International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 1(8), 82-84, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 82 Short Communication Desoxirrobonuclease Activity in Clinical Mycoplasma fermentans IsolateRivera Antonio1*, Cedillo Lilia , Hernandez Fernando and Giono SilviaCentro de Investigaciones en Ciencias Microbiológicas del Instituto de Ciencias de la Benemérita Universidad Autónoma de Puebla, MÉXICO Centro de Química del Instituto de Ciencias de la Benemérita Universidad Autónoma de Puebla, MÉXICO Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional, MÉXICO Available online at: www.isca.in Received 12th September 2012, revised 18th September 2012, accepted 21st September 2012Abstract Mycoplasmas are a heterogeneous group of the smallest organisms capable of self replication, some mycoplasmas cause respiratory or urogenital diseases in humans. However, mycoplasmas often chronically colonize our respiratory and urogenital tracts without apparent clinical significance. In this respect, wall free mycoplasmas are among the few prokaryotes that can grow silently in close interaction with mammalian cells for a long period of time. However, prolonged interactions with mycoplasmas of seemingly low virulence could, through a gradual and progressive course, significantly affect many biologic properties of mammalian cells. DNases are important constituents of mycoplasmal membranes and may be involved in the acquisition of host nucleic acids required for growth. The objective was detected DNase activity in clinical Mycoplasma fermentans isolate. Digestion products were analyzed by loading a Tris-borate agarose gel, absorbance and medium for DNase test. DNase activity was observed in strains tested and the levels of activity varied, Mycoplasma fermentans P140 showing greater activity. Results suggest DNase activities may be essential for growth and survival, this finding suggest that there is a mechanism of nucleotide acquisition. Key words: DNase, nucleotide acquisition, clinical sample, Mycoplasma fermentans. IntroductionMycoplasmas are a heterogeneous group of the smallest organisms capable of self-replication, some mycoplasmas cause respiratory or urogenital diseases in humans. However, mycoplasmas often chronically colonize our respiratory and urogenital tracts without apparent clinical significance. Wall-free mycoplasmas are among the few prokaryotes that can grow silently in close interaction with mammalian cells for a long period of time. However, prolonged interactions with mycoplasmas of seemingly low virulence could, through a gradual and progressive course, significantly affect may biologic properties of mammalian cells1-4. Mycoplasma fermentans commonly isolated from human genitourinary and respiratory tracts. It has been implicated in HIV pathogenesis, sexually transmitted genital tract infection, systemic infections, rheumatic disorders and chronic fatigue syndrome, notwithstanding the clinical significance, the roles it plays and the molecular mechanisms involved in various diseases have yet to be elucidated. Nuclease activity in mycoplasmas has been proposed as the mechanism by which these organisms acquire the precursors required for their nucleic acids. Therefore, mechanisms must be present to induce release, degradation, and transport of host nucleic acids to intracellular sites. Transports processes have been identified in mycoplasmas6,7, but DNA and RNA degradation processes have not received adequate attention. This study was undertaken to expand our knowledge about the desoxirribonuclease activity in clinical mycoplasmas isolate. Material and MethodsMycoplasma fermentans PG18 strain (ATCC 19989), and Mycoplasma fermentans P140 was isolated in our laboratory from the human respiratory tract. The organisms were grown in SP4 medium (mycoplasma broth base, tryptone, peptone, arginine, phenol red, DNA, supplemented with horse serum, CMRL 1066, yeast extract, yestolate, and glucose). The cultures were grown for 24-48 h at 37C. Growth was monitored by measuring the OD 640 of the culture and by recording pH changes. Mycoplasmas cultures were harvested at the mid exponential phase of growth by centrifugation at 12000 g for 20 min, washed once with 0.01 M sodium phosphate-0.14 M sodium chloride (pH 7.3) (PBS), and resuspended at a concentration of 100 µg of protein per ml in PBS containing 1% dextrose, 2 mM CaCl, and 2 mM MgCl (PBS-CM). Protein concentration was determined with Bio-Rad dye reagent, by using bovine serum albumin as a reference standard. To determine activity, 25 µl of suspended cells, representing 2.5 µg of protein, was diluted in twofold increments in a 96-well plate with PBS-CM. An equal volume lamda DNA (10µg/ml of PBS) was added to each well, and the plate was then incubating for 24 h at 37C. Lamda DNA International Research Journal of Biological Sciences ________________________________________________ Vol. 1(8), 82-84, December (2012) International Science Congress Association digestion was stopped by adding 7 µl of borate sample buffer ti each well. Digestion products were analyzed by loading a Tris borate agarose gel with 9 µl of sample per well, running the gel at 100 volts for 45 min, and then staining it with ethidium bromide. The end point was determined by analyzing the reaction mixtures on agarose gels and identifying the time at which the undigested, full- length lamda DNA first disappe Results and Discussion In the present study, we examined DNase activity has been proposed as a mechanism for nucleic acid precursors acquire free bases and/or oligonucleotides, so the capacity of some mycoplasma species cells suggests that invade th DNA serves as a substrate for the activity of nucleases and obtaining consistent precursors. DNase activity assays show that Mycoplasma fermentans reference strain DNA degrades by less than fermentans P140 clinical isolate. From 12 hours the Mycoplasma fermentans presents a more pronounced degradation gradient as they advance 24 hours as shown in figure- 1, increased DNase activity by the isolate (P140) is explained by the recent interaction with host, affects more virulent in some clinical samples. Figure-1 DNase activity products on agarose gel Tris- borate, lane 1 without digestion (control), lanes 2, 3, 4 and 5 with 6, 12, 18 and 24 h respectively in presence of Mycoplasma fermentans 7, 8 and 9 with 6, 12, 18 and 24 hours respectively in presence of Mycoplasmas fermentans P140. It is noted that at 24 hours (lane 9) Mycoplasma fermentans P140 DNase activity degrades in 95% DNA, and Mycoplasma fermentans PG18 with 45% degradation (lane 5) Absorbance readings confirm gradients observed in agarose gels of products from DNA degradation, DNA degradation due to the activity of DNase in Mycoplasma fermentans 45% and Mycoplasma fermentans P140 at 24 hours was 95% (figure-2). 1 2 3 4 5 6 7 8 International Research Journal of Biological Sciences ________________________________________________ Association digestion was stopped by adding 7 µl of borate sample buffer ti each well. Digestion products were analyzed by loading a Tris - well, running the gel at 100 volts for 45 min, and then staining it with ethidium bromide. The end point was determined by analyzing the reaction mixtures on agarose gels and identifying the time at length lamda DNA first disappe ared. In the present study, we examined DNase activity has been proposed as a mechanism for nucleic acid precursors acquire free bases and/or oligonucleotides, so the capacity of some mycoplasma species cells suggests that invade th e host cell DNA serves as a substrate for the activity of nucleases and Mycoplasma fermentans PG18 reference strain DNA degrades by less than Mycoplasma P140 clinical isolate. From 12 hours the presents a more pronounced degradation gradient as they advance 24 hours as shown in 1, increased DNase activity by the isolate (P140) is explained by the recent interaction with host, a situation that affects more virulent in some clinical samples. borate, lane 1 without digestion (control), lanes 2, 3, 4 and 5 with 6, 12, 18 and 24 h ours Mycoplasma fermentans PG18, lanes 6, 7, 8 and 9 with 6, 12, 18 and 24 hours respectively in presence of P140. It is noted that at 24 hours (lane 9) P140 DNase activity degrades in 95% PG18 with 45% degradation Absorbance readings confirm gradients observed in agarose gels of products from DNA degradation, DNA degradation due to Mycoplasma fermentans PG18 was P140 at 24 hours was 95% 200300 Absorbance Wavelength (nm) 200 300 0123456 Absorbance Wavelength (nm) Figure - Absorbance values of DNA degradation products by DNase activity, (A) Mycoplasma fermentans degradation at 24 hours and (B) P140 showed 95% DNA degradation In agar culture assay showed a clear zone on microbial growth, indicative of enzymatic digestion, still more evident in the samples from the clinical isolate of P140 (figure-3). These data suggest that the DNase activity is essential in the growth and survival of Mycoplasma fermentans the clinical isolate, representing nucleotide acquisition mecha nism, besides confirming that reported for pulmonis, Mycoplasma hyorhinis, Mycoplasma gallisepticum and Acholeplasma laidlawii that are not medical importance Isolation of Mycoplasma fermentans their involvement in the pathogenesis of human A B 1 2 3 4 5 6 7 8 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 83 400 Wavelength (nm) Control 6 h 12 h 18 h 24 h 300 400 Wavelength (nm) Control 6 h 12 h 18 h 24 h - 2 of DNA degradation products by DNase fermentans PG18 with 45% degradation at 24 hours and (B) Mycoplasma fermentans P140 showed 95% DNA degradation to 24 hours In agar culture assay showed a clear zone on microbial growth, indicative of enzymatic digestion, still more evident in the from the clinical isolate of Mycoplasma fermentans These data suggest that the DNase activity is essential in the Mycoplasma fermentans , principally in the clinical isolate, representing nucleotide acquisition nism, besides confirming that reported for Mycoplasma Mycoplasma hyorhinis, Mycoplasma gallisepticum that are not medical importance 9-12. Mycoplasma fermentans from different tissues and in the pathogenesis of human International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 82-84, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 84 immunodeficiency virus can give the clinical importance since their participation in different pathologies have not widely studied13,14, these data being a contribution in understanding some of the mechanisms that allow Mycoplasma fermentans be a successful parasite, besides representing a relevant data to carry out the etiology and in the determination of virulence of the bacterial strain isolated in the laboratory. (a) (b) Figure-3 DNase activity assay on agar showed a clear zone on microbial growth considered positive reaction, (A) Mycoplasma fermentans PG18 y (B) Mycoplasma fermentansP140 ConclusionData presented enable Mycoplasma fermentans to consider more as a microorganism with DNase activity, this helps us better understand and expand the understanding of the mechanisms by which this microorganism, and also to understand how survives in different human tissues which have been isolated and related etiologic agent. 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