Production of AI-2 by luxS
has been established for many bacterial species [31
]. While the involvement of the luxS
/AI-2 system in quorum-sensing remains to be proven for many of them, inactivation of luxS
results in altered virulence phenotypes in some pathogens [5
]. In the present study, we have shown (1) that H. ducreyi
contains a luxS
homolog, (2) that this bacterium is capable of producing an AI-2-like molecule, and (3) that the inactivation of luxS
results in modestly reduced infectivity in the human challenge model.
When tested in the V. harveyi
-based bioluminescence assay, cell-free culture supernatants from H. ducreyi
35000HP induced luminescence while 35000HP.303 had considerably less AI-2 activity, indicating that H. ducreyi
produces an AI-2-like molecule that is dependent on the presence of LuxS. Other Pasteurellaceae
family members have a functional LuxS/AI-2 regulatory system [8
]. When luxS
is inactivated in these bacteria, the virulence of the organisms is altered. An H. influenzae luxS
mutant has increased virulence in a chinchilla model of middle ear infection [37
] while A. pleuropneumoniae
and M. haemolytica
A1 mutants have reduced virulence in mouse [8
] and calf infection [9
] models, respectively. In the present study, we showed that inactivation of the luxS
gene resulted in a reduced pustule formation rate when compared to the parental strain, indicating that this mutant has a partially attenuated phenotype. This is the fourth report of a partially attenuated mutant in 35000HP.
The human model closely simulates the first two weeks of natural chancroid. Lesions are not allowed to progress beyond the pustular stage to ulcers, which are associated with lymphadenitis. In pustules, H. ducreyi
forms aggregates and co-localizes with collagen, fibrin, neutrophils and macrophages and is extracellular [13
]. It is possible that H. ducreyi
uses the production of AI-2 to alter its gene regulation profile and to aid in the initiation of infection. Since H. ducreyi
maintains similar relationships with host cells in experimental infection and naturally acquired chancroidal ulcers [39
], the expression of luxS
may also contribute to the ulcerative stage of disease.
We insertionally inactivated luxS gene by insertion of a cat cartridge. Complementation of the luxS mutant in trans partially restored AI-2 activity. The lower AI-2 activity obtained with the complemented luxS mutant () may be the result of the reduced extent of growth caused by the presence of kanamycin in the growth medium. We are precluded by biosafety considerations from testing complemented mutants in human volunteers. According to the human challenge protocols, we are only required to show, by Southern blot and PCR analyses, that the expected allelic exchange had occurred in the mutant. Subsequent to performing the human challenge trials, we used PCR to amplify the mutated luxS gene and some flanking DNA from the chromosome of the luxS mutant. Nucleotide sequence analysis showed two changes in the flanking ORFs, resulting in one amino acid substitution (S762N) in the HD0369 protein and one amino acid substitution (I28T) in the HD0371 protein.
We examined the role of LuxS in the virulence of 35000HP, a class I strain. There are two classes of H. ducreyi
strains, which express different variants of several outer membrane proteins and oligosaccharide components of LOS [40
]. Whether LuxS plays a role in the virulence of class II strains is unknown.
To our knowledge, this is the first report of a genital ulcer disease pathogen producing an AI-2-like molecule that likely plays a role in virulence. Further studies to determine (1) whether this autoinducer molecule is involved in gene regulation in H. ducreyi
, (2) the mechanism by which the luxS
/AI-2 system contributes to virulence of H. ducreyi
35000HP, and (3) whether the luxS
gene product plays a role in virulence of class II H. ducreyi
] are in progress.