Pathology of inhalational Francisella tularensis spp. tularensis SCHU S4 infection in African green monkeys (Chlorocebus aethiops).

Tularemia, caused by Francisella tularensis, is a sporadic zoonotic disease with the potential to be an agent of biowarfare or bioterrorism. We describe here the gross, histologic, immunohistochemical, and ultrastructural findings in a group of 5 African green monkeys (AGMs) that received an average inhaled dose of 729 colony-forming units of F. tularensis and died or were euthanatized between days 7 and 11 post infection. Clinical changes were evident by 48 hours post infection, and key physiologic abnormalities included increases in body temperature, heart rate, peak cardiac pressure, and mean blood pressure. Prominent gross changes in all cases included numerous pinpoint to 1-cm, well-demarcated, necrotic foci present consistently in the lungs, mediastinal lymph nodes, and spleen but also seen in the heart, mediastinum, diaphragm, liver, urinary bladder, urethra, and mesentery. The lungs, mediastinal lymph nodes, and spleen were most severely affected, with as much as 50% of the tissue replaced by necrotic foci. Histologic changes in all tissues consisted of well-delineated foci of necrosis and neutrophilic and histiocytic inflammation, with varying amounts of hemorrhage, edema, fibrin, and vasculitis. Some lesions were immature pyogranulomas. Strong immunoreactivity was identified primarily within macrophages. Ultrastructurally, bacteria were present within cytoplasmic vacuoles of alveolar macrophages, many of which were degenerate. In summary, AGMs infected with F. tularensis by aerosol develop lethal multisystemic disease that particularly targets the lungs and lymphoid tissues. Thus, AGMs should serve as a suitable and reliable animal model for further studies of tularemia.

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.

Lipid modification of the 17-kilodalton membrane immunogen of Treponema pallidum determines macrophage activation as well as amphiphilicity.

A murine monoclonal antibody specific for a 17-kDa major membrane immunogen of Treponema pallidum was used to select recombinant Escherichia coli clones expressing the molecule from a T. pallidum genomic library. Sequence analysis of the structural gene for the immunogen (designated tpp17) revealed a 468-bp open reading frame encoding a polypeptide of 156 amino acids with a calculated molecular mass of 16,441 Da. The deduced amino acid sequence included a putative leader peptide terminated by a consensus tetrapeptide for the modification and processing of prokaryotic lipoproteins. Immunoprecipitation of the cloned immunogen radiolabeled with [3H]palmitate confirmed that it was a lipoprotein. The amino acid sequence also predicted that the mature protein contains four cysteine residues in addition to the lipid-modified cysteine of the N terminus. The existence of disulfide-bonded multimeric forms of the native immunogen was demonstrated by immunoblotting T. pallidum solubilized in the presence and absence of 2-mercaptoethanol. Triton X-114 phase partitioning of a nonlipidated form of the 17-kDa immunogen cleaved from a glutathione S-transferase fusion protein demonstrated that lipid modification is responsible for the immunogen's hydrophobic character. The same nonlipidated form of the immunogen also was used to demonstrate that lipid modification is essential for the molecule's ability to stimulate production of tumor necrosis factor alpha by murine macrophages. We conclude that covalently attached fatty acids not only anchor T. pallidum lipoproteins to spirochetal membranes but also confer upon these molecules the ability to activate immune effector cells.

A temperature-dependent rabbit model for production of dermal lesions by Haemophilus ducreyi.

The fact that optimal in vitro growth of Haemophilus ducreyi occurs at 33 degrees C prompted evaluation of the effect of temperature on the ability of this organism to produce skin lesions in rabbits after intradermal inoculation. Animals housed at a reduced ambient temperature (15-17 degrees C) consistently developed necrotic lesions when injected intradermally with 10(5) cfu of H. ducreyi; this inoculum did not produce necrotic lesions in animals housed at normal room temperature (23-25 degrees C). Lesion production in this new model was dependent on both viability of the H. ducreyi inoculum and replication of these organisms after intradermal injection. Histopathologic examination of the lesions revealed that H. ducreyi infection of the rabbit dermis evolves from an acute inflammatory reaction to abscess formation. Evaluation of three additional strains of H. ducreyi in this model confirmed that lesion formation was not bacterial strain-dependent. This new temperature-dependent rabbit model for productive H. ducreyi infection will facilitate investigation of the molecular pathogenesis of chancroid.

Lipid modification of the 15 kiloDalton major membrane immunogen of Treponema pallidum.

The 15 kiloDalton major membrane immunogen was included among the Treponema pallidum polypeptides selectively labelled with [3H]-palmitate. The cloned gene for this immunogen, tpp15, encoded a signal peptide of 17 amino acids, a consensus signal peptidase II cleavage site, and a mature protein of 124 amino acids (13,967 Daltons). As predicted by the DNA sequence, the recombinant 15 kiloDalton immunogen labelled selectively with [3H]-palmitate, and globomycin inhibited processing of the precursor to the mature polypeptide. While the native and recombinant immunogens are amphiphilic, the 15 kiloDalton immunogen synthesized in a cell-free system was hydrophilic. The covalent attachment of fatty acids appears to be responsible for the amphiphilicity of the immunogen and its membrane attachment.

Molecular cloning and characterization of the 15-kilodalton major immunogen of Treponema pallidum.

Pathogen-specific membrane immunogens of Treponema pallidum subsp. pallidum (T. pallidum) have been identified previously by phase partitioning with the nonionic detergent Triton X-114. One of these antigens, a 15-kilodalton (kDa) polypeptide, is expressed in relatively small quantities in T. pallidum but is highly immunogenic in both human and experimental syphilis. The native T. pallidum antigen was purified to homogeneity from the mixture of Triton X-114 detergent-phase proteins by chromatofocusing. Recombinant Escherichia coli clones were selected from a T. pallidum genomic DNA library by using monoclonal antibodies specific to the 15-kDa antigen; immunoblotting and minicell analyses confirmed expression of the 15-kDa protein in the transformants. Southern hybridization with a 1.1-kilobase fragment of DNA encoding the 15-kDa-antigen gene indicated that the gene is probably present in a single copy within the genomes of both T. pallidum and T. pallidum subsp. pertenue (the agent of yaws), while it is absent from the genome of the nonpathogenic Treponema phagedenis biotype Reiter. Cell fractionation studies with Triton X-114 demonstrated that the recombinant polypeptide possesses hydrophobic properties similar to those of the native antigen and localized the cloned 15-kDa antigen to the inner membrane of E. coli. Protein processing experiments in minicells revealed that a precursor appears to be processed to the mature 15-kDa polypeptide.

Nucleotide sequences of the genes encoding type 1 fimbrial subunits of Klebsiella pneumoniae and Salmonella typhimurium.

The nucleotide sequences of the genes encoding the subunits of Klebsiella pneumoniae and Salmonella typhimurium type 1 fimbriae were determined. Comparison of the predicted amino acid sequences of the two subunits revealed domains in which the sequences were highly conserved. Both gene products possessed signal peptides, a fact consistent with the transport of the fimbrial subunit across the membrane, but these regions showed no amino acid homology between the two proteins. The predicted N-terminal amino acid sequences of the processed fimbrial subunits were in good agreement with those obtained by purification of the fimbrial subunits.

Characterization of genes encoding type 1 fimbriae of Klebsiella pneumoniae, Salmonella typhimurium, and Serratia marcescens.

With a minicell system, the organization of genes encoding type 1 fimbriae of Salmonella typhimurium, Klebsiella pneumoniae, and Serratia marcescens was determined. In all cases multiple gene products were necessary for the phenotypic expression of fimbriae; thus fimbrial expression in these strains is similar to that in Escherichia coli. The type 1 fimbrial subunit gene was detected by the ability of its product to react with specific antiserum. At least six genes were found to be involved in the expression of type 1 fimbriae by S. typhimurium, and at least four genes constituted the fimbrial gene cluster of K. pneumoniae. In the case of S. marcescens, a minimum of three detectable polypeptides was required for the production of fimbriae. Also, a gene probe consisting in part of nucleotide sequences from the E. coli fimbrial subunit gene hybridized to a discrete DNA fragment derived from the plasmid encoding K. pneumoniae fimbriae. Such a fragment was assumed to contain a gene encoding the structural component of the type 1 fimbriae. Each of the three cloned systems encoded a number of polypeptides which varied in size; thus, the organization and molecular weight of fimbrial accessory proteins of each genus were not identical.

Complementation analyses of recombinant plasmids encoding type 1 fimbriae of members of the family Enterobacteriaceae.

Insertion mutants of recombinant plasmids encoding type 1 fimbriae of four genera of enteric bacteria were used to detect genetic complementation. After transformation by pairs of plasmids, double transformants were screened for their ability to express type 1 fimbriae. Complementation was observed between genes derived from the same genus but was absent with chimeric molecules carrying genetic information from two different genera. The results indicate that diffusible gene products of the fim cluster are necessary for phenotypic expression of type 1 fimbriae.

Construction and expression of recombinant plasmids encoding type 1 fimbriae of a urinary Klebsiella pneumoniae isolate.

The type 1 fimbriae of Klebsiella pneumoniae have been implicated as important virulence factors in mediating Klebsiella urinary infections. The chromosomally encoded fimbrial genes were cloned by a cosmid cloning technique. Further subcloning was performed with the cloning vehicles pBR322 and pACYC184, and a recombinant plasmid containing the fimbrial genes was constructed. After transformation by this plasmid, both Escherichia coli and Salmonella typhimurium were shown to express fimbriae which reacted with Klebsiella fimbrial antiserum. The approximate location of the relevant genes on the chimeric plasmid was determined by insertion of the transposable element Tn5. Hemagglutination-negative phenotypes were used to estimate the minimum size of the DNA fragment necessary to encode fimbrial biosynthesis and expression. The size of the coding region of this fragment was found to be 5.5 kilobase pairs.