The RD2 Pathogenicity Island Modifies the Disease Potential of the Group A Streptococcus

Abstract

Serotype M28 isolates of the group A Streptococcus (GAS; Streptococcus pyogenes) are nonrandomly associated with cases of puerperal sepsis, a potentially life-threatening infection that can occur in women following childbirth. Previously, we discovered that the 36.3-kb RD2 pathogenicity island, which is present in serotype M28 isolates but lacking from most other isolates, promotes the ability of M28 GAS to colonize the female reproductive tract. Here, we performed a gain-of-function study in which we introduced RD2 into representative serotype M1, M49, and M59 isolates and assessed the phenotypic consequences of RD2 acquisition. All RD2-containing derivatives colonized a higher percentage of mice, and at higher CFU levels, than did the parental isolates in a mouse vaginal colonization model. However, for two additional phenotypes, survival in heparinized whole human blood and adherence to two human vaginal epithelial cell lines, there were serotype-specific differences from RD2 acquisition. Using transcriptomic comparisons, we identified that such differences may be a consequence of RD2 altering the abundance of transcripts from select core genome genes along serotype-specific lines. Our study is the first that interrogates RD2 function in GAS serotypes other than M28 isolates, shedding light on variability in the phenotypic consequences of RD2 acquisition and informing on why this mobile genetic element is not ubiquitous in the GAS population.

Keywords: Streptococcus pyogenes; mobile genetic elements; virulence.

FIG 1

Comparison of genes within RD2 of M28 GAS with genes present in GBS. Genes are represented by arrows and are color-coded based on putative open reading frame (ORF) function (blue, mobility; yellow, gene regulators; red, extracellular proteins; green, hypotheticals). Genes unique to GBS are colored white. Shading between GAS and GBS genes is based upon homology at the protein level. Percent positive amino acids were calculated by adding the percentage of identical and strongly similar amino acids together for each of the compared protein pairs. Note that the two diagonal lines on the GBS part of the figure signify that the two genes are not contiguous with the other genes in GBS but they are in the GAS. The black bar highlights the region of RD2 that was replaced with a spectinomycin resistance cassette in the RD2 derivative used in this study.

FIG 2

RD2 can be maintained in serotype M1, M49, and M59 GAS isolates. The presence of RD2 in strains M1::RD2, M49::RD2, and M59::RD2 was verified by PCR and Western blot approaches. (A) Schematic showing the locations of the 13 PCR products that were used to tile along RD2. (B) Results of the 13 RD2 PCRs for each of the three tested parental isolates (M1, M49, and M59) and their RD2-containing derivatives are shown. Note that the weak signal with PCR 11 is due to this region containing the r28 gene, which is difficult to amplify due to multiple repeat sequences. (C) Western blot analysis of cell wall-associated R28 expression in the six indicated GAS strains. Note the characteristic laddering pattern, which occurs due to the partial hydrolysis of acid‐labile Asp‐Pro sites within the repeat regions of R28 (47). The loading control was generated by Coomassie staining a gel that was identical to that used in the Western analysis.

FIG 3

Acquisition of RD2 leads to a serotype-specific enhancement in the ability of GAS to adhere to human epithelial cell lines. The six indicated strains were compared in tissue culture-based assays of adherence using VK2/E6E7 (A) and A431 (B) human cell lines. The experiment was performed on six occasions with each cell line, and mean values (± standard errors of the means) are shown. Statistical significance was determined via t test.

FIG 4

RD2 promotes the ability of serotype M1, M49, and M59 GAS isolates to colonize the murine vagina. Groups of 10 estradiol-treated mice (0.5 mg) were vaginally challenged with 1 × 10⁴ CFU of the indicated GAS strains. Shown is the mean number (± standard errors of the means) of CFU recovered from vaginal swabs over time for the two M1 isolates (A), the two M49 isolates (B), and the two M59 isolates (C). Repeated-measure analyses indicated that a statistically significantly higher number of CFU were recovered over time for each of the RD2-containing strains than their parental strains (the P values are shown on each graph). (D) Cumulative comparison (i.e., the 30 mice infected with a parental [M1, M49, or M59] GAS strain against the 30 mice infected with the RD2-containing derivatives) showing the percentage of mice that were colonized over time. Differences between the two data sets were tested for significance at individual time points (days 1, 3, 5, 7, and 10) by Fisher’s exact test (**, P < 0.01; *, P < 0.05; n.s., not significant).

FIG 5

In a serotype-specific manner, RD2 modifies the ability of GAS to survive and proliferate in human blood. The ability of the indicated strains to survive and replicate in human blood was tested. The data are shown as percentages of the number of CFU following 3 h of growth in blood relative to the inoculum (which is represented as the dashed line). The experiment was performed on six occasions, with mean values ± standard deviations being shown. Statistical significance was determined for pairs of isolates via t test.

FIG 6

RD2 variably modifies the transcriptomes of representative M1 and M49 GAS isolates. (A and B) Principal-component analysis (PCA) plots showing overall transcriptome variation between parental (pink/light green) and RD2-containing derivatives (red/dark green) of the M1 (A) and M49 (B) strains. PCA assesses the variance in a data set in terms of principal components. The percentages of the total variation that are accounted for by the 1st and 2nd principal components are shown on the x and y axis labels, respectively. Each oval represents one of the three triplicate samples grown and analyzed for each GAS strain. (C and D) Summary of RNA-seq data. Shown are pairwise comparisons of the transcriptomes between the parental and RD2-containing derivative M1 (C) and M49 (D) strains. The relative expression levels of genes present in the parental strains are graphed, with each gene represented by a circle. Select mRNAs of interest are colored and labeled. (E and F) TaqMan-based quantitative RT-PCR (qRT-PCR) data confirming the M1 (E) and M49 (F) RNA-seq data for the same set of select transcripts. Transcript abundances were determined from triplicate exponential-phase GAS cultures run in duplicate, with the mean (± standard deviation) fold change in transcript levels in the RD2-containing strains relative to their parental strains shown. Asterisks highlight data that are statistically significant (P < 0.05), as determined by t test.