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The identification of bacterial protein interaction partners pointed out to new intracellular functions of Francisella tularensis glyceraldehyde-3-phosphate dehydrogenase

ABSTRACT: Glyceraldehyde-3-phopshate dehydrogenase (GAPDH) is well-known for its involvement in numerous non-metabolic processes inside the mammalian cells. Alternative functions of prokaryotic GAPDH are mainly deduced from its extracellular localization and the ability to bind to selected host proteins. Data on its participation in intracellular bacterial processes are scarce as there is so far only one study dealing with this issue. We previously reported several evidence that the GAPDH homologue of Francisella tularensis, GapA, might also exert additional non-enzymatic functions. To follow up our former observation, here we decided to find out GapA’s interacting partners within the bacterial proteome to explore its new roles at the intracellular level. The quantitative proteomics approach based on stable isotope labelling of amino acids in cell culture (SILAC) in combination with affinity purification and mass spectrometry enabled us to identify 18 proteins potentially interacting with GapA, six of those interactions were further confirmed by alternative methods. Half of the identified proteins were involved in non-metabolic processes. Further analysis together with the quantitative label-free comparative analysis of proteomes isolated from the wild-type strain and strain with deleted gapA gene suggests that the GapA is implicated in DNA repair processes. The absence of GapA promotes secretion of its most potent interaction partner, the hypothetical protein with peptidase propeptide domain (PepSY) indicating its impact on subcellular distribution of some proteins.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Francisella Tularensis Subsp. Holarctica Fsc200 Bacteria

DISEASE(S): Tularemia

SUBMITTER: Pavel Rehulka

LAB HEAD: Jiri Stulik

PROVIDER: PXD019739 | Pride | 2020-08-31

REPOSITORIES: Pride

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			Action	DRS
	01162019_05_ML_MK_Pet1_1.raw	Raw
	01162019_07_ML_MK_Pet1_2.raw	Raw
	01162019_09_ML_MK_Pet1_3.raw	Raw
	01162019_11_ML_MK_Pet1_4.raw	Raw
	07292019_177_AF_15-19-IP_wtWCL_1.raw	Raw

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Project description:The intestinal microbiota plays a crucial role in protecting the host from pathogenic microbes, in modulating immunity and in regulating metabolic processes. We use the simplified human intestinal microbiota (SIHUMIx) consisting of eight bacterial species to study potential synergistic effects with a particular focus on detecting novel proteins with less than 100 amino acids (= sProteins), some of which may contribute to regulate the simplified human intestinal microbiota. Although several studies have shown that sProteins carry out a wide range of important functions, they are still often missed in genome annotations, and little is known about their structure and function in individual microbes and especially in microbial communities. In this study, we created a multi-species integrated proteogenomics search database (multi-species iPtgxDB) to enable a comprehensive identification of novel sProteins. Six of the eight SIHUMIx species, for which no complete genomes were available, were first sequenced and de novo assembled. Several proteomics approaches including two earlier optimized sProtein enrichment strategies were applied prior to mass spectrometric analysis to specifically increase the chances for novel sProtein discovery. Searching the MS/MS data against the multi-species iPtgxDB enabled us to identify 31 novel sProteins, of which the expression of 30 was supported by metatranscriptomics data. Using synthetic peptides, we were able to validate the expression of 25 novel sProteins. Importantly, when comparing the expression of these novel sProteins in single strain cultivations to the multi-species community grown in a bioreactor, we found that six of them were only identified in the SIHUMIx community, indicating a possible important role of sProteins in the organization of microbial communities. Furthermore, in silico prediction suggested that two of these novel sProteins have a potential antimicrobial function. We outline an integrated experimental and bioinformatics workflow for the discovery of novel sProteins in microbial communities that can be generically applied to other microbial communities. The further analysis of novel sProteins uniquely expressed in the multi-species community is expected to enable new insights into the structure, regulation and function of bacterial communities such as those of the human intestinal tract.

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The identification of bacterial protein interaction partners pointed out to new intracellular functions of Francisella tularensis glyceraldehyde-3-phosphate dehydrogenase

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