Rapid Divergence of Two Classes of Haemophilus ducreyi

Abstract

Haemophilus ducreyi, the etiologic agent of chancroid, expresses variants of several key virulence factors. While previous reports suggested that H. ducreyi strains formed two clonal populations, the differences between, and diversity within, these populations were unclear. To assess their variability, we examined sequence diversity at 11 H. ducreyi loci, including virulence and housekeeping genes, augmenting published data sets with PCR-amplified genes to acquire data for at least 10 strains at each locus. While sequences from all 11 loci place strains into two distinct groups, there was very little variation within each group. The difference between alleles of the two groups was variable and large at 3 loci encoding surface-exposed proteins (0.4 < K S < 1.3, where K S is divergence at synonymous sites) but consistently small at genes encoding cytoplasmic or periplasmic proteins (K S < 0.09). The data suggest that the two classes have recently diverged, that recombination has introduced variant alleles into at least 3 distinct loci, and that these alleles have been confined to one of the two classes. In addition, recombination is evident among alleles within, but not between, classes. Rather than clones of the same species, these properties indicate that the two classes may form distinct species.Haemophilus ducreyi is a Gram-negative coccobacillus in the family Pasteurellaceae (2) and the causative agent of chancroid, a sexually transmitted, genital ulcer disease which facilitates transmission of 14,23,44). While H. ducreyi was placed in the polyphyletic genus Haemophilus due to its requirement for hemin, Haemophilus species do not form a cohesive group within the Pasteurellaceae (6, 20). The species that are most closely related to H. ducreyi include Actinobacter pleuropneumoniae and Nicoletella semolina (see Fig. S1 in the supplemental material) (16).Previous attempts to examine the diversity within the species H. ducreyi using immunotyping (36) and outer membrane profiling (32) showed very few differences among strains. Molecular analyses showed some differences, e.g., ribotyping of strains from two outbreaks yielded multiple restriction fragment length polymorphism (RFLP) patterns (19), but designations based on these differences have not been widely adopted or used for epidemiologic studies, in part due to the difficulty in culturing this fastidious organism. The lack of a standard typing system makes epidemiologic studies more difficult, and epidemiologic data for chancroid are therefore limited.One way to study diversity in H. ducreyi is to assess its population structure. Whereas the lack of recombination in some bacterial species generates stable clones (5), others recombine sufficiently rapidly to obscure clonal relationships (10, 11). While many Haemophilus species are naturally competent, H. ducreyi is considered nontransformable due to defects in the DNA uptake apparatus (16,34). This would likely decrease rates of recombination and generate stable clones. Initial studies of the clonal structure of...

Haemophilus ducreyi Cutaneous Ulcer Strains Diverged from Both Class I and Class II Genital Ulcer Strains: Implications for Epidemiological Studies

“…In the present study, we performed phylogenetic analyses of all the available CU and GU strains whose genomes have been released. We also examined the utility of a multilocus sequence typing system developed by Humphreys and coworkers to classify the strains [6, 15]. As we had done previously [7], we also analyzed the genomes of the recently described CU strains for the presence of acquired antimicrobial resistance genes and genes required for the virulence of 35000HP in experimentally infected human volunteers.…”

Immunization with the Haemophilus ducreyi trimeric autotransporter adhesin DsrA with alum, CpG or imiquimod generates a persistent humoral immune response that recognizes the bacterial surface

“…H. ducreyi strains are grouped in classes, termed class I and II, according to polymorphisms in genome sequences [42, 43] and variant outer membrane determinants [17, 42, 44, 45], including DsrA. Although the DsrA proteins from the two classes of H. ducreyi strains share high amino acid homology in their C-terminal translocator domain, they vary greatly in their functional N-terminal passenger domain [17].…”

The Haemophilus ducreyi Fis Protein Is Involved in Controlling Expression of thelspB-lspA2Operon and Other Virulence Factors

“…Two apparently clonal populations of H. ducreyi (class I and class II) have been described (67) and confirmed by both proteomic methods (68) and additional genetic analyses (69). Whether a fis mutant of a class II strain of H. ducreyi would have a transcriptome profile different from that of 35000HP⌬fis remains to be determined but would seem unlikely in view of the conservation of Fis effects among closely related bacteria (i.e., enteric organisms).…”