Exploring the Bile Stress Response of Lactobacillus mucosae LM1 through Exoproteome Analysis.

Lactobacillus sp. have long been studied for their great potential in probiotic applications. Recently, proteomics analysis has become a useful tool for studies on potential lactobacilli probiotics. Specifically, proteomics has helped determine and describe the physiological changes that lactic acid bacteria undergo in specific conditions, especially in the host gut. In particular, the extracellular proteome, or exoproteome, of lactobacilli contains proteins specific to host- or environment-microbe interactions. Using gel-free, label-free ultra-high performance liquid chromatography tandem mass spectrometry, we explored the exoproteome of the probiotic candidate Lactobacillus mucosae LM1 subjected to bile treatment, to determine the proteins it may use against bile stress in the gut. Bile stress increased the size of the LM1 exoproteome, secreting ribosomal proteins (50S ribosomal protein L27 and L16) and metabolic proteins (lactate dehydrogenase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate dehydrogenases, among others) that might have moonlighting functions in the LM1 bile stress response. Interestingly, membrane-associated proteins (transporters, peptidase, ligase and cell division protein ftsH) were among the key proteins whose secretion were induced by the LM1 bile stress response. These specific proteins from LM1 exoproteome will be useful in observing the proposed bile response mechanisms via in vitro experiments. Our data also reveal the possible beneficial effects of LM1 to the host gut.

Protective effects of Bacillus subtilis against Salmonella infection in the microbiome of Hy-Line Brown layers.

OBJECTIVE: This study investigated the effects of Bacillus subtilis CSL2 (B. subtilis CSL2) administration before Salmonella challenge on the fecal microbiota and microbial functionality of Hy-line Brown (HLB) laying hens. METHODS: Fecal samples were collected from control (CON), Salmonella-infected (SAL) and Salmonella-infected, probiotic-treated (PRO) groups before and after Salmonella challenge for microbiome analysis using 16S rRNA gene pyrosequencing. RESULTS: Infection with Salmonella led to decreased microbial diversity in hen feces; diversity was recovered with Bacillus administration. In addition, Salmonella infection triggered significant alterations in the composition of the fecal microbiota. The abundance of the phylum Firmicutes decreased while that of Proteobacteria, which includes a wide variety of pathogens, increased significantly. Bacillus administration resulted in normal levels of abundance of Firmicutes and Proteobacteria. Analysis of bacterial genera showed that Salmonella challenge decreased the population of Lactobacillus, the most abundant genus, and increased populations of Pseudomonas and Flavobacterium genera by a factor of 3 to 5. On the other hand, Bacillus administration caused the abundance of the Lactobacillus genus to recover to control levels and decreased the population of Pseudomonas significantly. Further analysis of operational taxonomic units revealed a high abundance of genes associated with two-component systems and secretion systems in the SAL group, whereas the PRO group had more genes associated with ribosomes. CONCLUSION: The results of this study indicate that B. subtilis CSL2 administration can modulate the microbiota in HLB laying hens, potentially acting as a probiotic to protect against Salmonella Gallinarum infection.

Barcoded pyrosequencing-based metagenomic analysis of the faecal microbiome of three purebred pig lines after cohabitation.

The microbial communities in the pig gut perform a variety of beneficial functions. Along with host genetics and diet, farm management practices are an important aspect of agricultural animal production that could influence gut microbial diversity. In this study, we used barcoded pyrosequencing of the V1-V3 regions of the 16S ribosomal RNA (rRNA) genes to characterise the faecal microbiome of three common commercial purebred pig lines (Duroc, Landrace and Yorkshire) before and after cohabitation. The diversity of faecal microbiota was characterised by employing phylogenetic, distance-based and multivariate-clustering approaches. Bacterial diversity tended to become more uniform after mixing of the litters. Age-related shifts were also observed at various taxonomic levels, with an increase in the proportion of the phylum Firmicutes and a decrease in Bacteroidetes over time, regardless of the purebred group. Cohabitation had a detectable effect on the microbial shift among purebred pigs. We identified the bacterial genus Parasutterella as having utility in discriminating pigs according to time. Similarly, Dialister and Bacteroides can be used to differentiate the purebred lines used. The microbial communities of the three purebred pigs became more similar after cohabitation, but retained a certain degree of breed specificity, with the microbiota of Landrace and Yorkshire remaining distinct from that of their distant relative, Duroc.

Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing.

This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1-V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs.

Proteomic and transcriptional analysis of Lactobacillus johnsonii PF01 during bile salt exposure by iTRAQ shotgun proteomics and quantitative RT-PCR.

Lactobacillus johnsonii PF01 has been reported to be highly resistant to bile, a key property of probiotic microorganisms. Here, we examine the nature of the bile-salt tolerance of L. johnsonii PF01. Growth inhibition and surface morphology and physiology aberrations were observed after overnight exposure to bile stress. Quantitative proteomic profiles using iTRAQ-LC-MS/MS technology identified 8307 peptides from both untreated PF01 cells and those exposed to 0.1%, 0.2%, and 0.3% bile salts. Some 215 proteins exhibited changed levels in response to bile stress; of these, levels of 94 peptides increased while those of 121 decreased. These were classified into the following categories: stress responses, cell division, transcription, translation, nucleotide metabolism, carbohydrate transport and metabolism, cell wall biosynthesis, and amino acid biosynthesis, and 16 of unidentified function. Analysis of the mRNA expression of selected genes by quantitative reverse transcriptase-PCR verified the proteomic data. Both proteomic and mRNA data provided evidence for increased phosphotransferase activity and cell wall biosynthesis. In addition, three bile salt hydrolases were significantly upregulated by bile exposure. These findings provide a basis for future evaluations of the tolerance of potential probiotic strains toward the various gastrointestinal challenges, including bile stress.

Trace metals and animal health: Interplay of the gut microbiota with iron, manganese, zinc, and copper.

Metals such as iron, manganese, copper, and zinc are recognized as essential trace elements. These trace metals play critical roles in development, growth, and metabolism, participating in various metabolic processes by acting as cofactors of enzymes or providing structural support to proteins. Deficiency or toxicity of these metals can impact human and animal health, giving rise to a number of metabolic and neurological disorders. Proper breakdown, absorption, and elimination of these trace metals is a tightly regulated process that requires crosstalk between the host and these micronutrients. The gut is a complex system that serves as the interface between these components, but other factors that contribute to this delicate interaction are not well understood. The gut is home to trillions of microorganisms and microbial genes (the gut microbiome) that can regulate the metabolism and transport of micronutrients and contribute to the bioavailability of trace metals through their assimilation from food sources or by competing with the host. Furthermore, deficiency or toxicity of these metals can modulate the gut microenvironment, including microbiota, nutrient availability, stress, and immunity. Thus, understanding the role of the gut microbiota in the metabolism of manganese, iron, copper, and zinc, as well as in heavy metal deficiencies and toxicities, and vice versa, may provide insight into developing improved or alternative therapeutic strategies to address emerging health concerns. This review describes the current understanding of how the gut microbiome and trace metals interact and affect host health, particularly in pigs.

Exoproteome Perspective on the Bile Stress Response of Lactobacillus johnsonii.

Probiotics must not only exert a health-promoting effect but also be capable of adapting to the harsh environment of the gastrointestinal (GI) tract. Probiotics in the GI tract must survive the cell wall-disrupting effect of bile acids. We investigated the exoproteome of Lactobacillus johnsonii PF01 and C1-10 under bile stress. A comparative analysis revealed the similarities between the two L. johnsonii exoproteomes, as well as their different responses to bile. The large number of metabolic proteins in L. johnsonii revealed its metabolic adaptation to meet protein synthesis requirements under bile stress. In addition, cell wall modifications occurred in response to bile. Furthermore, some extracellular proteins of L. johnsonii may have moonlighting function in the presence of bile. Enolase, L-lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, 50s ribosomal protein L7/L12, and cellobiose-specific phosphotransferase system (PTS) sugar transporter were significantly upregulated under bile stress, suggesting a leading role in the collective bile stress response of L. johnsonii from its exoproteome perspective.

Association between the body weight of growing pigs and the functional capacity of their gut microbiota.

Gastrointestinal microbiota impact host's biological activities, including digestion of indigestible feed components, energy harvest, and immunity. In this study, fecal microbiota of high body weight (HW) and low body weight (LW) growing pigs at 103 days of age were compared. Principal coordinates analysis separated the HW and LW groups into two clusters, indicating their potential differences between microbial community composition. Although the abundances of two major phyla, Firmicutes and Bacteroidetes, did not significantly differ between the HW and LW groups, some genera showed significant differences. Among them, Peptococcus and Eubacterium exhibited strong positive correlations with body weight (BW) and average daily gain (ADG) (Rho > 0.40), whereas Treponema, Desulfovibrio, Parabacteroides, and Ruminococcaceae_unclassified exhibited strong negative correlations with BW and ADG (Rho < -0.40). Based on these results, the structure of intestinal microbiota may affect growth traits in pigs through host-microbe interactions. Further in-depth studies will provide insights into how best to reshape host-microbe interactions in pigs and other animals as well.

Valproate and sodium butyrate attenuate manganese-decreased locomotor activity and astrocytic glutamate transporters expression in mice.

Manganese (Mn) is an essential trace element, but chronic overexposure to this metal, either environmentally or occupationally may cause manganism, a disease analogous to Parkinson's disease. Inhibitors of histone deacetylases, such as valproic acid (VPA) and sodium butyrate (NaB) exert neuroprotective effects in various animal models of neurological disorders. Thus, the present study investigated whether VPA or NaB prevent Mn-induced neurotoxicity by assessing locomotor activities and expression of astrocytic glutamate transporters, glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST), in C57BL/6 mice. C57BL/6 mice were pretreated with VPA (200mg/kg, i.p.) or NaB (1200mg/kg, i.p.) prior to intranasal instillation of Mn (30mg/kg) continually for 21days, followed by open-field and rota-rod behavioral tests and analyses of astrocytic glutamate transporters GLT-1 and GLAST protein/mRNA levels. The results showed that Mn significantly decreased locomotor activity as determined by total distance travelled, stereotypic and ambulatory counts. Mn also significantly decreased rota-rod activity reflecting altered motor coordination. Pretreatment with VPA and NaB with Mn reversed the effects of Mn on the locomotor activity and motor coordination. VPA and NaB also attenuated the Mn-induced decrease in GLT-1 and GLAST mRNA and protein levels in the cerebral cortical and cerebellar regions of mice. These results suggest that VPA and NaB exert protective effects against Mn toxicity seem in vitro are also shown in vivo. VPA and NaB pretreatment in mice enhancing astrocytic glutamate transporter GLT-1 expression as well as locomotor activities. Future research endeavors are warranted to determine if the therapeutic potential of VPA and NaB is via common molecular mechanism, namely, inhibition of histone deacetylases.

Proteomic View of the Crosstalk between Lactobacillus mucosae and Intestinal Epithelial Cells in Co-culture Revealed by Q Exactive-Based Quantitative Proteomics.

Lactobacilli are bacteria that are beneficial to host health, but information on communication between Lactobacilli and host cells in the intestine is lacking. In this study, we examined the proteomes of the Lactobacillus mucosae strain LM1, as a model of beneficial bacteria, and the intestinal porcine epithelial cell line (IPEC-J2) after co-culture. Label-free proteomics demonstrated the high-throughput capability of the technique, and robust characterization of the functional profiles and changes in the bacteria and intestinal cells was achieved in pure and mixed cultures. After co-culture, we identified totals of 376 and 653 differentially expressed proteins in the LM1 and IPEC-J2 proteomes, respectively. The major proteomic changes in the LM1 strain occurred in the functional categories of transcription, general function, and translation, whereas those in IPEC-J2 cells involved metabolic and cellular processes, and cellular component organization/biogenesis. Among them, elongation factor Tu, glyceraldehyde 3-phosphate dehydrogenase, and phosphocarrier protein HPr, which are known to be involved in bacterial adhesion, were upregulated in LM1. In contrast, proteins involved in tight junction assembly, actin organization, and genetic information processing (i.e., histones and signaling pathways) were significantly upregulated in IPEC-J2 cells. Furthermore, we identified functional pathways that are possibly involved in host-microbe crosstalk and response. These findings will provide novel insights into host-bacteria communication and the molecular mechanism of probiotic establishment in the intestine.

Effects of Bacillus subtilis CSL2 on the composition and functional diversity of the faecal microbiota of broiler chickens challenged with Salmonella Gallinarum.

BACKGROUND: The chicken gastrointestinal tract contains a diverse microbiota whose composition and structure play important roles in gut functionality. In this study, microbial shifts resulting from feed supplementation with Bacillus subtilis CSL2 were evaluated in broilers challenged and unchallenged with Salmonella Gallinarum. To analyse bacterial community composition and functionality, 454 GS-FLX pyrosequencing of 16S rRNA gene amplicons was performed. RESULTS: The Quantitative Insights into Microbial Ecology (QIIME) pipeline was used to analyse changes in the faecal microbiota over a 24-h period. A total of 718,204 sequences from broiler chickens were recorded and analysed. At the phylum level, Firmicutes, Bacteroidetes, and Proteobacteria were the predominant bacterial taxa. In Salmonella-infected chickens (SC), Bacteroidetes were more highly abundant compared to control (NC) and Bacillus-treated (BT) chickens. At the genus level, in the NC and BT groups, Lactobacillus was present at high abundance, and the abundance of Turicibacter, unclassified Enterobacteriaceae, and Bacteroides increased in SC broilers. Furthermore, taxon-independent analysis showed that the SC and BT groups were compositionally distinct at the end of the 24-h period. Further analysis of functional properties showed that B. subtilis CSL2 administration increased gut-associated energy supply mechanisms (i.e. carbohydrate transport and metabolism) to maintain a stable microbiota and protect gut integrity. CONCLUSIONS: This study demonstrated that S. Gallinarum infection and B. subtilis CSL2 supplementation in the diet of broiler chickens influenced the diversity, composition, and functional diversity of the faecal microbiota. Moreover, the findings offer significant insights to understand potential mechanisms of Salmonella infection and the mode of action of probiotics in broiler chickens.

Revealing the combined effects of lactulose and probiotic enterococci on the swine faecal microbiota using 454 pyrosequencing.

Demand for the development of non-antibiotic growth promoters in animal production has increased in recent years. This report compared the faecal microbiota of weaned piglets under the administration of a basal diet (CON) or that containing prebiotic lactulose (LAC), probiotic Enterococcus faecium NCIMB 11181 (PRO) or their synbiotic combination (SYN). At the phylum level, the Firmicutes to Bacteroidetes ratio increased in the treatment groups compared with the CON group, and the lowest proportion of Proteobacteria was observed in the LAC group. At the family level, Enterobacteriaceae decreased in all treatments; more than a 10-fold reduction was observed in the LAC (0.99%) group compared with the CON group. At the genus level, the highest Oscillibacter proportion was detected in PRO, the highest Clostridium in LAC and the highest Lactobacillus in SYN; the abundance of Escherichia was lowest in the LAC group. Clustering in the discriminant analysis of principal components revealed distinct separation of the feeding groups (CON, LAC, PRO and SYN), showing different microbial compositions according to different feed additives or their combination. These results suggest that individual materials and their combination have unique actions and independent mechanisms for changes in the distal gut microbiota.

Constructing Proteome Reference Map of the Porcine Jejunal Cell Line (IPEC-J2) by Label-Free Mass Spectrometry.

In this study, the global proteome of the IPEC-J2 cell line was evaluated using ultra-high performance liquid chromatography coupled to a quadrupole Q Exactive™ Orbitrap mass spectrometer. Proteins were isolated from highly confluent IPEC-J2 cells in biological replicates and analyzed by label-free mass spectrometry prior to matching against a porcine genomic dataset. The results identified 1,517 proteins, accounting for 7.35% of all genes in the porcine genome. The highly abundant proteins detected, such as actin, annexin A2, and AHNAK nucleoprotein, are involved in structural integrity, signaling mechanisms, and cellular homeostasis. The high abundance of heat shock proteins indicated their significance in cellular defenses, barrier function, and gut homeostasis. Pathway analysis and annotation using the Kyoto Encyclopedia of Genes and Genomes database resulted in a putative protein network map of the regulation of immunological responses and structural integrity in the cell line. The comprehensive proteome analysis of IPEC-J2 cells provides fundamental insights into overall protein expression and pathway dynamics that might be useful in cell adhesion studies and immunological applications.

Quantitative Proteogenomics and the Reconstruction of the Metabolic Pathway in Lactobacillus mucosae LM1.

Lactobacillus mucosae is a natural resident of the gastrointestinal tract of humans and animals and a potential probiotic bacterium. To understand the global protein expression profile and metabolic features of L. mucosae LM1 in the early stationary phase, the QExactive(TM) Hybrid Quadrupole-Orbitrap Mass Spectrometer was used. Characterization of the intracellular proteome identified 842 proteins, accounting for approximately 35% of the 2,404 protein-coding sequences in the complete genome of L. mucosae LM1. Proteome quantification using QExactive(TM) Orbitrap MS detected 19 highly abundant proteins (> 1.0% of the intracellular proteome), including CysK (cysteine synthase, 5.41%) and EF-Tu (elongation factor Tu, 4.91%), which are involved in cell survival against environmental stresses. Metabolic pathway annotation of LM1 proteome using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that half of the proteins expressed are important for basic metabolic and biosynthetic processes, and the other half might be structurally important or involved in basic cellular processes. In addition, glycogen biosynthesis was activated in the early stationary phase, which is important for energy storage and maintenance. The proteogenomic data presented in this study provide a suitable reference to understand the protein expression pattern of lactobacilli in standard conditions.

Pyrosequencing-based analysis of fecal microbial communities in three purebred pig lines.

This study examined the fecal bacterial diversity of 15-week-old pigs from three purebred lines: Duroc, Landrace, and Yorkshire. Taxon-dependent and -independent analyses were performed to evaluate differences in the fecal bacterial communities and to identify bacterial genera that can be used to discriminate breeds, following high-throughput pyrosequencing of 16S rRNA genes. Among the breeds evaluated, Landrace had the most diverse bacterial community composition. Prevotella, Blautia, Oscillibacter, and Clostridium were detected in all samples regardless of breed. On the other hand, Catenibacterium, Blautia, Dialister, and Sphaerochaeta were differentially detected among breeds, as demonstrated by the canonical loading plot. The discriminant analysis of principal components plot also showed clear separation of the three purebred pig lines, with a certain degree of similarity between Landrace and Yorkshire pigs and a distinct separation between Duroc pigs and the other two breeds. Other factors not related to breed, such as season or time of sampling and pen effects, may contribute to shaping the gut microbiota of pigs.