Competition of Lactobacillus paracasei with Salmonella enterica for Adhesion to Caco-2 Cells

Competition of commensal and probiotic bacteria with pathogens for adhesion and colonization is one of the important protective mechanisms of gastrointestinal tract. In this study, we examined the ability of Lactobacillus paracasei to inhibit the adhesion of pathogenic Salmonella enterica to human colon adenocarcinoma Caco-2 cells. Caco-2 cells were grown for 6 or 21 days to obtain nondifferentiated or well-differentiated cells, respectively. In adhesion experiments, bacteria were added to the cells for 2 or 4 hours. The number of attached bacteria was expressed as colony-forming units (CFUs), Caco-2 cells were counted in hematocytometer. Both bacterial strains used adhered better to well-differentiated than to nondifferentiated Caco-2 cells, however, the amount of Salmonella adhered to Caco-2 after 2 hours of contact was 12-fold higher in comparison to L. paracasei and almost 27-fold higher after 4 hours of contact. Two types of experiments were done: coincubation (both bacteria were added to Caco-2 cells simultaneously), and preincubation (L. paracasei was incubated with Caco-2 cells first, and then S. enterica was added). In coincubation experiment, the presence of L. paracasei decreased S. enterica adhesion by 4-fold and in preincubation experiment even 7-fold. Generally, Lactobacillus spent culture supernatants (SCSs) acted weaker as inhibitors of Salmonella adhesion in comparison to the whole L. paracasei culture in coincubation experiment. In conclusion, the displacement of pathogens by lactic acid bacteria and its secretions showed here depends on the time of bacteria-epithelial cell contact, and also on the stage of Caco-2 differentiation

Dynamics of dark fermentation microbial communities in the light of lactate and butyrate production.

Abstract Background: This study focuses on the processes occurring during the acidogenic step of anaerobic digestion, especially resulting from nutritional interactions between dark fermentation (DF) bacteria and lactic acid bacteria (LAB). Previously, we have confirmed that DF microbial communities (MCs) that fed on molasses are able to convert lactate and acetate to butyrate. The aims of the study were to recognize the biodiversity of DF-MCs able and unable to convert lactate and acetate to butyrate and to define the conditions for the transformation. Results: MCs sampled from a DF bioreactor were grown anaerobically in mesophilic conditions on different media containing molasses or sucrose and/or lactate and acetate in five independent static batch experiments. The taxonomic composition (based on 16S_rRNA profiling) of each experimental MC was analysed in reference to its metabolites and pH of the digestive liquids. In the samples where the fermented media contained carbohydrates, the two main tendencies were observed: (i) a low pH (pH ≤ 4), lactate and ethanol as the main fermentation products, MCs dominated with Lactobacillus, Bifidobacterium, Leuconostoc and Fructobacillus was characterized by low biodiversity; (ii) pH in the range 5.0–6.0, butyrate dominated among the fermentation products, the MCs composed mainly of Clostridium (especially Clostridium_sensu_stricto_12), Lactobacillus, Bifidobacterium and Prevotella. The biodiversity increased with the ability to convert acetate and lactate to butyrate. The MC processing exclusively lactate and acetate showed the highest biodiversity and was dominated by Clostridium (especially Clostridium_sensu_stricto_12). LAB were reduced; other genera such as Terrisporobacter, Lachnoclostridium, Paraclostridium or Sutterella were found. Butyrate was the main metabolite and pH was 7. Shotgun metagenomic analysis of the selected butyrate-producing MCs independently on the substrate revealed C.tyrobutyricum as the dominant Clostridium species. Functional analysis confirmed the presence of genes encoding key enzymes of the fermentation routes. Conclusions: Batch tests revealed the dynamics of metabolic activity and composition of DF-MCs dependent on fermentation conditions. The balance between LAB and the butyrate producers and the pH values were shown to be the most relevant for the process of lactate and acetate conversion to butyrate. To close the knowledge gaps is to find signalling factors responsible for the metabolic shift of the DF-MCs towards lactate fermentation

Hyperarid soil microbial community response to simulated rainfall

The exceptionally long and protracted aridity in the Atacama Desert (AD), Chile, provides an extreme, terrestrial ecosystem that is ideal for studying microbial community dynamics under hyperarid conditions. Our aim was to characterize the temporal response of hyperarid soil AD microbial communities to ex situ simulated rainfall (5% g water/g dry soil for 4 weeks) without nutrient amendment. We conducted replicated microcosm experiments with surface soils from two previously well-characterized AD hyperarid locations near Yungay at 1242 and 1609 masl (YUN1242 and YUN1609) with distinct microbial community compositions and average soil relative humidity levels of 21 and 17%, respectively. The bacterial and archaeal response to soil wetting was evaluated by 16S rRNA gene qPCR, and amplicon sequencing. Initial YUN1242 bacterial and archaeal 16S rRNA gene copy numbers were significantly higher than for YUN1609. Over the next 4 weeks, qPCR results showed significant increases in viable bacterial abundance, whereas archaeal abundance decreased. Both communities were dominated by 10 prokaryotic phyla (Actinobacteriota, Proteobacteria, Chloroflexota, Gemmatimonadota, Firmicutes, Bacteroidota, Planctomycetota, Nitrospirota, Cyanobacteriota, and Crenarchaeota) but there were significant site differences in the relative abundances of Gemmatimonadota and Chloroflexota, and specific actinobacterial orders. The response to simulated rainfall was distinct for the two communities. The actinobacterial taxa in the YUN1242 community showed rapid changes while the same taxa in the YUN1609 community remained relatively stable until day 30. Analysis of inferred function of the YUN1242 microbiome response implied an increase in the relative abundance of known spore-forming taxa with the capacity for mixotrophy at the expense of more oligotrophic taxa, whereas the YUN1609 community retained a stable profile of oligotrophic, facultative chemolithoautotrophic and mixotrophic taxa. These results indicate that bacterial communities in extreme hyperarid soils have the capacity for growth in response to simulated rainfall; however, historic variations in long-term hyperaridity exposure produce communities with distinct putative metabolic capacities

Dynamics and Complexity of Dark Fermentation Microbial Communities Producing Hydrogen From Sugar Beet Molasses in Continuously Operating Packed Bed Reactors

This study describes the dynamics and complexity of microbial communities producing hydrogen-rich fermentation gas from sugar-beet molasses in five packed-bed reactors (PBRs). The bioreactors constitute a part of a system producing hydrogen from the by-products of the sugar-beet industry that has been operating continuously in one of the Polish sugar factories. PBRs with different working volumes, packing materials, construction and inocula were tested. This study focused on analysis (based on 16S rRNA profiling and shotgun metagenomics sequencing) of the microbial communities selected in the PBRs under the conditions of high (>100 cm3/g COD of molasses) and low (<50 cm3/g COD of molasses) efficiencies of hydrogen production. The stability and efficiency of the hydrogen production are determined by the composition of dark fermentation microbial communities. The most striking difference between the tested samples is the ratio of hydrogen producers to lactic acid bacteria. The highest efficiency of hydrogen production (130-160 cm3/g COD of molasses) was achieved at the ratios of HPB to LAB ≈ 4:2.5 or 2.5:1 as determined by 16S rRNA sequencing or shotgun metagenomics sequencing, respectively. The most abundant Clostridium species were C. pasteurianum and C. tyrobutyricum. A multiple predominance of LAB over HPB (3:1-4:1) or clostridia over LAB (5:1-60:1) results in decreased hydrogen production. Inhibition of hydrogen production was illustrated by overproduction of short chain fatty acids and ethanol. Furthermore, concentration of ethanol might be a relevant marker or factor promoting a metabolic shift in the DF bioreactors processing carbohydrates from hydrogen-yielding toward lactic acid fermentation or solventogenic pathways. The novelty of this study is identifying a community balance between hydrogen producers and lactic acid bacteria for stable hydrogen producing systems. The balance stems from long-term selection of hydrogen-producing microbial community, operating conditions such as bioreactor construction, packing material, hydraulic retention time and substrate concentration. This finding is confirmed by additional analysis of the proportions between HPB and LAB in dark fermentation bioreactors from other studies. The results contribute to the advance of knowledge in the area of relationships and nutritional interactions especially the cross-feeding of lactate between bacteria in dark fermentation microbial communities

Post-Translational Loss of Renal TRPV5 Calcium Channel Expression, Ca2+ Wasting, and Bone Loss in Experimental Colitis

Dysregulated Ca2+ homeostasis likely contributes to the etiology of IBD-associated loss of bone mineral density (BMD). Experimental colitis leads to decreased expression of Klotho, a protein which supports renal Ca2+ reabsorption by stabilizing TRPV5 channel on the apical membrane of distal tubule epithelial cells

Downregulation of aging-related Klotho gene in experimental colitis: the role of TNF and IFN-γ

Klotho deficiency in hypomorphic KL mice leads to premature senescence and phenotype consistent with impaired mineral homeostasis. Klotho has anti-inflammatory properties protecting from NO-induced endothelial dysfunction, reduces the expression of endothelial adhesion molecules, and may contribute to T-cell dysfunction. Since defective Ca2+/Pi homeostasis leading to osteopenia/osteoporosis is frequently associated with human IBD, we investigated the changes in Klotho gene expression as a consequence of experimental colitis

NHE3TcellMicrobiome

Data repository PONE-D-16-0424

Identification of protein partners in Mycobacteria using a single step affinity purification method

Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spreading and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method is attractively applicable to both medium- and highthroughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. From four tested epitopes, FLAG, eGFP, Protein A, and hemagglutinin, the eGFP tag was found most useful based on easily monitored expression and as a simultaneous tool for sub-cellular localization studies. It presents a relatively low background with cost effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as a bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells, treated with UV light were used to co-purify UvrA interaction partners

Pathophysiology of Intestinal Na+/H+ ExchangeSummary

Several members of the SLC9A family of Na+/H+ exchangers are expressed in the gut, with varying expression patterns and cellular localization. Not only do they participate in the regulation of basic epithelial cell functions, including control of transepithelial Na+ absorption, intracellular pH (pHi), cell volume, and nutrient absorption, but also in cellular proliferation, migration, and apoptosis. In addition, they modulate the extracellular milieu to facilitate other nutrient absorption and to regulate the intestinal microbial microenvironment. Na+/H+ exchangers are frequent targets of inhibition in gastrointestinal pathologies, either by intrinsic factors (eg, bile acids, inflammatory mediators) or infectious agents and associated microbial toxins. Based on emerging evidence, disruption of Na+/H+ exchange activity via impaired expression or function of respective isoforms may contribute not only to local and systemic electrolyte imbalance, but also to the disease severity via multiple mechanisms. Here, we review the current state of knowledge about the roles Na+/H+ exchangers play in the pathogenesis of disorders of diverse origin and affecting a range of gastrointestinal tissues. Keywords: Barrettâs Esophagus, Esophageal Adenocarcinoma, Epithelial Injury, Epithelial Restitution, Diarrhea, Infection, Inflammation, Microbiota, Inflammatory Bowel Disease, Hypertensio