B-cell epitopes in the immunodominant p34 antigen of mycobacterium avium ssp. paratuberculosis recognized by antibodies from infected cattle.

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) causes Johne's disease, a chronic and fatal enteritis in ruminants. In the last stage of the disease, antibody titres rise and levels of interferon-gamma decrease, suggesting that the host-immune response is switching from a T helper 1 (Th1) to a Th2 profile. In infected cattle, the membrane protein p34 elicits the predominant humoral response against M. paratuberculosis. To map the B-cell epitopes of this antigen, affinity-purified bovine antibodies against the carboxy-terminal region of p34 were used to screen a 12-mer phage display library. Several phage clones carrying peptides resembling fragments of p34 were affinity selected. Based on the predicted amino acid sequence, peptides were chemically synthesized, which demonstrated reactivity with serum from naturally infected and p34-vaccinated cattle. Immunization of mice with these peptides elicited an anti-p34 antibody response. Two B-cell epitopes were identified and characterized. Based on the reactivity and the type of immune response elicited, epitope A was determined to be conformational, whereas epitope B was demonstrated to be sequential. Both epitopes were shown to be present in p34 proteins from M. avium ssp. avium or M. paratuberculosis but absent from M. intracellulare, the other member of the M. avium complex. Furthermore, both epitopes were mapped to regions of p34 that display high variability when compared to homologous proteins from other mycobacterial species of public and animal health importance. We hypothesize that these variable regions of p34 may play a role in the immunobiology of M. paratuberculosis infections.

Cell sorting of formalin-treated pathogenic Mycobacterium paratuberculosis expressing GFP.

GFP is widely used as a molecular tool for the study of microbial pathogens. However, the manipulation of these pathogenic microorganisms poses a health threat to the laboratory worker, requiring biosafety level II or III containment. Although the GFPfluorophore is tolerant toformalin, a thorough analysis of this treatment on fluorescent output in prokaryotic systems has not been described. In addition, the analysis of microorganisms expressing GFP often depends on specialized equipment, which may not be housed in biosafety level II or III laboratories. Therefore, we sought to develop a safe and effective method for manipulating the GFP-expressing pathogenic bacterium Mycobacterium avium subsp, paratuberculosis (M. paratuberculosis) utilizing a formalin treatment that would permit the analysis of GFP fluorescence without requiring stringent biosafety containment. We demonstrate that formalin-treated M. paratuberculosis expresses 50% less fluorescence than viable cells, but this reduction is still compatible with spectrofluorometry and cell sorting. Furthermore, plasmid DNA that expresses GFP can be recovered efficiently from nonviable, sorted fluorescent cells. This approach is flexible, provides an additional margin of safety for laboratory personnel, and can be easily applied to other pathogenic microorganisms expressing GFP.

Identification of a secreted superoxide dismutase in Mycobacterium avium ssp. paratuberculosis.

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the causative agent of Johne's disease, is an important animal pathogen that has also been implicated in human disease. The major proteins expressed by M. paratuberculosis were analyzed by two-dimensional gel electrophoresis, and a superoxide dismutase (Sod) was identified from this protein profile. The M. paratuberculosis Sod has a molecular mass of 23 kDa and an isoelectric point of 6.1. Sequence analysis of the corresponding sodA gene from M. paratuberculosis indicates that this protein is a manganese-dependent enzyme. We show that the M. paratuberculosis Sod is actively secreted, suggesting that it may elicit a protective cellular immune response in the host during infection.

Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine.

Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the etiologic agent of a severe gastroenteritis in ruminants known as Johne's disease. Economic losses to the cattle industry in the United States are staggering, reaching $1.5 billion annually. A potential pathogenic role in humans in the etiology of Crohn's disease is under investigation. In this article, we review the epidemiology, pathogenesis, diagnostics, and disease control measures of this important veterinary pathogen. We emphasize molecular genetic aspects including the description of markers used for strain identification, diagnostics, and phylogenetic analysis. Recent important advances in the development of animal models and genetic systems to study M. paratuberculosis virulence determinants are also discussed. We conclude with proposals for the applications of these models and recombinant technology to the development of diagnostic, control, and therapeutic measures.

Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis, a slow-growing Mycobacterium, is the causative agent of Johne's disease. Although M. paratuberculosis is difficult to manipulate genetically, our laboratory has recently demonstrated the ability to introduce DNA into these bacteria by transformation and phage infection. In the current study we develop the first transposon mutagenesis system for M. paratuberculosis using the conditionally replicating mycobacteriophage phAE94 to introduce the mycobacterial transposon Tn5367. Southern blotting and sequence analysis demonstrated that the transposon insertion sites are distributed relatively randomly throughout the M. paratuberculosis genome. We constructed a comprehensive bank of 5620 insertion mutants using this transposon. The transposition frequency obtained using this delivery system was 1.0 x 10(-6) transposition events per recipient cell. Auxotrophic mutants were observed in this library at a frequency of 0.3%.

Development of a firefly luciferase-based assay for determining antimicrobial susceptibility of Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis (Johne's disease) is a fatal disease of ruminants for which no effective treatment is available. Presently, no drugs against Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the causative agent of Johne's disease, are approved for use in livestock. Additionally, M. paratuberculosis has been linked to a human chronic granulomatous ileitis (Crohn's disease). To assist in the evaluation of antimicrobial agents with potential activity against M. paratuberculosis, we have developed a firefly luciferase-based assay for the determination of drug susceptibilities. The microorganism used was M. paratuberculosis K-10(pYUB180), a clinical isolate carrying a plasmid with the firefly luciferase gene. The MICs determined by the broth macrodilution method were as follows: amikacin, 2 microg/ml; Bay y 3118, 0.015 microg/ml; clarithromycin, 1.25 microg/ml; D-cycloserine, 25 microg/ml; ethambutol, 20 microg/ml; and rifabutin, 0.5 microg/ml. The strain was resistant to isoniazid and kanamycin. The results obtained by the luciferase assay were identical or fell within 1 doubling dilution. These results suggest that a combination of amikacin, clarithromycin, and rifabutin may be the most efficacious therapy for the treatment of M. paratuberculosis infections and that the use of fluoroquinolone class of antibiotics deserves further consideration. We demonstrate that the luciferase drug susceptibility assay is reliable for M. paratuberculosis and gives results within 7 days, whereas the broth macrodilution method requires 14 days.

Regulation of the Escherichia coli sheA gene and characterization of its encoded hemolytic activity.

Escherichia coli K-12 carries the cryptic hemolysin gene sheA which is under the control of positive and negative transcriptional regulators. The objectives of the present study were to further analyze the regulation of the sheA gene in E. coli, to compare the sheA genes from E. coli K-12 and a pathogenic E. coli strain, and to characterize the SheA hemolytic activity. Northern blot analysis demonstrated that the transcriptional regulator SlyA activates the E. coli K-12 sheA gene. The main transcriptional start site of the sheA gene was 56 nucleotides upstream from the start codon as determined by primer extension analysis. The sheA genes from E. coli K-12 and a pathogenic E. coli strain were identical. SheA hemolytic activity was cell associated and Ca2+ independent.

Pathogenicity of an enterotoxigenic Escherichia coli hemolysin (hlyA) mutant in gnotobiotic piglets.

Pigs infected with hemolytic F4(+) strains of enterotoxigenic Escherichia coli often develop septicemia secondary to intestinal infection. We tested the hypothesis that inactivation of hemolysin would reduce the ability of F4(+) enterotoxigenic E. coli to cause septicemia in swine following oral inoculation. Inactivation of the hemolysin structural gene (hlyA) did not decrease the incidence of septicemia in the gnotobiotic piglet model.

Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis.

D-Cycloserine is an effective second-line drug against Mycobacterium avium and Mycobacterium tuberculosis. To analyze the genetic determinants of D-cycloserine resistance in mycobacteria, a library of a resistant Mycobacterium smegmatis mutant was constructed. A resistant clone harboring a recombinant plasmid with a 3.1-kb insert that contained the glutamate decarboxylase (gadA) and D-alanine racemase (alrA) genes was identified. Subcloning experiments demonstrated that alrA was necessary and sufficient to confer a D-cycloserine resistance phenotype. The D-alanine racemase activities of wild-type and recombinant M. smegmatis strains were inhibited by D-cycloserine in a concentration-dependent manner. The D-cycloserine resistance phenotype in the recombinant clone was due to the overexpression of the wild-type alrA gene in a multicopy vector. Analysis of a spontaneous resistant mutant also demonstrated overproduction of wild-type AlrA enzyme. Nucleotide sequence analysis of the overproducing mutant revealed a single transversion (G-->T) at the alrA promoter, which resulted in elevated beta-galactosidase reporter gene expression. Furthermore, transformants of Mycobacterium intracellulare and Mycobacterium bovis BCG carrying the M. smegmatis wild-type alrA gene in a multicopy vector were resistant to D-cycloserine, suggesting that AlrA overproduction is a potential mechanism of D-cycloserine resistance in clinical isolates of M. tuberculosis and other pathogenic mycobacteria. In conclusion, these results show that one of the mechanisms of D-cycloserine resistance in M. smegmatis involves the overexpression of the alrA gene due to a promoter-up mutation.

Bacterial vaccine vectors and bacillus Calmette-Guérin.

Recent advances in biotechnology now allow a more modern approach to the development of vaccines, particularly that of recombinant vaccines. Bacterial vaccine vectors have the advantage over viral vectors in that the former have the ability to express a greater number of antigens in different forms. Although no recombinant bacterial vaccines are currently in use, bacillus Calmette-Guérin (BCG), Salmonella species, and Escherichia coli are being developed as vaccine vectors. We review plasmid systems and mutant strains developed for the expression of foreign antigens, with particular emphasis on those developed for BCG. We describe the development of antigen expression systems as well as the immune response elicited by recombinant BCG vaccine strains to bacterial and human immunodeficiency virus (HIV) antigens. A modified recombinant BCG carrier with selection for the stable maintenance of rDNA is proposed.

Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages.

Effective chemotherapy of tuberculosis requires rapid assessment of drug sensitivity because of the emergence of multidrug-resistant Mycobacterium tuberculosis. Drug susceptibility was assessed by a simple method based on the efficient production of photons by viable mycobacteria infected with specific reporter phages expressing the firefly luciferase gene. Light production was dependent on phage infection, expression of the luciferase gene, and the level of cellular adenosine triphosphate. Signals could be detected within minutes after infection of virulent M. tuberculosis with reporter phages. Culture of conventional strains with antituberculosis drugs, including isoniazid or rifampicin, resulted in extinction of light production. In contrast, light signals after luciferase reporter phage infection of drug-resistant strains continued to be produced. Luciferase reporter phages may help to reduce the time required for establishing antibiotic sensitivity of M. tuberculosis strains from weeks to days and to accelerate screening for new antituberculosis drugs.

Identification of expression signals of the mycobacteriophages Bxb1, L1 and TM4 using the Escherichia-Mycobacterium shuttle plasmids pYUB75 and pYUB76 designed to create translational fusions to the lacZ gene.

Mycobacterial expression signals were cloned using specially constructed gene fusion shuttle plasmid probes carrying a truncated Escherichia coli lacZ (beta-galactosidase) gene which lacked a promoter, a ribosome binding site, and an ATG start codon. Libraries of mycobacteriophage Bxb1, L1 and TM4 DNAs were constructed, and introduced by electroporation into Mycobacterium smegmatis and the 'bacille Calmette-Guérin' (BCG). Clones carrying mycobacterial expression sequences were detected by their blue colour or characteristic fluorescence when plated on media containing chromogenic or fluorogenic substrates. Varying degrees of beta-galactosidase expression were observed, and one Bxb1 expression signal was identified where beta-galactosidase expression is repressed in phage lysogens.

A novel transposon trap for mycobacteria: isolation and characterization of IS1096.

In the course of developing strategies to obtain a mutation in the aspartate semialdehyde dehydrogenase (asd) gene of Mycobacterium smegmatis, an efficient transposon trap was constructed which may be generally useful for the identification of transposable elements in mycobacteria. A DNA fragment containing the asd gene was replaced with an aminoglycoside phosphotransferase gene (aph) to generate a delta asd::aph allele. Attempts to replace the wild-type asd gene with the delta asd::aph allele were unsuccessful, suggesting that this deletion was lethal to the growth of M. smegmatis. The plasmid, pYUB215, which contains beta-galactosidase expressed from a mycobacteriophage promoter and delta asd::aph, was integrated into the chromosome of M. smegmatis by a homologous, single-crossover, recombination event. Visual screening for inactivation of the beta-galactosidase gene in the resulting strain allowed the isolation of a novel mycobacterial insertion element from M. smegmatis. This insertion element, which is unique to M. smegmatis, was designated IS1096 and transposes at a frequency of 7.2 x 10(-5) per cell in an apparently random fashion. IS1096 is 2,275 bp in length and contains two open reading frames which are predicted to encode proteins involved in transposition. This insertion element exhibits several characteristics that suggest it may be a useful tool for genetic analysis of mycobacteria, possibly including the study of mechanisms of pathogenesis.

Recombinant BCG as a candidate oral vaccine vector.

Bacille Calmette-Guerin (BCG), currently the most widely used vaccine in the world, was originally administered for many years as an oral vaccine. The low frequency of serious complications, inexpensive production, and adjuvanticity make BCG an ideal candidate for a recombinant vaccine vehicle. Although mycobacteria are slow growing and not yet well characterized genetically, we have recently developed technology for the genetic manipulation of BCG and other mycobacteria. Phage and plasmid systems based on a shuttle strategy to manipulate DNA in Escherichia coli and transfer it to mycobacteria have been developed. We have established that the aminoglycoside phosphotransferase gene can be used as an effective selectable marker in the mycobacteria and that a foreign antigen from Mycobacterium leprae can be expressed in BCG. Furthermore, a thorough analysis of mycobacterial expression sequences has been undertaken to optimize the expression of foreign antigens in BCG. We constructed an expression probe shuttle plasmid with beta-galactosidase as reporter gene, and have used it successfully to identify multiple mycobacteriophage DNA sequences with varying levels of constitutive or regulable promoter activity. Further genetic advances required for development of recombinant BCG into an effective recombinant vaccine vehicle, including possibilities for oral administration, are adumbrated.

Impairment of melibiose utilization in Streptococcus mutans serotype c gtfA mutants.

The Streptococcus mutans serotype c gtfA gene encodes a 55-kilodalton sucrose-hydrolyzing enzyme. Analysis of S. mutans gtfA mutants revealed that the mutant strains were specifically impaired in the ability to use melibiose as a sole carbon source. S. mutans gtfA mutant strains synthesized less alpha-galactosidase activity inducible by raffinose than wild-type strains. Melibiose (an inducer in wild-type strains) failed to induce significant levels of alpha-galactosidase in the mutant strains. We hypothesize that melibiose use by S. mutans requires the interaction of the GtfA enzyme, or another gene product under the control of the gtfA promoter, with other gene product(s) involved in melibiose transport or hydrolysis.

Analysis of the virulence of Streptococcus mutans serotype c gtfA mutants in the rat model system.

The Streptococcus mutans serotype c gtfA gene encodes a 55-kilodalton protein which catalyzes the synthesis of a small glucan (1.5 kilodaltons) from sucrose (J.P. Robeson, R.G. Barletta, and R. Curtiss III, J. Bacteriol. 153:211-221, 1983). To investigate the role of the GtfA enzyme in virulence, we constructed S. mutans gtfA mutants from three cariogenic serotype c strains. A plasmid that carried an erythromycin resistance determinant and an internal fragment of the gtfA gene but that was unable to replicate in streptococci was used to transform S. mutans. The erythromycin-resistant transformants carried a partial duplication of the internal gtfA fragment, because of the integration of plasmid sequences within the S. mutans gtfA gene, which also resulted in the inactivation of the gtfA gene. This was verified by Southern DNA hybridization analysis and Western blot studies of cellular protein extracts of the mutant strains with GtfA antiserum. Mutants were fully virulent in both germfree and conventional rats. These results do not rule out the involvement of the GtfA protein in virulence. Pucci and Macrina (M.J. Pucci and F.L. Macrina, Infect. Immun. 54:77-84, 1986) have suggested that the GtfA enzyme synthesizes a primer for water-insoluble glucans. Another S. mutans protein, presumably a glucosyltransferase, may have a similar function and, thus, may obscure the relevance of the GtfA enzyme in pathogenesis.

Expression of a Streptococcus mutans glucosyltransferase gene in Escherichia coli.

Chromosomal DNA from Streptococcus mutans strain UAB90 (serotype c) was cloned into Escherichia coli K-12. The clone bank was screened for any sucrose-hydrolyzing activity by selection for growth on raffinose in the presence of isopropyl-beta-D-thiogalactoside. A clone expressing an S. mutans glucosyltransferase was identified. The S. mutans DNA encoding this enzyme is a 1.73-kilobase fragment cloned into the HindIII site of plasmid pBR322. We designated the gene gtfA. The plasmid-encoded gtfA enzyme, a 55,000-molecular-weight protein, is synthesized at 40% the level of pBR322-encoded beta-lactamase in E. coli minicells. Using sucrose as substrate, the gtfA enzyme catalyzes the formation of fructose and a glucan with an apparent molecular weight of 1,500. We detected the gtfA protein in S. mutans cells with antibody raised against the cloned gtfA enzyme. Immunologically identical gtfA protein appears to be present in S. mutans cells of serotypes c, e, and f, and a cross-reacting protein was made by serotype b cells. Proteins from serotype a, g, and d S. mutans cells did not react with antibody to gtfA enzyme. The gtfA activity was present in the periplasmic space of E. coli clones, since 15% of the total gtfA activity was released by cold osmotic shock and the clones were able to grow on sucrose as sole carbon source.