Vol. 174, No. 6

JOURNAL OF BACTERIOLOGY, Mar. 1992, P. 1965-1973

0021-9193/92/061965-09$02.00/0 Copyright © 1992, American Society for Microbiology

Structural Relatedness of Enteric Bacterial Porins Assessed with Monoclonal Antibodies to Salmonella typhimurium OmpD and OmpC SHIVA P. SINGH,1* YVONNE UPSHAW,1 TARIQ ABDULLAH,1 SHREE R. SINGH,1 AND PHILLIP E. KLEBBA2 Biomedical Research and Training Programs, Alabama State University, Montgomery, Alabama 36101-0271,1 and Department of Microbiology, Medical College of Wisconsin, Milwaukee, Wisconsin 532262 Received 4 December 1991/Accepted 10 January 1992

The immunochemistry and structure of enteric bacterial porins are critical to the understanding of the immune response to bacterial infection. We raised 41 monoclonal antibodies (MAbs) to SalmoneUla typhimurium OmpD and OmpC porin trimers and monomers. Enzyme-linked immunosorbent assays, immunoprecipitations, and/or Western immunoblot techniques indicated that 39 MAbs (11 anti-trimer and 28 anti-monomer) in the panel are porin specific and one binds to the lipopolysaccharide; the specificity of the remaining MAb probably lies in the porin-lipopolysaccharide complex. Among the porin-specific MAbs, 10 bound cellsurface-exposed epitopes, one reacted with a periplasmic epitope, and the remaining 28 recognized determinants that are buried within the outer membrane bilayer. Many of the MAbs reacting with surface-exposed epitopes were highly specific, recognizing only the homologous porin trimers; this suggests that the cellsurface-exposed regions of porins tend to be quite different among S. typhimurium OmpF, OmpC, and OmpD porins. Immunological cross-reaction showed that S. typhimurium OmpD was very closely related to Escherichia coli NmpC and to the Lc porin of bacteriophage PA-2. Immunologically, E. coli OmpG and protein K also appear to belong to the family of closely related porins including E. coli OmpF, OmpC, PhoE, and NmpC and S. typhimurium OmpF, OmpC, and OmpD. It appears, however, that S. typhimurium "PhoE" is not closely related to this group. Finally, about one-third of the MAbs that presumably recognize buried epitopes reacted with porin domains that are widely conserved in 13 species of the family Enterobacteriaceae, but apparently not in the seven nonenterobacterial species tested. These data are evaluated in relation to host immune response to infection by gram-negative bacteria. Lc porins show roughly 65% identity and 80% similarity (3, 35). The immunological properties of E. coli porins OmpF, OmpC, PhoE, and LamB have been studied with both polyclonal and monoclonal antibodies (MAbs). Purified porins are immunogenic in mice and rabbits (19, 43) as trimers or monomers. Although antisera to trimers usually do not recognize monomers and vice versa (19, 43, 46), MAbs raised to monomers can sometimes recognize epitopes on trimers (2). In this report, we describe the isolation and characterization of MAbs against OmpD and OmpC of S. typhimurium and the reactivity of these MAbs with the porins of Salmonella species, E. coli, and other bacteria in the family Enterobacteriaceae. We discovered a close structural relationship among S. typhimurium OmpD, E. coli NmpC, and the Lc porin of bacteriophage PA-2 and found that OmpG and protein K are immunologically similar to the OmpFOmpC-PhoE group of E. coli porins. Generally speaking, the Salmonella porins are like the porins of E. coli in that their surface epitopes are structurally diverse but their buried epitopes are structurally conserved.

The outer membrane (OM) of Salmonella typhimurium and other gram-negative bacteria contains a family of poreforming proteins called porins (40) that exist as homo- or heterotrimers (15) in vivo. The primary structure of porins is quite hydrophilic overall but contains short stretches (10 to 14 residues) that cross the OM bilayer as amphiphilic 13-strands (24, 59, 60). Porins have numerous (five or more) surface epitopes, 6 to 25 residues in length (24), that are at least partially obscured by the lipopolysaccharide (LPS) core and completely blocked by 0-antigen sugars (2, 32). The primary structure of porins varies significantly among different gram-negative bacteria (16), but it is likely that their predominant structural motif of amphiphilic ,B-strands forming a barrel is rather conserved (16, 22, 60). Generally, Escherichia coli K-12 produces two porins, OmpC and OmpF, whereas S. typhimurium LT-2 synthesizes three porin species, OmpC (36,000), OmpF (35,000), and OmpD (34,000) (28, 40). The relative amounts of OmpF and OmpC vary in response to changes in medium osmolarity (30, 52), and the production of other porins is limited either to strains with a special genetic background (NmpC, Lc, OmpG, protein K) or to cells cultured under special conditions (PhoE) (40). The OmpD porin of S. typhimurium is similar to the NmpC and Lc proteins of E. coli in that all three are subject to catabolite repression (28, 48). Unlike OmpC and OmpF, the biosynthesis of OmpD, NmpC, and Lc is not affected by the osmolarity of the growth medium (48, 52). The sequences of OmpF, OmpC, PhoE, NmpC, and

MATERIALS AND METHODS Bacterial strains and growth conditions. S. typhimurium LT-2 (wild-type) and SH 5014 were provided by H. Nikaido; the latter is an rfa mutant derived from strain SL 1027 that expresses all three porins (41); strains SH 7454, SH 7455, and SH 7457 (provided by P. H. Makela) are ompC::TnlO, ompD::TnlO ompC::TnlO, and ompD::TnlO gyrA deriva-

* Corresponding author. 1965

1966

SINGH ET AL.

tives of SH 6749 (54) and express the OmpD, OmpF, and OmpC proteins, respectively, as their sole porin when grown in appropriate medium (see below). Strain KB17 (provided by W. Boos) expresses "PhoE" as its major porin (1). E. coli JF 701 (provided by K. Gehring) expresses OmpF porin (39). JF 1047 and JF 1054 (provided by J. Foulds) express protein K and NmpC porin, respectively, while CE 1234 and CE 1230 (provided by J. Tommassen) express OmpC and PhoE porins, respectively (25). TNE 012 (provided by T. Nakae) expresses LamB protein. Strain PC 2086 (provided by J. Tommassen) is lysogenic for phage HK 253 and produces porin Lc (58). Strain RAM 456 (provided by S. Benson) is a derivative of MC 4100 that expresses OmpG (34). Clinical isolates of various enteric and nonenteric bacteria were generously provided by Dana Moore, Ronald Vance, and William Callan of the State Health Laboratory, Montgomery, Ala. The enteric bacteria (except Yersinia enterocolitica; see below) were grown at 37°C in L broth containing 1% tryptone (Difco), 0.5% yeast extract (Difco), 0.5% NaCl, and 0.5% glucose. To repress the synthesis of OmpF, L broth was adjusted to 1.5% NaCl. The production of LamB was induced by the addition of 0.5% maltose (in place of glucose) to the medium (38). The OmpF-expressing strains were grown in nutrient broth. Bordetella pertussis was grown on Jones-Kendrick medium with cephalothin, Legionella pneumophila was grown on buffered charcoal-yeast extract, Brucella suis was grown on brucella agar, Pseudomonas aeruginosa, Aeromonas hydrophila, and Y enterocolitica were grown on 5% sheep blood agar, Haemophilus influenzae was grown on enriched chocolate agar, and Neisseria gonorrhoeae was grown on Thayer-Martin medium. Exponentially growing cells were harvested by centrifugation (10,000 x g for 20 min at 4°C), washed with 50 mM Tris-HCl buffer (pH 7.2), and either used immediately for coating of the enzyme-linked immunosorbent assay (ELISA) microtiter plates or frozen at -85°C for later use in porin purification or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots (immuno-

blots). Isolation and purification of antigens. S. typhimurium SH 7454, SH 7455, and SH 7457 were used as the sources of OmpD, OmpF, and OmpC porins, respectively. Because SH 7454 and SH 7457 contained the ompF+ allele, its expression was repressed by growing cells in the presence of 1.5% NaCl (see above). Native porin trimers (trimer) were isolated and purified by solubilization in 1% SDS-0.5 M NaCl and then gel filtration (15, 38). Denatured monomeric porin (monomer) was prepared by boiling the trimer at 100°C for 5 min in 1% SDS (24). OM fragments were prepared by French pressure cell lysis and purified by sucrose density gradient centrifugation (53). LPS (R type) was isolated from S. typhimurium SH 7454 and SH 7457 by the method of Galanos et al. (14); the LPS from S. typhimurium LT-2 (S type) was purchased from Difco. Production of anti-porin MAbs. BALB/c mice were immunized in the foot pads with 80 p,g of monomer or trimer on days 1, 7, 10, 13, 16, and 19. The first injection was in complete Freund's adjuvant, while the remaining were given in Dulbecco's phosphate-buffered saline (PBS). Lymph node cells from immunized mice were fused with P3x63-Ag8.653 myeloma cells on day 20 by using polyethylene glycol 4000 (Accurate Chemical & Scientific Corp.) as described by Kearney (23). Hybridomas were selected in hypoxanthineaminopterin-thymidine medium containing peritoneal feeder cells (23). Culture fluids from wells containing hybridoma

J. BACTERIOL.

colonies were assayed for specificity by ELISA against monomer, trimer, OM, and LPS. Hybridomas of interest were cloned twice by limiting dilution and injected into BALB/c mice for production of ascitic tumors (23). Ascites fluids were clarified by centrifugation (300 x g for 6 min) and stored frozen at -85°C until use. ELISA titers of ascitic fluids varied from approximately 1035 to 106, with the majority of ascites titers being 104 to 105 (data not shown). Isotype and subclass determination. The class and subclass of MAbs in culture supernatants were determined by ELISA with goat antisera against mouse heavy and light chains jt, 'Yl' Y2a, Y2b, Y3, K, and X (Clonotyping Kit System I; Fisher Scientific Co.). ELISA. Intact, formaldehyde-fixed bacteria (5 x 108 cells per ml), OM (50 ,ug/ml), and purified porins (10 pg/ml) were suspended in 0.01 M ammonium acetate-0.01 M ammonium carbonate (pH 8.2) and used to coat the wells of microtiter plates (Immunol II; Dynatech Laboratories, Inc.). LPS was suspended in Dulbecco's PBS (at 100 ,ug/ml). The wells were blocked with 1% bovine serum albumin, washed with borate (0.1 M, pH 8.2)-buffered saline, and filled with 100 JLl of an appropriate dilution of ascitic fluid or hybridomna culture supernatant. After overnight incubation at 4°C, the plates were washed and developed with alkaline phosphataselabeled goat anti-mouse immunoglobulin (Ig) (1:500; Fisher)p-nitrophenylphosphate (1 mg/ml; Sigma). The reactions were quantitated by measurement of optical density at 405 nm on a BioTek model EL 310 ETA reader. Absorbance readings that were greater than twice the background absorbance were considered positive. Immunoprecipitation. Radiolabeled envelopes and OM were prepared from S. typhimurium LT-2 cells grown in MOPS (morpholinopropanesulfonic acid) minimal medium supplemented with 0.4% glucose as a carbon source (52). Exponentially growing cells (optical density at 420 nm = 0.2) were labeled with 200 p,Ci of [35S]methionine (1,162 Ci/ mmol) per ml of medium and allowed to grow to the mid-log phase. Cells were harvested and lysed in the French press, and the cell envelope was isolated as described previously (52). The OM was separated by sucrose density gradient centrifugation, and all subsequent procedures were carried out as described before (24) except that protein G coupled to Sepharose 4B (Zymed Laboratories) was used for immunoprecipitation of antigen-antibody complexes. SDS-PAGE. Electrophoretic separations were performed on 11% polyacrylamide gels by the method of Lugtenberg et al. (29). Whole cells (109 cells per ml), envelopes or OM (500 ,ug/ml), porins (100 p,g/ml), and LPS (250 jig/ml) were solubilized in SDS sample buffer by boiling at 100°C for 5 min. The samples were either loaded directly on top of the slab (1 ml per gel) or applied in individual lanes (20 pl per lane), and the electrophoresis was carried out at 4°C at 30 mA per gel. Molecular weight markers (Bio-Rad) were also included in one lane. Proteins were stained with Coomassie blue or ammoniacal silver (Bio-Rad); LPS was also stained with ammoniacal silver (55). Western immunoblot analysis. Proteins from SDS-polyacrylamide gels were transferred to nitrocellulose paper (BA 85, 0.45-,um pore size; Schleicher & Schuell) at 15 V for 12 to 16 h at 4°C. Following transfer, the paper was blocked with Tris-buffered saline containing 1% gelatin and 0.1% NaN3 for 1 h at 37°C and then cut into either 0.4-cm-wide vertical strips or a 3.2-cm horizontal strip containing proteins in the molecular mass range of 30 to 50 kDa. The strips were incubated overnight with a 1:50 dilution of ascites fluid at 4°C, and the subsequent blotting procedures were per-

ENTERIC BACTERIAL PORINS

VOL. 174, 1992

formed as described previously (2). Immunoblot reactions of anti-porin MAbs were evaluated in comparison with the reactions of normal mouse serum and ascites fluid from the cell fusion partner P3x63-Ag8.653, which is a nonsecretor of immunoglobulins (23). RESULTS AND DISCUSSION MAb specificities. Hybridomas were raised by immunizing BALB/c mice with S. typhimunum OmpD and OmpC trimers or monomers and subsequently fusing their immune lymphocytes with P3x63-Ag8.653 myeloma cells. After initial screening by ELISA, 303 hybridomas of interest were cloned twice by limiting dilution. Subsequently, 76 clones were injected into mice for ascites production. Further evaluation by ELISA and Western blot suggested that 41 of the 76 were unique antibodies. Among these, six IgGl, 12 IgG2a, 17 IgG2b, and six IgM heavy chains were identified (Table 1); all the MAbs contained K light chains. MAbs were classified into six distinct groups on the basis of their reactivity in ELISA with purified Salmonella porins, OM, LPS, and intact whole cells and their immunoblot reactivity with denatured whole-cell lysates (Table 1). (i) Group I. Ten MAbs recognized native porin epitopes that are exposed on the cell surface. Eight of these were raised against trimers, and among these, six MAbs reacted only with the immunogen (homologous) trimer, whether purified or as a component of OM or whole cells. These results, in agreement with previous studies (9, 24, 45, 57), identify antibodies that bind surface epitopes of the trimers. These determinants are presumably conformational rather than sequential, because they are not recognized if the trimers are denatured to monomers. The six antibodies that bind only homologous trimer (Table 1) demonstrate that sequence variability exists in the cell-surface-exposed parts of the three related Salmonella pore proteins. This is consistent with findings of several other studies which have shown that MAbs directed against native porins fail to detect common antigenic sites among, PhoE, OmpC, and OmpF pore proteins (2, 45, 57, 61), despite high sequence homology and cross-reactivity with polyclonal antisera (43). Since surface domains of OM proteins act as receptors for phages and colicins, the variability in their constituent residues may arise from selective pressure to evade such toxic agents and antibodies (4). Indeed, the study of an OM protein, FepA, among various species showed that the surface residues undergo a more rapid evolutionary change than polypeptides that are buried in the OM bilayer (49). Immunoprecipitations with 35S-labeled cell lysates revealed that MAbs raised to native porins reacted with both trimers and dimers (data not shown). Although MAbs 3, 10, 15, 42, 81, and 82 bound to whole cells of rough strains, none recognized smooth bacteria (Table 1), confirming that the surface epitopes of S. typhimunum are blocked by LPS in the same manner as are the surface epitopes of E. coli PhoE (57) and OmpF (2, 32) and P. aeruginosa protein F (37). The remaining four MAbs in group I (12, 46, 66, and 68) recognized some of the other (purified) Salmonella porins in their native or denatured forms (Table 1). These antibodies (two each raised against trimers and monomers) also bound to intact cells of homologous and heterologous strains, indicating conservation of the surface epitopes that they recognize. (ii) Group II. This category contained a single antibody (MAb 47) that reacted with homologous trimer, either puri-

1967

fied or in OM, but not in whole cells, suggesting that its epitope lies on the periplasmic face of the OmpC porin. (iii) Group III. MAbs 6 and 84 constituted a third distinct category of antibody that reacted with purified trimer but not with OM or intact whole cells (Table 1); these antibodies were positive only with the immunogen trimer in ELISA and presumably bind epitopes that are buried within the OM bilayer. (iv) Group IV. Twenty-six MAbs in group IV reacted strongly with homologous monomer in both ELISA (Table 1) and Western immunoblots. Approximately one-fifth of the MAbs in this group reacted with homologous monomer only (Table 1, and Fig. 1A and B), while the remainder also recognized heterologous (Salmonella) porins (Table 1). (v) Group V. One MAb (48) gave a weak positive reaction with purified OmpC trimer and monomer (Table 1) but a strong reaction with partially purified preparations of OmpC from which LPS had not been dissociated (data not shown). Furthermore, this antibody reacted with OM, purified LPS, and whole cells from the homologous or heterologous strains (Table 1). In Western blots of denatured OM, this antibody produced ladderlike bands in three different regions of the immunoblot (Fig. 1C). Intensity of the reaction was greater in the intermediate- and low-molecular-weight regions. The enhanced reactivity of this MAb with partially purified trimer, relative to purified trimer or LPS, suggests that the epitope it recognizes contains both porin residues and LPS sugars. It is known that LPS associates tightly with porins and other OM proteins, both in situ and during their extraction and purification from the OM (20). (vi) Group VI. One antibody (13) gave a strong positive reaction with LPS, partially purified porins (both homologous and heterologous), OM, and intact whole cells; in Western blots of whole-cell lysates, it recognized a lowmolecular-weight species that comigrated with purified LPS, near the dye front. Relatedness among S. typhimurium and E. coli porins. We studied the immunological cross-reaction among porins by using anti-OmpD and anti-OmpC MAbs that gave strong reactions in Western immunoblots. These antibodies were mostly generated by using denatured monomers as immunogens (Table 1). One focus of our study was to find porins similar to OmpD. As described in the Introduction, OmpD porin is regularly found in S. typhimurium but is absent from E. coli K-12. Western immunoblotting (Table 2) revealed that OmpD was closely related to the NmpC and Lc porins of E. coli, 16 of 17 anti-OmpD antibodies recognizing both these porins. This finding suggests very strongly that S. typhimurium OmpD is homologous to NmpC and Lc, a conclusion also consistent with the observation that all three are susceptible to catabolite repression. Lc is coded by a lambdoid phage genome, and NmpC also appears to be a part of the integrated defective phage genome (3). Perhaps OmpD is also a part of a phage genome, and this would be consistent with widely different chromosomal locations of the ompD gene (32 min) and nmpC gene (13 min) on the chromosomes of S. typhimurium and E. coli, respectively. Another focus was the relationship of S. typhimurium PhoE protein to other porins. This protein has so far been characterized as PhoE simply because it is derepressed by phosphate starvation and because it has a slight preference for anions in a black lipid membrane system. If it is indeed a homolog of E. coli PhoE, it should cross-react strongly with anti-OmpD and anti-OmpC MAbs, because E. coli PhoE protein is highly similar in its sequence to E. coli OmpF,

1968

SINGH ET AL.

J. BACTERIOL.

TABLE 1. Reactivity of anti-S. typhimunum MAbs with Salmonella porins, LPS, OM, and whole cells in ELISAa

Porine

Immunogenb

Isotype

DT DT DT CT

CT DT CT CM CM

IgG2b IgG2b IgG2b IgG2a IgG2b IgG2a IgM IgG2a IgG2a IgM

Group II (specific for periplasmic epitopes) 47

CT

IgG2a

Group III (specific for buried epitopes on native trimer) 6 84

DT CT

IgG2b

Group IV (specific for buried epitopes on denatured monomer) 18 19 20 22 23 24 25 26 27 28 29 31 32 33 34 35 36 54 55 56 57 58 61 62 72 73

DM DM DM DM DM DM DM DM DM DM DM DM DM DM DM DM DM CM CM CM CM CM CM CM CM CM

IgG2b IgM IgM IgG2b IgG2a IgGl IgGl IgG2a IgG2a IgGl IgG2b IgM IgG2b IgG2b IgG2b IgM IgG2b IgG2a IgG2a IgGl IgG2b IgG2a IgG2a IgM IgGl IgG2b

-

Group V (specific for porin + LPS epitopes) 48

CM

IgG2b

-

Group VI (specific for LPS epitopes) 13

DT

MAb

Group I (specific for cell surface epitopes) 3 10 15 81

82 42 12 46 66 68

IgG2b

CT

FT

Whole

D

C

cellse

F DCF

DM

+ + +

_-

-

+

+

_-

-

+

+

+

_-

-

+

+

-+

+

_-

+ _

+ + + +

FM

OMd

DT

-

CM

LPSc

+

_-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

+

+

+

+

+

-

-

+

+

+

+

+

+ +

+ +

+ +

+ +

-

+ +

+ +

+ +

+ +

+ +

_

+

+

+

-

-

+

+

+

+

+

+

_-

+

_-

+

-

+

+

-

+

-

+ + +

-

+

-

+

+ + + + +

+ +

-

-+

+ +

+ -

-

+

-

+

+

+

LT-2

+ +

_-

--_++ _-

-

-

+

+

+

+

-

+ + + + +

-

+ + +

+

-

+

+

--

+

+-

+

+ -

-

+---

+

--

+

-

+

+

+

-

+

-

+

+

-

+

-

+

-

_

_

_

_

_

+

+

+

+

+

---

+

-

+

+

-

+

--

_

+

+

-

-

+

_ + + + + + + + + + + IgG2b a Plates were coated with 100 pl of intact cells (5 x 101 cells per ml), OM (50 pug/ml), porins (10 at a dilution used were Antibodies (100 LPS and ,ug/ml). jig/ml),

which resulted in an optical density at 405 nm within the range of 1.0 to 1.5 when tested with the homologous antigen (immunogen). These dilutions were estimated from an initial titration of antibodies at 1:100, 1:1,000, 1:10,000, 1:50,000, 1:100,000, and 1:200,000 and later at more closely spaced dilutions with the homologous antigen in ELISA. Positive reactions were scored weakly positive (+) or strongly positive (+) if the absorbances were greater than two times (but less than three times) the background or greater than three times the background, respectively. b DT, OmpD (trimer); DM, OmpD (monomer); CT, OmpC (trimer); CM, OmpC (monomer); FT, OmpF (trimner); FM, OmpF (monomer). LPS isolated from the homologous strain. d OM from strain SH 5014, which expresses all three porins. e D, OmpD+ strain SH 7454; C, OmpC+ strain SH 7457; F, OmpF+ strain SH 7455; DCF, OmpD+ OmpC+ OmpF+ strain SH 5014 with Ra-type LPS; LT-2 strain (with S-type LPS).

ENTERIC BACTERIAL PORINS

VOL. 174, 1992

1969

OmpC, NmpC, and S. typhi OmpC (3, 35, 47). Crossreaction, however, was very weak, with only 3 anti-OmpC MAbs showing reaction among 11 MAbs tested (Table 2). This result suggests that the S. typhimurium PhoE is only distantly related to E. coli PhoE and other members of the porin family. Indeed, the gene for S. typhimurium PhoE does not appear to be located in the region where E. coli phoE is found (1). Finally, we were interested in seeing whether OmpG and protein K are related to the OmpF-OmpC-PhoE group of porins of E. coli. OmpG is a normally repressed protein that becomes derepressed in cog mutants (34), and protein K is an additional porin present in capsulated strains (44), but their immunological relatedness to other porins has not been studied so far. We found that both these proteins crossreacted extensively with both anti-OmpC and anti-OmpD antibodies, suggesting that they belong to the OmpF-OmpCPhoE family of porins. This result is also in agreement with the observation that the N-terminal sequence of protein K is nearly identical to that of E. coli OmpF (44). We also examined the E. coli LamB protein, which is a specific channel protein rather than a nonspecific porin, although it also exists as a stable trimer in the OM. LamB

FIG. 1. Specificity of anti-S. typhimurium MAbs. (A and B) Whole cells of strains SH 7455, SH 7454, and SH 7457 were applied to lanes 1, 2, and 3, respectively, subjected to SDS-PAGE, and transferred to nitrocellulose. The paper was cut into 3.5-cm-wide strips and incubated with anti-OmpD monomer MAb 34 (A) and anti-OmpC monomer MAb 58 (B), and the reaction was developed with alkaline phosphatase-labeled goat anti-mouse Ig-nitroblue tetrazolium plus bromochloroindolyl phosphate (see Table 2, footnote a). (C) OM from strain SH 7457 was subjected to SDS-PAGE and transferred to nitrocellulose, which was cut into 0.4-cm-wide strips and probed with MAb 48 as described above.

TABLE 2. Reactivity of anti-OmpD and anti-OmpC monomer MAbs with S. typhimunum and E. coli porins in Western immunoblotsa

Anti-OmpD 18 19 20 22 23 24 25 26 27 28 29 31 32 33 34 35 36

Total Anti-OmpC 54 55 56 57 58 61 62 66 68 72 73 Total

E. coli porin

Salmonella porin

MAb

OmpD

OmpC

OmpF

PhoE

OmpC

OmpF

PhoE

Protein K

NmpC

Lc

OmpG

LamB

Total

+ + + + + +

+ + + + + +

+ + +

+

+

+

+

+

+ + + +

+ + + +

+ + + + + +

+ + + + + +

+

+ + +

+ + + + + +

+ +

+ + +

+ + + + + +

+

+ +

+ + +

-

11 11 12 11 11 11 3 11 4

+

-

-

-

-

-

-

-

+

+

-

-

+ +

+ -

+ -

+ -

+

+

+

-

-

+ +

+ +

+

-

+ +

-

+

-

-+

+

+ + + +

+ + +

+ +

+

+ + + +

+

-

-

-

+ +

+ +

+ +

-

17

13

13

+

+

-

+

+ +

+ +

+ +

3

+ +

+ +

-

7 11 9

+

-

9

-

1 9 10

+ +

+ +

+ +

-

+

+ + +

-

-

-

-

-

-

-

+

+

+

+

+

+

+

+

+

+

+

+

+

-

9

11

12

12

12

16

16

12

1

+

-

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

+ +

+ +

+ +

+

+

+ +

+

-

+

+

-

+

+

+

-

-

+

-

-

+

+

+

-

-

-

-

-

+ + +

+ + +

+ + +

+ -

+ +

+ + +

+

+ + +

+

+ + +

+ +

-

+

+

-

+ +

+ +

+ +

-

+

+

-

-

+

+

+

-

-

-

-

-

+ +

+ +

+ +

-

+

+

-

+

+

+ +

+ +

+ +

+ +

+ +

-

9

11

8

3

10

10

9

8

7

7

6

0

10 1 7 11 4 10 10 10 5 10 10

Cell envelopes from bacterial strains selectively expressing the indicated porins were lysed with SDS, subjected to SDS-PAGE, and transferred to nitrocellulose. The paper was blocked with 1% gelatin and cut into vertical or horizontal strips as described under Materials and Methods. The strips were incubated overnight with anti-porin MAb, washed, incubated with alkaline phosphatase-labeled goat anti-mouse Ig for 3 h, washed, and developed with nitroblue tetrazolium-bromochloroindolyl phosphate. Reactions were scored as negative (-), weakly positive (+), or strongly positive (+) in comparison with the intensity of reaction with the immunogen porin and negative controls. a

1970

J. BACTERIOL.

SINGH ET AL. CU

c

0

t

m t

Co tf In nt rI en

CO

oo

e CO C-I

W

In

L.

nt r-C

0n Cr 00 (oCC en Cflt 0 + - 4+ ++ +

-

H; + I IIIII

+ ++++ +

0

+ I ++

+ I ++

C-'

+ I + I + I

'IC

+ I

*t

'at. U

'4K

.0

>0E

a

0

02

00 0

0

I ++

+ I

ai'

C-4

++

I1+

I+

I

I + + ++ +

I1+

I+

I

+ I + I ++

I1+

+ +

0

I +

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VOL. 174, 1992

showed little cross-reaction with anti-OmpD and anti-OmpC MAbs (Table 2). This is not surprising because there is little sequence similarity between LamB and porins, and it has been shown earlier that LamB does not react with polyclonal sera to OmpF, OmpC, or PhoE (43) or with MAbs to E. coli OmpF (24). The immunochemical data of the present study thus suggest that most of the trimeric (nonspecific) porins of E. coli and S. typhimurium belong to a large family. This is consistent with the easily observable sequence homology among E. coli OmpF, OmpC, PhoE, and NmpC and S. typhi OmpC, and the present study extended this family, on the basis of conservation of antigenic epitopes, to also include S. typhimunum OmpF, OmpC, and OmpD and E. coli OmpG and protein K. In some cases, subfamilies with an especially close relationship may be distinguished. One such subfamily contains E. coli NmpC, S. typhimurium OmpD, and the Lc porin coded by a phage genome. MAb reactivity with porins of Enterobacteriaceae. MAbs against OmpC and OmpD monomer were further tested by immunoblotting with 11 Salmonella serotypes (in seven different serogroups) and clinical isolates of Salmonella typhi and Salmonella paratyphi (Table 3). All the anti-OmpC MAbs and 12 of the 17 anti-OmpD MAbs reacted with all the isolates tested. The slightly higher variability of OmpD homologs becomes understandable if, as suggested above, OmpD is a prophage-coded porin. When these antibodies were tested similarly with seven nonenterobacterial species, including A. hydrophila, B. pertussis, Brucella suis, H. influenzae, L. pneumophila, N. gonorrhoeae, and P. aeruginosa, no clear cross-reactivity was observed, except for a protein inA. hydrophila showing a strong reaction with MAb 29, and weak reactions of a P. aeruginosa protein with MAbs 18 and 20 (data not shown). These results show that the porins of the nonenterobacterial species are not very closely related to the OmpF-OmpCPhoE porin family of enteric bacteria. Finally, the same antibodies were tested with 13 species of Enterobacteriaceae (Table 3). The reactions of anti-OmpC MAbs could be interpreted on the basis of phylogenetic relationship among these bacteria. Thus, a Citrobacter species, which is probably very closely related to Salmonella species (and all the Salmonella strains tested, as described above) reacted with all the anti-OmpC MAbs. Groups with fairly close relationship with Salmonella species, such as E. coli, Shigella boydii, and Klebsiella pneumoniae (7, 10), reacted with more than one-half of the MAbs. Groups that are most remote from Salmonella species, such as Proteus, Morganella, and Providencia species, showed the least cross-reaction. In contrast, the reactions of anti-OmpD MAbs were less predictable. Thus, even among Salmonella species, S. typhi failed to react to several MAbs, and Citrobacter amalonaticus showed the weakest cross-reaction. Again, this unpredictable variation can be explained if OmpD is indeed a prophage-coded porin. Overall, approximately one-third of the MAbs tested recognized porin epitopes that are widely conserved in the family Enterobacteriaceae. MAbs 23, 61, and 72 reacted with all 13 different enteric species and all 13 Salmonella serotypes tested. This finding suggests that appropriate natural or synthetic porin peptides may be effective vaccines against enteric bacterial pathogens. However, such evolutionarily preserved regions of OM proteins are known to consist primarily of polypeptides that are buried within the OM bilayer (24, 49) and are therefore refractory to immune recognition. The shielding of OM protein epitopes by LPS

ENTERIC BACTERIAL PORINS

1971

and capsule further obscures this question (2, 9, 18, 26, 32, 36, 37, 56, 57). The function of buried epitopes, or antisera against them, in host defense against bacterial infection is not known. Aside from this gap in the understanding of immunity to bacteria, it is known that humoral immunity to infection by the Enterobactenaceae consists in part of antibodies to OM proteins (6, 8, 13, 42) and that purified OM proteins elicit protective immunity in animals (5, 17, 21, 26, 27, 33, 51, 56). The physical shielding of surface epitopes by the LPS implies that the immunoprotection observed by immunization of mice with anti-porin antibodies in previous studies (26, 27, 33, 50) may have resulted from contaminating anti-LPS antibodies. OM protein epitopes may be transiently exposed at the cell surface, however, as a consequence of natural turnover of cell wall materials during certain periods (e.g., biogenesis of OM) in the life cycle of bacterial cells (12). Alternatively, in animals the bacteria may contain rough patches on their surface, either naturally or caused by enzymatic degradation (18), which permit the recognition of surface epitopes by anti-porin antibodies. Furthermore, during gram-negative cell division, blebs or fragments of OM containing LPS and proteins are released into the external milieu (31, 40), and these pieces of OM may be both crucial immunogens during the immune response to infection and partially responsible for the clinical manifestations of gramnegative sepsis. Although buried epitopes would seem ineffectual as protective antigens because of their localization within the OM bilayer, antibodies to these epitopes (and possibly to surface epitopes as well) may have therapeutic relevance to the clearance of bacterial debris from serum and tissues (11, 12) rather than in the initial recognition and opsonization of the pathogen. Recently, Cloeckaert et al. (9) reported that there was no relationship between the protection conferred on mice by MAbs directed against OM proteins of Brucella species and ability of these MAbs to bind to the challenge bacteria. ACKNOWLEDGMENTS We are gratefully indebted to Hiroshi Nikaido for helpful consultation throughout this work and for his critical reading of the manuscript; to John Kearney, Larry McDaniel, and Meenal Vakil for their help and valuable suggestions during the preparation of MAbs; to Suresh Pai, Daisy Lee, Lashun Bland, Ace Anglin, Audrey Napier, Lula Smith, Hiram Sims, Bridgette Person, Pearlene Brown, Katisha Terrell, Raegan Durant, and Thuy Tran for their participation in the isolation and characterization of some of the MAbs; to P. Helena Makela, Winfried Boos, Kalle Gehring, John Foulds, Jan Tommassen, Taiji Nakae, and Spencer Benson for the supply of bacterial strains which express specific porins; to Dana Moore, Ronald Vance, and William Callan for providing us with clinical isolates of most of the enterobacterial and nonenterobacterial strains; and to Valerie Davis for typing the manuscript. This work was supported by Public Health Service grants GM 08167 and GM 08219 (to S.P.S.) and Al 22608 (to P.E.K.). REFERENCES 1. Bauer, K., R. Benz, J. Brass, and W. Boos. 1985. Salmonella typhimurium contains an anion-selective outer membrane porin induced by phosphate starvation. J. Bacteriol. 161:813-816. 2. Bentley, A. T., and P. E. Klebba. 1988. Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface. J. Bacteriol. 170:1063-1068. 3. Blasband, A. J., W. R. Marcotte, Jr., and C. A. Schnaitman. 1986. Structure of the Ic and nmpC outer membrane porin protein genes of lambdoid bacteriophage. J. Biol. Chem. 261: 12723-12732. 4. Bloch, M.-A., and C. Desaymard. 1985. Antigenic polymor-

1972

SINGH ET AL.

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