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Demarcating SurA Activities Required for Outer Membrane Targeting of Yersinia pseudotuberculosis Adhesins Ikenna R. Obi and Matthew S. Francis Infect. Immun. 2013, 81(7):2296. DOI: 10.1128/IAI.01208-12. Published Ahead of Print 15 April 2013.

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Demarcating SurA Activities Required for Outer Membrane Targeting of Yersinia pseudotuberculosis Adhesins Ikenna R. Obi, Matthew S. Francis Department of Molecular Biology and Umea˚ Center for Microbial Research, Umea˚ University, Umea˚, Sweden

T

he Gram-negative bacterial outer membrane (OM) acts as a permeability and protective barrier to the external environment (1, 2). This asymmetric phospholipid and lipopolysaccharide (LPS) bilayer also acts as a scaffold for two major classes of membrane proteins: the integral outer membrane proteins (OMPs) and the lipoproteins (3). Integral OMPs are often referred to as ␤-barrel proteins and are assembled into the OM by the ␤-barrel assembly machinery (BAM) (4, 5, 6, 7, 8, 9). The BAM complex is composed of five proteins: BamA, which is itself an integral OMP, and the four lipoproteins BamB, BamC, BamD, and BamE, which localize to the inner leaflet of the OM (5, 6). The biogenesis of OMPs initiates in the bacterial cytoplasm and encompasses their translocation across the inner membrane through the aqueous periplasm to the OM site for assembly (3, 10, 11, 12). The passage through the periplasm requires protein quality control factors that assist in their piloting and folding, thereby preventing aggregation and inappropriate interactions with other proteins (13). These protein folding factors include peptidylprolyl cis/trans isomerases (PPIases), Skp, DegP, and disulfide bond isomerases. In Escherichia coli and related enteric bacteria, five periplasmic PPIases have been described: SurA, PpiD, PpiA, FkpA, and FklB (14, 15, 16). Among these proteins, SurA is particularly well studied. As an important folding factor, SurA is involved in the folding of many ␤-barrel OMPs (17, 18) and has been implicated as the major factor that traffics and targets OMPs to the BAM complex (5). Bacteria devoid of surA display OM perturbations that include aberrant cellular morphology, drastic alterations in OMP profile, susceptibility to detergents and antibiotics, and leakiness of LPS into the extracellular environment (16, 17). In addition, these bacteria display altered fatty acid and phospholipid compositions in agreement with a defective OM (16). Yersinia pseudotuberculosis is an enteropathogen commonly transmitted to humans through contaminated food or liquid (19, 20). This pathogen causes a gastrointestinal disorder that is usu-

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ally self-limiting and rarely results in a systemic disease (19). Important for Yersinia virulence is a plasmid-encoded Ysc-Yop type three secretion (T3S) system, which delivers toxic effector proteins from the bacterium into the cytosol of eukaryotic host cells (21, 22). In order to activate the type three secretion system (T3SS), bacterium-host cell contact is essential. To facilitate this, Yersinia produces several surface-localized adhesins (23). Most notable Yersinia adhesins are invasin, Ail, YadA, and the pH 6 antigen, incorporating the PsaA-dependent fibrillum. Invasin is an important enteropathogenic Yersinia adhesin that engages ␤-1 integrins on the host cell surface (24, 25). Although its secondary structure deviates from the classical autotransporter (AT) consensus, the fact that invasin anchors to the outer membrane with its N-terminal ␤-barrel domain is suggestive that invasin export occurs via type V secretion (26). A second important Yersinia adhesin is Ail, a small bacterial surface-associated protein that localizes in the OM. In addition to promoting the binding of Yersinia to host cells, Ail also contributes to serum resistance and the inhibition of host inflammatory responsiveness (27, 28, 29, 30). Another important Yersinia adhesin, PsaA, is a fimbrillar protein that plays an important role in Y. pseudotuberculosis binding to host cells (31). This protein is preferentially produced when Yersinia is grown at 37°C and can bind ␤1-linked galactosyl resi-

Received 31 October 2012 Returned for modification 4 December 2012 Accepted 4 April 2013 Published ahead of print 15 April 2013 Editor: J. B. Bliska Address correspondence to Matthew S. Francis, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.01208-12. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.01208-12

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SurA is a periplasmic protein folding factor involved in chaperoning and trafficking of outer membrane proteins across the Gram-negative bacterial periplasm. In addition, SurA also possesses peptidyl-prolyl cis/trans isomerase activity. We have previously reported that in enteropathogenic Yersinia pseudotuberculosis, SurA is needed for bacterial virulence and envelope integrity. In this study, we investigated the role of SurA in the assembly of important Yersinia adhesins. Using genetic mutation, biochemical characterization, and an in vitro-based bacterial host cell association assay, we confirmed that surface localization of the invasin adhesin is dependent on SurA. As a surA deletion also has some impact on the levels of individual components of the BAM complex in the Yersinia outer membrane, abolished invasin surface assembly could reflect both a direct loss of SurA-dependent periplasmic targeting and a potentially compromised BAM complex assembly platform in the outer membrane. To various degrees, the assembly of two other adhesins, Ail and the pH 6 antigen fibrillum PsaA, also depends on SurA. Consequently, loss of SurA leads to a dramatic reduction in Yersinia attachment to eukaryotic host cells. Genetic complementation of surA deletion mutants indicated a prominent role for SurA chaperone function in outer membrane protein assembly. Significantly, the N terminus of SurA contributed most of this SurA chaperone function. Despite a dominant chaperoning role, it was also evident that SurA isomerization activity did make a modest contribution to this assembly process.

Yersinia Adhesin Assembly Requires SurA

TABLE 1 Bacterial strains and plasmids used in this study Strain or plasmid Strains E. coli DH5 Y. pseudotuberculosis YPIII/pIB102 YPIII21/pIB102 YPIII73/pIB102 YPIII80/pIB102 YPIII/pIB619 Plasmids pWKS30 pIR005 pIR019

pIR021

Source or reference

F⫺ recA1 endA1 hsdR17 supE44 thi-1 gyrA96 relA1

Vicky Shingler

yadA::Tn5 (parent), inactive PhoP, Kmr surA in-frame deletion of codons 26 to 411, Kmr ppiA ppiD fklB fkpA in-frame deletion, Kmr surA ppiA ppiD fklB fkpA in-frame deletion, Kmr yopB and yopD in-frame deletion, Kmr

Hans Wolf-Watz 16 16 16 50

Low-copy-no. cloning plasmid, Cbr ⬃2,200-bp XbaI-XhoI PCR fragment of surA (full-length SurA) with its native promoter in pWKS30, Cbr ⬃1,849-bp XbaI-XhoI PCR fragment of surA allele (⌬N domain lacking amino acids 25 to 142) with intact surA native promoter in pWKS30, Cbr ⬃2,200-bp XbaI-XhoI PCR fragment of surA allele (His376Ala, Ile378Ala) with intact surA native promoter in pWKS30, Cbr ⬃1,573-bp XbaI-XhoI PCR fragment of surA allele (⌬P domains lacking amino acids 175 to 384) with intact surA native promoter in pWKS30, Cbr

Sidney Kushner 16 This study

dues in glycosphingolipids and phosphatidylcholine on the surface of host cells (32, 33). Exactly how these Yersinia adhesive proteins are targeted and folded into the OM is poorly understood. Limited information is available on which periplasmic proteins assist in their transport across the periplasm. Since a Y. pseudotuberculosis ⌬surA mutant is avirulent in a mouse infection model and also displays a reduction of proteins in the outer membrane, this study sought to investigate the role of SurA in the surface localization and assembly of invasin, Ail, and PsaA in Yersinia. We demonstrated that SurA is particularly required for proper assembly of invasin in the OM, although steady-state levels of Ail and PsaA are also compromised by loss of SurA. Defective assembly of these proteins leads to reduced Yersinia association with eukaryotic host cells. MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are summarized in Table 1. Y. pseudotuberculosis YPIII/pIB102 (serotype III) was used as the parental strain. This strain contains a Ysc-Yop T3SS encoded on the virulence plasmid pIB102, which is a variant of pIB1 having a kanamycin resistance cassette inserted into the yadA gene (34). Unless stated otherwise, cultivation of bacteria was performed in Luria-Bertani (LB) agar or broth at either 26°C (Y. pseudotuberculosis) or 37°C (E. coli), with aeration. When required, antibiotics were used at the following final concentrations: 50 ␮g/ml kanamycin, 25 ␮g/ml chloramphenicol, and 100 ␮g/ml carbenicillin. To assay for adhesin production, overnight cultures of Y. pseudotuberculosis grown either in LB broth at 26°C in the case of invasin and Ail or in brain heart infusion (BHI, Oxoid) supplemented with 2.5 mM CaCl2 at 37°C in the case of PsaA were subcultured (0.05 volume) into 2 ml fresh medium and were allowed to grow to an absorbance at 600 nm (A600) of ⬃0.75. After standardization and centrifugation of bacterial cultures, pellets were either resuspended in 1⫻ sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 5% ␤-mercaptoethanol, and 0.1% bromophenol blue) prior to SDS-polyacrylamide gel electrophoresis (SDSPAGE) or used in a cellular fractionation experiment. To assay for in vitro T3S, overnight cultures of Yersinia grown at 26°C in BHI supplemented with either 5 mM EGTA and 20 mM MgCl2 (medium without Ca2⫹) or 2.5 mM CaCl2 (medium with Ca2⫹) were subcultured into fresh medium and grown for 1 h at 26°C. To induce T3S-

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associated protein synthesis and secretion, the temperature of cultivation was then shifted to 37°C for 3 h. Bacterium-free supernatants containing secreted protein was treated with 4⫻ sample buffer and then analyzed by SDS-PAGE and Western blotting. Generation of surA complementation constructs. To generate the alleles coding for different SurA domains, Y. pseudotuberculosis YPIII/ pIB102 boiled lysate was used as a source of genomic template to amplify DNA fragments by overlap PCR. Every amplified surA allelic variant included endogenous upstream promoter elements. Primers used to generate each allele are listed in Table S1 in the supplemental material and were synthesized by Sigma-Aldrich Sweden AB (Stockholm, Sweden). Amplified fragments were cloned directly into pWKS30 and then confirmed by DNA sequencing (Eurofins MWG Operon, Ebersberg, Germany). Whole bacterial lysates and cellular fractionation. Generation and fractionation of bacterial cell lysates were performed as previously described (16). Initially, the robustness of the purification procedure was confirmed on parental material using OmpA and H-NS as protein markers for the OM and cytoplasmic fractions. When comparing with material sourced from mutant bacteria, we then routinely probed for DnaJ levels in the initial total cell extract and cytoplasmic fractions to confirm equal loading of protein in each lane. We relied on this information to control for loading of equal amounts of the OM samples in each lane since the same normalized total cell extract served as the initial starting material to purify both this fraction and the cytoplasmic fraction. We could not use any of our available antibodies to OMPs, for their levels might well be influenced by the functional status of one or more of the periplasmic PPIases. Western blot analysis. Proteins samples were separated by SDSPAGE on 12% to 15% polyacrylamide gels. Proteins were then transferred to a Protran nitrocellulose membrane (Whatman) using a wet transfer assembly (CBS Scientific). Proteins of interest were bound with specific rabbit polyclonal antibodies (anti-invasin, anti-Ail, anti-PsaA, anti-DnaJ, anti-OmpA, anti-H-NS, anti-YopD, anti-YopE, anti-LcrV, and antiYscO) or mouse polyclonal (anti-OmpF) or monoclonal (anti-␤-actin) antibodies and subsequently detected with an anti-rabbit or anti-mouse monoclonal antibody conjugated with horseradish peroxidase (GE Healthcare) and a homemade chemiluminescent solution. HeLa cell cytotoxicity and bacterium-HeLa cell association assays. Cultivation of HeLa cells was performed using standard methods (35). HeLa cell cytotoxicity was monitored after bacterial infection using a phase-contrast light microscope (Zeiss Telaval 31). Images were acquired

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pIR020

Important genotype or phenotype

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RESULTS

SurA is required for proper folding and OM insertion of invasin. SurA is a periplasmic protein required for the folding of many ␤-barrel OMPs (17, 18). We have previously reported a drastic reduction in OMPs of Y. pseudotuberculosis deficient in SurA (16). Based on this, we were keen to examine the impact of SurA on surface localization of functionally important Yersinia adhesins essential for bacterial attachment to host cells. Prominent adhesins produced by enteropathogenic Y. pseudotuberculosis include the chromosomally encoded invasin, Ail, and PsaA (pH 6 antigen) as well as the virulence plasmid-encoded YadA, which have all been implicated as important virulence determinants (37, 38, 39). The parental strain used in this study contains a kanamycin resistance cassette inserted into the virulence plasmid that disrupts the yadA gene (34). As a result, the effect of SurA on YadA

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assembly was not addressed in this analysis. To investigate the role of SurA in the folding and assembly of invasin, Ail, and PsaA, using specific rabbit antisera we initially determined their levels in total cell extract from parental, ⌬surA, ⌬ppiA ⌬ppiD ⌬fkpA ⌬fklB (⌬4), and ⌬ppiA ⌬ppiD ⌬fkpA ⌬fklB ⌬surA (⌬5) bacteria. Differences in total accumulated amounts of invasin among these bacterial strains were observed, with parental bacteria displaying the largest quantity of invasin, followed by the ⌬4 mutant (surA⫹), the single ⌬surA mutant, and then the quintuple ⌬5 mutant, showing the smallest amount (Fig. 1A and B). However, the total levels of Ail and PsaA were specifically reduced in total cell extracts of the two surA mutants (Fig. 1A and B). This suggests that only SurA activity affects the accumulated steady-state levels of Ail and PsaA. SurA activity also has a strong effect on invasin levels. However, these levels can also be influenced negatively by the cumulative loss of at least one other periplasmic PPIase. To investigate if the individual adhesins were more prone to degradation in the absence of surA, we used an assay to measure protein susceptibility to endogenous proteases. However, this assay could not distinguish any obvious stability differences in invasin, Ail, or PsaA when comparing SurA⫹ with SurA⫺ bacteria (see Fig. S1 in the supplemental material). To address how the loss of SurA impacted the surface assembly of these adhesins, we purified the OM fraction from the total cell lysates. To do so, we used our established cell fractionation assay (16), in which we first confirmed using rabbit antibodies to OmpA (OM protein marker) and H-NS (cytoplasmic protein marker) that the purified OM fraction is enriched in OMPs and is free from cytoplasmic protein contaminants (see Fig. S2 in the supplemental material). Critically, invasin levels in the purified OM fraction derived from surA-deficient bacteria were drastically reduced compared to those from the parental and ⌬4 (surA⫹) bacteria (Fig. 1A and B). As the OM level of invasin in the ⌬4 bacteria was lower than that of the parental bacteria, this also highlights a requirement of other PPIases in the overall OM assembly of invasin. The OM levels of PsaA and Ail were also reduced in the surA mutants (Fig. 1A and B), which reflected directly on their smaller amounts detected in the total cell lysate starting material. Since it could be argued that the reason for this decrease in OM localization is a reduction in their total levels, we determine whether reduced synthesis of these proteins in the surA mutants contributed to their decreased OM levels. To do this, we specifically examined the cytoplasmic levels of these Yersinia adhesins to identify the amount of protein that is readily available for export. Purified cytoplasmic fractions from ⌬surA and ⌬4 bacteria contained levels of invasin, Ail, and PsaA similar to those of the parental bacteria (Fig. 1A and B). However, the levels of these proteins were slightly reduced in the purified cytoplasmic fraction of ⌬5 bacteria (Fig. 1A and B). Next, we compared the levels of protein in the OM fraction to those in the cytoplasmic fraction; the ratio between them gave a value that we termed the percent assembly efficiency. With this approach, we could categorically determine that both ⌬surA and ⌬5 mutants showed about an 80% reduction in their ability to specifically export and/or assemble invasin in their OM compared to parental bacteria (Fig. 1C). In addition, the ⌬4 bacteria also exhibited lower assembly efficiency than the parental bacteria, amounting to ⬃35% reduction in their ability to assemble OM invasin (Fig. 1C). However, these bacteria assembled Ail and PsaA in their OM with an efficiency similar to that of the parental bacteria. In contrast,

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by direct capture of infected cell monolayers using a digital camera mounted directly to the microscope. Cytotoxicity was evaluated by a change in cell shape from oblong to round. To analyze the extent of bacterial association with eukaryotic cells, HeLa cells were grown to near-confluence in a 24-well tissue culture plate at 37°C in a humidified 5% CO2 environment. Prior to infection, bacteria were pregrown in tissue culture medium at 26°C for 30 min and then at 37°C for 1 h. Cells were infected with bacterial cultures, which were standardized according to equal A600, at a multiplicity of infection of 10 and were synchronized by centrifugation at 160 ⫻ g for 5 min. At 45 min postinfection, cells were gently washed three times with phosphate-buffered saline (PBS) to remove unattached and loosely attached bacteria. After 20 min of incubation in a solution of 0.1% (vol/vol) Triton and addition of LB medium, bacteria tightly bound to eukaryotic cells were recovered and quantified by viable-cell counting. Data are representative of multiple independent experiments and are expressed as the mean numbers of bacteria. RNA extraction and quantitative reverse transcription-PCR (qRTPCR). Yersiniae grown to an A600 of ⬃0.75 were immediately treated with RNAprotect bacterial reagent (Qiagen Nordic) to stabilize RNA. RNA was then extracted using the NucleoSpin RNA II method (Macherey-Nagel), which included an on-column DNase treatment. To fully remove DNA traces, extracted RNA was treated with a Turbo DNA free kit (Ambion) by following the manufacturer=s protocol. qRT-PCR was performed using the one-step method that incorporates cDNA synthesis and PCR amplification in the same tube. Specific internal primer combinations for inv, ail, psaA, ompA, bamA, bamC, and bamE were used in the qRT-PCR, and these are listed in Table S1 in the supplemental material. Experiments were carried out using an iScript one-step RT-PCR kit with SyBR green (Bio-Rad Laboratories). Each reaction mixture contained 200 nM primers, 4 ng RNA template, and 1⫻ SyBR green RT-PCR mix and was monitored in an iCycler iQ real-time PCR detection system (Bio-Rad). Cycling conditions used were as follows: cDNA synthesis at 50°C for 10 min; iScript reverse transcriptase inactivation at 95°C for 5 min; PCR cycling and detection at 95°C for 10 s (denaturation) and 55°C for 30 s (45 cycles); and melt curve analysis at 95°C for 1 min, 60°C for 1 min, and 55°C for 10 s. Multiple independent samples were tested in duplicate. For each sample, the mean cycle threshold of the test transcript was normalized to that of rpoA. To confirm the absence of contaminating DNA in the RNA preparations, a duplicate reaction lacking the RT enzyme was always performed in parallel. Protein stability measurements. Susceptibility to endogenous proteases of de novo-synthesized OMPs in SurA⫹ and SurA⫺ Yersinia bacteria was tested essentially by the method of Feldman and colleagues (36) following growth of the bacteria in LB broth for 1 h at 26°C or 37°C prior to the addition of chloramphenicol (50 ␮g/ml). Protein fractions collected at various time points after addition of the protein synthesis inhibitor were analyzed by SDS-PAGE and Western blotting.

Yersinia Adhesin Assembly Requires SurA

the surA mutants exhibited between 35 and 40% reduction in their efficiency to assemble Ail in their OM, while assembling PsaA with only a slight reduction in efficiency compared to that of the parental bacteria (Fig. 1C). Given the obvious reduction in percent OM assembly efficiencies for these three adhesins in the absence of SurA, we investigated if mistargeted protein accumulated in the periplasm in these bacteria. Indeed, we observed a truncated ⬃70-kDa invasin fragment in the periplasmic fraction of Yersinia lacking surA (see Fig. S3 in the supplemental material). Possibly, this fragment represents a proteolytic resistant remnant of full-length invasin. However, we could not detect any evidence of periplasmic Ail and PsaA accumulation under the same testing conditions (see Fig. S3). Taken together, these results suggest a key role for SurA in the targeting and OM assembly of invasin, while the biogenesis of Ail and, to a lesser degree, PsaA also appears somewhat linked to SurA. Our approach was to examine SurA-dependent effects on

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adhesin assembly following growth of bacteria to late logarithmic phase (A600 of ⬃0.75). It is possible that the extent of this SurA dependency might even be further influenced by the growth phase of the cultured bacteria. However, the potential for growth phase effects and thereby nutrient availability was not investigated in this study. Loss of surA induces high gene expression of inv, ail, and psaA. Given the reduced targeting of Yersinia adhesins to the OM in the absence of SurA, we were curious if this had any impact on their transcriptional regulation. Specific mRNA transcript levels derived from the inv, ail, and psaA genes were measured by qRTPCR. Interestingly, loss of surA prompted an elevation in mRNA transcripts derived from all three adhesin-encoding genes. Compared to the level in parental bacteria, the extents of elevation were ⬃1.5-fold for inv and psaA and ⬃2.5-fold for ail in the single surA mutant, and in the ⌬5 mutant they were ⬃1.5-, ⬃2-, and ⬃4-fold for inv, psaA, and ail, respectively (Fig. 2). As anticipated based on

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FIG 1 SurA-dependent assembly of Yersinia adhesins. (A) Immunoblot analysis of total cell lysate, OM, and cytoplasmic levels of invasin, Ail, and PsaA. Yersinia bacteria were grown to late exponential phase at 26°C (for invasin and Ail analysis) or 37°C (for PsaA analysis). Western blotting was performed on cell extract, purified OM, or cytoplasm from these bacteria. Protein level was probed with specific antisera raised against each protein. Equal loading of protein in each lane was confirmed by probing with DnaJ (indicated by arrows) levels in the cell extract and the purified cytoplasmic fraction. These served also as loading controls for the OM samples given that the same normalized cell extracts were the starting materials used for their purification. This approach is the most available means to ensure equal loading due to the high probability of reduced OMPs associated with surA mutants. Probable protein degradation products are indicated (¤) and are observed routinely in analyses of invasin by Western blotting (for examples, see references 69, 82, 83, and 84). Lanes: parent, YPIII/pIB102; ⌬surA, YPIII21/pIB102; ⌬4 (⌬ppiA ⌬ppiD ⌬fklB ⌬fkpA), YPIII73/pIB102; ⌬5 (⌬surA ⌬ppiA ⌬ppiD ⌬fklB ⌬fkpA), YPIII80/pIB102. (B) At least three independent experiments were used to quantify relative invasin, Ail, and PsaA levels using Quantity One software, version 4.52 (Bio-Rad). Values ⫾ standards deviations represent the ratio of protein level from each respective strain to that from the parental bacteria. (C) Percent assembly efficiency reflects the extent of protein assembly in the OM of each strain. This is calculated from the ratio of protein level in the OM (y) to stable accumulated levels in the cytoplasm (x).

Obi and Francis

previous reports (40, 41), we observed higher psaA expression at 37°C than at 26°C (Fig. 2). In surA⫹ bacteria represented by the ⌬4 mutant, only the ail transcript deviated from parental bacteria (⬃1.5-fold increase); the mRNA levels of inv and psaA remained

FIG 3 OM assembly of OmpA requires SurA. (A) Immunoblot analysis of cell extract, OM, and cytoplasmic levels of OmpA and OmpF. Yersinia bacteria were grown to late exponential phase at 26°C, prior to the fractionation of cell extract, purified OM, and cytoplasmic material. Western blotting was used to obtain protein levels by probing with specific antisera raised against each protein. Analysis with anti-DnaJ antisera to probe DnaJ (indicated by an arrow) levels was used to confirm equal loading of protein in each lane. This could be extrapolated to a loading control for the OM samples since the same normalized cell extract was the starting material used for purification of the OM fraction. This approach is the most reliable means to ensure equal loading of these samples given the high probability that OMPs will be specifically reduced in material derived from surA mutants. ¤, probable protein degradation product. (B) The quantification of relative OmpA and OmpF levels was calculated from at least three independent experiments using Quantity One software, version 4.52 (Bio-Rad). Values ⫾ standard deviations represent the ratio of protein from each respective strain to that from the parental bacteria. Complete quantification data for the DnaJ loading controls can be viewed in Fig. 1B. (C) Percent assembly efficiency of OmpA was detemined from the ratio of its level in the OM (y) to that stably maintained in the cytoplasm (x). n.d., not deterimined.

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FIG 2 Gene expression of inv, ail, and psaA by Y. pseudotuberculosis. mRNA isolated from bacteria grown to late exponential phase at 26°C (for invasin, Ail, and PsaA analysis) or 37°C (for PsaA analysis) was used in qRT-PCR. Each sample was normalized against the mean cycle threshold of rpoA for that sample. Results represent the averages of multiple independent experiments (⫾standard errors of the means). Data sets were analyzed using the nonparametric two-tailed Mann-Whitney U test. Analysis was performed using GraphPad Prism, version 5.00, for Windows. Differences with a P value of ⬍0.05 were considered significant. All data sets marked with asterisks are statistically significant and have P values of 0.0284. ns, nonsignificance.

unaltered (Fig. 2). Hence, it is possible that bacteria sense deficiencies in adhesin assembly and attempt to compensate for this by triggering an increase in their transcription output. SurA is needed for OmpA assembly in the OM of Y. pseudotuberculosis. OmpA is a ␤-barrel OMP that stabilizes the structure of the bacterial OM (4). Using mass spectrometric analysis, we have previously determined that OmpA is one of the reduced OMPs that result upon surA removal in Y. pseudotuberculosis (16). Interestingly, OmpA is assumed to be a true substrate of SurA (18, 42). Hence, we wanted to test this with our experimental setup by measuring the percent OM assembly efficiency of OmpA in Y. pseudotuberculosis with or without SurA. Our analysis also included one other ␤-barrel OMP, the general porin OmpF, that is also considered a SurA substrate (18, 42). Levels of OmpA and OmpF in surA mutant bacteria were determined using immunoblotting with specific rabbit- and mouse-derived antibodies, respectively. Total cell lysates of surA mutants contained less OmpA and even less OmpF than did the parental and ⌬4 bacteria (Fig. 3A and B). We do not believe this reduction to be due to unstable protein, since the two proteins resisted exposure to endogenous proteases equally well regardless of the presence or absence of SurA (see Fig. S4 in the supplemental material). Given this reduction in total OmpA and OmpF pools, we also observed a notable reduction in OmpA and OmpF levels in the purified OM fraction from the surA mutants only (Fig. 3A and B). However, OmpA pools in cytoplasm-derived material were the same in all the bacterial strains, whereas the cytoplasmic OmpF levels in the surA mutants were drastically reduced (Fig. 3A and B). Consequently, we could not determine the assembly efficiency for OmpF due to a severe defect in its synthesis. On the other hand, the efficiency of

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FIG 4 Effects of SurA on components of the BAM complex. (A) Determination of OM levels of BamA, BamC, and BamE in surA mutants. OM isolated from late exponentially grown bacteria was used to perform an immunoblot. Levels of BamA, BamC, and BamE were assessed using rabbit antisera raised against BamA, BamC, and BamE, respectively. DnaJ (arrow) in the total cell extract used as source material for OM purification was used for equal loading of protein in each lane. (B) BamA, BamC, and BamE levels were quantified from the results of at least three independent experiments using Quantity One software, version 4.52 (Bio-Rad). Values ⫾ standard deviations represent the ratio of protein from each respective strain to that from the parental bacteria. The tabulated quantification data for the DnaJ loading controls can be viewed in Fig. 1B. (C) Transcriptional analysis of bamA, bamC, and bamE expression. mRNA isolated from late exponentially grown bacteria was used in a one-step qRT-PCR that incorporates cDNA synthesis and PCR amplification in the same tube using specific primers for the genes. The level of rpoA transcript was used to normalize the mean cycle threshold of rpoA for each sample. Results represent the averages of multiple independent experiments (⫾standard errors of the means). Data sets were analyzed using the nonparametric two-tailed Mann-Whitney U test. Analysis was performed using GraphPad Prism, version 5.00, for Windows. Differences with a P value of ⬍0.05 were considered significant. All data set marked with asterisks are statistically significant and have P values of 0.0284. ns, nonsignificance.

enterica serovar Typhimurium. Amino acid sequence comparisons with SurA from Y. pseudotuberculosis revealed ⬃74% identity, whereas between E. coli and Salmonella the identity reaches ⬎91% (see Fig. S5 in the supplemental material). Hence, all three proteins boast considerable sequence conservation. Based on studies from E. coli and S. enterica, it is clear that SurA consists of four domains: two internal parvulin domains that are flanked by

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assembly of OmpA in the OM of the surA mutants was reduced by ⬃40% compared to that in the ⌬4 and parental bacteria (Fig. 3C). Therefore, OmpA is truly a substrate of SurA, which is needed to assist with OmpA assembly in the OM. At this stage, we do not know why OmpF synthesis is so specifically influenced by the SurA status in Y. pseudotuberculosis. In E. coli, both OmpA and OmpF are regulated at the posttranscriptional level by small regulatory RNA molecules, micA and micF, respectively (43, 44). It is well established that deletion of surA induces ␴E activity that, in turn, upregulates these small regulatory RNAs to feedback inhibit Omp synthesis (45, 46, 47). Although this is apparently not the case for OmpA control in SurA-defective Y. pseudotuberculosis, the presence of a similar micF-dependent feedback control mechanism in our surA mutants would at least explain why OmpF synthesis is so low. Expression and folding of BAM components in surA mutants. SurA delivers its ␤-barrel OMP substrates to the BAM complex, which then inserts this cargo into the OM (5). BamA itself is also an integral OMP. Given that this could also be a SurA substrate, we determined in surA mutants of Y. pseudotuberculosis the levels of OM-localized BamA, as well as two additional BAM complex constituents, the BamC and BamE lipoproteins. This was necessary in order to ascertain the extent of direct involvement by SurA in folding and assembly of OMPs. Therefore, we grew bacteria to late exponential phase (A600 of ⬃0.75) at 26°C and 37°C and isolated their OM. As revealed by specific rabbit polyclonal antibodies, removal of SurA resulted in decreased levels of OM BamA, BamE, and BamC compared to those in the parent or surA⫹ bacteria (Fig. 4A and B). This effect on the Bam proteins was the same regardless of bacterial growth at 26°C or 37°C. Hence, BAM complex assembly in Y. pseudotuberculosis likely requires SurA. This means that reduced OM assembly of invasin and, to a lesser extent, Ail, PsaA, and OmpA probably reflects both a loss of SurA-dependent trafficking across the periplasm as well as possibly reduced assembly capacity of the BAM complex in the OM. To further explore the effect of SurA on the BAM complex, we used qRT-PCR to examine whether the reduction in OM BamA, BamC, and BamE levels in surA mutants is due to a feedback inhibition of their gene transcription. Compared to those in the parental bacteria, however, the mRNA levels of bamA, bamC, and bamE were all increased in the single ⌬surA mutant ⬃2-, ⬃3-, and ⬃3.5-fold, respectively, and in the ⌬5 mutant ⬃2.5-, ⬃3.5-, and ⬃5.5-fold, respectively (Fig. 4C). On the other hand, surA⫹ bacteria that lacked all four other periplasmic PPIases showed similar levels of the bamA transcript and only a ⬃1.5-fold increase in bamC and bamE transcripts compared to the parent strain. Therefore, the decrease in OM levels of these proteins is not a result of reduced gene transcription. However, it does indicate that loss of SurA causes bacteria to compensate by adjusting transcriptional output from certain genes whose products influence OM biogenesis. At the same time, ompA transcript levels remained comparable in all four bacterial strains (Fig. 4C). Hence, despite documented evidence of feedback inhibition of ompA transcription via the small regulatory RNA micA (45, 46, 47), this type of reprogramming of ompA transcription following surA removal was apparently not instigated in Y. pseudotuberculosis. Contributions of individual SurA domains in the OM assembly of proteins by Y. pseudotuberculosis. The best-characterized SurA molecules are derived from Escherichia coli and Salmonella

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OmpA OM assembly was analyzed, complementation of surA mutants with the SurA full-length or 2A variant completely restored OM levels of these proteins (Fig. 5C and D). However, introduction of the ⌬P or ⌬N variant in these mutants produced only intermediate levels of Ail. On the other hand, PsaA and OmpA levels were completely restored by ectopic expression of the ⌬P variant, while expression of the ⌬N variant failed to restore the levels of these proteins (Fig. 5C and D). Based on these data, it seems feasible that the different SurA functional domains can both contribute to OMP targeting and assembly, although the relative importance of each depends on the individual substrate. surA mutant bacteria bind poorly to host cells. In previous work (16), we reported that SurA-deficient bacteria were attenuated in a murine infection model. While there are probably several reasons for this avirulence phenotype, loss of attachment to eukaryotic host cells could be a contributing factor, since attachment is an essential criterion for enteropathogenic Yersinia to initiate an infection. We have shown here that the important Yersinia adhesins invasin, Ail, and PsaA are all reduced in surA mutants, with reduced invasin surface localization being the most pronounced. Therefore, we were keen to determine the ability of these bacteria to associate with HeLa cells. Upon incubation with semiconfluent HeLa cell monolayers, we detected ⬃40- and ⬃60-fold decreases in the numbers of single ⌬surA and quintuple ⌬5 mutants, respectively, which associated with the host cells compared to the parental or surA⫹ bacteria (lacking the genes for all four other periplasmic PPIases) (Fig. 6). Critically, ⌬surA mutants transcomplemented with the PPIase-deficient variants ⌬P and 2A both fully restored Yersinia host cell contact. In stark contrast, ectopic expression of the SurA ⌬N variant still resulted in the two ⌬surA mutants having ⬃20- to ⬃40-fold reductions in host cell association (Fig. 6). Notably, this result correlated with our biochemical data that showed defective assembly of invasin, Ail, and PsaA in the surA mutants. Thus, SurA chaperone activity is indispensable for promoting Yersinia-host cell contact, while PPIase activity is not required. Yersinia lacking all five periplasmic PPIases is hindered in cytotoxicity toward eukaryotic host cells. Yersinia requires a structurally intact and functional plasmid-encoded Ysc-Yop T3SS to successfully infect eukaryotic host cells (21). Given that Yersinia surA mutants were deficient in host cell association, we investigated whether these bacteria can still assemble a functional T3S apparatus that can secrete and translocate Yersinia Yop effector proteins. Using an in vitro T3S secretion assay that measures YscYop protein synthesis and secretion under Ca2⫹-limiting conditions that presumably mimics host cell contact, we observed that the two surA mutants synthesized (data not shown) and secreted levels of Ysc-Yop substrates similar— exemplified by the structural proteins LcrV and YscO, the YopD translocator, and the translocated YopE effector toxin—to those synthesized and secreted by the parental and SurA⫹ bacteria (Fig. 7A). Hence, despite deficiencies in promoting surface localization of invasin, Ail, and PsaA, Yersinia surA mutants still assembled a functional T3SS during laboratory culturing. To determine if this T3SS maintains the ability to intoxicate eukaryotic cell monolayers, we performed a typical HeLa cell cytotoxicity assay (35). Cytotoxicity is characterized by the ability of cell-associated Yersinia to sense and inject principally the YopE cytotoxin into the cytosol of eukaryotic host cells, resulting in a change in morphology from oblong to rounded cells. Unlike parental bacteria, the ⌬yopB yopD mutant, unable to

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the N-and C-terminal domains. These N- and C-terminal domains can exert chaperone function (48), while the SurA PPIase activity resides in the second parvulin domain located in the Cterminal half of the protein (49). We have previously confirmed that purified SurA of Y. pseudotuberculosis also possesses PPIase activity (16). As yet, however, we do not yet have any understanding of the relative contributions of chaperone and PPIase activities to SurA-dependent periplasmic trafficking and OM assembly of proteins exported by Y. pseudotuberculosis. Therefore, to address which domains of SurA are necessary for folding and assembly of OMPs in Y. pseudotuberculosis, we PCR amplified three truncated surA alleles and cloned these into the low-copy-number plasmid pWKS30 with expression maintained under the control of the surA native promoter. The first allele codes for a SurA variant that lacks amino acids 25 to 142, which represents the N-terminal region of the protein (⌬N) (Fig. 5A; see also Fig. S5 in the supplemental material). The second allele encodes a SurA variant that lacks amino acids 175 to 384 covering the two parvulin domains of SurA (⌬P), while the third allele produces a SurA variant with the conserved His376 and Ile378 residues replaced with alanine (2A) (Fig. 5A; see also Fig. S5). In E. coli, alanine substitution of these conserved residues is reported to result in the loss of PPIase activity (48). We avoided constructs that encode the N-terminal domain devoid of the C-terminal domain since the resulting polypeptides were previously found to be unstable (48). Initially, we assessed the production of these SurA variants in the two surA mutants of Y. pseudotuberculosis, the first lacking only surA (⌬surA null mutant) and the second lacking all five periplasmic PPIases (⌬5 null mutant). With immunoblotting of cell extracts using rabbit antibodies raised against full-length SurA, we observed that all of the SurA variants were stably produced in the surA mutants, resulting in proteins of approximately 30 kDa (⌬N), 22 kDa (⌬P), and 47 kDa (2A), corresponding to their apparent molecular weights (Fig. 5B). The ⌬P variant could be detected only upon increased exposure time. As has been suggested for E. coli previously (48), this might be a reflection not of unstable protein but of an inability of SurA polyclonal antibodies to recognize this truncated variant. We then used trans-complementation of our surA mutants to investigate the contributions of individual SurA domains in the OM assembly of the Yersinia adhesins invasin, Ail, and PsaA as well as OmpA. Ectopic production of all the individual SurA variants in the surA mutants could, to differing degrees, restore OM localization of invasin (Fig. 5C and D). Critically, production of the full-length SurA variant in both surA mutants completely restored OM invasin assembly. This was true also of the single surA mutant complemented with the ⌬P or 2A variant (Fig. 5C and D). However, the same variants expressed in trans in the ⌬5 mutant restored only intermediate levels of invasin in the OM. Notably, complementation with the ⌬N variant permitted the least accumulation of OM-integrated invasin in either surA mutant (Fig. 5C and D). Moreover, the single ⌬surA mutant background complemented with any of the truncated or point-mutated SurA variants reproducibly had higher levels of OM-located invasin than with the same construct in the ⌬5 mutant background. Hence, these data suggest that to various degrees both chaperone and isomerase activities of SurA contribute to invasin OM assembly. They also suggest that one or more of the other four periplasmic PPIases can partially promote invasin assembly in the absence of SurA. When the involvement of the SurA domains in Ail, PsaA, and

Yersinia Adhesin Assembly Requires SurA

boxes show the N-terminal, first parvulin, second parvulin, and C-terminal domains of SurA, repsectively. The approximate positions of His376 and Ile378 replaced with alanines (“A”) are indicated on the 2A variant. Numbers indicate the amino acid positions in the pre-SurA protein. All the SurA variants contain the SurA signal peptide. SurA is full length (SurA1– 434), ⌬N is an N-terminally truncated form (SurA⌬25–175), ⌬P is a C-terminally truncated form (SurA⌬175–384), and 2A harbors two point mutations (SurAH376A and SurAI378A). (B) Production of SurA variants in Y. pseudotuberculosis. A cell extract from exponentially grown bacteria containing plasmids for the expression of SurA variants expressed from the native promoter was used to perform a Western blot. Production of proteins was detected using rabbit antisera raised against the full-length SurA. The ⌬P variant was detected only upon prolonged exposure, and the arrows indicate the banding positions depicting this protein. (C) Assessment of SurA domains for invasin, Ail, OmpA, and PsaA assembly. Western blotting was performed on OM fractions collected from SurA-positive or SurA-negative bacteria or surA mutants transformed with plasmids expressing the various SurA variants. Invasin, Ail, OmpA, and PsaA were detected using rabbit antisera raised against invasin, Ail, OmpA, and PsaA, respectively. Normalized cell extracts used for the OM purification were also probed with antisera specific for cytoplasmic DnaJ (indicated by an arrow) to confirm equal loading of invasin, Ail, and OmpA. A protein band marked with an asterisk that cross-reacted with anti-PsaA antisera could be used to confirm equal PsaA sample loading. ¤, probable protein degradation products. (D) Invasin, Ail, OmpA, and PsaA levels in the OM fraction were quantified from the results of at least three independent experiments using Quantity One software, version 4.52 (Bio-Rad). Values ⫾ standard deviations indicate the ratio of protein from each respective strain to that from the parental bacteria.

produce a T3S translocon pore in the host cell plasma membrane, cannot induce a cytotoxic response (50) (Fig. 7B). Intriguingly, the ⌬5 mutant was notably reduced in this cytotoxic effect toward HeLa cells, despite a normal capacity for in vitro Ysc-Yop T3SS synthesis and secretion (Fig. 7B). In contrast, the single ⌬surA

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mutant and SurA⫹ bacteria both induced rapid HeLa cell cytotoxicity indistinguishable from that of parental bacteria (Fig. 7B). These data suggest that via facilitating adhesin assembly at the bacterial surface, SurA is involved in ensuring that Yersinia is prepared for engaging with the target host cell. On its own, however,

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FIG 5 Analysis of SurA domains of Y. pseudotuberculosis. (A) Schematic depiction of SurA variants used in this study. The white, dark gray, light gray, and black

Obi and Francis

it is not necessarily involved in arming Yersinia with a functional Ysc-Yop T3SS that can intoxicate these targeted cells. However, the simultaneous absence of all periplasmic PPIases leads to an additional T3SS-dependent cell intoxication defect that indicates that cooperation between at least some of these PPIases is a requirement for full T3SS activity in the presence of eukaryotic cells. DISCUSSION

used to infect a monolayer of growing HeLa cells at 37°C. Unattached and loosely attached bacteria were removed by washing. Viable-cell counts were performed on the remaining tightly cell-associated bacteria. An immunoblot with monoclonal anti-␤-actin antibody (Sigma-Aldrich) was performed to probe actin levels to confirm equal seeding of HeLa cells. Results show the averages of at least four independent experiments (⫾standard deviations).

FIG 7 Analysis of T3SS function by Y. pseudotuberculosis. (A) Yersiniae were grown at 26°C in BHI broth either containing (⫹) or lacking (⫺) Ca2⫹ for 1 h and then at 37°C for 3 h. Protein samples were separated on a 12% SDS-polyacrylamide gel and then transferred onto a membrane. Secretion of T3S-associated structural components (LcrV and YscO), translocator (YopD), and effector (YopE) was detected by immunoblotting using specific antisera. (B) Quantification of YopD, YopE, LcrV, and YscO levels was performed from the results of at least three independent experiments using Quantity One software, version 4.52 (Bio-Rad). Values ⫾ standard deviations indicate the ratio of protein from each respective strain to that from the parental bacteria. (C) Yersinia bacteria were allowed to infect a monolayer of growing HeLa cells, and at 2 h postinfection, the effect of the bacteria on the HeLa cells was recorded by phase-contrast microscopy. Translocation of the YopE cytotoxin causes a distinct change in cell shape, from oblong to rounded (cytotoxicity), in affected cells. HeLa cells without YopE intoxication show normal uninfected cell morphology.

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FIG 6 HeLa cell association by Y. pseudotuberculosis. Yersinia bacteria were

SurA is an important periplasmic protein folding factor needed for proper assembly of proteins in the OM. We have shown in this study that SurA-deficient Y. pseudotuberculosis is defective in the assembly of several OMPs, although the extent of this defect varies with different OMPs. Most notable is the reduction in surface localization of three key adhesins, which, in turn, diminishes bacterial interaction with target eukaryotic cells. Reduced adhesin assembly occurs despite evidence of an elevation in their gene transcription. This is not a general defect in OMP assembly, however, since SurA is not required for the assembly of a functional T3SS. Finally, dissecting SurA into discrete functional entities highlighted the different requirements of SurA in OMP assembly. Based on the degree of OMP assembly defect in surA mutant

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altered lipid environment in our surA-negative bacteria is less amenable to the assembly of certain OMPs. Despite the fact that the details are still poorly understood, it is quite apparent that the mechanisms of assembly must differ among the three Yersinia adhesins analyzed in this study. Invasin is an AT, although its secretion utilizes an inverse mechanism compared to classical (type Va secretion) autotransport (57). Similar to type Va secretion substrates, invasin is translocated from the cytoplasm across the inner membrane via the Sec translocase (58). However, the N-terminal domain of invasin forms the ␤-barrel in the OM (57). Type V secretion is also thought to involve BamA and various periplasmic chaperones (1, 13, 52, 59, 60, 61). When we analyzed invasin assembly efficiency in surA mutant bacteria, we found that OM targeting of invasin was particularly poor. This could mean that SurA primarily performs a piloting function in trafficking invasin to the BAM complex. However, surA depletion did reduce BamA levels in the OM, albeit only subtly, while levels of BamC and BamE were further reduced. Hence, inferior invasin assembly may also be amplified by a compromised BAM complex assembly platform. This correlates with a recent study published while our manuscript was in preparation that reported how Y. enterocolitica-derived invasin ectopically expressed in an E. coli surA deletion mutant failed to assemble in the OM (57). Interestingly, in this surrogate experimental setup, BamA was also deemed to be necessary for surface localization of invasin. Moreover, it is also clear from our ability to demarcate SurA functional entities that chaperone activity of SurA is most important for invasin assembly, but PPIase activity is still involved. These data are all consistent with findings concerning the invasin-related adhesin of enteropathogenic E. coli known as intimin, which was also reported to depend on SurA for its OM assembly (59). While Ail is known to be an OMP forming an eight-stranded anti-parallel ␤-barrel with four extracellular loops (27), studies on the pathway needed for assembly of Ail are lacking. In our work, the steady-state level of accumulated Ail was reduced in both total cell extracts and OM fractions of surA mutants. However, its cytoplasmic level in these bacteria was basically unaffected, giving some credence to a direct role of SurA in Ail assembly in the OM. As Ail is a ␤-barrel OMP, the folding and/or periplasmic trafficking of Ail could depend on SurA, although other periplasmic folding factors probably contribute to its OM assembly. The fimbrial adhesin PsaA is assembled through the classical chaperone-usher pathway (62). This involves the periplasmic chaperone PsaB, which targets PsaA subunits to the OM-bound usher PsaC, which forms the platform for the assembly of these subunits on the bacterial surface (63). The observed effect of SurA on PsaA assembly could be indirect acting through the PsaB chaperone-PsaC usher system. Indeed, E. coli SurA is reported to function in the OM maturation of both PapC and FimD, respective ushers of P and type 1 pili (64, 65, 66). Reduction in usher level due to loss of SurA indirectly reduces the amount of the assembled pilin subunits (64). Thus, it is quite possible for SurA to aid in the OM assembly of PsaC, and this could partly explain the subtle deficiency in PsaA at the Yersinia surface when SurA was absent. With the exception of ompA mRNA transcript levels, it was evident that loss of SurA led to elevated transcription of the OMPencoding genes that we investigated in this study. Most remarkable was the high level of psaA mRNA transcript, and, to a lesser extent, the ail mRNA transcript, in the surA mutants grown at

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backgrounds, two SurA-dependent substrate classes were apparent. Assembly in the OM of one protein class that included PsaA and BamA was only minimally affected. On the other hand, another set comprising invasin, Ail, and OmpA was more severely impaired in the absence of SurA. As the total accumulated steadystate levels of many of these proteins were reduced, it was not clear how SurA exerts its effect. In the case of invasin, however, total accumulated levels were less dependent on SurA, making it possible to conclude that SurA played an important and direct role in its efficient OM assembly. In the periplasm of Gram-negative bacteria, two distinct dedicated protein folding pathways engage with OMPs as they emerge from the sec translocon to pilot them to the BAM complex for assembly in the membrane (17, 51). A SurA-dependent pathway is proposed to be the major route for trafficking ␤-barrel OMPs to the OM (52). The Skp- and DegP-dependent pathway is considered to be a backup pathway to transport OMPs that fall off the SurA route (52). It is therefore possible that those proteins with only a mild assembly defect in the absence of SurA could default to the Skp/DegP pathway. Such a notion has been proffered already (53), where it was suggested that other periplasmic chaperones could participate in the biogenesis of OMPs to ensure that their levels are maintained even in the absence of SurA. Conversely, proteins that displayed severe assembly defects upon SurA removal likely utilize only this pathway for their assembly; these proteins cannot be rescued by other periplasmic chaperones, including Skp and/or DegP, resulting in their gross misfolding and assembly defects. Indeed, studies with E. coli have identified true SurA substrates such as the OMPs LptD and FhuA, which cannot be folded by other periplasmic chaperones (53). Based upon our observations, invasin may represent a true substrate of SurA in enteropathogenic Yersinia. Additionally, an alternative, third chaperone pathway independent of SurA and Skp/DegP was recently suggested for the assembly of a number of proteins, including BamA and PldA (54). As this pathway might also function in Y. pseudotuberculosis, it offers an explanation for the mild assembly defects observed for BamA in the absence of SurA. Given that surA-depleted strains did display alterations in BAM complex proteins in the OM, further possibilities for the apparent observation of two distinct SurA substrate classes in Yersinia probably exist. For example, perhaps the minor effect of surA deletion on the BAM proteins might be amplified for proteins that depend upon them for integration into the OM. For such proteins, SurA depletion serves to deny them not only a route for periplasmic trafficking but also the means for assembly in the OM. In contrast, proteins not reliant upon the BAM complex for their OM assembly are less affected by the absence of SurA, especially if these substrates can be rescued by other periplasmic trafficking pathways. Furthermore, it is necessary to consider the gross abnormalities in bacterial envelope integrity and membrane lipid composition caused by lack of SurA. In particular, we know that Y. pseudotuberculosis lacking surA may struggle to efficiently assemble LPS in the outer leaflet of the OM, given how LPS is enriched in spent culture supernatant derived from these strains (16). Additionally, the same SurA⫺ strains also undergo clear compositional changes in phospholipid and fatty acid contents (16). Since the phospholipid environment actively assists in protein folding and assembly of membrane proteins (55, 56), it is conceivable that the

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nent of the T3S apparatus—the YscC secretin—is itself an OMP (73, 74, 75). During Ysc-Yop T3SS biogenesis, the YscC secretin is arguably the first protein to assemble in the bacterial envelope, where it serves as a platform for the subsequent assembly of several additional Ysc proteins (76). Secretins are a large OMP family functioning in several fundamental and diverse physiological processes (77, 78). They can be divided into several classes based upon their trafficking, multimerization, and OM assembly characteristics. While some secretins self-traffic and self-assemble, others are known to be dependent on auxiliary proteins for their efficient localization and assembly in the OM (77, 78). However, SurA is not yet known to be one of these auxiliary proteins. In contrast, the YscC secretin relies upon the customized auxiliary YscW lipoprotein to assist in its periplasmic trafficking and OM assembly (77, 78). There is also some evidence for a role for the periplasmic disulfide oxidoreductase DsbA in the process of YscC stabilization (79). We therefore speculate that perhaps it is the chaperoning and piloting function of YscW and the folding functions of DsbA that circumvent the need for SurA in OM assembly of the YscC secretin. Finally, we were able to confirm that the chaperone activity of SurA resides in its N-terminal domain and was most important for OMP assembly in Yersinia, corroborating earlier studies with E. coli (48, 65). However, the internal parvulin domains associated with PPIase activity also contributed to the assembly of OMPs. When expressed in isolation, this domain could restore intermediate levels of surface-localized invasin and Ail in the two surA mutants. This notion was clearly reinforced by the consistent observation that the trans-complemented single ⌬surA mutant produced larger invasin amounts in the OM than did the isogenic ⌬5 mutant, findings that implied contributions from the other periplasmic PPIases in folding of invasin particularly when SurA was absent. PPIase activity targets proline residues in proteins. The proline contents of invasin, Ail, and PsaA are in the order of ⬃4%, ⬃3%, and ⬃2%, respectively. Moreover, all these three adhesins contain the SurA recognition motif (aromatic-X-aromatic, where “X” is any amino acid that is bordered before and after by any aromatic amino acid) that is a characteristic of ␤-barrel proteins (42, 80, 81). Hence, a role for PPIase activity in OMP assembly is realistic. However, to directly demonstrate this might be elusive under standard laboratory conditions. ACKNOWLEDGMENTS This work was performed within the virtual framework of the Umea˚ Center for Microbial Research (UCMR)-Linnaeus Program (LP) and supported by the Swedish Research Council (2006-3869 and 2009-3660) and the Foundation for Medical Research at Umea˚ University. I.R.O. was supported in part by a UCMR-LP postdoctoral fellowship. We thank Sidney Kushner (University of Georgia, Athens, GA) for the gift of pWKS30. We express gratitude to the following researchers for their gifts of antiserum: Thomas Silhavy (Princeton University, Princeton, NJ—-anti-BamA, anti-BamC, and anti-BamE), Peng Chen (Peking University, Beijing, China—anti-OmpF), Hans Wolf-Watz (Umea˚ University, Umea˚, Sweden—anti-invasin, anti-DnaJ, anti-YopD, anti-YopE, anti-LcrV, and anti-YscO), Bernt Eric Uhlin (Umea˚ University—anti-H-NS and anti-OmpA), Åke Forsberg (Umea˚ University—anti-PsaA), and Gregory Plano (University of Miami, Miami, FL—anti-Ail). The authors have no competing interests to declare. I.R.O. and M.S.F. designed, performed, and analyzed experiments as well as wrote the paper. Both authors read and approved the final paper.

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37°C. We do not believe that this was an artifact, because higher expression of psaA was observed at 37°C than at 26°C, which corroborated earlier reports (40, 67). The psaA gene lies in a locus of five genes, psaEFABC; the products of psaEF are positive transcriptional regulators responsible for temperature- and pH-dependent regulation of psaA transcription (40, 67). The psaEFABC locus has also been recently identified as one of the most strongly affected members of the RovA regulon in Y. pestis (68). Interestingly, RovA is negatively regulated by the CpxR response regulator (69), which, along with ␴E, responds to extracytoplasmic stress (70). Since loss of SurA can induce the ␴E and Cpx stress response (I. R. Obi and M. S. Francis, unpublished data), we would have anticipated that increased CpxR levels in the surA mutants might have caused a repression of psaA transcription. However, this was not the case, as psaA transcription increased in these mutants. At present, therefore, we cannot elucidate the link leading to elevated gene expression upon loss of surA. Possibly, removal of SurA induces a signal that upregulates synthesis of these OMPs to restore their diminished OM levels. Considering the high levels of psaA and ail transcripts in the surA mutants, which are not commensurate with their total protein levels in cell extracts, an unknown posttranscriptional event could account for this effect. Our future goal will be to identify the mechanisms of regulation of these genes in the absence of surA. Our work shows that SurA-deficient bacteria associated poorly with eukaryotic host cells due to defective assembly of their Yersinia adhesins. This defect is likely to be a contributing factor in the impressive virulence attenuation of Yersinia surA mutants in a mouse model of infection (16). In other bacterial pathogens, SurA-dependent factors necessary for bacterial attachment to eukaryotic host cells have also been described (59, 64, 65, 71). Successful Yersinia infections also require close host cell contact so that the Ysc-Yop T3SS can disarm the host cell of its innate defense properties. Therefore, of additional significance is the observation that the ⌬surA, ⌬4, and ⌬5 mutants have differing phenotypes with regard to T3SS-induced cytotoxicity, which is in contrast to their respective Omp and adhesin assembly phenotypes. While the single ⌬surA mutant still successfully employed the Ysc-Yop T3SS to intoxicate host cells with antihost effectors, the ⌬5 displayed an obvious T3SS defect. Given the pleiotropic phenotypes associated with loss of surA (16, 17), this result is notable, as it describes the first specific defect of the quintuple mutant that distinguishes it from the single ⌬surA mutant. It suggests that combined loss of periplasmic PPIase activity affects T3SS function, although we do not yet know what this is given how T3S occurs normally in this strain in vitro. As T3SS activity is cell contact dependent (72), perhaps the quintuple mutant fails to productively engage with the host cell, disrupting contact-dependent signals that are necessary for prompt activation of the Ysc-Yop T3SS and/or polarized deployment of the toxins directly into the host cell cytosol. Future work will attempt to identify the mechanism behind this interesting discrepancy. In any event, it is indicative of an undisclosed role for the additional four periplasmic PPIases that are also absent in this quintuple mutant. Their role is clearly important for full Yersinia virulence, as the SurA⫹ strain lacking these four additional periplasmic PPIases is also attenuated in mouse infections (16). So far, however, the functions of these PPIases in Yersinia pathophysiology remain elusive. On a related point, we were surprised that SurA was not involved in T3S by Y. pseudotuberculosis, given how a core compo-

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