Functional and Structural Characterization of the Antiphagocytic Properties of a Novel Transglutaminase from Streptococcus suis*

Background: SsTGase was a newly identified secreted immunogenic protein of S. suis 2. Results: Anti-phagocytic ability of SsTGase-N was dependent on its TGase activity, and its crystal structure revealed that dimerization was crucial for maintaining functional activities. Conclusion: SsTGase was a novel virulence factor of Ss2 by acting as a TGase in dimer form. Significance: The presented research suggested that SsTGase could serve as a new therapeutic target. Streptococcus suis serotype 2 (Ss2) is an important swine and human zoonotic pathogen. In the present study, we identified a novel secreted immunogenic protein, SsTGase, containing a highly conserved eukaryotic-like transglutaminase (TGase) domain at the N terminus. We found that inactivation of SsTGase significantly reduced the virulence of Ss2 in a pig infection model and impaired its antiphagocytosis in human blood. We further solved the crystal structure of the N-terminal portion of the protein in homodimer form at 2.1 Å. Structure-based mutagenesis and biochemical studies suggested that disruption of the homodimer directly resulted in the loss of its TGase activity and antiphagocytic ability. Characterization of SsTGase as a novel virulence factor of Ss2 by acting as a TGase would be beneficial for developing new therapeutic agents against Ss2 infections.

Streptococcus suis serotype 2 (Ss2) is an important swine and human zoonotic pathogen. In the present study, we identified a novel secreted immunogenic protein, SsTGase, containing a highly conserved eukaryotic-like transglutaminase (TGase) domain at the N terminus. We found that inactivation of SsTGase significantly reduced the virulence of Ss2 in a pig infection model and impaired its antiphagocytosis in human blood. We further solved the crystal structure of the N-terminal portion of the protein in homodimer form at 2.1 Å. Structure-based mutagenesis and biochemical studies suggested that disruption of the homodimer directly resulted in the loss of its TGase activity and antiphagocytic ability. Characterization of SsTGase as a novel virulence factor of Ss2 by acting as a TGase would be beneficial for developing new therapeutic agents against Ss2 infections.
Streptococcus suis serotype 2 is an important swine and human zoonotic pathogen, causing septicemia, arthritis, endocarditis, meningitis, and even acute death in pigs and humans (1,2). Although several virulence factors have been identified, including capsule polysaccharide, extracellular protein factor, suilysin, factor H-binding surface protein, and adenosine synthase (3)(4)(5)(6)(7)(8)(9), the underlying mechanism of Ss2 pathogenesis remains unclear. In our previous study, we identified a new secreted protein from Ss2 culture supernatant encoded by SSU05_1815 locus with a high immunogenicity through an immunoproteomic approach (10). However, genetic studies and sequence analyses revealed that this protein was a transmembrane protein with a single transmembrane segment at the C terminus, suggesting that this protein was released into culture supernatant. Moreover, a highly conserved eukaryotic-like transglutaminase (TGase) 5 domain (residues 247-348) was found at the N terminus. The TGase domain belongs to the TGase-like superfamily (PF01841 in the PFAM database) (11), which contains a highly conserved catalytic triad Cys 302 -His 333 -Asp 348 . Therefore, we named this protein as SsTGase in Ss2. TGases, also named protein-glutamine ␥-glutamyltransferase, constitute a large superfamily of enzymes widely distributed in eukaryotes and prokaryotes and have been extensively studied because they were first extracted from animal liver (12)(13)(14)(15). The enzyme catalyzes an acyl transfer reaction between glutamine residues and lysine or other primary amine, leading to inter-or intramolecular cross-linking and polymerization of the proteins (16 -20). The catalytic reaction of TGases is based on a highly conserved catalytic center: a Cys-His-Asp triad or less frequently a Cys-His dyad. TGases are involved in regulation of a myriad of physiological processes by acting as biological glues, including blood clotting, wound healing, epidermal keratinization, neoplastic diseases, and membrane repair (13,21,22,24). Furthermore, the enzyme has been applied in the food, cosmetic, and textile industries as a biocatalyst (25).
To date, several crystal structures of TGases have been resolved in mammals (26 -31), including human factor XIII, fish-derived transglutaminase (FTGs), and human transglutaminase 2 (28,31). Previous studies have mainly focused on the biochemical characteristics of TGases in mammals, but the roles of TGases played in microorganisms remain largely unknown. Among microorganisms, only TGases from Streptoverticillium mobaraense (microbial TGase) and Phytophthora have been studied, and they represent a completely different structure fold compared with those in mammals (27,29). Therefore, bacterial TGases of the PF01841 superfamily are currently largely unknown for their structural features and specific activities.
In the present study, we showed that the SsTGase was secreted by Ss2 and developed strong antiphagocytic activity. Inactivation of SsTGase significantly reduced virulence in a pig infection model and impaired antiphagocytic resistance of Ss2 in human blood. To further investigate the molecular mechanism underlying the pathogenesis of SsTGase in Ss2, we determined the crystal structure of the N-terminal portion of SsTGase (residues 38 -437; referred to as SsTGase-N hereafter) that also included the TGase domain at 2.1 Å. The structure reveals that although the C-terminal domain of SsTGase-N contains a catalytic core region similar to other TGases its N-terminal domain displays a new structural fold. The overall folding of the SsTGase-N homodimer was novel and different from other known structures of TGases. Inactivation of the protein directly resulted in the loss of its antiphagocytic ability, indicating that antiphagocytic ability of SsTGase-N was dependent on its TGase activity. Furthermore, structure-based mutagenesis and biochemical studies suggested that dimerization of the protein was critical for its activation and antiphagocytic ability. These observations provide a novel insight into the activation mechanism and functions of SsTGase that would be valuable for the development of novel antibiotic strategies targeting SsTGase.

Experimental Procedures
Generation of the Mutant Strain ⌬SsTGase and the Complemented Strain C⌬SsTGase-The ⌬SsTGase mutant was obtained from the 05ZYH33 WT by in-frame deletion of the sstgase gene (SSU05_1815) as described previously (9). Briefly, DNA fragments corresponding to the upstream and downstream regions of the sstgase gene were amplified using primer pairs sstgase KOP1/sstgase KOP2 and sstgase KOP5/sstgase KOP6, respectively ( Table 1). The chloramphenicol cassette was amplified from plasmid pSET1 with primers CM-F and CM-R ( Table 1). The primer pairs sstgase KOP2/CM-F and CM-R/sstgase KOP5 were designed to be fused as an intact fragment by overlap extension PCR. PCR amplicons were cloned into the temperature-sensitive S. suis-Escherichia coli shuttle vector pSET4s, giving rise to the knock-out vector pSET4s::sstgase. The procedures for the selection of mutants by double crossover were described previously (32). The resulting mutant strain was verified by PCR using three pairs of primers, sstgase IN1/sstgase IN2, sstgase-F/sstgase-R, and sstgase KOP1/ sstgase KOP6 (Table 1), and direct DNA sequencing analysis of the mutation sites using genomic DNA as the template. For complementation assays, a DNA fragment containing the entire sstgase gene and its upstream promoter was amplified using primers C⌬sstgase-F and C⌬sstgase-R. The amplicon was subsequently cloned into the E. coli-S. suis shuttle vector pAT18 (33), resulting in the recombinant plasmid pAT18::sstgase. This plasmid was transformed into the ⌬SsTGase mutant, and the complemented ⌬SsTGase strain was screened on Todd-Hewitt broth agar with selective pressure by erythromycin. Reverse transcription-PCR (RT-PCR) analyses of the C⌬SsTGase, 05ZYH33, and ⌬SsTGase strains were used to further identify transcription of the gene sstgase in C⌬SsTGase.
Blood Survival Assay-Diluted strains of the wild type (05ZYH33), ⌬SsTGase, and C⌬SsTGase (50 l; 2 ϫ 10 4 colonyforming units (cfu)/ml) were added to fresh human blood (450 Samples were taken at time points and analyzed immediately. Colonies were counted, and the percentage of surviving bacteria was calculated as follows: (cfu PMNϩ /cfu PMNϪ ) ϫ 100%. The data are presented as means Ϯ S.D. from three or four separate experiments.
Experimental Infections of Piglets-To evaluate the effects of deletion of sstgase on the virulence of 05ZYH33, specific pathogen-free piglets (4 weeks old; six piglets/group) were challenged with 05ZYH33, ⌬SsTGase, and C⌬SsTGase strains and the avirulent strain 1330 (dose of 2 ϫ 10 8 cfu/piglet), respectively. Survival time, clinical signs, and bacterial loads in blood and tissue samples were recorded for 12 days postinoculation. To have a better understanding of the difference between 05ZYH33 and ⌬SsTGase and to reduce the individual differences in infection, groups of four specific pathogen-free piglets were inoculated intravenously with a 1:1 mixture of 05ZYH33/⌬SsTGase (dose of 10 8 cfu/piglet). When the infected piglets showed typical Ss2 infection symptoms, the capability of 05ZYH33/⌬SsTGase surviving in blood and colonizing the various tissues of piglets was analyzed by plating samples on plates without antibiotics or with chloramphenicol resistance selection. All animal experiments were performed in a biosafety level 3 facility and were approved by the local ethics committee.
Ethics Statement-The healthy donors who provided the blood in this study gave written informed consent in accordance with the Declaration of Helsinki. Approval was obtained from the medical ethics committee of the 307 hospital. This research was approved by the ethics committee on Animal Experimentation of the Chinese Association for the Accreditation of Laboratory Animals Care, including the relevant local animal welfare bodies in China. In addition, the permit number of all animal work was SCXK-(JUN) 2013-008, approved by the animal ethics committee of Beijing Institute of Microbiology and Epidemiology. All efforts were made to minimize suffering of animals used in this study.
Measurement of Transglutaminase Activity-TGase (EC 2.3.2.13) activity was assayed by a transglutaminase colorimetric microassay kit (TCM kit, Covalab), which uses immobilized N-carbobenzoxy-Gln-Gly as amine acceptor and biotin-conjugated cadaverine as amine donor. Protein samples were incubated in a 96-well microtiter plate coated with N-carbobenzoxy-Gln-Gly at 37°C for 15 min with calcium, DTT, and biotinylated cadaverine both in the presence and the absence of EDTA supplied in the kit. The wells were washed three times with phosphate buffer containing 0.1% Tween 20. To assay the formation of cadaverine covalently linked to N-carbobenzoxy-Gln-Gly (␥-glutamyl-cadaverine-biotin) by TGase, the wells were filled with streptavidin-labeled HRP and incubated for 15 min at 37°C. Following three washes with phosphate buffer containing 0.1% Tween 20, the wells were filled with HRP substrate/chromogen solution containing H 2 O 2 as the substrate and tetramethylbenzidine as the electron acceptor (chromogen). After incubation for 10 min at room temperature, 50 l of reaction blocking reagent was added, and the mixture was quantified by measuring A 450 . As a reference for the TGase activity, the kit includes the purified guinea pig TGase with a specific activity of 0.1 unit/mg. By definition, 1 unit of TGase catalyzes the formation of 1 mol of hydroxamate/min at pH 6.0 at 37°C using L-glutamic acid ␥-monohydroxamate as the standard.
Circular Dichroism (CD) Spectroscopic Analysis-For CD spectroscopic analysis, the purified proteins from peak 1 and peak 2 solubilized in buffer containing 20 mM Hepes, pH 7.5 and 200 mM NaCl were concentrated to 1 mg/ml. CD spectroscopy was carried out using an Applied Photophysics Chirascan Plus spectropolarimeter with a 10-mm-path length cell and a bandwidth of 1.0 nm. Spectra were recorded from 260 to 180 nm at an interval of 1 nm and were repeated three times. All resultant spectra were obtained by subtraction of the spectrum of the buffer.
Cloning, Expression, and Purification of SsTGase-N-The coding sequence of sstgase-N (residues 39 -437) was cloned into the pGEX6p-1 vector (Novagen), generating an N-terminal PreScission protease cleavage site following the GST tag that was confirmed by DNA sequencing. Overexpression of SsTGase-N was induced in E. coli BL21(DE3) strain by 0.5 mM isopropyl ␤-D-thiogalactoside when the cell density reached an A 600 nm of 1.2. After growth for 6 h at 30°C, the cells were collected, resuspended in buffer (25 mM Tris-HCl, pH 8.0, 200 mM NaCl), and lysed by sonication. Recombinant GST-tagged protein was purified by glutathione affinity chromatography and gel filtration chromatography with buffer containing 20 mM Hepes, pH 7.5, 200 mM NaCl, and 2 mM DTT. Notably, the GST tag of purified homodimer was cleaved off by PreScission protease (Amersham Biosciences) before gel filtration. The selenomethionine (Se-Met)-substituted SsTGase-N derivative was expressed in E. coli B834 strain, grown in selenomethionine medium (Molecular Dimensions Ltd.), and purified similarly (34).
According to procedures described previously (35), point mutations (C302S, H333S, D348S, T215A, and R311A) of SsTGase-N were generated by two-step PCR and confirmed by DNA sequencing. All the mutants were purified in the same way as wild-type protein.
Crystallization, Data Collection, and Structure Determination-Crystals of SsTGase-N were generated by mixing 1 l of protein solution with 1 l of well buffer using the hanging drop vapor diffusion method at 18°C. Crystals appeared after 2 weeks in the reservoir solution (0.2 M sodium chloride, 0.1 M Hepes, pH 7.2, 20% (w/v) polyethylene glycol 4000). The crystals were cryoprotected in reservoir solution plus 15-20% (v/v) glycerol and flash frozen in liquid nitrogen prior to data collection. JULY 31, 2015 • VOLUME 290 • NUMBER 31

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All the data were collected at the Shanghai Synchrotron Radiation Facility BL17U beamline and integrated and scaled using the HKL2000 package (36). Further processing was carried out using programs from the CCP4 suite (37). Data collection statistics are summarized in Table 2. The selenium sites were located using SHELXD from Bijvoet differences in the selenium single wavelength anomalous diffraction data (38). Heavy atom positions were defined, and the phases were calculated with the single wavelength anomalous diffraction experimental phasing module of Phaser (39). The real space constraints were applied to the electron density map in density modification. The final model rebuilding was performed with Coot (40), and the protein structure was refined with PHENIX (41) using non-crystallographic symmetry and stereochemistry information as restraints. Structural figures were generated in PyMOL (42). The structure factors of SsTGase-N have been deposited in the Protein Data Bank (accession code 4XZ7).

SsTGase Was Secreted by Ss2 and Possessed Strong
Antiphagocytic Ability-SsTGase is encoded by the SSU05_1815 locus of China pathogenic strain 05ZYH33 (NC_009442.1) isolated from a deceased streptococcal toxic shock syndrome patient. To investigate the specific roles of the protein, we constructed a mutant strain (⌬SsTGase) and a complemented strain (C⌬SsTGase) derived from wild-type strain 05ZYH33 (WT). First, we identified that SsTGase was secreted by Ss2 through Western blotting despite the fact it contained a predicted transmembrane segment (Fig. 1, A and B). This secreted form was possibly produced through proteolysis of the fulllength protein during biological processes. The results from the PMN killing assay suggested that the survival rates of the ⌬SsTGase strain were much lower than those of the WT and C⌬SsTGase strains by 1-3 h (Fig. 1C). Furthermore, to investi-gate the antiphagocytic activity of SsTGase, we carried out a blood survival assay with the culture supernatant of the WT, C⌬SsTGase, and ⌬SsTGase strains. The results showed that culture supernatant of WT and C⌬SsTGase, but not that of ⌬SsTGase, improved the survival rates of the three strains of Ss2 (WT, ⌬SsTGase, and C⌬SsTGase) in human blood ( Fig.  2A). In addition, the survival rate of the ⌬SsTGase strain was also improved significantly after incubating the strain with different amounts of SsTGase-N (residues 39 -437) protein in human blood (Fig. 2B), which suggested that both SsTGase and SsTGase-N possessed significant antiphagocytic activity.
SsTGase Was a New Virulence Factor of Ss2-To determine whether SsTGase is a potential virulence factor of Ss2, a piglet infection model was used to test the virulence of the WT, ⌬SsTGase, and C⌬SsTGase strains with the North American avirulent strain 1330 considered as a negative control. Each piglet was injected with 2 ϫ 10 8 cfu of bacteria, and the survival rates of infected piglets were measured over 12 days. All the  piglets infected with the WT strain died within 6 days after infection in contrast to a survival rate of 83.33% in the group infected by the ⌬SsTGase strain, 33.33% in the group infected by the C⌬SsTGase strain, and 100% in the group infected by the 1330 strain (Fig. 2C). Severe symptoms such as high fever, limping, swollen joints, shivering, and central nervous system failure were observed among the groups infected by the WT and C⌬SsTGase strains, whereas only mild symptoms were observed in the groups infected by the ⌬SsTGase strain. Additionally, the efficiencies of colonization by the WT and C⌬SsTGase strains in blood were much higher than that of the ⌬SsTGase strain from 12 to 132 h (Fig. 2D).  JULY 31, 2015 • VOLUME 290 • NUMBER 31

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To avoid the individual differences in piglets, the competitive infection assay was adopted to further compare the virulence of the WT and ⌬SsTGase strains in which a group of four piglets was challenged with a 1:1 mixture of the WT and ⌬SsTGase strains. Consequently, the cell numbers of the WT bacteria in blood and various tissue samples (heart, liver, kidney, spleen, lung, tonsil, and lymph) were much higher than those of the ⌬SsTGase bacteria (Fig. 2, E and F). Taken together, these data indicated that SsTGase was a new virulence factor of Ss2 with antiphagocytic activity.
Antiphagocytic Ability of SsTGase-N Was Dependent on Its TGase Activity-SsTGase-N corresponds to an active form with a high TGase activity (0.72971 unit/mg) compared with TGase from guinea pig liver (0.10789 unit/mg), whereas no activity could be detected after mixing it with monodansylcadaverine, a potent inhibitor of TGases (Fig. 3A). Similar to other TGases from microorganisms, the SsTGase-N enzyme activity is Ca 2ϩ -independent (Fig. 3A). In addition, we generated three point mutations of the catalytic residues (C302A, H333A, and D348A) to study the effect of each single active residue. All three point mutations displayed dramatically decreasing TGases activity, suggesting that these three residues played an important role in catalyzing the TGase reaction (Fig. 3C).
Notably, antiphagocytic abilities of the three point mutations also decreased to an extremely low level (Fig. 3B). Moreover, the survival rate of WT strain dropped dramatically after incubation with monodansylcadaverine (Fig. 1C). These results indicated that the antiphagocytic ability of SsTGase-N was dependent on its TGase activity.
Overall Structure of SsTGase-N-To better understand the biological functions of SsTGase, we solved the crystal structure of SsTGase-N that also included a TGase domain (residues 247-348). The structure reveals that SsTGase-N forms a homodimer in an antiparallel manner (Fig. 4A). In each protomer, the region spanning residues 353-363 is invisible probably due to intrinsic flexibility (Fig. 4B). Each protomer forms an elongated and twisted dumbbell-like fold, which can be divided into three regions: an N-terminal domain (residues 38 -208; referred to as NTD hereafter), a C-terminal TGaselike domain (residues 221-437; referred to as CTD hereafter), and a connecting helix (␣5 helix; residues 209 -220). The NTD consists of two antiparallel ␤-sheets in the center with four and three ␤-strands, respectively. Each of the two ␤-sheets is flanked by two helices. Similar to other solved structures of TGases, a deep cleft is generated at the edge of the CTD where catalytic residues Cys 302 , His 333 , and Asn 348 are located (Fig.  5B). Clusters of helices formed in the N terminus of the CTD are composed of ␣6, ␣6Ј, ␣7, and ␣7Ј followed by an antiparallel ␤-sheet comprising three ␤-strands (␤8, ␤9, and ␤10). In the C terminus of CTD, two parallel ␤-strands and two ␣-helices wrap around the whole domain. The two protomers are compacted together, and the CTD from one protomer lies opposite to the NTD of the other (Fig. 4A).
The interactions between the two protomers are distributed in two regions mainly composed of hydrogen-bonding interactions and water-mediated interactions. In one region, Thr 215 from one protomer interacts with Ser 218 of the other through . Antiphagocytic ability of SsTGase-N was dependent on its TGase activity. A, transglutaminase activity of SsTGase-N was Ca 2ϩ -independent. The assay of transglutaminase activity of purified SsTGase-N was performed using a transglutaminase colorimetric microassay kit (TCM kit). The TCM kit uses immobilized N-carbobenzoxy-Gln-Gly as the amine acceptor and biotin-conjugated cadaverine as the amine donor. As a reference for TGase activity, purified guinea pig TGase (gpTGase) with specific activity of 0.1 unit/mg was incubated under the same conditions. A representative result for each condition is shown. Error bars represent S.D. of three to four independent measurements. B, antiphagocytic abilities of SsTGase-N and mutants were demonstrated by blood survival assay. Error bars represent S.D. of three to four independent measurements. ***, p Ͻ 0.001. C, assay of transglutaminase activity of purified SsTGase-N and mutants using a transglutaminase colorimetric microassay kit (TCM kit). Error bars represent S.D. of three to four independent measurements. ***, p Ͻ 0.001.
hydrogen-bonding interactions, stabilizing the interactions between the two ␣5 helices in an antiparallel orientation (Fig.  6A). In the other region, water-mediated interactions could be observed among the main-chain carbonyl group of Leu 176 located at ␣4 helix (residues 172-179), the side-chain guanidine group of Arg 311 , and the carbonyl group of Leu 412 (Fig.  6A). Notably, Arg 311 sits on the same ␣-helix where the catalytic residue Cys 302 is located.
CTD of SsTGase-N Contains a Conserved Catalytic Core Region-Although SsTGase-N shares low primary sequence similarity with known TGases from mammals or bacteria, it possesses an active region consisting of a consensus sequence motif of thiol proteases (Fig. 5A). Cys 302 sits on the N terminus of ␣7Ј helix and is known to supply a thiolate ion for nucleophilic assault. The sulfhydryl group of Cys 302 forms a hydrogen bond with His 333 at a loop between ␤8 and ␤9 (Fig. 5B). Additionally, the imidazole ring of His 333 forms a hydrogen bond with Asp 348 , which is in a loop connecting ␤10 strand and ␣8 helix (Fig. 5B). Thr 350 and Tyr 377 also participate in the hydrogen bonding pattern in the active cavity (Fig. 5B). Specifically, both Tyr 377 and Thr 350 form hydrogen bonds with Asp 348 , whereas Tyr 560 in human factor XIII suppresses enzyme activity by forming a hydrogen bond with the active site residue Cys 314 (31). Hydrogen bonds formed by Cys 302 , His 333 , Asp 348 , Tyr 377 , and Thr 350 could enhance the stability of the entire active cavity, indicating that substrates approaching the enzyme might disrupt the stable state of the active cavity by breaking the hydrogen bonds mentioned above.
Structural alignment of the entire TGase domain of SsTGase-N with the corresponding regions of human factor XIII and FTG, which are representatives of TGases from mammals, reveals that overall folding of the active site region of SsTGase-N adopts a fold similar to that of human factor XIII and FTG with root mean square deviations of 3.24 and 0.517 Å, respectively (Fig. 5C). In addition, the three active residues of SsTGase superimpose well with the catalytic tri- ads of human factor XIII and FTG (28,31) (Fig. 5C). Therefore, CTD of SsTGase-N has a conserved catalytic core region similar to other TGases.
Interestingly, a Dali search with the NTD of SsTGase-N only returned entries with low Z-scores of 4.1-2.0, suggesting that no known structure was identified to share significant homo-logy with this domain (43). That is, the NTD of SsTGase-N likely represents a newly identified structural fold. Moreover, the overall folding of how SsTGase-N packs into homodimer is novel and differs from other known structures of TGases.
A New Activation Mechanism of SsTGase-N in Solution Environment-The zymogen forms of TGases require proteolytic activation or the presence of Ca 2ϩ to gain their activities. For instance, microbial TGase is secreted from the cytoplasmic membrane as a zymogen and is activated by proteolytic processing (44). During the purification of the recombinant protein, two peaks (peak 1 and peak 2) of SsTGase-N emerged in the gel filtration profile corresponding to the dimer and monomer forms, respectively, as deduced from the peak positions in the gel filtration assay (Fig. 6B). The protein from the peak 1 rerun on gel filtration assay remained a single peak, whereas the protein from the peak 2 rerun on gel filtration resulted in a shift into peak 1 position (Fig. 6B). Therefore, there is a dynamic equilibrium between these two forms, and the dimer form possibly represents the more stable state. Notably, although we have conducted crystallization experiments with proteins of both conformations, only the dimer form could be crystallized. Importantly, circular dichroism results showed that the protein from peak 2 shared a similar spectrogram profile with the protein from peak 1, indicating that the monomer form was also properly folded (Fig. 6C). Unexpectedly, the monomer form displayed extremely low TGase enzyme activity and antiphagocytic capacity compared with the dimer form (Fig. 3, B and C). All the protein we used for the activity assay, unless otherwise specified, came from peak 1. Thus, we proposed that dimerization of the protein could promote activation of the protein. To test that, we set out to examine the interface between the monomers. Because our structure indicated that interactions between the two monomers are mainly mediated by hydrogen-bonding interactions between Thr 215 from one monomer and Ser 218 of the other as well as water-mediated interactions between Leu 412 and Arg 311 from one monomer and Leu 176 of the other, we generated two point mutations, T215A and R311A. No conformational changes of the protein appeared after mutating Thr 215 to Ala, and the protein from peak 1 and peak 2 displayed a TGase activity and antiphagocytic ability similar to those of the wild type (Figs. 6B and 3, B and C). Interestingly, the SsTGase-N R311A protein only displayed a single peak at the monomer position in the gel filtration profile, implying that the conformation of the protein changed into a monomer completely and that Arg 311 played an important role in stabilizing the dimerization of the protein (Fig. 6B). TGase activity and antiphagocytic ability of SsTGase-N R311A decreased to quite a low level (Fig. 3, B and C), suggesting that dimerization of the protein is crucial for maintaining functional activities. Structural analysis revealed that Arg 311 and the cata-lytic amino acid Cys 302 from one monomer sit on the same ␣-helix (␣7Ј helix), which can be stabilized by water-mediated interactions mediated by ␣4 helix of the other monomer. To our knowledge, similar interactions have not been observed in other solved structures, such as human factor XIII and human TGase 3 (27,29,31). Taken together, we propose that the SsTGase-N monomer was not stable enough to catalyze the reaction in solution environment, whereas dimerization of the protein could promote its activation by stabilizing the architecture of the catalytic cavity.

Discussion
In this study, we identified SsTGase as a new virulence factor in the infection process with antiphagocytic function, which was dependent on its TGase activity. Structural analyses show that SsTGase-N shares a common feature of active site cavity with eukaryotic TGases but with a novel activation mechanism. Because of their ubiquitous distribution, TGases play important roles in physiological and pathological processes by posttranslational modifications of substrates. For instance, glycoprotein gp42 present in the cell wall of Phytophthora sojae can induce plenty of defense mechanisms, eliciting a hypersensitive response, resulting in death of the infected cells (21,45). Moreover, some bacterial toxins, including the cytotoxic factor 1 of E. coli, act as a TGase (46). Thus, we propose that SsTGase is secreted from the cytoplasmic membrane and activated by disruptions of the physiological homoeostatic environment after invasion, and then the mature SsTGase could modify the surface proteins of Ss2 and/or host to avoid phagocytosis. Obviously, the details of how SsTGase functions as a virulence factor in the infection process of Ss2 need further investigation.
Interestingly, mapping the electrostatic potential of SsTGase-N onto its surface revealed that the active site was mainly surrounded by highly negatively charged residues (Fig.  7). Indeed, a similar situation was also observed in the structure of GP42 in which a strong negative potential delineates a groove adjacent to the active site (29). To our knowledge, identification of the target proteins of TGase has been very challenging due to the highly cross-linked property and insolubility of the product (47). Therefore, to date, no substrates of SsTGase-N have been identified. This structural feature prompted us to propose that the enzyme likely interacts with positively charged substrates.
Structurally directed mutagenesis studies revealed that dimerization was required for the enzymatic activity of SsTGase-N. Based on this, we have proposed a new activation mechanism of SsTGase-N. Notably, human transglutaminase 2 undergoes a large conformational change upon activation, whereas overall structures of microbial TGase zymogen and mature microbial TGase are essentially the same (30,48).

Functional and Structural Study of a Novel Transglutaminase
Therefore, structural changes of SsTGase-N upon activation still await the determination of the structure of SsTGase-N in a monomer state. Detailed description of the activated mechanism will clearly require additional biochemical characterizations.
The characterization of SsTGase as a novel virulence factor of Ss2 by acting as a new TGase in TGase-like superfamily (PF01841) will facilitate the development of new therapeutic agents capable of efficiently interfering with Ss2 infection. In addition, compared with the high cost of transglutaminase of The purification profiles of the wild-type and mutant proteins, protein from peak 1, and the protein from peak 2 are shown after gel filtration. The sizes of the molecular markers are marked on top of the peaks. C, the protein in peak 2 is correctly folded like the protein in peak 1. The purified proteins (1 mg/ml) from peak 1 (black curve) and peak 2 (red curve) solubilized in buffer containing 20 mM Hepes, pH 7.5 and 200 mM NaCl were subjected to CD, and spectra are shown. mdeg, millidegrees.
animal origin (23,25), SsTGase might have the potential to be applied as a new biocatalyst in the biomedical and biotechnology fields.