Our understanding of the cellular and molecular mechanisms that comprise innate defense mechanisms of the mammalian male reproductive tract is rudimentary. In the present study we describe for the first time the identification and characterization of three novel β-defensins in the canine testis.
Typically, β-defensin genes are comprised of two exons and an intermediate intron. The first exon encodes the prepro-region, and the second exon encodes the mature peptide (43
). The pre-proproteins are processed into the mature peptides, which are generally composed of 36 to 47 amino acids. However, two groups of β-defensins do not fit this pattern. One group is comprised of the three alternate splicing variants of the human HE2/EP2 gene, EP2C, EP2D, and EP2E, which all share the conserved cysteine motif of β-defensins. Their peptide precursors are composed of 113, 133, and 80 amino acid residues, respectively (9
). The second group resides among the five newly identified human β-defensin genes (DEFB25
) clustered on chromosome 20p13; the longest, DEFB-29, is 183 amino acids, and DEFB-25 to −28 are at 156, 111, 99, and 93 amino acids, respectively (38
). The additional length of these β-defensin isoforms is generated mostly through extensions in the carboxy terminus following the consensus cysteine motifs. The three cBDs reported here exhibit similar C-terminal extensions. In comparison to other β-defensins, cBD-1
are within the average length of most; however, cBD-3 is longer due to its 34 additional amino acids at the C terminus. The function of these additional C-terminal amino acids is unknown. One possibility is that they serve as a regulatory domain to promote the attachment of the antimicrobial peptide to the membrane of bacteria. Alternatively, the two longer isoforms, i.e., cBD-2
, might be testis-specific storage antimicrobial peptides. We theorize that they may act as endogenous antimicrobial molecules by providing immediate access to a gene product that could be rapidly processed into a shorter active isoform, i.e., cBD-1, when it is required. In this case, the C-terminal extension may, in fact, protect the stored peptides from degradation or unnecessary antimicrobial activity.
The structural analysis revealed that the global folding of cBD assumes all of the common features of the β-defensin family, such as three disulfide bonds, three antiparallel β-sheets and an α-helix usually located near the N-terminal of the peptide (Fig. ). cBD also displayed another essential feature common in all β-defensins which was the high concentration of cationic residues (Fig. ). The number of positively charged residues (Arg, Lys, and His) reported in the mature peptides of other mammalian β-defensins (range, 6 to 14; average, 9) (43
) was in good agreement with the number of cationic amino acid residues (6 Lys, 2 Arg; n
= 8) found in the cBD sequence (Fig. and ). It has been proposed that this high cationic charge density enables β-defensins to bind and insert into the cellular membrane, leading to the killing of the microorganism by forming multiple membrane pores (55
). Our findings on the surface analysis of the cBD (Fig. ) established three well-defined cationic areas that may allow potent electrostatic interactions with bacterial membranes, furnishing cBD with the broad antimicrobial spectrum demonstrated in the in vitro assays.
Bacteria used in the present study were selected based on their ability to produce urinary tract infections (uropathogenic E. coli
and K. pneumonia
) and sexually transmitted diseases (N. gonorrhoeae
, C. albicans
, U. urealyticum
, and U. canigenitalium
). The antimicrobial effect of cBD was also evaluated against L. monocytogenes
, S. aureus
, and E. coli
according to previous reports that have used these bacteria in assays of antibacterial activity for novel antimicrobial peptides (25
). cBD effectively killed gram-positive and -negative bacteria, yeast, and Ureaplasma
spp. The antimicrobial activity of cBD was comparable to that reported for other synthetic or natural β-defensin peptides, such as mBD-12, hBD-1, and hBD-3 (22
). More importantly, synthetic cBD was more effective in killing C. albicans
isolated from vaginal epithelium than one isolated from hematogenous origin, strongly suggesting that cBD plays an essential role as an epithelium-derived antibiotic that functions as an immediate line of defense against genital pathogens. The MIC for cBD for C. albicans
is in agreement with previous reports (7
). Similar to other β-defensins (17
), the bactericidal activity of cBD is dependent on the salt concentration used in the in vitro assay (52
). Furthermore, our data suggest that the C termini of cBD-2 and cBD-3 may not be necessary for antimicrobial activity. Future studies will be required to determine differences in antimicrobial activities of the cBD isoforms and to investigate their individual role in relation to urogenital infections.
The biological activity of β-defensins is not limited to direct killing of microorganisms (1
). These epithelium-derived molecules also participate actively in other aspects of adaptive immunity, including chemotactic effects on immature dendritic cells and memory T cells exerted through the CCR6 receptor pathway (53
). Despite the lack of information about the RNA expression pattern of cBDs in other epithelial surfaces of the canine urogenital tract, the results from the present study allow us to propose a speculative scenario. Considering that the expression of the shortest isoform, cBD-1, was not restricted to the testis, it is likely that this β-defensin may work in conjunction with other epithelial components of the mucosal barrier of the collecting ducts, ureters, bladder, and urethra to counter bacterial invasion of the urogenital tract. Conversely, the two longer cBD isoforms (cBD-2 and cBD-3) may diffuse to the site of infection, actively orchestrating chemotactic signals that direct the migration of circulating cells needed to initiate an adaptive immune response at the site of inflammation. The fact that uropathogenic E. coli
and K. pneumoniae
(the two bacterial strains that account for up to 90% of the urinary tract infections in humans and dogs) were sensitive to cBD supports the hypothesis that this epithelium-derived defensin may be also expressed in urinary tract epithelia.
The existence of multiple β-defensin isoforms in the male reproductive tracts of humans, rats, and mice (10
) suggests that these antimicrobial cationic peptides might specifically and cooperatively contribute to protect the reproductive system against pathogenic microbes. The presence of three different testis-specific cBDs isoforms suggests synergistic peptide function might also occur in the male dog. cBD-1, which displayed a more ubiquitous tissue expression pattern, could be the primary endogenous antibiotic line of defense against bacterial invasion in testis and other epithelial surfaces (i.e., lung, and small intestine). The two testis-specific β-defensins (cBD-2 and cBD-3) could possibly interact with cBD
, providing a synergistic antimicrobial effect, thus rendering enhanced local protection to the canine reproductive system.
It is worth noting that only a few bacterial sexually transmitted diseases (Mycoplasma
, and Brucella
spp.) occur with any frequency in male dogs. Moreover, a direct relationship between these pathogens and clinical signs has not been fully documented and remains controversial (8
). Our results clearly showed that cBD possesses broad antibacterial effects against the sexually transmitted disease pathogens used in the present study. cBD also effectively killed two strains of C. albicans
; however, Ureaplasma
spp. appeared to be resistant to cBD. This fact may be explained by the cell membrane composition of Ureaplasma
spp. are among the few bacteria that possess a higher concentration of cholesterol within their cell membranes, protecting them from the antibacterial activity of defensins (21
). It has been suggested that eukaryotic cells are more resistant to the pore-forming effects of defensins due to the presence of cholesterol in the cellular membrane (56
). The lack of cBD killing effect may explain why Ureaplasma
is commonly isolated from the canine genital tract, where it is present as normal microflora (21
Previous studies have reported that N. gonorrhoeae
is very sensitive to protregrins (antimicrobial peptides derived from porcine neutrophils) but resistant to several human α-defensins (45
). Our results conclusively showed that N. gonorrhoeae
is killed by cBD. This inconsistency could reflect species-specific differences or more likely the site of defensin production; cBD is of epithelial origin (the first site of contact for a sexually transmitted disease pathogen), whereas most α-defensins are produced by circulating blood cells, which most likely are not the primary contact tissue for genital pathogens.
Several microbial pathogens are able to invade and colonize reproductive tract tissues and semen. If successful, they may cause serious clinical consequences to the reproductive status of the individual. Therefore, innate immunity should play a crucial role against bacterial invasion of the urethra, epididymis, and testes to protect and preserve the spermatozoa. Anatomically, the epididymis is the continuation of the urethra, exposing it to a permanent risk for ascending microbial infections. Our in situ hybridization results clearly showed that cBD-1
are expressed in Sertoli cells (supporting cells for the developing spermatozoa), suggesting that these epithelium-derived peptides may actively participate as local antimicrobial molecules keeping the environment sterile for adequate sperm development and maturation. Moreover, these results are in agreement with previous reports showing the expression of β-defensins in the epididymis and seminiferous tubules, chiefly Sertoli cells, of the reproductive tract of rats (10
In summary, we have cloned the full-length cDNA of three canine β-defensins—cBD-1, cBD-2, and cBD-3—from testicular tissues. Canine β-defensin has broad antimicrobial activity, particularly against pathogens of the urogenital tract. The expression pattern of cBDs in Sertoli and Leydig cells of the testis suggests that they play a role in host defense of the male reproductive tract. In light of the pattern of expression and activity of canine β-defensins, further studies are warranted to investigate the role of β-defensins in host defense and the physiological function of the male reproductive system.