Nasal-associated lymphoid tissue and olfactory epithelium as portals of entry for Burkholderia pseudomallei in murine melioidosis.

BACKGROUND: Burkholderia pseudomallei, the causative agent of melioidosis, is generally considered to be acquired via inhalation of dust or water droplets from the environment. In this study, we show that infection of the nasal mucosa is potentially an important portal of entry in melioidosis. METHODS: After intranasal inoculation of mice, infection was monitored by bioluminescence imaging and by immunohistological analysis of coronal sections. The bacterial loads in organ and tissue specimens were also monitored. RESULTS: Bioluminescence imaging showed colonization and replication in the nasal cavity, including the nasal-associated lymphoid tissue (NALT). Analysis of coronal sections and immunofluorescence microscopy further demonstrated the presence of infection in the respiratory epithelium and the olfactory epithelium (including associated nerve bundles), as well as in the NALT. Of significance, the olfactory epithelium and the brain were rapidly infected before bacteria were detected in blood, and a capsule-deficient mutant infected the brain without significantly infecting blood. CONCLUSIONS: These data suggest that the olfactory nerve is the route of entry into the brain and that this route of entry may be paralleled in cases of human neurologic melioidosis. This study focuses attention on the upper respiratory tract as a portal of entry, specifically focusing on NALT as a route for the development of systemic infection via the bloodstream and on the olfactory epithelium as a direct route to the brain.

Autofluorescence of mycobacteria as a tool for detection of Mycobacterium tuberculosis.

The diagnosis of tuberculosis in developing countries still relies on direct sputum examination by light microscopy, a method that is easy to perform and that is widely applied. However, because of its poor sensitivity and requirement for significant labor and training, light microscopy examination detects the bacilli in only 45 to 60% of all people whose specimens are culture positive for Mycobacterium tuberculosis. Therefore, new diagnostic methods that would enable the detection of the undiagnosed infected population and allow the early commencement of antituberculosis treatment are needed. In this work, the potential use of mycobacterial cyan autofluorescence for the detection of Mycobacterium tuberculosis was explored. The tubercle bacilli were easily visualized as brilliant fluorescent bacilli by microscopy and were easily tracked ex vivo during macrophage infection. Assays with seeded sputum and a 96-well microplate reader fluorimeter indicated that <10(6) bacilli ml(-1) of sputum could be detected. Moreover, the use of microplates allowed the examination of only 200 microl of sputum per sample without a loss of sensitivity. Treatment with heat or decontaminating chemical agents did not interfere with the autofluorescence assay; on the contrary, they improved the level of bacterial detection. Autofluorescence for the detection of bacilli is rapid and easy to perform compared to other methodologies and can be performed with minimal training, making this method suitable for implementation in developing countries.

IS6110 in oriC affects the morphology and growth of Mycobacterium tuberculosis and attenuates virulence in mice.

The IS6110 element is widely used in studies of molecular epidemiology of tuberculosis and it is considered the gold standard for genotyping Mycobacterium tuberculosis strains. Because of its high frequency of transposition, IS6110 is probably a major contributor to the evolution of M. tuberculosis. Nevertheless, very few studies of the effect of IS6110 insertions on the virulence of M. tuberculosis have been reported. We analysed two isogenic groups of M. tuberculosis strains isolated from the sputa of two patients. Strains belonging to the same isogenic group differed from one another by one IS6110-oriC hybridising band, but they showed identical spoligo and MIRU-VNTR profiles. Isogenic strains containing the IS6110 element in oriC exhibited a diminished growth rate and average dimensions of the bacilli were modified; moreover, they were less virulent in a mouse model.

par genes in Mycobacterium bovis and Mycobacterium smegmatis are arranged in an operon transcribed from "SigGC" promoters.

BACKGROUND: The ParA/Soj and ParB/Spo0J proteins, and the cis-acting parS site, participate actively in chromosome segregation and cell cycle progression. Genes homologous to parA and parB, and two putative parS copies, have been identified in the Mycobacterium bovis BCG and Mycobacterium smegmatis chromosomes. As in Mycobacterium tuberculosis, the parA and parB genes in these two non-pathogenic mycobacteria are located near the chromosomal origin of replication. The present work focused on the determination of the transcriptional organisation of the ~6 Kb orf60K-parB region of M. bovis BCG and M. smegmatis by primer extension, transcriptional fusions to the green fluorescence protein (GFP) and quantitative RT-PCR. RESULTS: The parAB genes were arranged in an operon. However, we also found promoters upstream of each one of these genes. Seven putative promoter sequences were identified in the orf60K-parB region of M. bovis BCG, whilst four were identified in the homologous region of M. smegmatis, one upstream of each open reading frame (ORF).Real-time PCR assays showed that in M. smegmatis, mRNA-parA and mRNA-parB levels decreased between the exponential and stationary phases. In M. bovis BCG, mRNA-parA levels also decreased between the exponential and stationary phases. However, parB expression was higher than parA expression and remained almost unchanged along the growth curve. CONCLUSION: The majority of the proposed promoter regions had features characteristic of Mycobacterium promoters previously denoted as Group D. The -10 hexamer of a strong E. coli sigma70-like promoter, located upstream of gidB of M. bovis BCG, overlapped with a putative parS sequence, suggesting that the transcription from this promoter might be regulated by the binding of ParB to parS.

Genetic tools for select-agent-compliant manipulation of Burkholderia pseudomallei.

Because of Burkholderia pseudomallei's classification as a select agent in the United States, genetic manipulation of this bacterium is strictly regulated. Only a few antibiotic selection markers, including gentamicin, kanamycin, and zeocin, are currently approved for use with this bacterium, but wild-type strains are highly resistant to these antibiotics. To facilitate routine genetic manipulations of wild-type strains, several new tools were developed. A temperature-sensitive pRO1600 broad-host-range replicon was isolated and used to construct curable plasmids where the Flp and Cre recombinase genes are expressed from the rhamnose-regulated Escherichia coli P(BAD) promoter and kanamycin (nptI) and zeocin (ble) selection markers from the constitutive Burkholderia thailandensis ribosomal P(S12) or synthetic bacterial P(EM7) promoter. Flp and Cre site-specific recombination systems allow in vivo excision and recycling of nptII and ble selection markers contained on FRT or loxP cassettes. Finally, expression of Tn7 site-specific transposase from the constitutive P1 integron promoter allowed development of an efficient site-specific chromosomal integration system for B. pseudomallei. In conjunction with a natural transformation method, the utility of these new tools was demonstrated by isolating an unmarked delta(amrRAB-oprA) efflux pump mutant. Exploiting natural transformation, chromosomal DNA fragments carrying this mutation marked with zeocin resistance were transferred between the genomes of two different B. pseudomallei strains. Lastly, the deletion mutation was complemented by a chromosomally integrated mini-Tn7 element carrying the amrAB-oprA operon. The new tools allow routine select-agent-compliant genetic manipulations of B. pseudomallei and other Burkholderia species.

Transcription analysis of the dnaA gene and oriC region of the chromosome of Mycobacterium smegmatis and Mycobacterium bovis BCG, and its regulation by the DnaA protein.

The regions flanking the Mycobacterium dnaA gene have extensive sequence conservation, and comprise various DnaA boxes. Comparative analysis of the dnaA promoter and oriC region from several mycobacterial species revealed that the localization, spacing and orientation of the DnaA boxes are conserved. Detailed transcriptional analysis in M. smegmatis and M. bovis BCG shows that the dnaN gene of both species and the dnaA gene of M. bovis BCG are transcribed from two promoters, whereas the dnaA gene of M. smegmatis is transcribed from a single promoter. RT-PCR with total RNA showed that dnaA and dnaN were expressed in both species at all growth stages. Analysis of the promoter activity using dnaA-gfp fusion plasmids and DnaA expression plasmids indicates that the dnaA gene is autoregulated, although the degree of transcriptional autorepression was moderate. Transcription was also detected in the vicinity of oriC of M. bovis BCG, but not of M. smegmatis. These results suggest that a more complex transcriptional mechanism may be involved in the slow-growing mycobacteria, which regulates the expression of dnaA and initiation of chromosomal DNA replication.

Caracterización de la expresión y función sigM de un factor sigma ECF en micobacterias / Characterization of the expression and function sigM of an ECF sigma factor in mycobacteriums

La supervivencia del bacilo Micobacteria tuberculosis dentro del macrófago depende de su capacidad de responder al stress oxidativa, y los factores sigma, subfamilia ECF, probablemente tienen un rol importante. Investigamos un factor sigma, sigM, cuyos gen se encuentran cerca del origen de replicación. Tanto en M.smegmatis como en M.bovis BCG, la expresión del gen esta inducida en temperaturas altas y en fase estacionaria. Cepas de M.smegmatis sin una copia activa de sigM muestran un defecto en la supervivencia en el stress oxidativo y también en la inducción de la actividad de thioredoxina reductasa, que reduce puentes disulfuricos formado en stress oxidativo. Estos datos sugieren que SigM regula los tioredoxinas y constituye parte de unas respuestas protectivas muy complejos de la bacteria