Artículo 1 SDS-PAGE

8/13/2019 Artculo 1 SDS-PAGE

1/5

Study on Outer Membrane Protein (OMP) Profile of AeromonasStrains using SDS-PAGE

1 2 1N Sachan *, RK Agarwal and VP Singh

1. Pt. D.D.U.Veterinary University & Go Anusandhan Sansthan, Mathura, U.P. India, 281001.2. Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P. India, 243122

*Corresponding author: email: [email protected]

Received: 03-09-2011, Accepted:09-10-2011, Published Online:17-12-2011doi: 10.5455/vetworld.2012.173-177

Abstract

Mesophilic aeromonads are being increasingly reported pathogen of humans and lower vertebrates. Water andfoods are considered to be the chief source ofAeromonasspp. At present there are several techniques availablefor the detection of Aeromonas spp. from water and foods. However, there is still need to developimmunodiagnostics for rapid detection ofAeromonasspp. irrespective of their species or serotype. To meet out

this requirement present study was undertaken to identify the common protein moiety in their OMPs by SDS-PAGE so, that immunoassays can be developed for efficient and rapid detection ofAeromonasspp. from foods.Key words:Aeromonas, OMP, SDS-PAGE, immunoassay, foods.

Introduction Chimnitz and named Bacillus punctatus(Zimmerman, 1890). A variety of foods and foodEmergence ofAeromonasspp. as an important

products including seafood, chicken and redhuman pathogen has led to a considerable interestmeat, vegetables, raw milk and its products havein the organism in last two decades. Aeromonads

been shown to harbourAeromonasspp. (Hunterare ubiquitous in nature being isolated from wideand Burke, 1987; Majeed et al., 1989; Palumbo etvariety of sources. They are normal flora ofal.,1989; Kirov et al., 1990). Cases ofAeromonasaquatic and terrestrial animals as well as etiologicassociated gastroenteritis have been reportedagents of disease in numerous cold- blooded andfrom all over the world including Australiawarm blooded animals (Cahill 1990; Joseph and(Burke et al., 1983), Bangladesh (Sack et al.,Carnahan, 1994). The aquatic environment is1987), Brazil (Schorling et al., 1990), Ethiopiaconsidered to be the principal reservoir of(Wadstrom et al., 1976).Aeromonas spp. (Von Graevenitz and Mensch,

In India, Chatterjee and Neogy (1972)1968, Davis et al.,1978; Burke et al., 1984a, b'reported 8% isolation ofAeromonasspp. from theWadstrom and Ljungh, 1991) and organism iscases of choleric diarrhoea in Calcutta. Since thenisolated from different waters includingsome reports of Aeromonas associatedchlorinated drinking water (Lechevallier et al.,gastroenteritis have appeared from different parts1982; Alvarez and Bitton, 1983; Boussaid et al.,of the country (Bhat et al., 1974; Sanyal et al.,1991; Kersters et al., 1995).

1983; De and Pal, 1984). Aeromonads are alsoThese bacteria are classified into; aknown to cause extra intestinal human infectionspsychrophilic, non motile group and a mesophilic,such as meningitis, endocarditis, peritonitis,motile group. Complexity of mesophilicseptic arthritis, eye and urinary tract infectionsAeromonas is further aggravated by various(Khardori and Fainstein, 1988; Krovacek et al.,serotypes which they contain. Ewing et al.,1983). SinceAeromonasspp. are resistant to the(1961) is credited with preliminary work onantibiotics commonly used for treatment ofserology ofA. hydrophila. The first isolation ofwound infections, wounds exposed to water areAeromonas was made in 1890 from tap water in

Vet. World, 2012, Vol.5(3):173-177 RESEARCH

www.veterinaryworld.org Veterinary World, Vol.5 No.3 March 2012 173

To cite this article:

Sachan N, Agarwal RK, Singh VP (2012) Study on Outer Membrane Protein (OMP) Profile ofAeromonasStrains

using SDS- PAGE, Vet. World. 5(3):173-177, doi: 10.5455/vetworld.2012.173-177.


2/5

Study on Outer Membrane Protein (OMP) Profile ofAeromonasStrains using SDS- PAGE

likely to be infected with Aeromonas spp. amount (25 l) of OMP was diluted with 400 l of(Skiendzielewski and O'Keefe, 1990). distilled water. Different concentrations (25, 50,

75, 100, and 125 g) of bovine serum albuminMaterials and Methods

(BSA) were prepared in distilled water to a totalvolume of 400 l. To all the tubes, 400 l of 2xAeromonas strains were isolated from

different sources such as chicken meat, fish, milk, lowry concentrate was added and incubated atroom temperature for a minimum of 10 min.egg etc. using Buffered Cephalothin DextrinThereafter, 200 l of the 0.2N Folin reagents wasBroth -10 (BCDB-10) as selective enrichment

oadded and incubated (55 C for 5 min). Thebroth. Isolates were serotyped by the courtesy ofAbsorbances were read at 650 nm usingDr. T. Shimada, Chief, Laboratory of Enteric

polystyrene cuvettes. A standard graph wasInfection 1, National Institute of Health, Tokyo,plotted against optical density (OD) of BSA in162, Japan (Table 1).different concentrations. Protein concentration of

Table-1: OMP strains and their concentrationsOMP was estimated using standard graph.

SDS-PAGE analysis of OMP: OMP fractions were

analysed by SDS-PAGE as per the method ofLaemmli (1970) employing 12.5% (w/v) acrylamide in the resolving gel and 5% (w/v) acrylamide in the stacking gel. The samples werediluted with sample buffer in a ratio of 4:1 andheated at 95C for 5 min. Approximately, 30lofthe sample containing 100g protein was loadedin each lane of the gel. The gel was run at 80V for12 hrs, then stained with coomassie brilliant blueR-250(sigma) staining solution for 8 hrs and

Separation of Outer membrane protein (OMP):finally, destained with destaining solution.OMP of 16Aeromonasstrains was separated asCalculation of molecular weight(s) (MW) of theper the method of Crosa and Hodges (1981) as

peptide(s) was done by extrapolation of relativemodified by Santos et al. (1996). Briefly, mobility of the unknown samples against that ofAeromonas grown overnight in 100 ml of tryptic

standard molecular weight markers. Details ofsoya broth (TSB, Difco) were recovered byreagents used for SDS-PAGE are given in table-2.centrifugation at 5000 rpm for 30 min. Cells were

resuspended in 3 ml of 10 m mol/l tris buffer Results and Discussioncontaining 0.3% (w/v) (pH 8.0) and sonicated

The separation of OMP was carried out bywith a sonifier (MSE, Ultrasonicator) (10 amplitude,culturing Aeromonas strains in brain heart

45 seconds, 4-5 times). After centrifugation at 0infusion broth at 37 C with shaking, followed by10,000 g for 2 min. the supernatant fluids wereultrasonification and centrifugation. Inner andtransferred to new tubes and centrifuged for 1 hr

0 outer membranes were separated by incubationat 17,000 g at 4 C. Cell envelop suspensions were

with sarkosyl. This separation was necessaryincubated with 3% sodium lauroyl sarcosinatebecause OMP profiles are reported to be

(sarcosyl) (w/v) in 10 m mol/l tris buffer at roominfluenced by temperature and air supply totemperature for 20 min. Outer membrane protein

bacterial cultures (Statner et al., 1988). Thiswas obtained by centrifugation at 17000 g for 1 hr

method was able to recover the whole OMPsand washed twice with distilled water. The OMP

from Aeromonas strains (Kuijper et al., 1989).owas stored at -20 C.

OMP of 16 strains were separated and proteinProtein estimation: The protein content was concentration estimated is presented in table 2. Indetermined by the method of Schaterle and this study protein concentrations were in thePollack (1973) with little modifications. A small range of 11.3 mg/ml to 16.2 mg/ml.

Strain no. (OMP) Serotype P rotein concentra tion ( mg/ml)

VPH 5 Rough 11.3

10 Rough 12.4Buff 1 Unknown 14.4C19 Unknown 16.269 Rough 12.2F2 O73 12.6F6 O6 11.5

AC5 O60 13.2G3 O38 12.9F17 O38 11.8F7 O38 12.2F14 O16 13.4Floor 2 O16 14.4C5 O16 11.8C11 O16 13.5C23 O16 13.2



3/5


SDS-PAGE analysis of OMPs: SDS-PAGE few minor bands in the lower molecular weightanalysis of OMP of differentAeromonasstrains range (10-12 kDa) were also observed in certainrevealed up to 4 major and 4 to 6 minor strains.

polypeptide bands (Fig. 1 & 2). The molecular Major polypeptides of 14 and 35 kDa andweight (MW) of the major polypeptide bands, minor polypeptide 25 kDa were common in mostestimated by comparison with standard MW of theAeromonasspp. irrespective of serogroupmarkers run parallel was in the range of 14 to or species. Heterogeneity in protein profile was55kDa. Large smearing of the bands in the MW observed amongAeromonasstrains of differentrange of 40 to 45 kDa was observed. In addition a serogroups as well as of same serogroups.


Reagent used Components of reagent Amount of components

1. 1M Tris HCl (pH 8.8) Tris 12.1g

Distilled water 50mlAdjustment of pH 8.8 with concentrated HCl and 100 ml volume was made with distilled water.2. 1M Tris HCl (pH 6.8) Tris 12.1g

Distilled water 50mlAdjustment of pH 8.8 with concentrated HCl and 100 ml volume was made with distilled water.

3. Electrophoresis buffer Tris 3.025 g(pH 8.3) Glycine 14.413 g

SDS 1.0 gDistilled water to make 1000 ml

4. Sample buffer (5X) 1 M Tris HCl (pH 6.8) 0.6 ml50% Glycerol 5 ml10% SDS (w/v) 2 ml2-Mercaptoethanol 0.5 ml1% Bromophenol blue 1 mlDistilled water 0.9 ml

5. Acrylamide solution Acrylamide 29.2 gBisacrylamide 0.8 gDistilled water to make 100 ml

6. Running Gel (12.5%) Acrylamide solution 12.5 ml1M Tris HCl (pH 8.8) 11.2 mlDistilled water 6.2 ml10% SDS 0.3 mlTEMED 20 l10% ammonium per sulphate (w/v) 100 l

7. Staking Gel Acrylamide solution 1.67 ml1M Tris HCl (pH 6.8) 1.25 mlDistilled water 7.03 ml10% SDS 0.4 mlTEMED 10 l10% ammonium per sulphate (w/v) 50 l

8. Staining solution Coomassie brilliant blue 0.15%Methanol 45%

Acetic acid 10%Distilled water 45%

9. Destaining solution Methanol 45%Acetic acid 10%Distilled water 45%

Table-2: SDS-PAGE reagents


4/5


AcknowledgementsHowever, some similarity was observed for somespecies of Aeromonas. Protein profile was

Authors are thankful to Director, Indianidentical for 2 (F 7 and F 17)A. caviaestrains and

Veterinary Research Institute, Izatnagar andfor 2 (VPH 5 and 10) A. hydrophila strains of Head, Division of Veterinary Public Health,rough serogroup.

Indian Veterinary Research Institute, IzatnagarThe SDSPAGE analysis revealed hetero-

for providing the necessary facilities for research.geneity in protein banding pattern both within

Conflict of interestand among the species and serotypes, except for afew exceptions. Similar variability in OMP profiles Authors declare that they have no conflict ofhas been described by Santos et al., (1996) who interest.observed that Aeromonas strains belonging to

Referencesdifferent serogroups and as well as to sameserogroup exhibited differences in their protein 1. Alavarej, R. J. and Bitton, G. (1983). Recovery

banding pattern. Aoki and Holland (1985) also and prevalence ofAeromonas hydrophilain 25Florida lakes.Florida Scientist, 46: 52-56 (citedobserved wide variation in the profiles of A.

from Kersters et al., 1996).hydrophilaproteins and reported them to be due 2. Aoki, J. and Holland, B. I. (1985). The outerto serogroup differences, variation in habitat, hostmembrane proteins of fish pathogensrange and virulence. They also reported that OMP

Aeromonas hydrophila, Aeromonas salmonicidaprofile ofA. hydrophiladiffered fromA. salmonicida. andEdwardsiella tarda. FEMS Microbiol.Lett. ,In an interesting study Kuijper et al., (1989) 27:299-305.analysed the OMP of 46 faecalAeromonasstrains 3. Bhat, P., Shanthakumari S. and Rajan, D. (1974).

The characterization and significance offrom hybridization groups (HGs) 1 (A. hydrophila;Plesiomonas shigelloides and Aeromonasn=10),4 (A. caviae, n=16) and 8(A. veronii;n=20)hydrophila isolated from an epidemic ofand reported that every isolate of HG-1 and HG-8diaarrhoea.Indian J Med. Res. 62:1051-1060.had a unique OMP profile, in contrast to isolates of

4. Boussaid, A., Baleux, B., Hassani L. and Lense,HG-4, which were separated into 5 different OMP

J. (1991). Aeromonas species in stabilizationtypes. Our study also revealed some homogeneity ponds in the arid region of marrakesh, Morocco

amongA .hydrophilastrains. and relation to fecal pollution and climaticIt may also be pointed out that 2 of the 3 factors.Microbial. Ecology, 21:367-370.rough strains tested in this study had identical 5. Burke, V., Gracey, M., Robinson, J. Peck, D.,

Beauman J. and Bundell, C. (1983). TheOMP profile. Detailed studies on more number ofMicrobiology of childhood gastroenteritis:strains are needed to confirm these findings. The

Aeromonasspecies and other infective agents.J.study revealed 4 major and 4 to 6 minor proteinInfect. Dis., 148:68-74.

bands within the MW range of 14 to 55 kDa. In6. Burke, V., Robinson, J., Gracey, M., Peterson,

addition some minor bands were also observed inD., Meyer N. and Haley, V. (1984). Isolation of

the range of 10 to 12 kDa and 66 kDa with a few Aeromonas hydrophila from a Metropolitanstrains. This is in agreement to reports of earlier water supply: seasonal correlation with clinicalresearchers who have noted major protein bands isolates.Appl. Environ.Microbiol., 48:361-366.

7. Cahill, M.M. (1990). Bacterial flora of fishes: ain the range of 30 and 45 kDa (Kuijper et al.,review. Microbial Ecology, 19:21-41.1989). Similarly Santos et al., (1996) observed

8. Chatterjee, B. D. and Neogy, K. N. (1972).major proteins to be in the range of 55 and 28Studies onAeromonasandPlesiomonas specieskDa. Dooley and Trust (1988) studied the OMP ofisolated from cases of choleraic diarrhoea.highly virulence group of strains. They observed

Indian J. Med. Res., 60:520-524.that major proteins of MW of 30 kDa and proteins

9. Crosa, J. H. and Hodges, L. L. (1981). Outerin the molecular weight range of 45 to 55 kDa to membrane proteins induced under conditions of

be predominant. These variations are not iron limitation in the marine fish pathogen Vibriounexpected due to the complex and diverse nature anguillarum.Infect. Immun. 31: 223-227.ofAeromonasspp. 10. Davis, W. A., Kane J. G. and Garagusi, V.F.



5/5


(1978). HumanAeromonasinfections: a review Ecology, 8: 325-333.of the literature and a case report of endocarditis. 23. Majeed, K., Egan, A. and MacRae, I. C. (1989).

Enterotoxigenic aeromonads on retail lamb meatMedicine, 57:267-277.

and offal.J. Appl. Bacteriol., 67: 166-170.11. De, G. and Pal, D. (1984). Transferable drug 24. Palumbo, S. A., Beneicengo, M. M., Delresistance inAeromonas species on enteric agar.Correl, F., Williams A. C. and Buchanan, R. L.J Clin. Microbial. 23; 1065-1067.(1989). Characterization of the Aeromonas12. Dooley, J. G. S. and Trust, T. J. (1988). Surfacehydrophilagroup isolated from retail foods ofprotein composition of Aeromonas hydrophilaanimal origin.J Clin. Microbiol. 27: 854-859.strains virulent for fish: Identification of

25. Sack, R. B., Lanata, C. and Kay, B. A. (1987).a surface array protein.J. Bacteriol., 170:499-506.Epidemiological studies of Aeromonas related13. Ewing, W. H., Hugh R. and Johnson, G. (1961).diarrhoel diseases.Experientia.43:364-365.Studies on the Aeromonas group .CDC

26. Santos, Y., Bandin I. and Toranzo, A. E. (1996).monograph, USDHEW, Communicable DiseaseImmunological analysis of extracellularCentre Atlanta (cited from Sakazaki andproducts and cell surface components of motileShimada, 1984).Aeromonas isolated from fish. J. Appl.14. Hunter, P. R. and Burke, S. H. (1987). IsolationBacteriol., 81:585-593.ofAeromonas caviaefrom ice-cream.Lett. Appl.

27. Sanyal, S. C., Agarwal A. K. and Annapurna, E.Microbiol., 4:45.(1983). Haemagglutination properties and15. Joseph, S. W. and Carnahan, A. (1994). Thefimbrigation in enterotoxigenic Aeromonasisolation, identification and systematics ofhydrophila strains. Indian J. Med. Res., 78:motileAeromonas species.Ann. Rev. Fish Dis.,(Suppl.): 324.4: 315-343.

28. Schaterle, G. R. and Pollack, R. L. (1973). A16. Kersters, I., Van Vooren, L., Huys, G., Janssen,simplified method for the quantitative assay ofP., Kersters K. and Verstraete, W. (1995). Influencesmall amounts of protein in biologicof temperature and process technology on thematerial.Anal. Biochem., 51:654-655.occurrence ofAeromonas species and hygienic

29. Schorling, J. B., Wanke, C. A., Schorling, S.K.,indicator organisms in drinking water productionMcAuliffe, J. F., Auxiliadora de Souza M. andplants.Microbial. Ecology, 30: 203-218.Cuerrant, R. L. (1990). A perspective study17. Khardori, N. and Fainstein, V. (1988).of persistent diarrhoea among children in anAeromonas and Plesiomonas as etiologicalurban Brazilian slum. Am. J. Epidemiol.,agents.Ann.Rev. Microbiol., 42:395-419.132:144-156.18. Kirov, S. M., Anderson M. J. and McMeekin, T.

30. Skiendzielewski, J. J. and O'Keefe, K P. (1990).A. (1990). A note on Aeromonas spp. from

Wound infection due to fresh water contaimi-chickens as possible food borne pathogens. J.

nation byAeromonas hydrophila.J.Emerg. Med.Appl. Bacteriol., 68:327-334.

8:701-703.19. Krovacek, K., Conte, M., Galderisi, P., Morelli,

31. Statner, B., Jones M. J. and Georg, W. L. (1988).G., Postiglione A. and Dumontet, S. (1983).

Effect of incubation temperature on growth andFatal septicaemia caused by Aeromonas

soluble protein profiles of motile Aeromonashydrophila in patients with cirrhosis. Comp.

strains. J. Clin. Microbiol.26:392-393.Immunol. Micribiol. Infect. Dis., 16:267-272.

32. Von Graevenitz, A. and Mensch, A. H. (1968).20. Kuijper, E. J., Van-Alphen, L., Leenders E. and

The genus Aeromonas in human bacteriology.Zanen, H. C. (1989). Typing ofAeromonasstrains Report of 30 cases and review of literature. Theby DNA restriction endonuclease analysis New Eng. J. Med., 278: 245-249.and polyacrylamide gel electrophoresis of cell 33. Wadstrom, T., Ljungh A. and Wretind, B. (1976).

envelops.J. Clin. Microbiol. 27: 1280-1285. Enterotoxin, haemolysin and cytotoxic protein21. Laemmli, U. K. (1970). Cleavage of structural inAeromonas hydrophilafrom human infection.

proteins during the assembly of the head of Acta Patho. Microbial. Scand. B., 84:112-114.bacteriophage T4. Nature (London). 227:680- 34. Wadstrom, T. and Ljungh, A. (1991).Aeromonas685. and Plesiomonas as food and waterborne

22. Lechevallier, M. W., Evans, T. M., Seidler, R. J., pathogens. Int. J. Food Microbiol. 12:303-312.Daily, O. P., Merrell, B. R., Rollins D. M. and 35. Zimmerman, O. E. R. (1890).Ber naturw. Ges.Joseph, S. W. (1982). Aeromonas sobria Chemnitz. 1:38 (cited from von Graevenitz,in hlorinated drinking water supplies.Microbial 1987). ********


Artículo 1 SDS-PAGE

Documents

Transcript of Artículo 1 SDS-PAGE