Molecular Cloning and Characterization of Three β-Defensins from Canine Testes

Abstract

Mammalian ␤-defensins are small cationic peptides possessing broad antimicrobial and physiological activities. Because dogs are particularly resilient to sexually transmitted diseases, it has been proposed that their antimicrobial peptide repertoire might provide insight into novel antimicrobial therapeutics and treatment regimens. To investigate this proposal, we cloned the full-length cDNA of three canine ␤-defensin isoforms (cBD-1, -2, and -3) from canine testicular tissues. Their predicted peptides share identical N-terminal 65-amino-acid residues, including the ␤-defensin consensus six-cysteine motif. The two longer isoforms, cBD-2 and -3, possess 4 and 34 additional amino acids, respectively, at the C terminus. To evaluate the antimicrobial activity of cBD, a 34-amino-acid peptide derived from the shared mature peptide region was synthesized. Canine ␤-defensin displayed broad antimicrobial activity against gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus; MICs of 6 and 100 g/ml, respectively), gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, and Neisseria gonorrhoeae; MICs of 20 to 50, 20, and 50 g/ml, respectively), and yeast (Candida albicans; MIC of 5 to 50 g/ml) and lower activity against Ureaplasma urealyticum and U. canigenitalium (MIC of 200 g/ml). Antimicrobial potency was significantly reduced at salt concentrations higher than 140 mM. All three canine ␤-defensins were highly expressed in testis. In situ hybridization indicated that cBD-1 was expressed primarily in Sertoli cells within the seminiferous tubules. In contrast, cBD-2 was located primarily within Leydig cells. The longest isoform, cBD-3, was detected in Sertoli cells and to a lesser extent in the interstitium. The tissue-specific expression and broad antimicrobial activity suggest that canine ␤-defensins play an important role in host defense and other physiological functions of the male reproductive system. The mucosal physical barrier is an important component protecting luminal surfaces from bacteria. Discovery of epithelium-derived antimicrobial peptides with activity at mucosal surfaces extends luminal protection from the traditional barrier effect to innate immune capabilities. These endogenous peptides are natural antibiotics that are widely distributed in nature and represent an important mechanism of innate defenses against microbial infection (1, 5). Among these naturally occurring antimicrobial peptides, defensins form a unique family of cysteine-rich, small cationic peptides (17).The defensin family is large and has been documented in mammals, plants, insects, and mollusks (6,27,29,39,47). In mammals, defensins are subdivided into ␣-, ␤-, and -defensins based on the organization of conserved six-cysteine motifs (43,50). With the exception of bovine neutrophils, ␤-defensins are synthesized primarily in epithelial tissues (18,27). Because epithelia of the male reproductive tract are essentially isolated from adaptive immune capabilities and because the luminal contents of the testis and...

Sertoli Cells Initiate Testicular Innate Immune Responses through TLR Activation

“…Obviously, the protection of germ cells from infections in the seminiferous tubules is of primary importance, because bacterial infections can seriously damage reproductive functions (2). A source of testicular infection is represented by some bacterial species, which are able to induce sexually transmitted diseases: Neisseria gonorrheae, Candida albicans, and Ureaplasma urealyticum (3). Several microbial pathogens are able to invade and colonize reproductive tract and semen.…”

“…The existence of multiple ␤-defensin isoforms in the male reproductive tract of humans, rats, mice and dogs has long been demonstrated (3, 9 -12). The canine ␤-defensin, expressed by Sertoli cells, could be the primary endogenous antibiotic line of defense against bacterial invasion in testis and other epithelial surfaces (i.e., lung and small intestine) (3).…”

Differences in the mode of phagocytosis of bacteria between macrophages and testicular Sertoli cells

“…We previously showed the existence of immunity in the seminiferous epithelium against invading bacteria (7). Sertoli cells possess some of the characteristics of immune cells: they engulf apoptotic spermatogenic cells (8), produce antimicrobial proteins (9)(10)(11)(12)(13), and express pattern recognition receptors such as Toll-like receptors (14)(15)(16)(17). The phagocytosis of apoptotic spermatogenic cells by Sertoli cells is achieved through the specifi c binding of phosphatidylserine exposed on the surface of target cells to class B scavenger receptor type I (SR-BI) of Sertoli cells (18)(19)(20).…”

Innate Immune Defense System of the Skin

“…4 Human BDs have a homology of about 51-71% with the first three cBDs. 67 Based on sequence comparisons, it appears that cBD1 is an orthologue of hBD1, cBD103 of hBD3, and that cBD102 and hBD2 have no equivalent orthologues. 65 The gene encoding cBD103 demonstrates two polymorphisms: a common 3 bp deletion allele and a gene copy-number variation.…”