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Negative N-terminal enrichment strategy for Mycobacterium tuberculosis H37Rv

ABSTRACT: Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB), which resulted millions of deaths worldwide every year. Although the type strain M. tuberculosis H37Rv was sequenced in 1998, annotation errors of encoding genes have emerged in an endless stream. The annotation errors are particularly severe in the N-terminal and start sites of the genes. In this study, we applied a dimethylation labeling combined with negative enrichment strategy on M. tuberculosis H37Rv to characterize the N-terminal start sites of its annotated encoding genes. Totally, 18,285 peptides and 1,641 N-terminal peptides were identified from all the 2,728 database annotated proteins. The negative N-terminal enrichment strategy allowed the re-annotation of 12 genes’ N-terminal and identification of 6 novel genes in H37Rv. The comparative genomics approach could provide possible help for the re-annotation of 403 mis-annotated genes from Mycobacteriaceae. In addition, we verified the novel gene Rv3409, which was identified with 3 high-confident peptides, with the synthetic peptides and the expression of recombinant protein Rv3409. Altogether, our study and findings contributed to a better understanding of the N-terminal annotation of H37Rv, which will contribute to further biological studies of other species of Mycobacteriaceae in the future.

ORGANISM(S): Mycobacterium Tuberculosis H37rv

SUBMITTER: Ping Xu

PROVIDER: PXD036249 | iProX | Wed Aug 24 00:00:00 BST 2022

REPOSITORIES: iProX

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Project description:Proteogenomics, the combination of proteomics, genomics and transcriptomics, has considerably improved genome annotation in under-studied phylogenetic groups, where homology information is missing. Yet, it can also be advantageous when re-investigating well-annotated genomes. Here, we apply an advanced proteogenomics approach, combining standard proteogenomics with peptide de novo sequencing, to refine annotation of the well-studied model fungus Sordaria macrospora. We investigated samples from different developmental and physiological conditions, resulting in detection of 104 hidden proteins and annotation changes in 575 genes, including 389 splice site refinements. Significantly, our approach provides peptide-level evidence for 113 single amino acid variations and 15 C-terminal protein elongations originating from A-to-I RNA editing, a phenomenon recently detected in fungi. Co-expression and phylostratigraphic analysis of the refined proteome suggests new functions in evolutionary young genes correlated with distinct developmental stages. In conclusion, our advanced proteogenomics approach is highly supportive to promote functional studies of model systems. Proteogenomics, the combination of proteomics, genomics and transcriptomics, has considerably improved genome annotation in under-studied phylogenetic groups, where homology information is missing. Yet, it can also be advantageous when re-investigating well-annotated genomes. Here, we apply an advanced proteogenomics approach, combining standard proteogenomics with peptide de novo sequencing, to refine annotation of the well-studied model fungus Sordaria macrospora. We investigated samples from different developmental and physiological conditions, resulting in detection of 104 hidden proteins and annotation changes in 575 genes, including 389 splice site refinements. Significantly, our approach provides peptide-level evidence for 113 single amino acid variations and 15 C-terminal protein elongations originating from A-to-I RNA editing, a phenomenon recently detected in fungi. Co-expression and phylostratigraphic analysis of the refined proteome suggests new functions in evolutionary young genes correlated with distinct developmental stages. In conclusion, our advanced proteogenomics approach is highly supportive to promote functional studies of model systems.

Project description:Background The generalist dipteran pupal parasitoid Nasonia vitripennis injects 79 venom peptides into the host before egg laying. This venom induces several important changes in the host, including developmental arrest, immunosuppression, and alterations to normal metabolism. It is hoped that diverse and potent bioactivities of N. vitripennis venom provide an opportunity for the design of novel acting drugs. However, currently very little is known about the individual functions of N. vitripennis venom peptides and less than half can be bioinformatically annotated. The paucity of annotation information complicates the design of studies that seek to better understand the potential mechanisms underlying the envenomation response. Although the RNA interference system of N. vitripennis provides an opportunity to functionally characterise venom encoding genes, with 79 candidates this represents a daunting task. For this reason we were interested in determining the expression levels of venom encoding genes in the venom gland, such that this information could be used to rank candidate venoms. To do this we carried out deep sequencing of the transcriptome of the venom gland and neighbouring ovary tissue and used RNA-seq to measure expression from the 79 venom encoding genes. The generation of a specific venom gland transcriptome dataset also provides further opportunities to investigate novel features of this highly specialised organ. Results High throughput sequencing and RNA-seq revealed that the highest expressed venom encoding gene in the venom gland was a serine protease called Nasvi2EG007167, which has previously been implicated in the apoptotic activity of N. vitripennis venom. As expected the RNA-seq confirmed that the N. vitripennis venom encoding genes are almost exclusively expressed in the venom gland relative to the neighbouring ovary tissue. Novel peptides appear to perform key roles in N. vitripennis venom function as only four of the highest 15 expressed venom encoding genes are bioinformatically annotationed. The high throughput sequencing data also provided evidence for the existence of an additional 471 novel genes in the Nasonia genome that are expressed in the venom gland and ovary. Finally, metagenomic analysis of venom gland transcripts identified viral transcripts that may play an important part in the N. vitripennis venom function. Conclusions The expression level information provided here for the 79 venom encoding genes provides an unbiased dataset that can be used by the N. vitripennis community to identify high value candidates for further functional characterisation. These candidates represent bioactive peptides that have value in drug development pipelines.

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Negative N-terminal enrichment strategy for Mycobacterium tuberculosis H37Rv

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