in-vitro-reconstitution-bacterial-ubiquitination-vcpp97-mediated

<em>In-vitro</em> Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

Ubiquitination is a versatile post-translational modification that plays a critical role in cytosolic immunity. Upon invasion of the host cells, ...

Indian Institute of Technology Bombay

 is a global priority respiratory pathogen that kills over a million people annually. The pore-forming cytotoxin, pneumolysin (PLY) is a major virulence factor. Here, we found that recombinant PLY as well as wild-type pneumococcal strains, but not the isogenic PLY mutant, upregulated the shedding of extracellular vesicles (EVs) harboring membrane-bound toxin from human THP-1 monocytes. PLY-EVs induced cytotoxicity and hemolysis dose-dependently upon internalization by recipient monocyte-derived dendritic cells. Proteomics analysis revealed that PLY-EVs are selectively enriched in key inflammatory host proteins such as IFI16, NLRC4, PTX3, and MMP9. EVs shed from PLY-challenged or infected cells induced dendritic cell maturation and primed them to infection. , zebrafish administered with PLY-EVs showed pericardial edema and mortality. Adoptive transfer of bronchoalveolar-lavage-derived EVs from infected mice to healthy recipients induced lung damage and inflammation in a PLY-dependent manner. Our findings identify that host EVs released during infection mediate pneumococcal pathogenesis.

Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection.

Cell-autonomous immunity prevents intracellular pathogen growth through mechanisms such as ubiquitination and proteasomal targeting of bacteria for degradation. However, how the proteasome eradicates ubiquitinated bacteria has remained unclear. Here we show that host AAA-ATPase, VCP/p97, associates with diverse cytosol-exposed ubiquitinated bacteria (Streptococcus pneumoniae, Salmonella enterica serovar Typhimurium, Streptococcus pyogenes) and requires the ATPase activity in its D2 domain to reduce intracellular bacterial loads. Combining optical trap approaches along with molecular dynamic simulations, in vitro reconstitution and immunogold transmission electron microscopy, we demonstrate that p97 applies mechanical force to extract ubiquitinated surface proteins, BgaA and PspA, from S. pneumoniae cell membranes. This causes extensive membrane lysis and release of cytosolic content, thereby killing the pathogen. Further, p97 also controls S. pneumoniae proliferation in mice, ultimately protecting from fatal sepsis. Overall, we discovered a distinct innate antimicrobial function of p97 that can protect the host against lethal bacterial infections.

Article	Year
Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection. iScience\| PubMed ID: 39211544	2024
Host AAA-ATPase VCP/p97 lyses ubiquitinated intracellular bacteria as an innate antimicrobial defence. Nature microbiology\| PubMed ID: 40217128	2025

Sumit Rakshit

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