Dataset Information

A Novel Luminescence-Based Serum Bactericidal Assay for Vibrio cholerae Reduces Assay Variation, Is Time- and Cost-Effective, and Directly Measures Continuous Titer Values.

ABSTRACT: Cholera remains a significant public health burden worldwide, and better methods for monitoring cholera incidence would enhance the effectiveness of public health interventions. The serum bactericidal assay (SBA) has been used extensively for Vibrio cholerae vaccine assessments and serosurveillance. Current SBA approaches for V. cholerae rely on colony enumeration or optical density (OD600nm) readings to measure viable bacteria following complement-mediated lysis. These methods provide titer values that are constrained to discrete dilution values and rely on bacterial outgrowth, which is time consuming and prone to variation. Detection of bacterial proteins following complement-mediated lysis presents a faster and potentially less variable alternative approach independent of bacterial outgrowth. Here, we present an SBA that measures luciferase luminescence driven by lysis-released adenylate kinase. This approach is faster and less variable than growth-dependent SBAs and directly measures continuous titer values. This novel SBA method can potentially be applied to other bacteria of interest.

SUBMITTER: Wahlig TA

PROVIDER: S-EPMC8592349 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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A Novel Luminescence-Based Serum Bactericidal Assay for Vibrio cholerae Reduces Assay Variation, Is Time- and Cost-Effective, and Directly Measures Continuous Titer Values.

Wahlig Taylor A TA Brintz Ben J BJ Prettyman Melanie M Azman Andrew S AS Leung Daniel T DT

The American journal of tropical medicine and hygiene 20210708 3

Cholera remains a significant public health burden worldwide, and better methods for monitoring cholera incidence would enhance the effectiveness of public health interventions. The serum bactericidal assay (SBA) has been used extensively for Vibrio cholerae vaccine assessments and serosurveillance. Current SBA approaches for V. cholerae rely on colony enumeration or optical density (OD600nm) readings to measure viable bacteria following complement-mediated lysis. These methods provide titer val ...[more]

PMID: 34237020

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Project description:Vibrio cholerae biofilm biogenesis, which is important for survival, dissemination, and persistence, requires multiple genes in the Vibrio polysaccharides (vps) operons I and II as well as the cluster of ribomatrix (rbm) genes. Transcriptional control of these genes is a complex process that requires several activators/repressors and the ubiquitous signaling molecule, cyclic di-GMP (c-di-GMP). Previously, we demonstrated that VpsR directly activates RNA polymerase containing σ70 (σ70-RNAP) at the vpsL promoter (P vpsL ), which precedes the vps-II operon, in a c-di-GMP-dependent manner by stimulating formation of the transcriptionally active, open complex. Using in vitro transcription, electrophoretic mobility shift assays, and DNase I footprinting, we show here that VpsR also directly activates σ70-RNAP transcription from other promoters within the biofilm formation cluster, including P vpsU , at the beginning of the vps-I operon, P rbmA , at the start of the rbm cluster, and P rbmF , which lies upstream of the divergent rbmF and rbmE genes. In this capacity, we find that VpsR is able to behave both as a class II activator, which functions immediately adjacent/overlapping the core promoter sequence (P vpsL and P vpsU ), and as a class I activator, which functions farther upstream (P rbmA and P rbmF ). Because these promoters vary in VpsR-DNA binding affinity in the absence and presence of c-di-GMP, we speculate that VpsR's mechanism of activation is dependent on both the concentration of VpsR and the level of c-di-GMP to increase transcription, resulting in finely tuned regulation.IMPORTANCEVibrio cholerae, the bacterial pathogen that is responsible for the disease cholera, uses biofilms to aid in survival, dissemination, and persistence. VpsR, which directly senses the second messenger c-di-GMP, is a major regulator of this process. Together with c-di-GMP, VpsR directly activates transcription by RNA polymerase containing σ70 from the vpsL biofilm biogenesis promoter. Using biochemical methods, we demonstrate for the first time that VpsR/c-di-GMP directly activates σ70-RNA polymerase at the first genes of the vps and ribomatrix operons. In this regard, it functions as either a class I or class II activator. Our results broaden the mechanism of c-di-GMP-dependent transcription activation and the specific role of VpsR in biofilm formation.

Project description:BackgroundGlobal estimates for cholera annually approximate 4 million cases worldwide with 95,000 deaths. Recent outbreaks, including Haiti and Yemen, are reminders that cholera is still a global health concern. Cholera outbreaks can rapidly induce high death tolls by overwhelming the capacity of health facilities, especially in remote areas or areas of civil unrest. Recent studies demonstrated that stool specimens preserved on filter paper facilitate molecular analysis of Vibrio cholerae in resource limited settings. Specimens preserved in a rapid, low-cost, safe and sustainable manner for sequencing provides previously unavailable data about circulating cholera strains. This may ultimately contribute new information to shape public policy response on cholera control and elimination.Methodology/principal findingsWhole genome sequencing (WGS) recovered close to a complete sequence of the V. cholerae O1 genome with satisfactory genome coverage from stool specimens enriched in alkaline peptone water (APW) and V. cholerae culture isolates, both spotted on filter paper. The minimum concentration of V. cholerae DNA sufficient to produce quality genomic information was 0.02 ng/μL. The genomic data confirmed the presence or absence of genes of epidemiological interest, including cholera toxin and pilus loci. WGS identified a variety of diarrheal pathogens from APW-enriched specimen spotted filter paper, highlighting the potential for this technique to explore the gut microbiome, potentially identifying co-infections, which may impact the severity of disease. WGS demonstrated that these specimens fit within the current global cholera phylogenetic tree, identifying the strains as the 7th pandemic El Tor.ConclusionsWGS results allowed for mapping of short reads from APW-enriched specimen and culture isolate spotted filter papers. This provided valuable molecular epidemiological sequence information on V. cholerae strains from remote, low-resource settings. These results identified the presence of co-infecting pathogens while providing rare insight into the specific V. cholerae strains causing outbreaks in cholera-endemic areas.

Dataset Information

A Novel Luminescence-Based Serum Bactericidal Assay for Vibrio cholerae Reduces Assay Variation, Is Time- and Cost-Effective, and Directly Measures Continuous Titer Values.

Publications

A Novel Luminescence-Based Serum Bactericidal Assay for Vibrio cholerae Reduces Assay Variation, Is Time- and Cost-Effective, and Directly Measures Continuous Titer Values.

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