Alnustone inhibits Streptococcus pneumoniae virulence by targeting pneumolysin and sortase A.

Streptococcus pneumoniae (S. pneumoniae) is a major Gram-positive opportunistic pathogen that causes pneumonia, bacteremia, and other fatal infections. This bacterium is responsible for more deaths than any other single pathogen in the world. Inexplicably, these symptoms persist despite the administration of effective antibiotics. Targeting pneumolysin (PLY) and sortase A (SrtA), the major virulence factors of S. pneumoniae, this study uncovered a novel resistance mechanism to S. pneumoniae infection. Using protein phenotype assays, we determined that the small molecule inhibitor alnustone is a potent drug that inhibits both PLY and SrtA. As essential virulence factors of S. pneumoniae, PLY and SrtA play a significant role in the occurrence of infection. Furthermore, evaluation using PLY-mediated hemolysis assay demonstrated alunstone had the potential to interrupt the haemolytic activity of PLY with treatment alunstone (4 µg/ml). Co-incubation of S. pneumoniae D39 SrtA with small-molecule inhibitors decreases cell wall-bound Nan A (pneumococcal-anchored surface protein SrtA), inhibits biofilm formation, and reduces biomass significantly. The protective effect of invasive pneumococcal disease (IPD) on murine S. pneumoniae was demonstrated further. Our study proposes a comprehensive bacteriostatic mechanism for S. pneumoniae and highlights the significant translational potential of targeting both PLY and SrtA to prevent pneumococcal infections. Our findings indicate that the antibacterial strategy of directly targeting PLY and SrtA with alnustone is a promising treatment option for S. pneumoniae and that alnustone is a potent inhibitor of PLY and SrtA.

Recent advances with Treg depleting fusion protein toxins for cancer immunotherapy.

T regulatory cells (Tregs) are an important T cell population for immune tolerance, prevention of autoimmune diseases and inhibition of antitumor immunity. The tumor-promoting role played by Tregs in cancer has prompted numerous approaches to develop immunotherapeutics targeting Tregs. One approach to depletion of Treg cells is retargeting the highly potent cytotoxic activity of bacterial toxins. These agents capitalize on the well-characterized bacterial toxins, diphtheria toxin and Pseudomonas aeruginosa exotoxin A-both of which harbor membrane translocation domains and enzymatic domains that catalytically halt protein synthesis within intoxicated eukaryotic cells and act at picomolar or subpicomolar concentrations. In this review, we summarize the preclinical and clinical development of several Treg-depleting cancer immunotherapies based on these two bacterial toxins.

An immunotoxin targeting the gH glycoprotein of KSHV for selective killing of cells in the lytic phase of infection.

Amongst the pathologies associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV), multicentric Castleman's disease is distinctive for involvement of the lytic phase of the virus replication cycle. This B cell lymphoproliferative disorder has shown clinical responsiveness not only to generalized immunotherapy and cytotoxic chemotherapy, but also to inhibitors of herpesvirus DNA replication, consistent with the involvement of lytic phase of replication. These findings suggest that selective killing of virus-producing cells might represent a novel therapeutic strategy. We designed an immunotoxin, YC15-PE38, containing a single chain variable region fragment of a monoclonal antibody against KSHV glycoprotein H (gH) linked to the effector domains of Pseudomonas aeruginosa exotoxin A. Purified YC15-PE38 displayed highly selective and potent killing of a gH-expressing transfectant cell line (subnanomolar IC(50)). The immunotoxin also strongly inhibited production of infectious KSHV virions from an induced chronically infected cell line, by virtue of selective killing of the virus-producing cells. Combination treatment studies indicated complementary activities between YC15-PE38 and the herpesviral DNA replication inhibitor ganciclovir. These results provide support for the development of anti-KSHV strategies based on targeted killing of infected cells expressing lytic phase genes.

Specific killing of CCR9 high-expressing acute T lymphocytic leukemia cells by CCL25 fused with PE38 toxin.

We have previously demonstrated that CCR9 plays a pivotal role in drug resistance and invasion in human acute T-lymphocytic leukemia (T-ALL). In this study, we investigated whether the MOLT4 cells, which naturally express CCR9 at high levels, can be successfully killed by the specific ligand, CCL25 fused to Pseudomonas exotoxin 38 (PE38) toxin. Our results demonstrated that CCL25-PE38 was able to specifically kill MOLT4 cells via apoptosis induction, and suppress the growth of CCR9(+) tumors. This work shows that CCR9 high-expressing human T-ALL cells can be successfully killed by delivering PE38 toxin fused to the ligand CCL25.