Virulence of Burkholderia mallei Quorum-Sensing Mutants

Abstract

bMany Proteobacteria use acyl-homoserine lactone-mediated quorum-sensing (QS) to activate specific sets of genes as a function of cell density. QS often controls the virulence of pathogenic species, and in fact a previous study indicated that QS was important for Burkholderia mallei mouse lung infections. To gain in-depth information on the role of QS in B. mallei virulence, we constructed and characterized a mutant of B. mallei strain GB8 that was unable to make acyl-homoserine lactones. The QS mutant showed virulence equal to that of its wild-type parent in an aerosol mouse infection model, and growth in macrophages was indistinguishable from that of the parent strain. Furthermore, we assessed the role of QS in B. mallei ATCC 23344 by constructing and characterizing a mutant strain producing AiiA, a lactonase enzyme that degrades acyl-homoserine lactones. Although acyl-homoserine lactone levels in cultures of this strain are very low, it showed full virulence. Contrary to the previous report, we conclude that QS is not required for acute B. mallei infections of mice. QS may be involved in some stage of chronic infections in the natural host of horses, or the QS genes may be remnants of the QS network in B. pseudomallei from which this host-adapted pathogen evolved.

Global Analysis of the Burkholderia thailandensis Quorum Sensing-Controlled Regulon

“…A global survey of QScontrolled factors in these pathogens has not yet been done despite the link between QS and virulence in both species (15,(63)(64)(65)(66). Conserved elements may provide insights about the role of QS during infections and how QS can be adapted from a system providing benefit in a saprophyte to one providing benefit in a pathogen.…”

“…We measured the AHLs in appropriate HPLC fractions by using bioassays as described elsewhere (16). Synthetic C 8 -L-HSL was purchased from Sigma Chemical Co. 3OHC 10 -L-HSL was purchased from the School of Molecular Medical Sciences at the University of Nottingham (http://www.nottingham.ac.uk /quorum/compounds.htm), and synthetic 3OHC 8 -L-HSL was synthesized as previously described (15).…”

“…To measure AHLs in B. thailandensis cultures, we twice extracted 5 ml of a culture grown to an OD 600 of 4.0 with acidified ethyl acetate. The analysis was performed as described elsewhere (15), except that high-performance liquid chromatography (HPLC) fractions containing 3OHC 10 -HSL were also collected. We measured the AHLs in appropriate HPLC fractions by using bioassays as described elsewhere (16).…”

Cross-Species Comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei Quorum-Sensing Regulons

“…For B. pseudomallei, 1.6 mM adenine sulfate and 0.005% thiamine-HCl were added to the growth medium. Antibiotics were used at the following concentrations: for Escherichia coli, 35 g/ml kanamycin (Kan) and 25 g/ml zeocin (Zeo); for B. pseudomallei, 2 mg/ml Zeo and 1 mg/ml Kan. Where stated, synthetic 3OHC 10 -HSL (4 M; University of Nottingham [http://www .nottingham.ac.uk/quorum/compounds.htm]), 3OHC 8 -HSL (2 M; synthesized as previously described [31]) and C 8 -HSL (2 M; Sigma Chemical Co.) were added. Except where indicated, bacteria were grown at 37°C with shaking.…”

“…These studies imply a requirement for QS in melioidosis, yet it is not known which QS-controlled factor or factors are used in the host. A current hypothesis is that QS regulates the acute-to-chronic disease shift in B. mallei (31). As is the case for B. pseudomallei, the QS-controlled factors utilized by B. mallei in the host are unknown.…”