Identification of the outer-membrane protein PagC required for the serum resistance phenotype in Salmonella enterica serovar Choleraesuis

Miki Nishio, Nobuhiko Okada, Tsuyoshi Miki, Takeshi Haneda and Hirofumi Danbara

Department of Microbiology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan

Correspondence
Nobuhiko Okada
okadan{at}pharm.kitasato-u.ac.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Serum resistance is a crucial virulence factor for the development of systemic infections, including bacteraemia, by many pathogenic bacteria. Salmonella enterica serovar Choleraesuis is an important enteric pathogen that causes serious systemic infections in swine and humans. Here, it was found that, when introduced into Escherichia coli, a recombinant plasmid carrying the pagC gene from a plasmid-based genomic library of S. enterica serovar Choleraesuis conferred a high-level resistance to the bactericidal activity of pooled normal swine serum. The resistance was equal to the level conferred by rck, a gene encoding a 17 kDa outer-membrane protein which promotes the serum resistance phenotype in S. enterica serovar Typhimurium. Insertional mutagenesis of the cloned pagC gene generated a mutation that resulted in the loss of the serum resistance phenotype in E. coli. When this mutation was introduced into the chromosome of S. enterica serovar Choleraesuis by homology recombination with the wild-type allele, the resulting strain could not produce PagC, and it showed a decreased level of resistance to complement-mediated killing. The mutation could be restored by introduction of the intact pagC gene on a plasmid, but not by introduction of the point-mutated pagC gene. In addition, PagC was able to promote serum resistance in the S. enterica serovar Choleraesuis LPS mutant strain, which is highly sensitive to serum killing. Although PagC is not thought to confer serum resistance directly, these results strongly suggest that PagC is an important outer-membrane protein that plays an important role in the serum resistance of S. enterica serovar Choleraesuis.


Abbreviations: DAPI, 4',6'-diamidino-2-phenylindole; MAC, membrane attack complex


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Salmonella enterica is a Gram-negative and facultative intracellular bacterium that causes a broad spectrum of diseases, such as gastroenteritis, bacteraemia and typhoid fever. The nature and severity of infections by Salmonella are generally dependent upon both the serovar and the host species. Typhoid and paratyphoid fevers result from systemic infection with the human-adapted serovars S. enterica serovar Typhi and S. enterica serovar Paratyphi A. In contrast, infection with the broad-host-range-adapted S. enterica serovar Typhimurium usually causes gastroenteritis in humans, but produces a systemic infection similar to typhoid fever in susceptible mice. Among more than 2000 Salmonella serovars, S. enterica serovar Choleraesuis is one of the most important swine pathogens, and a frequent cause of serious systemic infections, including typhoid disease and pneumonia. Although S. enterica serovar Choleraesuis is highly host adapted, the bacterium is a frequent cause of systemic infections, such as bacteraemia, in humans (Blaser & Feldman, 1981; Cohen et al., 1987).

The human complement system is an important effector system of the innate immunity in the first line of defence against invading micro-organisms (Joiner, 1988; Taylor, 1983). The complement system is a complex system of serum proteins that can be activated by three different pathways: a classical pathway, a lectin pathway and an alternative pathway. Activation of the complement system by any of the three pathways leads to a number of biological consequences resulting in killing of invading bacteria. Both Gram-negative and -positive bacteria are opsonized with complement activation products C3b and iC3b, while the membrane attack complex (MAC) can be formed on Gram-negative organisms only. Although opsonophagocytosis is the major antibacterial defence mechanism by the complement system, the formation of the MAC has been shown to be necessary for killing of some Gram-negative bacteria (Joiner et al., 1986; Tomlinson et al., 1989). Importantly, microbes that cause invasive infections have evolved a number of strategies to protect themselves against the bactericidal action of the complement (Joiner, 1988; Taylor, 1983).

Structures of the bacterial cell surface, which include capsules, LPS and outer-membrane proteins, are responsible for the complement resistance of bacteria (Joiner, 1988; Rautemaa & Meri, 1999; Taylor, 1983). In Salmonella, the presence of long O-antigen chains is required for resistance to the complement cascade (Grossman et al., 1990; Joiner et al., 1986; Murray et al., 2003; Tomas et al., 1988). Other potential components of complement resistance have been identified in S. enterica serovar Typhimurium, including rck (Heffernan et al., 1992b), rsk (Vandenbosch et al., 1989) and traT (Rhen & Sukupolvi, 1988). The complement resistance mediated by Rck is associated with a failure to form fully polymerized tubular MACs on the bacterial surface membrane (Heffernan et al., 1992b). The rsk region does not contain the coding sequence, but it appears to enhance the ability of S. enterica serovar Typhimurium to survive in human serum, perhaps by removing effector molecules which function to repress serum resistance (Vandenbosch et al., 1989). The TraT lipoprotein of Salmonella and Escherichia coli increases the resistance of the bacteria to complement killing by interfering with the formation of C5b6, and the correct assembly and membrane insertion of the MAC (Pramoonjago et al., 1992). These loci involved in the serum resistance phenotype are carried on the virulence plasmid of S. enterica serovar Typhimurium. However, S. enterica serovar Choleraesuis contains the traT and rsk loci, but not the rck gene, on its virulence plasmid (Haneda et al., 2001).

We undertook an investigation of the bacterial component(s) involved in the serum-resistance phenotype of S. enterica serovar Choleraesuis, and found that an outer-membrane protein, PagC, when expressed in E. coli, conferred a high level of serum resistance upon the bacteria, which was equal to that conferred by the wild-type Rck. Furthermore, in contrast to the wild-type parental strain, an isogenic pagC mutant showed a decrease in serum resistance. The results of this study provide direct evidence that, although PagC does not possess the functional activity of serum resistance (Heffernan et al., 1994), it is an important surface component of S. enterica serovar Choleraesuis required for escape from host complement-mediated killing.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. All strains were incubated at 37 °C in Luria–Bertani (LB) broth, with gentle shaking. When appropriate, antibiotics were used at the following concentrations: ampicillin at 100 µg ml–1, kanamycin at 25 µg ml–1, rifampicin at 50 µg ml–1 and spectinomycin at 50 µg ml–1.


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Table 1. Bacterial strains and plasmids

 
Construction of plasmids.
The gene rck was PCR-amplified from the purified 94 kb plasmid DNA of S. enterica serovar Typhimurium strain SL1344 (Hoiseth & Stocker, 1981) using primers harbouring restriction sites (underlined), rck-1 (5'-CGGGATCCATGAAGCGGCGTTACGGG-3') and rck-2 (5'-ACGCGTCGACATATCTGACCGGGAGCG-3'), and the PCR product was cloned into the pBR322 using BamHI and SalI to create prck. The genes pagC and waaG were amplified from S. enterica serovar Choleraesuis strain RF-1 chromosome using pagC-1 (5'-CGGGATCCGATGACATTGTAGAACCGG-3') and pagC-2 (5'-ACGCGTCGACACTGGTAAAGAAGCCCTG-3'), or waaG-1 (5'-AGGCAGATGTTAAAGCCGCT-3') and waaG-2 (5'-CGCTCACAGTCGATTAGGTA-3'), and the PCR products were cloned into the TA cloning vector pGEM-T Easy (Promega) to generate pGEM-pagC and pGEM-waaG, respectively. The SphI–SalI fragments of pGEM-pagC and pGEM-waaG were ligated with the same sites of pACYC184 and pMW118 to produce ppagC and pwaaG, respectively. pGEM-pagC1 was generated by insertion of a 2·0 kb SmaI fragment of the {Omega} interposon from pHP45{Omega} (Fellay et al., 1987), which contains a streptomycin/spectinomycin-resistance gene flanked by strong transcription and translation termination signals, at the blunted ClaI site of pGEM-pagC. pwaaG1 was produced by insertion of the SmaI-digested Kmr-encoding gene (kan) cassette from pUC18K (Menard et al., 1993) at the EcoRV site of pGEM-waaG. The SphI–SalI fragment of pGEM-pagC1 was ligated with the same sites of plasmid pACYC184 to create ppagC1.

Mutations in PagC were made by reverse-PCR-based site-directed mutagenesis. The PCR product was generated by using a set of primers harbouring XbaI sites (underlined), pagC-MT1 (5'-GGTCTAGAAAGTACGGTTCTTTAATGGTTG-3') and pagC-MT2 (5'-GGTCTAGAAAACTTGTCATGGTAATGAATA-3'), designed to replace Glu at position 89 and Val at position 90 in the PagC peptide, with Ser and Arg, respectively. pGEM-pagC was used as a template. After gel purification using a Qiagen gel extraction kit, the PCR-amplified segment was digested with XbaI, self-ligated, and transformed into E. coli DH5{alpha}. Sequence analysis verified that the mutation was generated correctly. The SphI–SalI fragment of the resulting plasmid containing mutagenized DNA that encodes PagCE89S/V90R was ligated with the same sites of pACYC184 to produce ppagC2.

Construction of S. enterica serovar Choleraesuis mutants.
To construct pagC and waaG mutants of S. enterica serovar Choleraeusis strain RF-1, a NotI fragment of pGEM-pagC1 containing the pagC : : {Omega} allele or pGEM-waaG1 containing waaG : : kan was ligated with the same sites of the suicide vector pWM91 (Metcalf et al., 1996), and the resulting plasmids were transferred from E. coli S17.1{lambda}pir to S. enterica serovar Choleraesuis strain RF-1 by conjugation. Exoconjugants were selected for sucrose resistance, and screened for ampicillin sensitivity, as described by Gotoh et al. (2003) and Metcalf et al. (1996). Modifications on the chromosome were confirmed by PCR.

Construction of Salmonella serovar Choleraesuis genome DNA library and isolation of serum-resistant E. coli transformants.
DNA from S. enterica serovar Choleraesuis strain RF-1 was partially digested by Sau3AI, and size-selected on a 5–20 % potassium acetate density gradient. Fractions containing DNA of 1–5 kb in size were purified and ligated into the BamHI site of the plasmid vector pBR322, and used to transform E. coli DH5{alpha}. To identify clones expressing serum resistance, a plasmid library prepared in E. coli DH5{alpha} was screened by a large-scale serum-killing assay. E. coli transformants grown on LB plates (~1000 c.f.u. per plate) were suspended in 5 ml PBS, harvested by centrifugation, and resuspended in 5 ml TGS medium [1 % (w/v) tryptone, 1 % (w/v) glucose and 30 % (v/v) swine serum]. After incubation for 1 h at 37 °C, bacteria were harvested by centrifugation, resuspended in 5 ml TGS medium, and incubated for a further 1 h at 37 °C. The serum-resistant clones were then isolated on selective LB plates. Insertion of the chromosomal DNA into the pBR322 was confirmed by PCR using oligonucleotide primers PBR-1 (5'-CTCTATCTACTACGCGAT-3') and PBR-2 (5'-ACCTGTGGCGCCGGTGAT-3'). DNA sequencing of insert DNA was performed as described previously (Haneda et al., 2001).

Serum sensitivity assay.
Normal swine serum was collected, pooled, and stored at –80 °C until use. Normal human serum was obtained from Sigma-Aldrich. Control serum was heat inactivated for 30 min at 56 °C. For the serum killing assay, bacteria were grown to an OD600 of 0·3, then 0·2 ml of the culture was removed and centrifuged for 2 min at 10 000 g. The bacterial pellet was then suspended in 1 ml PBS with 0·1 % gelatin, and the bacterial suspension (500 µl), containing 107 c.f.u., was added to the same volume of swine serum (final concentration 50 %). Samples were obtained at 0, 5, 15 and 30 min after incubation at 37 °C, and the number of viable bacteria was calculated by serial dilution and plating on LB agar.

Antibody production.
To obtain polyclonal antibody against PagC protein, rabbits were immunized with PagC-peptide, encompassing amino acid residues 32–46 (YAQSKVQDFKNIRGV), in accordance with the institutional guidelines for animal housing and handling. The IgG fraction specific for PagC peptide was separated from immunized sera with PagC-peptide-conjugated epoxy-activated Sepharose 6B (Amersham Pharmacia Biotech).

Outer membrane preparation, SDS-PAGE and Western blotting.
Triton-X-100-insoluble outer membranes were isolated from E. coli and Salmonella strains according to the method described by Heffernan et al. (1992a). Protein concentrations were determined by use of a Micro BCA protein assay kit (Pierce Biotechnology), with BSA as a standard. Equal amounts of protein were subjected to SDS-PAGE in 15 % polyacrylamide gel, under reducing conditions. For immunostaining of PagC protein, proteins were transferred to a PVDF membrane (Immobilon; Millipore), and labelled with rabbit anti-PagC antiserum diluted at 1 : 1000, and alkaline-phosphatase-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich) diluted at 1 : 5000, as described previously (Miki et al., 2004).

Detection of surface-exposed PagC protein by confocal laser microscopy.
E. coli strains expressing PagC and mutant PagC proteins were grown overnight in LB broth. Bacteria were fixed with 1·0 % formaldehyde in PBS for 10 min, and then washed three times in PBS. Fixed bacteria were incubated with antibody against PagC (1 : 1000) for 1 h at room temperature, and unbound antibody was removed by washing in PBS containing 0·05 % Tween 20 (PBST). The pellet was suspended in Alexa-Fluor-488-conjugated anti-rabbit IgG antibody diluted in 1 : 500 (Molecular Probes). The suspension was then spotted on poly-L-lysine-coated microscope slides, and mounted with Vectashield mounting medium (Vector Laboratories) containing 4',6'-di-amidino-2-phenylindole (DAPI; Molecular Probes). Samples were analysed by confocal laser microscopy (model LSM510 META; Carl Zeiss).

Whole-cell ELISA.
To provide quantitative information on the surface-exposed PagC protein of E. coli and Salmonella strains, a whole-cell ELISA was carried out. Briefly, bacteria grown to an OD600 of 0·5 were removed and centrifuged for 2 min at 10 000 g. The bacterial pellet was then suspended in 1 ml 50 mM carbonate buffer (pH 9·5), and the bacterial suspension (100 µl) containing 108 c.f.u. was coated onto the wells of a 96-well ELISA plate at 4 °C overnight. The wells were then washed, and excess binding sites were blocked with 2 % BSA in PBS containing 0·02 % sodium azide. Rabbit anti-PagC antibody (1 : 1000), the primary antibody, was then incubated in the wells for 2 h at 37 °C. Following three washes with PBST, alkaline-phosphatase-conjugated goat anti-rabbit IgG antibody (1 : 5000) was added to each well, and incubated for 1 h at 37 °C. After extensive washing with PBST, substrate (p-nitrophenyl phosphate; Sigma-Aldrich) was prepared at 1 mg ml–1 in 1 M diethanolamine buffer (pH 9·8) containing 0·5 mM MgCl2, and added to the wells for 30 min. The absorbance at 405 nm was then measured using an ELISA plate reader (Ultrospec visible plate reader II; Amersham-Pharmacia-Biotech).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of the genetic region responsible for serum resistance in S. enterica serovar Choleraesuis
To identify the gene(s) associated with serum resistance activity in S. enterica serovar Choleraesuis, a chromosomal gene library was made in E. coli DH5{alpha}, a strain that is quite sensitive to serum killing, from S. enterica serovar Choleraesuis strain RF-1. E. coli transformants carrying the chimeric plasmid DNAs were screened for their serum resistance phenotype. From a total of ~10 000 transformants tested, 12 clones were identified that expressed serum resistance. PCR-amplification of inserted DNA fragments from 12 of these chimeric plasmids demonstrated that all of them contained a 3·6 kb DNA fragment of the S. enterica serovar Choleraesuis chromosome. In addition, restriction enzyme analysis revealed that 12 of these chimeric plasmids were identical. Thus, only one of these 12 clones, plasmid pMK1 (Fig. 1), was chosen for further detailed characterization.



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Fig. 1. Restriction map and mutagenesis of the chimeric plasmid pMK1, which contains a 3584 bp fragment of the S. enterica serovar Choleraesuis strain RF-1 chromosome, including the gene encoding PagC (pagC). The top line represents the restriction sites on pMK1, with an adjacent region consisting of DNA from pBR322 (filled box). The boxes under the restriction map indicate the assignment of the putative coding regions identified by homology to the S. enterica serovar Typhimurium genome sequence (McClelland et al., 2001). Fragments of pMK1 were subcloned to further determine the region required for serum resistance, as indicated. The open boxes in the derivatives of pMK1 indicate deleted DNA segments. The filled arrowheads indicate the orientation of the promoter readthrough from the vector plasmid pBR322. Insertion of the {Omega}Km-2 fragment (Perez-Casal et al., 1991) is represented by an inverted open triangle. Serum resistance activity was determined as described for Fig. 2. + and –, resistance and sensitivity to pooled swine serum, respectively.

 
Characterization of the pagC region of S. enterica serovar Choleraesuis
A series of chimeric plasmids were constructed to determine the minimal DNA region required for serum resistance in E. coli DH5{alpha} (Fig. 1). To determine the serum resistance of E. coli transformants harbouring these plasmids, the kinetics of bacterial viability in swine serum was measured (Fig. 2). Plasmids pBR322 and prck were used for negative and positive controls, respectively. These data suggested that the 0·8 kb PstI–BglII fragment present in pMK6 was sufficient to make the bacteria serum resistant. Furthermore, insertional mutagenesis of the 1·6 kb PstI–EcoRI fragment, using an {Omega} element containing the kanamycin resistance gene ({Omega}Km2), showed that insertion into the ClaI site (pMK7), but not the BglII site (pMK8), abolished expression of the serum resistance activity of E. coli transformants, confirming the above results. Since inactivation of complement by heating at 56 °C for 30 min prior to bacterial inoculation permitted survival to the same extent as in PBS only, the bacterial killing by swine serum appeared to be mediated by the complement system.



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Fig. 2. Serum sensitivity assay for E. coli strains carrying the plasmid pMK1 and its derivatives. (a) E. coli DH5{alpha} strains containing pMK1 and its derivatives were tested for viability in swine serum. (b) E. coli HB101 strains containing pBR322 and ppagC were tested for viability in human serum. Bacteria grown to mid-exponential phase were incubated at 37 °C in 50 % serum ({bullet}) or heat-inactivated serum ({blacksquare}).

 
To further analyse the chromosomal region related to serum resistance in S. enterica serovar Choleraesuis, a 3·6 kb DNA fragment in pMK1 was sequenced. A homology search of the DNA database revealed that the 3584 bp DNA segment from S. enterica serovar Choleraesuis chromosome corresponded to that of bp 1 332 185–1 335 769 in the S. enterica serovar Typhimurium LT2 complete genome sequence (McClelland et al., 2001), which contains the five ORFs (STM1246–STM1250) and one truncated ORF (STM1251) (Fig. 1). A 558 bp coding region (STM1246, corresponding to pagC) between the PstI and BglII sites was identified as the minimal region necessary to confer serum resistance upon E. coli. The predicted amino acid sequence of PagC from S. enterica serovar Choleraesuis exhibited 100 % identity to the PagC from S. enterica serovar Typhimurium LT2 (McClelland et al., 2001), and 96 % identity (98 % homology) to the PagC from S. enterica serovar Typhi CT18 (Parkhill et al., 2001).

Heffernan et al. (1994) showed that PagC did not confer attachment, invasion or serum resistance. Thus, to examine whether expression of PagC on the outer membrane in E. coli DH5{alpha} could be responsible for the serum resistance phenotype, outer-membrane fractions, which had been partially purified by the treatment of whole cells with Triton X-100, were isolated and analysed for the presence of PagC by Western blotting, using an antibody specific for PagC. As shown in Fig. 3, a 17·8 kDa immunoreactive protein corresponding to the molecular mass of mature PagC was detected from E. coli harbouring pMK1, pMK3, pMK6 and pMK8, which showed serum resistance, but not from E. coli carrying pMK2, pMK4, pMK5 and pMK7, which showed serum sensitivity. The same results were obtained from E. coli strains containing ppagC with a cloned intact pagC gene, and ppagC1 with a pagC gene inactivated by {Omega} insertion (pagC : : {Omega}) (data not shown). These results strongly suggest that the serum resistance phenotype is conferred upon E. coli by expression of the outer-membrane protein PagC. Furthermore, since Heffernan et al. (1994), using E. coli strain HB101 and human serum, showed that cloned pagC does not encode a serum resistance phenotype, plasmid ppagC was transformed into E. coli strain HB101, and the transformant was tested for viability in normal human serum. As when expressing Rck, E. coli HB101 expressing PagC also exhibited serum resistance (Fig. 2b). Thus, the discrepancy between this and other studies regarding PagC function is not due to differences in the E. coli strain and the source of the serum.



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Fig. 3. Expression of PagC in the outer-membrane fraction prepared from E. coli strains carrying the indicated plasmids. Triton-X-100-insoluble outer membranes were isolated from E. coli strains, and equal amounts of protein (10 mg per lane) were separated in 15 % polyacrylamide gels by SDS-PAGE. Proteins were transferred to a PVDF membrane, and probed with affinity-purified rabbit anti-PagC antibody.

 
Serum resistance phenotype of PagC mutated in the surface-exposed loop 2 region
PagC protein belongs to a family of outer-membrane proteins containing eight transmembrane {beta} strands, and is highly homologous to Rck from S. enterica serovar Typhimurium, and Ail from Yersinia enterocolitica (Fig. 4a). Site-directed mutagenesis of Ail has revealed that amino acid residues Asp-68 and Val-69, in the C-terminal end of the second surface-exposed domain (loop 2), are active sites for serum resistance (Miller et al., 2001). Therefore, to test whether the functional region of PagC for serum resistance is similar to the homologous protein Ail, we constructed a mutant PagC protein (PagCE89S/V90R) in which Glu-89 and Val-90, corresponding to Asp-68 and Val-69 in Ail, were replaced with Ser and Arg, respectively. The mutant PagC containing double point mutations expressed by E. coli was detected in whole-cell extract at the same level as the wild-type PagC (data not shown). In addition, the amount of PagCE89S/V90R in Triton-X-100-insoluble membrane fractions analysed by immunoblotting, using a rabbit anti-PagC antibody, was found to be similar to the wild-type level, suggesting that PagCE89S/V90R is correctly located in the outer membrane (Fig. 5a). The surface expression of the PagC mutant protein was further confirmed quantitatively by whole-cell ELISA and immunofluorescence microscopy (Fig. 5b, c). Although the mutations in PagC did not affect the amount of PagC produced, or its proper location in the membrane, E. coli expressing mutant PagCE89S/V90R (ppagC2) showed complete loss of the serum resistance phenotype (Fig. 6). Thus, it is likely that the amino acid residues (Glu-89 and Val-90) in loop 2 (see Fig. 4b) are important for the function of the PagC protein.



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Fig. 4. (a) Alignment of the amino acid sequences for mature PagC, Rck and Ail proteins from S. enterica serovar Choleraesuis, S. enterica serovar Typhimurium and Y. enterocolitica, respectively. Amino acid residues conserved across all three proteins are indicated by an asterisk. The putative extracellular loop regions of these proteins are underlined. (b) A model of the topology of PagC in the outer membrane; the sequence of the extracellular loops is shown. Point-mutated amino acid residues of PagCE89S/V90R are indicated by the box.

 


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Fig. 5. Expression and localization of the wild-type PagC and point-mutated PagCE89S/V90R. (a) Western blot analysis of Triton-X-100-insoluble outer-membrane fractions from E. coli strains carrying ppagC, ppagC2 or pBR322 was performed by using affinity-purified rabbit anti-PagC antibody as described in Fig. 3. M, molecular mass standards. (b) E. coli strains expressing PagC (ppagC), PagCE89S/V90R (ppagC2), or nothing (pBR322) were grown to mid-exponential phase, and coated on the wells of a 96-well plate. The surface expression of PagC protein was then examined by an ELISA using a rabbit anti-PagC antibody. The data are means of triplicate samples ±SD. (c) Surface expression of E. coli strains expressing PagC (ppagC) and PagCE89S/V90R (ppagC2) was examined by immunofluorescence microscopy. Bacteria were fixed, immunostained using an antibody specific for PagC (green fluorescence), and counterstained with DAPI (blue fluorescence) to visualize bacterial DNA.

 


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Fig. 6. Serum sensitivity assay for E. coli strains carrying the plasmid ppagC ({bullet}) or ppagC2 ({circ}). Bacteria grown to mid-exponential phase were incubated at 37 °C in 50 % swine serum.

 
PagC is a determinant of bacterial resistance to serum complement in Salmonella serovar Choleraesuis
To further demonstrate whether the pagC gene is an important determinant of resistance to the host complement in S. enterica serovar Choleraesuis, we constructed an isogenic S. enterica serovar Choleraesuis pagC mutant strain, MK100, and compared its serum resistance activity with that of the wild-type strain RF-1, which was found to be highly resistant to serum-mediated killing. The surface expression of PagC protein in these strains was examined by Western blotting (Fig. 7a). As shown in Fig. 7(b), the pagC mutant strain MK100 could survive exposure to 50 % swine serum for 30 min; thereafter, the number of viable bacteria decreased, reaching less than 90 % of the inoculated dose at 60 min, whereas the wild-type RF-1 continued to show serum resistance at the same time point. Introduction of the intact pagC gene in plasmid ppagC to strain MK100 (strain MK101) restored the serum resistance to the wild-type level (Fig. 7b), but the vector alone had no effect (data not shown). In addition, no bactericidal activity was observed when these bacteria were incubated with heat-inactivated swine serum, as expected (data not shown).



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Fig. 7. PagC is required for full expression of the serum resistance phenotype in S. enterica serovar Choleraesuis. (a) Western blot analysis of Triton-X-100-insoluble outer-membrane fractions (20 mg per lane) from S. enterica serovar Choleraesus strains RF-1 (wild-type), MK100 (pagC : : {Omega}), and MK101 (MK100/ppagC) was performed by using an affinity-purified rabbit anti-PagC antibody. (b) Serum sensitivity assay for S. enterica serovar Choleraesuis strains RF-1 (wild-type; {bullet}), MK100 (pagC : : {Omega}; {blacksquare}) and MK101 (MK100/ppagC; {circ}). Bacteria grown to mid-exponential phase were incubated at 37 °C in 50 % swine serum. (c) Comparison of the viabilities of S. enterica serovar Choleraesuis strains RF-1 (wild-type; {bullet}, {circ}) and MK100 (pagC : : {Omega}; {blacksquare}, {square}) grown in TGM medium containing 30 % swine serum ({bullet}, {blacksquare}) or heat-inactivated serum ({circ}, {square}). The pagC mutant strain shows a significantly reduced growth rate compared to the parent strain.

 
We next tested the growth of S. enterica serovar Choleraesuis strains RF-1 and MK100 (pagC : : {Omega}) with TGS medium containing 30 % swine serum. Bacteria cultured at 37 °C without shaking were removed at regular intervals, and the OD600 was measured. The wild-type strain RF-1 grew at similar rates in TGS medium containing either serum or heat-inactivated serum, whereas the growth rate of the mutant strain decreased as compared with that of the wild-type strain (Fig. 7c). The growth rate in strain MK101 was similar to that of the wild-type strain (data not shown). These results suggested that pagC inactivation altered the serum resistance activity of S. enterica serovar Choleraesuis.

Since the LPS O-antigen has been implicated as a major determinant of the serum resistance of Salmonella (Grossman et al., 1990; Joiner et al., 1986; Murray et al., 2003; Tomas et al., 1988), the effect of pagC expression on the serum resistance of rough S. enterica serovar Choleraesuis strains was examined. The PagC expression of these strains was measured by immunoblotting and whole-cell ELISA using a rabbit anti-PagC antibody (Fig. 8a, b). The rough LPS mutant MK200 (waaG : : kan), which lacks an outer core region and O-antigen chains of LPS, showed a marked decrease in survival upon incubation with swine serum, although the level of PagC expression in its outer membrane was similar to that of the wild-type (Fig. 8a, b). Complementation with an intact waaG gene on the plasmid (pwaaG) restored the LPS structure as well as the serum resistance phenotype of the mutant strain MK200. Interestingly, overexpression of pagC by introduction of plasmid ppagC caused a marked increase in the serum resistance of the rough mutant MK200 (Fig. 8c). These results indicate that PagC plays an important role in the resistance of S. enterica serovar Choleraesuis to the bactericidal activity of serum complement. In addition, the waaG and pagC double mutant strain MK203 showed increased susceptibility to serum complement compared with the waaG single mutant strain MK200 at 60 min after incubation (Fig. 8c), indicating that the serum resistance activity encoded by pagC is expressed independently of the LPS structure.



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Fig. 8. Effect of PagC expression on the serum resistance of rough Salmonella strains. (a) Western blot analysis of Triton-X-100-insoluble outer-membrane fractions (20 mg per lane) from S. enterica serovar Choleraesus strains RF-1 (wild-type), MK200 (waaG : : kan), MK201 (MK200/pwaaG) and MK202 (MK200/ppagC) was performed by using a rabbit anti-PagC antibody. (b) S. enterica serovar Choleraesuis strains RF-1 (wild-type), MK 100 (pagC : : {Omega}), MK200 (waaG : : kan), MK201 (MK200/pwaaG) and MK202 (MK200/ppagC) were grown to mid-exponential phase, and coated on the wells of a 96-well plate. Surface expression of PagC protein was then examined by an ELISA using an affinity-purified rabbit anti-PagC antibody. The data are means±SD of triplicate samples. (c) Serum sensitivity assay for S. enterica serovar Choleraesuis strains RF-1 (wild-type; {bullet}), MK200 (waaG : : kan; {blacksquare}), MK201 (MK200/pwaaG; {blacktriangleup}), MK202 (MK200/ppagC; {circ}) and MK203 (pagC : : {Omega} waaG : : kan; {square}). Bacteria grown to mid-exponential phase were incubated at 37 °C in 50 % swine serum.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Resistance to complement-mediated killing or serum resistance is an important virulence determinant in Gram-negative bacteria. In this study, we found that the pagC gene of S. enterica serovar Choleraesuis chromosome confers a high level of resistance to the bactericidal activity of pooled normal swine serum when cloned into E. coli or Salmonella strains. In addition, insertion and deletion mutations of the cloned pagC gene abolished serum resistance in the host strains. pagC is a phoPQ-regulated gene required for intramacrophage survival and virulence of Salmonella in a mouse infection model (Miller et al., 1989). PagC belongs to a family of 17–19 kDa outer-membrane proteins that are predicted to have eight membrane-spanning amphipathic {beta}-strands and four loops (Heffernan et al., 1992a), i.e. Rck (S. enterica serovar Typhimurium), Ail (Y. enterocolitica), OmpX (Enterobacter cloacae), Lom (bacteriophage {lambda}) and Omp4 (Serratia marcescens). Despite the high level of homology among these outer-membrane proteins, they appear to be functionally distinct. While Rck and Ail share similar phenotypes that confer serum resistance, and attachment and invasion to tissue culture cells (Bliska & Falkow, 1992; Heffernan et al., 1994; Pierson & Falkow, 1993), expression of OmpX and Lom has no effect on these virulence-associated phenotypes (Heffernan et al., 1994). In contrast to the present findings, Heffernan et al. (1994) reported that PagC does not confer attachment, invasion or serum resistance. However, we found that a cloned pagC gene was able to promote resistance to complement-mediated lysis. Since both our study and that of Heffernan et al. (1994) used pBR322-based constructs in the same E. coli strain, and the same source of serum to assess the serum sensitivity, the reason for the discrepancy remains unclear. Recently, consistent with the high sequence similarity between the predicted extracellular loops of Rck and PagC, a conserved role of both proteins in binding to host tissue mediated by the extracellular matrix component laminin has been reported (Crago & Koronakis, 1999).

The lack of a common function shared among the members of this family of outer-membrane proteins has been attributed to the fact that the membrane-spanning regions are homologous (Beer & Miller, 1992), while the cell-surface domains, which appear to determine the specificity of bacteria–host-cell interactions, show higher levels of sequence divergence (Miller et al., 1992). Recently, Miller et al. (2001) determined the functional domain of Ail required for the invasion and serum resistance phenotypes. Analysis of the insertion, deletion and point mutations in the surface-exposed loops of Ail revealed that the second cell-exposed domain (loop 2), contains a sequence required for Ail function, and the first and fourth cell surface domains (loops 1 and 4) are not directly responsible for Ail-mediated phenotypes. In addition, D67G and V68G, the double point mutations in the C-terminal end of loop 2, completely eliminated both the serum resistance and invasion phenotypes. Consistently, the double mutations in the C-terminal end of the second cell-surface domain of PagC caused loss of serum resistance, demonstrating that a specific amino acid sequence in the second cell-surface-exposed domain is critical for the interaction of the protein with the complement components. Furthermore, in a study comparing the chimeric proteins Rck and PagC, Cirillo et al. (1996) showed that replacement of the C-terminal-half containing loops 3 and 4 of Rck with the corresponding region of PagC abolished the ability of Rck to confer serum resistance, while replacement of loop 4 alone had no effect on serum resistance. Thus, the third cell-surface-exposed domain of Rck is involved in the protein function (Cirillo et al., 1996). These data may also indicate that the third and fourth loops of PagC are not associated with PagC-mediated serum resistance.

In conclusion, although the basis of the serum resistance phenotype of PagC has not yet been elucidated, we have shown that the pagC gene from the S. enterica serovar Choleraesuis chromosome confers upon E. coli and Salmonella strains the ability to grow in swine serum at a rate comparable to their growth in control heat-inactivated serum. Interestingly, S. enterica serovar Typhimurium, but not serovar Choleraesuis, maintains two outer-membrane proteins that are responsible for serum resistance: Rck, encoded on the virulence plasmid, and PagC, encoded on the chromosome. Therefore, the conservation of the two copies of these proteins may be of selective advantage to S. enterica serovar Typhimurium, which has a wider range of host specificity than serovar Choleraesuis, because it offers an increased opportunity of survival during systemic infection of its host.


   ACKNOWLEDGEMENTS
 
We would like to thank Shinobu Imajoh-Ohmi for preparation of the synthetic peptides, and Hiroaki Shime and Takatoshi Kawakami for construction of the S. enterica serovar Choleraesuis genome DNA library. This work was supported in part by a Grant-in-Aid for Exploratory Research (15659105), and by a 21st Century COE Program Grant from the Japanese Ministry of Education, Culture, Sports, Sciences and Technology.


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Received 24 September 2004; revised 15 November 2004; accepted 16 November 2004.