Regulatory Characteristics of Vibrio vulnificus gbpA Gene Encoding a Mucin-binding Protein Essential for Pathogenesis.

Binding to mucin is the initial step for enteropathogens to establish pathogenesis. An open reading frame, gbpA, of Vibrio vulnificus was identified and characterized in this study. Compared with wild type, the gbpA mutant was impaired in binding to mucin-agar and the mucin-secreting HT29-methotrexate cells, and the impaired mucin binding was restored by the purified GbpA provided exogenously. The gbpA mutant had attenuated virulence and ability of intestinal colonization in a mouse model, indicating that GbpA is a mucin-binding protein and essential for pathogenesis of V. vulnificus. The gbpA transcription was growth phase-dependent, reaching a maximum during the exponential phase. The Fe-S cluster regulator (IscR) and the cyclic AMP receptor protein (CRP) coactivated, whereas SmcR, a LuxR homologue, repressed gbpA. The cellular levels of IscR, CRP, and SmcR were not significantly affected by one another, indicating that the regulator proteins function cooperatively to regulate gbpA rather than sequentially in a regulatory cascade. The regulatory proteins directly bind upstream of the gbpA promoter PgbpA. DNase I protection assays, together with the deletion analyses of PgbpA, demonstrated that IscR binds to two specific sequences centered at -164.5 and -106, and CRP and SmcR bind specifically to the sequences centered at -68 and -45, respectively. Furthermore, gbpA was induced by exposure to H2O2, and the induction appeared to be mediated by elevated intracellular levels of IscR. Consequently, the combined results indicated that IscR, CRP, and SmcR cooperate for precise regulation of gbpA during the V. vulnificus pathogenesis.

Safety Evaluation and Population Pharmacokinetics of Camostat Mesylate and Its Major Metabolites Using a Phase I Study.

Camostat mesylate is expected to be promising as a treatment option for COVID-19, in addition to other indications for which it is currently used. Furthermore, in vitro experiments have confirmed the potential of camostat and its metabolites to be effective against COVID-19. Therefore, clinical trials were conducted to evaluate the safety and pharmacokinetic characteristics of camostat after single-dose administration. Additionally, we aim to predict the pharmacokinetics of repeated dosing through modeling and simulation based on clinical trials. Clinical trials were conducted on healthy Korean adults, and an analysis was carried out of the metabolites of camostat, GBPA, and GBA. Pharmacokinetic modeling and simulation were performed using Monolix. There were no safety issues (AEs, physical examinations, clinical laboratory tests, vital sign measurements, and ECG) during the clinical trial. The pharmacokinetic characteristics at various doses were identified. It was confirmed that AUC last and Cmax increased in proportion to dose in both GBPA and GBA, and linearity was also confirmed in log-transformed power model regression. Additionally, the accumulation index was predicted (1.12 and 1.08 for GBPA and GBA). The pharmacokinetics of camostat for various dose administrations and indications can be predicted prior to clinical trials using the developed camostat model. Furthermore, it can be used for various indications by connecting it with pharmacodynamic information.

Secreted Aeromonas GlcNAc binding protein GbpA stimulates epithelial cell proliferation in the zebrafish intestine.

In response to microbiota colonization, the intestinal epithelia of many animals exhibit increased rates of cell proliferation. We used gnotobiotic larval zebrafish to identify a secreted factor from the mutualist Aeromonas veronii that is sufficient to promote intestinal epithelial cell proliferation. This secreted A. veronii protein is a homologue of the Vibrio cholerae GlcNAc binding protein GbpA, which was identified as a chitin-binding colonization factor in mice. GbpA was subsequently shown to be a lytic polysaccharide monooxygenase (LPMO) that can degrade recalcitrant chitin. Our phenotypic characterization of gbpA deficient A. veronii found no alterations in these cells' biogeography in the zebrafish intestine and only a modest competitive disadvantage in chitin-binding and colonization fitness when competed against the wild-type strain. These results argue against the model of GbpA being a secreted adhesin that binds simultaneously to bacterial cells and GlcNAc, and instead suggests that GbpA is part of a bacterial GlcNAc utilization program. We show that the host proliferative response to GbpA occurs in the absence of bacteria upon exposure of germ-free zebrafish to preparations of native GbpA secreted from either A. veronii or V. cholerae or recombinant A. veronii GbpA. Furthermore, domain 1 of A. veronii GbpA, containing the predicted LPMO activity, is sufficient to stimulate intestinal epithelial proliferation. We propose that intestinal epithelial tissues upregulate their rates of renewal in response to secreted bacterial GbpA proteins as an adaptive strategy for coexisting with bacteria that can degrade glycan constituents of the protective intestinal lining.

gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae.

Members of the bacterial genus Vibrio utilize chitin both as a metabolic substrate and a signal to activate natural competence. Vibrio cholerae is a bacterial enteric pathogen, sub-lineages of which can cause pandemic cholera. However, the chitin metabolic pathway in V. cholerae has been dissected using only a limited number of laboratory strains of this species. Here, we survey the complement of key chitin metabolism genes amongst 195 diverse V. cholerae. We show that the gene encoding GbpA, known to be an important colonization and virulence factor in pandemic isolates, is not ubiquitous amongst V. cholerae. We also identify a putatively novel chitinase, and present experimental evidence in support of its functionality. Our data indicate that the chitin metabolic pathway within V. cholerae is more complex than previously thought, and emphasize the importance of considering genes and functions in the context of a species in its entirety, rather than simply relying on traditional reference strains.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

BACKGROUND: Antivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed. Moreover, antiviral activity of Camostat mesylate in human lung tissue remains to be demonstrated. METHODS: We used recombinant TMPRSS2, reporter particles bearing the spike protein of SARS-CoV-2 or authentic SARS-CoV-2 to assess inhibition of TMPRSS2 and viral entry, respectively, by Camostat mesylate and its metabolite GBPA. FINDINGS: We show that several TMPRSS2-related proteases activate SARS-CoV-2 and that two, TMPRSS11D and TMPRSS13, are robustly expressed in the upper respiratory tract. However, entry mediated by these proteases was blocked by Camostat mesylate. The Camostat metabolite GBPA inhibited recombinant TMPRSS2 with reduced efficiency as compared to Camostat mesylate. In contrast, both inhibitors exhibited similar antiviral activity and this correlated with the rapid conversion of Camostat mesylate into GBPA in the presence of serum. Finally, Camostat mesylate and GBPA blocked SARS-CoV-2 spread in human lung tissue ex vivo and the related protease inhibitor Nafamostat mesylate exerted augmented antiviral activity. INTERPRETATION: Our results suggest that SARS-CoV-2 can use TMPRSS2 and closely related proteases for spread in the upper respiratory tract and that spread in the human lung can be blocked by Camostat mesylate and its metabolite GBPA. FUNDING: NIH, Damon Runyon Foundation, ACS, NYCT, DFG, EU, Berlin Mathematics center MATH+, BMBF, Lower Saxony, Lundbeck Foundation, Novo Nordisk Foundation.

Determination of camostat and its metabolites in human plasma - Preservation of samples and quantification by a validated UHPLC-MS/MS method.

OBJECTIVES: Camostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected. DESIGN AND METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds. RESULTS: Both diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 h of storage at room temperature or 4 months of storage at -20 °C and -80 °C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1-800 ng/mL. The trueness expressed as the relative bias of the test results was within ±3% at concentrations of 1-800 ng/mL. CONCLUSIONS: A methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.

Low risk of the TMPRSS2 inhibitor camostat mesylate and its metabolite GBPA to act as perpetrators of drug-drug interactions.

Camostat mesylate, a potent inhibitor of the human transmembrane protease, serine 2 (TMPRSS2), is currently under investigation for its effectiveness in COVID-19 patients. For its safe application, the risks of camostat mesylate to induce pharmacokinetic drug-drug interactions with co-administered drugs should be known. We therefore tested in vitro the potential inhibition of important efflux (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP, ABCG2)), and uptake transporters (organic anion transporting polypeptides OATP1B1, OATP1B3, OATP2B1) by camostat mesylate and its active metabolite 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA). Transporter inhibition was evaluated using fluorescent probe substrates in transporter over-expressing cell lines and compared to the respective parental cell lines. Moreover, possible mRNA induction of pharmacokinetically relevant genes regulated by the nuclear pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) was analysed in LS180 cells by quantitative real-time PCR. The results of our study for the first time demonstrated that camostat mesylate and GBPA do not relevantly inhibit P-gp, BCRP, OATP1B1 or OATP1B3. Only OATP2B1 was profoundly inhibited by GBPA with an IC50 of 11 µM. Induction experiments in LS180 cells excluded induction of PXR-regulated genes such as cytochrome P450 3A4 (CYP3A4) and ABCB1 and AhR-regulated genes such as CYP1A1 and CYP1A2 by camostat mesylate and GBPA. Together with the summary of product characteristics of camostat mesylate indicating no inhibition of CYP1A2, 2C9, 2C19, 2D6, and 3A4 in vitro, our data suggest a low potential of camostat mesylate to act as a perpetrator in pharmacokinetic drug-drug interactions. Only inhibition of OATP2B1 by GBPA warrants further investigation.

Host, pathogen and the environment: the case of Macrobrachium rosenbergii, Vibrio parahaemolyticus and magnesium.

Macrobrachium rosenbergii is well-known as the giant freshwater prawn, and is a commercially significant source of seafood. Its production can be affected by various bacterial contaminations. Among which, the genus Vibrio shows a higher prevalence in aquatic organisms, especially M. rosenbergii, causing food-borne illnesses. Vibrio parahaemolyticus, a species of Vibrio is reported as the main causative of the early mortality syndrome. Vibrio parahaemolyticus infection in M. rosenbergii was studied previously in relation to the prawn's differentially expressed immune genes. In the current review, we will discuss the growth conditions for both V. parahaemolyticus and M. rosenbergii and highlight the role of magnesium in common, which need to be fully understood. Till date, there has not been much research on this aspect of magnesium. We postulate a model that screens a magnesium-dependent pathway which probably might take effect in connection with N-acetylglucosamine binding protein and chitin from V. parahaemolyticus and M. rosenbergii, respectively. Further studies on magnesium as an environment for V. parahaemolyticus and M. rosenbergii interaction studies will provide seafood industry with completely new strategies to employ and to avoid seafood related contaminations.

A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase.

The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass polysaccharides. At the same time basic biochemical methods for characterizing LPMOs, such as activity assays are not well developed. Here we describe a method for quantification of C1-oxidized chitooligosaccharides (aldonic acids), and hence LPMO activity. The method was used to quantify the activity of a four-domain LPMO from Vibriocholerae, GbpA, which is a virulence factor with no obvious role in biomass processing.