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Development of a multiplexed opsonophagocytic killing assay (MOPA) for group B Streptococcus.

ABSTRACT: Group B Streptococcus (GBS) is a leading cause of sepsis in infants as well as chorioamnionitis in pregnant women. Opsonophagocytic killing assays (OPAs) are an essential technique in vaccine studies of encapsulated bacteria for estimating serotype-specific functional antibody levels in vitro. Here, we developed a three-fold multiplexed OPA (MOPA) to enable practical, large-scale assessment of GBS vaccine immunogenicity, including against serotypes Ia, III, and V. First, three target bacteria strains resistant to streptomycin, spectinomycin, or kanamycin were generated by natural selection through exposure to increasing antibiotic concentrations. Since a high level of nonspecific killing (NSK) of serotype V was observed in a 12.5% baby rabbit complement (BRC) solution, the BRC concentration was optimized. The final GBS-MOPA BRC concentration was 9%, which resulted in less than 20% NSK. The specificity was measured by preabsorbing serum with inactivated GBS. The opsonic index (OI) of preabsorbed serum with the homologous serotype GBS was significantly reduced in all three serotypes tested. The accuracy of the MOPA was compared with that of a single OPA (SOPA) with 35 serum samples. The OIs of the MOPA correlated well with those of the SOPA, and the r2 values were higher than 0.950 for all three serotypes. The precision of the MOPA assay was assessed in five independent experiments with five serum samples. The inter-assay precision of the GBS-MOPA was 12.5% of the average coefficient of variation. This is the first report to develop and standardize a GBS-MOPA, which will be useful for GBS vaccine development and evaluation.

SUBMITTER: Choi MJ

PROVIDER: S-EPMC5791567 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Development of a multiplexed opsonophagocytic killing assay (MOPA) for group B Streptococcus.

Choi Min Joo MJ Noh Ji Yun JY Cheong Hee Jin HJ Kim Woo Joo WJ Lin Shun-Mei SM Zhi Yong Y Lim Jae Hyang JH Lim Sangyong S Seo Ho Seong HS Song Joon Young JY

Human vaccines & immunotherapeutics 20171030 1

Group B Streptococcus (GBS) is a leading cause of sepsis in infants as well as chorioamnionitis in pregnant women. Opsonophagocytic killing assays (OPAs) are an essential technique in vaccine studies of encapsulated bacteria for estimating serotype-specific functional antibody levels in vitro. Here, we developed a three-fold multiplexed OPA (MOPA) to enable practical, large-scale assessment of GBS vaccine immunogenicity, including against serotypes Ia, III, and V. First, three target bacteria st ...[more]

PMID: 28933634

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Project description:Group A Streptococcus (Streptococcus pyogenes or GAS) causes pharyngitis, severe invasive infections, and the post-infectious syndromes of glomerulonephritis and rheumatic fever. GAS can be internalized and killed by epithelial cells in vitro, a process that may contribute to local innate defense against pharyngeal infection. Secretion of the pore-forming toxin streptolysin O (SLO) by GAS has been reported to stimulate targeted autophagy (xenophagy) upon internalization of the bacteria by epithelial cells. Whereas this process was associated with killing of GAS in HeLa cells, studies in human keratinocytes found SLO production enhanced intracellular survival. To reconcile these conflicting observations, we now report in-depth investigation of xenophagy in response to GAS infection of human oropharyngeal keratinocytes, the predominant cell type of the pharyngeal epithelium. We found that SLO expression was associated with prolonged intracellular survival; unexpectedly, expression of the co-toxin NADase was required for this effect. Enhanced intracellular survival was lost upon deletion of NADase or inactivation of its enzymatic activity. Shortly after internalization of GAS by keratinocytes, SLO-mediated damage to the bacteria-containing vacuole resulted in exposure to the cytosol, ubiquitination of GAS and/or associated vacuolar membrane remnants, and engulfment of GAS in LC3-positive vacuoles. We also found that production of streptolysin S could mediate targeting of GAS to autophagosomes in the absence of SLO, a process accompanied by galectin 8 binding to damaged GAS-containing endosomes. Maturation of GAS-containing autophagosome-like vacuoles to degradative autolysosomes was prevented by SLO pore-formation and by SLO-mediated translocation of enzymatically active NADase into the keratinocyte cytosol. We conclude that SLO stimulates xenophagy in pharyngeal keratinocytes, but the coordinated action of SLO and NADase prevent maturation of GAS-containing autophagosomes, thereby prolonging GAS intracellular survival. This novel activity of NADase to block autophagic killing of GAS in pharyngeal cells may contribute to pharyngitis treatment failure, relapse, and chronic carriage.

Dataset Information

Development of a multiplexed opsonophagocytic killing assay (MOPA) for group B Streptococcus.

Publications

Development of a multiplexed opsonophagocytic killing assay (MOPA) for group B Streptococcus.

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