Identification of potential pathogenic targets and survival strategies of Vibrio vulnificus through population genomics.

Vibrio vulnificus, a foodborne pathogen, has a high mortality rate. Despite its relevance to public health, the identification of virulence genes associated with the pathogenicity of currently known clinical isolates of V. vulnificus is incomplete and its synergistic pathogenesis remains unclear. Here, we integrate whole genome sequencing (WGS), genome-wide association studies (GWAS), and genome-wide epistasis studies (GWES), along with phenotype characterization to investigate the pathogenesis and survival strategies of V. vulnificus. GWAS and GWES identified a total of six genes (purH, gmr, yiaV, dsbD, ramA, and wbpA) associated with the pathogenicity of clinical isolates related to nucleotide/amino acid transport and metabolism, cell membrane biogenesis, signal transduction mechanisms, and protein turnover. Of these, five were newly discovered potential specific virulence genes of V. vulnificus in this study. Furthermore, GWES combined with phenotype experiments indicated that V. vulnificus isolates were clustered into two ecological groups (EGs) that shared distinct biotic and abiotic factors, and ecological strategies. Our study reveals pathogenic mechanisms and their evolution in V. vulnificus to provide a solid foundation for designing new vaccines and therapeutic targets.

Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis.

Francisella tularensis is a dangerous pathogen that causes an extremely contagious zoonosis in humans named tularemia. Given its low-dose morbidity, the potential to be fatal, and aerosol spread, it is regarded as a severe threat to public health. The US Centers for Disease Control and Prevention (CDC) has classified it as a category A potential agent for bioterrorism and a Tier 1 Select Agent. Herein, we combined recombinase polymerase amplification (RPA) with CRISPR/Cas12a system to select the F. tularensis target gene (TUL4), creating a two-pronged rapid and ultrasensitive diagnostic method for detecting F. tularensis. The real-time RPA (RT-RPA) assay detected F. tularensis within 10 min at a sensitivity of 5 copies/reaction, F. tularensis genomic DNA of 5 fg, and F. tularensis of 2 × 102 CFU/ml; the RPA-CRISPR/Cas12a assay detects F. tularensis within 40 min at a sensitivity of 0.5 copies/reaction, F. tularensis genomic DNA of 1 fg, and F. tularensis of 2 CFU/ml. Furthermore, the evaluation of specificity showed that both assays were highly specific to F. tularensis. More importantly, in a test of prepared simulated blood and sewage samples, the RT-RPA assay results were consistent with RT-PCR assay results, and the RPA-CRISPR/Cas12a assay could detect a minute amount of F. tularensis genomic DNA (2.5 fg). There was no nonspecific detection with blood samples and sewage samples, giving the tests a high practical application value. For example, in on-site and epidemic areas, the RT-RPA was used for rapid screening and the RPA-CRISPR/Cas12a assay was used for more accurate diagnosis.

Analysis of Gene Expression Profiles, Cytokines, and Bacterial Loads Relevant to Alcoholic Liver Disease Mice Infected With V. vulnificus.

Patients with liver disease are susceptible to infection with Vibrio vulnificus (V. vulnificus), but the specific reasons remain elusive. Through RNA-seq, we found that when mice with alcoholic liver disease (ALD) were infected with V. vulnificus by gavage, compared with the Pair group, the small intestinal genes affecting intestinal permeability were upregulated; and the number of differentially expressed genes related to immune functions (e.g., such as cell chemotaxis, leukocyte differentiation, and neutrophil degranulation) decreased in the liver, spleen, and blood. Further analysis showed that the number of white blood cells decreased in the Pair group, whereas those in the ALD mice did not change significantly. Interestingly, the blood bacterial load in the ALD mice was about 100 times higher than that of the Pair group. After the ALD mice were infected with V. vulnificus, the concentrations of T cell proliferation-promoting cytokines (IL-2, IL-23) decreased. Therefore, unlike the Pair group, ALD mice had weaker immune responses, lower T cell proliferation-promoting cytokines, and higher bacterial loads post-infection, possibly increasing their susceptibility to V. vulnificus infection. These new findings we presented here may help to advance the current understanding of the reasons why patients with liver disease are susceptible to V. vulnificus infection and provides potential targets for further investigation in the context of treatment options for V. vulnificus sepsis in liver disease patient.

Features of Ebola Virus Disease at the Late Outbreak Stage in Sierra Leone: Clinical, Virological, Immunological, and Evolutionary Analyses.

We performed Ebola virus disease diagnosis and viral load estimation for Ebola cases in Sierra Leone during the late stage of the 2014-2015 outbreak (January-March 2015) and analyzed antibody and cytokine levels and the viral genome sequences. Ebola virus disease was confirmed in 86 of 1001 (9.7%) patients, with an overall case fatality rate of 46.8%. Fatal cases exhibited significantly higher levels of viral loads, cytokines, and chemokines at late stages of infection versus early stage compared with survivors. The viruses converged in a new clade within sublineage 3.2.4, which had a significantly lower case fatality rate.

A recombinant mutant abrin A chain expressed in Escherichia coli can be used as an effective vaccine candidate.

We used site-directed mutagenesis to mutate two key amino acid residues, Glu164 and Arg167, of abrin A chain (ABRA), creating a mutant ABRA(E164AR167L). The mutant ABRA(mABRA) encoded by mABRA(E164AR167L) was expressed in the cytoplasm of Escherichia coli, and used to develop an effective vaccine to protect mice against native abrin intoxication. The cytotoxicity of mABRA was dramatically reduced as compared to that of recombinant ABRA(rABRA) and native abrin, but the antigenicity and immunogenicity remained the same. Balb/c mice were vaccinated with purified mABRA, and survival was evaluated after challenge with native abrin. Mice that were given three vaccinations developed a protective immune response that was 100% protective against an intraperitoneal (i.p.) administration of 10×LD50 of native abrin. Furthermore, the sera from immunized mice provided complete passive protection for naive mice. This study describes the generation of a substantial amount of mABRA from E. coli and the potential application of mABRA as an effective vaccine candidate for humans, to protect against a high-dose of native abrin.