Characterization of Surface Proteins of Cronobacter Muytjensii Using Monoclonal Antibodies and MALDI-TOF Mass Spectrometry Ziad W
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Purdue University Purdue e-Pubs Department of Food Science Faculty Publications Department of Food Science 6-25-2011 Characterization of Surface Proteins of Cronobacter Muytjensii Using Monoclonal Antibodies and MALDI-TOF Mass Spectrometry Ziad W. Jaradat Abrar M. Rashdan Qotaiba O. Ababneh Saied A. Jaradat Arun K. Bhunia Purdue University, [email protected] Follow this and additional works at: http://docs.lib.purdue.edu/foodscipubs Recommended Citation Jaradat, Ziad W.; Rashdan, Abrar M.; Ababneh, Qotaiba O.; Jaradat, Saied A.; and Bhunia, Arun K., "Characterization of Surface Proteins of Cronobacter Muytjensii Using Monoclonal Antibodies and MALDI-TOF Mass Spectrometry" (2011). Department of Food Science Faculty Publications. Paper 4. http://dx.doi.org/10.1186/1471-2180-11-148 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Jaradat et al. BMC Microbiology 2011, 11:148 http://www.biomedcentral.com/1471-2180/11/148 RESEARCH ARTICLE Open Access Characterization of surface proteins of Cronobacter muytjensii using monoclonal antibodies and MALDI-TOF Mass spectrometry Ziad W Jaradat1*, Abrar M Rashdan1, Qotaiba O Ababneh1,2, Saied A Jaradat1,3 and Arun K Bhunia4 Abstract Background: Cronobacter spp. is a newly emerging pathogen that causes meningitis in infants and other diseases in elderly and immunocompromised individuals. This study was undertaken to investigate surface antigenic determinants in Cronobacter spp. using monoclonal antibodies (MAbs) and MALDI-TOF Mass spectrometry. Results: Spleenocytes from mice that were immunized with heat-killed (20 min, 80°C) Cronobacter cells were fused with SP2 myeloma cells. Five desirable MAbs (A1, B5, 2C2, C5 and A4) were selected. MAbs A1, B5, 2C2 and C5 were of IgG2a isotype while A4 was an IgM. Specificity of the MAbs was determined by using immunoblotting with outer membrane protein preparations (OMPs) extracted from 12 Cronobacter and 6 non-Cronobacter bacteria. All MAbs recognized proteins with molecular weight ranging between 36 and 49 kDa except for one isolate (44) in which no OMPs were detected. In addition, MAbs recognized two bands (38-41 kDa) in four of the non- Cronobacter bacteria. Most of the proteins recognized by the MAbs were identified by MALDI-TOF peptide sequencing and appeared to be heterogeneous with the identities of some of them are still unknown. All MAbs recognized the same epitope as determined by an additive Index ELISA with their epitopes appeared to be conformational rather than sequential. Further, none of the MAbs recognized purified LPS from Cronobacter spp. Specificity of the MAbs toward OMPs was further confirmed by transmission electron microscopy. Conclusions: Results obtained in this study highlight the immunological cross-reactivity among Cronobacter OMPs and their Enterobacteriaceae counterparts. Nevertheless, the identity of the identified proteins appeared to be different as inferred from the MALDI-TOF sequencing and identification. Background More recent studies utilizing full length 16S rRNA Cronobacter spp. (formerly Enterobacter sakazakii)is gene sequencing, ribotyping, fluorescent-amplified frag- a non-spore forming, motile, facultative anaerobic Gram- ment length polymorphism and DNA-DNA hybridiza- negative bacillus and belongs to family Enterobacteriaceae tion have demonstrated that Cronobacter is a [1,2]. Initially isolates of Cronobacter spp. (Cronobacter) heterogenic genus exhibiting a high degree of genetic were identified as yellow pigment producing Enterobacter and phenotypic diversity among species and comprises cloacae. Later, Farmer et al., [3] reclassified them as a new six species: C. muytjensii, C. sakazakii, C. malonaticus, species and were given the name sakazakii based on C.turicensis,C.dublinensisand C. genomospecies I DNA-DNA homology, antibiotic susceptibility patterns [4-7]. Cronobacter is considered an emerging pathogen; and certain unique biochemical characteristics such as cat- though, little is known about its virulence properties alase production, the absence of oxidase and the produc- and antigenic determinants [8]. Recently, several studies tion of yellow pigment in all tested strains. have reported the involvement of an outer membrane protein (OMP), OmpA, in pathogenesis of C. sakazakii; however, nothing is known about its antigenicity. * Correspondence: [email protected] Besides, little is known about OMPs from other Crono- 1Department of Biotechnology and Genetic Engineering, P. O Box 3030, bacter species [8-10]. In contrast, the virulence and anti- Jordan University of Science and Technology, Irbid 22110, Jordan genic properties of OMPs of closely related Enterobacter Full list of author information is available at the end of the article © 2011 Jaradat et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jaradat et al. BMC Microbiology 2011, 11:148 Page 2 of 13 http://www.biomedcentral.com/1471-2180/11/148 species including E. aerogenes [11] and E. cloacae agar plates (HiMedia, India) at 4°C until use. The type [12,13] were studied well. strain C. muytjensii ATCC 51329 was grown overnight Prematurely born infants with low birth weights and at 37°C in nutrient broth (NB) (HiMedia). Cells were har- infants in neonatal intensive care units are highly sus- vested by centrifugation (5,000 × g, 10 min), washed twice ceptible to Cronobacter infections with the pathogen with 0.1 M PBS (pH 7.2) and adjusted to 108 CFU ml-1 being transmitted primarily from contaminated environ- using McFarland standard. Bacterial cells were heated at ments to the infant formula during the preparation 80°C for 20 min in a water bath and were subsequently [14-20]. In rare cases, nosocomial infections can happen used for immunization of mice and screening of hybri- in adults especially in immunocompromised ones [21]. doma cells for MAbs production using ELISA. Several In 2004, a joint FDA/WHO workshop raised an alert Cronobacter strains used in the study were isolated from concerning the presence of Cronobacter in powdered Jordan (Table 1). These isolates were identified and char- infant formula (PIF) and recommended applying higher acterized by several traditional and molecular methods microbiological standards during its manufacturing [22]. [19]. The 16S rRNA sequences of the isolates were depos- This warning culminated into increased research efforts ited in the GenBank (MD, USA) (Table 1), while the iso- to study Cronobacter including the development of lates were deposited in the Egyptian Microbial Culture improved isolation and identification methods, and Collection (Ain Shams University, Cairo, Egypt). understanding of the growth and survival characteristics. Antibodies are the most frequently used tools to study Lipopolysaccharide (LPS) extraction and antigen bacterial antigenic determinants; however, little is known preparation about the production of monoclonal antibodies that LPS was prepared following the method described by Jar- recognize Cronobacter antigenic determinants. In this adat and Zawistowski [23], with minor modifications. paper we describe the production and characterization of Briefly, C. muytjensii ATCC 51329 cells were harvested 5 MAbs that recognize outer membrane proteins of Cro- from an overnight culture by centrifugation (5,000 × g, nobacter. In addition, antigenic properties, identification, 10 min) and resuspended in 50 ml of 50 mM sodium distribution and cell surface localization of the MAbs- phosphate buffer, pH 7.0. The cells were sonicated 5 recognized OMPs were examined using electron micro- times for 45 s intervals at 300 Watts (Branson Sonifier). scopy and MALDI-TOF spectrometry. To our knowl- The sonicated suspension was incubated with pancreatic -1 edge, this is the first report on using monoclonal RNase and DNase (0.1 μgml ) in 20 mM MgCl2 at 37°C antibodies to study the surface antigens of this pathogen. for 10 min, followed by 10 min at 60°C and then mixed with an equal volume of preheated 90% phenol. Follow- Methods ing incubation (70°C for 15 min) with occasional mixing, Materials the mixture was centrifuged (18,000 × g, 1 h) and the Alkaline phosphatase-conjugated goat anti-mouse immu- resulting aqueous layer was collected and dialyzed using noglobulin, complete Freund’s adjuvant, incomplete dialysis tubing of 6,000-8,000 MW cutoff at room tem- Freund’s adjuvant, sarkosyl, DMSO, pancreatic RNase perature against several changes of distilled water until and DNase and a mouse subisotyping kit were from no detectable phenol odor remained. The dialysate was Sigma-Aldrich, USA. Gold-conjugated (18 nm) anti- treated with 20 μg/ml of Proteinase K in 0.1 M Tris-HCl mouse IgG was obtained from Jackson Immunochem- (pH 8.0) at 60°C for 1 h followed by overnight incubation icals, USA. Polyethelyene glycol 4000 was from Fluka, at 37°C. The samples were then lyophilized and stored at USA. Micro test plates, tissue culture plates and flasks -20°C until used. For antigen preparation, the extracted were from Griener, Germany. Coommassie Brilliant blue LPS from Cronobacter was mixed (1:1) with 30% (w/v) G-250 was from BDH chemicals, Ireland and BSA was polyacrylamide solution; ammonium persulfate (50 μl) from Biobasic, Canada;