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VapCs of Mycobacterium tuberculosis Cleave RNAs Essential for Translation

ABSTRACT: The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin-Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in Enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by site-specific cleavage of the universally conserved Sarcin-Ricin loop (SRL) in 23S rRNA. Here we identify cleavage targets for 12 VapCs from M. tuberculosis by applying UV-crosslinking and deep sequencing. Remarkably, these VapCs are all endoribo-nucleases that cleave RNA targets that are essential for decoding at the ribosomal A-site. Eleven VapCs cleave specific tRNAs while one exhibits SRL cleavage activity. These findings suggest that multiple vapBC modules contribute to the survival of M. tuberculosis in its human host by reducing the level of translation.

ORGANISM(S): Mycolicibacterium smegmatis

PROVIDER: GSE85905 | GEO | 2017/08/14

SECONDARY ACCESSION(S): PRJNA339732

REPOSITORIES: GEO

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Project description:Rickettsia spp. can cause mild to severe human disease. These intracellular bacteria are associated with arthropods, nematodes and trematodes, and usually, are efficiently transmitted transovarially to the progeny of the invertebrate host. We recently demonstrated foreign gene acquisition by lateral gene transfer in Rickettsia genomes. The unexpected presence of laterally transferred toxin-antitoxin (TA) genetic elements (including vapBC) in several Rickettsia genomes has not been connected with the pathogenic process or the host-bacteria relationship. We suspect that vapBC are selfish genetic elements that addict eukaryotic hosts to Rickettsia. We identified a statistical link between the transovarial transmission of Rickettsia in invertebrate hosts and the presence of TA operons, specifically vapBC, in the Rickettsia genome. These TA are neighboring to type IV secretion genes. Tunel assays and whole-genome expression of infected cells showed that antibiotic eradication of TA-containing Rickettsia from the host in cell culture initiates a proapoptotic program. Rickettsia VapC toxins inhibit the growth of transformed Escherichia coli and Saccharomyces cerevisiae. Rickettsia toxin presents in vitro RNase activity. Annexin-V staining and time-lapse video showed that intracytoplasmic injections of VapC toxins in cells cause apoptosis. These data demonstrate that host cells may develop a dependence on Rickettsia spp. expressing the vapBC operon. This would constitute a new evolutionary M-bM-^@M-^\mafia strategyM-bM-^@M-^] of intracellular bacteria based on host addiction. Fresh cells from the human microvascular endothelial cell line (HMEC-1) [26] were infected with R. felis California-2 strain in the presence and absence of antibiotics, at a rate of 5 bacteria per eukaryotic cell. Then, we added or not antibiotics (chloramphenicol 50 M-BM-5g/ml or doxycycline to 40 M-BM-5g/ml) in both experimental (R.felis-infected) and control, mock-infected cells for 6 hours. The cells were harvested and RNA was extracted using the RNeasy Mini Kit (Qiagen). DNA contamination was removed using the Turbo DNA-free Kit (Ambion). RNA were labeled using the Quick Amp Labeling Kit One-color (Agilent) and hybridized onto a Whole Human Genome Microarray, 4x44K (Agilent) as recommended by the manufacturer. Arrays were scanned with DNA Microarray Scanner (Agilent), and data were extracted using Feature Extractor (Agilent).

Project description:The ability of Mycobacterium tuberculosis to infect, proliferate, and survive during long periods in the human lungs largely depends on the rigorous control of gene expression. Transcriptome-wide analyses are key to understanding gene regulation on a global scale. Here, we combine 5’-end-directed libraries with RNAseq expression libraries to gain insight into the transcriptome organization and post-transcriptional mRNA cleavage landscape in mycobacteria during log phase growth and under hypoxia, a physiologically relevant stress condition. Using the model organism Mycobacterium smegmatis, we identified 6,090 transcription start sites (TSSs) with high confidence during log phase growth, of which 67% were categorized as primary TSSs for annotated genes, and the remaining were classified as internal, antisense or orphan, according to their genomic context. Interestingly, over 25% of the RNA transcripts lack a leader sequence, and of the coding sequences that do have leaders, 53% lack a strong consensus Shine-Dalgarno site. This indicates that like M. tuberculosis, M. smegmatis can initiate translation through multiple mechanisms. Our approach also allowed us to identify over 3,000 RNA cleavage sites, which occur at a novel sequence motif. To our knowledge, this represents the first report of a transcriptome-wide RNA cleavage site map in mycobacteria. The cleavage sites show a positional bias toward mRNA regulatory regions, highlighting the importance of post-transcriptional regulation in gene expression. We show that in low oxygen, a condition associated with the host environment during infection, mycobacteria change their transcriptomic profiles and endonucleolytic RNA cleavage is markedly reduced, suggesting a mechanistic explanation for previous reports of increased mRNA half-lives in response to stress. In addition, a number of TSSs were triggered in hypoxia, 56 of which contain the binding motif for the sigma factor SigF in their promoter regions. This suggests that SigF makes direct contributions to transcriptomic remodeling in hypoxia-challenged mycobacteria. Taken together, our data provide a foundation for further study of both transcriptional and posttranscriptional regulation in mycobacteria.

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VapCs of Mycobacterium tuberculosis Cleave RNAs Essential for Translation

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