Agonist-antagonist effectors manipulate host signaling 25 26
Diverse bacterial pathogens employ effector delivery systems to disrupt vital cellular processes 28 in the host (1). The type III secretion system 1 of the marine pathogen, Vibrio 29 parahaemolyticus, utilizes the sequential action of four effectors to induce a rapid, pro-30 inflammatory cell death uniquely characterized by a pro-survival host transcriptional response (2, 31
3). Herein, we show that this pro-survival response is caused by the action of the channel-32 forming effector VopQ that targets the host V-ATPase resulting in lysosomal deacidification and 33 inhibition of lysosome-autophagosome fusion. Recent structural studies have shown how VopQ 34 interacts with the V-ATPase and, while in the ER, a V-ATPase assembly intermediate can 35
interact with VopQ causing a disruption in membrane integrity. Additionally, we observe that 36
VopQ-mediated disruption of the V-ATPase activates the IRE1 branch of the unfolded protein 37 response (UPR) resulting in an IRE1-dependent activation of ERK1/2 MAPK signaling. We also 38 find that this early VopQ-dependent induction of ERK1/2 phosphorylation is terminated by the 39 VopS-mediated inhibitory AMPylation of Rho GTPase signaling. Since VopS dampens VopQ-40 induced IRE1-dependent ERK1/2 activation, we propose that IRE1 activates ERK1/2 41 phosphorylation at or above the level of Rho GTPases. This study illustrates how temporally 42 induced effectors can work as in tandem as agonist/antagonist to manipulate host signaling and 43 reveal new connections between V-ATPase function, UPR and MAPK signaling. The seafood-borne pathogen, Vibrio parahaemolyticus (V. para), uses two needle-like type III 62 secretion systems (T3SS1 and T3SS2) to inject effectors into host cells to manipulate signaling 63 and cellular processes during infection (2). The V. para T3SS2 is found in clinical isolates, is 64 linked to disease in humans and has been shown to mediate invasion into mammalian host cells 65 (4, 5). By contrast, the T3SS1 is present in all V. para isolates and is thus believed to be essential 66 for survival in its environmental niche. This niche has been rapidly expanding due to the 67 warming of coastal waters contributing to the resurgence of V. para as a significant cause of 68 gastroenteritis world-wide (6, 7). Together, the V. para T3SS1 effectors orchestrate a temporally 69 regulated non-apoptotic cell death in cultured cells (2). The specific cell type that T3SS1 has 70 evolved to target in the environment remains undefined; however, its effectors target processes 71 that are conserved from yeast to humans (2, (8) (9) (10) . These two activities inhibit autophagic flux, resulting in massive autophagosome accumulation 78 and contribute to a pro-inflammatory cell death within three hours(13). VPA0450 is a 79 phosphatidyl 5-phosphatase that hydrolyses PI(4,5)P 2 at about one hour after infection, resulting 80 in blebbing of the plasma membrane ( Fig. 1A) (14). Soon after, VPA0450-mediated blebbing is 81 observed, VopS, a Fic (filamentation induced by cAMP) domain-containing protein, covalently 82 attaches an adenosine monophosphate (AMP) to a threonine residue in the switch 1 region of 83
Rho guanosine triphosphatases (GTPases) Rho, Rac and Cdc42. This modification, termed 84
AMPylation, inactivates the Rho GTPases thereby precipitating cytoskeletal collapse and cell 85 rounding, as well as inactivation of nuclear factor-kappaB (NF-κB) and mitogen activated 86 protein kinase (MAPK) signaling pathways (Fig. 1A) Previously, it was found that VopQ was both necessary and sufficient for the accumulation of 96 LC3-positive autophagosomes as well as the deacidification of endolysosomal compartments. 97 of VopQ against the hydrophobic lipid environment (16). This disruption is predicted to lead to 103 the deacidification of the lysosomal membrane. Remarkably, a fully functioning or assembled V-104
ATPase at the vacuole is not necessary to induce VopQ toxicity in yeast. We found that VopQ 105
can interact with an assembly intermediate of the V-ATPase (V o c-ring) in the ER resulting in 106 cell death (16). As VopQ forms a pore in target membranes, the ER membrane is compromised, 107 and this could lead to the induction of host cell signaling events including the unfolded protein 108 response (UPR). 109
Herein, we find that VopQ activates the IRE1 branch of the UPR in yeast and cultured cells. We 110 demonstrate that the activation of IRE1 by VopQ results in an activation of extracellular singal-111 regulated protein kinase 1/2 (ERK1/2) signaling that is dependent on IRE1 kinase but not 112 nuclease activity. We also find that another T3SS1 effector VopS dampens VopQ-mediated 113 activation of ERK1/2 signaling by AMPylation-dependent inactivation of Rho GTPases, thereby 114 limiting the activation of ERK1/2 signaling to early infection time points. Taken together, our 115 work provides another example of the powerful interplay between T3SS effectors and how they 116 can temporally regulate host signaling pathways. We included VopS because it targets Rho GTPases that regulate MAPK signaling (15, 17). We 128 used V. para strain, POR3, a derivative of the clinical strain RIMD2210633 that does not 129 produce functional hemolysins or a functional T3SS2 (tdhASvcrD2), but maintains an active 130 T3SS1 (6). This strain and its vopQ and vopS derivatives will be referred to herein as T3SS1 + , 131 T3SS1 + vopQ and T3SS1 + vopS, respectively (Table S1 ). As observed with previously 132 characterized cell types, cytotoxicity of PHDFs occurring within the first four hours of infection 133 was completely dependent on VopQ (Fig. S1A,B) (2). We then performed RNA-sequencing on 134 the PHDFs after 90 minutes of infection with V. para T3SS1 + , T3SS1 + vopQ and T3SS1 + vopS 135 strains. The sequencing data passed statistical quality control tests, and principal component 136 analysis indicated tight clustering of replicates (Fig. S2A ). Complete differential expression data 137 is reported in Table S2 , but for this study we focused on the 398 host genes previously found to 138 be differentially expressed specifically in response to the T3SS1 (3). 139
The hierarchically clustered expression heatmap in Fig. 1B illustrates how the T3SS1 causes 140 changes in expression of these 398 genes in the absence of either VopQ or VopS. Of these 141 T3SS1-specific genes, 146 were similarly differentially expressed in the uninfected (UN) vs. 142 T3SS1 + and UN vs. T3SS1 + vopQ-infected cells, and 197 were similarly differentially expressed 143 in the UN vs. T3SS1 + and UN vs. T3SS1 + vopS-infected cells (Fig. S2B , Table S3 ). 252 and 201 144 T3SS1-specific genes were either not differentially expressed or changed direction during 145 T3SS1 + vopQ and T3SS1 + vopS infection, respectively. Expression of many genes, especially 146 those within clusters 1 and 4, was oppositely affected during infection with T3SS1 + vopQ 147 compared to during infection with T3SS1 + vopS (Fig. 1B, Table S3 ). Notably, expression of the 148 EGR1 and FOS transcription factors, which are known to be regulated by MAPK signaling 149 pathways, was reduced in T3SS1 + vopQ-infected cells compared to T3SS1 + -infected cells, and 150 highly elevated by T3SS1 + vopS infection (Table S3 )(18). We validated these findings by 151 quantitative RT-PCR and using V. para strains deleted for multiple effectors (T3SS1 + 152 vopQRvpa0450 and T3SS1 + vopRSvpa0450) and showed that VopQ is necessary and 153 sufficient for the elevated expression of both FOS and EGR1 (Fig. S2C,D) . 154
We next used Ingenuity Pathway Analysis (IPA) to understand how the activities of VopQ and 155
VopS contribute to the changes in host signaling events induced by the T3SS1 (Table S4 ). The 156 T3SS1-specific induction or repression of many pathways was dependent on VopQ and 157 enhanced in the absence of VopS (Fig. 1C ). For example, induction of NF-kB signaling, actin 158 cytoskeleton signaling and Rho GTPase signaling by T3SS1 was greatly reduced in the absence 159 of VopQ and enhanced in the absence of VopS. These observations are consistent with the 160 opposing effects on differential expression patterns observed in Fig. 1B . 161 162
To understand the relative contributions of VopQ and VopS to the host response to T3SS1 on the 164 network level, we used IPA to perform biological function network analysis. Previously, we had 165
shown that the T3SS1 activates cell survival networks and represses cell death networks(3). 166
Strikingly, this effect was completely lost during T3SS1 + vopQ infection and amplified during 167 Table S5 ). Specifically, we observed a loss in cell survival and 168 viability signaling network activation and death and mortality signaling network repression in 169
PHDFs infected with V. para T3SS1 + vopQ compared to V. para T3SS1 + while infection with 170 T3SS1 + vopS instead amplified these signaling changes (Fig. 1D) . Interestingly, the apoptosis 171 signalling network, which is normally repressed during T3SS1 + infection, was activated during 172 infection with T3SS1 + vopQ (Fig. 1D) . These data strongly a model in which the activity of 173
VopQ elicits significant transcriptional changes in the host cell that result in the activation of cell 174 survival and repression of cell death networks and that VopS functions to dampen this response. 175 176
To further dissect VopQ's effect on host signaling pathways in mammalian cells, we continued 178 with a more genetically tractable model, mouse embryonic fibroblasts (MEFs). We characterized 179 the cytotoxicity of the V. para T3SS1 + strain and its derivates in MEFs. The cell death induced 180 by the T3SS1 occurred over a similar timescale in MEFs as in PHDFs and was similarly 181 dependent on VopQ (Fig. S3A ). This result was expected because the mechanism of T3SS1-182 mediated cell death is conserved across diverse cell types (2, 3, 9, 15). We chose to examine the 183 ERK1/2 signaling pathway because the RNA-sequencing data suggested that VopQ activates 184 Rho GTPase signaling (Fig. 1C) and in previous work we demonstrated that T3SS1-induced 185 EGR1 and FOS expression requires active MEK1/2, the kinase upstream of ERK1/2 (3). 186
Furthermore, as we did not observe agonist and antagonist effects of VopQ and VopS, 187 respectively, on the expression of c-Jun N-terminal kinase (JNK) signaling target genes, we 188 focused our analysis on the ERK1/2 pathway (Table S6) . We repeated the infection time course with the V. para T3SS1 + vopQ strain and found that the 201 T3SS1-induced pulse of ERK1/2 phosphorylation was indeed dependent on VopQ as was the 202 increase in total Egr1 protein levels (Fig. 2C,D) . When MEFs were infected with T3SS1 + vopS, 203
we observed not only an amplified induction of ERK1/2 phosphorylation and Egr1 production, 204 but also an extended duration of ERK1/2 phosphorylation (Fig. 2C,D) If VopQ and VopS work together to fine-tune the host response, their co-occurrence in Vibrio 215 genomes containing the T3SS1 gene cluster would be predicted to be high. To test this, we used 216 the SyntTax server to identify all Vibrio strains that retained synteny in the T3SS1 gene 217 neighborhood (Table S7) . We identified 58 Vibrio strains representing 8 species containing the 218 T3SS1 gene cluster and found that 91.4% of genomes containing vopQ also contained vopS 219 (53/58) (Table S7 ). We found that these genes co-occur in diverse Vibrio species including V. 220 parahaemolyticus, V. diabolicus, V. antiquarius, V. campbelli and V. alginolyticus (Fig. S4) . 221
Interestingly, the 5 genomes containing vopQ but lacking vopS belonged to two Vibrio species, 222 V. harveyi and V. tubiashii. 223 224
Next, we wanted to understand how VopQ could activate ERK1/2 MAPK signaling in the host. 226
The VopQ channel de-acidifies vacuolar and lysosomal compartments, but also inhibits 227 homotypic fusion of yeast vacuoles, a model for Rab GTPase-and SNARE-dependent fusion 228 between the lysosome and autophagosome (9, 20). Mutation of serine 200 to a proline creates a 229 mutant, VopQ S200P , that is still able to neutralize the vacuole or lysosome, but can no longer 230 block fusion (9). This observation is likely due to reduced binding of VopQ S200P to the V-231
ATPase (16). To test if VopQ's activation of ERK1/2 MAPK signaling was caused by one or 232 both of these functions, we exchanged the chromosomal copy of the vopQ gene in the V. para 233 T3SS1 + strain with a version encoding VopQ S200P creating the V. para T3SS1 + vopQ S200P strain. 234
We tested the cytotoxicity of this strain during MEF infection and found that the VopQ S200P 235 mutant was no less lethal than wild-type VopQ (Fig. S3A) . However, unlike its parent strain, V. 236 para T3SS1 + vopQ S200P was unable to induce ERK1/2 phosphorylation and downstream 237 production of Egr1 in MEFs (Fig. 2E,F) . Notably, treatment of MEFs with chloroquine, a drug 238 that prevents lysosomal acidification, is not able to induce phosphorylation of ERK1/2 (Fig. 2E) . 239
These data suggest ERK1/2 MAPK signaling is not activated by lysosomal deacidification alone 240 and is instead dependent on VopQ's strong physical interaction with the V-ATPase. We 241 therefore propose two models by which VopQ could induce ERK1/2 MAPK signaling. In the 242 first, VopQ's inhibition of lysosome-autophagosome fusion directly activates ERK1/2 MAPK 243 signaling. In the second, VopQ manipulates another pathway upstream of both lysosome-244 autophagosome fusion and ERK1/2, thereby altering these two pathways in parallel. 245 246
To test these models, we aimed to identify a signaling pathway that could be upstream of both 248 autophagosome-lysosome fusion and ERK1/2 MAPK signaling. The unfolded protein response 249 has previously been linked to both of these processes through IRE1's connections to the UPR 250 and the ERAD pathway (21-24). Moreover, induction of ER stress with a proline analogue was 251 previously shown to partially stimulate IRE1-dependent ERK1/2 activation to promote cell 252 survival by an undefined mechanism (25). To examine if the UPR played a role, we tested if 253
VopQ's activation of ERK1/2 and downstream EGR1 production was dependent on any of the 254 three branches of UPR: IRE1, ATF6, or PERK(26). We infected IRE1 -/-MEFs, Atf6 -/-MEFs, (Fig. 3A,B) . Consistent with this observation, 260
accumulation of Egr1 was also not observed in IRE1 -/-MEFs infected with V. para T3SS1 + (Fig.  261   3A) . VopQ was still cytotoxic to IRE1 -/-MEFs (Fig. S5A) , consistent with the maintained 262 cytotoxicity of VopQ S200P in wild-type MEFs despite its inability to activate ERK1/2 (Fig. S3A) . 263
These data suggest that VopQ induces ERK1/2 phosphorylation by an IRE1-dependent 264
VopQ-mediated cytotoxicity was also conserved in the Atf6 -/and PERK -/-MEFs (Fig. S5B,C) . 266
Both the Atf6 -/and PERK -/cell lines exhibited the same pattern of ERK1/2 phosphorylation and 267
Egr1 accumulation upon T3SS1 + infection as wild-type MEFs (Fig. 3B) , indicating that VopQ's 268 activation of ERK1/2 was specifically IRE1-dependent. Finally, we stimulated wild-type, IRE1 -/-, 269
Atf6 -/and PERK -/-MEFs with fetal bovine serum (FBS) to assess whether the well described 270 growth factor-stimulated ERK1/2 MAPK signaling was functional in these cell lines (27). FBS-271 stimulated ERK1/2 phosphorylation and downstream Egr1 expression was observed in all cell 272 lines (Fig. 3C) . These data strongly support our model that VopQ's IRE1-dependent activation of 273 ERK1/2 occurs through pathway that is separate from the established growth factor-stimulated 274 pathway mediated by Ras and Raf (27). VopQ, a V-ATPase binding mutant VopQ S200P , and VopA were transformed into the BY4741 292 IRE1-GFP yeast strain. VopA is a V. para T3SS2 effector that kills yeast by a mechanism that is 293 distinct from VopQ and was included as a control (34). Upon galactose induction serial growth 294 assays showed that VopQ and VopA both inhibited growth in the BY4741 IRE1-GFP strain 295
while VopQ S200P and vector alone control did not (Fig. S6) . We then monitored IRE1p-GFP 296 clustering, or foci formation, at 30 and 45 minutes post galactose induction by confocal 297 microscopy. Treatment with dithiothreitol (DTT) for the same period was used as a positive 298 control for UPR stress. Yeast expressing VopQ, but not VopQ S200P or VopA induced IRE1p-GFP 299 foci formation (Fig. 3D) . IRE1p-GFP foci formation was observed, on average, in about 70% of 300 DTT treated cells compared to about 15% in cells expressing VopQ (Fig. 3E) . This difference 301 was expected because DTT treatment is homogeneous, whereas expression of VopQ is stochastic 302 (35). Our data indicate that expression of VopQ in yeast results in IRE1 activation. 303 304
We next asked if VopQ also activates IRE1 in mammalian cells during infection. IRE1 is 306 normally sequestered by BiP, but is released upon UPR activation when it then oligomerizes, 307 trans-autophosphorylates and activates its endoribonuclease activity, resulting in the non- 
Next, we wanted to further determine if the catalytic activities of IRE1 are required for VopQ-318 induced ERK1/2 signaling. IRE1 contains both protein kinase and an endoribonuclease domain 319 in its cytoplasmic region (33). To test if the kinase or endonuclease activity of IRE1 is required 320 for ERK signaling, MEFs were treated before V. para infection with KIRA6 or 4μ8c, an IRE1 321 specific kinase inhibitor and a potent inhibitor of IRE1 RNase activity, respectively (Fig. 4A) . By studying the function of two effectors of the T3SS1 of the seafood-borne pathogen V. para, 330
we have shown that T3SS effectors act together to systematically manipulate the host response. 331
We observe that the effector VopQ is responsible for the T3SS1-mediated activation of cell 332 survival and repression of cell death networks and another effector VopS is responsible for 333 dampening this response. Of note, VopS has been established to AMPylate and thereby 334 inactivate Rac, a known activator of MEK1/2 mediated ERK signaling(17). Furthermore, in 335
Vibrio alginolytcus, a Vibrio species closely related to V. para, VopS was found to be required 336
ATPase elicits early activation of ERK1/2 phosphorylation that is turned off by the delayed 338 temporal action of VopS (Fig. 4B) . 339
Manipulation of the V-ATPase to affect signaling in the cell has been previously observed with 340 oncogenic Ras, which induces a Rac-dependent plasma membrane ruffling and micropinocytosis. 341
However, in the case of oncogenic Ras, activation of Rac is dependent on the activity of the V-342
ATPase along with its relocation to the plasma membrane (40). Importantly, the activation 343 ERK1/2 MAPK signaling by VopQ does not occur through the growth factor inducible Ras-344 mediated pathway, but instead is dependent on the kinase activity of the ER stress sensor and 345 cell-fate executor, IRE1 (Fig. 4B) . Activation of ERK1/2 MAPK signaling specifically through 346 the IRE1-branch of the UPR by a bacterial effector has not previously been reported. The targeting of autophagy, UPR and MAPK signaling together is not unprecedented for 360 pathogens. For example, the mycotoxin Patulin was found to manipulate these pathways though 361 the inhibition of cathepsin B and cathepsin D, which leads to an accumulation of p62. Increased indicate that the kinase activity of IRE1 is required, but the mechanism by which IRE1 kinase 385 activity leads to ERK1/2 MAPK signaling is still poorly understood. Future experiments to 386 dissect the role of Nck and Rho GTPase activation in this process would be a valuable addition 387 to understanding this molecular mechanism of IRE1 induced, growth factor independent ERK1/2 388 MAPK signaling. 389 390
Bacterial strains and culture conditions 392
The Vibrio parahaemolyticus POR3 (POR1ΔvcrD2) and POR4 (POR1ΔvcrD1/vcrD2) strains 393 were generously provided by Drs. Tetsuya Iida and Takeshi Honda of Osaka University. Vibrio 394 strains were cultured at 30°C in MLB (Luria-Bertani broth +3% NaCl).All V. para strains except 395 V. para T3SS1 + vopQ S200P were from previous studies (Table S1 ). The T3SS1 + vopQ S200P strain 396 was created by cloning the vopQ S200P allele (9) flanked by the nucleotide sequences 1 kb 397 upstream and 1 kb downstream of vopQ (vp1680) into pDM4, a Cm R OriR6K suicide plasmid. E. 398 coli S17 ( pir) was used to conjugate the resulting plasmid into the POR3 strain and 399 transconjugants were selected on media containing 25mg/ml chloramphenicol. Bacteria were 400 then counter-selected on 15% sucrose and insertion of the vopQ S200P allele was confirmed by 401 All yeast genetic techniques were performed by standard procedures described previously (56). 414
All strains were cultured in either rich (YPD: 1% yeast extract, 2% peptone, and 2% dextrose) or 415 complete synthetic minimal (CSM) media (Sigma) lacking appropriate amino acids with 2% 416 dextrose, 2% raffinose, or 2% galactose. Yeast were serially diluted and spotted onto agar plates 417 to assay fitness and temperature sensitivity per standard technique. Yeast strains used in this 418 study were BY4741 (MATa his3∆0 leu2∆0 met15∆0 ura3∆0) and BY4741 IRE1-GFP (MATa 419 his3∆0 leu2∆0 met15∆0 ura3∆0 IRE1-GFP) (Thermo Fisher) as indicated. 420
Plasmids pRS416-Gal1-FLAG-VopQ and pRS416-Gal1-FLAG-VopQ S200P were generated by 421 sub-cloning pRS413-Gal1-VopQ and pRS413-Gal1-VopQ S200P (previously published (9)) into 422 the BamHI and EcoRV sites of pRS416-Gal1. Plasmid pRS416-Gal1-VopA-FLAG was 423 generated by sub-cloning pRS413-Gal1-VopA-FLAG (previously published (34)) into the EcoRI 424
and XhoI sites of pRS416-Gal1. 425 426
For RNA-sequencing, PHDFs were seeded onto 6-well plates at a density of 1x10 5 Cell Signaling Technologies (CST) p44/42 MAPK (137F5) and P-p44/42 MAPK T202/Y204 470 (197G2) primary antibodies, respectively. EGR1 was detected using CST EGR1 (15F7) and -471 actin was detected by Sigma-Aldrich A2228 Monoclonal Anti--actin. Total IRE1 and 472
Phospho-IRE1 were detected by CST IRE1 (14C10) and Novus IRE1 (pSer724), 473 respectively. -tubulin was detected by Santa Cruz -tubulin (B-7). Secondary HRP-conjugated 474 antibodies used were donkey anti-rabbit (GE Healthcare) and goat anti-mouse (Sigma-Aldrich). 475 476
Yeast strains were grown to mid-log phase (OD 600 ~0.5) in CSM media lacking uracil with 2% 478 raffinose. Cultures were then treated with 2% galactose, 2% raffinose, or 2% raffinose + 5mM 479 DTT for 30 or 45 minutes. Cultures were collected, resuspended in 1X PBS, and fixed with 4% 480 paraformaldehyde. Confocal images were acquired using a Zeiss LSM800 with Zen software. 481
Images were processed with ImageJ (National Institutes of Health) and Adobe Photoshop CS6. 482 Table S1 . Bacterial strains used in this work. 506 Table S2 . Complete RNA sequencing Differential Expression Data. 507 Table S3 . Differential Expression of T3SS1-specific genes. 508 Table S4 . IPA Canonical Pathway Analysis of Differential Expression Data. 509 Table S5 . IPA Network (Disease and Biofunction) Analysis of Differential Expression Data. 510 Table S6 . Expression of alternate MAPK pathway target genes 511 Table S7 . SyntTax VopQ and VopS cooccurence in Vibrio genomes with T3SS1 Synteny. 