Abstract
We employed a heterologous secretion assay to identify proteins potentially secreted by type III secretion systems (T3SSs) in Vibrio parahaemolyticus. N-terminal sequences from 32 proteins within T3SS genomic islands and seven proteins from elsewhere in the chromosome included proteins that were recognized for export by the Yersinia enterocolitica flagellar T3SS.
TEXT
Vibrio parahaemolyticus is a Gram-negative, food-borne pathogen that encodes two type III secretion systems (T3SS) (11). T3SS1 induces host cell autophagy (7, 23) and oncosis (22); T3SS2 is involved in enterotoxicity (14, 15); a flagellar system exists and could also be involved in secretion. Identifying secreted effector proteins is important to understand how T3SS contribute to pathogenesis. T3SS effector proteins typically include a “leader sequence” that signals export, a chaperone binding sequence, and a catalytic domain (1, 10). The leader sequence generally occurs within the first 30 amino acid residues (2–4, 8, 12, 16, 17), and while it is difficult to develop heuristic rules to identify leader sequences (5, 18), heterologous expression assays can be used to identify sequences of interest (6, 8, 20).
Christensen et al. developed a heterologous expression assay to identify leader sequences from Campylobacter jejuni (8). Chimeras were constructed between leader sequences from C. jejuni genes and a leaderless phospholipase gene (yplA). The chimeras were then transformed into a Yersinia enterocolitica ΔyplA strain (8). If the cloned sequence contained a recognizable T3SS leader sequence, then the chimeric protein was secreted by the Y. enterocolitica flagellar T3SS. Secreted YplA hydrolyzed Tween 80 contained in the indicator medium to produce a fatty acid precipitate that was easily detected by visual inspection. Christensen et al. screened 321 potential leader sequences, and 42 were recognized by the Y. enterocolitica T3SS (8).
To determine if sequences from V. parahaemolyticus are also recognized by Y. enterocolitica, we constructed chimeras for six genes using ∼100 bp of the N-terminal region with yplA and assayed for YplA secretion (Fig. 1) (8). TYE indicator plates (1% tryptone, 0.5% yeast extract, 1.5% agar, 1% Tween 80, and 1 mM CaCl2) permit activation of the flagellar T3SS (21). A precipitate was observed around strains with sequences from vp1680, vp1677, vpa1346, vp1656, and vp1657, indicating successful secretion. Both Vp1656 (24) and Vp1680 (13) are known to be secreted by V. parahaemolyticus. We included a sequence from a hypothetical chaperone (vp1684) and did not observe secretion as expected. Leader sequences from three uncharacterized proteins (Vp1657, homologous to YopB in Yersinia; Vp1677, a hypothetical protein; and Vpa1346, homologous to YopP in Yersinia) were positive for secretion. Positive secretion for both T3SS1-encoded proteins (Vp1656, Vp1657, Vp1680, and Vp1677) and the T3SS2-encoded protein (Vpa1346) demonstrated that this assay is suitable to identify proteins that are potentially secreted by V. parahaemolyticus.
Fig 1.
Putative leader sequences from V. parahaemolyticus genes vp1656, vp1657, vp1677, vp1680, vp1684, and vpa1346 were cloned in fusion with a leaderless yplA gene in the vector pCSP50. The leader sequence from ciaB (Campylobacter jejuni) was cloned in fusion with yplA as a positive control, and the leader sequence from cysM was included as negative control (8). Constructs were conjugated from E. coli S17 to a ΔyplA strain of Y. enterocolitica before being spot plated onto TYE medium. The precipitate (highlighted with bars) around the colonies was indicative of positive secretion.
We subsequently applied this assay to examine other open reading frames that are located in the T3SS1 and T3SS2 genomic islands. Potential leader sequences from 42 loci were PCR amplified and cloned upstream of yplA (8). Fifteen of the 42 cloned sequences were negative for secretion, while nine produced weak signal. Robust precipitate was observed for the remaining 18 clones. VopS (Vp1686), a known T3SS1-secreted protein (13), produced a clearly visible precipitate, while another known T3SS1-secreted protein (Vp1659) (23) produced a weaker response (Table 1).
Table 1.
YplA secretion assay results for putative leader sequences from the Vibrio parahaemolyticus T3SS1 (vp prefix) and T3SS2 (vpa prefix) genomic islands
| Gene name | Precipitate at 48 h on TYE platea | Predicted protein (11) |
|---|---|---|
| vp1656 | Positive | VopD |
| vp1657 | Positive | VopB |
| vp1658 | Positive | Low-calcium response locus protein H |
| vp1663 | Positive | Putative YscY |
| vp1664 | Positive | Putative type III secretion protein |
| vp1669 | Positive | Putative type III secretion protein YscO |
| vp1670 | Positive | Putative translocation protein in type III secretion |
| vp1677 | Positive | Hypothetical protein |
| vp1678 | Positive | Putative dienelactone hydrolase and related enzymes |
| vp1680 | Positive | VopQ |
| vp1683 | Positive | Hypothetical protein |
| vp1686 | Positive | VopS |
| vp1691 | Positive | Type III export protein |
| vp1692 | Positive | Putative type III export protein |
| vp1694 | Positive | Type III export protein YscF |
| vpa1336 | Positive | Hypothetical protein |
| vpa1345 | Positive | Hypothetical protein |
| vpa1346 | Positive | VopP |
| vpa1349 | Positive | Putative type III secretion protein Spa33 |
| vpa1350 | Positive | Hypothetical protein |
| vpa1360 | Positive | Hypothetical protein |
| vpa1361 | Positive | Hypothetical protein |
| vpa1362 | Positive | Putative secreted protein EspD |
| vp1659 | Weak | Putative LcrV |
| vp1682 | Weak | Hypothetical protein |
| vp1688 | Weak | Putative type III secretion protein |
| vp1693 | Weak | Putative type III secretion protein |
| vp1695 | Weak | Putative type III export protein PscD |
| vp1698 | Weak | ExsD |
| vpa1351 | Weak | Hypothetical protein |
| vpa1352 | Weak | Hypothetical protein |
| vpa1370 | Weak | Hypothetical protein |
| vp1661 | Negative | Putative LcrR |
| vp1665 | Negative | Putative type III secretion protein |
| vp1671 | Negative | Putative translocation protein in type III secretion |
| vp1672 | Negative | Translocation protein in type III secretion |
| vp1674 | Negative | Translocation protein in type III secretion |
| vp1675 | Negative | Translocation protein in type III secretion |
| vp1684 | Negative | Hypothetical protein |
| vp1689 | Negative | Putative type III secretion protein |
| vp1696 | Negative | Putative type III secretion protein YscC |
| vp1697 | Negative | Putative type III export apparatus protein NosA |
| vpa1339 | Negative | Putative type III secretion system EscC protein |
| vpa1342 | Negative | Putative type III secretion protein Spa24 |
| vpa1354 | Negative | Putative type III secretion system EscU protein |
| vpa1356 | Negative | Hypothetical protein |
| vpa1359 | Negative | Hypothetical protein |
| vpa1367 | Negative | Putative type III secretion system lipoprotein precursor EprK |
A precipitate zone was classified as “weak” if it was not visible by 24 h but visible by 48 h. Positive secretion was evident by a zone of precipitate after 24 h, and negative secretion indicates no precipitate after 48 h.
Among a total of 32 secretion-positive leader sequences (23 positive and 9 weakly positive), 21 and 11 were from genes carried on the T3SS1 and T3SS2 genomic islands, respectively. Sixteen of the T3SS1 genes encode potential homologues for structural or translocator proteins identified for Yersinia. Potential homologues for the remaining five proteins (Vp1678, Vp1680, Vp1682, Vp1683, and Vp1686) are not present in the T3SS of Yersinia. Among the 11 potential secretion proteins encoded by the T3SS2 island, eight proteins are hypothetical while the remaining three (Vpa1346, Vpa1349, and Vpa1362) are homologous to the structural or effector proteins present in other bacteria (9).
Because T3SS effector proteins can be distributed throughout the genome, we adapted the YplA assay to screen a whole-genome library for leader sequences. V. parahaemolyticus NY-4 genomic DNA was extracted and partially digested with 6 U, 1.5 U, or 0.75 U of Sau3AI (Promega, Madison, WI) for 15 min at 37°C. The DNA was then purified and ligated into three yplA expression vectors (representing three translation frames) that had been previously digested with BglII (Promega). Ligated products were transformed into Escherichia coli S17-λpir, and transformants were transferred into 384-well plates with 50 μl/well LB containing 10 μg/ml tetracycline (using a Genetix Qpix robot). Plasmids from the library were transferred into a Y. enterocolitica ΔyplA strain by conjugation on nonselective agar plates followed by selection for transformants on agar plates containing both nalidixic acid and tetracycline (nalidixic acid was used to select against E. coli, and tetracycline was used to select against plasmid-free Yersinia). The YplA assay was then used to identify insert sequences that functioned as secretion signals.
We screened approximately 136,000 clones and detected 127 positive clones of which 44 were confirmed as positive (2 times) for secretion and for containing in-frame insertions. Removing duplicated insert sequences left 38 clones, of which 26 had >45-bp sequences upstream from yplA and were not previously identified as T3SS substrates. Of these, half were then subjected to independent cloning and testing with the secretion assay. Ten of these clones had multiple inserts (chimeras) in the plasmid, but in these cases, the sequence that was in frame with the yplA gene was considered responsible for positive secretion. In total, this analysis identified seven unique loci with putative export signals (Table 2).
Table 2.
Loci from V. parahaemolyticus identified by genome-wide screening as having T3SS-compatible leader sequences
| TYE zone sizea | Multiple insertsb | Insert sequence | No. of in-frame nucleotides | Predicted protein (11) | GenBank IDc |
|---|---|---|---|---|---|
| 10 (10) | N | vp0873 | 733 | Septum site-determining protein MinD | JF913481 |
| 12 (11.5) | N | vp2162 | 258 | Hypothetical protein | JF913479 |
| 8 (7) | N | vp2633 | 315 | Putative phospho-beta-glucosidase | JF913474 |
| 9 (10) | Y | vp2650 | 103 | Conserved hypothetical protein | JF913475 |
| 9 (9) | Y | vpa0262 | 136 | LfgM | JF913480 |
| 8 (8) | N | vpa0273 | 201 | Putative flagellar hook-associated protein | JF913476 |
| 8 (9) | Y | vpa1744 | 301 | Putative phosphotransferase system, sucrose-specific IIBC component | JF913486 |
Zone size on TYE plate indicates halo diameter (mm) after incubation for 24 h or at 48 h (in parentheses).
Multiple inserts indicate evidence for chimeric sequences, but only the loci identified in the table were in frame with yplA. Y, yes; N, no.
Sequences associated with vp1656 (JF913482), vpa1421, and vpa1744 were recovered twice, and vpa0262 was recovered three times in the library. Sequence from vpa1361 (JF913484) (Table 1) was also recovered. Six other clones were positive by initial screening but failed to produce a halo after putative export regions were independently cloned and screened using the YplA assay (GenBank identifiers [IDs] JF913473, JF913477, JF913478, JF913483, JF913485, and JF913487).
Two of the export sequences (vpa0273 and vpa0262) are associated with the flagella. Both vp2633 and vpa1744 are interesting findings because of the possible role of T3SS in nutrient acquisition during infection. For example, Vpa1744 is considered part of a 3-component system used for carbohydrate transport and metabolism (19). Secretion of Vpa1744 is consistent with modification of sugar substrates in preparation for transport through the EIIC domain that spans the cell membrane.
Our strain of Y. enterocolitica does not express the flagellar T3SS when cultured at 37°C, and we confirmed that there was no evidence of secretion for the 15 clones when cultured at this temperature. As further confirmation, we PCR amplified the first ∼90 N-terminal nucleotides for the unique clones and made chimera constructs with yplA. Screening on indicator plates showed that seven clones were positive for secretion using this final confirmation (Table 2).
Our findings demonstrate that T3SS leader sequences from V. parahaemolyticus are recognized by the Y. enterocolitica flagellar T3SS. From a validation perspective, several known T3SS-secreted proteins were identified in this study (Vp1659, Vp1680, Vp1686, Vp1656, and Vpa1370) while two flagellum-associated export sequences were also identified (vpa0262 and vpa0273). We also demonstrated that the leader sequence for Vpa1346, a homologue of an extensively studied effector protein (YopP) in Yersinia, was secreted in the YplA assay. While several recognized substrates were identified in this study, false negatives would occur if fusion proteins interfered with export or function of the phospholipase. This problem is more likely to occur with the genomic screening protocol, where there is no control over the size of the fusion protein.
ACKNOWLEDGMENTS
This project was funded in part by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract number NO1-AI-30055, and by the Agricultural Research Center, Washington State University.
Footnotes
Published ahead of print 2 March 2012
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