文献 A rapid chromogenic microtitre assay of arginine aminopeptidase activity in Mycoplasma strains.

Department of Life Sciences, Kings College London, Franklin Wilkins Building, 150 Stamford Street, London SE1 9NH, UKARTICLE IN PRESSSystematic and Applied Microbiology 29 (2006) 589592www.elsevier.de/syapmSHORT COMMUNICATIONA rapid chromogenic microtitre assay of arginine aminopeptidase activityin Mycoplasma strainsYi-Ching Lina, Roger J. Milesa, , Robin A.J. Nicholasb, Ann P. Woodc,abMycoplasma Group, Veterinary Laboratories Agency (Weybridge), Addlestone, Surrey KT15 3NB, UKcDepartment of Microbiology, Kings College London Dental Institute at Guys, Kings College and St Thomas Hospitals,Floor 28 Guys Tower, Guys Campus, London SE1 9RT, UKReceived 19 December 2005AbstractArginine-utilizing strains of Mycoplasma can be screened by assay of their arginine aminopeptidase activity. Astandardized chromogenic method is described that enables enzyme detection in small volumes of cell suspension inless than 3 h. Cell suspensions (10 ml) in 96-well microtitre plates are incubated at 37 1C, pH 8.0, with 0.1 mM arginyl-b-naphthylamide (100 ml). This is hydrolysed to release b-naphthylamine, which gives a coloured product ondiazotization with fast garnet. M. alkalescens can be detected in this way with as few as 1.1 105 viable cells andM. fermentans with 2.3 106 cells. The method has been shown to enable division of 28 strains into three groups offermentative and arginine-hydrolysing mycoplasmas. This procedure has potential for routine laboratory use.r 2006 Elsevier GmbH. All rights reserved.Keywords: Mycoplasma fermentans; M. alkalescens; Arginine aminopeptidase; Chromogenic assay; Fast garnetMycoplasma is the largest genus of the class Molli-cutes, comprising over half of the approximately 200known species 4,11,12. They are characterized by theirsmall size (300800 nm diameter), low G+C content(2340 mol%), lack of a cell wall, and have smallgenomes (5801350 kb), which have apparently lostmost of the genes required for a functional tricarboxylicacid cycle, cytochrome-mediated electron transport, andbiosynthesis of amino acids, cofactors, fatty acids,purines and pyrimidines13,15. Consequently, Myco-plasma species are obligately parasitic and obtainessential metabolites from their hosts. The majority ofCorresponding author. Tel.: +44 20 7848 4344;fax: +44 20 7848 4500.E-mail address: ann.p.woodkcl.ac.uk (A.P. Wood). Deceased.0723-2020/$ - see front matter r 2006 Elsevier GmbH. All rights reserved.doi:10.1016/j.syapm.2006.01.005Mycoplasma species metabolize either glucose or argi-nine as energy substrates, and only a small number useboth substrates 14,19. Arginine is metabolized by thearginine dihydrolase pathway 16, but in vivo, exogen-ous arginine occurs in peptide-bound form, and itsacquisition depends the release of free arginine by amembrane-bound arginine aminopeptidase 1,2,10,17.This enzyme is thus diagnostic for arginine-using strains,and can be assayed chromogenically, using arginyl-b-naphthylamide as substrate 9. In this study, a simpleassay has been developed to provide a rapid test for thedetection of arginine-hydrolysing mycoplasmas.Twenty-eight test strains of Mycoplasma were used(Table 1). M. agalactiae NCTC 10123, M. alkalescensNCTC 10135, M. arginini NCTC 10129, M. bovigenita-lium NCTC 10122, M. bovis NCTC 10131,M. fermentans NCTC 10117, and M. verecundum NCTC590ARTICLE IN PRESSY.-C. Lin et al. / Systematic and Applied Microbiology 29 (2006) 589592Table 1. Change in culture medium pH, nal culture optical density (OD540 nm) at early stationary phase, and arginineaminopeptidase activity of Mycoplasma species grown in Eatons PPLO broth medium with arginine (2 g l 1)Mycoplasma species and strainsM. alkalescens NCTC 10135M. arginini NCTC 10129pH change inculture medium+0.91+0.59Final culture OD540 nm(mean7 SD; n 3)0.3477 0.0190.3817 0.016Arginine aminopeptidaseactivity (colour units)33M.M.M.M.M.M.M.M.M.M.M.fermentansfermentansfermentansfermentansfermentansfermentansfermentansfermentansfermentansfermentansfermentansM148M433NCTC 10117M640M39K7A6M641205928ACM671+0.69+0.67+0.65+0.65+0.51+0.44+0.39+0.39+0.35+0.34+0.300.1247 0.0100.1497 0.0040.1117 0.0100.1317 0.0030.1067 0.0040.0487 0.0000.0687 0.0040.0727 0.0000.0497 0.0040.0627 0.0050.0567 0.00211111111111M.M.M.M.M.M.M.M.M.M.M.M.M.M.M.agalactiae NCTC 10123bovigenitalium NCTC 10122bovigenitalium 102B00bovigenitalium 159B01bovigenitalium 434/81bovigenitalium 398/87bovis NCTC 10131ovine serogroup 11 2Dovine serogroup 11 52SR98ovine serogroup 11 47SR99ovine serogroup 11 52SR99ovine serogroup 11 95SR99ovine serogroup 11 96SR99ovine serogroup 11 126SR99verecundum NCTC 101450.610.280.080.260.310.140.390.340.260.270.240.310.320.300.110.1667 0.0170.0427 0.0210.0417 0.0000.0797 0.0040.0567 0.0010.0547 0.0010.1497 0.0030.0907 0.0010.0617 0.0030.0857 0.0030.0687 0.0010.0987 0.0020.1097 0.0010.0777 0.0030.0287 0.01200000000000000010145 were obtained from the National Collection ofType Cultures (London). Four isolates of M. bovigen-italium came from the UK (102B00, 159B01) andGermany (398/87, 434/81). Ten isolates of M. fermen-tans were from Dr. D. Pitcher (Respiratory andSystemic Infection Laboratory, Central Public HealthLaboratory, Colindale, UK) and Drs H. Windsor andP. Hannan (Mycoplasma Experience Ltd., Reigate,Surrey, UK). Five of these (2059, 28AC, A6, M148,M433) were isolated from cell lines, and ve fromhuman respiratory tract (M39, M640, M671), urethra(M641) and leukaemic bone marrow (K7). M. ovineserogroup 11 strain 2D and strains 52SR98, 47SR99,52SR99, 95SR99, 96SR99, 126SR99 were isolated froma UK sheep ock in 19982000.Mycoplasma strains were grown at 37 1C in a modiedEatons PPLO broth medium without crystal violet(Difco, BBL). The basal medium contained (g l 1):PPLO broth medium (21) and Difco yeast extract (10),adjusted to pH 7.0 or 7.6. After sterilization (121 1C,15 min), pre-sterilized additions were made of: 20% (v/v)heat inactivated (56 1C for 1 h) horse serum (Gibco),0.002% (w/v) DNA (Sigma), 0.0001% (w/v) ampicillin,and 0.0025% (v/v) phenol red. Ampicillin and DNAwere lter-sterilized. For fermentative Mycoplasmaspecies (grown at pH 7.6), lter-sterilized sodiumpyruvate (2 g l 1) and glucose (5 g l 1) were added. Fornon-fermentative, arginine-hydrolysing Mycoplasmaspecies (pH 7.0), arginine (2 g l 1) was added. Viablecounts, culture purity and viability were determinedusing Oxoid blood agar base No. 2 (40 g) in water,adjusted to pH 7.6, autoclaved, cooled to 45 1C, thenheat-inactivated Gibco horse serum added to give a nalconcentration 20% (v/v) in a total volume of one litre.Stock cultures in broth media, stored at 70 1C, werethawed and 30 ml inoculated into 5 ml of medium,incubated statically for 24 h, transferred into 45 mlmedium and incubated without shaking for assessmentof growth. Acid production by fermentative Mycoplasmaspecies was monitored by direct pH measurement (2 mlculture samples; Mettler Delta 350 pH meter), and wasindicated by the change from red to orange/yellow of theARTICLE IN PRESSY.-C. Lin et al. / Systematic and Applied Microbiology 29 (2006) 589592 591phenol red indicator. Release of ammonia during thegrowth of arginine-hydrolysing strains increased the pH,which was estimated by measuring pH and by theindicator changing from orange to red.Optical density (OD) measurement was used to assessgrowth, giving high and reproducible correlation withviable counts. OD of culture samples (540 nm; 1 cm lightpath) was read in a Gallenkamp Visi-Spec spectro-photometer. For early stationary phase cells, one ODunit was equivalent to 133 mg cell protein ml 1 and7 109 colony forming units (cfu) ml 1. Viable countswere determined by applying duplicate 10 ml drops ofserial decimal dilutions to the agar medium, drying atroom temperature, and incubating in a sealed plasticbox with a water-saturated atmosphere (37 1C for 3days, or until the development of visible colonies).Colonies were counted using a Kyowa (Tokyo, Japan)plate microscope, and viable counts expressed ascfu ml 1 18. The protein content of washed cells wasdetermined by a modied Lowry method 5,6: sodiumdodecyl sulfate in the alkali reagent, allowed its use formembrane lipoproteins without prior solubilization orlipid extraction. Absorbance was measured at 750 nm,with bovine serum albumin as the protein standard(0500 mg ml 1).Arginine aminopeptidase assay: Cultures were centri-fuged (13,400g, 4 min, in 1.5 ml microcentrifuge tubes;MSE Microcentaur centrifuge, Sanyo, Japan) and cellpellets washed twice and resuspended in dilute Ringer-HEPES (RH) buffer containing (g l 1): HEPES(N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid)buffer (18), NaCl (2.25), KCl (0.105), CaCl2 6H2O (0.12),and NaHCO3 (0.05). The aminopeptidase assay wasbased on that of Ball et al. 1,2,10, in whichb-naphthylamine released from arginyl-b-naphthylamidegives a coloured product when combined with thediazonium compound fast garnet (2-methyl-4-methylphe-nyl-azo benzenediazonium salt). Assays were done in at-bottomed 96-well microtitre plates (Nunc) with 10 ml ofMycoplasma cell suspension to which was added 100 mlarginyl-b-naphthylamide (10 4 M in 0.1 M Tris-HClbuffer, pH 8.0). After incubation at 37 1C for 3 h, releaseof b-naphthylamine was detected by adding 10 ml aqueous0.1% (w/v) aqueous fast garnet and the colour reactionassessed after 23 min at room temperature (20 1C). Thecolour was graded in intensity from 0 to 4 visual colourunits (CU): 0 yellow, 1 orange, 2 pale red, 3 red, 4 deepred. Three replicate assays were carried out for eachculture.Factors affecting the aminopeptidase assay: M. alka-lescens NCTC 10135 (high arginine aminopeptidaseactivity) and M. fermentans NCTC 10117 (low activity)were chosen as test strains. M. alkalescens does notferment glucose and uses arginine as a principalenergy source, while M. fermentans is one of thefew mycoplasmas having both a glucose fermentationpathway and the arginine dihydrolase pathway3,7,11,12. The basic protocol described above wasvaried to determine the effects of cell number, incuba-tion time, pH, and substrate concentration on activity.With M. alkalescens, b-naphthylamine was detectedafter 15 min (1 CU with 10 ml of a suspension of1.1 108 cfu ml 1, 2 CU with 1.1 109 cfu ml 1), inten-sifying, respectively, to 2 and 3 CU after 60 min and to 3and 4 CU after 180 min. With 10 ml of a suspension of1.1 107 cfu ml 1, 1 CU developed in 120 min. Nofurther increase in colour occurred between 180 and300 min incubation. With M. fermentans (10 ml of asuspension of 4.55 108 cfu ml 1) only 1 CU developedby 180 min, and did not increase after 300 min incuba-tion. The minimum cell concentrations required to givea reading of 1 CU after 3 h incubation at pH 8 werefound to be with 10 ml of a suspension of1.1 107 cfu ml 1 for M. alkalescens (no colour with2.2 106 cfu ml 1), and 2.27 108 cfu ml 1 for M. fer-mentans (no colour with 9.1 107 cfu ml 1). For routineassay of arginine aminopeptidase in growing cultures,cell suspensions were adjusted to an OD540 nm of 0.1,which corresponded to about 7 108 cfu ml 1.The effect of pH on aminopeptidase activity wasdetermined in 3 h incubation periods in seven differentbuffer systems: citrate, pH 4, pH 5; phosphate bufferedsaline, pH 6, pH 7; Tris-HCl, pH 8, and carbonate-bicarbonate buffer, pH 9, pH 10. With M. alkalescens(10 ml of 1.1 107, 1.1 108 and 1.1 109 cfu ml 1),readings of 1, 2 and 3 CU were obtained at pH 6 andpH 7. At pH 8, 9 and 10, CU values of 3 and 4 wereproduced by 1.1 108 and 1.1 109 cfu ml 1. No activitywith was found at pH 4 or 5. M. fermentans (10 ml of4.55 108 cfu ml 1) produced 1 CU only at pH 8 and 9.The effect of arginyl-b-naphthylamide concentrationwas assayed at six concentrations between 5 10 6and 10 3 M. Minimum concentrations required forM. alkalescens to develop 1 CU in 3 h were 5 10 6 Msubstrate (with 1.1 109 cfu ml 1), 10 5 M (1.1108 cfu ml 1) and 10 4 M (1.1 107 cfu ml 1). Produc-tion of 3 and 4 CU (with 1.1 109 cfu ml 1) required10 4, 5 10 4 or 10 3 M arginyl-b-naphthylamide.Production of 1 CU by M. fermentans (4.55108 cfu ml 1) was seen only with 10 4, 5 10 4 and10 3 M substrate.Growth and arginine aminopeptidase activity of 28strains: When supplemented with arginine (2 g l 1),cultures of M. alkalescens, M. arginini and ve strainsof M. fermentans gave signicant increases in nal cul-ture OD, relative to growth without arginine (Table 1),with values of 0.110.38 OD units. These correlated withsignicant increase in culture pH values (0.50.9 pHunit), indicating release of ammonia from arginine, andwith arginine aminopeptidase activities of 1 or 3CU (Table 1). The other six strains of M. fermentans(Table 1) all showed smaller increases in culture pH592ARTICLE IN PRESSY.-C. Lin et al. / Systematic and Applied Microbiology 29 (2006) 589592(by 0.30.44 pH unit) and expressed lower arginineaminopeptidase activity (1 CU). All the strains unable touse arginine showed typical decrease in culture pH andhad no arginine aminopeptidase activity (Table 1).Summary and conclusions: The standardized arginineaminopeptidase assay described enables the rapiddistinction of three physiological groups of mycoplas-mas: those like M. alkalescens NCTC 10135 andM. arginini NCTC 10129, which produce at least 3 CUin the assay; (2) M. fermentans strains, producing 1 CU;and (3) strains unable to hydrolyse arginine-peptides,and thus unlikely to make signicant use of arginine asan energy substrate in vivo. This simple, rapid andinexpensive procedure provides a means for the detec-tion of arginine-using strains, and helps distinguishbetween the three groups dened above. The procedureallows such physiological distinctions to be establishedin 3 h or less, and can be used in conjunction with moretime-consuming diagnostic procedures 8.References1 H.J. Ball, S.D. Neill, L.R. Reid, Use of arginineaminopeptidase activity in characterization of arginine-utilizing mycoplasmas, J. Clin. Microbiol. 15 (1982)2834.2 H.J. Ball, S.D. Neill, L.R. Reid, Aminopeptidase activityin arginine-utilizing Mycoplasma spp., J. Clin. Microbiol.21 (1985) 859860.3 E.A. Freundt, The mycoplasmas, In: N.E. Gibbons (Ed.),Bergeys Manual of Determinative Bacteriology, eighthed, The Williams & Wilkins Company, Baltimore, MD,USA, 1974, pp. 929950.4 K.-E. Johansson, B. Pettersson, Taxonomy of Mollicutes,In: S. Razin, R. Herrmann (Eds.), Molecular Biology andPathogenicity of Mycoplasmas, Kluwer Academic/Ple-num Publishers, New York, USA, 2002, pp. 129.5 O.H. Lowry, N.T. Rosebrough, N.T. Farr, A.L. Randall,Protein measurement with the Folin phenol reagent,J. Biol. Chem. 193 (1951) 265275.6 M.A.K. Markwell, S.M. Hass, L.L. Bieder, N.E. Tolbert,A modication of the Lowry procedure to simplify78910111213141516171819protein determination in membrane and lipoproteinsamples, Anal. Biochem. 87 (1978) 206210.R.J. Miles, Catabolism in mollicutes, J. Gen. Microbiol.138 (1992) 17731783.R. Miles, R. Nicholas (Eds.), Mycoplasma Protocols,Methods in Molecular MicrobiologyTM , vol. 104, Huma-na Press, Totowa, N