Characterisation and expression profile of the bovine cathelicidin gene repertoire in mammary tissue.
ABSTRACT: BACKGROUND: Cathelicidins comprise a major group of host-defence peptides. Conserved across a wide range of species, they have several functions related to host defence. Only one cathelicidin has been found in humans but several cathelicidin genes occur in the bovine genome. We propose that these molecules may have a protective role against mastitis. The aim of this study was to characterise the cathelicidin gene-cluster in the bovine genome and to identify sites of expression in the bovine mammary gland. RESULTS: Bioinformatic analysis of the bovine genome (BosTau7) revealed seven protein-coding cathelicidin genes, CATHL1-7, including two identical copies of CATHL4, as well as three additional putative cathelicidin genes, all clustered on the long arm of chromosome 22. Six of the seven protein-coding genes were expressed in leukocytes extracted from milk of high somatic cell count (SCC) cows. CATHL5 was expressed across several sites in the mammary gland, but did not increase in response to Staphylococcus aureus infection. CONCLUSIONS: Here, we characterise the bovine cathelicidin gene cluster and reconcile inconsistencies in the datasets of previous studies. Constitutive cathelicidin expression in the mammary gland suggests a possible role for these host defence peptides its protection.
Project description:Cathelicidins are important components of the innate immune system and have been identified in skin and epithelia of a range of mammals. In this study molecular techniques, including RACE-PCR, were used to identify the full cDNA sequence of a cathelicidin gene, MaeuCath8, from the Australian marsupial, the tammar wallaby, Macropus eugenii. This cathelicidin was not homologous to other such genes previously isolated from a tammar wallaby mammary gland EST library, however, it did contain 4 conserved cysteine residues which characterise the pre-propeptide and had 80% identity with a previously isolated bandicoot cathelicidin. Reverse transcriptase-PCR established the expression profile of MaeuCath8 in a range of tissues, including spleen, thymus, gastrointestinal tract, skin and liver, of the tammar wallaby from birth to adulthood. Expression of MaeuCath8 was observed in spleen and gastrointestinal tract of newborn animals and was observed in most tissues by 7 days post-partum. The results indicate that pouch young could synthesize their own antimicrobial peptides from an early age suggesting that this ability most likely plays a role in protecting the pouch young from infection prior to the development of immunocompetence.
Project description:Prototheca bovis (formerly P. zopfii genotype-II) is an opportunistic, achlorophyllous alga that causes mastitis in cows and skin disease in cats and dogs, as well as cutaneous lesions in both immunocompetent and immunosuppressed humans. Antifungal medications are commonly ineffective. This study aimed to investigate innate immune responses contributed by cathelicidins to P. bovis in the mammary gland using a mastitis model in mice deficient in the sole murine cathelicidin (Camp). We determined P. bovis caused acute mastitis in mice and induced Camp gene transcription. Whereas, Camp -/- and Camp +/+ littermates had similar local algae burden, Camp +/+ mice produced more pro-inflammatory cytokines, TNF-?, and Cxcl-1. Likewise, Camp +/+ bone marrow-derived macrophages were more responsive to P. bovis, producing more TNF-? and Cxcl-1. Human cathelicidin (LL-37) exhibited a different effect against P. bovis; it had direct algicidal activity against P. bovis and lowered TNF-?, Cxcl-1, and IL-1? production in both cultured murine macrophages and mammary epithelial cells exposed to the pathogenic algae. In conclusion, cathelicidins were involved in protothecosis pathogenesis, with unique roles among the diverse peptide family. Whereas, endogenous cathelicidin (Camp) was key in mammary gland innate defense against P. bovis, human LL-37 had algicidal and immunomodulatory functions.
Project description:BACKGROUND: Mastitis is still considered to be the most economically important infectious disease in dairy cattle breeding. The immune response in mammary gland tissues could help in developing support strategies to combat this disease. The role of neutrophils and macrophages in the innate response of mammary gland is well known. However, the immune response in mammary gland tissues, including levels of antimicrobial peptide transcripts, has not been well recognized. Moreover, most studies are conducted in vitro, on cell cultures, or on artificially infected animals, with analysis being done within a several dozen hours after infection.The aim of the study was to examine the in vivo transcript levels of beta-defensin and cathelicidins genes in cow mammary gland secretory tissue (parenchyma) with the chronic, recurrent and incurable mammary gland inflammation induced by coagulase-positive or coagulase-negative Staphyloccoci vs. bacteria-free tissue. RESULTS: The mRNA of DEFB1, BNBD4, BNBD5, BNBD10 and LAP genes, but not of TAP gene, were detected in all investigated samples regardless of the animals' age and microbiological status of the mammary gland, but at different levels. The expression of most of the beta-defensin genes was shown to be much higher in tissues derived from udders infected with bacteria (CoPS or CoNS) than from bacteria-free udders, regardless of parity. Cathelicidins (CATH4, CATH5 and CATH6) showed expression patterns contrasting those of ?-defensins, with the highest expression in tissues derived from bacteria-free udders. CONCLUSION: Increased expression of genes encoding ?-defensins in the infected udder confirms their crucial role in the defense of the cow mammary gland against mastitis. On the other hand, the elevated cathelicidin transcripts in non-infected tissues indicate their role in the maintenance of healthy mammary tissues. The expression levels of investigated genes are likely to depend on the duration of the infection and type of bacteria.
Project description:Cathelicidins are peptide components of the innate immune system of mammals. Apart from exerting a direct antibiotic activity, they can also trigger specific defense responses in the host. Their roles in various pathophysiological conditions have been studied, but there is a lack of published information on their expression and activities in the context of mastitis. The aims of this study were to investigate the expression of the bovine cathelicidins BMAP-27, BMAP-28, Bac5, and indolicidin in healthy and infected mammary tissue and in lipopolysaccharide (LPS)-treated cells, to determine their activities against bacteria isolated from bovine mastitis, and to examine their potentials to trigger defense responses in bovine mammary cells. The genes were found to be upregulated in LPS-stimulated neutrophils, but not in infected quarters or epithelial cells. All peptides showed a variably broad spectrum of activity against 28 bacterial isolates from bovine mastitis (MIC values, 0.5 to 32 microM), some of which were antibiotic resistant. The activity of each peptide was significantly enhanced when it was pairwise tested with the other peptides, reaching the synergy threshold when indolicidin was present. The bactericidal activity was sensitive to milk components; BMAP-27 and -28 were highly effective in mastitic bovine milk and inhibited in milk from healthy cows. Both peptides were also active in whey and in blood serum and triggered the expression of tumor necrosis factor alpha (TNF-alpha) in bovine mammary epithelial cells. Our results indicate multiple roles for the bovine cathelicidins in mastitis, with complementary and mutually enhanced antimicrobial activities against causative pathogens and the capacity to activate host cells.
Project description:Cathelicidins represent a major group of host defense peptides (HDPs) that share a highly conserved cathelin-like domain. In birds, this gene family has been identified in many species. However, no information was available in the goose until now. In this study, we present the molecular characterization of 2 goose cathelicidin genes, namely goose CATH2 and goose CATH3, for the first time. The complete cDNA of goose CATH2 and goose CATH3 were 571 bp and 573 bp in length, respectively, and the deduced amino acid sequences exhibited high similarity with other avian cathelicidins. Furthermore, evolutionary analyses indicated that all known cathelicidins form 3 distinct clusters from reptiles, while the oldest cathelicidin member, which is known as CATHB1, is very likely absent in the goose genome. Meanwhile, highly expressed goose CATH2 and goose CATH3 were also observed in primary and secondary lymphoid tissues, same as the observations in other avian species. In addition, chemically synthesized mature peptides of the 2 cathelicidins exerted optimal antimicrobial abilities to a range of gram-negative and gram-positive bacteria. The discovery and characterization of goose cathelicidins complete the knowledge for goose HDPs and might contribute to understanding the evolution of avian cathelicidins as well as for the development of antibacterial agents.
Project description:Cathelicidins play pivotal roles in host defense. The discovery of novel cathelicidins is important research; however, despite the identification of many cathelicidins in vertebrates, few have been reported in amphibians. Here we identified a novel cathelicidin (named cathelicidin-OA1) from the skin of an amphibian species, Odorrana andersonii. Produced by posttranslational processing of a 198-residue prepropeptide, cathelicidin-OA1 presented an amino acid sequence of 'IGRDPTWSHLAASCLKCIFDDLPKTHN' and a molecular mass of 3038.5?Da. Functional analysis showed that, unlike other cathelicidins, cathelicidin-OA1 demonstrated no direct microbe-killing, acute toxicity and hemolytic activity, but did exhibit antioxidant activity. Importantly, cathelicidin-OA1 accelerated wound healing against human keratinocytes (HaCaT) and skin fibroblasts (HSF) in both time- and dose-dependent manners. Notably, cathelicidin-OA1 also showed wound-healing promotion in a mouse model with full-thickness skin wounds, accelerating re-epithelialization and granulation tissue formation by enhancing the recruitment of macrophages to the wound site, inducing HaCaT cell proliferation and HSF cell migration. This is the first cathelicidin identified from an amphibian that shows potent wound-healing activity. These results will help in the development of new types of wound-healing agents and in our understanding of the biological functions of cathelicidins.
Project description:Apart from their role in the immune defence against pathogens evidence of a role of antimicrobial peptides (AMPs) in autoimmune diseases has accumulated in the past years. The aim of this project was to examine the functional impact of the human cathelicidin LL-37 and the mouse cathelicidin-related AMP (CRAMP) on the pathogenesis of lupus and arthritis. Serum LL-37 and anti-LL-37 levels were measured by ELISA in healthy donors and patients with Systemic Lupus Erythematosus (SLE) and Rheumatoid arthritis (RA). Pristane-induced lupus was induced in female wild type (WT) and cathelicidin-deficient (CRAMP-/-) mice. Serum levels of anti-Sm/RNP, anti-dsDNA, and anti-histone were determined via ELISA, cytokines in sera and peritoneal lavages were measured via Multiplex. Expression of Interferon I stimulated genes (ISG) was determined by real-time PCR. Collagen-induced arthritis (CIA) was induced in male WT and CRAMP-/- mice and arthritis severity was visually scored and analysed histomorphometrically by OsteoMeasure software. Serum levels of anti-LL-37 were higher in SLE-patients compared to healthy donors or patients with RA. However, no correlation to markers of disease activity or organ involvement was observed. No significant differences of autoantibody or cytokine/chemokine levels, or of expression of ISGs were observed between WT and CRAMP-/- mice after pristane-injection. Furthermore, lung and kidney pathology did not differ in the absence of CRAMP. Incidence and severity of CIA and histological parameters (inflammation, cartilage degradation, and bone erosion) were not different in WT and CRAMP-/- mice. Although cathelicidins are upregulated in mouse models of lupus and arthritis, cathelicidin-deficiency did not persistently affect the diseases. Also in patients with SLE, autoantibodies against cathelicidins did not correlate with disease manifestation. Reactivity against cathelicidins in lupus and arthritis could thus be an epiphenomenon caused by extensive overexpression in blood and affected tissues. In addition, other cationic AMPs could functionally compensate for the deficiency of cathelicidins.
Project description:Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis. The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals.
Project description:Pulmonary infections are a major global cause of morbidity, exacerbated by an increasing threat from antibiotic-resistant pathogens. In this context, therapeutic interventions aimed at protectively modulating host responses, to enhance defence against infection, take on ever greater significance. Pseudomonas aeruginosa is an important multidrug-resistant, opportunistic respiratory pathogen, the clearance of which can be enhanced in vivo by the innate immune modulatory properties of antimicrobial host defence peptides from the cathelicidin family, including human LL-37. Initially described primarily as bactericidal agents, cathelicidins are now recognised as multifunctional antimicrobial immunomodulators, modifying host responses to pathogens, but the key mechanisms involved in these protective functions are not yet defined. We demonstrate that P. aeruginosa infection of airway epithelial cells promotes extensive infected cell internalisation of LL-37, in a manner that is dependent upon epithelial cell interaction with live bacteria, but does not require bacterial Type 3 Secretion System (T3SS). Internalised LL-37 acts as a second signal to induce inflammasome activation in airway epithelial cells, which, in contrast to myeloid cells, are relatively unresponsive to P. aeruginosa. We demonstrate that this is mechanistically dependent upon cathepsin B release, and NLRP3-dependent activation of caspase 1. These result in LL-37-mediated release of IL-1? and IL-18 in a manner that is synergistic with P. aeruginosa infection, and can induce caspase 1-dependent death of infected epithelial cells, and promote neutrophil chemotaxis. We propose that cathelicidin can therefore act as a second signal, required by P. aeruginosa infected epithelial cells to promote an inflammasome-mediated altruistic cell death of infection-compromised epithelial cells and act as a "fire alarm" to enhance rapid escalation of protective inflammatory responses to an uncontrolled infection. Understanding this novel modulatory role for cathelicidins, has the potential to inform development of novel therapeutic strategies to antibiotic-resistant pathogens, harnessing innate immunity as a complementation or alternative to current interventions.
Project description:The forest musk deer (Moschus berezovskii) is a small-sized artiodactyl species famous for the musk secreted by adult males. In the captive population, this species is under the threat of infection diseases, which greatly limits the increase of individual numbers. In the present study, we computationally analyzed the repertoire of the cathelicidin (CATHL) family from the genome of forest musk deer and investigated their expression pattern by real-time PCR. Our results showed that the entire genome of forest musk deer encodes eight cathelicidins, including six functional genes and two pseudogenes. Phylogenetic analyses further revealed that all forest musk deer cathelicidin members have emerged before the split of the forest musk deer and cattle and that forest musk deer CATHL3L2 and CATHL9 are orthologous with two cattle pseudogenes. In addition, the gene expression results showed that the six functional genes are not only abundantly expressed in the spleen and lung, but are also differently expressed in response to abscesses, which suggests that forest musk deer cathelicidins may be involved in infections. Taken together, identification and characterization of the forest musk deer cathelicidins provide fundamental data for further investigating their evolutionary process and biological functions.