene encoding streptococcal proteinase B, a major RopB-regulated virulence factor. Comparison of parental, RopB-inactivated, and RopB isoallelic strains in mouse infection models demonstrated that ropB polymorphisms influence GAS virulence and disease manifestations. These data detail a paradigm in which unbiased, whole-genome sequence analysis of populations of clinical bacterial isolates creates new avenues of productive investigation into the pathogenesis of common human infections.

Authors

Ronan K. Carroll, Samuel A. Shelburne III, Randall J. Olsen, Bryce Suber, Pranoti Sahasrabhojane, Muthiah Kumaraswami, Stephen B. Beres, Patrick R. Shea, Anthony R. Flores, James M. Musser

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Figure 7

RopB single-amino-acid changes influence group A streptococcal virulence and gene transcript levels during infection.

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RopB single-amino-acid changes influence group A streptococcal virulence...
(A) Twenty outbred CD-1 mice were infected intraperitoneally with 1.0 × 107 CFUs of each indicated strain. Data shown are survival over time, with P values derived from Kaplan-Meier survival analysis. (B) Fifteen immunocompetent hairless mice were infected subcutaneously with 1.0 × 107 CFUs of each indicated strain. Lesion area was recorded daily and data graphed as mean ± SEM. P value refers to comparison of lesion size over time, performed using repeated measures analysis followed by Bonferroni’s adjustment for multiple comparisons. (C) Histologic analysis of lesions (original magnification, ×4) collected on day 9 following infection. Arrows indicate areas of ulceration and necrosis caused by the SpeB+ strains. Asterisks indicate focal abscess formation beneath an intact epithelium in mice infected with the SpeB– strains. (D) Bacterial density in skin/soft tissue over time for indicated strains. P value refers to repeated measures analysis of all strains. (E) In vivo gene expression of select group A streptococcal virulence factors was determined as described in Methods. For D and E, colors refer to strain inset, and data are graphed as mean ± SD of 3 biologic replicates. *P < 0.05 compared with parental strain MGAS10870. Gene names are referenced in the text.