Association between plant-based dietary index and gallstone disease: A cross sectional study from NHANES.

BACKGROUND: The relationship between plant-based diets and gallstone disease has been debated. This study aimed to shed light on the association between plant-based dietary index and the risk of developing gallstone disease. METHODS: Eligible participants were selected from National Health and Nutrition Examination Survey (NHANES) 2017-2020. Three plant-based diet indexes (PDI, healthy PDI, unhealthy PDI) were calculated using data from two NHANES 24-h dietary recall interviews. Restricted Cubic Spline and multivariate logistic regression were used to analyze the associations. Subgroup analysis was adopted to make the results more robust. RESULTS: A total of 5673 eligible participants were analyzed. After adjusting for various confounding variables, uPDI was positively associated with gallstone disease (OR = 1.53, 95%CI: 1.02-2.29). No association was found between PDI/hPDI and gallstone disease (p > 0.05). The results of subgroup analysis did not show any positive association between uPDI and gallstones in specific groups. CONCLUSION: Our study shows that the elevated uPDI are linked to a higher risk of gallstone disease.

A MYB-related transcription factor ZmMYBR29 is involved in grain filling.

BACKGROUND: The endosperm serves as the primary source of nutrients for maize (Zea mays L.) kernel embryo development and germination. Positioned at the base of the endosperm, the transfer cells (TCs) of the basal endosperm transfer layer (BETL) generate cell wall ingrowths, which enhance the connectivity between the maternal plant and the developing kernels. These TCs play a crucial role in nutrient transport and defense against pathogens. The molecular mechanism underlying BETL development in maize remains unraveled. RESULTS: This study demonstrated that the MYB-related transcription factor ZmMYBR29, exhibited specific expression in the basal cellularized endosperm, as evidenced by in situ hybridization analysis. Utilizing the CRISPR/Cas9 system, we successfully generated a loss-of-function homozygous zmmybr29 mutant, which presented with smaller kernel size. Observation of histological sections revealed abnormal development and disrupted morphology of the cell wall ingrowths in the BETL. The average grain filling rate decreased significantly by 26.7% in zmmybr29 mutant in comparison to the wild type, which impacted the dry matter accumulation within the kernels and ultimately led to a decrease in grain weight. Analysis of RNA-seq data revealed downregulated expression of genes associated with starch synthesis and carbohydrate metabolism in the mutant. Furthermore, transcriptomic profiling identified 23 genes that expressed specifically in BETL, and the majority of these genes exhibited altered expression patterns in zmmybr29 mutant. CONCLUSIONS: In summary, ZmMYBR29 encodes a MYB-related transcription factor that is expressed specifically in BETL, resulting in the downregulation of genes associated with kernel development. Furthermore, ZmMYBR29 influences kernels weight by affecting the grain filling rate, providing a new perspective for the complementation of the molecular regulatory network in maize endosperm development.

PlzD modifies Vibrio vulnificus foraging behavior and virulence in response to elevated c-di-GMP.

IMPORTANCE: Many free-swimming bacteria propel themselves through liquid using rotary flagella, and mounting evidence suggests that the inhibition of flagellar rotation initiates biofilm formation, a sessile lifestyle that is a nearly universal surface colonization paradigm in bacteria. In general, motility and biofilm formation are inversely regulated by the intracellular second messenger bis-(3´-5´)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we identify a protein, PlzD, bearing a conserved c-di-GMP binding PilZ domain that localizes to the flagellar pole in a c-di-GMP-dependent manner and alters the foraging behavior, biofilm, and virulence characteristics of the opportunistic human pathogen, Vibrio vulnificus. Our data suggest that PlzD interacts with components of the flagellar stator to decrease bacterial swimming speed and changes in swimming direction, and these activities are enhanced when cellular c-di-GMP levels are elevated. These results reveal a physical link between a second messenger (c-di-GMP) and an effector (PlzD) that promotes transition from a motile to a sessile state in V. vulnificus.

Ketyl Radical Anion Mediated Radical Polymerization and Anionic Ring-Opening Polymerization to Give Polymers with Low Molecular Weight Distribution.

The development of novel polymerization capable of yielding polymers with low molecular weight distribution (D) is essential and significant in polymer chemistry, where monofunctional initiator contains only one initiation site in these polymerizations generally. Here, ketyl radical anion species is introduced to develop a novel Ketyl Mediated Polymerization (KMP), which enables radical polymerization at carbon radical site and anionic ring-opening polymerization at oxygen anion site, respectively. Meanwhile, polymerization and corresponding organic synthesis generally couldn't be performed simultaneously in one pot. Through KMP, organic synthesis and polymerization are achieved in one pot, where small molecules (cyclopentane derivates) and polymers with low D are successfully prepared under mild condition simultaneously. At the initiation step, both organic synthesis and polymerization are initiated by single electron transfer reaction with ketyl radical anion formation. Cyclopentane derivates are synthesized through 3-3 coupling reaction and cyclization. Polystyrene and polycaprolactone with low D and a full monomer conversion are prepared by KMP via radical polymerization and anionic ring-opening polymerization, respectively. This work therefore enables both organic synthesis and two different polymerizations from same initiation system, which saves time, labour, resource and energy and expands the reaction mode and method libraries of organic chemistry and polymer chemistry.

The transcription factor POPEYE negatively regulates the expression of bHLH Ib genes to maintain iron homeostasis.

Iron (Fe) is an essential trace element for plants. When suffering from Fe deficiency, plants modulate the expression of Fe deficiency-responsive genes to promote Fe uptake. POPEYE (PYE) is a key bHLH (basic helix-loop-helix) transcription factor involved in Fe homeostasis. However, the molecular mechanism of PYE regulating the Fe deficiency response remains elusive in Arabidopsis. We found that the overexpression of PYE attenuates the expression of Fe deficiency-responsive genes. PYE directly represses the transcription of bHLH Ib genes (bHLH38, bHLH39, bHLH100, and bHLH101) by associating with their promoters. Although PYE contains an ethylene response factor-associated amphiphilic repression (EAR) motif, it does not interact with the transcriptional co-repressors TOPLESS/TOPLESS-RELATED (TPL/TPRs). Sub-cellular localization analysis indicated that PYE localizes in both the cytoplasm and nucleus. PYE contains a nuclear export signal (NES) which is required for the cytoplasmic localization of PYE. Mutation of the NES amplifies the repression function of PYE, resulting in down-regulation of Fe deficiency-responsive genes. Co-expression assays indicated that three bHLH IVc members (bHLH104, bHLH105/ILR3, and bHLH115) facilitate the nuclear accumulation of PYE. Conversely, PYE indirectly represses the transcription activation ability of bHLH IVc. Additionally, PYE directly negatively regulates its own transcription. This study provides new insights into the Fe deficiency response signalling pathway and enhances the understanding of PYE functions in Arabidopsis.

Intra-tumor heterogeneity and prognostic risk signature for hepatocellular carcinoma based on single-cell analysis.

Intra-tumor heterogeneity poses a serious challenge in the treatment of cancer, including hepatocellular carcinoma (HCC). Recent developments in single-cell RNA sequencing (scRNA-seq) make it possible to examine the heterogeneity of tumor cells. The Gene Expression Omnibus (GEO) database was retrieved to obtain scRNA-seq data of 13 HCC and 8 para cancer samples, and the cells were clustered using FindNeighbors and FindClusters functions. Cell subsets were defined using the "Enricher" function of the clusterProfiler package. Monocle was used to predict cell developmental trajectory. The LIMMA package included in the R program was utilized to detect differentially expressed genes (DEGs) between HCC and paracancerous tissues. Univariate Cox analysis and Least Absolute and Selection Operator (Lasso) Cox regression analysis were conducted to establish a risk assessment model. Thirteen cell subpopulations were identified from the sequencing data of 64,634 single cells. Four cell subgroups (dendritic cells, hepatocytes, liver bud hepatic cells, and liver progenitor cells) in tumor tissues were highly enriched. Between HCC and para cancer tissues, 3024 DEGs were identified, and 641 were specific markers of four cell subgroups. To develop a prognostic risk model, 9 genes among the 641 genes were selected. In the training and validation sets, the model demonstrated a higher 5-year AUC and independently served as a prognostic marker as confirmed by multivariate and univariate Cox analyses. This study revealed the characteristics of different cell subpopulations of immune cells and tumor cells from the HCC microenvironment. We established a novel nine-gene prognostic model to determine the death risk of HCC patients. The discoveries in this research improved the current knowledge of HCC heterogeneity and may inspire future research.

Living Covalent-Anionic-Radical Polymerization via a Barbier Strategy.

The developments of the living alkene polymerization method have achieved great progress and enabled the precise synthesis of important polyalkenes with controlled molecular weight, molecular weight distribution, and architecture through an anionic, cationic or radical strategy. However, it is still challenging to develop a living alkene polymerization method through an all-in-one strategy where anionic and radical characteristics are merged into one polymerization species. Here, a versatile living polymerization method is reported by introducing a well-established all-in-one covalent-anionic-radical Barbier strategy into a living polymerization. Through this living covalent-anionic-radical Barbier polymerization (Barbier CARP), narrow distributed polystyrenes, with D as low as 1.05, are successfully prepared under mild conditions with a full monomer conversion by using wide varieties of organohalides, for example, alkyl, benzyl, allyl, and phenyl halides, as initiators with Mg in one pot. This living covalent-anionic-radical polymerization via a Barbier strategy expands the methodology library of polymer chemistry and enables living polymerization with an unconventional polymerization mode.

Transcriptomic analysis of Streptococcus agalactiae periprosthetic joint infection.

Although Streptococcus agalactiae periprosthetic joint infection (PJI) is not as prevalent as staphylococcal PJI, invasive S. agalactiae infection is not uncommon. Here, RNA-seq was used to perform transcriptomic analysis of S. agalactiae PJI using fluid derived from sonication of explanted arthroplasties of subjects with S. agalactiae PJI, with results compared to those of S. agalactiae strain NEM316 grown in vitro. A total of 227 genes with outlier expression were found (164 upregulated and 63 downregulated) between PJI sonicate fluid and in vitro conditions. Functional enrichment analysis showed genes involved in mobilome and inorganic ion transport and metabolism to be most enriched. Genes involved in nickel, copper, and zinc transport, were upregulated. Among known virulence factors, cyl operon genes, encoding ß-hemolysin/cytolysin, were consistently highly expressed in PJI versus in vitro. The data presented provide insight into S. agalactiae PJI pathogenesis and may be a resource for identification of novel PJI therapeutics or vaccines against invasive S. agalactiae infections.

Effectiveness and Safety of Rituximab for Refractory Myasthenia Gravis: A Systematic Review and Single-Arm Meta-Analysis.

Background and Objective: Myasthenia gravis (MG) is an autoimmune neuromuscular disease. Nearly 10-30% of patients with MG are refractory to conventional therapy. Rituximab (RTX), a monoclonal antibody targeting CD20, is increasingly used in autoimmune disorders. We performed a systematic review and meta-analysis to evaluate the effectiveness and safety of RTX for refractory MG. Methods: Studies published between January 1, 2000 and January 17, 2021 were searched in PubMed, EMBASE, Cochrane Library, and ClincalTrails.gov. Primary outcomes included proportion of patients achieving minimal manifestation status (MMS) or better and quantitative MG (QMG) score change from baseline. Secondary outcomes were glucocorticoids (GC) doses change from baseline and proportion of patients discontinuing oral immunosuppressants. Results: A total of 24 studies involving 417 patients were included in the meta-analysis. An overall 64% (95% confidence interval, 49-77%) of patients achieved MMS or better. The estimated reduction of QMG score was 1.55 (95% confidence interval, 0.88-2.22). The mean reduction of GC doses was 1.46 (95% confidence interval, 1.10-1.82). The proportion of patients discontinuing oral immunosuppressants was 81% (95% confidence interval, 66-93%). Subgroup analyses showed that the proportion of patients achieving MMS or better and discontinuing oral immunosuppressants was higher in MuSK-MG group than those in AChR-MG group. Improvement was more pronounced in patients with mild to moderate MG compared to those with severe MG. Moreover, the efficacy appeared to be independent of the dose of RTX. 19.6% of patients experienced adverse events, most of which were mild to moderate. Only one patient developed progressive multifocal leukoencephalopathy. Conclusions: RTX can alleviate the symptom of weakness, decrease QMG score and reduce the doses of steroids and non-steroid immunosuppressive agents in refractory MG. It is well-tolerated with few severe adverse events. Randomized controlled trials are urgently needed to study the efficacy of RTX in treating refractory MG and to identify the characteristics of patients who might respond well to RTX.

Aminoglycosides with Anti-MRSA Activity: A Concise Review.

Methicillin-resistant Staphylococcus aureus (MRSA), a leading cause of infections in human being and is usually associated with a multidrug-resistant profile, represents a significant health threat and public burden globally. The limited options of effective antibiotics motivate the search for novel anti-MRSA agents. Aminoglycoside antibiotics have been extensively applied in the medical field due to their desirable broad-spectrum antibacterial activity, especially for systemic infections caused by Gram-negative organisms. Recent studies demonstrated that aminoglycosides also possessed potential activity against MRSA, so aminoglycosides may be useful weapons to fight against MRSA. The present work aims to summarize the current scenario of aminoglycosides with anti- MRSA potential, covering articles published between 2010 and 2020. The structure-activity relationship and the mechanism of action are also discussed for the further rational design of novel potential drug candidates.

IRON MAN interacts with BRUTUS to maintain iron homeostasis in Arabidopsis.

IRON MAN (IMA) peptides, a family of small peptides, control iron (Fe) transport in plants, but their roles in Fe signaling remain unclear. BRUTUS (BTS) is a potential Fe sensor that negatively regulates Fe homeostasis by promoting the ubiquitin-mediated degradation of bHLH105 and bHLH115, two positive regulators of the Fe deficiency response. Here, we show that IMA peptides interact with BTS. The C-terminal parts of IMA peptides contain a conserved BTS interaction domain (BID) that is responsible for their interaction with the C terminus of BTS. Arabidopsis thaliana plants constitutively expressing IMA genes phenocopy the bts-2 mutant. Moreover, IMA peptides are ubiquitinated and degraded by BTS. bHLH105 and bHLH115 also share a BID, which accounts for their interaction with BTS. IMA peptides compete with bHLH105/bHLH115 for interaction with BTS, thereby inhibiting the degradation of these transcription factors by BTS. Genetic analyses suggest that bHLH105/bHLH115 and IMA3 have additive roles and function downstream of BTS. Moreover, the transcription of both BTS and IMA3 is activated directly by bHLH105 and bHLH115 under Fe-deficient conditions. Our findings provide a conceptual framework for understanding the regulation of Fe homeostasis: IMA peptides protect bHLH105/bHLH115 from degradation by sequestering BTS, thereby activating the Fe deficiency response.

Traditional Chinese medicine for gouty arthritis: A protocol for meta-analysis.

BACKGROUND: Gouty arthritis (GA) is a chronic disease caused by monosodium urate crystal deposition. Repeated attacks of arthritis may lead to the deposition of urate to form gout stone, resulting in joint deformity and joint damage. Although GA is not fatal, it causes low work productivity and low quality of life. Western drug, such as febuxostat, colchicine, allopurinol, often cannot get satisfying curative effect, and may even lead to serious side effects, such as exfoliative dermatitis or uremia. However, the therapeutic effect of Traditional Chinese medicine is very satisfactory. The treatment effect of simiao powder, a Chinese patent medicine, combined with acupuncture was widely used on treatment of GA. Although it has been widely used in clinical practice, its relative effectiveness and safety have not been confirmed. Therefore, this study will use meta-analysis to verify the efficacy and safety of simiao powder combined with acupuncture in the treatment of GA. METHODS: All randomized controlled trial of simiao powder combined with acupuncture for the treatment of RA from their inception 29 October, 2020 will be searched form the China National Knowledge Infrastructure, Wanfang Database, Chinese Science and Technology Periodical Database, Chinese Biomedical Literature Database, Pubmed, Embase, Web of Science, and the Cochrane library. Two authors will independently select studies, extract data based on pre-designed inclusion and exclusion criteria. Methodological quality assessment and risk of bias will be assessed using Cochrane bias risk tool. All data analysis will be conducted using Revman5.3, WinBUGS 1.4.3, and Stata14.2 software. RESULTS: We will compare the different outcome indicators of various studies to provide a synthesis of the efficacy and safety of Simiao powder combined with acupuncture for GA patients. The main outcome measures included efficacy, remission rate (no drug symptoms), recurrence rate, clinical absolute score and relative score. Secondary outcome measures included related adverse reactions and uric acid concentration. CONCLUSION: The findings of the study will provide helpful evidence for the efficacy and safety of simiao powder combined with acupuncture in the treatment of GA. REGISTRATION NUMBER: This study protocol have been funded through a protocol registry. The registry number is INPLASY2020110028.

Role of gut microbiota in regulating gastrointestinal dysfunction and motor symptoms in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized primarily by motor and non-motor gastrointestinal (GI) deficits. GI symptoms' including compromised intestinal barrier function often accompanies altered gut microbiota composition and motor deficits in PD. Therefore, in this study, we set to investigate the role of gut microbiota and epithelial barrier dysfunction on motor symptom generation using a rotenone-induced mouse model of PD. We found that while six weeks of 10 mg/kg of chronic rotenone administration by oral gavage resulted in loss of tyrosine hydroxylase (TH) neurons in both germ-free (GF) and conventionally raised (CR) mice, the decrease in motor strength and coordination was observed only in CR mice. Chronic rotenone treatment did not disrupt intestinal permeability in GF mice but resulted in a significant change in gut microbiota composition and an increase in intestinal permeability in CR mice. These results highlight the potential role of gut microbiota in regulating barrier dysfunction and motor deficits in PD.

Clostridioides difficile Whole-genome Sequencing Differentiates Relapse With the Same Strain From Reinfection With a New Strain.

BACKGROUND: Current approaches in tracking Clostridioides difficile infection (CDI) and individualizing patient management are incompletely defined. METHODS: We recruited 468 subjects with CDI at Mayo Clinic Rochester between May and December 2016 and performed whole-genome sequencing (WGS) on C. difficile isolates from 397. WGS was also performed on isolates from a subset of the subjects at the time of a recurrence of infection. The sequence data were analyzed by determining core genome multilocus sequence type (cgMLST), with isolates grouped by allelic differences and the predicted ribotype. RESULTS: There were no correlations between C. difficile isolates based either on cgMLST or ribotype groupings and CDI outcome. An epidemiologic assessment of hospitalized subjects harboring C. difficile isolates with ≤2 allelic differences, based on standard infection prevention and control assessment, revealed no evidence of person-to-person transmission. Interestingly, community-acquired CDI subjects in 40% of groups with ≤2 allelic differences resided within the same zip code. Among 18 subjects clinically classified as having recurrent CDI, WGS revealed 14 with initial and subsequent isolates differing by ≤2 allelic differences, suggesting a relapse of infection with the same initial strain, and 4 with isolates differing by >50 allelic differences, suggesting reinfection. Among the 5 subjects classified as having a reinfection based on the timing of recurrence, 3 had isolates with ≤2 allelic differences between them, suggesting a relapse, and 2 had isolates differing by >50 allelic differences, suggesting reinfection. CONCLUSIONS: Our findings point to potential transmission of C. difficile in the community. WGS better differentiates relapse from reinfection than do definitions based on the timing of recurrence.

Plasmid Acquisition Alters Vancomycin Susceptibility in Clostridioides difficile.

The increasing incidence of primary and recurring Clostridioides difficile infections (CDI), which evade current treatment strategies, reflects the changing biology of C difficile. Here, we describe a putative plasmid-mediated mechanism potentially driving decreased sensitivity of C difficile to vancomycin treatment. We identified a broad host range transferable plasmid in a C difficile strain associated with lack of adequate response to vancomycin treatment. The transfer of this plasmid to a vancomycin-susceptible C difficile isolate decreased its susceptibility to vancomycin in vitro and resulted in more severe disease in a humanized mouse model. Our findings suggest plasmid acquisition in the gastrointestinal tract to be a possible mechanism underlying vancomycin treatment failure in patients with CDI, but further work is needed to characterize the mechanism by which plasmid genes determine vancomycin susceptibility in C difficile.

Bacterially Derived Tryptamine Increases Mucus Release by Activating a Host Receptor in a Mouse Model of Inflammatory Bowel Disease.

Recent studies emphasize the role of microbial metabolites in regulating gastrointestinal (GI) physiology through activation of host receptors, highlighting the potential for inter-kingdom signaling in treating GI disorders. In this study, we show that tryptamine, a tryptophan-derived bacterial metabolite, stimulates mucus release from goblet cells via activation of G-protein-coupled receptor (GPCR) 5-HT4R. Germ-free mice colonized with engineered Bacteroides thetaiotaomicron optimized to produce tryptamine (Trp D+) exhibit decreased weight loss and increased mucus release following dextran sodium sulfate treatment when compared with mice colonized with control B. thetaiotaomicron (Trp D-). Additional beneficial effects in preventing barrier disruption and lower disease activity index were seen only in female mice, highlighting sex-specific effects of the bacterial metabolite. This study demonstrates potential for the precise modulation of mucus release by microbially produced 5-HT4 GPCR agonist as a therapeutic strategy to treat inflammatory conditions of the GI tract.

The mRNA of TCTP functions as a sponge to maintain homeostasis of TCTP protein levels in hepatocellular carcinoma.

Translationally controlled tumor protein (TCTP) is a highly conserved protein that accumulated in the tumorigenesis of various malignancies. Despite the important role of TCTP protein in tumor progression, the precise function and underlying mechanistic regulation of TCTP mRNA in hepatocellular carcinoma (HCC) remain unclear. In this study, we found that TCTP protein was overexpressed in HCC patients but TCTP mRNA expression levels were reversed. TCTP knockout HCC cells exhibited attenuated abilities of proliferation, migration, and invasion. The knockdown of TCTP by siRNA effectively reduced TCTP mRNA levels but not protein levels in HCC cells. Moreover, although the constitutive knockdown of TCTP inhibited almost 80% of TCTP protein expression levels in tumors of wildtype transgenic mice (TCTP KD/WT), partial restoration of TCTP protein expression was observed in the tumors of heterozygous TCTP mice (TCTP KD/TCTP±). The blockage of mRNA synthesis with ActD stimulated TCTP protein expression in HCC cells. In contrast, combined treatment with ActD and CHX or MG132 treatment alone did not lead to the TCTP protein accumulation in cells. Furthermore, following the introduction of exogenous TCTP in cells and orthotopic HCC tumor models, the endogenous TCTP protein did not change with the recombinational TCTP expression and kept a rather stable level. Dual-luciferase assays revealed that the coding sequence of TCTP mRNA functions as a sponge to regulate the TCTP protein expression. Collectively, our results indicated that the TCTP mRNA and protein formed a closed regulatory circuit and works as a buffering system to keep the homeostasis of TCTP protein levels in HCC.

Traditional Chinese medicine for irritable bowel syndrome: A protocol for meta-analysis.

BACKGROUND: Irritable bowel syndrom (IBS) is a common functional gastrointestinal disorder which is characterized as recurrent abdominal pain, abdominal discomfort, and abnormal bowel habits such as diarrhea, constipation, both or alternate appear. Although IBS is not fatal, it seriously affects the patients' daily life and work. Western drug, such as antidiarrheals, gastrointestinal antispasmodic, often cannot get satisfying curative effects. However, the therapeutic effect of Traditional Chinese medicine (TCM) on IBS is very satisfactory which was shown in a large number of randomized controlled trials. Although TCM has been widely used in clinical practice, its relative effectiveness and safety have not been confirmed. Therefore, this study will use meta-analysis to verify the efficacy and safety of different types of TCM in the treatment of IBS. METHODS: We search the China National Knowledge Infrastructure, Wanfang Database, Chinese Science and Technology Periodical Database, Chinese Biomedical Literature Database, Pubmed, Embase, Web of Science, and the Cochrane library for all randomized controlled trial of TCM for the treatment of IBS from their inception to Oct 15, 2020. Two authors will independently select studies, extract data based on predesigned inclusion and exclusion criteria. Methodological quality assessment and risk of bias will be assessed using Cochrane bias risk tool. All data analysis will be conducted using Revman5.3, WinBUGS 1.4.3, and Stata14.2 software. RESULTS: This study will compare the different outcome indicators of various studies directly and indirectly, and provide a high-quality synthesis of effectiveness and safety of different TCM methods for patients with IBS. The main outcome indicators include effectiveness, remission rate (no drug symptoms), relapse rate, clinical absolute score, and relative score. Secondary outcome indicators included related adverse reactions and serum serotonin concentration. CONCLUSION: The conclusion of this systematic review will provide a high-quality evidence based on the efficacy and safety of different TCM treatment methods for IBS. REGISTRATION NUMBER: This study protocol has been funded through a protocol registry. The registry number is INPLASY2020100052.

Vibrio cholerae Type VI Activity Alters Motility Behavior in Mucin.

Motility is required for many bacterial pathogens to reach and colonize target sites. Vibrio cholerae traverses a thick mucus barrier coating the small intestine to reach the underlying epithelium. We screened a transposon library in motility medium containing mucin to identify factors that influence mucus transit. Lesions in structural genes of the type VI secretion system (T6SS) were among those recovered. Two-dimensional (2D) and 3D single-cell tracking was used to compare the motility behaviors of wild-type cells and a mutant that collectively lacked three essential T6SS structural genes (T6SS-). In the absence of mucin, wild-type and T6SS- cells exhibited similar speeds and run-reverse-flick (RRF) swimming patterns, in which forward-moving cells briefly backtrack before stochastically reorienting (flicking) in a new direction upon resuming forward movement. We show that mucin induced T6SS expression and activity in wild-type bacteria but significantly decreased their swimming speed and flicking, yielding curvilinear or near-surface circular traces for many cells. Conversely, mucin slowed T6SS- cells to a lesser extent, and many continued to flick and produce RRF-like traces. ΔcheY3 cells, which exclusively swim in the forward direction and thus cannot flick, also produced curvilinear traces with or without mucin present and, on occasion, near-surface circular traces in the presence of mucin. The dependence of flicking on swimming speed suggested that mucin-induced T6SS activity further decreased V. cholerae motility and thereby reduced flicking probability during reverse-to-forward transitions. We propose that this encourages cells to continue on their current trajectory rather than reorienting, which may benefit those tracking toward the epithelial surface.IMPORTANCEV. cholerae deploys an arsenal of virulence factors as it attempts to traverse a protective mucus layer and reach the epithelial surface of the distal small intestine. The T6SS used to cull bacterial competition during infection is induced by mucus. We show that this activity may serve an additional purpose by further decreasing motility in the presence of mucin, thereby reducing the probability of speed-dependent, near-perpendicular directional changes. We posit that this encourages cells to maintain course rather than change direction, which may aid those attempting to reach and colonize the epithelial surface.

Calcium-dependent site-switching regulates expression of the atypical iam pilus locus in Vibrio vulnificus.

The opportunistic human pathogen Vibrio vulnificus inhabits warm coastal waters and asymptomatically colonizes seafood, most commonly oysters. We previously characterized an isolate that exhibited greater biofilm formation, aggregation and oyster colonization than its parent. This was due, in part, to the production of a Type IV Tad pilus (Iam). However, the locus lacked key processing and regulatory genes required for pilus production. Here, we identify a pilin peptidase iamP, and LysR-type regulator (LRTR) iamR, that fulfil these roles and show that environmental calcium, which oysters enrich for shell repair and growth, regulates iam expression. The architecture of the iam locus differs from the classical LRTR paradigm and requires an additional promoter to be integrated into the regulatory network. IamR specifically recognized the iamR promoter (PiamR ) and the intergenic iamP-iamA region (PiamP-A ). PiamR exhibited classical negative auto-regulation but, strikingly, IamR inversely regulated the divergent iamP and iamA promoters in a calcium-dependent manner. Moreover, expression of the c-di-GMP and calcium-regulated, biofilm-promoting brp exopolysaccharide was IamA-dependent. These results support a scenario in which the calcium-enriched oyster environment triggers IamP-mediated processing of prepilin amassed in the periplasm for rapid pilin elaboration and subsequent BRP production to promote colonization.