Figure 4 Growth of strains in Middlebrook 7H9 broth. Duplicate log phase cultures of each strain were normalised to an O.D. of 0.05 and cultured with shaking Sunitinib datasheet with the O.D. repeated at intervals. No difference in the maximum rate of growth of the strains was observed. Cytokine secretion Human monocytes were infected with equal numbers of bacilli (moi 1:1) and co-cultured for 72 hours. During this period, the median secretion of IL-1β was significantly reduced by deletion of the 19 kDa gene (Figure 5A, p = 0.02). Introduction of the native

19 kDa gene as Δ19::19 restored secretion to wild type levels but the response to Δ19::19NA and Δ19::19NOG remained significantly less when compared to Δ19::19 (p = 0.031 in both cases). There was no difference between H37Rv, Δ19 and Δ19::19 in their ability to elicit IL-12p40 or TNF from monocytes (Figure 5B and 5C). Although the response to both the Δ19::19NA and Δ19::19NOG strains tended to be lower, these differences were also not significantly different from H37Rv. Figure 5 Secretion find more of IL-1β, IL-12p40 and TNF in response to strains of M. tuberculosis. Monocytes from 7 donors were infected with strains and co-cultured for 72 hours. The median secretion of IL-1β was significantly reduced by deletion of the 19 kDa gene (p = 0.02). Introduction of the native 19 kDa gene as Δ19::19 restored secretion to wild type levels but the response to Δ19::19NA and Δ19::19NOG remained significantly

less when compared to Δ19::19 (p = 0.031 in both cases). No differences existed between strains in their ability to induce the secretion of IL-12p40 or TNF. Induction of apoptosis Culture supernatants from 6

donors were also assayed for the presence of Histone associated DNA fragments, a marker of apoptosis. Results for each subject were normalised to unstimulated cells to generate an enrichment factor. The Δ19 and Δ19::19NA and Δ19::19NOG were associated with lower levels than H37Rv or the Δ19::19 strain. However responses varied considerably between donors and none of these trends attained statistical significance (Figure 6). Figure 6 Induction of apoptosis by strains of M. tuberculosis. Monocytes from 6 donors were infected with strains and co-cultured MRIP for 72 hours. Apoptosis was determined by ELISA for nucleosomal fractions in culture supernatants. Results for each subject were normalised to unstimulated cells to generate an enrichment factor. The mean + SD of this enrichment factor is shown. Although the Δ19 strain tended to induce less apoptosis than H37Rv and Δ19::19 none of the differences were statistically significant. Discussion We have investigated deletion of the 19 kDa lipoprotein (Rv3763) from M. tuberculosis and chromosomal complementation of the deletion mutant by the wild type gene and site directed mutagenised variants lacking motifs for acylation and O-glycosylation. We have determined that both acylation and O-glycosylation are necessary for the protein to remain within the cell.