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

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.

Fig 1. Endocytic uptake of S. agalactiae by H9C2 cells.

(a) Infection assay: A, B, C, D- merged images of bright field and Hoechst 33342 ‒stained H9C2 cells infected with live & heat‒killed S. agalactiae, S. gordonii ATCC 12403 and E. coli, respectively. A’, B’ C’, D’- merged images of H9C2 cells (counter-stained with Hoechst 33342 after infection) and endocytosed fluorescently labeled live‒ & heat‒killed S. agalactiae (AO & PI‒stained), S. gordonii ATCC 12403 (AO‒stained) and E. coli (AO‒stained), respectively. (b) Quantification of endocytic uptake of fluorescently‒labelled live & heat‒killed S. agalactiae, S. gordonii ATCC 12403 and E. coli by H9C2 cells. (c) Viable cell count of bacteria from infected H9C2 cells. H9C2 cells were infected with different inoculum of S. agalactiae, S. gordonii ATCC 12403 and E. coli (i.e., 10⁴, 10⁵ and 10⁶) for 2 h. The recovered intracellular bacteria from H9C2 cells are represented as CFU/well (Mean ± SD) obtained from three independent experiments. Statistically significant differences are indicated by an asterisk (* p<0.05 or **p<0.01).

Fig 2. Energy dependent endocytic uptake of S. agalactiae by H9C2 cells.

(a) Quantification of endocytic uptake of fluorescently‒labeled live S. agalactiae (AO-stained) by H9C2 cells in the presence (green) or absence (brown) of sodium azide by flow cytometry. P1-unstained population of H9C2 cells, P2- population of H9C2 cells internalized with fluorescently‒labelled S. agalactiae. (b) Determination of intracellular relative viable CFU of S. agalactiae recovered from H9C2 cells in the presence of different endocytic inhibitors such as genistein, chlorpromazine, cytochalasin B and nocodazole. The results were expressed in relative percentage compared with the experimental control. Statistically significant differences are indicated by an asterisk (* p<0.05 or **p<0.01).

Fig 3. S. agalactiae‒induced cytotoxicity in H9C2 cells.

(a) Morphological changes induced by S. agalactiae in H9C2 cells after 6 h of infection, Top panel: S. agalactiae-infected and uninfected H9C2 cells after 6 h of infection. Middle and bottom panel: (i), (ii), (iii) & (iv) are representative confocal micrographs showing changes in H9C2 cell morphology within 6 h of infection with live (ASG) and heat‒killed (PSG) S. agalactiae. Nuclei (N) of H9C2 cells were shown by Hoechst 33342 staining. (b) Determination of live and heat‒killed S. agalactiae‒induced cytotoxicity in H9C2 cells by MTT assay. Statistically significant differences are indicated by an asterisk (* p<0.05 or **p<0.01). (c) Quantification of live and heat‒killed S. agalactiae‒induced cytotoxicity in H9C2 cells by flow cytometry. Q3‒live population of H9C2 cells (blue dots); Q4‒early apoptotic H9C2 cells (green); Q2‒late apoptotic H9C2 cells (yellow).

Fig 4. S. agalactiae‒infection induced gene expression of Tubb5 and Acta1 in H9C2 cells.

(a) Semi quantitative RT-PCR analysis for expression of cytoskeleton forming genes, Tubb5 and Acta1 in S. agalactiae‒infected (B) and uninfected (A) H9C2 cells (b) RT Q-PCR analysis for expression of Tubb5 and Acta1 in S. agalactiae‒infected and uninfected H9C2 cells. Gene expression values of the uninfected H9C2 cells (control) were set equal to 1 and the relative change in gene expression in S. agalactiae‒infected H9C2 cells were calculated. RT Q-PCR was performed twice independently and the triplicate measurements of gene expression per gene were included, which yielded similar results. Data shown are the mean relative change in expression ± standard deviation from one such experiment. Statistically significant differences are indicated by an asterisk (* p<0.05 or **p<0.01).

Fig 5. S. agalactiae LTA‒induced cytotoxicity in H9C2 cells.

(a) Quantification of benzyl penicillin (BP)‒treated and untreated S. agalactiae internalization into H9C2 cells. Q3‒uninfected population of H9C2 cells (blue dots); Q4‒population of H9C2 cells internalized with AO‒stained S. agalactiae (green dots) (b) Cytotoxicity analysis of BP-treated and untreated S. agalactiae on H9C2 cells. S. agalactiae grown in the presence of benzyl penicillin showed reduced cytotoxicity in H9C2 cells compared to untreated control (c) Determination of LTA-induced cytotoxicity in H9C2 cells after 6 h by MTT assay. Statistically significant differences are indicated by an asterisk (* p<0.05 or **p<0.01).