Perfil proteómico de animales tolerantes a la paratuberculosis bovina.

Eva Blanco-Costales¹, Alejandra Isabel Navarro León¹, Susana Belén Bravo López², Nuria Adela Menéndez-Arias¹, Tania Iglesias³, Gerard Badía-Bringé⁴, Patricia Vázquez⁴, Joseba Mirena Garrido⁴, Ramón Juste⁴, Marta Alonso-Hearn⁴, Rosa Casais¹

(1)-Center for Animal Biotechnology, Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Asturias, Spain. (2)-Proteomic Unit, Instituto de Investigaciones Sanitarias de Santiago de Compostela – IDIS, Complejo Hospitalario Universitario de Santiago de Compostela (CHUS), Santiago de Compostela, Spain. (3)-Unidad de Consultoría Estadística, Servicios científico-técnicos, Universidad de Oviedo, Campus de Gijón, Asturias, Spain.
(4)-Department of Animal Health, NEIKER-Basque Institute for Agricultural Research and Development, Basque Research and Technology Alliance (BRTA), Derio, Spain.

Paratuberculosis (PTB) is a highly contagious chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). Most PTB control programs are based on testing and selective culling of test-positive cows combined with the implementation of appropriate sanitary and management practices. However, conventional diagnostic tests, used to identify infected animals, show limited sensitivity for detecting subclinical infections resulting in an underestimation of the actual PTB prevalence and in the persistence of MAP in the farms, compromising disease control.

Resilient animals used three defence strategies against pathogens: avoidance, resistance, and tolerance. The avoidance strategy relies on behavioural mechanisms that limit exposure by identifying and avoiding infection risks before exposure. Resistance refers to the elimination of the pathogen by preventing its invasion through the activation of innate immune and inflammatory responses. Disease tolerance, in turn, is genetically determined and reduces host susceptibility, reduces the negative impact of an infection on host fitness through tissue damage control without directly affecting the pathogen burden. One management strategy consists of retaining resilient individuals within the herd in order to enhance disease control. Maintaining clinically healthy animals capable of surviving exposure to MAP is economically advantageous and aligns with current animal health standards and welfare regulations.

In a previous genomic study, we identified a specific immunogenetic profile associated to PTB tolerance in cattle that was naturally infected with MAP but did not show PTB-associated lesions in gut tissue. Forty single nucleotide polymorphisms (SNPs) were identified, defining nine loci (Quantitative Trait Loci, QTL) and 98 genes. Most of the genes were involved in DNA repair, chromatin packaging, and innate immune response regulation. In this context, tolerant animals are unable to completely eliminate MAP, however, they are capable of limiting tissue damage and maintaining homeostasis through mechanisms of repair and immune regulation.

The aim of the present study was: 1) To identify biomarkers of tolerance to PTB; and 2) To elucidate the molecular mechanisms and protective pathways of tolerance that help to reduce the host vulnerability to tissue damage. High-throughput and label-free quantitative proteomics (SWATH-MS) was employed to analyse the proteomic profile of serum and ileocecal valve (ICV) of: 1) infected animals without PTB-associated histological lesions that tested positive for MAP by PCR and bacteriological culture (tolerant animals); 2) Animals presenting focal lesions but negative by PCR, culture and ELISA for the detection of anti-MAP antibodies (non-tolerant animals); and 3) Animals without lesions and negative by PCR, culture and ELISA (healthy controls). Proteins were identified using ProteinPilot software (SCIEX), only peptides passing FDR < 1% were considered reliable hits. Two comparisons were performed: Tolerant versus healthy controls and tolerant versus non-tolerant animals.

In the tolerant versus healthy control comparison, 347 and 116 differentially expressed proteins (DEPs; p < 0.05) were detected in ICV and serum, respectively, with 42 proteins shared between both types of samples. In the tolerant versus non-tolerant comparison, 263 and 149 DEPs were identified in ICV and serum, respectively, with 40 overlapping proteins between the two samples. Overall, the serum proteome and the intestinal tissue proteome show markedly different profiles in both comparisons. However, there is a group of proteins shared between the samples, suggesting the existence of a small number of common and conserved proteins strongly associated with tolerance.

To obtain information about key biological processes relevant to PTB tolerance STRING analysis was performed including the list of all the DEPs (p<0.05). Functional enrichment identified identical clusters in both comparisons, suggesting a consistent pattern in the tolerant phenotype. However, fold enrichment was consistently higher in the tolerant animals versus non-tolerant animals’ comparison indicating that tolerant animals have a profile more similar to healthy control individuals. The top networks identified were: 1) Mixed network, including extracellular matrix organization and ECM receptor interaction; 2) RNA recognition motif and K homology domain type 1; 3) Mixed network, including cell–extracellular matrix interactions and the alpha-catenin/vinculin-like superfamily; 4) Collagen formation and microfibril structures; 5) Fibrinolysis and serine protease inhibitor activity and 6) chaperone tailless complex polypeptide (TCP-1). Specifically, the most relevant functions and biological processes enriched in the comparison tolerant versus healthy controls involved extracellular matrix structural constituent, protein folding and acute-phase response

whereas in the comparison of tolerant versus non-tolerant animals, included protein folding chaperones, positive regulation of DNA biosynthetic processes, and regulation of extracellular matrix organization.

Finally, five DEPs have been selected as candidate biomarkers for PTB tolerance based on their cellular location (extracellular), biological function (maintaining tissue integrity and regulating immune responses), fold change (>1.5), and reagent availability. These selected DEPs will be validated by Enzyme-Linked Immunosorbent Assays (ELISA) in serum samples in order to confirm the proteomic results.

In summary, these findings provide insight into the molecular mechanisms and biological functions associated with PTB tolerance and contribute to the identification of potential biomarkers relevant to PTB tolerance and disease control.