Inactivation of DltA modulates virulence factor expression in Streptococcus pyogenes

Abstract

Background: D-alanylated lipoteichoic acid is a virtually ubiquitous component of gram-positive cell walls. Mutations in the dltABCD operon of numerous species exhibit pleiotropic effects, including reduced virulence, which has been attributed to increased binding of cationic antimicrobial peptides to the more negatively charged cell surface. In this study, we have further investigated the effects that mutating dltA has on virulence factor expression in Streptococcus pyogenes.

Methodology/principal findings: Isogenic Delta dltA mutants had previously been created in two distinct M1T1 isolates of S. pyogenes. Immunoblots, flow cytometry, and immunofluorescence were used to quantitate M protein levels in these strains, as well as to assess their ability to bind complement. Bacteria were tested for their ability to interact with human PMN and to grow in whole human blood. Message levels for emm, sic, and various regulatory elements were assessed by quantitative RT-PCR. Cell walls of Delta dltA mutants contained much less M protein than cell walls of parent strains and this correlated with reduced levels of emm transcripts, increased deposition of complement, increased association of bacteria with polymorphonuclear leukocytes, and reduced bacterial growth in whole human blood. Transcription of at least one other gene of the mga regulon, sic, which encodes a protein that inactivates antimicrobial peptides, was also dramatically reduced in Delta dltA mutants. Concomitantly, ccpA and rofA were unaffected, while rgg and arcA were up-regulated.

Conclusions/significance: This study has identified a novel mechanism for the reduced virulence of dltA mutants of Streptococcus pyogenes in which gene regulatory networks somehow sense and respond to the loss of DltA and lack of D-alanine esterification of lipoteichoic acid. The mechanism remains to be determined, but the data indicate that the status of D-alanine-lipoteichoic acid can significantly influence the expression of at least some streptococcal virulence factors and provide further impetus to targeting the dlt operon of gram-positive pathogens in the search for novel antimicrobial compounds.

Figure 1. Cell wall protein extracts of parent and ΔdltA mutants.

Bacteria were grown to mid-log phase and subjected either to lysozyme-mutanolysin digestion or hot acid extraction to obtain cell wall proteins. The material obtained was analyzed by SDS-PAGE followed by silver staining. Asterisks indicate positions where major M protein bands would migrate. Arrows indicate other proteins whose expression also appears to be altered in mutant compared to WT strains.

Figure 2. Immunoblot of cell wall extract with anti-M1 protein antibody.

Bacteria were grown to mid-log phase and their cell wall proteins were extracted with hot acid. The extracts were loaded exactly as in Fig. 1, which shows that total protein in each lane was virtually identical. After separating proteins by SDS-PAGE and blotting to nitrocellulose, the M protein containing bands were localized with an anti-M1 antibody.

Figure 3. Opsonophagocytosis test.

WT and ΔdltA mutant bacteria were grown to mid-log phase, washed and incubated in whole human blood for 10 or 40 minutes. Blood samples were placed on slides and stained. The percent of bacteria associated with neutrophils was determined by light microscope analysis.

Figure 4. Relative levels of C3 deposition on WT and ΔdltA mutant bacteria.

WT bacteria (open histogram) and ΔdltA mutants (filled histogram) were stained with a fluorescein-labeled antibody against human C3 and analyzed by flow cytometry.

Figure 5. Localization of C3 and M1 by immunofluorescence.

WT 8004 cells (Panels A and C) and 8004ΔdltA cells (Panels B and D) were stained either with human anti-C3 (Panels A and B) or anti-M1 (Panels C and D) antibodies. DAPI staining (blue) was used to visualize nuclei. Note that the fluorescence associated with M proteins (red) decreases in the dltA mutant and the fluorescence associated with C3 dramatically increases (green).

Figure 6. Comparing the relationship of complement deposition to M protein levels by flow cytometry.

WT cells, 5448 (panel A) and 8004 (panel C), and their isogenic mutants, 5448ΔdltA (panel B) and 8004ΔdltA (panel D), were labeled with anti-C3 (FITC-A) and anti-M1 (Cy5-A). Numbers are given representing the % of cells in each gated area.

Figure 7. Analysis of the amounts of M protein and SIC released into the medium.

Protein was precipitated from the supernatant of WT and dltA mutant bacteria grown to mid-log phase using TCA. A) Samples were separated by SDS-PAGE and blotted to nitrocellulose. Bands containing M1 protein were visualized using an anti-M1 antibody. B) Samples from 8004 and 8004ΔdltA were separated by 2-D gel electrophoresis. Only the portion of the gel surrounding SIC is shown. SIC was identified by mass spectrometry.