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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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. Sequence analysis verified that the mutation was generated correctly. The SphISalI 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 : : 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
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 520 % potassium acetate density gradient. Fractions containing DNA of 15 kb in size were purified and ligated into the BamHI site of the plasmid vector pBR322, and used to transform E. coli DH5. To identify clones expressing serum resistance, a plasmid library prepared in E. coli DH5
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 3246 (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 ml1 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).
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RESULTS |
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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
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
insertion (pagC : :
) (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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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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DISCUSSION |
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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 bacteriahost-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.
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ACKNOWLEDGEMENTS |
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Received 24 September 2004;
revised 15 November 2004;
accepted 16 November 2004.