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Genetic diversity of the O antigens of Proteus species and the development of a suspension array for molecular serotyping.

ABSTRACT: Proteus species are well-known opportunistic pathogens frequently associated with skin wound and urinary tract infections in humans and animals. O antigen diversity is important for bacteria to adapt to different hosts and environments, and has been used to identify serotypes of Proteus isolates. At present, 80 Proteus O-serotypes have been reported. Although the O antigen structures of most Proteus serotypes have been identified, the genetic features of these O antigens have not been well characterized. The O antigen gene clusters of Proteus species are located between the cpxA and secB genes. In this study, we identified 55 O antigen gene clusters of different Proteus serotypes. All clusters contain both the wzx and wzy genes and exhibit a high degree of heterogeneity. Potential functions of O antigen-related genes were proposed based on their similarity to genes in available databases. The O antigen gene clusters and structures were compared, and a number of glycosyltransferases were assigned to glycosidic linkages. In addition, an O serotype-specific suspension array was developed for detecting 31 Proteus serotypes frequently isolated from clinical specimens. To our knowledge, this is the first comprehensive report to describe the genetic features of Proteus O antigens and to develop a molecular technique to identify different Proteus serotypes.

SUBMITTER: Yu X

PROVIDER: S-EPMC5560731 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Genetic diversity of the O antigens of Proteus species and the development of a suspension array for molecular serotyping.

Yu Xiang X Torzewska Agnieszka A Zhang Xinjie X Yin Zhiqiu Z Drzewiecka Dominika D Cao Hengchun H Liu Bin B Knirel Yuriy A YA Rozalski Antoni A Rozalski Antoni A Wang Lei L

PloS one 20170817 8

Proteus species are well-known opportunistic pathogens frequently associated with skin wound and urinary tract infections in humans and animals. O antigen diversity is important for bacteria to adapt to different hosts and environments, and has been used to identify serotypes of Proteus isolates. At present, 80 Proteus O-serotypes have been reported. Although the O antigen structures of most Proteus serotypes have been identified, the genetic features of these O antigens have not been well chara ...[more]

PMID: 28817637

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Project description:BACKGROUND:Quantitative suspension arrays are powerful immunoassays to measure antibodies against multiple antigens in large numbers of samples in a short time and using few microliters. To identify antigen targets of immunity for vaccine development against complex microbes like Plasmodium falciparum, such technology allows the characterization of the magnitude and antigenic specificity of Ig isotypes and subclasses that are important for functional responses. However, standardized assays are not widely available. METHODS:We developed six quantitative suspension array assays to measure IgG1, IgG2, IgG3, IgG4, IgM and IgE specific to multiple P. falciparum antigens. Secondary and tertiary antibodies, as well as human purified antibodies for standard curves, were tested among several commercially available sources. Positive and negative controls included plasmas from malaria hyper-immune African adults and from malaria-naïve European adults, respectively. Reagents were selected and optimal antibody and test sample dilutions established according to sensitivity, specificity and performance of the standard curves. The variability between replicates and plates was assessed with 30 test samples and controls. RESULTS:Assays were able to detect P. falciparum antigen-specific antibodies for all isotypes and subclasses in samples from malaria-exposed individuals, with low background signal in blank wells. Levels detected in malaria-naïve individuals were overall low except for IgM. For the IgG2 and IgE assays, a triple sandwich was required for sensitivity. Standard curves with 5-parameter logistic fit were successfully obtained in all assays. The coefficients of variation for measurements performed in different days were all <30%, and <5% when comparing duplicates from the same plate. CONCLUSION:The isotype/subclass assays developed here were sensitive, specific, reproducible and of adequate quantification dynamic range. They allow performing detailed immuno-profiling to large panels of P. falciparum antigens to address naturally- and vaccine-induced Ig responses and elucidate correlates of malaria protection, and could also be applied to other antigenic panels.

Project description:BackgroundAntibody responses to Plasmodium falciparum play a critical role in disease control. Finding reliable IgG biomarkers of protection is complicated by a parasite proteome of over 5000 proteins, some with polymorphisms. Studies of anti-malarial naturally acquired and vaccine immunity would benefit from a standard high-throughput immunoassay to measure multiple antibodies. A multiplex quantitative suspension assay to measure antigen-specific IgGs was developed and its precision (reproducibility and repeatability), dynamic range, limits of detection and quantification, and non-specific binding to different P. falciparum proteins tested. A set of 288 human plasma samples from a malaria-endemic region were analysed twice by two different operators. Another set of samples from 9 malaria-naïve and 10 malaria-exposed individuals were repetitively assayed during 22 consecutive days. Positive controls, negative controls, blanks and microspheres coated with bovine serum albumin were included in all assays.ResultsThe multiplex quantitative suspension assay demonstrated low non-specific signal and good estimates of precision and reproducibility between operators. The overall mean of non-specific binding measured in 288 plasma samples was 32.83 to ± 44.81 median fluorescence intensity (MFI). Repeatability was 7.66% ± 15.89 between triplicates for all antigens and samples, being lower in samples from malaria-exposed than malaria-naïve individuals. No evidence of significantly different variance across days in MFI or arbitrary units (AU)/mL was found, assuming homogeneity of variance between days of analysis. Intra-class correlation coefficient between 22 days of analysis was 0.98 (0.97-0.98) for MFI units and 0.9 (0.87-0.93) for AU/mL. Reproducibility between operators for all samples and antigens had an overall adjusted correlation of 0.929 for MFI and 0.836 for AU/mL.ConclusionsThis high-throughput multiplex immunoassay is simple and highly reproducible. This represents an asset for malaria vaccine studies involving CSP-specific antibodies and selected antigens for sero-epidemiological purposes. Measuring a multiplex antigen panel in a single reaction will help to assess not only vaccine immunogenicity but also potential malaria vaccine effects on naturally acquired immune responses. This will accelerate the identification of immune correlates of protection, down-selection of vaccine formulations, antigen discovery and guide second-generation vaccine design.

Dataset Information

Genetic diversity of the O antigens of Proteus species and the development of a suspension array for molecular serotyping.

Publications

Genetic diversity of the O antigens of Proteus species and the development of a suspension array for molecular serotyping.

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