Characterization of a WaaF (RfaF) Homolog Expressed by Haemophilus ducreyi

Abstract

Haemophilus ducreyi lipooligosaccharide (LOS) is capable of inducing an inflammatory response in skin (A. A. Campagnari, L. M. Wild, G. Griffiths, R. J. Karalus, M. A. Wirth, and S. M. Spinola, Infect. Immun. 59:2601–2608, 1991) and likely contributes to the virulence of this sexually transmitted pathogen (B. A. Bauer, M. K. Stevens, and E. J. Hansen, Infect. Immun. 68:4290–4298, 1998). An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that was 59% identical to the protein product of the rfaF(waaF) gene of Salmonella typhimurium. TheH. ducreyi waaF gene was able to complement an S. typhimurium rfaF (waaF) mutant, a result which confirmed the identity of this gene. In contrast to therfaF (waaF) gene of enteric bacteria, theH. ducreyi waaF gene was not located adjacent to other genes involved in lipopolysaccharide expression. Inactivation of theH. ducreyi waaF gene by insertion mutagenesis resulted in expression of a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LOS of this mutant also did not bind a monoclonal antibody directed against a cell surface-exposed epitope of wild-type H. ducreyi LOS. Testing of the wild-type H. ducreyi strain and its isogenicwaaF mutant in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi revealed that thiswaaF mutant was less virulent than the wild-type parent strain. Complementation of the H. ducreyi waaF mutant with the wild-type H. ducreyi waaF gene resulted in expression of both wild-type LOS and wild-type virulence by this mutant.

Expression of Sialylated or Paragloboside-Like Lipooligosaccharides Are Not Required for Pustule Formation by Haemophilus ducreyi in Human Volunteers

“…However, in the temperature-dependent rabbit model, a lbgB (losB) mutant was as virulent as the parent strain, a result similar to our findings in humans (24). In the rabbit model, a gmhA mutant and a waaF mutant were less virulent than their isogenic parent (3,4). Although their LOS structures were not determined, the gmhA mutant probably produces an LOS composed of lipid A and KDO, while the waaF mutant makes lipid A-KDO-heptose.…”

“…Although their LOS structures were not determined, the gmhA mutant probably produces an LOS composed of lipid A and KDO, while the waaF mutant makes lipid A-KDO-heptose. However, these truncated LOS mutants also exhibited changes in their outer membrane protein profiles relative to the parent, as has been reported for enteric deep rough lipopolysaccharide mutants (3,4).…”

Molecular characterization of a gene region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum: cloning, sequencing and expression of rfaf gene

“…A similar contiguous arrangement of the rfaC and rfaF genes was observed in Pseudomonas aeruginosa [17]. However, a recent paper on the characterization of rfaF homolog in Haemophilus ducreyi reported that rfaF gene was not located adjacent to other genes involved in LPS expression [18].…”

Invited review: Lipopolysaccharide biosynthesis: which steps do bacteria need to survive?

“…131 So far, all waaF genes cloned from various bacteria have been characterized by their ability to complement waaFdefective S. enterica strains as shown in silver-stained SDS-polyacrylamide gels. [132][133][134][135][136][137] WaaF-defective mutants were constructed and their Rd 2 -chemotype was confirmed either in SDS-gels, 133,135 in immunoblots with monoclonal antibodies against LPS or determined by mass spectrometry. 132,134,137 All mutants showed a fast migrating LPS in SDS-PAGE and an impaired virulence when tested in vivo.…”

“…132,134,137 All mutants showed a fast migrating LPS in SDS-PAGE and an impaired virulence when tested in vivo. 132,133,138 In P. aeruginosa it was not possible to generate waaC or waaF null mutants, indicating that P. aeruginosa is not viable with a deep-rough LPS. 136 The reason for this is unclear, but since it is known that the core region of P. aeruginosa is phosphorylated to an unusual extent, the lack of these negatively charged groups in deep-rough LPS could be responsible for this observation.…”