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Title: The Conserved Tarp Actin Binding Domain Is Important for Chlamydial Invasion}
Article:
Abstract:
The translocated actin recruiting phosphoprotein( Tarp) is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.
Introduction:
The obligate intracellular gram negative bacterium, Chlamydia trachomatis, is the most frequently reported sexually transmitted disease in the United States and the leading cause of preventable blindness worldwide[1]. This genetically intractable microorganism undergoes a developmental cycle that involves an extracellular infectious form referred to as an elementary body( EB) and an intracellular replicative reticulate body( RB). EB to RB and RB to EB transitions occur within the protective confines of a membrane bound parasitophorous vacuole termed an inclusion[2]. The exact mechanisms of chlamydial attachment and entry of nonphagocytic cells are unclear, but certain features, such as the recruitment of actin to the site of attachment, are conserved among all chlamydial species examined thus far[3]. Furthermore, drugs such as cytochalasin D that prevent actin polymerization inhibit infection[3]–[7]. Like many intracellular microorganisms, chlamydial pathogenesis is partially mediated by the translocation of secreted effectors at the time of bacterium-host-cell contact[8]–[13]. Recent studies have shown that C. trachomatis directly induces actin polymerization via the type III secreted effector, Tarp[14]. C. trachomatis elementary bodies harbor presynthesized Tarp effector protein which is tyrosine phosphorylated upon translocation to the cytosol of the host cell and has been implicated in the recruitment of actin observed at the site of chlamydial attachment in vivo[9]. Tarp was subsequently shown to polymerize actin independently of host factors in vitro[14]. The mechanism of Tarp actin nucleation appears to be distinct from known eukaryotic actin nucleators[15]. Chlamydia trachomatis L2 Tarp harbors at least three functionally distinct domains; an N-terminal tyrosine-rich repeat domain of unknown function, a proline rich domain required for Tarp oligomerization and a single Wiskott-Aldrich syndrome protein( WASP)-Homology-2( WH2) G-actin binding domain[9],[14]. The proline rich domain and actin binding domain are harbored within the minimum Tarp peptide required for Tarp mediated actin nucleation. Oligomerization mediated by the proline-rich domain presumably brings multiple actin monomers into apposition to nucleate actin filament formation[14]. Sequence analysis of all known Tarp orthologs indicate that the proline rich domain and actin binding alpha helix are conserved although the tyrosine-rich repeat domain is absent from C. caviae, C. muridarum, and C. pneumoniae[16]. Overall, Tarp orthologs show a low level of sequence identity[16], however the actin binding domains appears to be a conserved feature. We demonstrate here that Tarp orthologs from Chlamydia pneumonia, C. caviae, C. muridarum and various C. trachomatis serovars all harbor at least one and up to four functional actin binding domains and that purified recombinant Tarps from all chlamydial species were capable of nucleating actin filament formation in vitro. Furthermore, a peptide antiserum with affinity to the actin binding domain prevented Tarp mediated actin nucleation and, when delivered into host cells, significantly reduced host cell susceptibility to chlamydial infection. Our findings show that Tarp mediated actin polymerization is a conserved feature among diverse chlamydial species and suggest that the actin binding domain plays an important role in bacterial entry.
Discussion:
Many bacterial pathogens modulate actin dynamics of eukaryotic host cells as a virulence mechanism. These pathogenic processes may involve manipulation of the cytoskeleton to promote internalization, inhibition of actin polymerization to prevent phagocytosis, or utilization of actin filamentation to promote intracellular movement and cell-to-cell spread of cytosolic pathogens. In most cases, bacteria that stimulate actin filament formation directly or indirectly do so through the recruitment of host actin nucleating machinery[25]. Recently, it has been recognized that certain bacterial proteins display actin nucleating activity independently of host factors[14],[15],[26]–[28]. The mechanism of chlamydial Tarp actin nucleation appears to be distinct from known eukaryotic or bacterial actin nucleators[14]. C. trachomatis L2 Tarp contains a single WH2 actin binding domain and requires an upstream proline-rich domain for oligomerization, which presumably brings multiple actin monomers into apposition to nucleate actin filament formation[14]. Surprisingly, Tarp of some chlamydial strains and species contains multiple actin binding domains on a single polypeptide thus may utilize a hybrid mechanism of actin nucleation involving binding of multiple actin monomers to a single polypeptide but potentially synergistic with oligomerization mediated by the upstream proline-rich domain. Notably, the actin binding function of Tarp appears to be essential to chlamydial pathogenesis as inhibition of its activity by cytosolic anti-ABD antibody is inhibitory to internalization of EBs. Factors that nucleate actin filament formation do so through the stabilization of at least three actin monomers to overcome the kinetic barrier to nucleation. Eukaryotic cells possess three general classes of actin nucleation factors; the Arp2/3 complex, formins, and Spire[29],[30]. Each of these initiates actin filament formation by distinct mechanisms. The Arp2/3 complex nucleates branched filaments from existing actin filaments[31]. This is in contrast to Tarp, which nucleates linear filaments[14]. Formins nucleate formation of linear actin filaments and possess two characteristic formin homology domains, FH1 and FH2[32],[33], which are not found in Tarp. The Spire protein of Drosophila also nucleates formation of linear actin filaments but does so through a mechanism involving four sequential WH2 domains on a single peptide[24]. More recently, it has been shown that Spire functions in close association with the Drosophila formin, Cappuccino[34]. As additional eukaryotic and bacterial proteins with actin nucleating activity have been discovered, it has become apparent that many of these novel nucleators do not fall clearly into of the above classes but may possess a combination of functional domains to create an actin nucleator employing hybrid mechanisms of actin nucleation[15]. For example, Cordon-Bleu, a vertebrate-specific nucleator important in neuronal development, contains three WH2 domains and six proline-rich domains[35]. Leiomodin-2, another recently recognized nucleator found in heart and skeletal muscle, is characterized by an N-terminal actin binding helix similar to that of tropomodulins, a leucine-rich repeat domain that binds actin, and a C-terminal WH2 domain[36]. Bacterial proteins with actin nucleating activity include Salmonella SipC[26], Vibrio VopF[28], and VopL[27], and chlamydial Tarp[14]. Although the mechanisms of actin nucleation differ, all but SipC share a common theme of the presence of one or more WASP Homology 2( WH2) actin binding domains[15]. VopF and VopL each contain three WH2 domains and two proline-rich domains with the most N-terminal of these showing similarity to formin homology domain 1( FH1)[27],[28]. Chlamydial Tarp shows no homology to formins but, as we demonstrate here, possesses a variable number of WH2 domains. Comparison of the sequenced Tarp orthologs reveals a surprising diversity of isomers. C. trachomatis L2 Tarp contains six tyrosine-rich repeats while serovars D and A contain only three[9],[18],. Chlamydia pneumoniae, C. caviae, C. muridarum Tarp proteins lack the tyrosine-rich repeat domain and are not tyrosine phosphorylated[16]. Sequence analysis of all known Tarp orthologs indicates that the proline rich domain and actin binding alpha helix are conserved[16]. However, some Tarp orthologs harbor multiple actin binding domains. All Tarp orthologs examined harbored at least one actin binding domain that associated with actin derived from HeLa cell extracts. Furthermore, all Tarp orthologs increased initial rates of actin polymerization in vitro compared to actin alone and GST controls. Surprisingly, the C. caviae and C. trachomatis serovars A and D Tarp orthologs contained multiple functional actin binding domains suggesting a spire-like actin nucleation mechanism involving multiple WH2 domains on a single peptide may be employed by some Tarp orthologs. The presence of multiple ABDs could potentially allow one Tarp protein to align multiple actin monomers together to initiate an actin filament. While Tarp fragments harboring multiple actin binding domains nucleated actin in vitro even in the absence of the proline rich oligomerization domain, this domain appears to be conserved thus the proline-rich domain likely plays an essential role in promoting actin nucleation in vivo.. The biological advantage to strains bearing different numbers of Tarp WH2 domains is unclear. Indeed, the extent of variation within known C. trachomatis serovars or between clinical isolates is unknown. Intrinsic differences in Tarp structure are perhaps not surprising given that chlamydiae differ both clinically and biologically, demonstrating diverse tissue tropisms and varying degrees of localized and systemic infection[1],[37]. To confirm the role of the Tarp effector in vivo, an antibody was raised to a peptide overlapping the C. trachomatis L2 Tarp WH2 actin binding domain.. Antibody specific for the Tarp actin binding domain completely neutralized Tarp mediated actin nucleation in vitro. Furthermore, delivery of the ABD serum into host cells prior to a chlamydial infection significantly reduced the number of invasive bacteria. These data suggest that intracellular ABD antibody is capable of neutralizing the Tarp actin nucleating activity following translocation into the host but prior to bacterial entry. Defining essential virulence determinants in pathogens like chlamydiae that lack tractable genetic systems is often difficult. The inhibition of entry by intracellular antibody to a functional domain implies a role for Tarp as a virulence determinant of chlamydiae. In addition to Tarp, chlamydial entry is also dependent on signal transduction cascades initiated following activation of host Rho family GTPases culminating in the activation of host cell Arp2/3 complex[38]–[41]. Because host cell Arp2/3 complex is also required for C. trachomatis invasion, we have proposed that the chlamydial and cellular actin nucleating activities function in concert to promote chlamydial invasion[14],[40]. Tarp nucleates the formation of linear actin filaments[14], whereas host Arp2/3 nucleates actin filaments that grow from the side of existing linear filaments, thus the two mechanisms may function synergistically. Although the role of phosphorylated Tarp in entry and the recruitment of Arp2/3 is somewhat controversial[16],[17],, Tarp proteins secreted from C. pneumoniae and C. caviae are not phosphorylated and therefore presumably either do not activate the Arp2/3 complex or activate Arp2/3 by an alternate signaling pathway. Furthermore, inhibition of C. trachomatis L2 Tarp tyrosine phosphorylation had no adverse effect on entry[17]. The signaling cascades initiated by the different chlamydiae appear to be species specific. C. trachomatis invasion requires the Rac GTPase while C. caviae requires both Rac and Cdc42[39],[41]. Potentially related to the biodiversity exhibited by various chlamydial species, Tarp orthologs harbor domains that are species specific but variable in number such as the tyrosine rich repeat domain and conserved domains like the actin binding alpha helical domain that also varies in number[16]. Despite the observed differences in phosphorylation of the tyrosine rich repeat domain, the actin binding domain identified in all Tarp orthologs examined is conserved and a testament to the significance of this domain in chlamydial biology. The Tarp effectors from various chlamydial species harbor distinct and conserved features such as the tyrosine rich repeat domain and the actin binding domain, respectively. In this study we demonstrate that all Tarp orthologs examined share the ability to bind and nucleate actin directly. This conserved feature is required for efficient invasion of host cells. Further elucidation of how Tarp and the Arp2/3 complex cooperate to produce a successful invasion is required to fully characterize the chlamydial entry mechanism. Careful analysis of the unique features of various Tarp orthologs may provide insight into differences observed within chlamydial species regarding tissue tropism and dissemination. Taken together our findings suggest that the type III secreted effector protein Tarp plays a vital role in chlamydial entry of human cells.
Chlamydiae are bacterial obligate intracellular pathogens responsible for multiple human and veterinary diseases . The induction of cytoskeletal rearrangements to promote chlamydial internalization is partially mediated by a type III secreted effector protein called Tarp that is translocated upon contact with host cells and independently nucleates actin filament formation . Tarp from a C . trachomatis lymphogranuloma venereum ( LGV ) strain consists of a tyrosine-rich repeat domain , a proline-rich domain required for oligomerization , and a single actin binding domain . Oligomerization is required to bring multiple actin monomers together to initiate actin filament formation by a mechanism distinct from host actin nucleators . Here we have examined Tarp from several other strains of chlamydiae and find that certain of these contain up to four actin binding domains . Tarp fragments bearing multiple actin binding domains nucleate actin in in vitro assays even in the absence of the oligomerization domain . This suggests that Tarp from different chlamydial species may utilize hybrid mechanisms to induce actin nucleation . Determination of virulence determinants in chlamydiae is challenging due to the lack of tractable genetic systems . The direct introduction of anti-Tarp actin binding domain antibodies into the cytosol of host cells inhibited entry and thus demonstrates an essential role for Tarp in chlamydial pathogenesis .