Fecal microbiota transplantation: current challenges and future landscapes.

SUMMARYGiven the importance of gut microbial homeostasis in maintaining health, there has been considerable interest in developing innovative therapeutic strategies for restoring gut microbiota. One such approach, fecal microbiota transplantation (FMT), is the main "whole gut microbiome replacement" strategy and has been integrated into clinical practice guidelines for treating recurrent Clostridioides difficile infection (rCDI). Furthermore, the potential application of FMT in other indications such as inflammatory bowel disease (IBD), metabolic syndrome, and solid tumor malignancies is an area of intense interest and active research. However, the complex and variable nature of FMT makes it challenging to address its precise functionality and to assess clinical efficacy and safety in different disease contexts. In this review, we outline clinical applications, efficacy, durability, and safety of FMT and provide a comprehensive assessment of its procedural and administration aspects. The clinical applications of FMT in children and cancer immunotherapy are also described. We focus on data from human studies in IBD in contrast with rCDI to delineate the putative mechanisms of this treatment in IBD as a model, including colonization resistance and functional restoration through bacterial engraftment, modulating effects of virome/phageome, gut metabolome and host interactions, and immunoregulatory actions of FMT. Furthermore, we comprehensively review omics technologies, metagenomic approaches, and bioinformatics pipelines to characterize complex microbial communities and discuss their limitations. FMT regulatory challenges, ethical considerations, and pharmacomicrobiomics are also highlighted to shed light on future development of tailored microbiome-based therapeutics.

Future Nanotechnology-Based Strategies for Improved Management of Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) is a recalcitrant pathogen, which can cause gastric disorders. During the past decades, polypharmacy-based regimens, such as triple and quadruple therapies have been widely used against H. pylori. However, polyantibiotic therapies can disturb the host gastric/gut microbiota and lead to antibiotic resistance. Thus, simpler but more effective approaches should be developed. Here, some recent advances in nanostructured drug delivery systems to treat H. pylori infection are summarized. Also, for the first time, a drug release paradigm is proposed to prevent H. pylori antibiotic resistance along with an IVIVC model in order to connect the drug release profile with a reduction in bacterial colony counts. Then, local delivery systems including mucoadhesive, mucopenetrating, and cytoadhesive nanobiomaterials are discussed in the battle against H. pylori infection. Afterward, engineered delivery platforms including polymer-coated nanoemulsions and polymer-coated nanoliposomes are poposed. These bioinspired platforms can contain an antimicrobial agent enclosed within smart multifunctional nanoformulations. These bioplatforms can prevent the development of antibiotic resistance, as well as specifically killing H. pylori with no or only slight negative effects on the host gastrointestinal microbiota. Finally, the essential checkpoints that should be passed to confirm the potential effectiveness of anti-H. pylori nanosystems are discussed.

The modulatory effect of Lactobacillus gasseri ATCC 33323 on autophagy induced by extracellular vesicles of Helicobacter pylori in gastric epithelial cells in vitro.

Helicobacter pylori has been recognized as a true pathogen, which is associated with various gastroduodenal diseases, and gastric adenocarcinoma. The crosstalk between H. pylori virulence factors and host autophagy remains challenging. H. pylori can produce extracellular vesicles (EVs) that contribute to gastric inflammation and malignancy. Some probiotic strains have been documented to modulate cell autophagy process. This study was aimed to investigate the modulatory effect of cell-free supernatant (CFS) obtained from Lactobacillus gasseri ATCC 33323 on autophagy induced by H. pylori-derived EVs. EVs were isolated from two clinical H. pylori strains (BY-1 and OC824), and characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The viability of AGS cells was assessed after exposure to different concentrations of H. pylori EVs, and L. gasseri CFS. Based on MTT assay and Annexin V-FITC/PI staining, 50 µg/ml of H. pylori EVs and 10 % v/v of L. gasseri CFS were used for further cell treatment experiments. Autophagy was examined using acridin orange (AO) staining, RT-qPCR analysis for autophagy mediators (LC3B, ATG5, ATG12, ATG16L1, BECN1, MTOR, and NOD1), and western blotting for LC3B expression. H. pylori EVs were detected to range in size from 50 to 200 nm. EVs of both H. pylori strains and L. gasseri CFS showed no significant effect on cell viability as compared to untreated cells. H. pylori EVs promoted the development of acidic vesicular organelles and the expression of autophagy-related genes (LC3B, ATG5, ATG12, ATG16L1, BECN1, and NOD1), and decreased the expression of MTOR in AGS cells at 12 and 24 h time periods. In addition, the production of LC3B was increased following 12 h of treatment in AGS cells. In contrast, L. gasseri CFS effectively inhibited EVs-induced autophagy, as evidenced by reduced acidic vesicular organelle formation and modulation of autophagy markers. Our study indicated that L. gasseri CFS can effectively suppress H. pylori EV-induced autophagy in AGS cells. Further investigations are required to decipher the mechanism of action L. gasseri CFS and its metabolites on autophagy inhibition induced by H. pylori.

The inhibitory effects of live and UV-killed Akkermansia muciniphila and its derivatives on cytotoxicity and inflammatory response induced by Clostridioides difficile RT001 in vitro.

Clostridioides difficile infection (CDI) is the leading cause of healthcare-acquired infections worldwide. Probiotics are widely recommended to prevent CDI and its recurrences. Akkermansia muciniphila, as a therapeutic symbiont colonizing the intestinal mucosal layer, is considered to be a promising next-generation probiotic. In this work, we assessed the inhibitory effects of A. muciniphila MucT and its derivatives on cytotoxicity and inflammatory response induced by C. difficile RT001 in Caco-2 cells. The results obtained from SEM revealed that the morphology of UV-killed A. muciniphila remained unchanged after UV inactivation. TEM analysis showed that A. muciniphila-isolated extracellular vesicles (EVs) were spherical and ranged from 50 to 200 nm in size. Toxigenic supernatant (Tox-S) of C. difficile RT001 (500 µg/ml) significantly (P <0.01) reduced the cell viability of Caco-2 cells. Caco-2 cells treated with live (MOI 10), UV-killed (MOI 10), cell-free supernatant (CFS, 106 cfu/ml), and EVs (20 µg/ml) of A. muciniphila exhibited over 90% viability in comparison to untreated control. The neutralized CFS preparation using A. muciniphila and its derivatives could notably reduce the expression level of inflammatory markers. Additionally, A. muciniphila and its derivatives modulated the production of IL-1ß, TNF-α, and IL-10 in Tox-S stimulated Caco-2 cells. We demonstrated that A. muciniphila and its derivatives can modulate changes in the gut barrier-related genes and inflammatory response caused by C. difficile Tox-S in Caco-2 cells.

A reconstructed genome-scale metabolic model of Helicobacter pylori for predicting putative drug targets in clarithromycin and rifampicin resistance conditions.

BACKGROUND: Helicobacter pylori is considered a true human pathogen for which rising drug resistance constitutes a drastic concern globally. The present study aimed to reconstruct a genome-scale metabolic model (GSMM) to decipher the metabolic capability of H. pylori strains in response to clarithromycin and rifampicin along with identification of novel drug targets. MATERIALS AND METHODS: The iIT341 model of H. pylori was updated based on genome annotation data, and biochemical knowledge from literature and databases. Context-specific models were generated by integrating the transcriptomic data of clarithromycin and rifampicin resistance into the model. Flux balance analysis was employed for identifying essential genes in each strain, which were further prioritized upon being nonhomologs to humans, virulence factor analysis, druggability, and broad-spectrum analysis. Additionally, metabolic differences between sensitive and resistant strains were also investigated based on flux variability analysis and pathway enrichment analysis of transcriptomic data. RESULTS: The reconstructed GSMM was named as HpM485 model. Pathway enrichment and flux variability analyses demonstrated reduced activity in the ribosomal pathway in both clarithromycin- and rifampicin-resistant strains. Also, a significant decrease was detected in the activity of metabolic pathways of clarithromycin-resistant strain. Moreover, 23 and 16 essential genes were exclusively detected in clarithromycin- and rifampicin-resistant strains, respectively. Based on prioritization analysis, cyclopropane fatty acid synthase and phosphoenolpyruvate synthase were identified as putative drug targets in clarithromycin- and rifampicin-resistant strains, respectively. CONCLUSIONS: We present a robust and reliable metabolic model of H. pylori. This model can predict novel drug targets to combat drug resistance and explore the metabolic capability of H. pylori in various conditions.

Alterations in the gut microbiota and their metabolites in human intestinal epithelial cells of patients with colorectal cancer.

BACKGROUND: The gut microbiota has become one of the main risk factors for the formation and development of colorectal cancer (CRC). CRC intensification may be due to the microbial pathogens' colonization and their released metabolites. Here, we analyzed Bacteroidetes and Clostridia bacteria in CRC patients and studied bacterial metabolome in cancerous tissues compared to their adjacent normal tissues. METHODS AND RESULTS: The population of selected bacteria in biopsy specimens of 30 patients with CRC was studied by RT-qPCR. The mutagenicity and cytotoxicity effects of microbiota metabolites were evaluated by Ames test and MTT Assay, respectively. Moreover, gene expression in carcinogenic pathways was studied by RT-qPCR, and genes with different expressions in tumor and non-tumor tissues were diagnosed. Based on microbiota analysis, the relative abundance of Clostridia and C. difficile was significantly higher in CRC tissue, whereas C. perfringens showed higher relative abundance in normal tissue. AIMES test confirmed the proliferation and mutagenicity effects of the bacterial metabolites in CRC patients. Significant upregulation of C-Myc, GRB2, IL-8, EGFR, PI3K, and AKT and downregulation of ATM were observed in CRC samples compared to the control. CONCLUSIONS: The influence of bacterial metabolites on inflammation and altered expression of genes in the cell signaling pathways was observed. The findings confirm the role gut microbiota composition and bacterial metabolites as key players in CRC onset and development.

Identification of Candida albicans and non-MRSA Staphylococcus aureus in free-living amoebae isolated from the hospital wards; an alarm for distribution of nosocomial infections via FLA.

Free-living amoebae (FLA) are isolated from the hospital environments and known as Trojan horses for medical essential microorganisms. This study aimed to investigate the prevalence and the presence of FLA and two critical agents of nosocomial infections, in the hospital wards. Sixty samples were collected from four communities and cultured onto non-nutrient agar (NNA). After total DNA extraction, FLA were characterized using PCR and sequencing. The presence of Candida albicans and Staphylococcus aureus was evaluated using real-time and conventional PCR, respectively. Acanthamoeba sp. was characterized in 30 (50%) samples. Two (6.6%) and one (3.3%) samples were positive for Vahlkampfiidae and Vermamoeba vermiformis, respectively . S. aureus was detected in 13 (43.3%) of samples, while none of them were positive for methicillin-resistant gene. C. albicans DNA was detected in one (3.3%) FLA-positive sample. The isolation of FLA from hospital suggests an essential role these eukaryotes in the inter-ward circulation of nosocomial infections.

Outer Membrane Vesicles Derived from Adherent-Invasive Escherichia coli Induce Inflammatory Response and Alter the Gene Expression of Junction-Associated Proteins in Human Intestinal Epithelial Cells.

Adherent-invasive Escherichia coli (AIEC) pathobionts, which are characterized by their ability to adhere to and invade intestinal epithelial cells, are associated with the etiopathogenesis of inflammatory bowel diseases (IBDs). Outer membrane vesicles (OMVs) released by AIEC strains can facilitate the interaction of these bacteria with host cells through delivering bacterial effectors. The aim of this study was to determine the ability of OMVs derived from AIEC strain LF82 to induce the host immune response, leading to production of proinflammatory cytokines and also altering the gene expression of junction-associated proteins in the human epithelial colorectal adenocarcinoma Caco-2 cell line. OMVs were extracted from AIEC strain LF82, and the cell viability of Caco-2 cells treated with these vesicles was assessed by MTT assay. The morphology and size distribution of vesicles were analyzed using transmission electron microscopy and dynamic light scattering, respectively. Gene expression of occludin, ZO-1, claudin-2, E-cadherin, TLR-2, and TLR-4 in response to OMVs was assessed in Caco-2 cells by RT-qPCR. Moreover, the secretion of IL-8 and TNF-α into the supernatant of Caco-2 cells upon treatment with OMVs was measured using ELISA. Our results demonstrated that OMVs upregulated the gene expression level of TLRs and also altered the gene expression level of junction-associated proteins. OMVs derived from AIEC may play a major role in the promotion of intestinal inflammation and epithelial barrier dysfunction. However, further investigations are needed to elucidate the putative role of OMVs in the pathogenesis of AIEC and IBD.

Helicobacter pylori-derived outer membrane vesicles suppress liver autophagy: A novel mechanism for H. pylori-mediated hepatic disorder.

BACKGROUND: Helicobacter pylori outer membrane vesicles (OMVs) are nano-sized structures, which have been recently suggested to play a crucial role in H. pylori pathogenesis. There are growing evidence indicating the relationship of H. pylori infection with extra-gastroduodenal diseases, especially liver-related disorders. This study was aimed to investigate the effects of H. pylori-derived OMVs on autophagy in hepatic stellate cells (HSCs). MATERIAL AND METHODS: A selection of five clinical strains of H. pylori with different virulence genotypes were included. The OMVs were isolated by ultracentrifugation and characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The protein concentration of OMVs was measured by BCA assay. MTT assay was used to determine the viability of LX-2 cells (human HSCs) treated with OMVs. The expression level of MTOR, AKT, PI3K, BECN1, ATG16 and LC3B genes was assessed in OMVs-treated LX-2 cells using quantitative real-time PCR. Moreover, immunocytochemistry was performed to evaluate the protein expression of MTOR and LC3B autophagy markers. RESULTS: H. pylori strains produced round shape nano-vesicles ranging from 50 to 500 nm. Treatment of HSCs with H. pylori-derived OMVs at concentration of 10 µg/mL for 24 h significantly elevated the expression of autophagy inhibitory markers (PI3K, AKT, and MTOR) and suppressed the mRNA expression level of autophagy core proteins (BECN1, ATG16 and LC3B). Immunocytochemistry also presented a substantial reduction in the concentration of LC3B autophagy core protein, and a marked elevation in the amount of MTOR autophagy inhibitory protein. CONCLUSION: This study revealed that H. pylori-derived OMVs could potentially suppress autophagy flux in HSCs as a novel mechanism for H. pylori-mediated liver autophagy impairment and liver disease development. Further studies are required to elucidate the exact role of OMV-carried contents in liver autophagy, and liver-associated disorders.

Clostridioides difficile PCR ribotypes 001 and 084 can trigger autophagy process in human intestinal Caco-2 cells.

Autophagy is a homeostatic process that can promote cell survival or death. However, the exact role of autophagy in Clostridioides difficile infection (CDI) is still not precisely elucidated. Here, we investigate the role of distinct C. difficile ribotypes (RTs) in autophagy induction using Caco-2 cells. The expression analysis of autophagy-associated genes and related miRNAs were examined following treatment of Caco-2 cells with C. difficile after 4 and 8 h using RT-qPCR. Toxin production was assessed using enzyme-linked immunosorbent assay (ELISA). Immunofluorescence analysis was performed to detect MAP1LC3B/LC3B, followed by an autophagic flux analysis. C. difficile significantly reduced the viability of Caco-2 cells in comparison with untreated cells. Elevated levels of LC3-II and SQSTM1/p62 by C. difficile RT001 and RT084 in the presence of E64d/leupeptin confirmed the induction of autophagy activity. Similarly, the immunofluorescence analysis demonstrated that C. difficile RT001 and RT084 significantly increased the amount of LC3-positive structures in Caco-2 cells. The induction of autophagy was further demonstrated by increased levels of LC3B, ULK1, ATG12, PIK3C3/VPS34, BECN1 (beclin 1), ATG5, and ATG16L1 transcripts and reduced levels of AKT and MTOR gene expression. The expression levels of MIR21 and MIR30B, microRNAs that suppress autophagy, were differentially affected by C. difficile. In conclusion, the present work revealed that C. difficile bacteria can induce autophagy through both toxin-dependent and -independent mechanisms. Also, our results suggest the potential role of other C. difficile virulence factors in autophagy modulation using intestinal cells in vitro.

Evaluation of gut microbiota of iranian patients with celiac disease, non-celiac wheat sensitivity, and irritable bowel syndrome: are there any similarities?

BACKGROUND AND AIMS: Individuals with celiac disease (CD), non-celiac wheat sensitivity (NCWS), and irritable bowel syndrome (IBS), show overlapping clinical symptoms and experience gut dysbiosis. A limited number of studies so far compared the gut microbiota among these intestinal conditions. This study aimed to investigate the similarities in the gut microbiota among patients with CD, NCWS, and IBS in comparison to healthy controls (HC). MATERIALS AND METHODS: In this prospective study, in total 72 adult subjects, including CD (n = 15), NCWS (n = 12), IBS (n = 30), and HC (n = 15) were recruited. Fecal samples were collected from each individual. A quantitative real-time PCR (qPCR) test using 16S ribosomal RNA was conducted on stool samples to assess the relative abundance of Firmicutes, Bacteroidetes, Bifidobacterium spp., and Lactobacillus spp. RESULTS: In all groups, Firmicutes and Lactobacillus spp. had the highest and lowest relative abundance respectively. The phylum Firmicutes had a higher relative abundance in CD patients than other groups. On the other hand, the phylum Bacteroidetes had the highest relative abundance among healthy subjects but the lowest in patients with NCWS. The relative abundance of Bifidobacterium spp. was lower in subjects with CD (P = 0.035) and IBS (P = 0.001) compared to the HCs. Also, the alteration of Firmicutes to Bacteroidetes ratio (F/B ratio) was statistically significant in NCWS and CD patients compared to the HCs (P = 0.05). CONCLUSION: The principal coordinate analysis (PCoA), as a powerful multivariate analysis, suggested that the investigated gut microbial profile of patients with IBS and NCWS share more similarities to the HCs. In contrast, patients with CD had the most dissimilarity compared to the other groups in the context of the studied gut microbiota.

Immunomodulatory effects of live and pasteurized Lactobacillus crispatus strain RIGLD-1 on Helicobacter pylori-triggered inflammation in gastric epithelial cells in vitro.

BACKGROUND: Helicobacter pylori infection is considered as the major risk factor for gastric adenocarcinoma. Today, the increasing emergence of antibiotic-resistant strains has drastically decreased the eradication rate of H. pylori infection. This study was aimed to investigate the inhibitory and modulatory effects of live and pasteurized Lactobacillus crispatus strain RIGLD-1 on H. pylori adhesion, invasion, and inflammatory response in AGS cell line. METHODS AND RESULTS: The probiotic potential and properties of L. crispatus were evaluated using several functional and safety tests. Cell viability of AGS cells exposed to varying concentrations of live and pasteurized L. crispatus was assessed by MTT assay. The adhesion and invasion abilities of H. pylori exposed to either live or pasteurized L. crispatus were examined by gentamycin protection assay. The mRNA expression of IL-1ß, IL-6, IL-8, TNF-α, IL-10, and TGF-ß genes was determined by RT-qPCR from coinfected AGS cells. ELISA was used for the detection of IL-8 secretion from treated cells. Both live and pasteurized L. crispatus significantly decreased H. pylori adhesion/invasion to AGS cells. In addition, both live and pasteurized L. crispatus modulated H. pylori-induced inflammation by downregulating the mRNA expression of IL-1ß, IL-6, IL-8, and TNF-α and upregulating the expression of IL-10, and TGF-ß cytokines in AGS cells. Furthermore, H. pylori-induced IL-8 production was dramatically decreased after treatment with live and pasteurized L. crispatus. CONCLUSIONS: In conclusion, our findings demonstrated that live and pasteurized L. crispatus strain RIGLD-1 are safe, and could be suggested as a potential probiotic candidate against H. pylori colonization and inflammation.

Characterization of the mucosal microbiota in patients with nodular lymphoid hyperplasia with concurrent irritable bowel syndrome compared to healthy controls.

BACKGROUND: Nodular lymphoid hyperplasia (NLH) is known as a lymphoproliferative lesion in which multiple small nodules appear on the intestinal wall. It has been documented that patients who struggle with irritable bowel syndrome (IBS) are at greater risk of developing NLH. Here, we aimed to investigate the previously reported pathogens and the abundance of a selection of mucosal microbiota in IBS + NLH patients compared to IBS, and healthy controls. METHODS AND RESULTS: Terminal ileum biopsies were collected from 37 IBS + NLH, 37 IBS, and 29 healthy controls. Bacterial culture and PCR was performed to detect the presence of pathogens in biopsies. A qPCR assay was applied to assess the abundance of a selection of bacterial taxa. Totally, five bacterial isolates including two enteropathogenic and one enteroaggregative Escherichia coli (EPEC, EAEC), one enterotoxigenic Staphylococcus aureus (SEA), and one Yersinia enterocolitica strains were detected among the IBS + NLH cases. The relative abundance of Bacteroidetes and Streptococcus spp. in IBS + NLH patients was significantly less than IBS and healthy controls. Firmicutes, Pseudomonas spp., Haemophilus spp., and Campylobacter spp. were notably more abundant in IBS + NLH than in IBS patients. The abundance of Verrucomicrobia was higher in NLH + IBS than in healthy controls. Actinobacteria was also significantly more abundant among NLH + IBS patients than the controls. CONCLUSION: Our results demonstrated that mucosal microbiota composition in NLH + IBS patients slightly differs from that of IBS patients and healthy controls. Further research using large-scale cohorts are needed to enhance current understanding of the contribution of the mucosal microbiota to NLH pathogenesis with concurrent IBS.

Characterization of vaginal Lactobacillus species as a predictor of fertility among Iranian women with unexplained recurrent miscarriage and fertile women without miscarriage history using machine learning modeling.

BACKGROUND: Lactobacillus spp. are the predominant bacteria of the vaginal tract, the alteration of which has been previously linked to miscarriage. Here, we investigated differences between selected vaginal Lactobacillus species of women with a history of recurrent miscarriages and fertile women without a history of miscarriage in Iran. METHODS AND RESULTS: Vaginal swabs were taken from 29 fertile and 24 infertile women and quantitative real-time PCR (qPCR) assay was used to determine a selection of vaginal Lactobacillus species in both groups. The logistic regression (LR) model, Naive Bayes (NB) model, support vector machine model (SVM), and neural network model (NN) were developed to predict disease outcome by selected variables. LR analysis was used to construct a nomogram indicating predictions of the risk of miscarriage. The most abundant species among the patients were L. rhamnosus, L. ruminis, and L. acidophilus, while L. gasseri, L. vaginalis, L. fermentum, and L. iners were more abundant in healthy subjects. The distribution of L. ruminis, L. iners, and L. rhamnosus was higher in patients, while L. acidophilus, L. gasseri, and L. fermentum were highly distributed among healthy subjects. Higher AUC in predicting the disease outcome was observed for L. gasseri, L. rhamnosus, L. fermentum, and L. plantarum. CONCLUSION: Our findings provide experimental evidence of vaginal Lactobacillus imbalance in infertile women and a suitable predictor for miscarriage based on the AUC algorithms. Further studies with larger sample size and using high-throughput technologies are needed to boost our understanding of the role of lactobacilli in miscarriage.

Modulation of microRNAs and claudin-7 in Caco-2 cell line treated with Blastocystis sp., subtype 3 soluble total antigen.

BACKGROUND: Blastocystis sp., is a eukaryote of the large intestine, which is reported from almost all countries. The pathogenesis of this protist is not clear. The current study aimed to analyze the effects of Blastocystis sp., ST3 soluble total antigen (B3STA) on the microRNAs (miRNAs) involved in the gut permeability and also pro-inflammatory cytokines, occludin, and claudin-7. METHODS: Blastocystis sp., ST3 isolated from stool sample was purified, and its soluble total antigen was extracted using freeze and thawing. The Caco-2 cell line was treated with B3STA for 24 h and the expression levels of mir-16, mir-21, mir-29a, mir-223, and mir-874 were analyzed. In addition, the expression levels of il-8, il-15, occludin, and claudin-7 genes were assessed. RESULTS: B3STA significantly upregulated the expression of mir-223, and mir-874, and downregulated mir-29a. The expression of mir-16 and mir-21 was not significant. In addition, the expression of il-8 and il-15 was not significant. B3STA significantly decreased the expression level of claudin-7 (P-value < 0.0001), but the expression of occludin was not significant. Our results showed significant correlation between all studied miRNAs, except mir-29a, with downregulation of claudin-7. CONCLUSIONS: This is the first study investigating the effects of Blastocystis sp., ST3 isolated from symptomatic subjects on the expression levels of miRNAs involved in the gut permeability. Our results demonstrated that B3STA may change miRNA expression, which are involved in the gut barrier integrity, and downregulates claudin-7, which is known as sealing factor.

Surface layer protein A from hypervirulent Clostridioides difficile ribotypes induce significant changes in the gene expression of tight junctions and inflammatory response in human intestinal epithelial cells.

BACKGROUND: Surface layer protein A (SlpA), the primary outermost structure of Clostridioides difficile, plays an essential role in C. difficile pathogenesis, although its interaction with host intestinal cells are yet to be understood. The aim of this study was to investigate the effects of SlpA extracted from C. difficile on tight junction (TJ) proteins expression and induction of pro-inflammatory cytokines in human colon carcinoma cell line HT-29. SlpA was extracted from three toxigenic C. difficile clinical strains including RT126, RT001, RT084 as well as C. difficile ATCC 700057 as non-toxigenic strain. Cell viability was performed by MTT assay, and the mRNA expression of TJ proteins and inflammation-associated genes was determined using quantitative RT-PCR. Additionally, the secretion of IL-8, IL-1ß and TNF-α cytokines was measured by ELISA. RESULTS: C. difficile SlpA from selected RTs variably downregulated the expression level of TJs-assassinated genes and increased the expression level of TLR-4 and pro-inflammatory cytokines in HT-29 treated cells. SlpA from RT126 significantly (padj<0.05) decreased the gene expression level of claudins family and JAM-A and increased the secretion of IL-8, TNF-α and IL1-ß as compared to untreated cells. Moreover, only SlpA from RT001 could significantly induce the expression of IL-6 (padj<0.05). CONCLUSION: The results of the present study highlighted the importance of SlpA in the pathogenesis of CDI and C. difficile-induced inflammatory response in the gut. Further studies are required to unravel the significance of the observed results in promoting the intestinal inflammation and immune response induced by C. difficile SlpA from different RTs.

Surface layer protein A from hypervirulent Clostridioides difficile ribotype 001 can induce autophagy process in human intestinal epithelial cells.

Clostridioides difficile is the leading cause of nosocomial diarrhea with high morbidity and mortality worldwide. C. difficile strains produce a crystalline surface layer protein A (SlpA), which is an absolute necessity for its pathogenesis. However, its pathogenic mechanisms and its pro-inflammatory behavior are not yet fully elucidated. Herein, we report for the first time that SlpA extracted from C. difficile can induce autophagy process in Caco-2 cells. SlpA protein was purified from two C. difficile strains (RT001 and ATCC 700075). The cell viability of Caco-2 cells after exposure with different concentrations (15, 20, 25 µg/mL) of SlpA at various time points (3, 6, 12, 24 h) was measured by MTT assay. Acridine orange staining was used to visualize the hypothetical acidic vesicular organelles. The gene expression of autophagy mediators including LC3B, Atg5, Atg16L, and Beclin-1 was determined by quantitative real-time PCR assay. Western blotting assay was used to detect the expression of LC3B protein. MTT assay showed that different concentrations of SlpA did not induce significant changes in the viability of Caco-2 cells. SlpA at concentration of 20 µg/mL enhanced the formation of acidic vesicular organelles in Caco-2 cells after 12 h of exposure. Moreover, SlpA treatment significantly increased the expression of autophagy-associated genes, and increased the expression of LC3B protein in Caco-2 cells. In conclusion, our study demonstrated that SlpA is capable to induce autophagy in intestinal epithelial cells. These findings reveal a novel mechanism for the pathogenesis of C. difficile mediated by its SLPs.

Curcumin and capsaicin regulate apoptosis and alleviate intestinal inflammation induced by Clostridioides difficile in vitro.

BACKGROUND: The dramatic upsurge of Clostridioides difficile infection (CDI) by hypervirulent isolates along with the paucity of effective conventional treatment call for the development of new alternative medicines against CDI. The inhibitory effects of curcumin (CCM) and capsaicin (CAP) were investigated on the activity of toxigenic cell-free supernatants (Tox-S) of C. difficile RT 001, RT 126 and RT 084, and culture-filtrate of C. difficile ATCC 700057. METHODS: Cell viability of HT-29 cells exposed to varying concentrations of CCM, CAP, C. difficile Tox-S and culture-filtrate was assessed by MTT assay. Anti-inflammatory and anti-apoptotic effects of CCM and CAP were examined by treatment of HT-29 cells with C. difficile Tox-S and culture-filtrate. Expression of BCL-2, SMAD3, NF-κB, TGF-ß and TNF-α genes in stimulated HT-29 cells was measured using RT-qPCR. RESULTS: C. difficile Tox-S significantly (P < 0.05) reduced the cell viability of HT-29 cells in comparison with untreated cells. Both CAP and CCM significantly (P < 0.05) downregulated the gene expression level of BCL-2, SMAD3, NF-κB and TNF-α in Tox-S treated HT-29 cells. Moreover, the gene expression of TGF-ß decreased in Tox-S stimulated HT-29 cells by both CAP and CCM, although these reductions were not significantly different (P > 0.05). CONCLUSION: The results of the present study highlighted that CCM and CAP can modulate the inflammatory response and apoptotic effects induced by Tox-S from different clinical C. difficile strains in vitro. Further studies are required to accurately explore the anti-toxin activity of natural components, and their probable adverse risks in clinical practice.

Helicobacter pylori Infection Mediates Inflammation and Tumorigenesis-Associated Genes Through miR-155-5p: An Integrative Omics and Bioinformatics-Based Investigation.

Helicobacter pylori (H. pylori) is a major human pathogenic bacterium that survives in the gastric mucosa. The aim of this study is to evaluate the expression of the target gene network of miR-155-5p in H. pylori-related gastritis using a combination of public gene expression datasets and web-based platforms. To evaluate the expression of genes related to gastritis, we used two datasets from Gene Expression Omnibus (GEO) database. Then, we determined the overlaps between the predicted miR-155-5p target genes and gastritis-dysregulated GEO datasets genes; in the next step, we identified the possible miR-155-5p target-DEGs (Target-Differentially Expressed Genes). Also, we performed multiple bioinformatics analyses to identify the most important targets and downstream pathways associated with this miRNA. Using the UCSC cancer genomic browser analysis tool, we investigated the expression of hub genes in relation to gastric cancer and H. pylori infection, as well as the potential role of hub genes in gastritis, inflammation, and cancer. In this regard, 28 differentially expressed target genes of miR-155-5p were identified. Most of the captured target genes were correlated with the host immune response and inflammation. Based on the specific patterns of expression in gastritis and cancer, CD9, MST1R, and ADAM10 were candidates for the most probable targets of miR-155-5p. Although the focus of this study is primarily on bioinformatics, we think that our findings should be experimentally validated before they can be used as potential therapeutic and diagnostic tools.

Impact of Nutritional Profile on Gut Microbiota Diversity in Patients with Celiac Disease.

The pathogenesis of celiac disease (CD) is significantly influenced by gut microbiota. Daily nutritional profile influences the diversity of gut microbiota. This study was aimed to compare the abundance of gut microbiota in CD patients compared to normal control (NC), and to investigate the impact of nutritional factors on their fecal microbiota diversity. In this study, a selected panel of intestinal bacteria was assessed in 31 confirmed CD patients adhering to gluten-free diet (GFD) for more than 6 months and in 20 NC subjects. Stool samples were collected from each participant, DNA was extracted, and absolute quantitative real-time PCR (qPCR) was carried out. The gut microbiota including Bacteroidetes, Bifidobacterium, Clostridium, Staphylococcus, Enterobacteiaceae, Firmicutes, and Lactobacillus were assessed. The quantities of fruits, vegetables, meat, liquids, sugar and gluten-free candy/bread consumption were evaluated using a questionnaire. The proportion of Bifidobacterium, Firmicutes, and Lactobacillus in CD cases was significantly lower than NC (P < 0.005). Significant correlation coefficients between Bifidobacterium and Lactobacillus (P < 0.001), and also Firmicutes and Lactobacillus (P < 0.001) were recorded. Moreover, a significant association between medium amount of meat and bean consumptions and low abundance of Lactobacillus and Firmicutes (P = 0.024 and P = 0.027, respectively), and also high amount of bean consumptions and low abundance of Lactobacillus (P = 0.027) in CD were observed. The results showed that meat and bean consumptions could reduce the beneficial bacteria including Firmicutes and Lactobacillus in CD patients. Therefore, changes in the gut microbiota abundance may contribute to dietary changes and unimproved CD symptoms.