Outer membrane protein P4 is not required for virulence in the human challenge model of Haemophilus ducreyi infection

Diane M Janowicz 0 Beth W Zwickl 0 Kate R Fortney 2 Barry P Katz 0 1 Margaret E Bauer 2 0 Department of Medicine, Indiana University School of Medicine , 545 Barnhill Drive Room EH-435, Indianapolis, IN 46202 , USA 1 Department of Biostatistics, Indiana University School of Medicine , 635 Barnhill Drive Room MS-218, Indianapolis, IN 46202 , USA 2 Department of Microbiology and Immunology, Indiana University School of Medicine , 635 Barnhill Drive Room MS-218, Indianapolis, IN 46202 , USA Background: Bacterial lipoproteins often play important roles in pathogenesis and can stimulate protective immune responses. Such lipoproteins are viable vaccine candidates. Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, expresses a number of lipoproteins during human infection. One such lipoprotein, OmpP4, is homologous to the outer membrane lipoprotein e (P4) of H. influenzae. In H. influenzae, e (P4) stimulates production of bactericidal and protective antibodies and contributes to pathogenesis by facilitating acquisition of the essential nutrients heme and nicotinamide adenine dinucleotide (NAD). Here, we tested the hypothesis that, like its homolog, H. ducreyi OmpP4 contributes to virulence and stimulates production of bactericidal antibodies. Results: We determined that OmpP4 is broadly conserved among clinical isolates of H. ducreyi. We next constructed and characterized an isogenic ompP4 mutant, designated 35000HPompP4, in H. ducreyi strain 35000HP. To test whether OmpP4 was necessary for virulence in humans, eight healthy adults were experimentally infected. Each subject was inoculated with a fixed dose of 35000HP on one arm and three doses of 35000HPompP4 on the other arm. The overall parent and mutant pustule formation rates were 52.4% and 47.6%, respectively (P = 0.74). These results indicate that expression of OmpP4 in not necessary for H. ducreyi to initiate disease or progress to pustule formation in humans. Hyperimmune mouse serum raised against purified, recombinant OmpP4 did not promote bactericidal killing of 35000HP or phagocytosis by J774A.1 mouse macrophages in serum bactericidal and phagocytosis assays, respectively. Conclusions: Our data suggest that, unlike e (P4), H. ducreyi OmpP4 is not a suitable vaccine candidate. OmpP4 may be dispensable for virulence because of redundant mechanisms in H. ducreyi for heme acquisition and NAD utilization. - Background Bacterial genomes usually contain a significant portion of open reading frames (ORFs) that encode lipoproteins. For example, the genome of Neisseria meningitidis group B strain MC58 has 70 ORFs that encode surface-exposed or exported putative lipoproteins [1]. Approximately 8% of the ORFs of Borrelia burgdorferi encode putative lipoproteins [2]. The presence of numerous lipoproteins in bacterial genomes suggests their importance for bacterial survival and pathogenesis. Lipoproteins have been demonstrated to have roles in preserving membrane structure, functioning as enzymes, and serving as transporters or toxins. Lipoproteins also serve as immunogens; for example, the lipoprotein outer surface protein A (OspA), which plays important roles in B. burgdorferis biology, was used to develop an OspA-based vaccine [3,4]. Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has the capacity to express 67 putative lipoproteins (GenBank accession number AE017143), only four of which have been well characterized: the peptidoglycan associated lipoprotein (PAL), the fibrinogen binding protein (FgbA), the ducreyi lectin A (DltA), and H. ducreyi lipoprotein (Hlp) [5-7]. PAL is conserved among H. ducreyi strains and contains a surface-exposed epitope defined by the monoclonal antibody 3B9 [8]. An isogenic PAL mutant is unable to cause pustules in the human infection model [9]. FgbA and DltA also contribute to H. ducreyi virulence in humans [5,10]. The roles of other lipoproteins in H. ducreyi pathogenesis have not yet been delineated. In order to better understand the bacterial factors that contribute to the pathogenesis of H. ducreyi, an experimental human model of infection was developed [11,12]. In this model, adult volunteers are inoculated with H. ducreyi strain 35000HP, or its isogenic derivatives, on the skin overlying the upper deltoid. Within 24 h of inoculation, volunteers develop papules that either resolve or progress into pustules over the ensuing 2 weeks. Experimental infection mimics natural infection both clinically and histologically and has allowed identification of H. ducreyi genes that are expressed in vivo [13]. One of the genes identified as being expressed in multiple volunteers was HD1170. HD1170 encodes a putative lipoprotein, designated outer membrane protein P4 (OmpP4). OmpP4 is a homolog of the outer membrane lipoprotein e (P4) of H. influenzae. e (P4) is broadly conserved among typeable and nontypeable H. influenzae (NTHI) strains and is expressed as an abundant, immunodominant 28 kDa lipoprotein in outer membrane protein (OMP) fractions [14]. e (P4) was shown to play a role in virulence in an infant rat model of infection with H. influenzae type b [15]. Mechanistically, e (P4) is a phosphomonoesterase that facilitates the transport of two essential nutrients, heme and nicotinamide nucleotides, across the outer membrane of NTHI [16,17]. Monoclonal anti-e (P4) antibodies are highly reactive with a surface exposed epitope of e (P4), and anti-e (P4) serum is bactericidal against NTHI strains [14,18]. Immunization with e (P4) afforded protection against colonization in a mouse model of NTHI infection [19]. Thus, e (P4) is being actively investigated as a vaccine candidate against NTHI [18-20]. The predicted H. ducreyi OmpP4 shares 61% identity with e (P4), including conservation of the functional motifs required for enzymatic activity and for heme binding in e (P4) [21]. Because of its significant homology with e (P4) and its in vivo expression, we hypothesized that H. ducreyi OmpP4 may play an important role during human infection. Here, we found that ompP4 is conserved among clinical isolates of H. ducreyi. To investigate its role in virulence and its utility as a vaccine candidate for H. ducreyi, we constructed and tested an isogenic ompP4 mutant in H. ducreyi 35000HP for virulence in human volunteers. We also tested whether mouse serum elicited against H. ducreyi OmpP4 promoted complement-mediated bactericidal activity or phagocytic uptake. Results Identification of the ompP4 gene Analysis of the 35000HP genome identified an 831 bp open reading frame (ORF) that encoded an OmpP4 homologue. Sequence analysis of ompP4 demonstrated an N-terminal signal II peptide and a consensus lipidation sequence, N-VLSGC-C (Figure 1). Based on sorting signals described for Escherichia coli, the presence of a tyrosine at position 2 suggests that OmpP4 sorts to the outer membrane [22,23]. The ompP4 ORF lies within a putative op (...truncated)