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The rumen microbiome: an underexplored resource for novel antimicrobial discovery.

ABSTRACT: Antimicrobial peptides (AMPs) are promising drug candidates to target multi-drug resistant bacteria. The rumen microbiome presents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including AMPs. Using functional screening and computational approaches, we identified 181 potentially novel AMPs from a rumen bacterial metagenome. Here, we show that three of the selected AMPs (Lynronne-1, Lynronne-2 and Lynronne-3) were effective against numerous bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). No decrease in MRSA susceptibility was observed after 25 days of sub-lethal exposure to these AMPs. The AMPs bound preferentially to bacterial membrane lipids and induced membrane permeability leading to cytoplasmic leakage. Topical administration of Lynronne-1 (10% w/v) to a mouse model of MRSA wound infection elicited a significant reduction in bacterial counts, which was comparable to treatment with 2% mupirocin ointment. Our findings indicate that the rumen microbiome may provide viable alternative antimicrobials for future therapeutic application.

SUBMITTER: Oyama LB

PROVIDER: S-EPMC5711939 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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The rumen microbiome: an underexplored resource for novel antimicrobial discovery.

Oyama Linda B LB Girdwood Susan E SE Cookson Alan R AR Fernandez-Fuentes Narcis N Privé Florence F Vallin Hannah E HE Wilkinson Toby J TJ Golyshin Peter N PN Golyshina Olga V OV Mikut Ralf R Hilpert Kai K Richards Jennifer J Wootton Mandy M Edwards Joan E JE Maresca Marc M Perrier Josette J Lundy Fionnuala T FT Luo Yu Y Zhou Mei M Hess Matthias M Mantovani Hilario C HC Creevey Christopher J CJ Huws Sharon A SA

NPJ biofilms and microbiomes 20171201

Antimicrobial peptides (AMPs) are promising drug candidates to target multi-drug resistant bacteria. The rumen microbiome presents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including AMPs. Using functional screening and computational approaches, we identified 181 potentially novel AMPs from a rumen bacterial metagenome. Here, we show that three of the selected AMPs (Lynronne-1, Lynronne-2 and Lynronne-3) were effective against numerous bacterial path ...[more]

PMID: 29214045

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Project description:BackgroundCarbohydrate-active enzymes (CAZymes) form the most widespread and structurally diverse set of enzymes involved in the breakdown, biosynthesis, or modification of lignocellulose that can be found in living organisms. However, the structural diversity of CAZymes has rendered the targeted discovery of novel enzymes extremely challenging, as these proteins catalyze many different chemical reactions and are sourced by a vast array of microbes. Consequently, many uncharacterized members of CAZyme families of interest have been overlooked by current methodologies (e.g., metagenomic screening) used to discover lignocellulolytic enzymes.ResultsIn the present study, we combined phenotype-based selective pressure on the rumen microbiota with targeted functional profiling to guide the discovery of unknown CAZymes. In this study, we found 61 families of glycoside hydrolases (GH) (out of 182 CAZymes) from protein sequences deposited in the CAZy database-currently associated with more than 20,324 microbial genomes. Phenotype-based selective pressure on the rumen microbiome showed that lignocellulolytic bacteria (e.g., Fibrobacter succinogenes, Butyrivibrio proteoclasticus) and three GH families (e.g., GH11, GH13, GH45) exhibited an increased relative abundance in the rumen of feed efficient cattle when compared to their inefficient counterparts. These results paved the way for the application of targeted functional profiling to screen members of the GH11 and GH45 families against a de novo protein reference database comprised of 1184 uncharacterized enzymes, which led to the identification of 18 putative xylanases (GH11) and three putative endoglucanases (GH45). The biochemical proof of the xylanolytic activity of the newly discovered enzyme validated the computational simulations and demonstrated the stability of the most abundant xylanase.ConclusionsThese findings contribute to the discovery of novel enzymes for the breakdown, biosynthesis, or modification of lignocellulose and demonstrate that the rumen microbiome is a source of promising enzyme candidates for the biotechnology industry. The combined approaches conceptualized in this study can be adapted to any microbial environment, provided that the targeted microbiome is easy to manipulate and facilitates enrichment for the microbes of interest. Video Abstract.

Project description:Background:Cellulosic biomass has great potential as a renewable biofuel resource. Robust, high-performance enzymes are needed to effectively utilize this valuable resource. In this study, metatranscriptomics was used to explore the carbohydrate-active enzymes (CAZymes), especially glycoside hydrolases (GHs), present in the rumen microbiome of Hu sheep. Select CAZymes were experimentally verified and characterized after cloning and expression in E. coli. Results:The metatranscriptomes of six Hu sheep rumen microbiomes yielded 42.3 Gbp of quality-checked sequence data that represented in total 2,380,783 unigenes after de novo assembling using Trinity and clustered with CD-HIT-EST. Annotation using the CAZy database revealed that 2.65% of the unigenes encoded GHs, which were assigned to 111 different CAZymes families. Firmicutes (18.7%) and Bacteroidetes (13.8%) were the major phyla to which the unigenes were taxonomically assigned. In total, 14,489 unigenes were annotated to 15 cellulase-containing GH families, with GH3, GH5 and GH9 being the predominant. From these putative cellulase-encoding unigenes, 4225 open reading frames (ORFs) were predicted to contain 2151 potential cellulase catalytic modules. Additionally, 147 ORFs were found to encode proteins that contain carbohydrate-binding modules (CBMs). Heterogeneous expression of 30 candidate cDNAs from the GH5 family in E. coli BL21 showed that 17 of the tested proteins had endoglucanase activity, while 7 exhibited exoglucanase activity. Interestingly, two of the GH5 proteins (Cel5A-h28 and Cel5A-h11) showed high specific activity against carboxymethylcellulose (CMC) and p-nitrophenyl-?-d-cellobioside (pNPC) (222.2 and 142.8 U/mg), respectively. The optimal pH value for activity of Cel5A-h11 and Cel5A-h28 was 6.0 for both enzymes, and optimal temperatures were 40 and 50 °C, respectively. Both enzymes retained over 70 and 60%, respectively, of their original activities after incubation at 40 °C for 60 min. However, their activities were rapidly diminished upon exposure to higher temperatures. Cel5A-h11 and Cel5A-h28 retained more than 80 and 60% of their maximal enzymatic activities after incubation for 16 h in buffered solutions in the pH range from 4.0 to 9.0. Conclusion:The metatranscriptomic results revealed that the rumen microbiome of Hu sheep encoded a repertoire of new enzymes capable of cellulose degradation and metatranscriptomics was an effective method to discover novel cellulases for biotechnological applications.

Project description:BackgroundMany countries have already banned the use of antibiotics in animal husbandry, making it extremely difficult to maintain animal health in livestock breeding. In the livestock industry, there is an urgent need to develop alternatives to antibiotics which will not lead to drug resistance on prolonged use. In this study, eighteen castrated bulls were randomly divided into two groups. The control group (CK) was fed the basal diet, while the antimicrobial peptide group (AP) was fed the basal diet supplemented with 8 g of antimicrobial peptides in the basal diet for the experimental period of 270 d. They were then slaughtered to measure production performance, and the ruminal contents were isolated for metagenomic and metabolome sequencing analysis.ResultThe results showed that antimicrobial peptides could improve the daily weight, carcass weight, and net meat weight of the experimental animals. Additionally, the rumen papillae diameter and the micropapillary density in the AP were significantly greater than those in the CK. Furthermore, the determination of digestive enzymes and fermentation parameters showed that the contents of protease, xylanase, and β-glucoside in the AP were greater than those in the CK. However, lipase content in the CK was greater than that in the AP. Moreover, the content of acetate, propionate, butyrate, and valerate was found to be greater in AP than those in CK. The metagenomic analysis annotated 1993 differential microorganisms at the species level. The KEGG enrichment of these microorganisms revealed that the enrichment of drug resistance-related pathways was dramatically decreased in the AP, whereas the enrichment of immune-related pathways was significantly increased. There was also a significant reduction in the types of viruses in the AP. 187 probiotics with significant differences were found, 135 of which were higher in AP than in CK. It was also found that the antimicrobial mechanism of the antimicrobial peptides was quite specific. Seven low-abundance microorganisms (Acinetobacter_sp._Ac_1271, Aequorivita soesokkakensis, Bacillus lacisalsi, Haloferax larsenii, Lysinibacillus_sp._3DF0063, Parabacteroides_sp._2_1_7, Streptomyces_sp._So13.3) were found to regulate growth performance of the bull negatively. Metabolome analysis identified 45 differentially differential metabolites that significantly different between the CK and the AP groups. Seven upregulated metabolites (4-pyridoxic acid, Ala-Phe, 3-ureidopropionate, hippuric acid, terephthalic acid, L-alanine, uridine 5-monophosphate) improve the growth performance of the experimental animals. To detect the interactions between the rumen microbiome and metabolism, we associated the rumen microbiome with the metabolome and found that negative regulation between the above 7 microorganisms and 7 metabolites.ConclusionsThis study shows that antimicrobial peptides can improve the growth performance of animals while resisting viruses and harmful bacteria and are expected to become healthy alternatives to antibiotics. We demonstrated a new antimicrobial peptides pharmacological model. We demonstrated low-abundance microorganisms may play a role by regulating the content of metabolites.

Dataset Information

The rumen microbiome: an underexplored resource for novel antimicrobial discovery.

Publications

The rumen microbiome: an underexplored resource for novel antimicrobial discovery.

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