Figures & data
Table 1. M. avium A5 genes associated with the rapid apoptosis in secondary-infected human macrophages.
Table 2. THP-1 cell apoptosis in response to M. avium secondary infection measured by TUNEL assay.
Figure 1. In vivo survival of M. avium A5 wild type and gene knockout mutants expressing delayed apoptotic phenotype in secondary infected macrophages after 24h (A) and 12 days (B) post-infection. ***, p < 0.0001 between the wild type control and all mutant infections at day12.
Figure 2. M. avium survival rates in (A) primary and (B) secondary-infected macrophages. THP-1 monolayers were infected with M. avium A5 wild type, the gene knockout mutant MAVA5_06970(-) or complemented MAVA5_06970(+) clone as described in materials and methods. (C) The colony forming units of extracellular bacterial were also evaluated in the supernatants of THP-1 macrophages at 5 days post-infection. *, p < 0.05 and **, p < 0.001 compared with either the wild type M. avium A5 strain or the complemented clone.
Figure 3. Percent of apoptosis of (A) primary and (B) secondary-infected THP-1 cells following 48h infection with M. avium A5 wild type, the MAVA5_06970(-) mutant and MAVA5_06970(+) clone. The percentage of early and late apoptotic THP-1 cells labeled with the Annexin V/FITC were determined by evaluating fifty thousand cells by flow cytometry in three separate experiments.
Figure 4. M. avium MAVA5_06970 gene expression following exposure or infection of primary and secondary macrophages. Total RNAs from broth-grown bacteria, as well as from macrophage-exposed and intracellular bacteria, were used to determine the copy numbers of cDNAs for target and reference genes. Quantitation of the expression of the MAVA5_06970 gene was carried out with SYBR Green I assay by Real-Time PCR detection system using gene-specific primers. Results were analyzed on Ct values basis for each sample and normalized with an internal housekeeping gene control 16S rRNA. Data represents mean and standard deviation values from two independent experiments.
Table 3. Host proteins interacting with M. avium recombinant MAVA5_06970.
Figure 5. (A) The yeast two-hybrid interaction of MAVA5_06970 with the host target SPP1 protein. The open reading frame of SPP1 encoding a 301 amino acid protein was amplified from the cDNA of THP-1 cells amplified from the total RNA. The yeast two-hybrid screening of MAVA5_06970 established positive interaction with SPP1 (1). The known interaction between pGBKT7-lam and pGADT7-T served as a positive control (3), and pGBKT7-53 and pGADT7-T as a control for a negative interaction (2). (B) OPN levels of secondary THP-1 cells infected with the wild type M. avium, the gene knockout MAVA5_06970(-) mutant and complemented MAVA5_06970(+) clone. After 2h of infection cell lysates were subjected to OPN immunoprecipitation under denaturing conditions by using an agarose-conjugated primary antibody and analyzed via anti-OPN antibody (a) or phosphoserine/threonine/tyrosine antibody (b). The photon emission means were recorded for each band to quantify the signal intensity on the Odyssey Imager (Li-Cor). The β–actin served as a loading control (c).
Table 4. IL-12 synthesis by primary- and secondary-infected THP-1 cells.
Figure 6. The recombinant MAVA5_06970 protein restores the MAVA5_06970(-) mutant phenotype in secondary-infected macrophages. (A) THP-1 macrophage monolayers were seeded in 8-chammber glass slides and infected either with the wild-type, MAVA5_06970 gene knockout mutant, complemented clone or MAVA5_06970(-) exposed to 100μg/ml recombinant MAVA5_06970 protein. Infection was carried out for 2h with the MOI of 10:1, and then monolayers were washed 3 times with PBS. After 48h, apoptosis were analyzed using the TUNEL assay. The percentage of apoptosis was calculated in 200 secondary-infected THP-1 cells. *, p < 0.05 compared with the wild type M. avium A5, complemented and MAVA5_06970(-) infection in combination with the MAVA5_06970 recombinant protein at 48h post-infection. (B) THP-1 macrophage monolayers seeded in 24-well plate were infected/exposed to recombinant protein as describe above and supernatants were collected at 48h post-infection for IL-12 measurement using ELISA assay. *P < 0.05 between mutant and the wild type, complemented and mutant infection in combination with the MAVA5_06970 recombinant protein at 48h post-infection. Data represents mean and standard deviation values from two independent experiments.
