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Proteomics analysis of virulent and avirulent strains of mycobacterium

ABSTRACT: label free quantification analysis was performed on four strains of mycobacterium tuberculosis. The four strains are H37Rv, H37Ra, BND and JAL. The purpose of this study is to identify strain specific proteome expression pattern among the four strains.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mycobacterium Tuberculosis

SUBMITTER: gagan jhingan

LAB HEAD: Vinay Kumar Nandicoori

PROVIDER: PXD001188 | Pride | 2016-05-10

REPOSITORIES: Pride

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			Action	DRS
	170813_Top10HCD_Mtb_BND1_SK_94.raw	Raw
	170813_Top10HCD_Mtb_BND2_SK_95.raw	Raw
	170813_Top10HCD_Mtb_BND3_SK_96.raw	Raw
	170813_Top10HCD_Mtb_BND4_SK_97.raw	Raw
	170813_Top10HCD_Mtb_JAL1_SK_98.raw	Raw

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Publications

Comparative Proteomic Analyses of Avirulent, Virulent, and Clinical Strains of Mycobacterium tuberculosis Identify Strain-specific Patterns.

Jhingan Gagan Deep GD Kumari Sangeeta S Jamwal Shilpa V SV Kalam Haroon H Arora Divya D Jain Neharika N Kumaar Lakshmi Krishna LK Samal Areejit A Rao Kanury V S KVS Kumar Dhiraj D Nandicoori Vinay Kumar VK

The Journal of biological chemistry 20160505 27

Mycobacterium tuberculosis is an adaptable intracellular pathogen, existing in both dormant as well as active disease-causing states. Here, we report systematic proteomic analyses of four strains, H37Ra, H37Rv, and clinical isolates BND and JAL, to determine the differences in protein expression patterns that contribute to their virulence and drug resistance. Resolution of lysates of the four strains by liquid chromatography, coupled to mass spectrometry analysis, identified a total of 2161 prot ...[more]

PMID: 27151218

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Project description:THP-1 macrophages were infected with four strains of Mycobacterium tuberculosis to study the temporal dynamics of newly synthesized proteins in the secretome. Temporal snapshots of secretome reflect the macrophage response to pathogenicity which in combination with intracellular events, completes the disease picture. However, such studies are compromised by limitations of quantitative proteomics. Metabolic labeling by SILAC allows a 3-plex experiment while isobaric chemical labeling by iTRAQ/TMT allows up to 8 to 10-plex respectively. This makes studying temporal proteome dynamics an intangible and elusive proposition. We have developed a new variant of hyperplexing method, combining triplex SILAC with 6-plex iTRAQ to achieve 18-plex quantitation in a single MS run. THP-1 macrophages were infected with H37Ra, H37Rv, BND433 and JAL2287 and the newly synthesized secreted host proteins were studied over six temporal frame still 30 hours post infection, at a difference of 4 hours each. For quantitation, the strains were encoded with two sets of triple SILAC- H37Ra & H37Rv in one and BND433 & JAL2287 in another with a control in each. These sets were then iTRAQ labeled to encode for temporal profiles across six time points in 6-plex iTRAQ. Effectively a 36-plex design with 4 replicates of each set, these experiments were completed within few days on the mass spectrometer. Using MaxQuant and in house developed tools and pipelines, we have analysed the data to map the temporal and strain specific dynamics of newly synthesized proteins in host. Hyperplexing enables large scale spatio-temporal systems biology studies where large number of samples can be processed simultaneously and in quantitative manner.

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Project description:M. tuberculosis H37Ra, an avirulent tubercle bacillus, is a commonly used model to investigate virulence attenuation in M. tuberculosis. Comparative high-throughput studies have earlier correlated its avirulence to the presence of specific mutations or absence of certain proteins. However, a recent sequencing study of H37Ra has disproved several genomic differences earlier reported to be associated with virulence. This warrants further investigations on the H37Ra proteome as well. In this study, we carried out an integrated analysis of the genome, transcriptome and proteome of H37Ra. In addition to confirming single nucleotide variations and insertion-deletions that were reported earlier, our study provides novel insights into the mutation spectrum in the promoter regions of 7 genes. In all, we provide protein coding evidence for 3,199 proteins representing ~79% of the total predicted gene count. Transcriptome analysis revealed the expression of 3,945 high-confidence transcripts including several transcripts mapping to the genome that were previously thought to be non-coding. We identified 9 genes whose coding potential was hitherto reported to be absent in H37Ra . These include 2 putative virulence factors belonging to ESAT-6 like family of proteins. Furthermore, proteogenomic analysis enabled us to identify 63 novel proteins coding genes and correct 25 existing gene models in H37Ra genome. A majority of these were found to be conserved in the virulent strain H37Rv as well as in other mycobacterial sp. suggesting that that the differences in the virulent and avirulent strains of M. tuberculosis are not entirely dependent on the expression of certain proteins or their absence but may possibly be ascertained to functional changes.

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