Pyroptosis induced by the N-terminal gasdermin domain (GSDM^NT) holds great potential for anti-tumor therapy. However, due to the extreme cytoxicity of GSDM^NT, it is challenging to efficiently produce and deliver GSDM^NT into tumor cells. Here, we report the development of two strategies to package recombinant adeno-associated virus (rAAV) expressing GSDM^NT: 1) drive the expression of GSDM^NT by a mammal specific promoter and package the virus in Sf9 insect cells to avoid its expression; 2) co-infect rAAV-Cre to revert and express the double-floxed inverted GSDM^NT. We demonstrate that these rAAVs can induce pyroptosis and prolong survival in preclinical cancer models. The oncolytic-viruses induce pyroptosis and evoke a robust immune-response. In a glioblastoma model, rAAVs temporarily open the blood-brain barrier and recruit tumor infiltrating lymphocytes into the brain. The oncolytic effect is further improved in combination with anti-PD-L1. Together, our strategies efficiently produce and deliver GSDM^NT into tumor cells and successfully induce pyroptosis, which can be exploited for anti-tumor therapy.

The essential role of pathogens in host metabolism is widely recognized, yet the mechanisms by which they affect host physiology remain to be fully defined. Here, we found that NleB, an enteropathogenic Escherichia coli (EPEC) type III secretion system effector known to possess N-acetylglucosamine (GlcNAc) transferase activity, GlcNAcylates HIF-1α, a master regulator of cellular O₂ homeostasis. We determined that NleB-mediated GlcNAcylation at a conserved arginine 18 (Arg18) at the N-terminus of HIF-1α enhanced HIF-1α transcriptional activity, thereby inducing HIF-1α downstream gene expression to alter host glucose metabolism. The arginine transferase activity of NleB was required for its enhancement of HIF-1α transactivity and the subsequent effect on glucose metabolism in a mouse model of EPEC infection. In addition, HIF-1α acted as a mediator to transact NleB-mediated induction of glucose metabolism-associated gene expression under hypoxia. Thus, our results further show a causal link between pathogen infection and host glucose metabolism, and we propose a new mechanism by which this occurs. Accumulating evidence shows that pathogens can affect host metabolism, resulting in human diseases such as obesity and type 2 diabetes. However, how pathogens influence their hosts is still not clear, and this results in a lack of effective and specific clinical treatments. Further investigations into the causes of pathogen disturbance of host metabolism are urgently needed. In this study, we show that a protein molecule, NleB, secreted by enteropathogenic bacteria (EPEC) can get into host cells and modify the function of a master regulator of cellular O₂ homeostasis, HIF-1α, thereby altering host glucose metabolism. We show that HIF-1α acts as a mediator to transact NleB-mediated induction of glucose metabolism-associated gene expression under hypoxia. Our results reveal a causal link between pathogen infection and host glucose metabolism, which may provide a new explanation for the causes of human diseases related to metabolic disturbance.