Indian Journal of Experimental Biology Vol. 42, May 2004, pp. 508-514

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotl!lberculosis strains isr)lated from India

Rekha Khushiramani, Urmil Tuteja, lyoti Shukla & Harsh Vardhan Batra*

Division of Microbiology, Defence R&D Establishment, Jhansi Road, Gwalior 474002, India

Received 17 June 2003; Revised 21 January 2004

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pesris isolates recovered durillg 1994 plague outbreak and rodent surveillance samples of Southern "tates of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersillia species by SDS­PAGE. Western blotting with the polyclonal antisera raised against tllese Omp preparations revealed few immuno-reactive band, that appeared to be shared among Y. pestis, Y. pseudotuberculosis, Y. ellterocolitica, Y. fredmcksellii, Y. illtermcdia. Y. kris/ellsen ii ~nd E. coli. Thrce recombinant Yap proteins namely, YopM, YopB and LcrV were produced and antisera to these pr0teiils could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monocionaj antibodies against Omp preparatioll. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, Olher Ye!:~h:ia species and the rnecbers of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP 1, exhibited reactior. to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. illferllledia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross­reactive within the family Enterobacteriaceae.

Keywords: Outer membrane proteins, Yersillia pestis, Yersinia pseudotuberculosis

Medically significant yersiniae consist of Yersinia pestis, the causative agent of bubonic plague, and the closely related enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica. Wild type cells of these 3 species share a 70 kb low calcium response (Lcr) plasmid encoding V antigen and a group of highly L:onserved Yersinia outer membrane p~c(eins (Yops) known [() be essential for virulence l

.

l)lasrr:id bearing Y. ellferocolitica and y~

IFel~ :lufi'beYCl!l(}sis ha\(' the potf.'ntiai to alter JrarG·tic(111y til';!!r anter r:1em~rane protein profile::; as a n:srdt (:J plm;mid gt!U'; cxy>ress:on. R.el2tive to the chro!l;c somally encoded majo.r (·uter mcml:lrm'e fr·.)i d~l S th~ r·1(1"micl m(';(Eat~d protp.!ns are e'\prc:s~; ::d

in l'j(;!,h u)l1centration in the outer membrane. The fact th'lt :h~ Y'!rs:ni·::; plasmij-enc:oded (lutec mejnb:'~' ne probins are only expre<;sed Gil the l:Jacterizl wrfac~ ;At 37°C and the.] only ;n Cf~rt(lin r;mwth er.vironro.~nt,

*COfft'sponctent 3.uthor Phone: 0751-341550 fax hu: :175\-341148 .::. ·maL; "_ '.'_bacr.:@rediffmai;.com, drde@san,:harnet.in

suggests that they are regulated by both temperature and nutritional factors2

• Investigation of 1994 plague outbreak in the country resulted in 11 Y. pestis isolates from Surat pneumonic patients and 7 from rodents3

• The initial 3 isolates from the Surat pneumonic patients when independently tested in WHO collaboration Centre, Fort Collins, USA and Stavrapol Antiplague Research Institute, Russia, b!ought out at least one factor that the infectivity dose to bdn~ out mortality in experimental mice was :r>latively high even though 3 known virulence as~ociated plasmids were demonstrated in these ~.':cl2.tes (Tac, R'!port). PresenC'~ of these 3 plasm ids 'was alsa observed in rest of the Y. pestis isolates from cJUtbreak regions4 Furthe~' surveillance work yielded a lot of 8 rr:ore Y. pe.~tis and 4 Y. pseudctubercu{osis ;r;olates from rodents of known plague foci of Deccan plateau of Southern India.

Char::'cterintion of important Y ops in these isolates \¥as attempte-Ai in the present stl'.dy by analyzing ~he Omp preparations on SDS-PAGE and by immuno­react;'vity testing .

KHUSHIRAMANI et al.: OMP IN YERSINIA STRAINS ISOLATED FROM INDIA 509

Materials and Methods Bacterial strains - The standard strain of Y. pestis

AI122 and Y. pseudotuberculosis IA were procured from Dr. May C Chu (WHO Collaborating Centre at CDC, Fort Collins, USA). Yersinia enterocolitica strain 0:8, Y. kristensenni, Y. fredricksenni, and Y. intermedia were received from a laboratory in Norway.

List of Y. pestis and Y. pseudotuberculosis isolates and their sources is given in Table 1. Cultures of Klebsiella pneumoniae, Escherichia coli, Salmonella typhi, Salmonella abortus-equi, and Staphylococcus aureus, used in present study were obtained from DRDE, Gwalior. All these strains were maintained in tryptic soya agar slants.

Outer membrane preparation - Outer membrane proteins (Omps) were prepared as per the method described by Bolin5

. Briefly, cultures of all bacteria including Yersinia and non-Yersinia species were grown on calcium deficient agar (Brain heart infusion agar containing 0.04 mol of sodium oxalate and 2 mM MgCh) plates and incubated at 28°C for 2 days and further shifted to 37°C for another one day. After harvesting bacterial cell from agar plates, the cells

. were washed twice with sterile PBS and were suspended in IOmM Tris-hydrochloride (PH 7.8), 5 mM EDTA and 1 mM 2-mercaptoethanol. The organisms were sonicated at 6 micron output using Microson Sonicator for 10 cycles per min. The sonicates were centrifuged at 5000 rpm for 10 min. The membrane pellet was suspended in a solution containing 1% Triton-X 100 and 1 mM 2-mercaptoethanol, incubated overnight at 4°C and centrifuged at 100,000 g for 1 hr to obtain the outer membrane containing pellet. The proteins were

Table l-Yersinia pestis isolates

Yersinia isolates Recovered from Species Place

Outbreak 4,8,9,101,102,103, Humans Surat region 104,105.106,107 &108

111,112,114,115 &116 Rattus rattus Mamla 113 Rattus raUus Surat 117 Tetera indica Mamla

Surveillance 2H, 3H, 12H, 18H, 24 Teteraindica Hosur region

&25H 9R& lOR Tetera indica Palamner

Yersinia pseudo-tuberculosis isolates

A87 Tetera indica Atti,bele K174 Tetera indica Kolar Ps 1122 Tetera indica Palamner 12R Tetera indica Palamner

analyzed by SDS-PAGE as described by Laemmli6.

Outer membrane samples were solubilized in the sample buffer and boiled for 5 min. Separation and detection of Y. pestis proteins were obtained followed by Coomassie blue staining. Protein contents were measured by the method of Lowryet aF.

Recombinant protein preparations- Recombinant Y op proteins i. e. Y opM, YopB . and LcrV were prepared employing pQE series of . cloning ,and expression vectors followed by purification usin:g Ni­NTA column chromatography.

Immunization with outer membrane proteins and recombinant proteins - Outer membrane proteins were prepared from a Y. pestis isolate from Surat pneumonic patient and the BALB/c mice " were immunized at weekly intervals with 50 !J.gof this protein preparation. Similarly, the BALB/c mice were also immunized with each of the 'recombinant proteins. Titres of hyperimmune sera thus produced were checked by dot ELISA ; using 2-fold sera dilutions starting from 1: 1000. Hyperimmune sera to purified recombinant Y opM, YopB and LcrV proteins was utilized to detect Y ops in Omp preparation by Western blotting. '

Monoclonal , antibodies - Immunized BALB/c mice were sensitized by 3 successive intraperitoneal injections prior to the fusion and sensitized spleen cells were fused with a mouse myeloma cell line Sp2/0-Ag 14 according to the method of Kohlet: and Milstein8

. ' ~

Isotyping ; of monoclonal antibodies -Immunoglobulin heavy chain class .(IgG, IgM and IgA) and light chain type (lambda and kappa) of monoclonal antibodies were determined by dot­ELISA using horse radish peroxidase labelled rabbit anti-mouse IgG, IgM and IgA conjugates (Mouse Typer Sub-Isotyping kit, Biorad).

Specificity testing of rrIonoclonal , 'antibodies­Specificity of MAbs· was checked by dot-ELISA using sonicated antigen preparatK>n~ ,of standard strain of Y. pestis A1122, Y. pseudotuberculosis lA, Y. enterocolitica strain 0:8, .Y.,' kriste'nsenni, Y. fredricksenni , Y. intermedia, E. coli, S. typhi, S. aureus, S. abortus and K. p~eumoni~t . For this bacterial cells harvested from brain heart infusion agar plates, were washed thrice, with sterile PBS and were suspended 'in sterile distil~d water contaiding 20 mM PMSF and~sonicated using Microson Sonicator (Misonix Ltd, USA).

Whole cell lysate blotted membranes were used for Western blotting9

• Purified recombinant YopM, YopB

510 INDIAN J EXP BIOL, MA Y 2004

and LcrV proteins were also used for testing the immuno-reactivity of monoclonal antibodies in Western blotting. Coated combs and blotted membranes were incubated with monoclonal antibodies and the conjugate used was rabbit anti­mouse polyvalent-HRP (Dakopats, Germany).

Testing of Y. pestis and Y. pseudotuberculosis isolates - Western blot was performed on all the isolates of Y. pestis and Y. pseudotuberculosis using polyc1onal antibodies to Omps.

Whole cell antigens of all the isolates of Y. pestis and Y. pseudotuberculosis were coated on nitro­cellulose combs for testing by dot-ELISA using monoclonal antibodies to Omps.

Results Outer membralle proteins - Outer membrane

proteins (Omps) were made following extraction of bacierial culture from the standard strains of Yersinia species, certain non-yersiniae enterobacteriaceae organism and from all the 26 Y. pestis and 4 Y. pseudotuberculosis isolates grown in Ca2

+ deficient BHIA media. On SDS-PAGE, 3-4 prominent bands were observed in the region of 34-42 kDa, 2-3 faint bands at low molecular weight re[,ion of 17-25 kDa and few clear bands at higher molecular weight region of 60-70 kDa. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to various Y ops were seen in all of the isolates. In few isolates protein bands either at lower or higher molecular weight regions were observed. Species related distinct patterns of Omp were observed mainly in the regions of 30-45kDa in Yersillia species and other members of the enterobacteriaceae family tested. Two thick prominent bands at the region of 38-40 and 45 kDa region were characteristic identification marker for Y. entemcolitica. A prominent band at 30 kDa in Y. pseudotuberculosis, bands at 30 and 65 kDa region in Y. intermedia, a 45kDa band in Y. kristensenii and bands at 40 and 44 kDa in Y. fredrecksenii were found specific. A doublet at the region of 48-50 kDa, two bands at 65-75 kDa in E. coli and a cluster of 3 bands at the 38-42 kDa in S. typhi were observed unique. Though expression of proteins by non-pathogenic Yersinia species was relatively low compared to the pathogenic Yersinia species. Isolates of Y. pestis and that of Y. pseudotuberculosis had patterns nearly similar to those of the standard strains (Fig. lA, B, C, D and E).

Titration of hyjJerim11lulle sera - In the mice hyperimmune sera dot-ELISA titre of 1: 1 ,28000 was

exhibited against outer membrane protein antigen obtained from the Y. pestis isolate of Surat pneumonic patient. A titre of 1: 64,000 was observed for both the recombinant proteins, YopM and YopB. Titre of mice HIS to recombinant LcrV protein was found to be 1:32,000.

Testing of hyperimmune sera to recombinant proteins by Western blotting - Hyperimmune sera prepared against recombinant Y opM, YopB and LcrV when tcsted on Omp preparations from Y. pestis strains in Western blotting, had immuno-reactivity at expected protein masses of 42 kDa for YopM, 41 kDa for YopB and 37 kDa for LcrV proteins (data not shown).

Monoclonal antibodies - A total of 4 monoclonal antibodies producing clones could be stabilized and preserved against outer membrane protein antigen. Immunoglobulin class of the monoclonal antibodies is presented in Table 2.

Specificity testing of monoclonal antibodies­Specificity of monoclonal antibodies was checked by

dot-ELISA using sonicated antigens and by Western blotting using whole cell lysate antigens of Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. kristensenii, Y. intermedia, Y. fredricksenii, E. coli, K. . plleu11loniae, S. typhi, S. abortus-equi and S. aureus.

Monoclonal antibody YPI was found reactive to Y. pestis, Y. pseudotuberculosis, K. pneul1loniae, Y. enterocolitica, Y. intermedia, Y. kristensenii, S. typhi and S. abortus-equi but not to E. coli, Y. fredrecksenii, and S. aureus in dot-ELISA, The rest of the 3 monoclonal antibodies namely, YP2, YP3 and YP4 exhibited reactions with 10 of the 11 species except S. aureus in dot-ELISA. In Western blotting YPI monoclonal antibody exhibited reaction with Y. pestis and other Yersinia species at 2 different regions. Western blotting reaction of Y. pestis was similar to that of Y. intermedia and K. pneu11loniae with this monoclonal antibody but different from that of Y. pseudotuberculosis and Y. enterocolitica. The rest of 3 monoclonal antibodies had reaction to one antigen of Yersinia species and the E. coli strain tested. However, this reactivity within Yersinia species and with E. coli was at different molecular weight regions '

Table 2-Isotyping of monoclonal antibodies to Omp preparation

Isotype Clone

YPI YP2 YP3 YP4

Heavy chain

IgG2b IgG2a IgG2b IgG2b

Light chain" ..•

Kappa Kappa

Lambda Kappa

KHUSHIRAMANI et al.: OMP IN YERSINIA STRAINS ISOLATED FROM INDIA 511

Fig. I-A) SDS-PAGE of Omp preparations of Y. pestis isolates from Surat pneumonic patients. Lane 1-9: Y. pestis isolates (4, 8, 9, 101 , 102, 103, 104, 105 & 106); Lane 10: Standard Y. pestis A1l22. B) SDS-PAGE of Omp preparations of Y. pestis isolates of rodents from 96 outbreak regions. Lane I & 2: Y. pestis isolates (107 & 108); Lane 3-9: Y. pestis isolates (lll, 112, 113, 114, lIS, 116, 117); Lane 10: Standard Y. pestis A 1122. C) SDS-PAGE of Omp preparations of Y. pestis isolates from rodents of surveillance regions. Lane I : Y. pestis isolate 112; Lane 2-9: Y. pestis isolates (2H, 3H, 12H, 18H, 24H, 25H, 9R & lOR); Lane 10: Standard Y. pestis Al122. D) SDS-PAGE of Omp preparations of Yersinia species. Lane 1: Standard Y. pestis Ali22; Lane 2: Standard Y. pseudotuberculosis lA; Lane 3-6: Y. pseudotuberculosis1so1ates (A87, K174, Ps1l22 & 12R); Lane 7: Y. enterocolitica; Lane 8: Y.fredricksenii; Lane 9: Y. intermedia; Lane 10: Y. kristensenii. E) SDS-PAGE of Omp preparations of certain Enterobacteriaceae organisms species. Lane I: Standard Y. pestis A1122; Lane 2: Standard Y. pseudotuberculosis IA; Lane 3: Y. ellterocolitica; Lane 4: Y. fredrecksenii; Lane 5: Y. illtermedia; Lane 6: Y. kristellsenii; Lane 7: E. coli; Lane 8: S. typhi; Lane 9 & 10: Y. pestis isolates 8 & 114·

512 INDIAN J EXP BIOL, MAY 2004

in Western blotting. Antigens from K. pneul1loniae. S. typlzi. S. abortus-equi and S. aureus were not tested by Western blotting. None of the 4 monoclonal antibodies had reaction with the 3 recombinant proteins namely, YopM, YopB and LcrV in Western blotting (Fig. 2A and B). Results · are presented in Table 3.

2 3 4 5 6 7 8 9 10

Fig. 2-A) Spec ific ity testing of monoclonal an tibody YP 2 in Western blotting. Lane I : Y. kristellSellii ; Lane 2: Y. fredricksellii ; Lane 3: Y. illterllledia; Lane 4 : Y. enterocolitica; Lane 5: Y. pselldof!lberculosis ; Lane 6: Y. pestis; Lane 7: Recombinant YopB protein; Lane 8: Recombinant LcrV protein; Lane 9: Recombinant YopM protein; Lane 10: Omp preparation. B) Specific ity testing of monoclonal antibody YP 3 in Western blotting. Lane I: Y. kristensellii ; Lane 2: Y. fredrecksellii ; Lane 3: Y. illlerllledia; Lane 4: Y. ellterocolitica; Lane 5: Y. pseudotuberculosis ; Lane 6: Y. pestis; Lane 7: Recombinant YopB protein; Lane 8: Recombinant LcrV protein; Lane 9: Recombinant YopM protein; Lane 10: Omp preparation

Testing of Y. pestis and Y. pseudotuberculosis isolates Polyclonal antibodies - Polyclonal antibodies

against,Omps had the reactions with all the isolates of Y. pestis at 4 to 5 prominent outer membrane proteins at 34-42 kDa regions by Western blotting. Few isolates exhibited protein bands either at lower or higher ~egion. A prominent immuno-reactive band at approximate 70 kDa region was observed only in USA Y. pestis strain A1122 (Fig. 3).

Mon.oclonal antibodies - All the 4 monoclonal antibodies produced reaction with all the isolates of outbreak regions as well as from surveillance regions in dot-ELISA.

Discussion The Y ops are shown to be necessary for full

virulence in Y. pestis or the other pathogenic Yersillia species and cor~prise 10 Omps that get translocated into the eukaryotic cytosol I. Further, a plague convalescent-phase serum recognized two of these cross-reactive Yops, a finding indicating that Y. pestis expresses Y ops during human infection 10.

Identification of these Y ops in Indian Y. pestis strain was attempted by the conventional procedure of partially purifying Omps after cultivation in calcium deficient media at 28°C with further induction at 37°C and then analyzing the Omps by SDS-PAGE. The maximal expression of these Y ops is observed I I at 37°C in absence of Ca2

+. This procedure would have permitted isolation of Omps containing the Yops mainly specific to pathogenic Yersinia. Prominent protein bands numbering 4-5 between 34-42 kDa region were seen in all the isolates as well as in other Yersillia and non- Yersinia species though their patterns distinct in pathogenic yerS1I1Jae. The prominent bands observed in 34-42 kDa region could have been the representations of reported Yops like YopJ (32.5 kDa), YopD (34.5 kDa), YopT (35.5

Table 3-Specificity testing of monoc lonal antibodies

Organisms

l.Y. pestis 2. Y. pseudotuberculosis 3. Y: ellterocolitica 4. Y. fredrecksellii 5. Y. illtennedia 6. Y. kristellsellii 7. S. fyphi 8. S. abortus-equi 9. K. pllellllloniae 10, E. coli II. S. aurem

YPI

+ + +

+ + + + +

Monoclonal antibodies YP2 YP3 YP4

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

KHUSHIRAMANI et al.: aMP IN YERSINIA STRAINS ISOLATED FROM INDIA 513

Fig. 3-Characterization of amps wilh hyperimrnune sera by Western blotting. Lane I: Standard Y. pseudotuberculosis lA; Lane 2: Standard Y. pestis A1l22; Lane 3-6: Y. pseudotuberculosis isolates (A87, K174, Psl122 & 12R); Lane 7: Y. enterocolitica; Lane 8: Y. kristellsenii; Lane 9: E. coli; Lane 10: Y. pestis isolate 8 '

kDa), IcrV (37 kDa), YopB (41.5 kDa), YopM (42 kDa), and YopH (45 kDa), expressed in pathogenic Yersinia species '2. In all the 3 pathogenic Yersinia species both in standard strains and isolates, number and intensity of protein bands demonstrable in SDS­PAGE were relatively more compared to non­pathogenic Yersinia species and other enterobacteriaceae organisms suggesting their relationships to the pathogenic yersiniae. Recent reports have suggested that including the Yops, a set of nearly 40 different Y. pestis proteins (YPP), were expressed after growth in calcium deficient media and temperature induction. Several of these proteins could also be identified in a Y. pestis strain devoid of plasmids 11. It may therefore, be difficult to identify Y ops only on the basis of molecular mass because certain YPPs can overlap Y ops, for example, Y opH could be 45 kDa YPP 10, 46 kDa YPP 11, 53 kDa YPP 7 or 54 kDa YPP 8 and Y opN could be 35 kDa YPP 15 or 34 kDa YPP 16 (ref. 12). Demonstration of 3-6 prominent bands in 30-42 kDa region in Y. pestis isolates in SDS-PAGE observed in present work could have been the YPPs and/or the Yops. In contrast to Y. pseudotuberculosis strains, protein bands in certain Y. pestis isolates had varying patterns, suggesting the possibility of differential expression of YPPs and/or Yops in different Y. pestis strains.

Western blotting with the polyclonal antisera raised against these Omp preparations revealed a few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotuberculosis, Y. ellferocolitic([, Y. fredrecksell.ii, Y. illtermedia, Y.

kristensenii and E. coli. Hyperimmune sera prepared against recombinant YopM, YopB and LcrV when tested on Omp preparations from Y. pestis in Western blotting had immuno-reactivity at expected protein masses. These 3 important Y ops-Y opM, which binds to a-thrombin and blocks platelet activation; YopB, involved in controlling Yop translocation; and LcrV, has an antihost function and is involved in regulating the low calcium response, therefore appears to be present in the Omp preparation but probably failed to elicit antibody response.

Interestingly, Y. pestis strain A1l22 had a unique immuno-reactive band at about 70 kDa region which was not found in any of the Y. pestis isolates as well as other Yersillia and non-Yersinia species. The behaviour of Y. pestis All22 by colony morphology, culture characteristics, detailed biochemical analysis, bacteriophage lysis, SDS-PAGE and plasmid profile was identical to Y. pestis isolates3

.4 . This observation is suggestive of strain specific expression of Omps that too with immunodominant antigen under Ca2

+

deficient conditions. Further studies at epitope levels may provide useful information on the possibilities of existence of strain variation among Y. pestis isolates of different parts of world. Ribotyping using 16S-23S rRNA probe from E. coli following restriction digestion with EcoRI and EcoRV is the only molecular typing procedure reported for Y. pestis isolates where 16 ribotypes have been documented ' 3.

In order to further characterize the important Y ops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Four stable reactive clones that were obtained when tested

,514 INDIAN J EXP BIOL, MAY 2004

for specificity showed extensive cross-reactivity among the members of Enterobacteriaceae. Monoclonal antibody YP 1 was reactive with antigens shared among Y. pestis, K. pneumoniae, Y. pseudotuberculosis, Y. enteFOcoiitica, Y. intermedia, Y. kristensenii, S. typhi and S. abortus-equi but was negative to E. coli, Y. fredrecksenii, and S. aureus whereas the rest ' 3 clones YP2, YP3 and YP4 exhibited reactions with all the Enterobacteriaceae organisms tested. None of the 4 monoclonal antibodies had reaction with the 3 recombinant proteins namely, YopM, YopB and LcrV in Western blotting. All the 4 monoclonal antibodies produced reaction with all the 26 Y. pestis and 4 Y. pseudotuberculosis isolates by dot-ELISA.

On the basis of reaction observed with polyclonal and monoclonal antibodies, it was found that Omp

, preparation following cultivation of Y. pestis in calcium deficient media did contain Yersinia specific Yops. May be in the presence of other dominant YPPs, these Y ops could not evoke detectable antibody response. Results also proved that under low calcium response, the Y. pestis not only activates secretion of Y ops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae and these cross-reactions are reflected even at the epitope levels. Existence of the common epitopes at different antigens in these organisms suggests ' the possibility of shared homology among the proteins expressed under low calcium response.

Acknowledgement The authors are grateful to Director, DRDE,

Gwalior for encouragement.

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