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Intracellular MRSA (Phosphorylation dynamics in S.aureus (Hochgräfe et al,…

- - - - Only minor changes in protien levels were observed but phosphorylation events showed dynamic alterations mainly in the pathways associated with cytoskeletal dynamics, cell-cell matrix contacts and GTPase signalling
        
        Methods include: analysis of host protein kinases by substrate mapping, active regulatory site immunoblotting and predicition algorith highlighted known and novel host kinases with putative critical roles in S. aureus infection and targeted pharmacological inhibitoion of these kinases
        
        Methods also involve creating immortal cells but introducing a viral vector , must wonder if this will affect the integrity of the cells.
    - - increased activation of FAK and ERK1/2 was confirmed by western blot
    - - Altered levels of CDC42
      - Altered levels of Rho (RhoA, CDC42, Rac)- cytoskeletal dynamics
  - - - De,pmstrated in macrophages, Brucella / Salmonella #
  - - - Bacterial pathogens have devised mutliple ways to interfere with NFkB signalling
  - - - Receptor activation leads to recruitment of FAK which then recuits cytoplasmic compounds of the receptor linked signalling complex including the tyrosine kinase scr, the adaptor protein Crk and P130Cas (Crk subastances). #
        
        Crk phosphorylation leads to strong activation of the small GTPase Rac, which is required for cell motility #
        
        Src phosphorylation triggers the association of PL3K with the siganlling complex
        
        PI3L phosphorylates PIP2-->PIP3
        
        Activated PIP 3 recruits Ser /Thr kinase Akt (PKB) to the membrane via its pleckstring homology domain which binds to PIP with high affinity ( Harlam et al, 1994) . Akt regulates cell migration, metabolism, proliferation and survival.
      - 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
      - Activation of PI3K can also activate small GTPases such as Sas and Rac which are linked to proliferation and cytoskeletal dynamics respectively ( Reedijke et al 1990, Pleiman et al 1993)
        
        Can also activate cdc42 which activates N-wasp
    - - Ibrutinib inhibits BTK and possibly TEC in platelets
        
        Side effect of treatment = bleeding
      - Quek LS, Bolen J, Watson SP. A role for Bruton’s tyrosine kinase (Btk) in platelet activation by collagen. Current Biol 1998;8: 1137–40.
        79 Oda A, Ikeda Y, Ochs HD et al. Rapid tyrosine phosphorylation and activation of Bruton’s tyrosine/Tec kinases in platelets induced by collagen
        
        BTK and Tec expressed by platelets are both involved in collagen activation
        
        Atkinson BT, Ellmeier W, Watson SP. Tec regulates platelet activation by GPVI in the absence of Btk. Blood 2003;102:3592– 9., Oda A, Ikeda Y, Ochs HD et al. Rapid tyrosine phosphorylation and activation of Bruton’s tyrosine/Tec kinases in platelets induced by collagen binding or CD32 cross-linking. Blood 2000;95:1663–70.
        80
        
        binding og collagen to the receptor BTK or Tec leads to P13K phosphiorylation
        
        Atkinson BT, Ellmeier W, Watson SP. Tec regulates platelet activation by GPVI in the absence of Btk. Blood 2003;102:3592–
        9.
        Quek LS, Bolen J, Watson SP. A role for Bruton’s tyrosine kinase (Btk) in platelet activation by collagen. Current Biol 1998;8: 1137–40.
        
        Following activation of P13K and they in turn activate PLC2
        
        Autophosphorylation of BTK and phosphorylation of PLCc2 in response to collagen are both reduced in ibrutinib-treated platelets [20].
        
        Levade M, David E, Garcia C et al. Ibrutinib treatment affects collagen and von Willebrand factor-dependent platelet functions. Blood 2014;124:3991–5.
- - - - Filament branching is typically mediated by the Arp 2/3 complex
    - - 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
    - - 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.
  - - - Shigella flexneri recruits host N-WASP to its surface causing the formation of comet tails that push it through the host cytosplasm
      - Listeria uses its own protein ActA to activate the Arp2/3 complex
  - - - Cytosol replaced by mixture of purified products
    - - Using artifical objects such as beads to mimic the bacteria
    - - Beads or lipid vesicles coated with ActA
    - - Beads could also be coated with NPF such as N-WASP
    - - Pulling gently on the load object actually accelerates actin network growth
    - - Systematic Mutagenisis and the discovery that WH 2 is the imporant component for
    - - CONFLICT: Arp 2/3 as mediator of actin attachment. Hanein's structural ecvidence shows that NPF does not remain attached after bracnch formation.
- - - - BTK is responsible for activating WASP by activating VAC, a guanine nucleotide exchnage factor for Cdc42, stimulating production of PIP and inducing the phosphorylation of WASP
- - - - additional factors beyond Arp2/3 and NPFs were required for motility, as purified Arp2/3 only promoted actin assembly but not movement (Welch et al., 1997
    - - Resolving these hypotheses will await further experimentation. The remaining essential factor, ADF/cofilin, severs and depolymerizes actin filaments, enabling monomer recycling for further polymerization (Carlier et al., 1997; Rosenblatt et al., 1997). Thus, motility requires factors that both enhance actin assembly and disassembly.
        Although
    - - Profilin is thought to promote filament elongation at barbed ends through several mechanisms including enhancing ADP/ATP exchange, displacing monomers from sequestering proteins, and enhancing the local concentration of actin monomers available for assembly. Moreover, it has been shown to be important for enhancing actin elongation during Listeria motility (Grenklo et al., 2003). Ena/ VASP proteins, which specifically bind to the proline-rich repeat regions of ActA (but not IcsA or N-WASP), enhance motility by recruiting profilin (Auerbuch et al., 2003; Geese et al., 2002) (Figure 4) and by enabling processive barbed end elongation while antagonizing the activity of capping proteins (Breitsprecher et al., 2011; Hansen and Mullins, 2010). These factors synergize with Arp2/3 to enable rapid filament elongation and actin-based motility.
        
        The reconstitution of actin-based bacterial motility demonstrates that the process is driven by a core set of proteins that regulate actin assembly dynamics. It also provides insights into the basic biochemical mechanisms that underlie host processes like lamellipodia protrusion during cell migration, which are driven by the same set of factors (Campellone and Welch, 2010). The reconstitution approach also highlights the fact that Listeria and Shigella motility rely on a short list of components, whereas the corresponding processes in host cells are more complex. Because they represent a stripped-down system, these pathogens have served as very useful models for cell biologists and biophysicists to study the basic mechanisms that control actin-based movement. Future studies will continue to make use of pathogens to study the biochemical basis of actin-based movement and how the biochemical properties of the system enable force generation to drive motility.
        Pathogen
- - - - 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
    - - Btk depletion led to a striking decrease in the number of bacteria displaying actin-based motility in HT-29 cells. The
        
        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
  - - - 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