The M1 protein of Streptococcus pyogenes triggers an innate uptake mechanism into polarized human endothelial cells

Abstract

Serotype M1 Streptococcus pyogenes is a major human pathogen associated with severe invasive diseases causing high morbidity and mortality. In a substantial number of cases, invasive disease develops in previously healthy individuals with no obvious port of entry. This has led to the hypothesis that the source of streptococci in these cases is a transient bacteraemia. This study focuses on the analysis of interaction of tissue-invasive serotype M1 S. pyogenes with human endothelial cells (EC) of the vascular system. We identify the M1 surface protein of S. pyogenes as the EC invasin which is recognised by polarized human blood EC, thereby triggering rapid, phagocytosis-like uptake of streptococci into polarized EC layers. Upon internalization, the M1 S. pyogenes serotype is incorporated into phagosomes which traffic via the endosomal/lysosomal pathway. However, some of the streptococci successfully evade this innate killing process and hereby mediate their escape into the cytoplasm of the host cell. The results of this study demonstrate that blood EC possess an efficient uptake mechanism for serotype M1 S. pyogenes. Despite efficient phagocytosis, streptococcal survival within EC constitutes one potential mechanism which favours intracellular persistence and thus facilitates continuous infection and dissemination from the primary side of infection into deep tissue.

Fig. 1

Serotype M1 S. pyogenes clinical isolates trigger uptake into polarized EC. The figure shows immunofluorescence analysis of the invasion potential of 4 different M1 GAS strains on polarized confluent EC. HUVEC were infected with the M1 GAS strains A527 (a), A302 SF370 (b), A270 (c) and A271 (d) for 5 h. After washing and fixation, cells were stained differentially for extracellular (green-yellow) and intracellular (red) streptococci. The actin cytoskeleton stained with phalloidin is depicted in green. Representative images of 3 independent experiments are shown. a-d Bars: 5 µm. e Quantification of internalization rates of different M1 GAS strains on HUVEC 4 h after infection. The graph represents mean values ± SD from 1 of 3 independent experiments.

Fig. 2

M1 S. pyogenes induces actin cytoskeleton rearrangements and traffics along the endocytic pathway. a Field-emission scanning electron microscopy image showing uptake of streptococci (GAS A527) into EC 3 h after infection. Upon infection, EC form membrane protrusions that tightly engulf the streptococcal chain, representing a zipper-like uptake process. Bar: 600 nm. Representative images of 3 independent experiments are shown. b Immunofluorescence staining of β₁-integrins on the surface of EC. β₁-integrin clustering (green) is detectable (3 h after infection) in the vicinity of internalized bacteria (GAS A527) (red). The inserts show split channels for GAS and β₁-integrin of the indicated area of the merged image. EC nuclei are stained in blue. Representative images of 3 independent experiments are shown. Bar: 5 µm. c Involvement of F-actin in the internalization process of M1 GAS A527. Internalized streptococci (red) co-localize with F-actin (green) 1 h after infection. Inserts show enlarged sections of split channels of the indicated area from the merged image. Representative images of 3 independent experiments are shown. Bar: 5 µm. d Aggregation of the Arp2/3 complex (green) is detectable in the near vicinity of internalized streptococci (blue) 1 h after infection. Inserts show enlarged sections of split channels of the indicated area from the merged image. Representative images of 3 independent experiments are shown. Bar: 5 µm. e Use of specific inhibitors, which interfere with host-cell signalling molecules involved in classic phagocytic uptake processes, blocks M1 GAS-mediated uptake into EC (3 h after infection). Prior to infection with M1 GAS A527, HUVEC were pre-treated with edelfosine (20 µM), BAPTA-AM (25 µM) and calphostin C (2 µM) to inhibit phospholipase C, intracellular calcium release and PKC, respectively. Internalization rates are expressed as % intracellular bacteria compared to a DMSO-treated control with 100% internalization. The graph shows the mean values ± SD of 1 representative of 3 independent experiments. f M1 GAS A527 (red) co-localizes with the Lamp-1-positive (green) late endosomal/lysosomal compartments 3 h after infection. The inserts show split channels for Lamp-1 and GAS of the indicated area in the merged image. Representative images of 3 independent experiments are shown. Bar: 5 µm. g TEM analysis of ultra-thin sections of infected EC layers. A close association of streptococci with gold particles is detectable (arrows), indicating fusion of the M1 GAS-containing phagosome with BSA-gold-loaded terminal lysosomes. Representative images of 3 independent experiments are shown. Bar: 500 nm.

Fig. 3

The M1 knock-out mutant is deficient for internalization into EC. a Analysis of the ability of the M1 wt and mutant strains to promote internalization into HUVEC. The rate of internalization is indicated by + or +++ or ++++++ and no internalization is indicated by -. b Quantification of internalization rates of the M1 wt GAS strain 90-226 (A766), the M1 knock-out mutant ΔM1 90-226 (A767) and the ΔM1 mutant complemented with emm1 (Compl. ΔM1 GAS) into HUVEC (4 h after infection). In contrast to the M1 wt strain, the ΔM1 knock-out mutant is deficient for internalization into HUVEC. Complementation of ΔM1 GAS strain (Compl. ΔM1 GAS) restores the invasive phenotype, yielding uptake rates comparable to those of the M1 wt GAS strain. The graph represents mean values ± SD of 1 representative of 3 independent experiments. c, d Fluorescence images of HUVEC infected with the ΔM1 knock-out mutant (c) and the complemented ΔM1 knock-out mutant (d) 4 h after infection. Intracellular bacteria are stained in red, extracellular bacteria are depicted in green-yellow and EC nuclei are shown in blue. Representative images of 3 independent experiments are shown. Bars: 20 µm. e Analysis of the invasion potential of L. lactis strains on HUVEC 3 h after infection. The parental L. lactis strain (-M1), lacking the M1 protein on its surface, is not internalized. In contrast, heterologous expression of M1 on the lactococcal surface converts L. lactis into an efficiently internalized strain (+M1).

Fig. 4

Viable M1 S. pyogenes escapes from the lysosomal compartment. a TEM analysis of ultra-thin sections of infected HUVEC layers shows streptococcal escape (arrow) from the phagolysosomal compartment into the cytoplasm of the host cell (4 h after infection). Representative images of 2 independent experiments are shown. Bar: 1 µm. b Free M1 GAS A527 (white arrows) reside within the cytoplasm of the host cell (5 h after infection) and are localized in close proximity to the endoplasmic reticulum (black arrow). Representative images of 2 independent experiments are shown. Bar: 500 nm. c, d Immunofluorescence analysis of the viability of M1 GAS. Viable intracellular M1 GAS (green) is detectable 5 h (c) and 10 h (d) after infection. EC nuclei also absorb the green dye and, therefore, also appear green. Representative images of 3 independent experiments are shown. Bars: 5 µm. e Quantification of intracellular survival of M1 GAS A527 in HUVEC 2, 4, 6 and 9 h after infection. The graph shows mean values ± SD of triplicates from 1 representative experiment.