Hemolysin Coregulated Protein (HCP) from Vibrio Cholerae Interacts with the Host Cell Actin Cytoskeleton.

Vibrio cholerae (V. cholerae), the etiological agent of cholera, employs various virulence factors to adapt and thrive within both aquatic and human host environments. Among these factors, the type VI secretion system (T6SS) stands out as one of the crucial determinants of its pathogenicity. Valine glycine repeat protein G1 (VgrG1) and hemolysin coregulated protein (HCP) are considered major effector molecules of T6SS. Previous studies have highlighted that VgrG1 interacts with HCP proteins. Additionally, it has been shown that VgrG1 possesses an actin cross-linking domain (ACD) with actin-binding activity. Interestingly, it was reported that purified HCP protein treatment increased the stress fibers within cells. Therefore, we hypothesize that HCP may interact with host cell actin, potentially playing a role in the cytoskeletal rearrangement during V. cholerae infection. To test this hypothesis, we characterized HCP from the V. cholerae O139 serotype and demonstrated its interaction with actin monomers. In silico analysis and experimental validation revealed the presence of an actin-binding site within HCP. Furthermore, overexpression of HCP resulted in its colocalization with actin stress fibers in host cells. Our findings establish HCP as an effector molecule for potent host cell actin cytoskeleton remodeling during V. cholerae infection, providing new insights into bacterial pathogenicity mechanisms. Understanding the interplay between bacterial effectors and host cell components is crucial for developing targeted therapeutic interventions against cholera and related infectious diseases.

Probing the ligand binding specificity of FNBP4 WW domains and interaction with FH1 domain of FMN1.

Formins are a group of actin-binding proteins that mediate nascent actin filament polymerization, filament elongation, and barbed end-capping function, thereby regulating different cellular and developmental processes. Developmental processes like vertebrate gastrulation, neural growth cone dynamics, and limb development require formins functioning in a regulated manner. Formin-binding proteins like Rho GTPase regulate the activation of auto-inhibited conformation of diaphanous formins. Unlike other diaphanous formins, Formin1 (FMN1) a non-diaphanous formin is not regulated by Rho GTPase. FMN1 acts as an antagonist of the Bone Morphogenetic Protein (BMP) signaling pathway during limb development. Several previous reports demonstrated that WW domain-containing proteins can interact with poly-proline-rich amino acid stretches of formins and play a crucial role in developmental processes. In contrast, WW domain-containing Formin-binding Protein 4 (FNBP4) protein plays an essential role in limb development. It has been hypothesized that the interaction between FNBP4 and FMN1 can further attribute to the role in limb development through the BMP signaling pathway. In this study, we have elucidated the binding kinetics of FNBP4 and FMN1 using surface plasmon resonance (SPR) and enzyme-linked immunosorbent assays (ELISA). Our findings confirm that the FNBP4 exhibits interaction with the poly-proline-rich formin homology 1 (FH1) domain of FMN1. Furthermore, only the first WW1 domains are involved in the interaction between the two domains. Thus, this study sheds light on the binding potentialities of WW domains of FNBP4 that might contribute to the regulation of FMN1 function.

Zwitterionic Polysulfobetaine Inhibits Cancer Cell Migration Owing to Actin Cytoskeleton Dynamics.

Cell migration is an essential dynamic process for most living cells, mainly driven by the reorganization of actin cytoskeleton. To control actin dynamics, a molecular architecture that can serve as a nucleator has been designed by polymerizing sulfobetaine methacrylate. The synthesized zwitterionic polymer, poly(sulfobetaine methacrylate) (PZI), effectively nucleates the polymerization process of G-actin and substantially accelerates the rate of polymerization. Isothermal titration calorimetry (ITC) and bioinformatics analysis indicated binding between PZI and monomeric G-actin. Thus, in vitro actin dynamics was studied by dynamic light scattering (DLS), pyrene-actin polymerization assay, and total internal reflection fluorescence microscopy (TIRFM). Furthermore, a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore-containing monomeric unit was incorporated into the sulfobetaine zwitterionic architecture to visualize the effect of polymer in the cellular environment. The BODIPY-containing zwitterionic sulfobetaine polymer (PZI-F) successfully penetrated the cell and remained in the lysosome with minimal cytotoxicity. Confocal microscopy revealed the influence of this polymer on the cellular actin cytoskeleton dynamics. The PZI-F polymer was successfully able to inhibit the collective migration of the human cervical cancer cell line (HeLa cell) and breast cancer cell line (MDA-MB-231 cell), as confirmed by a wound healing assay. Therefore, polyzwitterionic sulfobetaine could be explored as an inhibitor of cancer cell migration.