For this study, we focused on whether the expression of the Flp proteins was necessary for virulence of H. ducreyi. We constructed an unmarked, in frame deletion mutant lacking the flp1flp2flp3 genes in 35000HP using a recombineering strategy [8, 9] and found that 35000HPΔflp1-3 was significantly impaired in its ability to cause disease in the human model of infection. flp1-3 joins hgbA, dsrA, ncaA, lspA1-lspA2, pal, tadA sapBC and cpxA as the ninth gene required for full virulence by H. ducreyi in the human inoculation experiments [9, 10] (unpublished observations).
The flp-tad gene cluster is constitutively transcribed as a single polycistronic operon in vitro . Relative to its expression during in vitro growth, tadA transcripts are enriched in experimental pustules, suggesting that the flp-tad operon is upregulated in vivo . CpxRA is the only obvious intact two-component signal transduction system contained in H. ducreyi. Transcription of flp1-3 and several other major virulence determinants are negatively regulated by conditions that favor phosphorylation of CpxR [9, 12, 13]. Purified recombinant CpxR interacts with the promoter regions of the flp operon in electrophoretic mobility shift assays . Deletion of cpxA leads to loss of CpxA phosphatase activity, activates CpxR, and cripples the ability of H. ducreyi to infect humans . In contrast, a cpxR deletion mutant has no effect on or upregulates the expression of virulence determinants and is fully virulent in human volunteers . Taken together, the data suggest that the flp-tad operon may be upregulated in vivo due to downregulation of CpxRA.
The human inoculation experiments are limited in that we are precluded by several regulatory bodies from testing trans-complemented mutants in humans. However, complementation of 35000HPΔflp1-3 in trans restored the ability of the mutant to form microcolonies and bind to HFF cells, suggesting that the phenotype of the mutant is due to the deletion of the flp genes. In the human inoculation experiments, we use 35000HP to examine the role of virulence factors in H. ducreyi pathogenesis. There are two classes of H. ducreyi strains, which express different immunotypes and proteomes [14, 15]. Although we were able to amplify flp1-3 alleles from six class I and three class II strains (data not shown), attempts to sequence the amplicons were unsuccessful, so we do not know if there is a difference in the flp genes in the class I and class II strains. 35000HP is a class I strain; whether the Flp proteins play a role in the virulence of class II strains is unknown.
We previously reported that a tadA mutant is attenuated for pustule formation in the human challenge model . However, the tadA mutant, but not a flp1flp2 double mutant, is attenuated in the rabbit model of chancroid [4, 5]. Nika et al previously reported that both the flp1flp2 mutant and the tadA mutant demonstrate decreased abilities to attach to HFF cells and form fewer microcolonies on HFF cells . These data suggested that microcolony formation by itself is not a virulence factor for H. ducreyi. Although H. ducreyi does not appear to co-localize with fibroblasts in experimental or natural chancroid [16, 17], our data indicate that adherence to HFF cells in vitro correlates with the virulence of H. ducreyi in humans. Similarly, both flp1 and tadA mutants fail to colonize or cause disease in a rat infection model with A. actinomycetemcomitans , and both flp1 mutant and tadD mutants of Pasteurella multocida are attenuated in the septicemic mouse model [18, 19].
In A. actinomycetemcomitans, Flp pili are assembled as bundles of long fibers in which Flp1 is the major structural component [3, 20]. However, there is no evidence that the Flp proteins are assembled into a pilus-like structure in H. ducreyi . Several bacterial species including A. actinomycetemcomitans have two flp genes . H. ducreyi contains three flp genes, which have between 50-80% similarity to one another . Deletion of flp1 and flp2 results in decreased adherence of H. ducreyi to HFF cells and subsequent microcolony formation ; the function of Flp3 is unclear.
In vitro, H. ducreyi forms microcolonies, a key step in biofilm formation. In vivo, H. ducreyi forms aggregates and colocalizes with macrophages, PMNs, collagen and fibrin [16, 17]. H. ducreyi contains a luxS homologue that has autoinducer (AI-2) activity in a Vibrio harveyi-based reporter system, and a luxS mutant is partially attenuated for virulence in human volunteers . Taken together, these data suggest that the formation of microcolonies, aggregates and quorum sensing mechanisms may be important for H. ducreyi pathogenesis. Whether the Flp proteins contribute to this process by mediating attachment to host cells or initiating microcolony formation in the skin remains a subject for future investigation.