Mannose-sensitive Hemagglutinin Research Articles

PA-MSHA improves prognosis of patients undergoing radical cystectomy: a retrospective cohort study using inverse probability of treatment weighting.

To observe the effect of Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) on the prognosis and the incidence of lymphatic leakage in patients undergoing radical cystectomy (RC). A total of 129 patients who underwent RC in Lanzhou University Second Hospital from 2013 to 2022 were enrolled in this study. They were divided into 43 patients treated with PA-MSHA and 86 patients in the control group. Inverse probability of treatment weighting (IPTW) was applied to reduce potential selection bias. Kaplan-Meier method and Cox regression analysis were used to analyze the effect of PA-MSHA on the survival of patients and the incidence of postoperative lymphatic leakage. The PA-MSHA group exhibited improved overall survival (OS) and cancer-specific survival (CSS) rates compared to the control group. The 3-year and 5-year overall survival (OS) rates for the PA-MSHA group were 69.1% and 53.2%, respectively, compared to 55.6% and 45.3% for the control group (Log-rank=3.218, P=0.072). The 3-year and 5-year cancer-specific survival (CSS) rates for the PA-MSHA group were 73.3% and 56.5%, respectively, compared to 58.0% and 47.3% for the control group (Log-rank=3.218, P=0.072). Additionally, the 3-year and 5-year progression-free survival (PFS) rates for the PA-MSHA group were 74.4% and 56.8%, respectively, compared to 57.1% and 52.2% for the control group (Log-rank=2.016, P=0.156). Multivariate Cox regression analysis indicates that lymph node metastasis and distant metastasis are poor prognostic factors for patients, while the use of PA-MSHA can improve patients' OS (HR: 0.547, 95%CI: 0.304-0.983, P=0.044), PFS (HR: 0.469, 95%CI: 0.229-0.959, P=0.038) and CSS (HR: 0.484, 95%CI: 0.257-0.908, P=0.024). The same trend was observed in the cohort After IPTW adjustment. Although there was no significant difference in the incidence of postoperative lymphatic leakage [18.6% (8/35) vs. 15.1% (84.9%), P=0.613] and pelvic drainage volume [470 (440) ml vs. 462.5 (430) ml, P=0.814] between PA-MSHA group and control group, PA-MSHA could shorten the median retention time of drainage tube (7.0 d vs 9.0 d) (P=0.021). PA-MSHA may improve radical cystectomy in patients with OS, PFS, and CSS, shorten the pelvic drainage tube retention time.

Insight into the antibacterial properties of terpinen-4-ol on planktonic growth and biofilm-formation of Vibrio parahaemolyticus

Vibrio parahaemolyticus outbreaks emphasize the urgency of highly efficient prevention strategies to control this foodborne pathogen. This study investigated the antibacterial effects of terpinen-4-ol on V. parahaemolyticus RIMD2210633 and explored the underlying mechanism via RNA-seq analysis. The results showed that terpinen-4-ol inhibited V. parahaemolyticus efficiently with minimum inhibitory concentration and minimum bactericidal concentration values at 0.8 μL/mL. Scanning electron microscopy and PI/SYTO 9 staining assay demonstrated that cell morphology and cell membrane integrity were dose-dependently destructed. Additionally, biofilm formation was significantly inhibited according to crystal violet and MTT assay analysis. Notably, terpinen-4-ol showed good inhibitory activity against V. parahaemolyticus on salmon fillets via fumigation at 4 °C. Transcriptional analysis revealed significant downregulation of genes participated in cell wall formation (oppB, oppF), cell membrane integrity, pilus assembly protein, biofilm formation and cell motility (ToxR , LuxR), capsular polysaccharide biosynthesis protein, type IV pilin MshA and mannose-sensitive haemagglutinin type-IV pili (MSHA) biogenesis protein (MshJ, MshJ, MshK, MshL, MshN). The results demonstrated that terpinen-4-ol can efficiently inhibit the growth and pathogenicity of V. parahaemolyticus. With its safety and pleasant odor, terpinen-4-ol can be developed as a natural antibacterial substance to tackle V. parahaemolyticus contamination in food preservation and prevent associated diseases.

Unveiling novel threats: Urban river isolation of Aeromonas veronii with unusual VEB-28 extended-spectrum β-lactamase and distinct mcr variants

Aeromonas spp. are frequently encountered in aquatic environments, with Aeromonas veronii emerging as an opportunistic pathogen causing a range of diseases in both humans and animals. Recent reports have raised public health concerns due to the emergence of multidrug-resistant Aeromonas spp. This is particularly noteworthy as these species have demonstrated the ability to acquire and transmit antimicrobial resistance genes (ARGs). In this study, we report the genomic and phenotypic characteristics of the A. veronii TR112 strain, which harbors a novel variant of the Vietnamese Extended-spectrum β-lactamase-encoding gene, blaVEB-28, and two mcr variants recovered from an urban river located in the Metropolitan Region of São Paulo, Brazil. A. veronii TR112 strain exhibited high minimum inhibitory concentrations (MICs) for ceftazidime (64 μg/mL), polymyxin (8 μg/mL), and ciprofloxacin (64 μg/mL). Furthermore, the TR112 strain demonstrated adherence to HeLa and Caco-2 cells within 3 h, cytotoxicity to HeLa cells after 24 h of interaction, and high mortality rates to the Galleria mellonella model. Genomic analysis showed that the TR112 strain belongs to ST257 and presented a range of ARGs conferring resistance to β-lactams (blaVEB-28, blaCphA3, blaOXA-912) and polymyxins (mcr-3 and mcr-3.6). Additionally, we identified a diversity of virulence factor-encoding genes, including those encoding mannose-sensitive hemagglutinin (Msh) pilus, polar flagella, type IV pili, type II secretion system (T2SS), aerolysin (AerA), cytotoxic enterotoxin (Act), hemolysin (HlyA), hemolysin III (HlyIII), thermostable hemolysin (TH), and capsular polysaccharide (CPS). In conclusion, our findings suggest that A. veronii may serve as an environmental reservoir for ARGs and virulence factors, highlighting its importance as a potential pathogen in public health.

Autoinducer-2 promotes adherence of Aeromonas veronii through facilitating the expression of MSHA type IV pili genes mediated by c-di-GMP.

Aeromonas veronii can adhere to host cells through different adherence factors including outer-membrane proteins (OMPs), lipopolysaccharide (LPS), and pili, but its adherence mechanisms are still unclear. Here, we evaluated the effect of autoinducer-2 (AI-2) on adherence of A. veronii and its regulation mechanism. After determination of the promotion effect of AI-2 on adherence, we investigated which adherence factor was regulated by AI-2, and the results show that AI-2 only limits the formation of pili. Among the four distinct pili systems, only the mannose-sensitive hemagglutinin (MSHA) type IV pili genes were significantly downregulated after deficiency of AI-2. MshE, an ATPase belonged to MSHA type IV pilin, was confirmed as c-di-GMP receptor, that can bind with c-di-GMP which is positively regulated by AI-2, and the increase of c-di-GMP can promote the expression of MSHA type IV pili genes and adherence of A. veronii. Therefore, this study confirms that c-di-GMP positively regulated by AI-2 binds with MshE, then increases the expression of MSHA pili genes, finally promoting adherence of A. veronii, suggesting a multilevel positive regulatory adhesion mechanism that is responsible for A. veronii adherence.

Transcriptomic Profiles of Vibrio parahaemolyticus During Biofilm Formation.

Vibrio parahaemolyticus, the leading cause of bacterial seafood-associated gastroenteritis, can form biofilms. In this work, the gene expression profiles of V. parahaemolyticus during biofilm formation were investigated by transcriptome sequencing. A total of 183, 503, and 729 genes were significantly differentially expressed in the bacterial cells at 12, 24 and 48h, respectively, compared with that at 6h. Of these, 92 genes were consistently activated or repressed from 6 to 48h. The genes involved in polar flagellum, chemotaxis, mannose-sensitive haemagglutinin type IV pili, capsular polysaccharide, type III secretion system 1 (T3SS1), T3SS2, thermostable direct hemolysin (TDH), type VI secretion system 1 (T6SS1) and T6SS2 were downregulated, whereas those involved in V. parahaemolyticus pathogenicity island (Vp-PAI) (except for T3SS2 and TDH) and membrane fusion proteins were upregulated. Three extracellular protease genes (vppC, prtA and VPA1071) and a dozen of outer membrane protein encoding genes were also significantly differentially expressed during biofilm formation. In addition, five putative c-di-GMP metabolism-associated genes were significantly differentially expressed, which may account for the drop in c-di-GMP levels after the beginning of biofilm formation. Moreover, many putative regulatory genes were significantly differentially expressed, and more than 1000 putative small non-coding RNAs were detected, suggesting that biofilm formation was tightly regulated by complex regulatory networks. The data provided a global view of gene expression profiles during biofilm formation, showing that the significantly differentially expressed genes were involved in multiple cellular pathways, including virulence, biofilm formation, metabolism, and regulation.

Effect of sublethal dose of chloramphenicol on biofilm formation and virulence in Vibrio parahaemolyticus.

Vibrio parahaemolyticus isolates are generally very sensitive to chloramphenicol. However, it is usually necessary to transfer a plasmid carrying a chloramphenicol resistance gene into V. parahaemolyticus to investigate the function of a specific gene, and the effects of chloramphenicol on bacterial physiology have not been investigated. In this work, the effects of sublethal dose of chloramphenicol on V. parahaemolyticus were investigated by combined utilization of various phenotypic assays and RNA sequencing (RNA-seq). The results showed that the growth rate, biofilm formation capcity, c-di-GMP synthesis, motility, cytoxicity and adherence activity of V. parahaemolyticus were remarkably downregulated by the sublethal dose of chloramphenicol. The RNA-seq data revealed that the expression levels of 650 genes were significantly differentially expressed in the response to chloramphenicol stress, including antibiotic resistance genes, major virulence genes, biofilm-associated genes and putative regulatory genes. Majority of genes involved in the synthesis of polar flagellum, exopolysaccharide (EPS), mannose-sensitive haemagglutinin type IV pilus (MSHA), type III secretion systems (T3SS1 and T3SS2) and type VI secretion system 2 (T6SS2) were downregulated by the sublethal dose of chloramphenicol. Five putative c-di-GMP metabolism genes were significantly differentially expressed, which may be the reason for the decrease in intracellular c-di-GMP levels in the response of chloramphenicol stress. In addition, 23 genes encoding putative regulators were also significantly differentially expressed, suggesting that these regulators may be involved in the resistance of V. parahaemolyticus to chloramphenicol stress. This work helps us to understand how chloramphenicol effect on the physiology of V. parahaemolyticus.

Regulatory effect of Pseudomonas aeruginosa mannose-sensitive hemagglutinin on inflammation and immune function in percutaneous nephrolithotomy patients with upper urinary tract calculi complicated with infection.

To study the effect of an injection of Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) on inflammation and immune function in patients with upper urinary tract calculi complicated by infection who have undergone percutaneous nephrolithotomy. We retrospectively recorded the clinical data of patients with upper urinary tract calculi complicated by infection who have undergone Percutaneous nephrolithotomy(PCNL) in the Department of Urology, 2nd Affiliation Hospital of Kunming Medical University, from March to December 2021. Clinical data include general condition, laboratory index, CT, postoperative body temperature, heart rate, respiration, SIRS, sepsis, etc. Patients were divided into treated and control groups according to whether they had received a preoperative PA-MSHA injection. The two groups were compared for indices of inflammation and complications of infection after PCNL. Pre- and post-operative lymphocyte subsets and immunoglobulin changes were compared. 115 patients were included in the study, including 43 in the treatment group and 72 in the control group. After Propensity Score Matching, 90 patients were divided into treatment (n=35) and control (n=55) groups. The postoperative inflammation index was higher in the treatment group than in the control group (P<0.05). The incidence of postoperative SIRS was higher in the treatment group than control (P<0.05). There were no cases of sepsis in either group. The double-positive T cells lymphocyte subsets were higher in the treatment group than in the control group ((P<0.05). Pre- and post-operative changes in immune function: total T lymphocyte count reduced, NK and NKT cell count increased in the control group, double-positive T cell count increased in the treatment group, IgG, IgA, IgM, complement C3 and C4 count reduced in both groups post-operatively. This study found that patients with upper urinary tract calculi and infection treated with antibiotic-based PA-MSHA before percutaneous nephrolithotomy had an increased inflammatory response after surgery, which may play a role in the prevention and treatment of sepsis. The percentage of double-positive T cells in the peripheral blood was increased after PA-MSHA treatment, which may have an immunomodulatory and protective effect in PCNL patients with stones complicated by infection.

Biofilm formation on human immune cells is a multicellular predation strategy of Vibrio cholerae

Biofilm formation is generally recognized as a bacterial defense mechanism against environmental threats, including antibiotics, bacteriophages, and leukocytes of the human immune system. Here, we show that for the human pathogen Vibrio cholerae, biofilm formation is not only a protective trait but also an aggressive trait to collectively predate different immune cells. We find that V.cholerae forms biofilms on the eukaryotic cell surface using an extracellular matrix comprising primarily mannose-sensitive hemagglutinin pili, toxin-coregulated pili, and the secreted colonization factor TcpF, which differs from the matrix composition of biofilms on other surfaces. These biofilms encase immune cells and establish a high local concentration of a secreted hemolysin to kill the immune cells before the biofilms disperse in a c-di-GMP-dependent manner. Together, these results uncover how bacteria employ biofilm formation as a multicellular strategy to invert the typical relationship between human immune cells as the hunters and bacteria as the hunted.

Plastics select for distinct early colonizing microbial populations with reproducible traits across environmental gradients.

Little is known about early plastic biofilm assemblage dynamics and successional changes over time. By incubating virgin microplastics along oceanic transects and comparing adhered microbial communities with those of naturally occurring plastic litter at the same locations, we constructed gene catalogues to contrast the metabolic differences between early and mature biofilm communities. Early colonization incubations were reproducibly dominated by Alteromonadaceae and harboured significantly higher proportions of genes associated with adhesion, biofilm formation, chemotaxis, hydrocarbon degradation and motility. Comparative genomic analyses among the Alteromonadaceae metagenome assembled genomes (MAGs) highlighted the importance of the mannose-sensitive hemagglutinin (MSHA) operon, recognized as a key factor for intestinal colonization, for early colonization of hydrophobic plastic surfaces. Synteny alignments of MSHA also demonstrated positive selection for mshA alleles across all MAGs, suggesting that mshA provides a competitive advantage for surface colonization and nutrient acquisition. Large-scale genomic characteristics of early colonizers varied little, despite environmental variability. Mature plastic biofilms were composed of predominantly Rhodobacteraceae and displayed significantly higher proportions of carbohydrate hydrolysis enzymes and genes for photosynthesis and secondary metabolism. Our metagenomic analyses provide insight into early biofilm formation on plastics in the ocean and how early colonizers self-assemble, compared to mature, phylogenetically and metabolically diverse biofilms.

Neoadjuvant Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) and chemotherapy versus placebo plus chemotherapy in patients with HER2-negative breast cancer: a randomized, controlled, double-blind trial.

According to preclinical experiments, Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) exerts antiproliferative effects against breast cancer cells. It has been approved by the State Food and Drug Administration in China for complementary cancer treatment, and its safety has been confirmed in previous clinical trials. The present randomized, controlled, double-blind clinical trial was conducted to investigate the efficacy and safety of neoadjuvant PA-MSHA and placebo with chemotherapy in patients with human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Eligible patients aged 18 years or older with previously untreated HER2-negative stage II-III breast cancer were enrolled and randomly assigned at a 1:1 ratio to receive neoadjuvant chemotherapy with PA-MSHA or a placebo. The Response Evaluation Criteria in Solid Tumors (RECIST) was used to assess clinical response every 2 cycles. The primary endpoint was the objective response rate (ORR) based on the clinical response following neoadjuvant chemotherapy. A total of 75 patients were randomly assigned to either the PA-MSHA group (37 patients) or the control group (38 patients). The ORR was found to be significantly higher in the PA-MSHA group compared with the control group [86.5% versus 60.5%; rate difference 26.0; 95% confidence interval (CI): 5.9-43.5%; P=0.011]. The pathological complete response (pCR) and survival outcomes did not differ significantly between the 2 groups. Patients with immune-related adverse events (irAEs) appeared to benefit from the PA-MSHA treatment, with greater disease-free, relapse-free, and overall survival. The application of PA-MSHA to neoadjuvant chemotherapy did not increase the incidence of severe adverse events. Moreover, the addition of PA-MSHA increased serum interferon-γ levels and the percentage of peripheral blood T cells, CD8+/CD4+ T cells, CD8+CD28+ T cells, and natural killer (NK) cells, and decreased serum interleukin 4 levels. The addition of PA-MSHA to neoadjuvant chemotherapy is an effective alternative regimen for HER2-negative breast cancer. Patients with irAEs caused by PA-MSHA may obtain more benefits from this treatment. Chinese Clinical Trial Registry ChiCTR-TRC-10000794.

PA-MSHA Regulates PD-L1 Expression in Hepatoma Cells

ABSTRACT Background Programmed death ligand 1 (PD-L1) is expressed in hepatocellular carcinoma (HCC) cells. PD-L1 function and structure are regulated through glycosylation and various signaling pathways. However, the relationship between Pseudomonas aeruginosa mannose sensitive hemagglutinin (PA-MSHA), glycosylation and PD-L1 warrants further study. In this study, we investigated the effects of PA-MSHA on the regulation of mannosyl and N-glycosylation to identify the mechanisms underlying its function. Methods PD-L1, β-catenin, c-Myc, mannosyl, MGAT1 and mannosidase II in HCC were identified by postoperative specimens from the HCC cohort with immunohistochemistry and immunofluorescence. PA-MSHA was used to suppress tumor progression. Alterations to the expression of PD-L1, β-catenin, c-Myc, MGAT1, and mannosidase II at the gene and protein levels were detected by qRT-PCR and Western blot analysis. Soluble PD-L1 (sPD-L1) were detected using enzyme-linked immunosorbent assay. Results Mannosyl and mannosidase II expression levels increased, whereas those of MGAT1 decreased in the HCC cells. The glycosylation-related pathway proteins, namely, β-catenin, c-Myc and PD-L1, had increased expression levels. Moreover, proliferation in the HCC cells was inhibited after PA-MSHA treatment, PD-L1 function was significantly inhibited. Transmission electron microscopy showed that PA-MSHA penetrated into the HCC cytoplasm through the cytomembrane, resulting in apoptosis. Here, PA-MSHA significantly reduced sPD-L1 expression levels in the tumor cells. Conclusions PA-MSHA plays the role of a lectin, affecting receptors on the cytomembrane. This strain inhibits mannosyl by suppressing β-catenin signaling. We hypothesized that PA-MSHA suppresses PD-L1 by: 1. Inhibiting the glycosylation process; and 2. Suppressing β-catenin and c-Myc, thereby reducing the transcription of this protein.

Toxic potential of Pseudomonas aeruginosa mannose-sensitive hemagglutinin: in silico investigation of adverse outcomes in cancer patients

Toxic potential of Pseudomonas aeruginosa mannose-sensitive hemagglutinin: in silico investigation of adverse outcomes in cancer patients

The PilT retraction ATPase promotes both extension and retraction of the MSHA type IVa pilus in Vibrio cholerae.

Diverse bacterial species use type IVa pili (T4aP) to interact with their environments. The dynamic extension and retraction of T4aP is critical for their function, but the mechanisms that regulate this dynamic activity remain poorly understood. T4aP are typically extended via the activity of a dedicated extension motor ATPase and retracted via the action of an antagonistic retraction motor ATPase called PilT. These motors are generally functionally independent, and loss of PilT commonly results in T4aP hyperpiliation due to undeterred pilus extension. However, for the mannose-sensitive hemagglutinin (MSHA) T4aP of Vibrio cholerae, the loss of PilT unexpectedly results in a loss of surface piliation. Here, we employ a combination of genetic and cell biological approaches to dissect the underlying mechanism. Our results demonstrate that PilT is necessary for MSHA pilus extension in addition to its well-established role in promoting MSHA pilus retraction. Through a suppressor screen, we also provide genetic evidence that the MshA major pilin impacts pilus extension. Together, these findings contribute to our understanding of the factors that regulate pilus extension and describe a previously uncharacterized function for the PilT motor ATPase.

An Increase of Seawater Temperature Upregulates the Expression of Vibrio parahaemolyticus Virulence Factors Implicated in Adhesion and Biofilm Formation.

Climate change driven seawater temperature (SWT) increases results in greater abundance and geographical expansion of marine pathogens, among which Vibrio parahaemolyticus (Vp) causes serious economic and health issues. In addition, plastic pollution in the ocean constitutes a vector for harmful pathogens dissemination. We investigate the effect of elevated SWT on the expression of genes implicated in adhesion and biofilm formation on abiotic surfaces in the clinical Vp strain RIMD2210633, which expresses hemolysins. Among the genes studied, the multivalent adhesion molecule-7 and the GlcNAc-binding protein A were involved in the adhesion of Vp to abiotic and biotic surfaces, whereas the type IV pili, the mannose-sensitive hemagglutinin, and the chitin-regulated pilins facilitate attachment and biofilm formation. Data presented here show that at 21°C, Vp is still viable but does not either proliferate or express the virulence factors studied. Interestingly, at 27°C and as early as 1 h of incubation, all factors are transiently expressed in free-living bacteria only and even more upregulated at 31°C. These results clearly show that increased SWT has an important impact on the adhesion properties of free-living Vp to plastic support and thus emphasize the role of climate change in the spread of this pathogenic bacteria.

Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. The dynamic extension and retraction of pili are often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To address this question, we study the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we use a combination of genetic and cell biological approaches to describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. Specifically, we show that V. cholerae cells retract MSHA pili and detach from a surface in a diffusion-limited, enclosed environment. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP to induce MSHA pilus retraction. CdpA contains a putative nitric oxide (NO)-sensing NosP domain, and we demonstrate that NO is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that the endogenous production of NO (or an NO-like molecule) in V. cholerae stimulates the retraction of MSHA pili. These results extend our understanding of how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

Quorum sensing regulates transcription of the pilin gene mshA1 of MSHA pilus in Vibrio parahaemolyticus.

Vibrio parahaemolyticus produces two types of IV pili: mannose-sensitive haemagglutinin type IV pili (MSHA) and chitin-regulated pili (ChiRP). Both of them are required for biofilm formation and the pathogen persistence in hosts. However, there are few reports on the regulation of their expression. In the present study, we showed that the master quorum sensing (QS) regulators AphA and OpaR oppositely regulated the transcription of mshA1 encoding the pilin of MSHA pilus in V. parahaemolyticus. At low cell density (LCD), AphA indirectly repressed mshA1 transcription. In contrast, at high cell density (HCD), OpaR bound to the regulatory DNA region of mshA1 to activate its transcription. Oppositely regulation of mshA1 by AphA and OpaR led to a gradual increase in the expression level of mshA1 from LCD to HCD. Thus, regulation of type IV pili production was one of the mechanisms that V. parahaemolyticus adopted to control biofilm formation.

Intravesical Pseudomonas aeruginosa mannose-sensitive Hemagglutinin vaccine triggers a tumor-preventing immune environment in an orthotopic mouse bladder cancer model.

Bacillus Calmette-Guerin (BCG) immunotherapy can prevent recurrence and progression in selected patients with non-muscle-invasive bladder cancer (NMIBC); however, significant adverse events and treatment failure suggest the need for alternative agents. A commercial anti-infection vaccine comprises a genetically engineered heat-killed Pseudomonas aeruginosa (PA) expressing many mannose-sensitive hemagglutination (MSHA) fimbriae, termed PA-MSHA, which could be a candidate for bladder cancer intravesical therapy. In an immunocompetent orthotopic MB49 bladder cancer model, we characterized the antitumor effects and mechanisms of PA-MSHA compared with those of BCG. Three weekly intravesical PA-MSHA or BCG treatments reduced tumor involvement; however, only PA-MSHA prolonged survival against MB49 implantation significantly. In non-tumor-bearing mice after treatment, flow-cytometry analysis showed PA-MSHA and BCG induced an increased CD4/CD8 ratio, the levels of effector memory T cell phenotypes (CD44, CXCR-3, and IFN-γ), and the proportion of CD11b+Ly6G-Ly6C-IA/IE+ mature macrophages, but a decrease in the proportion of CD11b+Ly6G-Ly6C+IA/IE- monocytic myeloid-derived suppressor cells (Mo-MDSCs) and the expression of suppressive molecules on immune cells (PD-L1, PD-1, TIM-3, and LAG-3). Notably, PA-MSHA, but not BCG, significantly reduced PD-1 and TIM-3 expression on CD4+ T cells, which might account for the better effects of PA-MSHA than BCG. However, in tumor-bearing mice after treatment, the increased proportion of Mo-MDSCs and high expression of PD-L1 might be involved in treatment failure. Thus, modulating the balance among adaptive and innate immune responses was identified as a key process underlying PA-MSHA-mediated treatment efficacy. The results demonstrated mechanisms underlying intravesical PA-MSHA therapy, pointing at its potential as an alternative effective treatment for NMIBC.

Two regulatory factors of Vibrio cholerae activating the mannose-sensitive haemagglutinin pilus expression is important for biofilm formation and colonization in mice.

Vibrio cholerae the causative agent of cholera, uses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host intestine, which is its preferred colonization site. Transcription of the mannose-sensitive haemagglutinin pilus (MSHA), which aids the persistence of V. cholerae in aquatic environments, but causes its clearance by host immune defenses, was found to be regulated by a yet unknown mechanism during the infection cycle of V. cholerae. In this study, genomic expression library screening revealed that two regulators, VC1371 and VcRfaH, are able to positively activate the transcription of MSHA operon. VC1371 is localized and active in the cell membrane. Deletion of vc1371 or VcrfaH genes in V. cholerae resulted in less MshA protein production and less efficiency of biofilm formation compared to that in the wild-type strain. An adult mouse model showed that the mutants with vc1371 or VcrfaH deletion colonized less efficiently than the wild-type; the VcrfaH deletion mutant showed less colonization efficiency in the infant mouse model. The findings strongly suggested that the two regulators, namely VC1371 and VcRfaH, which are involved in the regulation of MSHA expression, play an important role in V. cholerae biofilm formation and colonization in mice.

Crash landing of Vibrio cholerae by MSHA pili-assisted braking and anchoring in a viscoelastic environment.

Mannose-sensitive hemagglutinin (MSHA) pili and flagellum are critical for the surface attachment of Vibrio cholerae, the first step of V. cholerae colonization on host surfaces. However, the cell landing mechanism remains largely unknown, particularly in viscoelastic environments such as the mucus layers of intestines. Here, combining the cysteine-substitution-based labeling method with single-cell tracking techniques, we quantitatively characterized the landing of V. cholerae by directly observing both pili and flagellum of cells in a viscoelastic non-Newtonian solution consisting of 2% Luria-Bertani and 1% methylcellulose (LB+MC). The results show that MSHA pili are evenly distributed along the cell length and can stick to surfaces at any point along the filament. With such properties, MSHA pili are observed to act as a brake and anchor during cell landing which includes three phases: running, lingering, and attaching. Importantly, loss of MSHA pili results in a more dramatic increase in mean path length in LB+MC than in 2% LB only or in 20% Ficoll solutions, indicating that the role of MSHA pili during cell landing is more apparent in viscoelastic non-Newtonian fluids than viscous Newtonian ones. Our work provides a detailed picture of the landing dynamics of V. cholerae under viscoelastic conditions, which can provide insights into ways to better control V. cholerae infections in a real mucus-like environment.

Nanocochleates containing N-Octylglicoside extracted Vibrio cholerae antigens elicited high vibriocidal antibodies titers after intragastric immunization in a mice model

Biological detergents are used in research laboratories, to extract or solubilize proteins from cell membranes. In order to evaluate the ability to extract antigens from the bacterial cell surface of the wild Vibrio cholerae strain C7258 and study their immunogenic potential by forming proteoliposomes and cochleate and preserving their immunogenicity, the non-ionic detergent, n-Octylglucoside (n-OG), and the Zwitterionic detergent (3-cholamidopropyl dimethylammonio 1-propanesulfonate; CHAPS) were tested in concentrations between 5 and 15%. The anionic detergent sodium deoxycholate (DOC) was used as a reference. Electrophoretic, immunochemical and electron microscopy techniques have characterized the extracts and their chromatographic fractions. With CHAPS and n-OG detergents in concentrations between 5 and 15%, a higher yield was obtained in the extraction of proteins and lipopolysaccharides (LPS) and other components from the bacterial surface compared to 10% DOC. When using 10% DOC, 15% CHAPS and n-OG between 5 and 15%, stable proteoliposomes were formed, of average size between 82 and 93 nm in diameter, with known proportions of proteins, LPS and other components. In some of the concentrations, liposomes were formed with almost pure proteins. Some cholera outer membrane proteins like the 17 kDa protein, which corresponds to the mannose-sensitive hemagglutinin (MSHA), which mediates the adhesion to the brush border of the small intestine and the outer membrane protein U (OMPU) were identified with monoclonal antibodies (mAbs) and purified. The fundamental components of liposomes, proteins and LPS, retained their molecular weights, when compared with known standards and by processing programs of electrophoretic profiles and their antigenicity, without alterations due to the extraction procedure, as could be verified by immune identification techniques with monoclonal antibodies in the case of LPS, significant antigens in this pathogen. The main purpose of the present work was to show that a new anticholera vaccine formulation based on cochleates, containing selected protein and LPS fraction extracted by detergents, is able to elicit protective high titers of bactericidal antibodies after intragastric immunization in the mice model. The objective was achieved.