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Impairment by sperm agglutinating factor isolated from Escherichia coli: receptor specific interactions.

ABSTRACT: In an earlier work done in our laboratory, we have been able to isolate a sperm agglutinating strain of Escherichia coli from the semen sample of a male attending infertility clinic. Further, factor responsible for sperm agglutination (SAF) was isolated and purified, and, using SAF as a tool, corresponding SAF binding receptor from human spermatozoa has been purified. Characterization of SAF and SAF binding receptor using MALDI-TOF showed homology to glutamate decarboxylase and MHC class I molecule, respectively. Coincubation of SAF with spermatozoa not only resulted in spermagglutination but could also compromise other sperm parameters, namely, Mg(2+) dependent ATPase activity and apoptosis. Intravaginal administration of SAF could lead to infertility in Balb/c mice. SAF induced impairment of sperm parameters, and infertility was observed to be due to interaction of SAF with sperm surface receptor component as, when purified receptor was introduced, receptor completely inhibited all the detrimental effects induced by SAF. From these results, it could be concluded that interaction of SAF with spermatozoa is receptor mediated.

SUBMITTER: Kaur K

PROVIDER: S-EPMC3730385 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Impairment by sperm agglutinating factor isolated from Escherichia coli: receptor specific interactions.

Kaur Kiranjeet K Prabha Vijay V

BioMed research international 20130717

In an earlier work done in our laboratory, we have been able to isolate a sperm agglutinating strain of Escherichia coli from the semen sample of a male attending infertility clinic. Further, factor responsible for sperm agglutination (SAF) was isolated and purified, and, using SAF as a tool, corresponding SAF binding receptor from human spermatozoa has been purified. Characterization of SAF and SAF binding receptor using MALDI-TOF showed homology to glutamate decarboxylase and MHC class I molec ...[more]

PMID: 23956989

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Project description:BACKGROUND:Voluntary control of fertility is of paramount importance to the modern society. But since the contraceptive methods available for women have their limitations such as urinary tract infections, allergies, cervical erosion and discomfort, a desperate need exists to develop safe methods. Vaginal contraceptives may be the answer to this problem, as these are the oldest ways of fertility regulation, practiced over the centuries. With minimal systemic involvement, these are also the safest. Natural substances blocking or impairing the sperm motility offer as valuable non-cytotoxic vaginal contraceptives. Antimicrobial peptides (AMPs) isolated from plants, animals and microorganisms are known to possess sperm immobilizing and spermicidal properties. Following this, in the quest for alternative means, we have cloned, over expressed and purified the recombinant sperm agglutinating factor (SAF) from Staphylococcus warneri, isolated from the cervix of a woman with unexplained infertility. METHODS:Genomic library of Staphylococcus warneri was generated in Escherichia coli using pSMART vector and screened for sperm agglutinating factor (SAF). The insert in sperm agglutinating transformant was sequenced and was found to express ribonucleotide-diphosphate reductase-? sub unit. The ORF was sub-cloned in pET28a vector, expressed and purified. The effect of rSAF on motility, viability, morphology, Mg++-dependent ATPase activity and acrosome status of human sperms was analyzed in vitro and contraceptive efficacy was evaluated in vivo in female BALB/c mice. RESULTS:The 80?kDa rSAF showed complete sperm agglutination, inhibited its Mg2+-ATPase activity, caused premature sperm acrosomal loss in vitro and mimicked the pattern in vivo showing 100% contraception in BALB/c mice resulting in prevention of pregnancy. The FITC labeled SAF was found to bind the entire surface of spermatozoa. Vaginal application and oral administration of rSAF to mice for 14 successive days did not demonstrate any significant change in vaginal cell morphology, organ weight and tissue histology of reproductive and non-reproductive organs and had no negative impact in the dermal and penile irritation tests. CONCLUSION:The Sperm Agglutinating Factor from Staphylococcus warneri, natural microflora of human cervix, showed extensive potential to be employed as a safe vaginal contraceptive.

Project description:BackgroundTo control the overpopulation and unintended pregnancies, vaginal contraceptives have gained recent surge of interest because of its topical application with possible avoidance of systemic effects. However non-specific cytotoxicity associated with detergent-based synthetic vaginal contraceptive agents limits their use and generates considerable interest in the development of vaginal contraceptives of biological origin for controlling reproduction and ultimately growing population. In this study, we have cloned, over-expressed an Escherichia coli gene encoding a sperm immobilizing factor (SIF) that inhibits sperm motility for the development of vaginal contraceptive from a biological source i.e. E. coli. The contraceptive efficacy of the Escherichia coli recombinant sperm immobilizing factor (r-SIF) was also determined.MethodsGenomic DNA library of an E. coli strain isolated from semen sample of an infertile male was constructed for the identification and cloning of E. coli SIF coding gene. This gene was sub-cloned in pBADmycHisB for over-expression and the r-SIF was purified using Ni-NTA affinity chromatography. Effect of r-SIF on mouse sperm motility, viability and on morphology was evaluated. Binding of r-SIF to mouse sperm was demonstrated by fluorescent labeling. Contraceptive efficacy of r-SIF was checked in murine model.ResultsGenomic library resulted in five hundred transformants; five clones were found positive for sperm immobilizing activity. The protein product of the insert DNA sequence in one of the transformants showed maximum sperm immobilizing activity. Sequence analysis of ORFs in the insert revealed homology to recX on both nucleotide and protein level. 40 μg of the purified r-SIF showed immediate spermicidal activity in vitro for mouse sperm. Scanning electron micrograph of the r-SIF treated sperm showed intense morphological damage to sperm. FITC labeled r-SIF showed highest fluorescence at the head region of the sperm. 5 μg of purified r-SIF exhibited a complete contraceptive effect in mouse model.Conclusionr-SIF could be seen as potential target to be developed as potent and safe vaginal contraceptive in future.

Project description:BackgroundThe leading cause of surface water impairment in United States' rivers and streams is pathogen contamination. Although use of fecal indicators has reduced human health risk, current approaches to identify and reduce exposure can be improved. One important knowledge gap within exposure assessment is characterization of complex fate and transport processes of fecal pollution. Novel modeling processes can inform watershed decision-making to improve exposure assessment.MethodsWe used the ecological model, Maxent, and the fecal indicator bacterium Escherichia coli to identify environmental factors associated with surface water impairment. Samples were collected August, November, February, and May for 8 years on Sinking Creek in Northeast Tennessee and analyzed for 10 water quality parameters and E. coli concentrations. Univariate and multivariate models estimated probability of impairment given the water quality parameters. Model performance was assessed using area under the receiving operating characteristic (AUC) and prediction accuracy, defined as the model's ability to predict both true positives (impairment) and true negatives (compliance). Univariate models generated action values, or environmental thresholds, to indicate potential E. coli impairment based on a single parameter. Multivariate models predicted probability of impairment given a suite of environmental variables, and jack-knife sensitivity analysis removed unresponsive variables to elicit a set of the most responsive parameters.ResultsWater temperature univariate models performed best as indicated by AUC, but alkalinity models were the most accurate at correctly classifying impairment. Sensitivity analysis revealed that models were most sensitive to removal of specific conductance. Other sensitive variables included water temperature, dissolved oxygen, discharge, and NO3. The removal of dissolved oxygen improved model performance based on testing AUC, justifying development of two optimized multivariate models; a 5-variable model including all sensitive parameters, and a 4-variable model that excluded dissolved oxygen.DiscussionResults suggest that E. coli impairment in Sinking Creek is influenced by seasonality and agricultural run-off, stressing the need for multi-month sampling along a stream continuum. Although discharge was not predictive of E. coli impairment alone, its interactive effect stresses the importance of both flow dependent and independent processes associated with E. coli impairment. This research also highlights the interactions between nutrient and fecal pollution, a key consideration for watersheds with multiple synergistic impairments. Although one indicator cannot mimic theplethora of existing pathogens in water, incorporating modeling can fine tune an indicator's utility, providing information concerning fate, transport, and source of fecal pollution while prioritizing resources and increasing confidence in decision making.

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Impairment by sperm agglutinating factor isolated from Escherichia coli: receptor specific interactions.

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Impairment by sperm agglutinating factor isolated from Escherichia coli: receptor specific interactions.

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