Mutations in the control of virulence sensor gene from Streptococcus pyogenes after infection in mice lead to clonal bacterial variants with altered gene regulatory activity and virulence.

The cluster of virulence sensor (CovS)/responder (CovR) two-component operon (CovRS) regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS) genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448), containing wild-type (WT) CovRS (5448/CovR+S+), or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS- was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection

PCR of gDNA.

<p>Primers specific for the (A) <i>sda1</i> gene and (B) <i>speA</i> gene were used for PCR analysis of genomic DNA. The GAS strains employed are indicated in the figure along with the <i>emm</i> serotype for each of these strains (in parenthesis). M refers to the molecular size marker.</p

Mutations found in <i>covS</i> gene of GAS strain 5448/<i>covS</i>⁺ during infection in mice.^a

a<p>Putative domains of CovS as defined elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100698#pone.0100698-Walker1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100698#pone.0100698-Tatsuno1" target="_blank">[11]</a>, where mutations occur: CovS: TMI (22–33); EC Loop (34–180); TMII (181–205); HAMP (208–266); HisKA (269–333); HATPase (386–492).</p>b<p>Tissue from which clones were obtained.</p>c<p>Nucleotide (NT) position of change, or range of change, based on position of the <i>covS</i> start codon.</p>d<p>Number of nucleotides of WT-<i>covS</i> replaced, inserted, or deleted.</p>e<p>Number of clones sequenced.</p>f<p>Effect on CovS protein.</p

GAS strain 5448 dissemination from skin to deep tissue.

<p>Analysis of bacterial loads in liver, spleen, and skin wound tissue. C57Bl/6[hPg(Tg)] mice were injected with a dose of ∼1–2×10⁸ CFU/100 µl of 5448 WT-5448/CovR⁺S⁺. At 3 days post-infection, organs were harvested and bacterial counts obtained from serial dilutions of homogenized tissues. Asterisks indicate statistical significance determined by unpaired two-tailed students t-test, P<0.001.</p

PCR of GAS 5448/covR⁺S⁺ inoculum prior to infection.

<p>Primers specific for regions of the <i>covS</i> gene that did not undergo mutations, but whose products would reveal the presence of a mutation, were used for PCR analysis. Genomic DNA isolated from overnight cultures of four different colonies of 5448/CovR⁺S⁺ was tested to determine if any mutations were present prior to injection. Mutant strains identified in this study were used as controls in lanes 2–5.</p

<i>Streptococcus pyogenes</i> strains used in this study.

<p><i>Streptococcus pyogenes</i> strains used in this study.</p

Mutations found in <i>covS</i> gene of GAS strain AP53/<i>covS⁺</i> during infection.

a<p>Nucleotide (NT) position of change based on position of <i>covS</i> start codon, ATG.</p>b<p>Number of nucleotides of WT-<i>covS</i> replaced, inserted, or deleted.</p>c<p>Effect on CovS protein.</p>d<p>1 and 2 were isolated from wounds in the same mouse.</p

DNase activity and transcript levels in WT-5448/CovR⁺S⁺ and mouse-passaged clones of 5448/CovR⁺S⁻ in culture supernatants.

<p>(A) Cultures of WT-5448/CovR⁺S⁺ and clonal mouse-passaged 5448/CovR⁺S⁻ isolates #12 (skin) and #14 (skin) were grown to mid-log phase (LP; A_600nm ∼0.6) and their supernatants monitored for DNase activity. Supernatants were incubated with 1 µg of calf thymus DNA for 5 min at 37°C. DNase activity was confirmed by visualizing DNA fragmentation by ethidium bromide staining and UV-light detection. THY in lane 5 represents the negative control. <i>B</i>, The effect of mouse-passaged GAS 5448 on <i>sda1</i> gene expression is shown using mRNA isolated from washed cells harvested at mid-log phase (LP; A_600nm ∼0.6) and early stationary phase (SP; A_600nm ∼1.2) growth. WT-5448/CovR⁺S⁺ and mouse-passaged isolates (5448/CovR⁺S⁻) #12 (skin) and #14 (skin) were employed. The relative % gene expression levels of the mouse-passaged strains are relative to those in WT-5448/CovR⁺S⁺.</p

Survival of hPg-transgenic mice after infection with mouse-passaged GAS strains.

<p>C57Bl/6[hPg(Tg)] male mice (6–10 weeks of age) containing the hPg transgene were injected subcutaneously with GAS cells and the mice were monitored for death as an end-point. (A) 5448/CovR⁺S⁺/M1⁺, and mouse-passaged 5448/CovR⁺S⁻/M1⁺ strains #12 (skin) and #14 (skin). A total of 1.2×10⁷ cells were injected. P<0.05 when comparing the wild-type strain to the two MP strains. (B) AP53/CovR⁺S⁻/M53⁺ (human passaged) and AP53/CovR⁺S⁻/M53⁺ clonal strain #4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100698#pone-0100698-t003" target="_blank">Table 3</a>) obtained after mouse passaging of AP53/CovR⁺S⁺/M53⁺. A dose of 1×10⁸ cells was injected. P<0.05 when comparing the covS⁺ strain to the human passaged or mouse passaged covS mutants. (C) Manfredo/CovR⁺S⁺/M5⁺ and Manfredo/CovR⁺S⁻/M5⁺ clonal strain #3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100698#pone-0100698-t004" target="_blank">Table 4</a>) obtained after mouse passaging of Manfredo/CovR⁺S⁺/M5⁺. A total of 1.4×10⁷ cells were injected. These two curves were not statistically different. N = 4–8 mice for each tested strain.</p

Mutations found in <i>covS</i> gene of GAS strain Manfredo M5/<i>covS⁺</i> during infection.

a<p>Nucleotide (NT) position of change based on position of <i>covS</i> start codon, ATG.</p>b<p>Number of nucleotides of WT-<i>covS</i> replaced, inserted, or deleted.</p>c<p>Effect on CovS protein.</p