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. 2008 Sep 5;4(9):e1000145.
doi: 10.1371/journal.ppat.1000145.

V体育安卓版 - PerR confers phagocytic killing resistance and allows pharyngeal colonization by group A Streptococcus

Ioannis Gryllos  1 , Renata Grifantini, Annalisa Colaprico, Max E Cary, Anders Hakansson, David W Carey, Maria Suarez-Chavez, Leslie A Kalish, Paul D Mitchell, Gary L White, Michael R Wessels
Affiliations

PerR confers phagocytic killing resistance and allows pharyngeal colonization by group A Streptococcus

Ioannis Gryllos et al. PLoS Pathog. .

"V体育平台登录" Abstract

The peroxide response transcriptional regulator, PerR, is thought to contribute to virulence of group A Streptococcus (GAS); however, the specific mechanism through which it enhances adaptation for survival in the human host remains unknown. Here, we identify a critical role of PerR-regulated gene expression in GAS phagocytosis resistance and in virulence during pharyngeal infection VSports手机版. Deletion of perR in M-type 3 strain 003Sm was associated with reduced resistance to phagocytic killing in human blood and by murine macrophages in vitro. The increased phagocytic killing of the perR mutant was abrogated in the presence of the general oxidative burst inhibitor diphenyleneiodonium chloride (DPI), a result that suggests PerR-dependent gene expression counteracts the phagocyte oxidative burst. Moreover, an isogenic perR mutant was severely attenuated in a baboon model of GAS pharyngitis. In competitive infection experiments, the perR mutant was cleared from two animals at 24 h and from four of five animals by day 14, in sharp contrast to wild-type bacteria that persisted in the same five animals for 28 to 42 d. GAS genomic microarrays were used to compare wild-type and perR mutant transcriptomes in order to characterize the PerR regulon of GAS. These studies identified 42 PerR-dependent loci, the majority of which had not been previously recognized. Surprisingly, a large proportion of these loci are involved in sugar utilization and transport, in addition to oxidative stress adaptive responses and virulence. This finding suggests a novel role for PerR in mediating sugar uptake and utilization that, together with phagocytic killing resistance, may contribute to GAS fitness in the infected host. We conclude that PerR controls expression of a diverse regulon that enhances GAS resistance to phagocytic killing and allows adaptation for survival in the pharynx. .

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Mutagenesis of perR (spyM3_0147) in M-type 3 GAS strain 003Sm.
Chromosomal integration of the temperature-sensitive plasmid pJLperRΔ upstream or downstream of perR resulted in integrant strains 003Sm-Int-perRΔ and 003Sm-Int-WT that exhibited perR mutant and wild-type phenotype, respectively. In addition to the truncated perR copy, the deletion construct on pJLperRΔ encompassed the complete coding sequence of spyM3_0146 located upstream of perR and the 3′-end sequence of spyM3_0148 located downstream of perR in opposite orientation. The recombination event (X1) in perR mutant strain 003Sm-Int-perRΔ separated the wild-type perR copy, still present on the chromosome, from its native promoter (depicted with an arrow upstream of spyM3_0146) resulting in loss of PerR expression. In strain 003Sm-Int-WT, recombination in the 3′-end of spyM3_0148 (X2) did not separate perR from its natural promoter, thereby allowing PerR expression and wild-type phenotype. To derive the deletion mutant strain 003SmperRΔ, excision of the plasmid from the chromosome with simultaneous recombination downstream of perR was achieved during growth of integrant strain 003Sm-Int-perRΔ at 30°C.
Figure 2
Figure 2. Attenuated survival of the GAS perR mutant inside macrophages is rescued by inhibition of the macrophage oxidative burst.
Mouse RAW246.7 macrophages were infected for 1 h with wild-type strain 003Sm (black bars) or perR mutant strain 003SmperRΔ (white bars) and then treated with penicillin-gentamicin for an additional hour in the absence (untreated) or presence (DPI-treated, 10 µM) of the general respiratory burst inhibitor DPI. Following the 1 h treatment, intracellular GAS were recovered and enumerated by quantitative culture. Note that DPI treatment rescues attenuated survival of the perR mutant evident in the absence of the inhibitor. **p<0.04 for comparison of 003SmperRΔ versus 003Sm cfu (Student's t-test).
Figure 3
Figure 3. PerR expression in GAS integrant strains 003Sm-Int-perRΔ and 003Sm-Int-WT and the perR deletion mutant strain 003SmperRΔ as determined by immunoblotting.
Cell lysates of the three strains, as well as of parent strain 003Sm, were fractionated by SDS-PAGE and immunoblotted with PerR-specific rabbit antiserum. Note the loss of PerR in both 003SmperRΔand 003Sm-Int-perRΔ in contrast to wild-type PerR expression levels in 003Sm-Int-WT.
Figure 4
Figure 4. Competitive pharyngeal colonization in baboons following co-infection with wild-type GAS strain 003Sm and PerR mutant 003Sm-Int-perRΔ (top panel), or with 003Sm and PerR-expressing strain 003Sm-Int-WT (bottom panel).
Symbols represent competitive colonization indices (CCIs) calculated for each infected animal at each of eleven time points. Black lines indicate the median CCI values in the two groups of infected animals over time. CCI values are shown for samples that yielded at least one of two co-infecting strains. A CCI equal to 1, indicated by a broken horizontal line in both panels, represents identical counts for the two co-infecting strains.
Figure 5
Figure 5. Time-to-clearance of wild-type strain 003Sm versus PerR mutant strain 003Sm-Int-perRΔ (left panel) or 003Sm versus PerR-expressing strain 003Sm-Int-WT (right panel) in two groups of five co-infected baboons, respectively.
The proportion of animals that yielded positive throat cultures for each of the two co-infecting strains in each group is shown. In the left panel, note the rapid clearance of the perR mutant strain 003Sm-Int-perRΔ (closed triangles) versus the persistence of the wild-type parent strain (open squares) in the same animals. In the control group shown in the right panel, persistence of the PerR-expressing strain 003Sm-Int-WT (closed squares) was similar to that of the wild-type strain 003Sm (open squares).
Figure 6
Figure 6. Genes controlled by PerR in GAS strain 003Sm during growth in mid-exponential (A600nm = 0.25) or late-exponential (A600nm = 0.60) phase growth.
Values represent relative expression levels (fold-change) in wild-type bacteria compared to those in the perR mutant strain 003SmperRΔ. Numbers on y-axis indicate SPy or spyM3 ORF numbers corresponding to M-type 1 strain SF370 and M-type 3 GAS strain MGAS315 , respectively. *Genes regulated in similar fashion at both growth phases.
Figure 7
Figure 7. Genetic organization of four PerR-dependent predicted operons in strain 003Sm.
The ribonucleotide reductase system (A), the putative lactose/galactose PTSs SPy1709-SPy1711 (B) and SPy1923-SPy1916 (C) with their putative cognate regulators in opposite orientation, and the alkyl hydroperoxidase/alkyl hydroperoxide reductase AhpCF (D) loci are shown. The genes and ORF numbers for each locus in M-type 1 GAS strain SF370 are indicated. Numbers below the genes indicate relative expression levels (fold-change) in wild-type strain 003Sm compared to perR mutant strain 003SmperRΔ recorded in the microarray experiments.
Figure 8
Figure 8. PerR binds to pmtA (spyM3_1093 or SPy1434) and ahpC (spyM3_1770 or SPy2079) promoters (242 bp and 219 bp, respectively) encompassing canonical Per boxes.
Each of the two promoter fragments, or the negative control guaB promoter (246 bp), was incubated with recombinant his6-PerR at the concentrations indicated for 15 min at room temperature. Following analysis on native polyacrylamide gels, electrophoretic mobility shift of each promoter fragment was visualized by ethidium bromide staining.
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