hsdS
A

regulated extracellular vesicle-associated PLY to protect
Streptococcus pneumoniae
from macrophage killing via LAPosomes
Authors: Wang Liping , Liu Mengyuan , Qi Yixin , Wang Jian , Shi Qixue , Xie Xiaolin , Zhou Changlin , Ma Lingman 
Published: November 2023

Streptococcus pneumoniae
is a notorious human opportunistic pathogen which undergoes a spontaneous and reversible phenotypic change in response to the host environment. We demonstrated that the regulatory gene

hsdS
A

of DNA methylation in the type I restriction modification system altered colony transparency and substantially contributed to
S. pneumoniae
virulence. Most importantly,

hsdS
A

regulated the production of extracellular vesicles (EVs) which package cytosolic, surface, and secreted proteins, including pneumolysin (PLY). Interestingly, we confirmed that EV-associated PLY utilized internalization into macrophages to prolong the survival of intracellular bacteria as a major immune evasion strategy; that is, EV-associated PLY produced by the D39 strain (EVs-D39) could induce the formation of LC3-associated monolayer vacuoles [LC3-associated phagocytosis (LAP)] and co-localize with the NADPH oxidase 2 (NOX2) complex but not ULK1 when macrophages were infected with the D39Δ
ply
strain. In addition, EV-associated PLY derived from the EVs-D39 promoted macrophages to release more reactive oxygen species (ROS) and expression of p-p70s6k than EV-associated PLY derived from the D39Δ

hsdS
A

strain (EVs-D39Δ

hsdS
A

), whereas the expression of p-ULK1 was reversed, indicating that EVs-D39Δ

hsdS
A

was more likely to induce conventional xenophagy. Furthermore, we identified the β1 integrin receptor as a crucial inducer of ROS to mediate LAP activation. Bacterial evasion of host clearance is closely related to insufficient acidification after the fusion of autophagosomes or LAPosomes with lysosomes. Of note, we found EV-associated PLY damaged the integrity of the lysosome membrane and changed the pH gradient, resulting in lysosomes being unable to remove intracellular bacteria and ultimately prolonging the survival of
S. pneumoniae
in macrophages. Finally, the extracted mouse alveolar macrophages and mouse intranasal infection models were employed to further verify the above findings.

IMPORTANCE

S. pneumoniae
is a major human pathogen that undergoes a spontaneous and reversible phase variation that allows it to survive in different host environments. Interestingly, we found

hsdS
A

, a gene that manipulated the phase variation, promoted the survival and replication of
S. pneumoniae
in macrophages by regulating EV production and EV-associated PLY. More importantly, here we provided the first evidence that higher EV-associated PLY (produced by D39) could form LAPosomes that were single membrane compartments containing
S. pneumoniae
, which are induced by integrin β1/NOX2/ROS pathway. At the same time, EV-associated PLY increased the permeability of lysosome membrane and induced an insufficient acidification to escape the host killing, and ultimately prolonged the survival of
S. pneumoniae
in macrophages. In contrast, lower EV-associated PLY (produced by D39Δ

hsdS
A

) activated ULK1 recruitment to form double-layered autophagosomes to eliminate bacteria.