Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG.

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18 kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.

Outer membrane pore protein prediction in mycobacteria using genomic comparison.

Proteins responsible for outer membrane transport across the unique membrane structure of Mycobacterium spp. are attractive drug targets in the treatment of human diseases caused by the mycobacterial pathogens, Mycobacterium tuberculosis, M. bovis, M. leprae and M. ulcerans. In contrast with Escherichia coli, relatively few outer-membrane proteins (OMPs) have been identified in Mycobacterium spp., largely due to the difficulties in isolating mycobacterial membrane proteins and our incomplete understanding of secretion mechanisms and cell wall structure in these organisms. To further expand our knowledge of these elusive proteins in mycobacteria, we have improved upon our previous method of OMP prediction in mycobacteria by taking advantage of genomic data from seven mycobacteria species. Our improved algorithm suggests 4333 sequences as putative OMPs in seven species with varying degrees of confidence. The most virulent pathogenic mycobacterial species are slightly enriched in these selected sequences. We present examples of predicted OMPs involved in horizontal transfer and paralogy expansion. Analysis of local secondary structure content allowed identification of small domains predicted to perform as OMPs; some examples show their involvement in events of tandem duplication and domain rearrangements. We discuss the taxonomic distribution of these discovered families and architectures, often specific to mycobacteria or the wider taxonomic class of Actinobacteria. Our results suggest that OMP functionality in mycobacteria is richer than expected and provide a resource to guide future research of these understudied proteins.

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.

Functional characterization of a small heat shock protein from Mycobacterium leprae.

BACKGROUND: Small heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen. M. leprae, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloning M. leprae genes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen of M. leprae is scanty. RESULTS: The gene encoding Mycobacterium leprae small heat shock protein (sHsp18) was amplified from biopsy material of leprosy patients, and cloned and expressed in E. coli. The localization and in vitro characterization of the protein are detailed in this report. Data show that major portion of the protein is localized in the outer membrane of E. coli. The purified sHsp18 functions as an efficient chaperone as shown by their ability to prevent thermal inactivation of restriction enzymes SmaI and NdeI. Physical interaction of the chaperone with target protein is also demonstrated. Size exclusion chromatography of purified protein shows that the protein can form multimeric complexes under in vitro conditions as is demonstrated for several small heat shock proteins. CONCLUSION: The small heat shock protein sHsp18 of M. leprae is a chaperone and shows several properties associated with other small heat shock proteins. Membrane association and in vitro chaperone function of sHsp18 shows that the protein may play a role in the virulence and survival of M. leprae in infected host.

Expression of the Moraxella catarrhalis UspA1 protein undergoes phase variation and is regulated at the transcriptional level.

The UspA1 protein of Moraxella catarrhalis has been shown to function as an adhesin that mediates adherence to human epithelial cell lines in vitro (E. R. Lafontaine, L. D. Cope, C. Aebi, J. L. Latimer, G. H. McCracken, Jr., and E. J. Hansen, J. Bacteriol. 182:1364-1373, 2000). In the present study, cell lysates prepared from individual colonies of several M. catarrhalis wild-type strains were analyzed by Western blot analysis using monoclonal antibodies (MAbs) specific for the UspA1 protein. Expression of UspA1 was shown to exhibit phase variation that was correlated with both adherence ability in vitro and the number of guanine (G) residues contained within a homopolymeric [poly(G)]tract located upstream of the uspA1 open reading frame (ORF). Nucleotide sequence analysis revealed that isolates expressing relatively high levels of UspA1 had 10 G residues in their uspA1 poly(G)tracts, whereas isolates that expressed much lower levels of UspA1 had 9 G residues. This poly(G) tract was located 30 nucleotides (nt) upstream of the uspA1 ORF and 168 nt downstream of the uspA1 transcriptional start site. Primer extension experiments, RNA slot blot analysis, and cat reporter constructs were used to demonstrate that M. catarrhalis isolates with 10 G residues in their uspA1 poly(G) tracts expressed two-to threefold more uspA1 mRNA than did isolates which had 9 G residues in their poly(G)tracts. Northern hybridization analysis revealed that an intact uspA1 mRNA was readily detectable in RNA from M. catarrhalis isolates that had 10 G residues in their uspA1 poly(G) tracts, whereas no full-length uspA1 mRNA was observed in isolates whose poly(G)tracts contained 9 G residues. M. catarrhalis strain O35E uspA1 genes that contained wild-type and mutated poly(G) tracts were expressed in Haemophilus influenzae to demonstrate that the length and composition of the poly(G)tract affected expression of UspA1.

Detection of phase variation in expression of proteins involved in hemoglobin and hemoglobin-haptoglobin binding by nontypeable Haemophilus influenzae.

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study (I. Maciver, J. L. Latimer, H. H. Liem, U. Muller-Eberhard, Z. Hrkal, and E. J. Hansen. Infect. Immun. 64:3703-3712, 1996) indicated that nontypeable H. influenzae (NTHI) strain TN106 expressed a protein that bound hemoglobin-haptoglobin and was encoded by an open reading frame (ORF) that contained a CCAA nucleotide repeat. Southern blot analysis revealed that several NTHI strains contained between three and five chromosomal DNA fragments that bound an oligonucleotide probe for CCAA repeats. Three ORFs containing CCAA repeats were identified in NTHI strain N182; two of these ORFs were arranged in tandem. The use of translational fusions involving these three ORFs and the beta-lactamase gene from pBR322 revealed that these three ORFs, designated hgbA, hgbB, and hgbC, encoded proteins that could bind hemoglobin, hemoglobin-haptoglobin, or both compounds. Monoclonal antibodies (MAbs) specific for the HgbA, HgbB, and HgbC proteins were produced by immunizing mice with synthetic peptides unique to each protein. Both HgbA and HgbB were readily detected by Western blot analysis in N182 cells grown in the presence of hemoglobin as the sole source of heme, whereas expression of HgbC was found to be much less abundant than that of HgbA and HgbB. The use of these MAbs in a colony blot radioimmunoassay analysis revealed that expression of both HgbA and HgbB was subject to phase variation. PCR and nucleotide sequence analysis were used in conjunction with Western blot analyses to demonstrate that this phase variation involved the CCAA repeats in the hgbA and hgbB ORFs.

The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro.

The UspA1 and UspA2 proteins of Moraxella catarrhalis are structurally related, are exposed on the bacterial cell surface, and migrate as very high-molecular-weight complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Previous analysis of uspA1 and uspA2 mutants of M. catarrhalis strain 035E indicated that UspA1 was involved in adherence of this organism to Chang conjunctival epithelial cells in vitro and that expression of UspA2 was essential for resistance of this strain to killing by normal human serum (C. Aebi, E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 66:3113-3119, 1998). In the present study, isogenic uspA1, uspA2, and uspA1 uspA2 mutations were constructed in three additional M. catarrhalis strains: 012E, TTA37, and 046E. The uspA1 mutant of strain 012E had a decreased ability to attach to Chang cells. However, inactivation of the uspA1 gene in both strain TTA37 and strain 046E did not cause a significant decrease in attachment ability. Inactivation of the uspA2 gene of strain TTA37 did result in a loss of attachment ability. Nucleotide sequence analysis revealed that the predicted protein encoded by the uspA2 genes of both strains TTA37 and 046E had a N-terminal half that resembled the N-terminal half of UspA1 proteins, whereas the C-terminal half of this protein was nearly identical to those of previously characterized UspA2 proteins. The gene encoding this "hybrid" protein was designated uspA2H. PCR-based analysis revealed that approximately 20% of M. catarrhalis strains apparently possess a uspA2H gene instead of a uspA2 gene. The M. catarrhalis uspA1, uspA2, and uspA2H genes were cloned and expressed in Haemophilus influenzae cells, which were used to prove that both the UspA1 and UspA2H proteins can function as adhesins in vitro.

Characterization of the Moraxella catarrhalis uspA1 and uspA2 genes and their encoded products.

The uspA1 and uspA2 genes of M. catarrhalis O35E encode two different surface-exposed proteins which were previously shown to share a 140-amino-acid region 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). The N-terminal amino acid sequences of the mature forms of both UspA1 and UspA2 from strain O35E were determined after enzymatic treatment to remove the N-terminal pyroglutamyl residue that had blocked Edman degradation. Mass spectrometric analysis indicated that the molecular mass of UspA1 from M. catarrhalis O35E was 83,500 +/- 116 Da. Nucleotide sequence analysis of the uspA1 and uspA2 genes from three other M. catarrhalis strains (TTA24, ATCC 25238, and V1171) revealed that the encoded protein products were very similar to those from strain O35E. Western blot analysis was used to confirm that each of these three strains of M. catarrhalis expressed both UspA1 and UspA2 proteins. Several different and repetitive amino acid motifs were present in both UspA1 and UspA2 from these four strains, and some of these were predicted to form coiled coils. Linear DNA templates were used in an in vitro transcription-translation system to determine the sizes of the monomeric forms of the UspA1 and UspA2 proteins from strains O35E and TTA24.

Molecular characterization of a surface-exposed superoxide dismutase of Mycobacterium avium.

Mycobacterium avium is an intracellular pathogen capable of growing inside the phagosomal compartment of macrophages. In this work, we characterized the superoxide dismutase of M. avium, as a putative candidate to resist the oxidative stress. The gene sodA encoding superoxide dismutase (SOD:EC1.15.1.1) from Mycobacterium avium TMC724 was cloned and sequenced. It encodes a 23 kDa protein (207 aminoacids) showing identity with the Mycobacterium leprae SOD (91%) and the M. tuberculosis SOD (83%). This enzyme was functionally expressed in both Escherichia coli and Mycobacterium smegmatis, and identified as a manganese (Mn) SOD on the basis of sequence comparison with other MnSODs from different organisms, and by activity inhibition studies. By indirect immunogold labeling of M. avium with a mAb directed against M. leprae SOD, the enzyme was found to be exposed at the cell surface of M. avium. It was also shown that SOD was released in supernates of M. avium TMV724 during exponential growth, suggesting a role of this enzyme during interactions with the environment. When SOD was expressed in the non-pathogenic M. smegmatis, it was also exposed at the surface of bacteria and released in supernates, but this was not sufficient to protect this recombinant mycobacterium from the killing mechanisms of macrophages.

A gene cluster involved in the utilization of both free heme and heme:hemopexin by Haemophilus influenzae type b.

The utilization of heme bound to the serum glycoprotein hemopexin by Haemophilus influenzae type b (Hib) strain DL42 requires the presence of the 100-kDa heme:hemopexin-binding protein encoded by the hxuA gene (M. S. Hanson, S. E. Pelzel, J. Latimer, U. Muller-Eberhard, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). Nucleotide sequence analysis of a 5-kb region immediately upstream from the hxuA gene revealed the presence of two genes, designated hxuC and hxuB, which encoded outer membrane proteins. The 78-kDa HxuC protein had similarity to TonB-dependent outer membrane proteins of other organisms, whereas the 60-kDa HxuB molecule most closely resembled the ShlB protein of Serratia marcescens. A set of three isogenic Hib mutants with cat cartridges inserted individually into their hxuA, hxuB, and hxuC genes was constructed. None of these mutants could utilize heme:hemopexin. The hxuC mutant was also unable to utilize low levels of free heme, whereas both the hxuA and hxuB mutants could utilize free heme. When the wild-type hxuC gene was present in trans, the hxuC mutant regained its ability to utilize low levels of free heme but still could not utilize heme:hemopexin. The hxuA mutant could utilize heme:hemopexin when a functional hxuA gene from a nontypeable H. influenzae strain was present in trans. Complementation analysis using this cloned nontypeable H. influenzae hxuA gene also indicated that the HxuB protein likely functions in the release of soluble HxuA from the Hib cell. These studies indicate that at least two and possible three gene products are required for utilization of heme bound to hemopexin by Hib strain DL42.

Cloning and sequencing of a Bordetella pertussis serum resistance locus.

We have characterized a new virulence factor in Bordetella pertussis: serum resistance. Compared with Escherichia coli HB101, wild-type B. pertussis was relatively resistant to classical-pathway, complement-dependent killing by normal human serum. However, a mutant of B. pertussis (BPM2041) which is less virulent in mice and which has Tn5 lac inserted in a previously uncharacterized bvg-regulated gene was found to be at least 10-fold more susceptible to serum killing than the wild type. We have named this locus brk, for Bordetella resistance to killing. We have cloned and sequenced the brk locus, and it encodes two divergently transcribed open reading frames (ORFs), termed BrkA and BrkB. Both ORFs are necessary for serum resistance. Within the 300 bases which separate the two ORFs and upstream of each ORF are putative sites for BvgA binding. BrkA shows 29% identity to pertactin and has two RGD motifs in addition to a conserved proteolytic processing site and an outer membrane targeting signal. Like pertactin, BrkA is involved in adherence and invasion. Despite the similarities, a pertactin mutant was found to be not as sensitive to serum killing as the BrkA or BrkB mutants. BrkB is similar to ORFs in E. coli and Mycobacterium leprae and displays domains of homology to various transporters. On the basis of its hydropathy profile, BrkB is predicted to be a cytoplasmic membrane protein. By Southern blot, brk sequences were found in Bordetella bronchiseptica and Bordetella parapertussis but not in Bordetella avium.