Listeria surface (Theriot et al., 1994; Welch et al., 1997). A host factor sufficient for actin assembly was purified and identified as the Arp2/3 complex. Arp2/3 had previously been identified in amoebae, and was proposed to function in actin nucleation (Machesky et al., 1994), although no activity was detected in in vitro actin assembly assays (Kelleher et al., 1995). The Arp2/3 complex has since been shown to be necessary for Listeria actin assembly (Loisel et al., 1999; May et al., 1999; Yarar et al., 1999). Notably, although Arp2/3 could promote the assembly of actin by bacteria, it was not sufficient to enable motility, indicating that additional host components were required for full reconstitution of motility (Welch et al., 1997). Moreover, actin polymerization by Arp2/3 complex at the Listeria surface required ActA (Welch et al., 1997), consistent with the essential nature of ActA in actin assembly during infection.
The fact that actin assembly by Listeria requires both ActA and Arp2/3 complex suggested that these factors act together to nucleate actin assembly. Subsequent experiments using purified ActA and Arp2/3 complex demonstrated that, although neither factor alone was sufficient, together the proteins formed an efficient nucleator (Welch et al., 1998). Based on the subunit composition of Arp2/3 complex and the presence of actin related proteins Arp2 and Arp3, it was proposed that ActA is an activator or NPF for Arp2/3. Subsequent work showed that ActA was indeed the first identified member of a broad class of NPF proteins, which are characterized by the presence of actin-binding WH2 domains (W), along with Arp2/3-binding C and A motifs (collectively called WCA) (Campellone and Welch, 2010). The WCA domain is the minimal region of NPF proteins that stimulates Arp2/3-dependent actin nucleation. The NPF family also includes other pathogen proteins such as baculovirus p78/83 (see later), Rickettsia spp. RickA (Gouin et al., 2004; Jeng et al., 2004) and Burkholderia thailandensis BimA (Sitthidet et al., 2010) (Figure 4). Thus, expressing proteins that mimic NPFs is a conserved mechanism of pathogenesis, and studying how pathogens deploy their NPFs will shed light on both pathogenic strategies as well as the function and regulation of actin assembly in uninfected cells.
It is noteworthy that Listeria actin-based motility appears to occur largely independently of regulation by host signaling pathways (tyrosine kinases and GTPases) that control actin assembly (Ebel et al., 1999; Marchand et al., 1995). However, it has been shown that the serine-threonine kinase CK2 phosphorylates ActA, enhancing Arp2/3 binding and Listeria motility, similar to the function for CK2 in phosphorylating host NPFs WASP and WAVE (Chong et al., 2009). Notably, despite the activity of CK2, bacterially expressed and purified ActA is active (Skoble et al., 2000; Welch et al., 1998). Thus, Listeria has evolved the ability to bypass the requirement for many host cell actin regulatory pathways, which differs from the behavior of other pathogens including Shigella and vaccinia virus.