Novel secreted STPKLRR from Vibrio splendidus AJ01 promotes pathogen internalization via mediating tropomodulin phosphorylation dependent cytoskeleton rearrangement

We previously demonstrated that the flagellin of intracellular Vibrio splendidus AJ01 could be specifically identified by tropomodulin (Tmod) and further mediate p53-dependent coelomocyte apoptosis in the sea cucumber Apostichopus japonicus. In higher animals, Tmod serves as a regulator in stabilizing the actin cytoskeleton. However, the mechanism on how AJ01 breaks the AjTmod-stabilized cytoskeleton for internalization remains unclear. Here, we identified a novel AJ01 Type III secretion system (T3SS) effector of leucine-rich repeat-containing serine/threonine-protein kinase (STPKLRR) with five LRR domains and a serine/threonine kinase (STYKc) domain, which could specifically interact with tropomodulin domain of AjTmod. Furthermore, we found that STPKLRR directly phosphorylated AjTmod at serine 52 (S52) to reduce the binding stability between AjTmod and actin. After AjTmod dissociated from actin, the F-actin/G-actin ratio decreased to induce cytoskeletal rearrangement, which in turn promoted the internalization of AJ01. The STPKLRR knocked out strain could not phosphorylated AjTmod and displayed lower internalization capacity and pathogenic effect compared to AJ01. Overall, we demonstrated for the first time that the T3SS effector STPKLRR with kinase activity was a novel virulence factor in Vibrio and mediated self-internalization by targeting host AjTmod phosphorylation dependent cytoskeleton rearrangement, which provided a candidate target to control AJ01 infection in practice.

1 Summary 23 We previously demonstrated that the flagellin of intracellular Vibrio splendidus 24 AJ01 could be specifically identified by tropomodulin (Tmod) and further mediate 25 p53-dependent coelomocyte apoptosis in the sea cucumber Apostichopus japonicus. 26 In higher animals, Tmod serves as a regulator in stabilizing the actin cytoskeleton. 27 However, the mechanism on how AJ01 breaks the AjTmod-stabilized cytoskeleton for 28 internalization remains unclear. Here, we identified a novel AJ01 Type III secretion 29 system (T3SS) effector of leucine-rich repeat-containing serine/threonine-protein 30 kinase (STPKLRR) with five LRR domains and a serine/threonine kinase (STYKc) 31 domain, which could specifically interact with tropomodulin domain of AjTmod. 32 Furthermore, we found that STPKLRR directly phosphorylated AjTmod at serine 52 regulate microfilament assembly (Welch et al., 1997). Tmod, the only known capping 77 protein of F-actin, constitutes and stabilizes the actin cytoskeleton and is also an ideal 78 target for pathogen internalization (Weber et al., 1994). However, to our knowledge, 79 pathogen effectors targeting Tmod have not been identified, and their corresponding 80 mechanism remains unclear. 81 Vibrio splendidus AJ01 is a facultative intracellular bacterium that is a common 82 However, the mechanism via which AJ01 breaks through the AjTmod-stabilized 90 cytoskeleton for internalization remains unclear. In this study, we identified a novel A. intestine, which presented dense muscle fibers with holes and damaged intestinal 156 mucosa (Fig. 2D, black arrows). However, sea cucumbers from the found sequences identical to the AJ01 sequence (data not shown). Clonal counting 165 revealed that the numbers of intracellular AJ01 in groups AJ01 and 166 ΔSTPKLRR::pMP-STPKLRR reached 3 × 10 5 and 2.1 × 10 5 CFU/mL, respectively, 167 whereas that in the ΔSTPKLRR and ΔSTPKLRR::pMP2444 groups were only 2 × 168 10 4 and 2.7 × 10 4 CFU/mL, respectively (Fig. 2G) infection group under the same condition (Fig. 3C). Importantly, fluorescence signals 188 between STPKLRR and AjTmod were found to colocalize in coelomocytes (Fig. 3C).

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Consistently, the number of AJ01 in coelomocytes was increased accompanied with  could depress STPKLRR-mediated AjTmod phosphorylation (Fig. 4B). The serine 235 residues are the major target for protein phosphorylation and are also critical to signal 236 transduction (Barford, 1996). Given that STPKLRR targets the tropomodulin domain 237 of AjTmod, we selected five serine sites in the tropomodulin domain for mutation into 238 alanine, namely, S12A, S44A, S49A, S52A, and S144A. We found that GSTTmod 239 and the four mutants S12A, S44A, S49A, and S144A could be phosphorylated by 240 recombinant STPKLRR in vitro. However, the S52A mutant lost the ability for 241 phosphorylation by STPKLRR (Fig. 4C). Surprisingly, the phosphorylation site was 242 unique to AjTmod and was not found in several other invertebrates and higher 243 mammals. (Fig. 4D).

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To confirm STPKLRR phosphorylating AjTmod rather than intracellular AJ01, 245 we treated coelomocytes with recombinant STPKLRR or His tag protein for 12 h. As 246 shown in Figure S4A, only one phosphorylated site of AjTmod was detected in the 247 STPKLRR treated group. Furthermore, the FITC-labeled fluorescent microspheres (2 248 μm in diameter, Sigma) was used to assay the coelomocyte phagocytosis activity 249 under the same condition. The results showed that phagocytosis rate in STPKLRR 250 treated group was significantly induced compared to the His tag treated group ( Figure  S4B and S4C). These results supported that phosphorylation of AjTmod was modified 252 by STPKLRR rather than intracellular AJ01.

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In metazoans under normal conditions, Tmod could specifically bind to F-actin 254 to stabilize the cytoskeleton (Bennett et al., 2001;Fowler, 1996). The cellular location    showed no significant changes (Fig. 5C, and 5D). All these results indicated that 335 STPKLRR was critical for AJ01 internalization by targeting AjTmod.

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In our previous work, we found that AJ01 could enter coelomocytes through  and 92 aa and is the main region of Tmod binding to actin (Kostyukova, 2008).   with elution buffer (pH = 7.9 -8.1). Different truncated AjTmods, including GSTTro and GSTTLRR, were also generated to verify their STPKLRR binding region. The

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GST tag was used as a control. The eluent was detected by using SDS-PAGE, and 528 differential proteins were characterized by mass spectrometry. The pull-down assay 529 was also performed to screen the AjTmod interactive protein from sea cucumber 530 coelomocytes with 500 µg of total protein as the load with similar procedure.