Inactivation of DltA modulates virulence factor expression in Streptococcus pyogenes

Kathleen H. Cox, Eduardo Ruiz-Bustos, Harry Courtney, James Dale, Morgan A. Pence, Victor Nizet, Ramy K. Aziz, Ivan Gerling, Susan M. Price, David L. Hasty

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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 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/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 Dalanine 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.

Original languageEnglish (US)
Article numbere5366
JournalPloS one
Volume4
Issue number4
DOIs
StatePublished - Apr 29 2009

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Streptococcus pyogenes
Virulence Factors
Cell Wall
inactivation
virulence
Cells
Operon
mutants
Virulence
Bacteria
cell walls
Blood
operon
Genes
Antimicrobial Cationic Peptides
Regulon
complement
Proteins
Flow cytometry
Gene Regulatory Networks

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Cox, K. H., Ruiz-Bustos, E., Courtney, H., Dale, J., Pence, M. A., Nizet, V., ... Hasty, D. L. (2009). Inactivation of DltA modulates virulence factor expression in Streptococcus pyogenes. PloS one, 4(4), [e5366]. https://doi.org/10.1371/journal.pone.0005366

Inactivation of DltA modulates virulence factor expression in Streptococcus pyogenes. / Cox, Kathleen H.; Ruiz-Bustos, Eduardo; Courtney, Harry; Dale, James; Pence, Morgan A.; Nizet, Victor; Aziz, Ramy K.; Gerling, Ivan; Price, Susan M.; Hasty, David L.

In: PloS one, Vol. 4, No. 4, e5366, 29.04.2009.

Research output: Contribution to journalArticle

Cox, KH, Ruiz-Bustos, E, Courtney, H, Dale, J, Pence, MA, Nizet, V, Aziz, RK, Gerling, I, Price, SM & Hasty, DL 2009, 'Inactivation of DltA modulates virulence factor expression in Streptococcus pyogenes', PloS one, vol. 4, no. 4, e5366. https://doi.org/10.1371/journal.pone.0005366
Cox, Kathleen H. ; Ruiz-Bustos, Eduardo ; Courtney, Harry ; Dale, James ; Pence, Morgan A. ; Nizet, Victor ; Aziz, Ramy K. ; Gerling, Ivan ; Price, Susan M. ; Hasty, David L. / Inactivation of DltA modulates virulence factor expression in Streptococcus pyogenes. In: PloS one. 2009 ; Vol. 4, No. 4.
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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 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/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 Dalanine 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.",
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AU - Ruiz-Bustos, Eduardo

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AU - Dale, James

AU - Pence, Morgan A.

AU - Nizet, Victor

AU - Aziz, Ramy K.

AU - Gerling, Ivan

AU - Price, Susan M.

AU - Hasty, David L.

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