The globally disseminated M1T1 clone of group A Streptococcus evades autophagy for intracellular replication

Abstract

Autophagy is reported to be an important innate immune defense against the intracellular bacterial pathogen Group A Streptococcus (GAS). However, the GAS strains examined to date belong to serotypes infrequently associated with human disease. We find that the globally disseminated serotype M1T1 clone of GAS can evade autophagy and replicate efficiently in the cytosol of infected cells. Cytosolic M1T1 GAS (strain 5448), but not M6 GAS (strain JRS4), avoids ubiquitylation and recognition by the host autophagy marker LC3 and ubiquitin-LC3 adaptor proteins NDP52, p62, and NBR1. Expression of SpeB, a streptococcal cysteine protease, is critical for this process, as an isogenic M1T1 ΔspeB mutant is targeted to autophagy and attenuated for intracellular replication. SpeB degrades p62, NDP52, and NBR1 in vitro and within the host cell cytosol. These results uncover a proteolytic mechanism utilized by GAS to escape the host autophagy pathway that may underpin the success of the M1T1 clone.

Figure 1. M1T1⁵⁴⁴⁸ replicates within epithelial cells and avoids autophagy

(A) Ability of M1T1⁵⁴⁴⁸ and M6^JRS4 GAS to survive following internalization into HEp-2 epithelial cells. Data is represented as mean ± SEM of three independent experiments. *, p < 0.05; **, p < 0.01; one-tailed paired t-test. (B) Association of intracellular M6^JRS4 and M1T1⁵⁴⁴⁸ with GFP-LC3 at 4 h post-infection. Arrows indicate intracellular M6^JRS4 associated with GFP-LC3 and intracellular M1T1⁵⁴⁴⁸ devoid of GFP-LC3. Bar = 5 μm. (C) Percentage of intracellular GAS contained within LC3-positive compartments. Values represent the mean ± SEM for three independent experiments. *, p < 0.05; **, p < 0.01; one-tailed unpaired t-test.

Figure 2. M1T1⁵⁴⁴⁸ replicate efficiently in the cytosol of epithelial cells

(A and B) Transmission electron micrographs of HEp-2 cells infected with M1T1⁵⁴⁴⁸ (A) and M6^JRS4 (B) GAS at 6 h post-infection. The M6^JRS4 GAS is entrapped within a membrane compartment with arrowheads indicating the membrane (zoomed micrograph on right); In contrast, the M1T1⁵⁴⁴⁸ GAS is entirely exposed to the cytosol. (C) Percentage of intracellular M1T1⁵⁴⁴⁸ contained within EEA1/Lamp1-positive compartments. Values represent the mean ± SEM for three technical replicates. (D and E) Susceptibility of intracellular M1T1⁵⁴⁴⁸ (D) and M6^JRS4 (E) GAS to penicillin. Values represent the mean ± SEM of three independent experiments. *, p < 0.05; **, p < 0.01; one-tailed paired t-test. See also Figure S1.

Figure 3. Intracellular M1T1⁵⁴⁴⁸ avoids ubiquitylation and the ubiquitin-LC3 adaptor proteins NDP52, p62 and NBR1

(A to D) Association of M1T1⁵⁴⁴⁸ and M6^JRS4 GAS with ubiquitylated proteins (A), NDP52 (B), p62 (C) and NBR1 (D). Quantitative data at 2, 4, 6 and 8 h post-infection is provided in Table S1. Arrows indicate intracellular M6^JRS4 associated with GFP-LC3 and adaptors and intracellular M1T1⁵⁴⁴⁸ devoid of GFP-LC3 and adaptors at 4 h post-infection. Bar = 5 μm. See also Table S1.

Figure 4. SpeB cysteine protease degrades ubiquitin-LC3 adaptor proteins and is required for efficient intracellular replication of GAS

(A) Ability of M1T1⁵⁴⁴⁸ ΔspeB to replicate within HEp-2 epithelial cells. Data is represented as mean ± SEM of three independent experiments. *, p < 0.05; **, p < 0.01; one-tailed paired t-test. (B) Association of M1T1⁵⁴⁴⁸ ΔspeB with GFP-LC3 at 4 h post-infection. Arrows indicate intracellular M1T1⁵⁴⁴⁸ ΔspeB mutant associated with GFP-LC3. Bar = 5 μm. (C) Percentage of intracellular GAS contained within LC3-positive compartments. Values represent the mean ± SEM of three independent experiments. *, p < 0.05; one-tailed paired t-test. The M1T1⁵⁴⁴⁸ data was taken from Figure 1C and is included for comparison. (D) Intracellular replication of the SpeB-expressing M6^JRS4 + pSpeB strain compared with the SpeB-negative M6^JRS4 strain. Data is represented as mean ± SEM of three technical replicates. *, p < 0.05; **, p < 0.01; one-tailed unpaired t-test. (E) Western immunoblot showing that purified SpeB cysteine protease degrades NDP52, p62 and NBR1 in HEp-2 cell lysates. (F) Ectopically expressed SpeB degrades cytosolic NDP52, p62 and NBR1. Bars represent the number of puncta ± SEM in at least 15 transfected cells and is representative of 2 experimental replicates. **, p < 0.01; ***, p < 0.001; one-tailed paired t-test. Representative images used for the quantitation are shown in Figure S2H to J. See also Figure S2.