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Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL−10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium–host interaction. [ABSTRACT FROM AUTHOR]

The polymerase chain reaction (PCR) was evaluated in a trial which, with respect to the positive-to-negative ratio, approximated the situation of a diagnostic laboratory in a tuberculosis-endemic area. Three hundred sputum samples were included in the study, of which one-third were known to contain mycobacteria as judged by direct microscopy. The repetitive insertion sequence IS 6110/IS 986 of Mycobacterium tuberculosis was used as a target. The samples were spiked with DNA from a modified IS 6110/IS 986 sequence, which gives rise to PCR products easily distinguished from PCR products amplified from chromosomal Mycobacterium tuberculosis DNA. This allowed identification of samples that contained substances inhibitory to the Taq polymerase. The detection limit of the assay was 0.05 pg to 0.5 pg of purified Mycobacterium tuberculosis DNA, corresponding to 10 to 100 organisms. The sensitivity and specificity of the PCR was compared with that of conventional microscopy and culture. It was concluded that this method is fast and sensitive, but that culture currently is crucial for assessing viability and thus infectivity. [ABSTRACT FROM AUTHOR]

Histidine-requiring auxotrophs of Mycobacterium smegmatis were isolated following W-methyl-W-nitro-N'-nitrosoguanidine treatment. One of these mutants, his5, was transformed with an M. smegmatis shuttle cosmid library, and complementing clones were isolated at a frequency of approximately 1%. A 2.3 kb fragment was subcloned and sequenced, and found to contain the start of an operon including the hisD gene and part of the hisC gene. No hisG gene was detected upstream of hisD, suggesting that the regulation of histidine biosynthesis in mycobacteria may differ from that of Escherichia coli. The strategy used here will allow the molecular genetics of complex mycobacterial-specific biosynthetic pathways involved in the virulence of pathogenic species to be studied. [ABSTRACT FROM AUTHOR]