ABSTRACT
Objective To compare the sub-cellular proteome of isoniazid ( INH)-resistant Mycobacterium tuberculosis (MTB) with that of sensitive strains for identifying of unique proteins of these strains and discussing their preliminary application in clinical diagnosis. MethodsProteins of cell wall and membrane of 5 INH-resistant strains and 5 INH-sensitive strains were extracted by density gradient centrifugation.The extracts were subsequently analyzed using weak cation exchange (WCX) liquid chromatography ( LC )followed reverse phase (RP) liquid chromatography to compare the sub-cellular protein patterns. A total of 1280 fractions were collected and identified by matrix assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOF MS/MS). The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for cell component and biological process analysis. Normalized Spectral Abundance Factors (NASF) was used for semi-quantity of protein expression. 5 proteins significantly up-regulated in INH-resistant strains and 2 proteins significantly up-regulated in INH-sensitive strains were selected for ELISA analysis with autologous sera respectively. Results A total of 347 proteins were identified. Cell component analysis showed that 58％ proteins were cells well or membrane proteins. Biological process analysis showed that 31％ proteins involved in carboxylic/monocarboxylic acid biosynthetic and metabolic process, 26％ and 15％ proteins involved in organic acid or fatty acid biosynthetic and metabolic process,while 28％ proteins involved in lipid biosynthetic , metabolic, transport and localization process. O-succinylbenzoate synthase, monooxygenase, hypothetical protein Rv2255c, nicotinate-nucleotide--dimethylbenzimidazole phosphoribosyltransferase and membrane phosphatidate cytidylyltransferase cdsA were up-regulated in INH-resistant strains and fractions contained these proteins could elicit specific antibody response with autologous sera. The A450 was higher than that with INH-sensitive sera. The differences between the INH-resistant sera and the INH-sensitive sera were significant ( t = 0.028, 0.044, 0.066, 0.064, 0.083, all P＜0.01 ). Chain A of Rv2002 Gene Product and Chain A of Crystal Structure Of Rv2632c were up-regulated in INH-sensitive strains and fractions contained these proteins could elicit specific antibody response with autologous sera. The A450 was higher than that with INH-resistant sera. The differences between the INH-sensitive sera and the INH-resistant sera were significant (t=0.053, 0.073, both P＜0.05). ConclusionThe combination of density gradient centrifugation and 2D-LC MS/MS technology is useful in enrichment and identification of differential expressed proteins between INH-resistant and INH-sensitive strains at sub-cellular level. It is useful in finding antigens associated with INH-resistant MTB infection, which may prove useful for further study in the mechanism of INH resistant, as well as interaction between MTB and host.