LXIX Convegno
Use of proteomic analyses to improve immunoblotting test for serological diagnosis of Contagious Bovine Pleuropneumonia
Perugia, 15-17 giugno 2015
T. Di Febo1, I. Krasteva1, G.M. Muuka2, D.G.E. Smith3, N.F. Inglis3, M. Scacchia1, A. Pini1, F. Sacchini1* Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Teramo, Italy. * Corresponding author:
[email protected] 2 Central Veterinary Research Institute, Lusaka, Zambia 3 Moredun Proteomics Facility, Moredun Research Institute, Penicuik, Midlothian, United Kingdom 1
Introduction Contagious Bovine Pleuropneumonia (CBPP) is a highly contagious disease of cattle caused by Mycoplasma mycoides subsp. mycoides (Mmm) that causes high morbidity and mortality in affected herds (1). Diagnosis of CBPP is based on isolation and identification of the causal agent, post-mortem examination of lungs of affected animals and serological methods such as the complement-fixation test (CFT), c-ELISA and immunoblotting (IB) (2). Despite offering high levels of sensitivity and specificity, IB, by its very nature, is not considered suitable for mass screening and is used mainly to confirm doubtful results obtained using CFT and/or c-ELISA. Qualitative protein electropherotypic differences observed amongst Mmm strains (3) and the use of unfractionated whole mycoplasma cell lysates have made IB difficult to standardise. Moreover, accurate interpretation of results is often confounded by the limitations of conventional one-dimensional SDS-PAGE through its failure to adequately resolve proteins of similar molecular weight. Thus there is a clear need for refinements that would simplify IB standardisation and facilitate unambiguous interpretation of test results.
Keywords
Scope
Figure 1. LC-ESI-MS/MS analysis results of the 5 specific immunogenic bands recognised by IgG of CBPP infected animals when tested using immunoblotting according to the OIE protocol. MSC_0089, MSC_0160, MSC_0253, MSC_0263, MSC_0266, MSC_0476,MSC_0500, MSC_0532, MSC_0679, MSC_0830, MSC_0855, MSC_0860 110 kDa 98 kDa 95 kDa
MSC_0139, MSC_0160, MSC_0253, MSC_0256, MSC_0263, MSC_0265, MSC_0266, MSC_0303, MSC_0454, MSC_0532, MSC_0544, MSC_0625, MSC_0627, MSC_0679, MSC_0855, MSC_0860
62/60 kDa
48 kDa
MSC_0007, MSC_0139, MSC_0160, MSC_0253, MSC_0256, MSC_0263, MSC_0265, MSC_0266, MSC_0352, MSC_0532, MSC_0588, MSC_0625, MSC_0679, MSC_0830, MSC_0860
MSC_0011, MSC_0160, MSC_0237, MSC_0253, MSC_0256, MSC_0263, MSC_0266, MSC_0273, MSC_0300, MSC_0519, MSC_0532, MSC_0610, MSC_0679, MSC_0860
MSC_0066, MSC_0160, MSC_0253, MSC_0263, MSC_0505, MSC_0509, MSC_0544, MSC_0554, MSC_0588, MSC_0830, MSC_0850, MSC_1019
Figure 2. Comassie (A) and western blotting of CBPP positive (B) and negative (C) sera using Mmm recombinant proteins as antigens. Lane M: Novex Sharp Pre-Stained Protein Standards (Life Technologies); lane 1: MSC_0011; lane 2: MSC_0139; lane 3: MSC_0160; lane 4: MSC_0266; lane 5: MSC_0011, MSC_0139, MSC_0160 and MSC_0266 mixed. A
B
Materials and methods
The aim of this study was to investigate the protein composition of the 5 common immunogenic bands observed at 110, 98, 95, 62/60 and 48 kDa in IB protein electropherotypes of Mmm and use this data to design a new generation IB test based on a selection of specific recombinant antigens.
Results and conclusions Mass spectrometry analyses of the 5 immunogenic bands identified a total of 35 different proteins, 15 of which were detected in more than one band. Twelve proteins were found in the 110 kDa band, 16 in the 98 kDa band, 15 in the 95 kDa band, 14 in the 62/60 kDa band and 12 in the 48 kDa band (Figure 1, Table 1). Among these 35 proteins, 17 have previously been reported in the literature as immunogenic. IB analyses using the four recombinant proteins (Ribose/galactose ABC transporter, substrate-binding component, MSC_0011, 61 kDa; Fructose-bisphosphate aldolase class II, MSC_0139, 32.8 kDa; Translation elongation factor Tu, MSC_0160, 43 kDa; Pyruvate dehydrogenase -lipoamide-, beta chain, MSC_0266, 36 kDa) (Table 1) revealed clear qualitative differences between CBPP positive and negative sera (Figure 2A, 2B, 2C). The preliminary data presented here demonstrate that the use of a carefully selected panel of specific recombinant Mmm reference proteins offers the potential for optimising the IB test and improving the serological diagnosis of CBPP.
Contagious Bovine Pleuropneumonia, Diagnosis, Immunoblotting, LC-ESI-MS/MS
C
References 1. Thiaucourt F. et al. 2007. Contagious Bovine Pleuropneumonia. In Coetzer W.A.J., Thomson R.G., Tustin C.R. (eds), Infectious diseases of livestock with reference to Southern Africa, 3rd Ed, Oxford University Press. 2. World Organisation for Animal Health (OIE). 2014. Contagious Bovine Pleuropneumonia. In Manual of diagnostic tests and vaccines for terrestrial animals, 6th Ed, OIE, Paris, 1-16. 3. Gonçalves R. et al. 1998. Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins. Vet Microbiol, 63, 13-28.
SDS-PAGE of Mmm was carried out in accordance with prescribed OIE guidelines (2) and the gel stained with Comassie Blue. Five protein bands corresponding to immunogenic bands of approx 110, 98, 95, 62/60 and 48 kDa, as recognised by IgG of sera from CBPP infected animals using IB, were characterised by liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Four of the identified proteins, reported as immunogenic, were expressed as recombinant antigens in E. coli and tested by IB against positive and negative CBPP reference sera. Table 1. Proteins identified by LC-ESI-MS/MS analysis in the 5 specific immunogenic bands recognised by IgG of CBPP infected animals when tested using immunoblotting according to the OIE protocol. RP: recombinant proteins used in IB test. I: proteins reported in the literature as immunogenic. Locus tag 1 MSC_0089 MSC_0160 MSC_0253 MSC_0263 MSC_0266 MSC_0476 110 kDa MSC_0500 MSC_0532 MSC_0679 MSC_0830 MSC_0855 MSC_0860 MSC_0139 MSC_0160 MSC_0253 MSC_0256 MSC_0263 MSC_0265 MSC_0266 MSC_0303 98 kDa MSC_0454 MSC_0532 MSC_0544 MSC_0625 MSC_0627 MSC_0679 MSC_0855 MSC_0860 MSC_0007 MSC_0139 MSC_0160 MSC_0253 MSC_0256 MSC_0263 MSC_0265 95 kDa MSC_0266 MSC_0352 MSC_0532 MSC_0588 MSC_0625 MSC_0679 MSC_0830 MSC_0860 MSC_0011 MSC_0160 MSC_0237 MSC_0253 MSC_0256 MSC_0263 MSC_0266 62/60 kDa MSC_0273 MSC_0300 MSC_0519 MSC_0532 MSC_0610 MSC_0679 MSC_0860 MSC_0066 MSC_0160 MSC_0253 MSC_0263 MSC_0505 MSC_0509 48 kDa MSC_0544 MSC_0554 MSC_0588 MSC_0830 MSC_0850 MSC_1019
Protein 2
IB band
1 2
Translocase Translation elongation factor Tu Phosphopyruvate hydratase NADH oxidase Pyruvate dehydrogenase (lipoamide), beta chain P115-like protein with SMC_C motif Hypothetical prolipoprotein L-lactate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Thymidine phosphorylase Ribonucleotide-diphosphate reductase beta subunit PTS system, glucose-specific IIBC component Fructose-bisphosphate aldolase class II Translation elongation factor Tu Phosphopyruvate hydratase Hypoxanthine phosphoribosyltransferase NADH oxidase Pyruvate dehydrogenase (lipoamide), alpha chain Pyruvate dehydrogenase (lipoamide), beta chain Valine-tRNA ligase Endopeptidase La L-lactate dehydrogenase Dihydrolipoamide dehydrogenase Prolipoprotein Prolipoprotein Glyceraldehyde-3-phosphate dehydrogenase Ribonucleotide-diphosphate reductase beta subunit PTS system, glucose-specific IIBC component DNA Gyrase Subunit A Fructose-bisphosphate aldolase class II Translation elongation factor Tu Phosphopyruvate hydratase Hypoxanthine phosphoribosyltransferase NADH oxidase Pyruvate dehydrogenase (lipoamide), alpha chain Pyruvate dehydrogenase (lipoamide), beta chain Transcription elongation factor NusA L-lactate dehydrogenase Cell division protein FtsZ Prolipoprotein Glyceraldehyde-3-phosphate dehydrogenase Thymidine phosphorylase PTS system, glucose-specific IIBC component Ribose/galactose ABC transporter, substrate-binding component Translation elongation factor Tu Oligoendopeptidase F Phosphopyruvate hydratase Hypoxanthine phosphoribosyltransferase NADH oxidase Pyruvate dehydrogenase (lipoamide), beta chain Phosphoenolpyruvate-protein phosphotransferase Hypothetical protein MSC_0300 Prolipoprotein B L-lactate dehydrogenase Heat shock protein 70 (chaperone) Glyceraldehyde-3-phosphate dehydrogenase PTS system, glucose-specific IIBC component Seryl-tRNA synthetase Translation elongation factor Tu Phosphopyruvate hydratase NADH oxidase Glucose-6-phosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase(NADP) Dihydrolipoamide dehydrogenase Xaa-His dipeptidase Cell division protein FtsZ Thymidine phosphorylase Adenylosuccinate synthase NADH oxidase
I I I I
RP
RP
I I I I I I I I I I I
RP RP
RP
I I
I I I I I I I I I
RP RP
RP
I
I I I I
RP RP
I I I I
RP
I I I I I I I I I I I
I I
RP
MW 107.7 43.3 49.5 50.1 36.2 112.9 108.0 34.6 37.0 48.9 39.1 73.3 32.8 43.3 49.5 21.8 50.1 41.8 36.2 103.2 90.4 34.6 50.8 98.3 96.7 37.0 39.1 73.3 94.5 32.8 43.3 49.5 21.8 50.1 41.8 36.2 67.6 34.6 41.5 98.3 37.0 48.9 73.3 60.9 43.3 70.0 49.5 21.8 50.1 36.2 64.5 65.6 69.8 34.6 63.9 37.0 73.3 48.6 43.3 49.5 50.1 48.5 51.9 50.8 52.2 41.5 48.9 49.1 53.0
pI 5.7 5.0 5.3 6.1 5.9 5.7 9.6 5.8 7.7 5.5 4.9 9.0 6.4 5.0 5.3 5.7 6.1 5.2 5.9 7.2 5.6 5.8 8.9 9.1 8.8 7.7 4.9 9.0 6.4 6.4 5.0 5.3 5.7 6.1 5.2 5.9 4.6 5.8 4.3 9.1 7.7 5.5 9.0 9.4 5.0 5.5 5.3 5.7 6.1 5.9 5.3 9.2 9.2 5.8 4.8 7.7 9.0 6.7 5.0 5.3 6.1 6.1 9.0 8.9 5.3 4.3 5.5 7.8 7.6
locus tag as defined for M. mycoides subsp. mycoides SC strain PG1 (accession number NC_005364.2) protein annotation as defined for M. mycoides subsp. mycoides SC strain PG1 (accession number NC_005364.2)
Acknowledgements This work was funded by Italian Ministry of Health (grant codes IZS AM 01/09 RC and IZS AM 07/11 RC).
