Dataset Information

Small proteins in a simplified human gut microbiome

ABSTRACT: Small proteins (sProteins) with a size of 100 amino acids and less are involved in major biological processes and play an important role in different bacteria. Despite the increasing interest in them, the data on sProteins in bacterial communities, like the human gut microbiome is sparse. A reason for this is the technical challenge in the detection and identification of sProteins using bottom-up proteomics. In this study, we are using the simplified human gut microbiome (SIHUMIx) as model system of the human gut microbiome to detect sProteins and to compare different sProtein enrichment methods. We observed that with our tested methods, the C8-cartridge enrichment resulted in the highest number of detected sProteins (n=295) with high reproducibility among replication analysis. However, in order to further increase the total number of sProteins, the combination of C8 cartridge enrichment with GelFree enrichment is favored because the latter complemented n=48 more sProteins compared to the C8 cartridge approach resulting in n= 343 sProteins. Among all detected sProteins we were able to identify 79 so far uncharacterized sProteins, with no described protein evidence in the current released database. In total, 34 of those uncharacterized sProteins are localized in gene clusters conserved between different bacteria species allowing functional predictions. This study improves the assessment of sProtein detection and enables their functional characterization in future experiments.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Erysipelatoclostridium Ramosum Dsm 1402 Blautia Producta Atcc 27340 = Dsm 2950 Clostridium Butyricum Dsm 10702 Lactobacillus Plantarum Subsp. Plantarum Anaerostipes Caccae Dsm 14662 Bifidobacterium Longum Ncc2705 Escherichia Coli Str. K-12 Substr. Mg1655 Bacteroides Thetaiotaomicron (strain Atcc 29148 / Dsm 2079 / Nctc 10582 / E50 / Vpi-5482)

SUBMITTER: Nico Jehmlich

LAB HEAD: Nico Jehmlich

PROVIDER: PXD016298 | Pride | 2020-01-07

REPOSITORIES: Pride

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Enrichment and identification of small proteins in a simplified human gut microbiome.

Petruschke Hannes H Anders John J Stadler Peter F PF Jehmlich Nico N von Bergen Martin M

Journal of proteomics 20191210

Small proteins (sProteins) with a size of 100 amino acids and less are involved in major biological processes and play an important role in different bacteria. Despite the increasing interest in them, the data on sProteins in bacterial communities, like the human gut microbiome is sparse. In this study, we are using the extended simplified human intestinal microbiota (SIHUMIx) as model system of the human gut microbiome to detect sProteins and to compare different sProtein enrichment methods. We ...[more]

PMID: 31841667

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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.

Project description:Abstract: The crenarchaeal order Sulfolobales collectively contains at least five major terminal oxidase complexes. Based on genome sequence information, all five complexes are found only in Metallosphaera sedula and Sulfolobus tokodaii, the two sequenced Sulfolobales capable of iron oxidization. While specific respiratory complexes in certain Sulfolobales have been characterized previously as proton pumps for maintaining intracellular pH and generating proton motive force (pmf), their contribution to sulfur and iron biooxidation has not been considered. For M. sedula growing in the presence of ferrous iron and reduced inorganic sulfur compounds (RISCs), global transcriptional analysis was used to track the response of specific genes associated with these complexes, as well as other known and putative respiratory electron transport chain elements. ORFs from all five terminal oxidase or bc1-like complexes were stimulated on one or more conditions tested. Components of the fox (Msed0467-0489) and soxNL-cbsABA (Msed0500-0505) terminal/quinol oxidase clusters were triggered by ferrous iron, while the soxABCDD' terminal oxidase cluster (Msed0285-0291) were induced by tetrathionate and S°. Chemolithotrophic electron transport elements, including a putative tetrathionate hydrolase (Msed0804), a novel polysulfide/sulfur/DMSO reductase-like complex (Msed0812-0818), and a novel heterodisulfide reductase-like complex (Msed1542-1550), were also stimulated by RISCs. Furthermore, several hypothetical proteins were found to have strong responses to ferrous iron or RISCs, suggesting additional candidates in iron or sulfur oxidation-related pathways. From this analysis, a comprehensive model for electron transport in M. sedula could be proposed as the basis for examining specific details of iron and sulfur oxidation in this bioleaching archaeon. 5-slide loop of Mse cells includes 5 conditions tested: yeast exact (Y), yeast extract + ferrous sulfate (YFS), yeast extract + potassium sulfate (YKS), yeast extract + potassium tetrathionate (YKT), and yeast extract + elemental sulfur (YS). Half of an RNA sample for one condition was labeled with Cy3 while the other half was labeled with Cy5. The two differently labeled samples were run on different slides. Each probe is spotted on each slide 5 times (5 replicates; spot intensities for all replicates on slide provided in associated raw data file).

			Action	DRS
	09-02-2019_S8_1_C8_F.msf	Msf
	09-02-2019_S8_1_C8_F1.raw	Raw
	09-02-2019_S8_1_C8_F2.raw	Raw
	09-02-2019_S8_1_SP3.msf	Msf
	09-02-2019_S8_1_SP3.raw	Raw