Project description:After performing multiplex PCR, we analysed extracted DNA (500 ng ssDNA) from 9 Mycobacterium tuberculosis clinical isolates to detect multidrug resistance. In addition, a mixed strain situation was simulated by mixing wild type Mtb CDC1551 (20 ng) with 4 concentrations of Mtb mutant DNA (1 ng, 250 pg, 62.5 pg, and 15.6 pg), which is equivalent to relative concentrations of 5%, 1.25%, 0.31% and 0.08% Mtb mutant DNA.
Project description:This project describes the isoniazid (INH) resistance acquisition event in Mycobacterium tuberculosis (Mtb) from the proteomics perspective. In this way, an exploration of the protein differences, comparing clonal INH susceptible (INHs) and INHr pairs of Mtb were evaluated. One clonal clinical and one clonal laboratory-derived Mtb pair with different susceptibility profiles to INH were studied. The laboratory INHr strain had one katG mutation (V1A), while the clinical INHr strain had two (V1A and E3V). Large-scale bacterial cultures were grown in triplicate to obtain secreted proteins as well as proteins from cell fractions. The resulting peptide solutions from all fractions were analyzed using liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS). LC-MS/MS spectra were compared against an Mtb database to determine the protein abundance. Protein abundance differences were tested by Student’s t test. Looking at the same cellular fractions, there were 25 commonly altered Mtb proteins after acquiring INH resistance. These proteins were involved in ATP synthase machinery, lipid metabolism, regulatory events, virulence, detoxification and adaptation processes.