The Role of Actin Depolymerizing Factor in Regulating Actin Dynamics in Toxoplasma gondii

Apicomplexan parasites utilize a unique process of rapid motility termed gliding, which is coupled to their invasion of host cells. Gliding and invasion are dependent on parasite actin filaments, yet parasite actin is mostly non-filamentous. Filaments have been detected only transiently during gliding, suggesting that parasite actin filaments are rapidly assembled and disassembled during gliding motility. Little is known about what regulates the turnover of parasite actin filaments. In higher eukaryotes the Actin Depolymerizing Factor: ADF)/Cofilin proteins are essential regulators of actin filament turnover. ADF is one of the few actin binding proteins conserved in apicomplexan parasites. To investigate the role of ADF in regulating actin dynamics in apicomplexan parasites, Toxoplasma gondii was used as a model apicomplexan, and the activities of T. gondii ADF: TgADF) were analyzed in vitro and in vivo. We found that TgADF engaged in dual activities. In contrast to most ADF/Cofilin proteins, TgADF was found to be a potent actin monomer sequestering protein that strongly inhibited actin polymerization, suggesting that it likely functions to maintain high G-actin concentrations in the cytoplasm of non-motile parasites. This role was reflected in its molecular structure, in which conserved G-actin binding sites were maintained, while key F-actin binding residues were absent. Despite this, TgADF demonstrated the ability to promote actin filament disassembly via a severing mechanism. Using a conditional knockout system we examined the function of TgADF in the parasite. TgADF was found to be essential for controlling productive gliding motility, and its absence lead to defects in host cell invasion, parasite egress, and parasite dispersal. Detailed analysis of motility revealed that parasites were unable to engage in sustained helical gliding, and moved at markedly reduced speeds. These defects are predicted to arise from the presence of more stable actin filaments in the parasite. Overall both the monomer sequestering and filament severing activities of TgADF are predicted to serve important functions in vivo for maintaining high G-actin concentrations for rapid filament assembly, and disassembling actin filaments for rapid filament turnover, respectively. These studies demonstrated that ADF is essential for regulating actin dynamics in T. gondii

Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

Actin-based motility is vital for host cell invasion by protozoan parasites such as Toxoplasma, which provides a model for studying actin-based motility in parasites. Our study reveals that, in addition to intrinsic differences in actin dynamics, regulatory proteins like actin depolymerizing factor are required to regulate this process in vivo

<i>Mycobacterium tuberculosis</i> universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth

<div><p>We have previously shown that the <i>Mycobacterium tuberculosis</i> universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623_WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623_T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an <i>M</i>. <i>tuberculosis Rv2623</i> null mutant (Δ<i>Rv2623</i>) displays enhanced expression of phosphatidyl-<i>myo</i>-inositol mannosides (PIMs), while the Δ<i>Rv1747</i> mutant expresses decreased levels of PIMs. Animal studies have previously shown that Δ<i>Rv2623</i> is hypervirulent, while Δ<i>Rv1747</i> is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing <i>in vivo</i> growth phenotypes of Δ<i>Rv2623</i> and Δ<i>Rv1747</i>, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of <i>M</i>. <i>tuberculosis in vivo</i>.</p></div

Rv2623 is post-translationally modified and phosphorylated at T237.

<p><b>(A)</b> Immunoblot of 2D-gel electrophoretically-resolved BCG lysates with anti-Rv2623 monoclonal antibody revealed three isoforms with differing isoelectric pH values, thus providing evidence for post-translational modification of the USP; <b>(B)</b> Dot-blot analysis of affinity-purified Rv2623 from <i>M</i>. <i>smegmatis</i> mc²155 (Top panel); <i>M</i>. <i>tuberculosis</i> Erdman (Mid panel), and <i>M</i>. <i>bovis</i> BCG (Bottom panel) demonstrating immunoreactivity with an anti-pT antibody. “+” indicate positive pT control; soy bean trypsin inhibitor serves as negative controls (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.s010" target="_blank">S9 Fig</a>). <b>(C)</b> Mass spectrometry-based phosphomapping of Rv2623. The sequence coverage of the protein was ~90%. Manual examination of the appropriate MS/MS spectra was conducted to verify the phosphopeptides identified via software programs. The graph shown represents MS/MS spectra of m/z 507.24(2+) corresponding to peptide spanning amino acid residues 231–238 of Rv2623. Labelled are b- and y- fragment ions produced from Collisional Induced Dissociation (CID) in an LTQ-Orbitrap Elite LC MS/MS instrument. Phosphorylation on T237 of Rv2623 was determined based on the MS/MS fragmentation patterns; in particular are the observed loss of phosphoric acid (indicated as "y(<i>n</i>)-98Da") for fragment ions y(3), y(4), y(5), y(6), y(7), and b(7) under CID conditions [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref076" target="_blank">76</a>]. The "^<b>++</b>" sign indicates that the assigned fragment ion is doubly charged. The graph shown represents results derived from analysis of <i>in vitro</i> phosphorylated <i>M smegmatis-</i>expressed recombinant Rv2623. Analysis of vitro phosphorylated <i>E</i>. <i>coli-</i>expressed recombinant Rv2623 yielded the same results. <b>(D)</b> Molecular docking was employed to depict the interaction of the pTRVV-containing FHA domain-binding motif of Rv2623 with Rv1747 FHA I [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref038" target="_blank">38</a>] (<a href="https://life.bsc.es/servlet/pydock/home/" target="_blank">https://life.bsc.es/servlet/pydock/home/</a>). This was performed with the [amino acids 152 to 294] domain of Rv2623 (with T237 phosphorylated) on the [amino acids 1 to 90] domain of Rv1747 FHA I, using PyDockWeb, with the d(N64-V240) constrained. The phosphate was built into T237 by the PyTMs plugin in Pymol prior to the docking. The PDB code of Rv2623 is 3cis. The structure of the FHA domain of Rv1747 is taken from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.g002" target="_blank">Fig 2</a> (B and C). The Threonine residue and the pT+3 residues in the binding motifs of Rv2623 are labeled in yellow and cyan, respectively.</p

The Rv2623 threonine residues.

<p><b>(A)</b> The solvent accessible surface (SAS) of threonine residues and the corresponding OH group in each monomer of the dimeric Rv2623 was calculated based on the crystal structure of the USP (PDB ID 3CIS) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref006" target="_blank">6</a>] using AREAIMOL program from CCP4 suite [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref035" target="_blank">35</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref036" target="_blank">36</a>]. Among the 9 Thr residues, 5 have solvent accessible hydroxyl oxygen atoms (Shaded: T90, T103, T212, T237, T280). T90, T103, T212, and T237 also have a previously reported phosphorylation motif concerning the pT+3 residues (bracketed): pTXX(S) for T90, pTXX(D) for T103, pTXX(M) for T212, and pTXX(V) for T237 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref024" target="_blank">24</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref032" target="_blank">32</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref033" target="_blank">33</a>]. KAPP: kinase associated protein phosphatase of <i>Arabidopsis thaliana</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref072" target="_blank">72</a>]; KIAA: also known as KIAA0710/NFBD1 (nuclear factor BRCT domain 1) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref073" target="_blank">73</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref074" target="_blank">74</a>]; CDS-1: Checkpoint DNA synthesis protein kinase [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref075" target="_blank">75</a>]; Y127_MYCTU: Cy1A11.16C [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref032" target="_blank">32</a>], <i>M</i>. <i>tuberculosis</i> GarA [tuberculist.epfl.ch]. <b>(B)</b> The surface of the Rv2623 protein is displayed using PYMOL (<a href="http://www.pymol.org/" target="_blank">www.pymol.org</a>). The subunits A and B are colored in blue cyan and green respectively. The solvent accessible Threonine residues (T90, T103, T212, T237) are colored in yellow. <b>(C)</b> PYMOL display of a ribbon representation of an Rv2623 monomer based on previously solved structure [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref006" target="_blank">6</a>] depicting the position of the four solvent accessible threonine residues with the corresponding pT+3 residues that have been shown to promote interaction with FHA domains [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref024" target="_blank">24</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref032" target="_blank">32</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref033" target="_blank">33</a>]. The threonine and the pT+3 residues are labeled in yellow and cyan, respectively.</p

<i>M</i>. <i>tuberculosis</i> Rv2623 interacts with Rv1747.

<p><b>(A)</b> The primary structure of Rv1747 with 2 FHA domains (Orange: FHA I & FHA II); elements typical of ABC transporters: NBD (<u><b><i>n</i></b></u>ucleoside-<u><b><i>b</i></b></u>inding <u><b><i>d</i></b></u>omain; 1–559 amino acids; Walker A&B (Yellow; the ATP-binding domain), and transmembrane domain (Blue bars: transmembrane helices). <b>(B)</b> The GAL4-based Matchmaker Gold Yeast Two-Hybrid system was used to identify interacting partners of Rv2623, which was cloned into the pGBKT7 vector as a fusion to the GAL4 DNA-binding domain (pGBKT7::Rv2623). A DNA library of <i>M</i>. <i>tuberculosis</i> Erdman prey proteins were expressed as fusions to the Gal4 activation domain using pGADT7AD. The screen revealed that Rv2623 interacts with the N-terminal FHA I domain of Rv1747. Analysis of a re-cloned full-length Rv1747 FHA I domain validated the interaction (B, bottom panel: pGBKT7::Rv2623/pGADT7AD::FHA I). Rv2623 dimerization was exploited to serve as positive control (B, top panel; pGBKT7::Rv2623/pGADT7AD::Rv2623). pGBKT7::Rv2623/pGADT7AD and pGBKT7/pGADT7AD::FHA I represent negative controls. The interaction was further confirmed by affinity chromatography study <b>(C)</b>. Purified histidine (His₆)-tagged Rv2623 (Rv2623) and FLAG-tagged FHA I (FHA I: first 100 amino acids of Rv1747) were expressed in <i>M</i>. <i>smegmatis</i> mc²155. Purified FLAG-tagged FHA I was passed over columns with or without Rv2623 immobilized onto the Nickel (Ni)-NTA resin. Western analyses of the appropriate elution fractions using anti-Rv2623 and anti-FLAG antibodies revealed that Rv2623 and Rv1747 FHA I co-eluted—upper and lower panels of lane 3 represent the results of probing eluents from column containing both (Ni)-NTA resin-immobilized (His₆)-tagged Rv2623 and FLAG-tagged Rv1747 FHA I with anti-Rv2623 and anti-FLAG antibody, respectively—thus demonstrating interaction of these two mycobacterial components. Lane 1: upper panel and lower panel represent results of reacting eluents from column with only Rv2623 with the appropriate antibody. The upper and lower panels of Lane 2 depict reactivity of eluents from column harboring only FHA I with the appropriate antibody. Lane 4 of upper and lower panels represent recombinant Rv2623 (+ve Rv) and FLAG-FHA I (+FHA); respectively, loaded as positive controls. α-Rv2623 and α-FLAG: anti-Rv2623 and anti-FLAG antibodies; respectively. Arrows indicated the molecular weight of His-tagged Rv2623 (~32.31 kDa)) and FLAG-tagged Rv1747 FHA I (~12 kDa; expressed as the first 100 amino acids of Rv1747).</p

Schematic of the regulation of Rv1747 putative PIM transport by Rv2623.

<p>In response to certain signals encountered in the host <b>(1)</b>, the threonine residue of Rv2623 of <i>M</i>. <i>tuberculosis</i> (a universal stress protein) at position 237 (red “T” in purple sphere) is phosphorylated <b>(2).</b> This results in the formation of a conserved phosphothreonine (pT237)-containing motif that enables the engagement of Rv2623 with the FHA I domain of Rv1747 <b>(3).</b> This interaction negatively modulates the function of the putative transporter Rv1747, turning it off <b>(4)</b>. In the absence of the signals operative in step <b>(1)</b>, or in the presence of additional signals <b>(5)</b>, dephosphorylation of the phosphorylated Rv2623 occurs <b>(6)</b>, leading to disengagement of Rv2623 from Rv1747 FHA I <b>(7)</b>. This disengagement releases the inhibitory effect of the phosphorylated Rv2623, allowing Rv1747 to transport the putative substrates PIMs <b>(8)</b>. Whether PIMs are the substrates for Rv1747 remains to be proven. The signals that induce the phosphorylation of Rv2623 are presently unclear–potential candidates include hypoxia and nitrosative stress, as well as nutritional restriction. The nature of the kinase that phosphorylates Rv2623 <i>in vivo</i> is also unknown. The Rv1747 is depicted in its monomeric form except for its transmembrane domain (TMD) for clarity. Rv2623 tethered to an orange circle with a red P represents the phosphorylated form. Red “T” in purple sphere: T237. Small red spheres represent the substrates transported by the Rv1747 transporter.</p