The Salmonella effector protein SopA is translocated into host cells via the SPI-1 type III secretion system [TTSS] and contributes to enteric disease . We found that the chaperone InvB binds toSopA and slightly stabilizes it in the bacterial cytosol andthat it is required for its transport via the SPI-1 TTSS.

 
Type III secretion systems [TTSS] are found in many pathogenic gram-negative bacteria and mediate the injection of an arrayof effector proteins into the host cell cytoplasm . Once injected,the effector proteins modulate host cell signaling cascadesfor the benefit of the pathogen . The secretion mechanism ofthese effector proteins and their secretion signals are stillpoorly understood . It has been shown that secretion and translocationof many effector proteins require a cognate chaperone [9, 17]. These chaperones usually bind to the N-terminal region and exert various functions on their cognate effector protein, i.e., cytosolic stability [10, 20, 21], transcriptional regulation [4, 5, 19],prevention of premature interactions [7, 12, 13], maintenanceof the effector in a secretion-competent state [18, 21], andrecognition by the TTSS [1] . Type III secretion [TTS] chaperonesdo not exhibit sequence similarities but share some common features.They are generally small, acidic proteins with an amphipathicC-terminal -helix and are often encoded next to or in closevicinity to the effector protein [9, 17] . In contrast, the chaperone Spa15 of Shigella spp . is encoded within an operon encoding essential components of the TTS apparatus and binds to not just one but several effector proteins which do not show sequence similarities [16] . Due to these special features, Spa15 is thoughtto represent a new class of TTS chaperone [16, 17].

The Salmonella pathogenicity island 1 [SPI-1] of Salmonella enterica serovar Typhimurium encodes the protein InvB, which is homologous to Spa15 of Shigella spp . InvB is a chaperone for the SPI-1-encoded effector SipA/SspA [2] . Recently membersof our group have shown that InvB also binds to SopE and SopE2,two effector proteins encoded outside of SPI-1 but secreted in a SPI-1-dependent manner [7a] . Secretion and translocationof SipA, SopE, and SopE2 depend on InvB . Based on this observation,we hypothesized that InvB might be required for secretion ofadditional effector proteins of serovar Typhimurium.

To address this question, we expressed a glutathione S-transferase [GST]-InvB fusion protein [pM672] [7a] in the mutant strainM574 [invB::aphT sopE sopE2 sopB sipA] [7a], which lacks allknown InvB binding effector proteins and the chromosomally encodedinvB . This strain also lacks the effector protein gene sopB.However, SopB/SigD is transported via its own cognate chaperone,PipC/SigE [6] . Therefore, the sopB mutation was not expectedto affect any InvB-effector protein interactions . M574 [pM672]was grown overnight in Luria broth containing 0.3 M NaCl, diluted1:20 into fresh medium, and grown for another 4 h at 37°C[referred to as SPI-1 inducing conditions] . Cells were lysedin a French pressure cell, and GST-InvB and bound proteins werepurified on glutathione-Sepharose beads from the cleared celllysate . Aliquots from every step of the purification procedurewere analyzed on a Coomassie brilliant blue-stained SDS gel.A polypeptide with an apparent molecular weight of 80 kDa was copurified with GST-InvB [Fig . 1] . The band was excised fromthe gel, trypsin digested, and eluted as described recently [7a] . The protein was identified by matrix-assisted laser desorptionionization-mass spectrometry fingerprint analysis as SopA [12matching peptides, 21% covered sequence], a known effector protein,which is encoded outside of SPI-1 but translocated in a SPI-1-dependentmanner [22] . Although the biochemical activity of SopA is stillunknown, it was shown to play a role in bovine enterocolitismodels [22, 23].

 
The binding of SopA to InvB was verified by a coimmunoprecipitation experiment . For this purpose, a suicide vector [pM261] encodinga C-terminally M45-tagged version of sopA was integrated into the chromosome of SL1344 to generate M612, which expresses sopA_M45 under its native promoter [Table 1].

 
M612 was grown under SPI-1 inducing conditions and lysed ina French pressure cell . SopA_M45 binding proteins were precipitated with a mouse monoclonal anti-M45 antibody from cleared bacterial lysates as described previously [7a] . Aliquots from every stepof the precipitation procedure were analyzed by Western blotting using a polyclonal anti-InvB antiserum [7a] and a mouse monoclonalanti-M45 antibody [14] . InvB was coimmunoprecipitated with SopA_M45from M612 but not from the control lysate of an isogenic strain[M712] lacking amino acids 2 to 782 of sopA [Fig . 2, lane f].This result supports the notion that InvB binds [directly orindirectly] to SopA.

 
InvB has been described as a chaperone necessary for secretionof the effector proteins SipA [2], SopE, and SopE2 [7a] . Thissuggested that InvB might also be required for secretion of SopA . To explore this hypothesis, we have constructed the isogenic serovar Typhimurium ATCC 14028 strains M619 [wild type] [2], M618 [invB] [2], and M623 [invC::aphT], which all harbor an M45 epitope-tagged sopA gene in the chromosome [Table 1] . Thestrains were grown under SPI-1 inducing conditions, and SopA_M45secretion was analyzed by Western blotting as described elsewhere[7a] . SopA_M45 was secreted from the wild-type strain M619 butnot from the secretion-deficient strain M623 [invC::aphT], lacking the ATPase InvC [8], and the invB strain M618 [Fig . 3, upperpanel] . The latter secretion defect could be complemented usingthe invB expression vector pM250, which expresses invB undercontrol of an arabinose-inducible promoter [7a] . Reprobing with a rabbit polyclonal antiserum raised against amino acids 49to 543 of the SPI-1 effector protein SptP verified that theinvB deletion had no general effect on the TTSS [Fig . 3, middle panel].

 
The cytoplasmic SopA_M45 pool was slightly lower in the invB strain M618 than in the wild-type strain, M619 [Fig . 3] . Thisindicated that InvB might play a role in stabilization or expressionof SopA . To examine the cytoplasmic stability of SopA_M45, onehas to consider that InvB might have two functions: stabilization of cytoplasmic SopA and transport of SopA via the SPI-1 TTSS. If significant amounts of SopA protein become transported tothe outside during the course of the assay, this fraction mightbecome protected from degradation by Salmonella proteases . Toexclude this, we have analyzed the role of invB in stabilizationof cytoplasmic SopA_M45 in secretion-deficient strains . The invB open reading frame is overlapping with the invC open reading frame . Therefore, it was not possible to combine the invB andinvC::aphT alleles [Fig . 3; Table 1] by P22 transduction . Forthis reason we constructed the secretion-deficient spaO::aphTstrain [M629], lacking an essential subunit of the export apparatus[3] encoded 2.7 kb downstream of invB, and the invB spaO::aphTdouble mutant [M630].

We then analyzed the cytosolic stability of SopA_M45 in the invB strain M618 [sopA_M45 invB; does not secrete SopA_M45 [Fig . 3]],the secretion-deficient mutant M629 [sopA_M45 spaO::aphT], andthe double mutant M630 [sopA_M45 spaO::aphT invB] [Table 1].As a control we also examined the cytosolic stability of SopA_M45in the wild-type strain M619 [sopA_M45] . M619, M618, M629, andM630 were grown under SPI-1 inducing conditions, and proteinbiosynthesis was inhibited by addition of spectinomycin [finalconcentration, 200 µg/ml] . Aliquots were removed 0, 5,20, 40, and 90 min after spectinomycin addition . Western blotanalysis of bacterial pellets revealed that SopA_M45 degradationwas slightly accelerated in the absence of InvB [Fig . 4A, compareM629 and M630 or M618 and M629] . In the wild-type strain background[M619], the amount of bacterium-associated SopA_M45 was slightly higher than in the secretion-deficient strain M629 at the beginning of the experiment [0' to 20'] but decreased faster [Fig . 4A]. As discussed above, this is probably due to cumulative effects of secretion of SopA_M45 from M619 into the culture supernatant and degradation . For this reason we could not base any conclusions about the role of InvB in SopA stabilization on this strain.

 
The stability of the effector protein SptP [cognate chaperoneis SicP] was not affected by the invB mutation [Fig . 4] . Altogether,these data suggested that InvB has a slight effect on stabilizationof SopA in the bacterial cytosol.

To verify that the invB expression level is not altered in the secretion-deficient mutants, we performed a Western blot analysis using the strains M619 [sopA_M45], M618 [sopA_M45 invB], M629[sopA_M45 spaO::aphT], M630 [sopA_M45 spaO::aphT invB], and M623[sopA_M45 invC::aphT] [Table 1] . This analysis confirmed thatthe amount of cytosolic InvB is not altered in the secretion-deficientmutants M629 [sopA_M45 spaO::aphT] and M623 [sopA_M45 invC::aphT]and that InvB is absent from the invB deletion strains M618[sopA_M45 invB] and [sopA_M45 spaO::aphT invB] [Fig . 5].

 
To analyze whether invB reduces transcription of sopA, we constructedlacZ transcriptional reporter strains . The lacZ cassette ofpSB1040 [kindly provided by D . Zhou and J . E . Galán] was cloned downstream of the sopA_M45 stop codon into pM261 [seeabove] to create the suicide vector pM265 [Table 1] . pM265 wasintegrated into the chromosome of CS401, yielding M635 [sopA_M45lacZ] . The control strains M636 [invB], M637 [spaO::aphT], M638[invB spaO::aphT], and M639 [invC::aphT] were constructed by P22 transduction of the sopA::pM265 allele [Table 1].

Thus, we could use ß-galactosidase assays to studysopA promoter activity . The ß-galactosidase activitywas determined in at least eight independent experiments, andstatistical analysis was performed using the exact Mann-WhitneyU test . We found that sopA transcription was in the same orderof magnitude for all strains, analyzed [Fig . 6] . Disruptionof invB did not decrease ß-galactosidase activity.Rather, ß-galactosidase activity was slightly butsignificantly increased in M636 [invB] [P < 0.001], M639[invC::aphT] [P = 0.001], and M638 [invB spaO::aphT] [P <0.001] [Fig . 6] . Therefore, the decreased SopA_M45 protein levelsin the cytoplasm of an invB mutant [Fig . 3] are attributable to a slightly decreased protein stability but not to transcriptional down regulation . However, the reasons for the slight augmentation of transcription in M636, M638, and M639 remain to be analyzed.

 
In summary, the copurification and coimmunoprecipitation experiments demonstrate that InvB binds directly or indirectly to SopA.In the absence of InvB, SopA is not secreted and its intracellularstability is decreased . Although SipA, SopE/SopE2, and SopAdo not share sequence similarities, they all require InvB fortheir transport via the SPI-1 TTSS [2, 7a; also this study].

TTS chaperones have been divided into three classes: class I, chaperones which associate with effector proteins; class II, chaperones which associate with translocators; and class III, chaperones of the flagellar system [17] . Due to their unique features, InvB and its homologs Spa15 [Shigella spp.], YsaK [Yersinia spp.], and InvB [Sodalis spp.] are thought to representa new family of TTS chaperones . Therefore, they have been assignedto the new subclass IB, which represents chaperones that bindseveral different effectors [17] . This classification was basedon experimental evidence from Shigella flexneri [16] . Interestingly,Page and Parsot have hypothesized that InvB, like Spa15, mightalso associate with different unrelated proteins [15] . Thiswas confirmed by our findings that InvB is a chaperone not onlyfor SipA [2] but also for SopE, SopE2 [7a], and SopA [this work].

 
We thank Günther Paesold, Markus Schlumberger, and CosimaPelludat for critically reviewing the manuscript, Samuel I.Miller for providing strains, Shiva P . Singh for providing theanti-OmpC antibody, and Rene Brunisholz for the matrix-assistedlaser desorption ionization-mass spectrometry analysis.

The project was funded in part by the Swiss National Foundation.

 
* Corresponding author . Mailing address: Institute of Microbiology, ETH Zürich, Schmelzbergstrasse 7, 8092 Zürich, Switzerland . Fax: 41-1-632-1129 . Phone: 41-1-632-5143 . E-mail: hardt@micro.biol.ethz.ch.

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