Ugarova, T. | bioRxiv Science

bioRxiv2026

TARGETING THE CAPSULE OF KLEBSIELLA PNEUMONIAE WITH A CATIONIC CR3-BINDING PROTEIN ENHANCES PHAGOCYTOSIS AND PROMOTES BACTERIAL CLEARANCE AND SURVIVAL IN A MOUSE SEPSIS MODEL

Platelet Factor 4 (PF4), a cationic antimicrobial peptide, serves as a ligand for the myeloid-specific phagocytic receptor CR3 (Mac-1, CD11b/CD18). We previously demonstrated that recombinant dimeric PF4 (rdPF4) functions as a bacterial opsonin, enhancing phagocytosis of Gram-positive Staphylococcus aureus and facilitating clearance of both antibiotic-susceptible and methicillin-resistant S. aureus in a mouse model of infectious peritonitis. In this study, we examined whether rdPF4 is pathogen-agnostic by assessing its effect on phagocytosis of Gram-negative encapsulated Klebsiella pneumoniae, a WHO Bacterial Priority Pathogen. We demonstrate that rdPF4 enhances CR3-mediated phagocytosis of both live and heat-inactivated high-virulence K2 and low-virulence K3 strains of K. pneumoniae by various mouse and human macrophage cell lines, as well as primary neutrophils and macrophages. It also increased phagocytosis of carbapenem-resistant K. pneumoniae. rdPF4 did not directly kill bacteria but acted as an opsonin binding to the negatively charged bacterial capsule and creating recognition sites for CR3 on leukocytes. In a mouse sepsis model, a single dose of rdPF4 significantly enhanced bacterial clearance from the lungs, liver, and peritoneum and reduced bacteremia. Histological analyses showed that rdPF4 provided substantial protection to lung and liver tissues against K. pneumoniae-induced damage. Consistent with these findings, rdPF4 treatment increased the survival rates of infected mice. These results show that rdPF4 effectively targets the capsule, a key virulence factor of K. pneumoniae, thereby reducing the bacteriums ability to evade the host immune response. Overall, the data suggest a common mechanism in which cationic rdPF4, by binding to the negatively charged surfaces of both Gram-negative and Gram-positive bacteria, diminishes their antiphagocytic properties.

microbiology↗

bioRxiv2019

An actin-based protrusion originating from a podosome-enrichedregion initiates macrophage fusion

Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating macrophage fusion is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase contrast video microscopy demonstrated that in the majority of events, short protrusions (3 {+/-} 1 m) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (16 {+/-} 7 m). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent actin-based protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data indicate that an actin-based protrusion formed at the leading edge initiates macrophage fusion.

cell biology↗