Molecular cloning, sequencing, expression, and characterization of an immunogenic 43-kilodalton lipoprotein of Bartonella bacilliformis that has homology to NlpD/LppB.

A recombinant clone expressing an immunoreactive antigen of Bartonella bacilliformis was isolated by screening a genomic DNA library with serum from a patient with the chronic verruga phase of bartonellosis. The clone, pBIPIM-17, contained a partial open reading frame that expressed an immunoreactive fusion protein. Subsequent rescreening of the library by plaque hybridization resulted in the isolation of recombinant clones that contain the entire open reading frame. The open reading frame (ORF-401) is capable of encoding a protein of 401 amino acids with a predicted molecular mass of 43 kDa. The deduced amino acid sequence of the encoded protein was found to be highly homologous to a recently identified bacterial lipoprotein (LppB/NlpD) which has been associated with virulence. Evidence has been provided to show that the 43-kDa antigen of B. bacilliformis is a lipoprotein and that it is likely to use the same biosynthetic pathway as other bacterial lipoproteins. This is the first report to date that characterizes a lipoprotein of B. bacilliformis. The immunogenicity of the B. bacilliformis LppB homologue was demonstrated by Western blot analysis using sera from patients with clinical bartonellosis. Sera from patients who had a high titer for Bartonella henselae, the causative agent of bacillary angiomatosis and cat scratch disease, also recognized the recombinant 43-kDa antigen, suggesting that a homologue of this antigen is present in B. henselae. Using a cocktail of synthetic peptides corresponding to predicted major antigenic sites, polyclonal antiserum specific for the LppB homologue of B. bacilliformis was generated. This antiserum did not recognize the NlpD homologue of Escherichia coli or the 43-kDa antigen of B. henselae.

The gene encoding the 17-kDa antigen of Bartonella henselae is located within a cluster of genes homologous to the virB virulence operon.

A Bartonella henselae genomic A library was screened with antiserum generated in mice against live B. henselae. One of the immunoreactive clones expressed a 17-kDa antigen that was characterized previously as an immunodominant protein of B. henselae. Sequence analysis of the recombinant clone, pBHIM-2, revealed that the open reading frame (ORF) encoding the 17-kDa antigen was situated between homologs of virB4 and virB6, two genes that belong to the virB operon. The virB operon has been associated with the transfer of oncogenic T-DNA in Agrobacterium tumefaciens and with secretion of the pertussis toxin in Bordetella pertussis. Downstream of the virB6 gene within pBHIM-2 was a partial open reading frame that was homologous to the virB8 gene. Rescreening of the library by plaque hybridization using probes specific to the 5' and 3' ends of the pBHIM-2 insert resulted in the isolation of recombinant clones containing additional virB genes. Assembly of the sequences obtained from the recombinant clones revealed that eight of the open reading frames encode homologs of the VirB proteins. The homology and colinearity with the virB genes suggest that the gene encoding the 17-kDa antigen is expressed within the virB locus of B. henselae.

Use of the cell division protein FtsZ as a means of differentiating among Bartonella species.

Genes coding for homologs of the highly conserved cell division protein FtsZ were isolated from Bartonella henselae and Bartonella quintana, the causative agents of cat scratch disease and trench fever, respectively. DNA fragments coding for the ftsZ open reading frames (ORFs) were cloned into Escherichia coli following PCR amplification with primers based on the ftsZ sequence of the closely related species Bartonella bacilliformis. The amino acid sequences predicted from the cloned B. henselae and B. quintana ftsZ ORFs are 81 to 83% identical to the corresponding protein in B. bacilliformis. Like the FtsZ protein of B. bacilliformis, the B. henselae and B. quintana homologs are about twice as large as the FtsZ proteins reported in most other organisms. Localized sequence differences within the C-terminal coding regions of the Bartonella ftsZ genes were used as the basis for species-specific identification of these organisms at both the DNA and protein levels. Oligonucleotide primers which permit the amplification of an ftsZ fragment from each of the Bartonella species without amplifying DNA from the other two species were designed. Anti-FtsZ antisera raised in rabbits against synthetic peptides corresponding to the relatively divergent C-terminal regions were shown via Western blot analysis to react only with the FtsZ protein from the cognate Bartonella species. These observations raise the possibility that the differences in ftsZ sequences can be used as the basis for diagnostic tests to differentiate among these closely related pathogens.

The 75-kilodalton antigen of Bartonella bacilliformis is a structural homolog of the cell division protein FtsZ.

A genomic library of Bartonella bacilliformis was constructed and screened with human anti-Bartonella serum from a patient with the chronic, verruga peruana phase of bartonellosis. An immunoreactive clone isolated from this library was found to code for a 591-amino-acid protein with a high degree of sequence similarity to the FtsZ family of proteins. The degree of amino acid identity between the B. bacilliformis protein (FtsZ[Bb]) and the other FtsZ proteins is especially pronounced over the N-terminal 321 amino acids (N-terminal domain) of the sequence, with values ranging from 45% identity for the homolog from Micrococcus luteus (FtsZ[Ml]) to 91% identity for the homolog from Rhizobium melliloti, (FtsZ[Rm1]). All of the functional domains required for FtsZ activity are conserved in FtsZ(Bb) and are located within the N-terminal domain of the protein. FtsZ(Bb) is approximately twice as large as most of the other FtsZ proteins previously reported, a property it shares with FtsZ(Rm1). Like the Rhizobium homolog, FtsZ(Bb) has a C-terminal region of approximately 256 amino acids that is absent in the other FtsZ proteins. Evidence is presented that implicates this region in the protein's antigenicity and suggests that, unlike most other FtsZ homologs, FtsZ(Bb) is at least partly exposed at the cell surface. PCR analysis revealed that an ftsZ gene similar in size to the B. bacilliformis gene is present in Bartonella henselae, a bacterium that is closely related to B. bacilliformis.

Bacteriophage-like particle of Rochalimaea henselae.

An extracellular particle approximately 40 nM in diameter was detected in culture supernatant from the fastidious bacterium Rochalimaea henselae. This particle has at least three associated proteins and contains 14 kbp linear DNA segments that are heterogeneous in sequence. The 14 kbp DNA was also present in R. henselae cells as an extrachromosomal element for all 14 strains tested. Despite attempts to induce lysis of R. henselae, plaque formation was not observed. A similar particle, also containing 14 kbp DNA, was observed in Bartonella bacilliformis, and may be analogous to a bacteriophage that has been described elsewhere for B. bacilliformis.

Anthropometric studies in diabetes in the Tropics.

Anthropometric studies were carried out in three groups of diabetics seen in southern India, namely fibrocalculous pancreatic diabetes (FCPD) (n = 49) (a subtype of malnutrition related diabetes), insulin dependent diabetes mellitus (IDDM) (n = 55) and non-insulin dependent diabetes mellitus (NIDDM) (n = 104). Both FCPD and IDDM had significantly lower body mass index, skinfold thickness (triceps, biceps, subscapular and suprailiac), mid-arm circumference and fat mass compared to controls and NIDDM patients, (p less than 0.001 for all parameters). FCPD and IDDM males did not show any significant differences in any of the anthropometric parameters studied. Among the females, FCPD had lower triceps skinfold measurements (p = 0.007) and mid-arm circumferences (p less than 0.05) compared to IDDM patients. Patients with NIDDM did not show any significant difference compared to the control group. This study shows that both FCPD and IDDM patients have lower body mass and fat mass compared to NIDDM patients and control subjects.