Contemporary Pharyngeal and Invasive <i>emm</i>1 and Invasive <i>emm</i>12 Group A <i>Streptococcus</i> Isolates Exhibit Similar <i>In Vivo</i> Selection for CovRS Mutants in Mice

<div><p>Group A <i>Streptococcus</i> (GAS) causes diverse infections ranging from common pharyngitis to rare severe invasive infections. Invasive GAS isolates can have natural mutations in the virulence regulator CovRS, which result in enhanced expression of multiple virulence genes, suppressed the expression of the protease SpeB, and increased virulence. It is believed that CovRS mutations arise during human infections with GAS carrying wild-type CovRS and are not transmissible. CovRS mutants of invasive GAS of the <i>emm</i>1 genotype arise readily during experimental infection in mice. It is possible that invasive GAS arises from pharyngeal GAS through rare genetic mutations that confer the capacity of mutated GAS to acquire <i>covRS</i> mutations during infection. The objective of this study was to determine whether contemporary pharyngeal <i>emm</i>1 GAS isolates have a reduced propensity to acquire CovRS mutations <i>in vivo</i> compared with invasive <i>emm</i>1 GAS and whether <i>emm</i>3, <i>emm</i>12, and <i>emm</i>28 GAS acquire CovRS mutants in mouse infection. The propensity of invasive and pharyngeal <i>emm</i>1 and invasive <i>emm</i>3, <i>emm</i>12, and <i>emm</i>28 SpeB^A+ isolates to acquire variants with the SpeB^A- phenotype was determined during subcutaneous infection of mice. The majority of both invasive and pharyngeal <i>emm</i>1 SpeB^A+ isolates and two of three <i>emm</i>12 isolates, but not <i>emm</i>3 and <i>emm</i>28 isolates, were found to acquire SpeB^A- variants during skin infection in mice. All analyzed SpeB^A- variants of <i>emm</i>1 and <i>emm</i>12 GAS from the mouse infection acquired <i>covRS</i> mutations and produced more platelet-activating factor acetylhydrolase SsE. Thus, contemporary invasive and pharyngeal <i>emm</i>1 GAS isolates and <i>emm</i>12 GAS have a similar capacity to acquire <i>covRS</i> mutations <i>in vivo</i>. The rarity of severe invasive infections caused by GAS does not appear to be attributable to a reduced ability of pharyngeal isolates to acquire CovRS mutations.</p></div

Selection of CovRS mutants of <i>emm</i>12 GAS in subcutaneous mouse infection.

<p>(A) The capacity of SpeB^A+ <i>emm</i>3, <i>emm</i>12, and <i>emm</i>28 invasive isolates to give rise to SpeB^A- variants in mice. Shown are SpeB^A−% among GAS bacteria recovered from skin infection sites of mice at day 4 after subcutaneous inoculation with ~10⁸ cfu of each of the indicated 6 <i>emm</i>3, 3 <i>emm</i>12, and 4 <i>emm</i>28 invasive isolates. (B) Levels of SsE platelet-activating factor acetylhydrolase activity in culture supernatant at the exponential growth phase of 6 SpeB^A- variants and their 2 <i>emm</i>12 SpeB^A+ parent strains listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162742#pone.0162742.t004" target="_blank">Table 4</a>.</p

Average percentages of SpeB^A- variants selected from invasive and pharyngeal <i>emm</i>1 SpeB^A+ isolates and P values in statistical analysis.

<p>Average percentages of SpeB^A- variants selected from invasive and pharyngeal <i>emm</i>1 SpeB^A+ isolates and P values in statistical analysis.</p

Distribution of <i>emm</i> genotypes and %SpeB^A- among the HMC pharyngeal isolates.

<p>Distribution of <i>emm</i> genotypes and %SpeB^A- among the HMC pharyngeal isolates.</p

Selection of SpeB^A- variants of SpeB^A+ invasive <i>emm</i>1 isolates in subcutaneous infection of mice.

<p>Five or 10 mice were subcutaneously inoculated with ~10⁸ cfu of each of the indicated 24 <i>emm</i>1 invasive isolates. Four days later, GAS bacteria were recovered from skin infection sites, and 48 colonies from each mouse were tested for SpeB activity in overnight culture supernatant by the casein hydrolysis assay. Presented are percentages of variants without detectable SpeB activity among the 48 colonies.</p

Selection of SpeB^A- variants of SpeB^A+ <i>emm</i>1 pharyngeal isolates in mice.

<p>Shown are SpeB^A−% among GAS bacteria recovered from skin infection sites of mice at day 4 after subcutaneous inoculation with ~10⁸ cfu of each of the indicated 8 <i>emm</i>1 pharyngeal isolates. The SpeB^A-% values were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162742#pone.0162742.g001" target="_blank">Fig 1</a> legend.</p

Evidence for CovRS mutations as the basis for the SpeB^A- phenotype of variants selected in subcutaneous infection of mice.

<p>(A) MGAS2221 and its <i>rocA</i> deletion mutant, but not its <i>covS</i> deletion mutant, had SpeB protease activity in overnight culture supernatant as determined by the casein hydrolysis assay. (B) Levels of SsE platelet-activating factor acetylhydrolase activity in culture supernatant at the exponential growth phase of 10 SpeB^A- variants and their 5 <i>emm</i>1 SpeB^A+ parent strains listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162742#pone.0162742.t004" target="_blank">Table 4</a>. The SsE activity was measured using the colorimetric assay as described in the Methods.</p