Inactivation of DltA Modulates Virulence Factor Expression in Streptococcus pyogenes

Abstract

BackgroundD-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 FindingsIsogenic ΔdltA 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 ΔdltA 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 ΔdltA mutants. Concomitantly, ccpA and rofA were unaffected, while rgg and arcA were up-regulated.Conclusions/SignificanceThis 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.

d -Alanine Modification of a Protease-Susceptible Outer Membrane Component by the Bordetella pertussis dra Locus Promotes Resistance to Antimicrobial Peptides and Polymorphonuclear Leukocyte-Mediated Killing

“…1). In Gram-positive bacteria, the dltABCD loci catalyze the incorporation of D-alanine esters into teichoic acid (32)(33)(34)(35). Gram-negative bacteria lack teichoic acid.…”

Disease Manifestations and Pathogenic Mechanisms of Group A Streptococcus

“…The D-alanine esterification of LTA, coordinated by the dltABCD operon (263), increases the net positive surface charge, improving GAS resistance to LL-37, acidic pH, lysozyme, and neutrophil-mediated killing (264). Targeted mutagenesis of dltA reduces expression levels of the M protein and SIC, rendering the bacteria more susceptible to C3b complement deposition and killing in whole human blood (265).…”

The Transcriptional Regulator CpsY Is Important for Innate Immune Evasion in Streptococcus pyogenes

“…M protein is a known antiphagocytic factor of GAS (36)(37)(38) that is also involved in resistance to antimicrobial peptides (39). Likewise, modification of the cell wall is important for immune cell evasion in multiple streptococcal species (8,(40)(41)(42). Cell wall-modifying enzymes have been linked to the expression of antiphagocytic factors in GAS.…”

“…Cell wall-modifying enzymes have been linked to the expression of antiphagocytic factors in GAS. Eliminating D-alanine esterification of surface lipoteichoic acid resulted in reduced expression of emm transcripts in 5448 and a related covS mutant strain (8004) (41). The underlying mechanism was not described, but Cox et al speculated that unidentified regulatory pathways may be responsible.…”