Fimbrial ghfGene Cluster of Genital Strains of Haemophilusspp

ABSTRACTWe analyzed the LKP fimbrial gene clusters of six piliated strains of a cryptic genospecies of Haemophilusisolated from the genital tracts of adult patients (five strains) and from an infected neonate. In a group of 19 genital strains, LKP-like genes have been found in only these 6 strains. In addition to the ghfA, ghfD, and ghfEgenes previously described, we characterized two genes, designated ghfBand ghfC, encoding the putative chaperone and assembly platform proteins. All six strains had a complete and unique LKP-like gene cluster consisting of the five genes ghfAto ghfE, homologous to genes hifAto hifEof Haemophilus influenzae. The sequences of the coding and intergenic regions of the ghfclusters of the six strains were remarkably homologous. Unlike hifclusters, which are inserted between purEand pepN, the ghfcluster was inserted between purKand pepNon the chromosome. Analysis of the flanking regions of the ghfcluster identified a large deletion, identical in the 5′ end regions of all strains, including the whole purEgene and much of the purKgene. Ultrastructural observations, an attempt at enriching LKP fimbriae, and hemagglutination experiments demonstrated that none of the strains had LKP-type fimbriae. Nevertheless, reverse transcription (RT)-PCR showed that ghfgenes were transcribed in four of the six strains. Sequencing of the intergenic ghfA-ghfBregions, including the ghfgene promoters, showed that the absence of transcripts in the remaining two strains was due to a decrease in the number of TA repeats (4 or 9 repeats rather than 10) between the −10 and −35 boxes of the two overlapping and divergent promoters. The other four strains, which had ghftranscripts, had the optimal 10 TA repeats (one strain) or 5 repeats associated with putative alternative −35 boxes (three strains). The absence of 10 repeated palindromic sequences of 44 or 45 nucleotides upstream of ghfBinduces an increased instability of mRNA, as quantified by real-time RT-PCR, and may explain why the LKP fimbrial gene cluster is not expressed in these strains.