2.5. Bioinformatics analysis
Data generated by mass spectrometry analyses were then submitted for bioinformatics analysis for protein identification and selection. As first step a combination of softwares were applied to identify cytosolic and non-cytosolic proteins. LipoP 1.0 Server [37] was used for prediction of lipoprotein signal peptides; TMHMM Server version 2.0 was predictive of transmembrane helices [38, 39] and SignalP 4.1 Server [40] was applied for signal peptides prediction. PSORTb version 3.0.2 [41] and CELLO version 2.5 [42, 43] predicted subcellular localization. The data obtained from the above software analyses were combined to discard cytosolic proteins.
Non–cytosolic proteins were further analyzed to predict B-cell linear epitopes by BepiPred version 1.0 Server [44] by setting a threshold equal or higher than 0.35 and a minimum length of 4 residues. NetSurfP version 1.1 server [45] was used to predict the surface accessibility of an amino acid and protein secondary structure. The epitopes not exposed to the solvent were discarded. The proteins with B-cell solvent-exposed epitopes were further analyzed by Vaxign [46-47] and VaxiJen tools [49] for prediction of protective antigens. Only the proteins with adhesion score greater than 0.5 (Vaxign tool) and those with threshold greater than 0.4 (VaxiJen) were considered as candidate antigens.
As a final step, the potential antigenic proteins of B. canisresulting from the above bioinformatics analyses were screened by BLASTp to check for sequence similarity with other Brucella species and cross-reactive bacteria. Among the genus Brucella , B. melitensis , B. ovis , B. abortus and B. suis were considered. Cross-reactive bacteria included Pseudomonas aeruginosa , Bordetella bronchiseptica , Actinobacillus equuli , Streptococcus spp., Staphyloccus spp.,Moraxella type, Salmonella spp. and Campylobacterspp. [50], the environmental bacterium Ochrobactrum anthropiand the plant pathogens or symbionts Rhizobium leguminosarum ,Rhizobium /Agrobacterium group and Rhizobium tropici[1]. The criteria used to identify non-homologous proteins were: identity and/or coverage lower than 95% for Brucella species and 35% for cross reactive bacteria.
3. Results
3.1 Western blotting
Serum antibodies from 31 out of 32 B. canis infected animals identified common bands ranging from 7 to 30 kDa, in contrast to serum antibodies from non-infected animals, where no bands or bands ranging from 40-200 kDa (3 animals only) were observed (Figure 1).
3.2 Mass spectrometry (nLC-ESI-MS/MS) and bioinformatics analysis
Two gel slices containing B. canis proteins ranging from 7 to 30 kDa were excised and analyzed by mass spectrometry analysis (Figure 1) and 398 B. canis proteins were identified. Some proteins were present in more than one band, therefore the repeated proteins were discarded. The workflow adopted for the prediction of protein candidates is shown in Figure 2.
Among the 398 identified proteins, 245 (61.3%) proteins were cytoplasmic and 153 (38.7%) non-cytoplasmic. Hence, the study focused on non-cytoplasmic proteins, as they are involved in pathogenesis and survival of Brucella in macrophages [51].
These proteins were examined to identify B-cell solvent-exposed epitopes (Supplementary Table 1): 145 proteins were identified and further investigated by Vaxign and VaxiJen tools to predict antigens. Forty-seven proteins had adhesion score greater than 0.5 when analyzed by Vaxign tool, and 123 proteins had threshold greater than 0.4 by VaxiJen tool. Overall, 126 proteins were predicted as potential antigens: 44 proteins were predicted as protective antigens by both softwares, 3 proteins only by Vaxign and 79 proteins only by VaxiJen tool.
Then BLAST was used to verify similarity among the 126 B. canis potential antigen proteins and proteins of other species of Brucella as well as cross-reactive bacteria.
As expected, all B. canis proteins resulted homologous to B. abortus and B. suis. Nine B. canis proteins are non-homologous to B. ovis and, among them, one was found non-homologous to B. melitensis. As the sequence homology present among the Brucella species is very high, the criterion used to identify non-homologous proteins were 95% identity.
Sixteen B. canis proteins were found to be non-homologous to all cross-reactive bacteria examined (P. aeruginosa, B. bronchiseptica, A. equuli, Streptococcus spp., Staphyloccus spp., Moraxella type, Salmonella spp. and Campylobacter spp,). According to Uniprot, 7 proteins are included in the following categories: one is an integral component of membrane, one has oxidoreductase activity, one is mitochondrial respiratory chain complex I assembly, one is a membrane protein, one has phosphatidylserine decarboxylase activity and phosphatidylethanolamine biosynthetic process and for two proteins no category was assigned. Nine proteins are uncharacterized, even if for two of them it was possible to assign gene ontology (integral membrane components). Regarding environmental and plant pathogens/symbionts cross-reactive bacteria (Rhizobium and Agrobacterium), 2 proteins are non-homologous to all cross-reactive bacteria examinated and among them one is also non-homologous to all cross-reactive bacteria; the other one is uncharacterized protein.
4. Discussion
In this study a western blotting assay was set up in order to identify the B. canis protein pattern recognized by serum antibodies from infected dogs. The test clearly showed that IgGs of infected animals selectively bind to some B. canis proteins of low molecular weight (7-30 KDa) not recognized by antibodies of non-infected dogs, so the western blotting may serve to distinguish infected from non-infected animals.
Use of western blotting method as diagnostic test, mainly confirmatory test, has been reported for serological diagnosis of other animal diseases, such as Contagious Bovine Pleuropneumonia in cattle [52, 53] or Dourine in horses [54]. The use of western blotting to characterize antibody response against B. canis antigen has been described in the past [55] and more recently Barkha et al. (2011) [56] showed that dog anti-B. canis hyperimmune sera identified low molecular weight immune reactive bands of B. canisexternal (12, 28, 39 and 45 kDa) and internal antigens fractions (20-24 kDa). Results obtained in the present work also support these findings with some differences in the molecular range of the immune reactive bands identified that in our case was restricted to 7-30 kDa. The difference in B. canis strain, the antigen preparation procedure used in this study together with the application of chemiluminescence to reveal immunoreactivity might have contributed to the observed variations. Though these encouraging results, western blotting was never applied for serological diagnosis of B. canis on a large scale. Our results, in addition to previous findings, encourage a field applicability of western blotting, mainly as confirmatory test of doubtful cases, where epidemiological evidences of B. canisinfection do not support serological positivity to other indirect tests.
The second step of this study was focused on characterizing the protein composition of immunodominant bands identified by IgGs antibodies ofB. canis infected dogs, in order to find potential diagnostic antigenic biomarkers to be used as antigens for new recombinant diagnostic tests specific for canine brucellosis. The low molecular weight protein pattern specifically recognized by sera of infected dogs was then characterized by mass spectrometry, identifying 398 B. canis proteins. Among them, an ad hoc developed bionformatics pipeline identified 126 potential antigens and then 16 B. canispotential specific targets were selected after screening for non-cytosolic, immunogenic, non-cross-reactive proteins.
In a recent study, Jimenez and coworkers (2020) [17] carried out identification and characterization of immunoreactive proteins focusing on the cytoplasmic (internal) fraction of B. canis that led to the expression of two recombinant target antigens with limited sensitivity and specificity. In our study, we targeted non-cytosolic proteins located on the membrane/external part of the bacteria that have higher chance to be involved in host-pathogen interactions and to be immunogenic. Starting from the set of proteins identified by mass spectrometry, bioinformatics analyses recognized 126 non-cytosolic proteins potentially immunogenic, with some proteins already describe in the literature. One of the protein identified was the outer membrane protein assembly factor BamD (A9M681), a conserved multi-component protein complex that is responsible for the biogenesis of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. BamD deletion causes lethality in E. coli and Neisseria meningitidis , and Bam has a role in the production of OMPs for survival and pathogenesis [57]. Proteins Omp25, Omp31 and SodC were also identified: these proteins have been well characterized as virulence factors or immunogenic proteins in Brucella ; further these proteins were identified in outer membrane vesicles (OMVs) in B. canis[58]. The protein Sod (Superoxide dismutase [Cu-Zn]) is associated to virulence in a number of microorganisms [31]. Omp31 appears as an immunodominant antigen in the course of “rough” (R)B. ovis infection in rams and as important protective antigen forB. ovis infection in a mouse model. Omp25 is involved in virulence of B. melitensis [59], moreover B. suisOmp25 suppresses production of TNFα, crucial to clear B. suisinfection [60]. It was shown that Omp25 and Omp31 induce protection against Brucella in vivo and could be a potential subunit brucellosis vaccines candidate [61]. The proteins SodC, Omp25 and Omp31 were also identified on membrane blebs isolated from B. abortus 2308 and RB51. Mice vaccinated with membrane blebs from rough or smooth B. abortus showed a protective immune response similar to the one elicited by vaccine B. abortus RB51 after the challenge with virulent strain B. abortus 2308, suggesting that these proteins could be good candidate for vaccine against brucellosis [62]. In another study in mice, Clausse et al. (2014) [63] showed that immunization with Omp31 is effective againstB. canis infection.
Recently, in a study of Paci et al. (2020) [34] B. ovis Omp31 and B. melitensis Omp25 were indicated as good candidate antigens for development of Brucella specific serological tests and vaccines.
One of the major drawbacks of current serological tests for B. canis is the cross-reactivity with other bacteria that results in false positive reactions in the course of serological testing [6, 22]. Thus, it is important to assess the cross-reactivity of potential target antigens. In theory, an experimental laboratory approach would have required the screening of all candidate antigens identified, expressed as recombinant antigens, against cross-reactive sera. However, the high number of antigens identified and the lack of reference hyperimmune sera against the different cross-reactive bacteria imposed an alternative, time-saving and economically sustainable strategy. Thus, bioinformatics analyses were used to discard all the non-cytosolic immunogenic proteins showing an identity higher than 35% with any of the cross-reactive bacteria. This led to the exclusion of 87% of potential candidate proteins ascertained, narrowing the number of optimal targets but also, confirming the high homology of several B. canis proteins with the bacteria responsible for cross-reactive immunity. Among the 16B. canis specific proteins finally predicted, chaperone surA protein was identified, that is reported to be a protective antigen ofB. abortus 104M [64]. For the remaining proteins, no functional information are described in the literature and some of them resulted uncharacterized. One of the major limitations of the in silico approach described in this study is that, despite the accuracy adopted in combining the different bioinformatics softwares, results generated are predictive and requires subsequent laboratory confirmation.Canine brucellosis caused by B. canis is nowadays considered an emerging and zoonotic disease and the increased trade and movement of dogs worldwide is imposing the application of measures to prevent, monitor and control disease spread within and across Countries. Diagnosis of B. canis relies on the analysis and interpretation of epidemiological data and together with laboratory results of direct and indirect tests. However, serological tests still represent the most cost/effective tools for disease surveillance and the diagnosis ofB. canis in humans are lacking. Based on the results of the present study the western blotting test is able to distinguish between infected and uninfected animals and could be used as confirmatory test for the serological diagnosis of B. canis . The mass spectrometry and in silico results lead to the identification of a set ofB. canis specific candidate antigens that pave the way for the development of more efficient diagnostic tests.
Author Contributions: Conceptualization, L.M. and I.K.; methodology, L.M., T.D.F., F.D.O. and I.K.; software, I.K.; validation, I.K.; formal analysis and investigation, L.M., T.D.F., F.D.O.., F.P. and I.K.; resources, F.S., F.D.M. and M.T.; data curation, L.M., T.D.F., F.P. and I.K.; writing—original draft preparation, M.L., I.K., T.D.F.; writing—review and editing, F.S., M.T. and F.D.M.; supervision, F.S., M.T. and F.D.M.; project administration, F.S., M.T. N.D.A. and F.D.M.; funding acquisition, F.S., M.T. N.D.A. and F.D.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Italian Ministry of Health, grant number IZSAM 02/20 RC (BruM-Life project).
Data Availability Statement: The data presented in this study are openly available in UniProt.
Conflicts of Interest: The authors declare no conflict of interest.
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Figure 1. SDS-PAGE and western blot analyses. Representative image of SDS-PAGE and Coomassie stain of B. canis total proteins (lane 1) and immunoblot using sera from B. canis infected (lane 2) and non-infected dogs (lane 3). The proteins were separated on a 4–12% Bis-tris gel (Life Technologies). M: molecular weight marker 10-260 KDa (Novex Sharp Prestained Protein Standard, Life Technologies).
Figure 2. Overview of bioinformatics tools used for prediction of protein candidates.