Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems.

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900-3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

Identification of the znuA-encoded periplasmic zinc transport protein of Haemophilus ducreyi.

The znuA gene of Haemophilus ducreyi encodes a 32-kDa (mature) protein that has homology to both the ZnuA protein of Escherichia coli and the Pzp1 protein of H. influenzae; both of these latter proteins are members of a growing family of prokaryotic zinc transporters. Inactivation of the H. ducreyi 35000 znuA gene by insertional mutagenesis resulted in a mutant that grew more slowly than the wild-type parent strain in vitro unless ZnCl(2) was provided at a final concentration of 100 microM. Other cations tested did not restore growth of this H. ducreyi mutant to wild-type levels. The H. ducreyi ZnuA protein was localized to the periplasm, where it is believed to function as the binding component of a zinc transport system. Complementation of the znuA mutation with the wild-type H. ducreyi znuA gene provided in trans restored the ability of this H. ducreyi mutant to grow normally in the absence of exogenously added ZnCl2. The wild-type H. ducreyi znuA gene was also able to complement a H. influenzae pzp1 mutation. The H. ducreyi znuA isogenic mutant exhibited significantly decreased virulence (P = 0.0001) when tested in the temperature-dependent rabbit model for experimental chancroid. This decreased virulence was not observed when the znuA mutant was complemented with the wild-type H. ducreyi znuA gene provided in trans.

Characterization of a Haemophilus ducreyi mutant deficient in expression of cytolethal distending toxin.

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that kills HeLa, HEp-2, and other human epithelial cells in vitro. H. ducreyi CDT activity is encoded by a three-gene cluster (cdtABC), and antibody to the cdtC gene product can neutralize CDT activity in vitro (L. D. Cope, S. R. Lumbley, J. L. Latimer, J. Klesney-Tait, M. K. Stevens, L. S. Johnson, M. Purven, R. S. Munson, Jr., T. Lagergard, J. D. Radolf, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 94:4056-4061, 1997). Culture supernatant fluid from a recombinant Escherichia coli strain containing the H. ducreyi cdtABC gene cluster readily killed both HeLa cells and HaCaT keratinocytes and had a modest inhibitory effect on the growth of human foreskin fibroblasts. Insertional inactivation of the cdtC gene in this recombinant E. coli strain eliminated the ability of this strain to kill HeLa cells and HaCaT keratinocytes. This mutated H. ducreyi cdtABC gene cluster was used to construct an isogenic H. ducreyi cdtC mutant. Monoclonal antibodies against the H. ducreyi CdtA, CdtB, and CdtC proteins were used to characterize protein expression by this cdtC mutant. Culture supernatant fluid from this H. ducreyi cdtC mutant did not detectably affect any of the human cells used in this study. The presence of the wild-type H. ducreyi cdtC gene in trans in this H. ducreyi mutant restored its ability to express a CDT that killed both HeLa cells and HaCaT keratinocytes. The isogenic H. ducreyi cdtC mutant was shown to be as virulent as its wild-type parent strain in the temperature-dependent rabbit model for experimental chancroid. Lack of expression of the H. ducreyi CdtC protein also did not affect the ability of this H. ducreyi mutant to survive in the skin of rabbits.

Characterization of a WaaF (RfaF) homolog expressed by Haemophilus ducreyi.

Haemophilus ducreyi lipooligosaccharide (LOS) is capable of inducing an inflammatory response in skin (A. A. Campagnari, L. M. Wild, G. Griffiths, R. J. Karalus, M. A. Wirth, and S. M. Spinola, Infect. Immun. 59:2601-2608, 1991) and likely contributes to the virulence of this sexually transmitted pathogen (B. A. Bauer, M. K. Stevens, and E. J. Hansen, Infect. Immun. 68:4290-4298, 1998). An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that was 59% identical to the protein product of the rfaF (waaF) gene of Salmonella typhimurium. The H. ducreyi waaF gene was able to complement an S. typhimurium rfaF (waaF) mutant, a result which confirmed the identity of this gene. In contrast to the rfaF (waaF) gene of enteric bacteria, the H. ducreyi waaF gene was not located adjacent to other genes involved in lipopolysaccharide expression. Inactivation of the H. ducreyi waaF gene by insertion mutagenesis resulted in expression of a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LOS of this mutant also did not bind a monoclonal antibody directed against a cell surface-exposed epitope of wild-type H. ducreyi LOS. Testing of the wild-type H. ducreyi strain and its isogenic waaF mutant in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi revealed that this waaF mutant was less virulent than the wild-type parent strain. Complementation of the H. ducreyi waaF mutant with the wild-type H. ducreyi waaF gene resulted in expression of both wild-type LOS and wild-type virulence by this mutant.

Haemophilus ducreyi secretes a filamentous hemagglutinin-like protein.

We have identified two extremely large open reading frames (ORFs) in Haemophilus ducreyi 35000, lspA1 and lspA2, each of which encodes a predicted protein product whose N-terminal half is approximately 43% similar to the N-terminal half of Bordetella pertussis filamentous hemagglutinin (FhaB). To the best of our knowledge, lspA1 (12,500 nucleotides [nt]) and lspA2 (14,800 nt) are among the largest prokaryotic ORFs identified to date. The predicted proteins, LspA1 and LspA2, are 86% identical overall to each other and also have limited amino acid sequence similarity at their N termini to other secreted bacterial proteins, including certain hemolysins. Southern blot analysis indicated that lspA1 and lspA2 sequences were present in 15 other geographically diverse H. ducreyi strains. Reverse transcriptase PCR analysis of total RNA isolated from H. ducreyi 35000 grown in liquid medium, grown on solid agar medium, and isolated from lesions of H. ducreyi-infected rabbits indicated that lspA1 and lspA2 were transcribed both in vitro and in vivo. A 260-kDa protein present in culture supernatant from eight virulent H. ducreyi strains reacted with both polyclonal serum from rabbits infected with H. ducreyi 35000 and a monoclonal antibody predicted to bind both LspA1 and LspA2. This 260-kDa protein in H. ducreyi 35000 culture supernatant was shown to be the protein product of the lspA1 ORF based on its reactivity with a monoclonal antibody specific for LspA1. Four H. ducreyi strains, previously shown to be avirulent in the temperature-dependent rabbit model for chancroid, did not produce either LspA1 or LspA2 in vitro. This finding raised the possibility that LspA1, LspA2, or both may be involved in the ability of H. ducreyi to cause lesions in this animal model.

Phenotypic effect of isogenic uspA1 and uspA2 mutations on Moraxella catarrhalis 035E.

The UspA surface antigen of Moraxella catarrhalis was recently shown to be comprised of two different proteins (UspA1 and UspA2) which share an internal region containing 140 amino acids with 93% identity (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367-4377, 1997). Isogenic uspA1, uspA2, and uspA1 uspA2 mutants were tested in a number of in vitro systems to determine what effect these mutations, either individually or together, might exert on the phenotype of M. catarrhalis 035E. Monoclonal antibodies specific for UspA1 or UspA2 were used in an indirect antibody accessibility assay to prove that both of these proteins were expressed on the surface of M. catarrhalis. All three mutants grew in vitro at the same rate and did not exhibit autoagglutination or hemagglutination properties that were detectably different from those of the wild-type parent strain. When tested for the ability to adhere to human epithelial cells, the wild-type parent strain and the uspA2 mutant readily attached to Chang conjunctival cells. In contrast, the uspA1 mutant and the uspA1 uspA2 double mutant both attached to these epithelial cells at a level nearly 2 orders of magnitude lower than that obtained with the wild-type parent strain, a result which suggested that expression of UspA1 by M. catarrhalis is essential for attachment to these epithelial cells. Both the wild-type parent strain and the uspA1 mutant were resistant to the bactericidal activity of normal human serum, whereas the uspA2 mutant and the uspA1 uspA2 double mutant were readily killed by this serum. This latter result indicated that the presence of UspA2 is essential for expression of serum resistance by M. catarrhalis.

A diffusible cytotoxin of Haemophilus ducreyi.

Little is known about the virulence mechanisms employed by Haemophilus ducreyi in the production of genital ulcers. This Gram-negative bacterium previously has been shown to produce a soluble cytotoxic activity that kills HeLa and HEp-2 cells. We have now identified a cluster of three H. ducreyi genes that encode this cytotoxic activity. The predicted proteins encoded by these genes are most similar to the products of the Escherichia coli cdtABC genes that comprise the cytolethal distending toxin (CDT) of this enteric pathogen. Eleven of 12 H. ducreyi strains were shown to possess this gene cluster and culture supernatants from these strains readily killed HeLa cells. The culture supernatant from a single strain of H. ducreyi that lacked these genes was unable to kill HeLa cells. When the H. ducreyi cdtABC gene cluster was cloned into E. coli, culture supernatant from the recombinant E. coli clone killed HeLa cells. A monoclonal antibody that neutralized this soluble cytotoxic activity of H. ducreyi was shown to bind to the H. ducreyi cdtC gene product. This soluble H. ducreyi cytotoxin may play a role in the development or persistence of the ulcerative lesions characteristic of chancroid.

Identification of tandem genes involved in lipooligosaccharide expression by Haemophilus ducreyi.

A transposon insertion mutant of Haemophilus ducreyi 35000 possessing a truncated lipooligosaccharide (LOS) failed to bind the LOS-specific monoclonal antibody 3E6 (M. K. Stevens, L. D. Cope, J. D. Radolf, and E. J. Hansen, Infect. Immun. 63:2976-2982, 1995). This transposon was found to have inserted into the first of two tandem genes and also caused a deletion of chromosomal DNA upstream of this gene. These two genes, designated lbgA and lbgB, encoded predicted proteins with molecular masses of 25,788 and 40,236 Da which showed homology with proteins which function in lipopolysaccharide biosynthetic in other gram-negative bacteria. The tandem arrangement of the lbgA and lbgB genes was found to be conserved among H. ducreyi strains. Isogenic LOS mutants, constructed by the insertion of a cat cartridge into either the lbgA or the lbgB gene, expressed an LOS phenotype indistinguishable from that of the original transposon-derived LOS mutant. The wild-type LOS phenotype could be restored by complementation with the appropriate wild-type allele. These two LOS mutants proved to be as virulent as the wild-type parent strain in an animal model. A double mutant with a deletion of the lbgA and lbgB genes yielded equivocal results when its virulence was tested in an animal model.

A hemoglobin-binding outer membrane protein is involved in virulence expression by Haemophilus ducreyi in an animal model.

Haemophilus ducreyi exhibits a requirement for exogenously supplied heme for aerobic growth in vitro. Nine of ten wild-type isolates of H. ducreyi were shown to contain a readily detectable hemoglobin-binding activity. Spontaneous hemoglobin-binding-negative mutants of two of these wild-type isolates lost the ability to express an outer membrane protein with an apparent molecular mass of approximately 100 kDa. Similarly, the single wild-type isolate that lacked the ability to bind hemoglobin also appeared to lack expression of this same 100-kDa protein. A monoclonal antibody (5A9) to this 100-kDa protein was used to identify a recombinant clone which possessed an H. ducreyi chromosomal fragment containing the gene encoding the 100-kDa protein; this protein was designated hemoglobin utilization protein A (HupA). Nucleotide sequence analysis of the hupA gene revealed that the predicted protein, with a calculated molecular mass of 108 kDa, was similar to TonB-dependent outer membrane proteins of other bacteria. Increasing the concentration of heme in the growth medium resulted in decreased expression of the HupA protein. Mutant analysis was used to prove that the HupA protein was essential for the utilization by H. ducreyi of both hemoglobin and hemoglobin-haptoglobin as sources of heme in vitro. In addition, it was found that an isogenic hupA mutant was less virulent than the wild-type parent strain in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi.

Detection of Haemophilus ducreyi lipooligosaccharide by means of an immunolimulus assay.

A murine monoclonal antibody (MAb) directed against a surface-exposed epitope of the lipooligosaccharide (LOS) of Haemophilus ducreyi strain 35000 was shown to be reactive with all 37 strains of this pathogen tested in a colony blot-radioimmunoassay. The LOS epitope bound by this MAb appeared to be stably expressed by H. ducreyi growing in vitro. The use of this MAb in the immunolimulus system revealed that it could detect purified H. ducreyi LOS at a level of 25 pg/ml. Similarly, this immunolimulus system could detect as few as 1000 colony forming units of in vitro-grown H. ducreyi cells per ml of buffer. When this MAb was utilized in the immunolimulus system together with lesion material from rabbits infected with two different H. ducreyi strains, a positive reaction was obtained with every sample tested, even when no viable organisms were present in the lesion material. In contrast, this MAb yielded consistently negative results when used in the immunolimulus system with lesion material from animals infected with Staphylococcus aureus.

Induction of protective immunity to Haemophilus ducreyi in the temperature-dependent rabbit model of experimental chancroid.

The temperature-dependent rabbit model for chancroid, a sexually transmitted disease caused by the fastidious Gram-negative bacterium Haemophilus ducreyi, was used to investigate the abilities of previous infection and immunization with an acellular preparation of H. ducreyi to induce protective immunity. In the first set of experiments, animals were infected intradermally with either the 35000 or Cha-1 strains of H. ducreyi and then rechallenged 30 days later with both the homologous and heterologous strains. In animals infected with the 35000 strain, statistically significant protective immunity occurred only against the homologous strain, whereas protection against both homologous and heterologous challenge was obtained in rabbits previously infected with strain Cha-1. In a separate series of experiments, rabbits were immunized with cell envelopes from either strain 35000 or strain Cha-1 and then challenged with both the homologous and heterologous strains. In rabbits immunized with strain 35000 cell envelopes, significant protective immunity was observed only against challenge with the homologous strain. In animals immunized with strain Cha-1 cell envelopes, protection was obtained against both homologous and heterologous challenge. Histopathologic analysis of sites inoculated with strain 35000 (10(5) CFU) demonstrated that the inflammatory response in control animals was predominantly suppurative (i.e., heterophilic), whereas that of immunized animals was predominantly mononuclear and, at later time points, largely histiocytic. ELISA and Western blot analyses revealed that immunization produced a better humoral immune response than did infection and provided evidence for antigenic cross-reactivity between these two strains. These results provide the experimental basis for continued efforts to identify potential H. ducreyi vaccinogens.

A mutation affecting expression of a major outer membrane protein of Moraxella catarrhalis alters serum resistance and survival in vivo.

A major outer membrane protein (CopB) of Moraxella catarrhalis is a target for antibodies that enhance clearance of this organism from the lungs of mice. A mini-Tn10kan transposon was inserted into the cloned copB gene from M. catarrhalis O35E, and an isogenic mutant unable to express the CopB protein was constructed by transforming this mutated gene into the wild-type strain. The mutant grew at the same rate as the wild-type parent strain in broth. Unlike the serum-resistant parent strain, this mutant was sensitive to killing by normal human serum, and its ability to survive and grow in the lungs of animals was impaired. Genetic restoration of CopB protein expression resulted in the simultaneous acquisition of wild-type levels of serum resistance and the ability to resist pulmonary clearance in vivo. Thus, the CopB protein of M. catarrhalis may be important in the interaction between this organism and the defense mechanisms of the respiratory tract.