Endocytosis‒Mediated Invasion and Pathogenicity of Streptococcus agalactiae in Rat Cardiomyocyte (H9C2)

Masaharu Seno,Jeyaprakash Rajendhran,Muthuirulan Pushpanathan,Paramasamy Gunasekaran,Sharma Pooja

doi:10.1371/journal.pone.0139733

Abstract

Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae internalization, we chemically inhibited discrete parts of cellular uptake system in H9C2 cells using genistein, chlorpromazine, nocodazole and cytochalasin B. Chemical inhibition of microtubule and actin formation by nocodazole and cytochalasin B impaired S. agalactiae internalization into H9C2 cells. Consistently, reverse‒ transcription PCR (RT‒PCR) and quantitative real time‒PCR (RT-qPCR) analyses also detected higher levels of transcripts for cytoskeleton forming genes, Acta1 and Tubb5 in S. agalactiae‒infected H9C2 cells, suggesting the requirement of functional cytoskeleton in pathogenesis. Host survival assay demonstrated that S. agalactiae internalization induced cytotoxicity in H9C2 cells. S. agalactiae cells grown with benzyl penicillin reduced its ability to internalize and induce cytotoxicity in H9C2 cells, which could be attributed with the removal of surface lipoteichoic acid (LTA) from S. agalactiae. Further, the LTA extracted from S. agalactiae also exhibited dose‒dependent cytotoxicity in H9C2 cells. Taken together, our data suggest that S. agalactiae cells internalized H9C2 cells through energy‒dependent endocytic processes and the LTA of S. agalactiae play major role in host cell internalization and cytotoxicity induction.

Highlights

Group B Streptococci (GBS), or Streptococcus agalactiae is considered as a leading cause of life‒threatening invasive bacterial infections in pregnant women, infants, adults and immunocompromised individuals [1]
The intracellular viable colony forming units (CFUs) recovered from S. agalactiae‒infected H9C2 cells were found to be increased with increase in multiplicity of infection (MOI)
The total intracellular CFUs recovered from S. agalactiae‒infected H9C2 cells at MOI of 1:10, 1: 100, 1: 1000 were found to be 3.3 x 103, 1.5 x104, and 3.3 x 105 ml-1 respectively

Summary

Introduction

Group B Streptococci (GBS), or Streptococcus agalactiae is considered as a leading cause of life‒threatening invasive bacterial infections in pregnant women, infants, adults and immunocompromised individuals [1]. These bacteria are Gram‒positive, β‒hemolytic, chain‒forming cocci that are normal residents of the vaginal microflora of 25% of healthy women. The S. agalactiae colonize genital tracts of pregnant women and approximately 50% of newborn babies to these infected mothers are highly susceptible to GBS infection [5, 6]. S. agalactiae infection causes high mortality (34–50%) in cardiovascular disease (CVD) patients especially in case of setting prosthetic valve during cardiac surgery [19]. There is high risk of S. agalactiae infection to CDV individuals, the mechanisms underlying the colonization and pathogenesis of S. agalactiae in cardiac cells has not been well studied

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Conclusion