O-Polysaccharide of Actinobacillus actinomycetemcomitans (Aa) Affects Expression of Virulence Determinants

The ubiquitous localization of lipopolysaccharide (LPS) on the outer membrane (OM) of the bacterium implies an intimate association with membrane-associated secretion systems.  Some large autotransporters of Gram-negative bacilli translocate across the periplasm through a pole-secretion pathway.  Localization to the pole requires a complete LPS structure.  EmaA is a large putative autotransporter adhesin of Aa, orthologous to Yersinia virulence factor, YadA.  Leukotoxin of Aa is secreted via ABC transport machinery.  Objective: The purpose of this study is to determine if translocation of proteins through the membrane of Aa is affected by LPS, particularly the O-polysaccharide (O-PS).  Methods: O-PS mutants (rmlC^-, abcA^- and rfbH^-) and an emaA^- mutant of a serotype b strain were identified from a transposon mutant library.  The LPS was purified using hot phenol-water or a modified proteinase K-digestion method.  Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assays (ELISAs), using antibody against the whole cell extract of the wild-type strain, were performed to determine the changes to the O-PS in these mutants.  Real-time reverse transcriptional PCR (RT-RTPCR) was performed to quantitate the EmaA and leukotoxin expression of the mutants and the wild type at the transcriptional level.  Results: The O-PS mutants showed different growth characteristics and higher sensitivity to antibiotics, including ampicillin, kanamycin and chloramphenicol due to deficient permeability barrier of the OM.  The LPS profiles from SDS-PAGE and ELISAs clearly showed defects in O-PS of the mutants.  The RT-RTPCR showed down-regulation of the transcription of emaA in the rlmC^- mutant to the level of the emaA^- mutant.  In comparison, leukotoxin expression was up-regulated in the same mutants.  Conclusion: The deficiency in O-PS appears to regulate the transcription of emaA.  The intact expression of EmaA on the OM surface may require complete O-PS to stabilize and maintain the membrane architecture.  (Supported by NIH-NIDCR grant RO1-DE13824).