The effect of deletion and complementation on IL-12p40 and

TNF secretion was less marked with no statistically significant differences between strains. Although deletion of the 19 kDa reduced apoptosis, an effect that could also only be reversed by complementation with the wild type gene, the MM-102 results were variable between donors and did not attain statistical significance. An interesting finding was that 19 kDa protein was only detected in the supernatant of cultures of the non-acylated (NA) and non-O-glycosylated complemented strains, whereas the Δ19::19 strain expressed the molecule in both pellet and supernatant. This suggests that in order to be retained within the cell wall both acylation

and glycosylation are necessary for anchoring within the cell wall. Whether this relates to a specific physicochemical interaction or merely reflects the recognised hydrophobiCity of the mycobacterial cell membrane Epacadostat concentration remains to be determined. Citarinostat Sartain and Belisle have recently shown that o-glycosylation affects the positioning in the cell wall but not the enzymatic activity of the superoxide dismuase sodC [30]. In a previous study overexpression of the 19 kDa in M. smegmatis reduced its capaCity to induce the secretion of IL-12p40 and TNF[18]. This effect was dependent on acylation and glycosylation, as tranformation of, M. smegmatis with NA and NOG variants of the 19 kDa did not reduce the secretion of these cytokines. By contrast overexpression of the native 19 kDa molecule in Δ19 strain of virulent M. tuberculosis had precisely the opposite effect, with the production of IL-12p40 and TNF increased irrespective

of phagocyte maturity [22]. In this study we reintroduced the 19 kDa gene as a single copy into the chromosome of H37Rv under the control of its own promoter. We precisely reproduced our previous findings with respect to the effect of deletion of the 19 kDa on the cytokine response of monocytes. We have shown that the 19 kDa mediated induction of IL-1β is dependent on acylation and glycosylation. Taken together these and other studies suggest a consistent effect of acylation and O-glycosylation on the cytokine response to the 19 kDa, but that the the genetic background and level of expression are also important. Further evidence in favour of this hypothesis is our recent finding that a naturally occuring M. tuberculosis strain that lacks the 19 kDa gene does not have the same in vitro phenotype as the engineered knock out on the Rv background (data not shown). This potentially important finding requires further investigation as much of our knowledge about gene function in M. tuberculosis is inferred from studies of isogenic mutants on the H37Rv background. Considerable evidence now points to the protective role of macrophage apoptosis in tuberculosis.