MicroRNAomes of Cattle Intestinal Tissues Revealed Possible miRNA Regulated Mechanisms Involved in Escherichia coli O157 Fecal Shedding.

Cattle have been suggested as the primary reservoirs of E. coli O157 mainly as a result of colonization of the recto-anal junction (RAJ) and subsequent shedding into the environment. Although a recent study reported different gene expression at RAJ between super-shedders (SS) and non-shedders (NS), the regulatory mechanisms of altered gene expression is unknown. This study aimed to investigate whether bovine non-coding RNAs play a role in regulating the differentially expressed (DE) genes between SS and NS, thus further influencing E. coli O157 shedding behavior in the animals through studying miRNAomes of the whole gastrointestinal tract including duodenum, proximal jejunum, distal jejunum, cecum, spiral colon, descending colon and rectum. The number of miRNAs detected in each intestinal region ranged from 390 ± 13 (duodenum) to 413 ± 49 (descending colon). Comparison between SS and NS revealed the number of differentially expressed (DE) miRNAs ranged from one (in descending colon) to eight (in distal jejunum), and through the whole gut, seven miRNAs were up-regulated and seven were down-regulated in SS. The distal jejunum and rectum were the regions where the most DE miRNAs were identified (eight and seven, respectively). The miRNAs, bta-miR-378b, bta-miR-2284j, and bta-miR-2284d were down-regulated in both distal jejunum and rectum of SS (log2fold-change: -2.7 to -3.8), bta-miR-2887 was down-regulated in the rectum of SS (log2fold-change: -3.2), and bta-miR-211 and bta-miR-29d-3p were up-regulated in the rectum of SS (log2fold-change: 4.5 and 2.2). Functional analysis of these miRNAs indicated their potential regulatory role in host immune functions, including hematological system development and immune cell trafficking. Our findings suggest that altered expression of miRNA in the gut of SS may lead to differential regulation of immune functions involved in E. coli O157 super-shedding in cattle.

Engineering dual-glycan responsive expression systems for tunable production of heterologous proteins in Bacteroides thetaiotaomicron.

Genetically engineering intestinal bacteria, such as Bacteroides thetaiotaomicron (B. theta), holds potential for creating new classes of biological devices, such as diagnostics or therapeutic delivery systems. Here, we have developed a series of B. theta strains that produce functional transgenic enzymes in response to dextran and arabinogalactan, two chemically distinct glycans. Expression systems for single glycan induction, and a novel "dual-glycan" expression system, requiring the presence of both dextran and arabinogalactan, have been developed. In addition, we have created two different chromosomal integration systems and one episomal vector system, compatible with engineered recipient strains, to improve the throughput and flexibility of gene cloning, integration, and expression in B. theta. To monitor activity, we have demonstrated the functionality of two different transgenic enzymes: NanoLuc, a luciferase, and BuGH16C, an agarase from the human intestinal bacterium, Bacteroides uniforms NP1. Together this expression platform provides a new collection of glycan-responsive tools to improve the strength and fidelity of transgene expression in B. theta and provides proof-of-concept for engineering more complex multi-glycan expression systems.

Interactions of the Hindgut Mucosa-Associated Microbiome with Its Host Regulate Shedding of Escherichia coli O157:H7 by Cattle.

Cattle are the primary carrier of Escherichia coli O157:H7, a foodborne human pathogen, and those shedding >104 CFU/gram of feces of E. coli O157:H7 are defined as supershedders (SS). This study investigated the rectoanal junction (RAJ) mucosa-associated microbiota and its relationship with host gene expression in SS and in cattle from which E. coli O157:H7 was not detected (nonshedders [NS]), aiming to elucidate the mechanisms involved in supershedding. In total, 14 phyla, 66 families, and 101 genera of RAJ mucosa-associated bacteria were identified and Firmicutes (61.5 ± 7.5%), Bacteroidetes (27.9 ± 6.4%), and Proteobacteria (5.5 ± 2.1%) were the predominant phyla. Differential abundance analysis of operational taxonomic units (OTUs) identified 2 OTUs unique to SS which were members of Bacteroides and Clostridium and 7 OTUs unique to NS which were members of Coprococcus, Prevotella, Clostridium, and Paludibacter Differential abundance analysis of predicted microbial functions (using PICRUSt [phylogenetic investigation of communities by reconstruction of unobserved states]) revealed that 3 pathways had higher abundance (log2 fold change, 0.10 to 0.23) whereas 12 pathways had lower abundance (log2 fold change, -0.36 to -0.20) in SS. In addition, we identified significant correlations between expression of 19 differentially expressed genes and the relative abundance of predicted microbial functions, including nucleic acid polymerization and carbohydrate and amino acid metabolism. Our findings suggest that differences in RAJ microbiota at both the compositional and functional levels may be associated with E. coli O157:H7 supershedding and that certain microbial groups and microbial functions may influence RAJ physiology of SS by affecting host gene expression.IMPORTANCE Cattle with fecal E. coli O157:H7 at >104 CFU per gram of feces have been defined as the supershedders, and they are responsible for the most of the E. coli O157:H7 spread into farm environment. Currently, no method is available for beef producers to eliminate shedding of E. coli O157:H7 in cattle, and the lack of information about the mechanisms of supershedding greatly impedes the development of effective methods. This study investigated the role of the rectoanal junction (RAJ) mucosa-associated microbiome in E. coli O157:H7 shedding, and our results indicated that the compositions and functions of RAJ microbiota differed between supershedders and nonshedders. The identified relationship between the differentially abundant microbes and 19 previously identified differentially expressed genes suggests the role of host-microbial interactions involved in E. coli O157:H7 supershedding. Our findings provide a fundamental understanding of the supershedding phenomenon which is essential for the development of strategies, such as the use of directly fed microbials, to reduce E. coli O157:H7 shedding in cattle.

Host mechanisms involved in cattle Escherichia coli O157 shedding: a fundamental understanding for reducing foodborne pathogen in food animal production.

The host mechanisms involved in Escherichia coli O157 super-shedding in cattle is largely unknown. In this study, the comparison of transcriptomes of intestinal tissues between super-shedders (SS) and cattle negative for E. coli O157 (NS) was performed, aiming to identify genes that are potentially associated with super-shedding. In total, 16,846 ± 639 (cecum) to 18,137 ± 696 (distal jejunum) were expressed throughout the intestine, with the expressed genes associated with immune functions more pronounced in the small intestine. In total, 351 differentially expressed (DE) genes were identified throughout the intestine between SS and NS, with 101 being up-regulated and 250 down-regulated in SS. Functional analysis revealed DE genes were involved in increased T-cell responses and cholesterol absorption in the distal jejunum and descending colon, and decreased B-cell maturation in the distal jejunum of SS. RNA-Seq based SNP discovery revealed that the mutations in seven DE genes involved in leukocyte activation and cholesterol transportation were associated with E. coli O157 shedding. Our findings suggest that T-cell responses and cholesterol metabolism in the intestinal tract may be associated with super-shedding phenomenon, and the SNPs in the DE genes are possibly associated with the observed gene expression difference between SS and NS.

Comparative Transcriptomic Analysis of Rectal Tissue from Beef Steers Revealed Reduced Host Immunity in Escherichia coli O157:H7 Super-Shedders.

Super-shedder cattle are a major disseminator of E. coli O157:H7 into the environment, and the terminal rectum has been proposed as the primary E. coli O157:H7 colonization site. This study aimed to identify host factors that are associated with the super-shedding process by comparing transcriptomic profiles in rectal tissue collected from 5 super-shedder cattle and 4 non-shedder cattle using RNA-Seq. In total, 17,859 ± 354 genes and 399 ± 16 miRNAs were detected, and 11,773 genes were expressed in all animals. Fifty-eight differentially expressed (DE) genes (false discovery rate < 0.05) including 11 up-regulated and 47 down-regulated (log 2 (fold change) ranged from -5.5 to 4.2), and 2 up-regulated DE miRNAs (log 2 (fold change) = 2.1 and 2.5, respectively) were identified in super-shedders compared to non-shedders. Functional analysis of DE genes revealed that 31 down-regulated genes were potentially associated with reduced innate and adaptive immune functions in super-shedders, including 13 lymphocytes membrane receptors, 3 transcription factors and 5 cytokines, suggesting the decreased key host immune functions in the rectal tissue of super-shedders, including decreased quantity and migration of immune cells such as lymphocytes, neutrophils and dendritic cells. The up-regulation of bta-miR-29d-3p and the down regulation of its predicted target gene, regulator of G-protein signaling 13, suggested a potential regulatory role of this miRNA in decreased migration of lymphocytes in super-shedders. Based on these findings, the rectal tissue of super-shedders may inherently exhibit less effective innate and adaptive immune protection. Further study is required to confirm if such effect on host immunity is due to the nature of the host itself or due to actions mediated by E. coli O157:H7.

Structural and biochemical analysis of a unique phosphatase from Bdellovibrio bacteriovorus reveals its structural and functional relationship with the protein tyrosine phosphatase class of phytase.

Bdellovibrio bacteriovorus is an unusual δ-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Longitudinal characterization of antimicrobial resistance genes in feces shed from cattle fed different subtherapeutic antibiotics.

BACKGROUND: Environmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA. RESULTS: The concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments. CONCLUSION: The abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.

Structures of Selenomonas ruminantium phytase in complex with persulfated phytate: DSP phytase fold and mechanism for sequential substrate hydrolysis.

Various inositide phosphatases participate in the regulation of inositol polyphosphate signaling molecules. Plant phytases are phosphatases that hydrolyze phytate to less-phosphorylated myo-inositol derivatives and phosphate. The phytase from Selenomonas ruminantium shares no sequence homology with other microbial phytases. Its crystal structure revealed a phytase fold of the dual-specificity phosphatase type. The active site is located near a conserved cysteine-containing (Cys241) P loop. We also solved two other crystal forms in which an inhibitor, myo-inositol hexasulfate, is cocrystallized with the enzyme. In the "standby" and the "inhibited" crystal forms, the inhibitor is bound, respectively, in a pocket slightly away from Cys241 and at the substrate binding site where the phosphate group to be hydrolyzed is held close to the -SH group of Cys241. Our structural and mutagenesis studies allow us to visualize the way in which the P loop-containing phytase attracts and hydrolyzes the substrate (phytate) sequentially.