Results MDP1 is essential for adaptation of BCG to low pH Bacteria present in activated macrophages have to face low phagosomal pH conditions. We therefore tested the ability to

adapt to low pH of M. bovis BCG, containing the empty cloning vector pMV261 [BCG (pMV261)], EVP4593 chemical structure and of M. bovis BCG with the MDP1-antisense-plasmid pAS-MDP1 [BCG (pAS-MDP1)], by comparing the growth without and with pH stress. Bacteria were grown to optical density (OD) 3 [600 nm], then diluted and inoculated into fresh Middlebrook 7H9 (Mb) /Oleic Acid-Dextrose-Catalase (OADC) medium PRI-724 nmr adjusted to pH 7 and pH 5.3, respectively, and growth was monitored by measurement of OD and ATP content. As shown in Figure 1A, BCG (pAS-MDP1) reached a slightly higher OD in medium with neutral pH in comparison to BCG (pMV261) and also a higher maximal amount of ATP (Figure 1B). In medium adjusted to pH 5.3 only BCG (pMV261) was able to grow (Figure 1C, D). The growth rate of

BCG (pMV261) in low pH medium was slightly below its growth rate in neutral medium if determined by OD measurement. In medium adjusted to pH 7 BCG (pMV261) grew to an OD of 3.6 after 42 days (Figure 1A). In contrast the OD of cultures grown in medium adjusted to pH 5.3 was only 2.9 after 42 days (Figure 1C). The strain BCG (pAS-MDP1) behaved very find protocol differently at low pH. It was not able to adapt to the low pH conditions and showed no growth at pH 5.3 (Figure 1C, D). Figure 1 Growth in acidic medium. BCG (pMV261) and BCG (pAS-MDP1) were grown in Mb/OADC medium adjusted to pH 7 (A, B) or pH 5.3 (C, D), respectively, and the growth of the mycobacteria was monitored by measurement of the OD [600 nm] (A, C) and the amount of ATP in the cultures (B, D). The ATP amount was measured using a luminescence assay and is reported as relative light

units (RLU). Each value MycoClean Mycoplasma Removal Kit represents the mean of three cultures with the standard deviation. The results of a paired student’s t test are shown by asterisks (*: P < 0.05, **: P < 0.01). MDP1 plays a role in persistence of BCG in human blood monocytes The alveolar macrophages represent the first line of defence the mycobacteria have to overcome in order to establish a successful long-lasting infection. We therefore analysed the ability of our BCG strains to survive in human blood monocytes. Monocytes were infected with BCG (pMV261) and BCG (pAS-MDP1) grown to OD 2 at an MOI of 1 and the amount of intracellular bacteria was quantified one, two, three and five days after infection by quantitative real-time PCR. As shown in Figure 2, the BCG with the empty plasmid started multiplying after one day post infection. After five days, 3.8 times more cells of BCG (pMV261) were present than after the initial infection period of four hours.