Molecules involves are highly complex and dynamic in relation to their environment

The Arp 2/3 complex binds to the side of an exisiting mother filament when activated and nucleates the polymerisation of a new daughter filament

Continuous extension is thought to occur through filament nucleation at the leading edge, balanced by depolymierisation at the posterior zone.

Actin assembly and dissembly occurs throughout the whole structure

Filament branching is typically mediated by the Arp 2/3 complex

Nucelation is enhanced at the leading edge by specific NPFs such as proteins of the WASP/ WAVE family which are capable of activating the Arp 2/3 residue on the leading edge membrane.

Biochemical dissection of actin based protrusion machinary has been greatly facilitated by the existence of motile intracellular pathogens that hijack host cell actin systems to propel themselves by leading edge machinary

Accumulating evidence indicates that S. aureus is a facultative intracellular pathogen that can
invade and survive in different types of non-professional phagocytic cells, such as airway epithelial cells

**Garzoni, C.; Kelley, W. L., Staphylococcus aureus: new evidence for intracellular persistence. Trends in microbiology 2009, 17, 59-65.

6. Sendi, P.; Proctor, R. A., Staphylococcus aureus as an intracellular pathogen: the role of small colony variants. Trends in microbiology 2009, 17, 54-8.
7. Loffler, B.; Tuchscherr, L.; Niemann, S.; Peters, G., Staphylococcus aureus persistence in non-professional phagocytes. International journal of medical microbiology : IJMM 2014, 304, 170-6.**

n the ongoing evolutionary arms race between pathogens and their hosts, pathogens tend to “think big,” generally targeting path- ways that control important cellular functions like cytoskeletal dynamics

estimated that roughly 30% of all cellular proteins may be modified by protein kinase activity #

kinases give rise to a vast network of interwoven signaling pathways, and the sheer complexity of eukaryotic kinase signaling networks still baffles scientists, biochemists, and system biologists alike

In many cases seemingly discreet signalling pathways are communicating but we lack deeper knowledge based on this

It is an increasingly popular strategy to attempt to understand complex eukaryotic pathways by studying the mechanisms that pathogens use to subvert them.

This pathway leading to the recruitment of FAK is targeted by many pathogens including S.aureus (Sinha, 2012). the pathogen will either reinforce of destroy the focal adhesion

Fibronectin (Fn) is a multidomain glycoprotein found in blood plasma, other bodily fluids and the extra-cellular matrix (ECM) and serves as a natural ligand for several integrins including α5β1, αvβ3, and α4β1Leiss M, Beckmann K, Giros A, Costell M, Fassler R. The role of integrin binding sites in fibronectin matrix assembly in vivo. Curr Opin Cell Biol. 2008; 20: 502–507. doi: 10.1016/j.ceb.2008.06.001 PMID: 18586094
2.

Adherence ofmicrobes to host tissues is a hallmark of infectious disease and is often medi- ated by a class of adhesins termedMSCRAMMs (Microbial Surface Components Recogniz- ing AdhesiveMatrixMolecules)

integrin- initiated invasion of S. aureus critically depends on additional host cell factors and we have identified cellular Src PTKs to be essential for this process.

Kim AS, Kakalis LT, Abdul-Manan N, Liu GA, Rosen MK. 2000. Autoinhibition and activation mechanisms of the Wiskott-Aldrich syn- drome protein. Nature 404:151–158.

Rohatgi R, Ma L, Miki H, Lopez M, Kirchhausen T, Takenawa T, Kirschner MW. 1999. The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97:221– 231.
15.

Many unrelated bacterial and viral pathogens mobilize the Arp2/3 complex to nucleate actin by mimicking or exploiting molecules ranging from tyrosine kinase substrates to NPFs, proving the flexibility of evolutionary strategies to hijack the cytoskeleton (Haglund and Welch, 2011)

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.
Shigella

Welch and Way Page 6
motility before it was demonstrated to be an NPF for Arp2/3, and thus studies with Shigella were among the first to implicate N-WASP in actin nucleation.
The mechanism of N-WASP recruitment to Shigella involves direct binding to the IcsA protein. In particular, the glycine-rich repeats of IcsA, which are implicated in actin assembly, bind N-WASP in vitro (Suzuki et al., 1998) (Figure 4). In an influential study, it was shown that IcsA binding causes N-WASP to shift from an inactive auto-inhibited conformation to an open state where the WCA domain of N-WASP activates Arp2/3 complex (Egile et al., 1999) (Figure 4). Consistent with this, Arp2/3 complex was required for Shigella motility. This finding was remarkable because it built on a contemporary report
that the host signaling molecules Cdc42 and PIP2 also bind to N-WASP and cause a change from an inactive to an active NPF (Rohatgi et al., 1999). Thus, it appears that Shigella IcsA evolved as a mimic of host signaling pathways that recruit and activate N-WASP, in contrast with Listeria ActA, which mimics activated N-WASP.
IcsA is not sufficient to activate N-WASP in host cells entirely independently of host signaling proteins or pathways. It has been reported that the activities of Abl kinase (Burton et al., 2005) and Bruton’s tyrosine kinase (Btk) (Dragoi et al., 2013) are important for N- WASP phosphorylation and Shigella motility. N-WASP activation and actin assembly by Shigella also requires Toca-1 (Leung et al., 2008), a cellular cofactor needed for N-WASP
activation by Cdc42 and PIP2 (Ho et al., 2004) (Figure 4). However, N-WASP activation is independent of other cellular factors needed for N-WASP activity including Cdc42 or the WASP-interacting protein (WIP) (Moreau et al., 2000). Thus, IcsA bypasses the need for
Cdc42 and PIP2 in N-WASP activation, but not the requirement for other N-WASP activating inputs.
Reconstitu

The effect on actin tail formation was matched by a decrease in the number of bacteria displaying N-WASP recruit- ment

Altogether, our results support the notion that, in HT-29 intestinal cells, S. flexneri actin-based motility is facilitated by the Btk-mediated phosphorylation of N-WASP on Y256, which sup- ports N-WASP recruitment to the bacterial surface and the effi- ciency of actin tail formation

To our knowledge, this is the first report of a role for Btk-mediated phosphorylation of N-WASP in a nonhematopoietic cell type. Interestingly, we observed a striking increase in Btk-dependent N-WASP phosphorylation upon S. flexneri infection, suggesting that the infection process modulates Btk activity