Genome-wide analysis of group a streptococci reveals a mutation that modulates global phenotype and disease specificity

Abstract

Many human pathogens produce phenotypic variants as a means to circumvent the host immune system and enhance survival and, as a potential consequence, exhibit increased virulence. For example, it has been known for almost 90 y that clinical isolates of the human bacterial pathogen group A streptococci (GAS) have extensive phenotypic heterogeneity linked to variation in virulence. However, the complete underlying molecular mechanism(s) have not been defined. Expression microarray analysis of nine clinical isolates identified two fundamentally different transcriptomes, designated pharyngeal transcriptome profile (PTP) and invasive transcriptome profile (ITP). PTP and ITP GAS differed in approximately 10% of the transcriptome, including at least 23 proven or putative virulence factor genes. ITP organisms were recovered from skin lesions of mice infected subcutaneously with PTP GAS and were significantly more able to survive phagocytosis and killing by human polymorphonuclear leukocytes. Complete genome resequencing of a mouse-derived ITP GAS revealed that the organism differed from its precursor by only a 7-bp frameshift mutation in the gene (covS) encoding the sensor kinase component of a two-component signal transduction system implicated in virulence. Genetic complementation, and sequence analysis of covR/S in 42 GAS isolates confirmed the central role of covR/S in transcriptome, exoproteome, and virulence modulation. Genome-wide analysis provides a heretofore unattained understanding of phenotypic variation and disease specificity in microbial pathogens, resulting in new avenues for vaccine and therapeutics research.

Figure 1. Differential Gene Expression of ITP and PTP GAS

(A) Principal component analysis plot showing transcriptome differences between invasive (red) and pharyngeal (blue) isolates. Principal component analysis assesses the variance in a dataset in terms of principal components. The two most significant principal components are displayed on the x- and y-axes. Ellipses (calculated using 2X standard deviation for each gene per group) have been superimposed to highlight group differences between ITP and PTP strains. Numbered data points refer to the MGAS strain number from which the RNA sample was isolated. The percentages of the total variation that are accounted for by the 1st and 2nd principal components are shown on the x- and y-axes labels. (B) Log₁₀-fold transcript differences between the three ITP and six PTP isolates shown in (A) for select virulence genes and virulence gene regulators. Genes that are expressed higher in ITP isolates are shown in red; those expressed higher in PTP isolates are shown in blue. All genes shown are statistically significant (t-test followed by a false discovery rate correction, Q < 0.05).

Figure 2. Mouse Passage Results in Recovery of ITP Derivatives from Infecting PTP GAS

(A) Western immunoblots of supernatant proteins obtained from overnight cultures of ITP and PTP GAS isolates, before and after mouse passage. The amount of immunoreactive SpeA, SpeB, SLO/SPN, Spd3, and Mac was unaffected by mouse passage of the ITP strain MGAS5005, whereas they were affected by all spleen and some lesion-isolated GAS after mouse passage of the PTP GAS isolate MGAS2221. Strains with a mucoid colony morphology are indicated with an asterisk (*), while nonmucoid strains are indicated by a hash (#). (B) Principal component analysis plot portraying PTP to ITP transition for several mouse-passaged derivatives of strain MGAS2221. The 2221 NM (green) and 2221 M (purple) data points are mouse-passaged derivatives of strain MGAS2221 with distinct nonmucoid and mucoid colony morphologies, respectively. The 5005 M (orange) data points are mouse-passaged derivatives of strain MGAS5005 that retain the mucoid colony morphology of the parental strain. Ellipses (calculated using 2X standard deviation for each gene per group) have been superimposed to highlight differences between ITP and PTP GAS. The percentages of the total variation that are accounted for by the 1st and 2nd principal components are shown on the x- and y-axes labels. (C) Log₁₀-fold differences in transcripts for select virulence genes and virulence gene regulators between all the PTP and ITP strains shown in (B). Genes expressed higher in ITP derivatives are shown in red; those expressed higher in PTP derivatives are shown in blue. RNA was isolated from bacteria grown to the exponential phase of growth in THY media. All genes shown are statistically significant as assessed by a t-test followed by a false discovery rate correction of Q < 0.05.

Figure 3. Differential Virulence of ITP and PTP GAS in Mouse Models of Invasive Disease

(A) ITP GAS are significantly more virulent than PTP GAS in a mouse model of bacteremia (p < 0.0001, logrank test). Female CD-1 mice were injected intraperitoneally with 2.5 × 10⁷ CFU of GAS and lethality was monitored. Four ITP and four PTP strains were each used to infect 20 mice. Survival curves were generated by pooling data from mice infected with GAS strains of the same transcriptome profile. (B) PTP GAS produce significantly larger lesion volumes than ITP GAS in a mouse soft-tissue infection model (p < 0.01, mixed-model repeated-measures analysis). Female Crl:SKH1-hrBR mice were injected subcutaneously with 1 × 10⁷ CFU of GAS and monitored for skin lesion formation. Shown are the average lesion volumes (± standard error of the mean) of mice pooled together based on the transcriptome profile of the infecting strain. Fifteen mice were infected per GAS strain, with four ITP and four PTP strains being used.

Figure 4. ITP and PTP GAS Show Differential Resistance to PMN-Mediated Killing

Killing of ITP GAS strains MGAS5005 and 26PL1 by human PMNs occurred at significantly lower levels than the PTP GAS strains MGAS2221 and 21PS1 (p < 0.001, analysis of variance with Bonferroni's posttest for multiple comparisons). Results shown are the mean ± standard error of the mean of four experiments.

Figure 5. The ITP of MGAS5005 Is Due to a Variant covS Allele

Strain MGAS5005 was transformed with a derivative of plasmid pDC123 encoding a functional covS allele (pCovComp), and the supernatants from overnight cultures were analyzed by Western immunoblot for SpeA, SpeB, SLO/SPN, Mac, and Spd3 reactivity. Strain MGAS5005 transformed with vector pDC123 retained the pattern of protein secretion identified for MGAS5005 alone. In contrast, strain MGAS5005 transformed with pCovComp had an altered exoprotein secretion pattern, which was identical to that observed for PTP GAS (e.g., strain MGAS2221). Curing plasmid pCovComp from complemented MGAS5005 returned the secretion pattern to an ITP form.