Role of the WASP family proteins for Mycobacterium marinum actin tail formation

Abstract

Mycobacterium marinum, a natural pathogen of fish and frogs and an occasional pathogen of humans, is capable of inducing actin tail formation within the cytoplasm of macrophages, leading to actinbased motility and intercellular spread. Actin tail formation by M. marinum is markedly reduced in macrophages deficient in the Wiskott-Aldrich syndrome protein (WASP), which still contain the closely related and ubiquitously expressed protein N-WASP (neuronal WASP). In fibroblasts lacking both WASP and N-WASP, M. marinum is incapable of efficient actin polymerization and of intercellular spread. By reconstituting these cells, we find that M. marinum is able to use either WASP or N-WASP to induce actin polymerization. Inhibition or genetic deletion of tyrosine phosphorylation, Nck, WASP-interacting protein, and Cdc42 does not affect M. marinum actin tail formation, excluding the participation of these molecules as upstream activators of N-WASP in the initiation of actin-based motility. In contrast, deletion of the phosphatidylinositol 4,5-bisphosphate-binding basic motif in N-WASP eliminates M. marinum actin tail formation. Together, these data demonstrate that M. marinum subversion of host actin polymerization is most similar to distantly related Gram-negative organisms but that its mechanism for activating WASP family proteins is unique. Binding of specific molecules including phosphatidylinositol 4,5-bisphosphate (PIP 2 ), Cdc42, Nck, and Grb2 to N-WASP disrupts this inhibition and unmasks the WASP homology 2 and acidic (WA) domain, allowing the Arp2͞3 complex to initiate de novo actin polymerization (2-6). WASP, a protein found only in hematopoietic cells, is closely related to N-WASP, sharing 50% sequence similarity. The two proteins are similarly organized and are controlled by the same host cell inputs. Less related to N-WASP are the WAVE proteins that contain the WA domain output region but have different activating inputs (7).Diverse pathogens have evolved mechanisms to hijack this Arp2͞ 3-dependent pathway of actin polymerization for their own benefit (reviewed in ref. 8). For vaccinia virus, actin polymerization is required for viral egress from infected cells. Enterohemorrhagic and enteropathogenic Escherichia coli species' actin polymerization leads to pedestal formation, causing attachment and effacement lesions on gut epithelia. For Listeria, Shigella, Burkholderia, Rickettsia, and Mycobacterium marinum, actin polymerization on the surface of cytoplasmic bacteria results in actin tails, intracellular motility, and direct intercellular spread. Interestingly, these pathogens use independently evolved proteins to target different steps leading to Arp2͞3 activation. Listeria, Rickettsia, and perhaps Burkholderia mimic host cell N-WASP to activate the Arp2͞3 complex directly, and actin tail formation by these species is independent of host cell N-WASP (9-14). The other pathogens depend on the WASP family for actin polymerization: Shigella and enterohemorrhagic E. coli (EHEC) have molecules that directly recruit...

Cdc42 Regulates FcγReceptor-mediated Phagocytosis through the Activation and Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) and Neural-WASP

“…Furthermore, both WASP and N-WASP were activated during phagocytosis in a Cdc42-dependent manner ( Figure 5, B and C). N-WASP is a ubiquitously expressed homologue of WASP that is expressed in macrophages in addition to WASP (Stamm et al, 2005). A role for N-WASP in the internalization of pathogens in nonhematopoietic cells has been reported recently (McGee et al, 2001;Bierne et al, 2005).…”

“…Consistent with results reported using human monocyte-derived macrophages (Lorenzi et al, 2000), BMMs from WASP-deficient mice showed a 50% decrease in phagocytosis ( Figure 3A). The residual phagocytosis observed in the absence of WASP may be due to the presence of N-WASP in macrophages (O'Sullivan et al, 1999;Stamm et al, 2005). To address the role of this WASP homologue, the phagocytic ability of cells in the presence of wiskostatin was assessed.…”

Acting on Actin During Bacterial Infection

“…The actin polymerization system Arp2/3 complex has been manipulated by several pathogens differently. Some mimics the Wiskott-Aldrich syndrome protein (WASP) family [56], while other's recruit WASP directly to activate Arp2/3 [57]. Examples of the first include the actA protein of listeria and RickA of riquetsia.…”

“…Examples of the first include the actA protein of listeria and RickA of riquetsia. For the second examples exist as is the case of IcsA of S. flexneri and nondetermined factors of M. marinum but dependent on the ESAT-6 secretion system 1 [57]. M. marinum is a water-borne bacterium that naturally infects fish and amphibians and is an opportunistic pathogen for humans causing tuberculosis while Rickettsia conorii belongs to the spotted fever group of Rickettsia species transmitted by ticks [55].…”

Interaction of the Mycobacterial Heparin-Binding Hemagglutinin with Actin, as Evidenced by Single-Molecule Force Spectroscopy

“…However, recent reports have indicated that free M. tuberculosis, Mycobacterium marinum, and Mycobacterium leprae can be present in the cytoplasm (27,28,30) and demonstrated that intracellular M. marinum not only enters the cytoplasm of infected macrophages but also has the ability to be propelled by actin-based motility (27,28), as observed for numerous intracellular pathogens, including Listeria monocytogenes, Shigella flexneri, and Rickettsia rickettsii (11,27,28,29). Upon cell invasion, mycobacteria may thus be in contact with cytosolic components, including actin, which may be involved in epithelial cell trafficking.…”