Transcriptome analysis of mycobacteria in sputum samples of pulmonary tuberculosis patients

Abstract

Pulmonary tuberculosis, the disease caused by Mycobacterium tuberculosis, still retains a top rank among the deadliest communicable diseases. Sputum expectorated during the disease continues to be a primary diagnostic specimen and also serves as a reservoir of bacteria. The expression pattern of mycobacteria in sputum will lead to an insight into bacterial adaptation at the most highly transmissible stage of infection and can also help in identifying newer diagnostic as well as drug targets. Thus, in the present study, a whole genome microarray of Mycobacterium tuberculosis was used to elucidate the transcriptional profile of mycobacteria in the sputum samples of smear positive pulmonary tuberculosis patients. Overall, the mycobacteria in sputum appeared to be in a low energy and low replicative state as compared to in vitro grown log phase M. tb with downregulation of genes involved in ATP synthesis, aerobic respiration and translational machinery. Simultaneously, downregulation was also seen in the genes involved in secretion machinery of mycobacteria along with the downregulation of genes involved in the synthesis of phthiocerol dimycocerosate and phenol glycolipids. In contrast, the majority of the genes which showed an upregulation in sputum mycobacteria were of unknown function. Further identification of these genes may provide new insights into the mycobacterial behavior during this phase of infection and may help in deciphering candidates for development of better diagnostic and drug candidates.

Fig 1. Heat map of the differentially expressed genes in sputum from PTB patients using a mycobacterial whole genome microarray.

A) Panel A shows the upregulated genes and B) panel B shows the downregulated genes in sputum from PTB patients as compared to in vitro grown M.tb H37Rv. Red and green coloured triangles represent the amount of fold upregulation or downregulation respectively.

Fig 2. Venn diagram indicating overlap of M. tb genes detected as upregulated in sputum in TB patients and in sputum from lung cancer patients.

163 genes were upregulated in sputum from PTB patients and 26 genes also showed upregulation in sputum from lung cancer patients out of which 19 genes were common between the two and hence were eliminated from the list of diagnostic candidates

Fig 3. Validation of microarray data on real time PCR by analyzing the relative expression of 5 genes in smear positive PTB samples as compared to in vitro grown M.tb H37Rv.

16s rRNA was used as reference gene for normalization. Y-axis values (Log2 fold change) of ≥1 indicate upregulation and values ≤ -1 indicate down-regulation. Each bar represents mean ± SD values for each of the genes with three technical replicates. *p<0.05; **p<0.01 by student’s t test.