Rickettsia Sca2 has evolved formin-like activity through a different molecular mechanism

Abstract

Sca2 (surface cell antigen 2) is the only bacterial protein known to promote both actin filament nucleation and profilin-dependent elongation, mimicking eukaryotic formins to assemble actin comet tails for Rickettsia motility. We show that Sca2's functional mimicry of formins is achieved through a unique mechanism. Unlike formins, Sca2 is monomeric, but has N-and C-terminal repeat domains (NRD and CRD) that interact with each other for processive barbed-end elongation. The crystal structure of NRD reveals a previously undescribed fold, consisting of helix-loop-helix repeats arranged into an overall crescent shape. CRD is predicted to share this fold and might form together with NRD, a doughnut-shaped formin-like structure. In between NRD and CRD, proline-rich sequences mediate the incorporation of profilin-actin for elongation, and WASP-homology 2 (WH2) domains recruit actin monomers for nucleation. Sca2's α-helical fold is unusual among Gram-negative autotransporters, which overwhelmingly fold as β-solenoids. Rickettsia has therefore "rediscovered" formin-like actin nucleation and elongation.passenger domain | translocator domain | spotted fever M any bacterial pathogens use the actin cytoskeleton of host eukaryotic cells for invasion and motility (1, 2). In so doing, bacteria often resort to mimicry by expressing proteins that adopt core functions of key actin cytoskeletal components, particularly actin filament nucleation and elongation factors. However, bacterial proteins tend to bypass the elaborate regulatory networks characteristic of their eukaryotic counterparts, offering a rare opportunity to dissect their functions within a simplified system (2, 3), with implications for our understanding of pathogenicity and the eukaryotic actin cytoskeleton alike.Rickettsiae are obligate intracellular Gram-negative pathogens that are transmitted to humans via arthropod vectors, such as ticks, fleas, and lice (4). Rickettsia species are responsible for a number of severe human diseases, including typhus and spotted fever (5). The spotted fever group, including Rickettsia parkeri, Rickettsia conorii, Rickettsia rickettsii, and over 20 other species throughout the world, uses the host-cell actin cytoskeleton to spread inter-and intracellularly. Similar to Listeria and Shigella, Rickettsia forms actin comet tails to propel its movement. However, the actin tails of Rickettsia consist of long and unbranched actin filaments, whereas those of Listeria and Shigella contain shorter and densely branched filaments (6, 7). These morphological differences stem from different molecular mechanisms for comet tail formation by these pathogens. Listeria and Shigella rely heavily on the activity of the host Arp2/3 complex that localizes uniformly along their tails (6). Although the Arp2/3 complex, activated by either host nucleation promoting factors (8) or the Rickettsia surface protein RickA (9, 10), is necessary for Rickettsia invasion (11), it is absent from Rickettsia tails (6). Another protein, Sca2 (surface cell antigen 2), has ...

Actin Dynamics, Architecture, and Mechanics in Cell Motility

“…Reconstitution in vitro of bacterial and virus motility demonstrates that the minimum machinery for pathogen motility consists of actin, the Arp2/3 complex, and a capping factor with profilin and ADF/cofilin to ensure actin monomer recycling (91,181). This minimum machinery has recently been extended to include formins since Ricketssia Sca2, a bacterial-formin-like protein, drives the motility of this bacteria, taking the place of the Arp2/3 complex as nucleator (108,188). Interestingly, another formin (INF2) localized at the endoplasmic reticulum of mammalian cells is reported to modulate mitochondria fission and mutations in INF2 can result in Charcot-Marie-Tooth neuropathy (157).…”

The WH2 Domain and Actin Nucleation: Necessary but Insufficient

“…Sca2 has a central repeat of three WH2 domains ( Figure 1 ), but this repeat is only partially responsible for actin assembly by Sca2. In what could be considered an evolutionary tour de force, Sca2 combines properties of both tandem WH2 domain-based nucleators and formins into a new fold, suitable for passage across the narrow pore formed by its translocator domain in the outer membrane of the bacterium [39]. Unlike formins, Sca2 is monomeric, but has N- and C-terminal repeat domains (NRD and CRD) that interact with each other for processive barbed-end elongation.…”

“…The WH2 repeat, which is located in between the two PRDs, sequesters actin monomers when in isolation, analogous to Cobl's repeat [30]. However, disabling the WH2 domain through mutagenesis shows that within the full-length protein the WH2 repeat is essential for nucleation, although it does not participate in elongation [39]. Although the existing evidence allows to propose a tentative model of Sca2's nucleation and elongation mechanism ( Figure 3 ), several features of this model remain untested and should inspire further studies on this protein (see Outstanding Questions).…”

Secretome of obligate intracellularRickettsia

“…The basis for this formin mimicry was recently discovered, as a crystal structure of the R . conorii formin homology 2 (FH2) domain revealed a crescent-like fold similar to half of the dimeric structure of formin (Madasu et al ., 2013) . Together with a similar structure in the CT region of the Sca2 passenger, a doughnut-shaped structure analogous to formins is predicted to elongate actin, with intervening Pro-rich regions (PRRs) and Wiskott-Aldrich syndrome protein (WASP) homology 2 (WH2) domains incorporating profilin-actin for elongation and recruiting actin monomers for nucleation, respectively (Madasu et al ., 2013) .…”

“…conorii formin homology 2 (FH2) domain revealed a crescent-like fold similar to half of the dimeric structure of formin (Madasu et al ., 2013) . Together with a similar structure in the CT region of the Sca2 passenger, a doughnut-shaped structure analogous to formins is predicted to elongate actin, with intervening Pro-rich regions (PRRs) and Wiskott-Aldrich syndrome protein (WASP) homology 2 (WH2) domains incorporating profilin-actin for elongation and recruiting actin monomers for nucleation, respectively (Madasu et al ., 2013) . Importantly, while conserved in most spotted fever group (SFG) rickettsiae Sca2 proteins, the FH2 domains contain deletions or are entirely absent in Sca2 proteins of rickettsial species that either form minimal actin tails ( R. typhi ) or entirely lack actin-based motility (ABM) ( R .…”