Suresh Yenugu

Antimicrobial Activity of Human EPPIN, an Androgen-Regulated, Sperm-Bound Protein with a Whey Acidic Protein Motif

Abstract The role of epididymal sperm-binding proteins in reproductive tract immunity is now well recognized in addition to their role in sperm maturation. Spermatozoa acquire forward motility and fertilizing ability during their passage through the epididymis, where they acquire a wide variety of proteins belonging to different classes. Previously, we demonstrated that EPPIN (epididymal protease inhibitor), an androgen-regulated, sperm-binding protein containing protease-inhibitory motifs, is expressed specifically in the testis and epididymis. In the present study, we investigated the antibacterial activity of EPPIN against Escherichia coli and the mechanism of antimicrobial action. EPPIN exhibited dose- and time-dependent antibacterial activity that was relatively insensitive to salt. However, EPPIN lost its antibacterial activity completely on reduction and alkylation of its cysteines, indicating the importance of disulfide bonds for its activity. EPPIN permeabilized the outer and inner membranes of E. coli, which is consistent with its ability to induce striking morphological alterations of E. coli membranes as shown by scanning electron microscopy. EPPIN did not cause disruption of eukaryotic membranes in the rat erythrocyte hemolytic assay. The present results indicate that EPPIN has a role in the innate immune system of human epididymis.

Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence and their interaction with outer and cytoplasmic membranes of Escherichia coli.

During passage through the epididymis, sperm interact with secreted epididymal proteins that promote maturation, including the acquisition of motility and fertilization competence. Viewed previously as distinct from sperm maturation, host defence capabilities are now recognized functions of the human epididymis 2 (HE2) family of sperm-binding proteins. We analysed the potent dose and time-dependent bactericidal activity of recombinant HE2alpha, HE2beta1 and HE2beta2 and found that the full-length proteins (10 microg/ml or approximately 1 microM) caused more than a 50% decrease in Escherichia coli colony forming units within 15 min. By contrast, human beta-defensin-1, at a similar concentration, required more than 90 min to exhibit similar antibacterial activity. The epididymis-specific lipocalin, LCN6, failed to kill bacteria. Higher concentrations (25-100 microg/ml) of HE2 proteins and a longer duration of treatment resulted in near total inhibition of bacterial growth. The C-terminal peptides of HE2alpha, HEbeta1 and HEbeta2 proteins exhibited antibacterial activity similar to their full-length counterparts, indicating that the antibacterial activity of HE2 proteins resides in these C-terminal regions. Antibacterial activities of HE2 proteins and peptides were slightly inhibited by NaCl concentrations of up to 150 mM, while human beta-defensin-1 activity was nearly eliminated. Reduction and alkylation of disulphide bonds in HE2 proteins and their C-terminal peptides abolished their antibacterial activity. Consistent with the ability to kill bacteria, full-length HE2 proteins and C-terminal peptides caused rapid dose-dependent permeabilization of outer and cytoplasmic E. coli membranes. A much longer exposure time was required for human beta-defensin-1-mediated permeabilization of membranes, suggesting a possible difference in mode of action compared with the HE2 antibacterial peptides.

Antimicrobial actions of the human epididymis 2 (HE2) protein isoforms, HE2alpha, HE2beta1 and HE2beta2

BackgroundThe HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis.MethodsE. coli treated with 50 micro g/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 micro g/ml of the individual peptides for 0–60 min and measuring the incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N)]leucine into DNA, RNA and protein. Statistical analyses using Students t-test were performed using Sigma Plot software. Values shown are Mean ± S.D.ResultsE. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis.ConclusionsThe morphological changes observed in E. coli treated with epididymal HE2 peptides provide further evidence for their membrane dependent mechanism of antibacterial action. HE2 C-terminal peptides can inhibit E. coli macromolecular synthesis, suggesting an additional mechanism of bacterial killing supplementary to membrane permeabilization.

Identification, characterization, and evolution of a primate β-defensin gene cluster

Defensins are members of a large diverse family of cationic antimicrobial peptides that share a signature pattern consisting of six conserved cysteine residues. Defensins have a wide variety of functions and their disruption has been implicated in various human diseases. Here we report the characterization of DEFB119–DEFB123, five genes in the human β-defensin cluster locus on chromosome 20q11.1. The genomic structures of DEFB121 and DEFB122 were determined in silico. Sequences of the five macaque orthologs were obtained and expression patterns of the genes were analyzed in humans and macaque by semiquantitative reverse transcription polymerase chain reaction. Expression was restricted to the male reproductive tract. The genes in this cluster are differentially regulated by androgens. Evolutionary analyses suggest that this cluster originated by a series of duplication events and by positive selection. The evolutionary forces driving the proliferation and diversification of these defensins may be related to reproductive specialization and/or the host–parasite coevolutionary process.

Differential Expression and Antibacterial Activity of Epididymis Protein 2 Isoforms in the Male Reproductive Tract of Human and Rhesus Monkey (Macaca mulatta)

Abstract The epididymis protein 2 (EP2) gene, the fusion of two ancestral β-defensin genes, is highly expressed in the epididymis and subject to species-specific regulation at the levels of promoter selection, transcription, and mRNA splicing. EP2 mRNA expression is also androgen dependent, and at least two of the secreted proteins bind spermatozoa. Alternative splicing produces more than 17 different EP2 mRNA variants. In this article, the expression of EP2 variants was profiled in different tissues from the human and rhesus monkey (Macaca mulatta) male reproductive tract using reverse transcriptase-polymerase chain reaction. Different EP2 mRNA variants were identified not only in human and rhesus testis and epididymis but also in the novel sites, seminal vesicle and prostate. Immunolocalization of EP2 protein in epithelial cells from rhesus and human seminal vesicle demonstrated that EP2 transcripts are translated in these tissues. In addition, two novel splicing variants, named EP2R and EP2S, were discovered. EP2C was the only splice variant expressed in all tissues tested from rhesus monkey. However, expression was not detected in human testis or seminal vesicle. For the first time, bactericidal function was demonstrated for EP2C, EP2K, and EP2L. Taken together, the results indicate that EP2 expression is more widespread in the male reproductive tract than realized previously. Whereas the activity of every EP2 variant tested thus far is antibacterial, further investigation may reveal additional physiological roles for EP2 peptides in the primate male reproductive tract.

LCN6, a novel human epididymal lipocalin

BackgroundThe lipocalin (LCN) family of structurally conserved hydrophobic ligand binding proteins is represented in all major taxonomic groups from prokaryotes to primates. The importance of lipocalins in reproduction and the similarity to known epididymal lipocalins prompted us to characterize the novel human epididymal LCN6.Methods and ResultsLCN6 cDNA was identified by database analysis in a comprehensive human library sequencing program. Macaca mulatta (rhesus monkey) cDNA was obtained from an epididymis cDNA library and is 93% homologous to the human. The gene is located on chromosome 9q34 adjacent LCN8 and LCN5. LCN6 amino acid sequence is most closely related to LCN5, but the LCN6 beta-barrel structure is best modeled on mouse major urinary protein 1, a pheromone binding protein. Northern blot analysis of RNAs isolated from 25 human tissues revealed predominant expression of a 1.0 kb mRNA in the epididymis. No other transcript was detected except for weak expression of a larger hybridizing mRNA in urinary bladder. Northern hybridization analysis of LCN6 mRNA expression in sham-operated, castrated and testosterone replaced rhesus monkeys suggests mRNA levels are little affected 6 days after castration. Immunohistochemical staining revealed that LCN6 protein is abundant in the caput epithelium and lumen. Immunofluorescent staining of human spermatozoa shows LCN6 located on the head and tail of spermatozoa with the highest concentration of LCN6 on the post-acrosomal region of the head, where it appeared aggregated into large patches.ConclusionsLCN6 is a novel lipocalin closely related to Lcn5 and Lcn8 and these three genes are likely products of gene duplication events that predate rodent-primate divergence. Predominant expression in the epididymis and location on sperm surface are consistent with a role for LCN6 in male fertility.

Identification, cloning and functional characterization of novel beta-defensins in the rat (Rattus norvegicus)

Backgroundbeta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation.MethodsIn silico rat genomic analyses were used to identify novel beta-defensins related to human defensins 118–123. RNAs isolated from male reproductive tract tissues of rat were reverse transcribed and PCR amplified using gene specific primers for defensins. PCR products were sequenced to confirm their identity. RT-PCR analysis was performed to analyze the tissue distribution, developmental expression and androgen regulation of these defensins. Recombinant defensins were tested against E. coli in a colony forming unit assay to analyze their antimicrobial activities.ResultsNovel beta-defensins, Defb21, Defb24, Defb27, Defb30 and Defb36 were identified in the rat male reproductive tract. Defb30 and Defb36 were the most restricted in expression, whereas the others were expressed in a variety of tissues including the female reproductive tract. Early onset of defensin expression was observed in the epididymides of 10–60 day old rats. Defb21-Defb36 expression in castrated rats was down regulated and maintained at normal levels in testosterone supplemented animals. DEFB24 and DEFB30 proteins showed potent dose and time dependent antibacterial activity.ConclusionRat Defb21, Defb24, Defb27, Defb30 and Defb36 are abundantly expressed in the male reproductive tract where they most likely protect against microbial invasion. They are developmentally regulated and androgen is required for full expression in the adult epididymis.

Antimicrobial Actions of Human and Macaque Sperm Associated Antigen (SPAG) 11 Isoforms: influence of the N-terminal peptide

In addition to their role in sperm maturation, recent evidence has indicated that epididymal proteins have a role in male reproductive tract innate immunity. Herein we demonstrate that human and macaque epididymal protein isoforms in the SPAG (sperm associated antigen) 11 family, full length SPAG11C, K and L exhibit potent antibacterial activity against E. coli. Analysis of activities of the N- and C-terminal domains revealed that the human N-terminal peptide is bactericidal, while the C-terminal domains that contain the defensin-like 6 cysteine array in SPAG11C and partial arrays in SPAG11K and SPAG11L, lack antibacterial activity. The N-terminal peptide does not appear to contain all the determinants of activity since full-length human SPAG11C is more active than the isolated N-terminal peptide and since sulfhydryl reduction and alkylation, which would affect primarily the C-terminal peptides, completely abolished activities of the whole proteins. These results suggest that the structure conferred by the disulfide bonds in human SPAG11C contributes to the antibacterial activity of the whole molecule. The activities of the N-terminal peptide and of full length human SPAG11C were somewhat reduced in increasing NaCl concentrations. In contrast, the antibacterial activities of full length macaque SPAG11C, K and L were unaffected by the presence of NaCl suggesting a mechanism in the macaque that is less dependent upon electrostatic interactions. SPAG11C, K and L disrupted E. coli membranes but had no effect on erythrocyte membranes. Inhibition of E. coli RNA, DNA and protein synthesis by nonlethal concentrations of SPAG11 isoforms indicated an additional mechanism of bacterial killing.

Identification, cloning and functional characterization of novel sperm associated antigen 11 (SPAG11) isoforms in the rat

BackgroundSperm binding proteins and their C-terminal peptides of the Sperm Associated Antigen 11 (SPAG11) family were found to play an important role in epididymal innate immunity in addition to their role in sperm maturation. However, the expression of Spag11 transcripts in rodents is not well documented.MethodsComputational analysis was employed to identify novel Spag11 isoforms in the rat. RT-PCR analyses were carried out on RNAs isolated from the male reproductive tract tissues of rat using gene specific primers for Spag11c and Spag11t. The identities of PCR products were confirmed by sequencing. Tissue distribution, developmental expression and androgen regulation of Spag11t and Spag11c were studied using RT-PCR. The antimicrobial activities of recombinant Spag11t and Spag11c were tested against E coli in a colony forming unit assay.ResultsIn this study, we identified two novel Spag11 transcripts, namely, Spag11t and Spag11c derived from the long arm of chromosome 16 in the rat (Rattus norvegicus), using both in silico and molecular biology approaches. Spag11c is expressed in all three regions of the epididymis, in testis and in ovary but is absent from the seminal vesicle. Spag11t expression is confined to the caput and it is not expressed in the testis, seminal vesicle or ovary. Age dependent expression of Spag11t and Spag11c was observed in the epididymides of rats (10–60 day old). Their expression was found to be most abundant in the adult rat (60 day) suggesting roles in mature reproductive function. Further, both Spag11t and Spag11c expression was down regulated in castrated rat epididymides and the expression was maintained in the testosterone replaced castrated rats. SPAG11C is a potent antibacterial agent. SPAG11T also displayed bactericidal capacity although weaker than SPAG11C and SPAG11E.ConclusionThe abundant expression of Spag11t and Spag11c in the male reproductive tract suggests an important role in male reproductive tract immunity. Their expression is developmentally regulated and androgen dependent. Characterization of novel SPAG11 isoforms will contribute to our understanding of the role of epididymal proteins in sperm maturation and innate immunity.

Regulation and functional impact of lipopolysaccharide induced Nod2 gene expression in the murine epididymal epithelial cell line PC1.

Epididymitis represents a serious threat to male fertility and usually develops following secondary bacterial infection of the epididymis such as urinary tract infections or sexually transmitted diseases. Surprisingly, very little is known about the innate host response triggered by bacterial infection in the male reproductive tract. In this study we investigated the regulation and function of Nod2 in epididymal epithelial cells following lipopolysaccharide (LPS) stimulation. The immortalized epididymal epithelial cell line PC1 (proximal caput 1) constitutively expressed Toll‐like receptor 4, MD‐2, CD‐14 but not Nod2 messenger RNA. Lipopolysaccharide (LPS; 0·5 μg/ml) rapidly induced IκB phosphorylation and degradation, RelA nuclear translocation and phosphorylation, which correlated with enhanced transcriptional activity (four‐fold) in PC1 cells. The LPS and lipid A rapidly (1 hr) induced Nod2 messenger RNA accumulation in a dose‐dependent manner. RelA and RNApolII recruitment to the Nod2 gene promoter was enhanced in LPS‐stimulated cells. Molecular blockade of nuclear factor‐κB signalling with adenovirus 5 (Ad5) IκBAA or adenovirus 5 double‐negative (Ad5dn) IKKβ prevented LPS‐induced Nod2 gene expression. Functionally, Nod2 upregulation enhanced muramyl dipeptide (MDP) ‐induced tumour necrosis factor messenger RNA accumulation in PC1 cells. We conclude that epididymal epithelial cells mount an innate response following LPS exposure which leads to upregulation of Nod2 and enhanced responsiveness to the microbial product MDP. The rapid Nod2 upregulation in epididymal epithelial cells is probably part of a complex innate host response aimed at protecting the male reproductive tract from the deleterious impact of bacteria.