Magnetic chitin beads (MCB) coated with Vibrio cholerae reveals transcriptome dynamics in adult mice with a complex gut microbiota.

Vibrio cholerae adapts to the host environment by altering gene expression. Because of the complexity of the gut microbiome, current in vivo V. cholerae transcriptome studies have focused on microbiota-undeveloped conditions, neglecting the interaction between the host's commensal gut microbiota and V. cholerae. In this study, we analyzed the transcriptome of fully colonized adult mice in vivo using V. cholerae coated-magnetic chitin beads (vcMCB). This provides a simple yet powerful method for obtaining high-quality RNA from V. cholerae during colonization in mice. The transcriptome of V. cholerae recovered from adult mice infected with vcMCB shows differential expression of several genes when compared to V. cholerae recovered from the infant mouse and infant rabbit model. Some of these genes were also observed to be differentially expressed in previous studies of V. cholera recovered from human infection when compared to V. cholerae grown in vitro. In particular, we confirmed that V. cholerae resists the inhibitory effects of low pH and formic acid from gut microbiota, such as Anaerostipes caccae and Dorea formicigenerans, by downregulating vc1080. We propose that the vc1080 product may protect V. cholerae from formic acid stress through a novel acid tolerance response mechanism. Transcriptomic data obtained using the vcMCB system provide new perspectives on the interaction between V. cholerae and the gut microbiota, and this approach can also be applied to studies of other pathogenic bacteria.

Thiol-Based Modification of MarR Protein VnrR Regulates Resistance Toward Nitrofuran in Vibrio cholerae By Promoting the Expression of a Novel Nitroreductase VnrA and of NO-Detoxifying Enzyme HmpA.

Aims: Epidemiological investigations have indicated low resistance toward nitrofuran in clinical isolates, suggesting its potential application in the treatment of multidrug-resistant bacteria. Therefore, it is valuable to explore the mechanism of bacterial resistance to nitrofuran. Results: Through phenotypic screening of ten multiple antibiotic resistance regulator (MarR) proteins in Vibrio cholerae, we discovered that the regulator VnrR (VCA1058) plays a crucial role in defending against nitrofuran, specifically furazolidone (FZ). Our findings demonstrate that VnrR responds to FZ metabolites, such as hydroxylamine, methylglyoxal, hydrogen peroxide (H2O2), ß-hydroxyethylhydrazine. Notably, VnrR exhibits reversible responses to the addition of H2O2 through three cysteine residues (Cys180, Cys223, Cys247), leading to the derepression of its upstream gene, vnrA (vca1057). Gene vnrA encodes a novel nitroreductase, which directly contributes to the degradation of FZ. Our study reveals that V. cholerae metabolizes FZ via the vnrR-vnrA system and achieves resistance to FZ with the assistance of the classical reactive oxygen/nitrogen species scavenging pathway. Innovation and Conclusion: This study represents a significant advancement in understanding the antibiotic resistance mechanisms of V. cholerae and other pathogens. Our findings demonstrate that the MarR family regulator, VnrR, responds to the FZ metabolite H2O2, facilitating the degradation and detoxification of this antibiotic in a thiol-dependent manner. These insights not only enrich our knowledge of antibiotic resistance but also provide new perspectives for the control and prevention of multidrug-resistant bacteria.

Cross-Platform Transcriptomic Data Integration, Profiling, and Mining in Vibrio cholerae.

A large number of transcriptome studies generate important data and information for the study of pathogenic mechanisms of pathogens, including Vibrio cholerae. V. cholerae transcriptome data include RNA-seq and microarray: microarray data mainly include clinical human and environmental samples, and RNA-seq data mainly focus on laboratory processing conditions, including different stresses and experimental animals in vivo. In this study, we integrated the data sets of both platforms using Rank-in and the Limma R package normalized Between Arrays function, achieving the first cross-platform transcriptome data integration of V. cholerae. By integrating the entire transcriptome data, we obtained the profiles of the most active or silent genes. By transferring the integrated expression profiles into the weighted correlation network analysis (WGCNA) pipeline, we identified the important functional modules of V. cholerae in vitro stress treatment, gene manipulation, and in vitro culture as DNA transposon, chemotaxis and signaling, signal transduction, and secondary metabolic pathways, respectively. The analysis of functional module hub genes revealed the uniqueness of clinical human samples; however, under specific expression patterning, the Δhns, ΔoxyR1 strains, and tobramycin treatment group showed high expression profile similarity with human samples. By constructing a protein-protein interaction (PPI) interaction network, we discovered several unreported novel protein interactions within transposon functional modules. IMPORTANCE We used two techniques to integrate RNA-seq data for laboratory studies with clinical microarray data for the first time. The interactions between V. cholerae genes were obtained from a global perspective, as well as comparing the similarity between clinical human samples and the current experimental conditions, and uncovering the functional modules that play a major role under different conditions. We believe that this data integration can provide us with some insight and basis for elucidating the pathogenesis and clinical control of V. cholerae.

Dps-dependent in vivo mutation enhances long-term host adaptation in Vibrio cholerae.

As one of the most successful pathogenic organisms, Vibrio cholerae (V. cholerae) has evolved sophisticated regulatory mechanisms to overcome host stress. During long-term colonization by V. cholerae in adult mice, many spontaneous nonmotile mutants (approximately 10% at the fifth day post-infection) were identified. These mutations occurred primarily in conserved regions of the flagellar regulator genes flrA, flrC, and rpoN, as shown by Sanger and next-generation sequencing, and significantly increased fitness during colonization in adult mice. Intriguingly, instead of key genes in DNA repair systems (mutS, nfo, xthA, uvrA) or ROS and RNS scavenging systems (katG, prxA, hmpA), which were generally thought to be associated with bacterial mutagenesis, we found that deletion of the cyclin gene dps significantly increased the mutation rate (up to 53% at the fifth day post-infection) in V. cholerae. We further determined that the dpsD65A and dpsF46E point mutants showed a similar mutagenesis profile as the Δdps mutant during long-term colonization in mice, which strongly indicated that the antioxidative function of Dps directly contributes to the development of V. cholerae nonmotile mutants. Methionine metabolism pathway may be one of the mechanism for ΔflrA, ΔflrC and ΔrpoN mutant increased colonization in adult mice. Our results revealed a new phenotype in which increased fitness of V. cholerae in the host gut via spontaneous production nonmotile mutants regulated by cyclin Dps, which may represent a novel adaptation strategy for directed evolution of pathogens in the host.

Assembly encapsulation of BSA and CCCH-ZAP in the sodium alginate/atractylodis macrocephalae system.

Zinc finger antiviral proteins (ZAP) can significantly inhibit the replication of avian leukosis virus subgroup J (ALV-J), but the traditional method of ZAP administration is by injection, which can easily cause stress effects in chickens. In this work, we established a sodium alginate/atractylodis macrocephalae system for the encapsulation of CCCH-type zinc finger antiviral protein (CCCH-ZAP). Because of the high cost of ZAP, we first chose bovine serum albumin (BSA) as a model protein to investigate the encapsulation performance. The SEM images clearly confirmed that BSA and the sodium alginate/atractylodis macrocephalae system can assemble easily to form relatively stable nanostructures, and the encapsulation amount of BSA can reach 68%. Subsequently, the encapsulation of ZAP was studied. The SEM and the encapsulation experiments confirmed that ZAP can also be assembly encapsulated in the sodium alginate/atractylodis macrocephalae system with the encapsulation amount of 80%. Release studies showed that the SA/AM-ZAP nanocomposite was able to achieve a release rate of 32% of ZAP. This work successfully confirms the assembly encapsulation of ZAP, which will be beneficial for the usage of ZAP-based animal drugs.

CBS-derived H2S facilitates host colonization of Vibrio cholerae by promoting the iron-dependent catalase activity of KatB.

Sensing and resisting oxidative stress is critical for Vibrio cholerae to survive in either the aquatic environment or the gastrointestinal tract. Previous studies mainly focused on the mechanisms of oxidative stress response regulation that rely on enzymatic antioxidant systems, while functions of non-enzymatic antioxidants are rarely discussed in V. cholerae. For the first time, we investigated the role of hydrogen sulfide (H2S), the simplest thiol compound, in protecting V. cholerae against oxidative stress. We found that degradation of L-cysteine by putative cystathionine ß-synthase (CBS) is the major source of endogenous H2S in V. cholerae. Our results indicate that intracellular H2S level has a positive correlation with cbs expression, while the enhanced H2S production can render V. cholerae cells less susceptible to H2O2 in vitro. Using proteome analysis and real-time qPCR assay, we found that cbs expression could stimulate the expression of several enzymatic antioxidants, including reactive oxygen species (ROS) detoxifying enzymes SodB, KatG and AhpC, the DNA protective protein DPS and the protein redox regulator Trx1. Assays of ROS detoxification capacities revealed that CBS-derived H2S could promote catalase activity at the post-translational level, especially for KatB, which serves as an important way that endogenous H2S participates in H2O2 detoxification. The enhancement of catalase activity by H2S is achieved through facilitating the uptake of iron. Adult mice experiments showed that cbs mutant has colonization defect, while either complementation of cbs or exogenous supplement of N-Acetyl-L-Cysteine restores its fitness in the host environment. Herein, we proposed that V. cholerae regulates CBS-dependent H2S production for better survival and proliferation under ROS stress.