Type VII secretion systems participate in protein export, virulence, conjugation, and metabolic regulation. Five subtypes (ESX-1 to ESX-5) exist, each with specific roles and well-characterized secretion profiles in various mycobacterial species. Mycobacterium abscessus, encodes only ESX-3 and ESX-4. Here, single and double M. abscessus mutants lacking the main ATPases EccC3 and EccC4 were used to define ESX-3 and ESX-4 contributions to substrate secretion and virulence. Our results demonstrate that EsxG/H secretion depends entirely on ESX-3, whereas both ESX-3 and ESX-4 secrete EsxU/T. Furthermore, two newly identified PE/PPE substrates (MAB_0046/MAB_0047) require ESX-3 for secretion. Functional complementation restored secretion and revealed subpolar localization of these systems. Macrophage infections showed that ESX-3 and ESX-4 contribute to bacterial internalization, phagosomal escape, and intracellular survival. In mice, infections with eccC3- and/or eccC4-deletion mutants resulted in complete survival and reduced bacterial loads in the lungs. These findings demonstrate that both ESX systems drive M. abscessus pathogenicity.
© 2025. The Author(s).

Antibody-drug conjugates (ADCs) combining monoclonal antibodies with cytotoxic payloads are a rapidly emerging class of immune-based therapeutics with the potential to improve the treatment of cancer, including children with relapse/refractory acute lymphoblastic leukemia (ALL). CD123, the α subunit of the interleukin-3 receptor, is overexpressed in ALL and is a potential therapeutic target. Here, we show that pivekimab sunirine (PVEK), a recently developed ADC comprising the CD123-targeting antibody, G4723A, and the cytotoxic payload, DGN549, was highly effective in vivo against a large panel of pediatric ALL patient-derived xenograft (PDX) models (n = 39). PVEK administered once weekly for 3 weeks resulted in a median event-free survival (EFS) of 57.2 days across all PDXs. CD123 mRNA and protein expression was significantly higher in B-lineage (n = 65) compared with T-lineage (n = 25) ALL PDXs (p < 0.0001), and mice engrafted with B-lineage PDXs achieved significantly longer EFS than those engrafted with T-lineage PDXs (p < 0.0001). PVEK treatment also resulted in significant clearance of human leukemia cells in hematolymphoid organs in mice engrafted with B-ALL PDXs. Notably, our results showed no direct correlation between CD123 expression and mouse EFS, indicating that CD123 is necessary but not sufficient for in vivo PVEK activity. Importantly, a PDX with very high CD123 cell surface expression but resistant to in vivo PVEK treatment, failed to internalize the G4723A antibody while remaining sensitive to the PVEK payload, DGN549, suggesting a novel mechanism of resistance. In conclusion, PVEK was highly effective against a large panel of B-ALL PDXs supporting its clinical translation for B-lineage pediatric ALL.
© 2025 The Author(s). HemaSphere published by John Wiley & Sons Ltd on behalf of European Hematology Association.

Mycobacteria produce a large repertoire of surface-exposed lipids with major biological functions. Among these lipids, trehalose polyphleates (TPPs) are instrumental in the infection of Mycobacterium abscessus by the therapeutic phage BPs. However, while the biosynthesis and transport of TPPs across the membrane by MmpL10 have been reported, the role of TPPs in host infection remains enigmatic. Here, we addressed whether the loss of TPPs influences interactions with macrophages and the virulence of M. abscessus. As anticipated, the deletion of mmpL10 in smooth (S) and rough (R) variants of M. abscessus abrogated TPP production, which was rescued upon gene complementation. Importantly, infection of human THP-1 cells with the mmpL10 mutants was associated with decreased intramacrophage survival and a reduced proportion of infected cells. The rough mmpL10 mutant showed an impaired capacity to block phagosomal acidification and was unable to co-localize with Galectin-3, a marker of phagosomal membrane damage. This suggests that TPPs participate, directly or indirectly, in phagolysosomal fusion and in phagosomal membrane damage to establish cytosolic communication. The TPP defect that affects the fitness and virulence of M. abscessus was further demonstrated in zebrafish embryos using a rough clinical strain resistant to phage BPs and harboring a frameshift mutation in mmpL10. Infection with this strain was correlated with a slight decrease in embryo survival and a reduced bacterial burden as compared to the corresponding parental and complemented derivatives. Together, these results indicate that TPPs are important surface lipids contributing to the pathogenicity of M. abscessus.IMPORTANCETrehalose polyphleates (TPPs) are complex lipids associated with the mycobacterial cell surface and were identified 50 years ago. While the TPP biosynthetic pathway has been described recently, the role of these lipids in the biology of mycobacteria remains yet to be established. The wide distribution of TPPs across mycobacterial species suggests that they may exhibit important functions in these actinobacteria. Here, we demonstrate that Mycobacterium abscessus, an emerging multidrug-resistant pathogen that causes severe lung diseases in cystic fibrosis patients, requires TPPs for survival in macrophages and virulence in a zebrafish model of infection. These findings support the importance of this underexplored family of lipids in mycobacterial pathogenesis.

Human CD81 and CD9 are members of the tetraspanin family of proteins characterized by a canonical structure of four transmembrane domains and two extracellular loop domains. Tetraspanins are known as molecular facilitators, which assemble and organize cell surface receptors and partner molecules forming clusters known as tetraspanin-enriched microdomains. They have been implicated to play various biological roles including an involvement in infections with microbial pathogens. Here, we demonstrate an important role of CD81 for the invasion of epithelial cells by Salmonella enterica. We show that the overexpression of CD81 in HepG2 cells enhances invasion of various typhoidal and non-typhoidal Salmonella serovars. Deletion of CD81 by CRISPR/Cas9 in intestinal epithelial cells (C2BBe1 and HT29-MTX-E12) reduces S. Typhimurium invasion. In addition, the effect of human CD81 is species-specific as only human but not rat CD81 facilitates Salmonella invasion. Finally, immunofluorescence microscopy and proximity ligation assay revealed that both human tetraspanins CD81 and CD9 are recruited to the entry site of S. Typhimurium during invasion but not during adhesion to the host cell surface. Overall, we demonstrate that the human tetraspanin CD81 facilitates Salmonella invasion into epithelial host cells.

mRNA incorporated in lipid nanoparticles (LNPs) became a new class of vaccine modality for induction of immunity against COVID-19 and ushered in a new era in vaccine development. Here, we report a novel, easy-to-execute, and cost effective engineered extracellular vesicles (EVs)-based combined mRNA and protein vaccine platform (EVX-M+P vaccine) and explore its utility in proof-of-concept immunity studies in the settings of cancer and infectious disease. As a first example, we engineered EVs, natural nanoparticle carriers shed by all cells, to contain ovalbumin mRNA and protein (EVOvaM+P vaccine) to serve as cancer vaccine against ovalbumin-expressing melanoma tumors. EVOvaM+P administration to mice with established melanoma tumors resulted in tumor regression associated with effective humoral and adaptive immune responses. As a second example, we generated engineered EVs that contain Spike (S) mRNA and protein to serve as a combined mRNA and protein vaccine (EVSpikeM+P vaccine) against SARS-CoV-2 infection. EVSpikeM+P vaccine administration in mice and baboons elicited robust production of neutralizing IgG antibodies against RBD (receptor binding domain) of S protein and S protein specific T cell responses. Our proof-of-concept study describes a new platform with an ability for rapid development of combination mRNA and protein vaccines employing EVs for deployment against cancer and other diseases.
Copyright © 2024. Published by Elsevier B.V.