Transcriptional analysis of Mycobacterium fortuitum cultures upon hydrogen peroxide treatment using the novel standard rrnA-P1.
BACKGROUND
The ability of an intracellular pathogen to establish infection depends on the capacity of the organism to survive and replicate inside the host. Mycobacterium fortuitum is a bacteria that contains genes involved in the detoxification of the oxygen reactive species such as those produced by the host during the infection. In this work, we investigate the effects of hydrogen peroxide on the transcription and expression of these genes by developing a real time quantitative PCR technique (qRT-PCR) using the ribosomal promoter region (rrnA-P1) as reference product for quantification of the mRNA levels.


RESULTS
M. fortuitum cultures were treated with different hydrogen peroxide concentrations (0.02 to 20 mM) during several periods of time (30 to 120 minutes). The activity of the enzymes KatGII and SodA, and the transcription of corresponding genes were evaluated. The transcriptional regulator furAII gene was also studied. The ribosomal promoter region rrnA-P1 was validated as referential product under the stress conditions checked by qRT-PCR. Minor changes were observed under the conditions tested except when bacteria were incubated in the presence of 20 mM hydrogen peroxide. Under those conditions, the levels of transcription of the three genes under study increased at 30 minutes of treatment. The viability of the bacteria was not influenced under the conditions tested.


CONCLUSIONS
In this work, we have quantified transcriptional responses to stress suggesting that, the opportunistic pathogen M. fortuitum is more resistant and differs in behaviour in the presence of hydrogen peroxide, when compared to the major pathogen Mycobacterium tuberculosis and the saprophyte Mycobacterium smegmatis. Besides, we demonstrate the mycobacterial non-coding region rrnA-P1 to be a suitable reference product in the analysis of qRT-PCR transcriptional data of M. fortuitum.
Departamento de Medicina Preventiva, Facultad de Medicina, Universidad Autónoma, c/Arzobispo Morcillo, 4, 28029-Madrid, Spain. cnserrano@inia.es
María Carmen
MC
Núñez
María Carmen
MC
Menéndez
María José
MJ
Rebollo
María J
MJ
García
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D050296:Microbial Viability / Q000187:drug effects; D019907:Mycobacterium fortuitum / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D006861:Hydrogen Peroxide