Thesis (doctoral)--München, 1934.

Thesis (doctoral)--München, 1934.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Haemophilus ducreyi is a gram-negative and heme-dependent bactreia. H. ducreyi is the responsible of causing chancroid, a sexually transmitted infection forming genital ulcers. Infection with H. ducreyi is associated with an increased risk of acquiring HIV-1 as well as increasing the risk of the HIV-1 transmission. Heme acquisition in H. ducreyi occur through a receptor mediated process in which it start with binding of hemoglobin and heme to their cognate outer membrane receptors, HgbA and TdhA, respectively. The receptors are energized by the TonB complex. Following that the deposition of heme into the periplasmic area is unclear. Profiling of the periplasmic proteome of the H. ducreyi resulted in the identification of a periplasmic- binding protein that highly expressed in heme limitation conditions, and it has been called hHbp. This protein is encoded by a gene resides in a locus of four genes displaying genetic features of an ABC transporter. The gene cluster is organized as an operon comprising an internal membrane protein (IntPro), a sulphate reductase gamma subunit (dsvC), a heme dependant periplasmic bind-ing protein (hHBP), and an ATPase. The purified periplasmic binding protein, hHbp, bind heme in a dose-dependent and saturable manner. Moreover, the binding between heme and hHbp was specifically competitively inhibited by heme. The proposal planned to cre-ate an isogenic hhbp mutant by insertional inactivation using a kanamycin cassette, to genotypically and phenotypically characterize the mutant and thereby to confirm the cru-cial role of the hhbp gene in heme transport in H. ducreyi. Several attempts to ligate a kanamycin resistance cassette into hhbp to construct such a mutant were unsuccessful de-spite the systematic alteration of the ligation conditions and the use of kanamycin re-sistant genes derived from a variety of different plasmids. The explanations for this fail-ure are uncertain. In future work, two other approaches to construct an hhbp mutant in-clude the FRT-FLP recombinase technology and the use of overlapping extension PCR with a chloramphenicol cassette.

Indiana University-Purdue University Indianapolis (IUPUI) / Haemophilus ducreyi is the causative agent of the genital ulcer disease chancroid, which has been shown to facilitate the transmission of HIV. H. ducreyi is likely exposed to multiple sources of antimicrobial peptides in vivo. APs are small, cationic molecules with both bactericidal and immunomodulatory functions. Because H. ducreyi is able to establish and maintain an infection in an environment rich with antimicrobial peptides, we hypothesized that the bacterium was resistant to the bactericidal effects of these peptides. Using a 96-well AP bactericidal assay, we examined H. ducreyi susceptibility to eight human APs likely to be encountered at the site of infection, including the α-defensins human neutrophil peptide-1, human neutrophil peptide-2, human neutrophil peptide-3, and human defensin 5, the β-defensins human β defensin-2, human beta defensin-3, and human beta defensin-4, and the human cathelicidin, LL-37. H. ducreyi survival was compared to the survival of Escherichia coli ML35, a strain known to be susceptible to several antimicrobial peptides. H. ducreyi was significantly more resistant than E. coli ML35 to the bactericidal effects of all peptides tested. Furthermore, we found that representative class I and class II strains of H. ducreyi were each resistant to APs of each functional category, indicating that resistance to antimicrobial peptides could represent a conserved method of pathogenesis for H. ducreyi as a species. The H. ducreyi genome contains a homolog for the Sap influx transporter. To study the role of the H. ducreyi Sap transporter in AP resistance, we generated an isogenic sapA mutant and used the 96-well AP bactericidal assay to compare the AP susceptibility profiles of wild-type H. ducreyi, the sapA mutant and the sapA trans-complement to α-defensins, β-defensins, and LL-37. We observed a 25% decrease in the survival of the sapA mutant when it was exposed to LL-37. These findings suggest that the H. ducreyi Sap transporter plays a role in H. ducreyi resistance to LL-37, but it is likely that other AP resistance mechanisms co-exist within the bacterium.

Indiana University-Purdue University Indianapolis (IUPUI) / Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcerative disease that facilitates the transmission of HIV-1. H. ducreyi also causes non-sexually transmitted cutaneous ulcers in children in tropical regions. During human infection, H. ducreyi is subject to a variety of stresses. The stringent response is a bacterial stress response system induced by nutrient limiting conditions and mediated by guanosine tetra- and pentaphosphate [(p)ppGpp] and the transcriptional regulator DksA. (p)ppGpp and DksA jointly interact with RNA polymerase to regulate genes critical for bacterial survival. We hypothesized that the stringent response is required for H. ducreyi virulence in humans. A ΔrelAΔspoT mutant, which is unable to synthesize (p)ppGpp, was partially attenuated for abscess formation in human volunteers. Loss of (p)ppGpp increased bacterial resistance to phagocytosis and stationary phase survival; however, the mutant was more sensitive to oxidative stress. A ΔdksA mutant was also partially attenuated in humans. The ΔdksA mutant behaved like the (p)ppGpp mutant in stationary phase survival and sensitivity to oxidative stress, but exhibited decreased resistance to phagocytosis. Both mutants had decreased adherence to fibroblasts, but the mechanisms underlying the adherence defect were distinct. To better understand the roles of (p)ppGpp and DksA in regulating gene expression, we performed transcriptome analysis of the parent and mutant strains. (p)ppGpp and DksA deficiency resulted in dysregulation of multiple genes including several known virulence determinants. At stationary phase, (p)ppGpp and DksA targets were not identical but significantly overlapped; as the mutants were phenotypically distinct, this finding underscores both the unique and joint roles DksA and (p)ppGpp play in regulation of H. ducreyi virulence. We conclude that (p)ppGpp and DksA play significant roles in H. ducreyi pathogenesis. This is the first study to show that the stringent response has a direct role in the ability of a bacterial pathogen to cause disease in humans.