In Vitro Influence of Specific Bacteroidales Strains on Gut and Liver Health Related to Metabolic Dysfunction-Associated Fatty Liver Disease.

Metabolic dysfunction-associated fatty liver disease (MAFLD) has become a major health risk and a serious worldwide issue. MAFLD typically arises from aberrant lipid metabolism, insulin resistance, oxidative stress, and inflammation. However, subjacent causes are multifactorial. The gut has been proposed as a major factor in health and disease, and over the last decade, bacterial strains with potentially beneficial effects on the host have been identified. In vitro cell models have been commonly used as an early step before in vivo drug assessment and can confer complementary advantages in gut and liver health research. In this study, several selected strains of the order Bacteroidales were used in a three-cell line in vitro analysis (HT-29, Caco-2, and HepG2 cell lines) to investigate their potential as new-generation probiotics and microbiota therapeutics. Antimicrobial activity, a potentially useful trait, was studied, and the results showed that Bacteroidales can be a source of either wide- or narrow-spectrum antimicrobials targeting other closely related strains. Moreover, Bacteroides sp. 4_1_36 induced a significant decrease in gut permeability, as evidenced by the high TEER values in the Caco-2 monolayer assay, as well as a reduction in free fatty acid accumulation and improved fatty acid clearance in a steatosis HepG2 model. These results suggest that Bacteroidales may spearhead the next generation of probiotics to prevent or diminish MAFLD.

Bioinformatic mining for RiPP biosynthetic gene clusters in Bacteroidales reveals possible new subfamily architectures and novel natural products.

The Bacteroidales order, widely distributed among diverse human populations, constitutes a key component of the human microbiota. Members of this Gram-negative order have been shown to modulate the host immune system, play a fundamental role in the gut's microbial food webs, or be involved in pathogenesis. Bacteria inhabiting such a complex environment as the human microbiome are expected to display social behaviors and, hence, possess factors that mediate cooperative and competitive interactions. Different types of molecules can mediate interference competition, including non-ribosomal peptides (NRPs), polyketides, and bacteriocins. The present study investigates the potential of Bacteroidales bacteria to biosynthesize class I bacteriocins, which are ribosomally synthesized and post-translationally modified peptides (RiPPs). For this purpose, 1,136 genome-sequenced strains from this order were mined using BAGEL4. A total of 1,340 areas of interest (AOIs) were detected. The most commonly identified enzymes involved in RiPP biosynthesis were radical S-adenosylmethionine (rSAM), either alone or in combination with other biosynthetic enzymes such as YcaO. A more comprehensive analysis of a subset of 9 biosynthetic gene clusters (BGCs) revealed a consistent association in Bacteroidales BGCs between peptidase-containing ATP-binding transporters (PCATs) and precursor peptides with GG-motifs. This finding suggests a possibly shared mechanism for leader peptide cleavage and transport of mature products. Notably, human metagenomic studies showed a high prevalence and abundance of the RiPP BGCs from Phocaeicola vulgatus and Porphyromonas gulae. The mature product of P. gulae BGC is hypothesized to display γ-thioether linkages and a C-terminal backbone amidine, a potential new combination of post-translational modifications (PTM). All these findings highlight the RiPP biosynthetic potential of Bacteroidales bacteria, as a rich source of novel peptide structures of possible relevance in the human microbiome context.

Role of microbiota and related metabolites in gastrointestinal tract barrier function in NAFLD.

The Gastrointestinal (GI) tract is composed of four main barriers: microbiological, chemical, physical and immunological. These barriers play important roles in maintaining GI tract homeostasis. In the crosstalk between these barriers, microbiota and related metabolites have been shown to influence GI tract barrier integrity, and alterations of the gut microbiome might lead to an increase in intestinal permeability. As a consequence, translocation of bacteria and their products into the circulatory system increases, reaching proximal and distal tissues, such as the liver. One of the most prevalent chronic liver diseases, Nonalcoholic Fatty Liver Disease (NAFLD), has been associated with an altered gut microbiota and barrier integrity. However, the causal link between them has not been fully elucidated yet. In this review, we aim to highlight relevant bacterial taxa and their related metabolites affecting the GI tract barriers in the context of NAFLD, discussing their implications in gut homeostasis and in disease.

Combining Modules for Versatile and Optimal Labeling of Lactic Acid Bacteria: Two pMV158-Family Promiscuous Replicons, a Pneumococcal System for Constitutive or Inducible Gene Expression, and Two Fluorescent Proteins.

Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis, we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter PX or PM from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying PM also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (∼115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (∼8-45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.