Distribution of ecto 5P-nucleotidase on Mycoplasma speciesassociated with arthritis

Sheena Johnson a;*, David Pitcher b

a Biochemistry Division, Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, UKb Respiratory and Systemic Infection Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK

Received 6 June 2000; received in revised form 23 August 2000; accepted 29 August 2000

Abstract

The enzyme ecto 5P-nucleotidase (5PN) was found to be active on 8/14 strains of Mycoplasma fermentans, Km ( þ S.D.) 3.8 þ 2.8 WM 5P-AMP, and on the type strain of Mycoplasma pulmonis, Km 0.63 WM 5P-AMP. The six M. fermentans strains lacking 5PN activity were relatedby restriction fragment length polymorphism typing. At pH 8.5, the type strains of Mycoplasma arthritidis, Mycoplasma buccale andUreaplasma urealyticum showed a relatively non-specific phosphatase activity against 5P-AMP but no activity was shown by the type strainsof Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasmasalivarium at this pH. M. fermentans has been reported from rheumatoid joints, which show a raised 5PN activity on their synovial cells andin their fluid which may be associated directly or indirectly with the mycoplasma. ß 2000 Federation of European MicrobiologicalSocieties. Published by Elsevier Science B.V. All rights reserved.

Keywords: Mycoplasma; Rheumatoid arthritis ; 5P-Nucleotidase; Mycoplasma fermentans

1. Introduction

The enzyme ecto 5P-nucleotidase EC 3.1.3.5 and CD73(5PN) is raised in the synovial £uid and on the synoviallining cells of rheumatoid arthritis (RA) patients but notin osteoarthritis patients [1,2]. Macrophages release their5PN on activation [3], which could explain the increase ofthe enzyme activity in the RA synovial £uid, but not theincrease on the macrophage-like synovial lining cells.

Various mycoplasma species have been detected in hu-man synovial £uids, but Mycoplasma fermentans in partic-ular has been associated with RA or other in£ammatoryarthritides [4^6]. Several other species are proven causes ofarthritis in animals. Little is known about the occurrenceof 5PN on the surface of mycoplasmas, therefore we mea-sured ecto 5PN on 14 strains of M. fermentans from var-ious sources, other mycoplasmas that have been reportedfrom human arthritic joints [7^10], and on two mycoplas-ma species which are proven causes of arthritis in rodents[11].

A wide variety of mammalian cells possess 5PN which

occurs as a dimer on the plasma membrane, and as amonomer in the serum. In circulating blood cells, it isfound only on some subgroups of B and T lymphocytes.The enzyme is active at neutral or slightly alkaline pH, isactivated by Mg2�, and removes the 5P-phosphate fromnucleoside 5P-monophosphates. It is strongly and speci¢-cally inhibited by ADP and ATP, or by the non-metabo-lisable analogue of ADP, K,L-methylene adenosine diphos-phate (AMPCP), which distinguishes it from non-speci¢calkaline or acid phosphatases [12,13]. The active site of5PN therefore recognises nucleosides, whereas non-speci¢cphosphatases recognise phosphate groups. In mammals,the enzyme is anchored to the cell plasma membrane bya glycosyl-phosphatidyl inositol link, and although thistype of linkage has not been reported from mycoplasmas,the enzyme may still be a lipoprotein.

During a study of 5PN activity on CESS human lym-phoblastoid cells, it was discovered that the culture wascontaminated with M. fermentans. The mycoplasma wasisolated and designated strain M106. The presence of themycoplasma was found to increase the activity of 5PNassociated with the cells about 5-fold. In subsequent ex-periments, washed cell suspensions of the mycoplasmaalone were found to possess considerable 5PN activity.

M. fermentans was originally isolated from a male gen-

0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 4 0 9 - 2

* Corresponding author. Tel. : +44 (020) 8725-5779;Fax: +44 (020) 8725-2992; E-mail : sjohnson@sghms.ac.uk

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ital ulcer [14], although its natural habitat has never beencon¢rmed. It is a common contaminant of cell lines [15].The organism has only rarely been cultured from rheuma-toid joints [16] but, using a PCR method, M. fermentanshas been reported from the synovial £uid of patients withRA and other in£ammatory arthritides [5]. The primersused were for the insertion sequence-like element (ISLE)found in M. fermentans [17]. However, a similar sizedproduct can be obtained from Mycoplasma orale, possiblyleading to the misidenti¢cation of the mycoplasma in somecases [18]. The presence of M. fermentans DNA in thejoints of 88% of RA and other in£ammatory arthritis pa-tients has been unambiguously con¢rmed by sequencingthe amplicon obtained from a PCR reaction using primersdirected against part of the 16S rRNA genes of M. fer-mentans [4]. It was not found in osteoarthritis patients.

It has been suggested that, unlike many species of my-coplasma, M. fermentans is genetically heterogeneous [17].On the basis of the restriction fragment length polymor-phism (RFLP) patterns using the ISLE as a hybridisationprobe, 14 strains can be classi¢ed into four groups [19].

In view of the apparent links between M. fermentans,5PN and in£ammatory arthritis, it was decided to examine

the 14 strains to see if they showed any signi¢cant diver-sity in their 5PN activity and if so, whether there was anycorrelation with RFLP type. In addition, we tested a num-ber of other Mycoplasma species for their ability to express5PN. These were: M. genitaliumT, M. hominisT, M. oraleT,M. pneumoniaeT, M. salivariumT and Ureaplasma urea-lyticumT, species which on occasion have been found injoints. Other human species included were MycoplasmabuccaleT and Mycoplasma penetransT. Two species knownto be arthritogenic in rodents, Mycoplasma arthritidisT

and Mycoplasma pulmonisT, were also tested.

2. Materials and methods

2.1. Strains and culture conditions

A list of M. fermentans strains and their origins used inthese experiments is shown in Table 1. The other myco-plasmas were: M. arthritidis NCTC 10162T, M. buccaleNCTC 10136T, M. genitalium NCTC 10195T, M. hominisNCTC 10111T, M. orale NCTC 10112T, M. penetransGTU 54-6A1T, M. pneumoniae NCTC 10119T, M. pulmo-

Table 1Sources of mycoplasma strains and the kinetic parameters for 5P-nucleotidase activity on washed cells of M. fermentans and M. pulmonis

Species andRFLP group [19]

Strainreference

Receivedfrom:

Location Isolation site Km

(WM 5P-AMP)Vmax (kat315

(CFU)31)

M. fermentansII M70 J. Tully ex. R.

DularCancer Research Facility,Frederick, MD, USA,originally Canada.

Respiratory tract ^ 0

II M64 J. Tully ex. R.Dular

Cancer Research Facility,Frederick, MD, USA,originally Canada.

Respiratory tract ^ 0

II M52 J. Tully ex. R.Dular

Cancer Research Facility,Frederick, MD, USA,originally Canada.

Respiratory tract ^ 0

II PG18T NCTC10117T

NCTC Central Public HealthLaboratory, London, UK.

Male genital ulcer ^ 0

II M433 P. Walker Guy's Hospital, London, UK. Tissue culture ^ 0II M148 S. Hancock Royal London Hospital,

London, UK.Tissue culture ^ 0

IV KL4 M.H. Williams Institute of Rheumatology,London, UK.

Synovial £uid 1.8 4.5

IV KL8 M.H. Williams Institute of Rheumatology,London, UK.

Synovial £uid 2.6 17

I GIM C. Bebear University of Bordeaux,France.

Synovial £uid 4.1 4.5

I BRO C. Bebear University of Bordeaux,France.

Urethra 2.6 1.8

I M106 S. Johnson St. George's HospitalMedical School, London, UK.

Tissue culture 2.6 15

I incognitus S.-C. Lo Armed Forces Institute,Washington, DC, USA.

Kaposi's sarcomatissue

2.2 3.5

III #5 R.B. Kundsin Brigham and Womens'Hospital, Boston, MA, USA.

Urine (AIDS) 10.4 22

III #29 R.B. Kundsin Brigham and Womens'Hospital, Boston, MA, USA.

Urine (AIDS) 4.2 7

M. pulmonis NCTC 10139T NCTC Central Public HealthLaboratory, UK.

Rat lung 0.63 3

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nis NCTC 10139T, M. salivarium NCTC 10113T, and U.urealyticum NCTC 10177T.

Stock cultures were maintained in the freeze-dried state,cultured routinely on SP4 agar and SP4 broth [20] andincubated at 37³C. Agar cultures were incubated in 5%CO2 :95% N2. Broth cultures were examined daily. Whengrowth was evident as indicated by a pH indicator change,the culture was transferred to 10 ml of SP4 broth and re-incubated until growth was observed. A 100-Wl aliquot ofbroth culture was serially diluted from 1031 to 1036 into1 ml fresh broth, 100 Wl from each dilution was spreadover a 5-cm petri dish containing SP4 agar allowed to dryat room temperature and incubated. The broth dilutionswere immediately stored at 370³C for the RNA assay.

After incubation, counts were carried out on dilutionplates and expressed as the number of colony-formingunits per ml (CFU ml31).

The remainder of the 10-ml broth culture was centri-fuged at 10 000Ug for 20 min. The cell pellet was washedtwice in cold sterile TS bu¡er (150 mM NaCl, 10 mMTris^HCl, pH 7.5), resuspended in 10 ml TS bu¡er andstored at 4³C until the 5PN assay was carried out.

2.2. Assay for 5P-nucleotidase

5P-AMP, 5P-AMPCP, concanavalin A, levamisole and L-glycerophosphate were supplied by Sigma.

The 5PN assay used was adapted from Rowe et al. [21]who used it to measure 5PN on the surface of humanlymphocytes. For the reactions, 0.5 ml of a mixture con-taining 40 WM AMP with 0.4 WCi (1.48U104 Bq) ml31

[2-3H]AMP, 20 mM MgCl2 and 200 mM glycine/NaOH,pH 8.5 was diluted with an equal volume of TS bu¡er,containing the (20 Wl) sample and the relevant inhibitor.Acid phosphatase was inhibited by the pH and 5PN wasinhibited by AMPCP. The reaction mixtures in 1.5-mlEppendorf tubes were incubated for 15 min at 37³C. Afterincubation, the tubes were cooled on ice to stop the reac-tion, and unreacted substrate precipitated with 0.15 ml0.15 M ZnSO4 followed by 0.5 ml 0.15 M Ba(OH)2 solu-tion. The tubes were centrifuged at 8500Ug for 1 min,then 0.5 ml supernatant was added to 5 ml Cytoscint(ICN Biomedicals), and counted for 3H on a scintillationcounter. After subtraction of the reagent blank, the frac-tion of AMP hydrolysed was calculated. To determine theKm and Vmax, the initial rate of reaction, Vi, was measuredin duplicate at six dilutions of AMP ranging from 1 to 40WM and Km and Vmax were calculated by the Lineweaver^Burke method, from the reciprocal plot of the Michaelis^Menten equation.

2.3. Estimation of concanavalin A binding

Three human B lymphoblastoid cell lines were grown inRPMI 1640 medium (Flow) and 10% foetal calf serum.Cells were centrifuged at 330Ug for 10 min, washed

with 25 ml TS bu¡er and the ¢nal pellet suspended inTS at a concentration of approximately 1U106 ml31.Cell suspensions or mycoplasma suspensions (20 Wl) wereadded to the reaction mixture with or without 20 Wg ml31

concanavalin A, and cooled on ice for 0.5 h before com-mencing the assay. The ratio of AMP hydrolysed with andwithout concanavalin A was calculated.

2.4. Measurement of mycoplasmal RNA

The concentration of mycoplasmal RNA was measuredusing the Gen-probe1 kit (Eurogenetics), a rapid detec-tion system for mycoplasmas in tissue culture [22]. Theprocedure was carried out according to the manufacturer'sinstructions, except that one ¢fth of the recommendedvolumes was used and quantitative measurements wereconducted in duplicate in 1.5-ml Eppendorf tubes. A cor-rection to the hybridisation reading was necessary if serumwas present, as this decreased the percentage hybridisationapproximately 2-fold. To determine the correction re-quired, hybridisation assays were carried out on a concen-tration range of mycoplasma suspensions in TS with andwithout 5 Wl broth. The corrected % RNA hybridisationwas plotted against cell density (CFU ml31). The maxi-mum achievable hybridisation varied with di¡erentbatches of probe, and the hybridisation was calculatedas a fraction of the maximum for that batch. This max-imum was adjusted to 21%, so it could be compared withthe initial values obtained for M. fermentans.

3. Results and discussion

3.1. Accuracy of cell counts

Fig. 1 shows the relationship between the % RNA hy-

Fig. 1. The relationship between the % RNA hybridisation and the cellcount for M. fermentans, M. arthritidis and M. pulmonis. a M. fermen-tans, F M. arthritidis, R M. pulmonis. The maximum % RNA hybrid-isation is approximately 21%.

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bridisation and the CFU ml31 for the eight 5PN positivestrains and the type strain of M. fermentans, M. pulmonisT

and M. arthritidisT. The regression line is

y � 0:25265x� 1:0892 �1�where y refers to the % hybridisation and x the CFU ml31.The correlation coe¤cient is 0.85 (P = 0.001). The scatterin the points at the four highest CFU ml31 values prob-ably represents colony-counting errors. The results indi-cate that the RNA hybridisation is an e¡ective measureof the concentration of mycoplasmas. The % RNA hybrid-isation was measured on all the mycoplasmas and theureaplasma.

3.2. Nature of the enzymes

Table 1 summarises the 5PN results for the 14 strainsof M. fermentans, and other species, of which onlyM. pulmonisT showed 5PN activity. The 5PN activity onM. pulmonisT and eight M. fermentans strains was con-¢rmed by the speci¢c inhibition of the assay by 5P-AMPCP whereas L-glycerophosphate, an inhibitor ofmost non-speci¢c phosphatases [23], had no e¡ect (Fig. 2).

The strain M106 grew poorly in broth and showed avery high Km for 5PN of 23 WM AMP. Because of the

problems of growing M106 in broth, it was not possibleto repeat this result. However, M106 grew more readily onSP4 agar, and two determinations on cell suspensions pre-pared from this medium showed a Km of 2.6 þ 1.0 WMAMP, which does not di¡er signi¢cantly from that ob-tained for the mean ( þ S.D.) of 4.0 þ 3.0 for the seven5PN positive clinical strains of M. fermentans which didgrow in broth. These values are consistent with those ofmammalian 5P-nucleotidases reported by Zimmerman [13].

Values of the apparent Vmax were calculated using the %RNA hybridisation, and translated into CFU ml31 usingEq. 1, therefore minimising colony-counting errors.

The Km for the 5PN on M. pulmonisT (0.63 WM AMP)was lower than that for 5PN positive strains of M. fermen-tans and the 50% inhibitory concentration of AMPCP wasalso lower, about 8 WM, as opposed to about 29 WM forM. fermentans.

Table 2 shows that U. urealyticumT, M. buccaleT and M.arthritidisT exhibited non-speci¢c phosphatase activity atpH 8.5, that on M. buccaleT being particularly active.Phosphatase activity in these organisms has previouslybeen described [24^26]. The mycoplasmal non-speci¢cphosphatases were not a¡ected by 1 mM levamisole, aninhibitor of mammalian alkaline phosphatase activity [27].

In contrast to 5PN, the phosphatase activity on M. ar-

Fig. 2. The e¡ect of putative inhibitors on the 5P-nucleotidase activity of M. fermentans and M. pulmonis. (a) M. fermentans ; (b) M. pulmonis. b

AMPCP (speci¢c inhibitor), S L-glycerophosphate (non-speci¢c inhibitor). The error bars show the mean þ S.D. for the eight strains of M. fermentanstesting positive for 5P-nucleotidase. Only one strain of M. pulmonis was tested, the points shown are the mean of duplicates.

Table 2Kinetic parameters for the type strains of mycoplasmas showing non-AMPCP inhibitable phosphatase activity against 5P-AMP at pH 8.5

Species Isolation site Km (WM 5P-AMP) Vmax (kat315 (CFU)31)

M. buccaleT Oropharynx NT (77)U. urealyticumT Human urogenital tract NT (11)M. arthritidisT Rat joint 1.0 7.5

The values in brackets for the apparent Vmax are only approximate as they were calculated from the initial reaction velocity when the 5P-AMP substrateconcentration was 40 WM. NT, not tested.

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thritidisT showed enhanced activity in the presence ofAMPCP (Fig. 3), showing that AMPCP is stimulatingthe phosphatase activity in this organism, and that theenzyme could not be 5PN. However, the activity was notblocked by L-glycerophosphate as expected, di¡ering inthis respect from most non-speci¢c phosphatases. TheKm for the phosphatase was 1.04 WM AMP, which is com-parable to that of the 5PN on M. pulmonisT.

M. genitaliumT, M. hominisT, M. oraleT, M. penetransT,M. pneumoniaeT and M. salivariumT were found tohave no 5PN or phosphatase activity against 5P-AMP atpH 8.5.

3.3. Binding of 5PN with concanavalin A

Mammalian 5PN is a glycosylated enzyme possessing amannose moiety and is inactivated by concanavalin A [28].We compared the e¡ect of concanavalin A on the 5PNpositive M. pulmonisT and M. fermentans strains withthat on the 5PN of three human B lymphoblastoid celllines, one of which was the IgG-secreting CESS cell line,and the other two were an IgM- and an IgG-secretingclone from Epstein-Barr virus-transformed peripheralblood B cells.

The 5PN activity on the human cells was reduced to28 þ 7% of its original value but concanavalin A failedto inhibit the 5PN on M. pulmonisT or any of the M.fermentans strains (106% and 99 þ 4% of the original, re-spectively). This shows that the enzyme on the mycoplas-mas lacks mannose groups.

3.4. Phosphatases on mycoplasmas

Only washed cell suspensions were tested for ecto 5PN.

It is possible that intracellular 5PN could occur. A 5PN hasbeen reported from a lysed preparation of Acholeplasmalaidlawii, but it is not known whether the enzyme wasactive on the surface of the organism [29].

An acid phosphatase, optimum pH 5.5, which willhydrolyse p-nitrophenyl phosphate and has a proteintyrosine phosphatase-like activity, has been reported onM. fermentans strain 11D 812 and on strain incognitus[30], and a similar enzyme which will hydrolyse p-nitro-phenyl phosphate at pH 5.0 but not pH 8.5 has beenobserved on strain M106 (S. Johnson, unpublished). Itis not known if the 5PN becomes a non-speci¢c acidphosphatase at low pH. M. hominis, M. salivarium andM. orale have also been reported to show relativelyweak activity against p-nitrophenyl phosphate at acidicpH [25].

3.5. Distribution of 5PN among mycoplasmas

It appears that 5PN activity does occur on mycoplasmasbut is less widely distributed than phosphatase activity.However, only the type strains of mycoplasmas otherthan M. fermentans were tested. This was in part becausethe numbers of representative strains of the species in cul-ture collections are few in most cases and, in some, onlythe type strain is available. It is possible that 5PN or phos-phatase activity may appear on other strains of these spe-cies. However, most other species exhibit less inter-strainvariability than M. fermentans. In addition, we chose tostudy only those species that have been associated withhuman joint pathology or were relatively commonly de-tected from humans, and the two arthritogenic rodent spe-cies.

3.6. Distribution of 5PN among M. fermentans strains

The 14 strains of M. fermentans used in this study havebeen classi¢ed into four groups on the basis of RFLPpatterns [19] (Table 1). All six strains which did not pos-sess 5PN activity, including the type strain, PG18T, andthree respiratory tract isolates were members of groupII, whereas 5PN positive strains belong to groups I, IIIand IV and included three isolates from synovial £uid.The probability that the six strains without 5PN shouldfall into the same group by chance can be calculated usingFisher's exact test, from which P = 0.001, and therefore itis highly probable that the absence of 5PN is linked to aparticular evolutionary line. In group II isolates, it is pos-sible that the ISLE DNA is inserted into the 5PN gene orits promoter region, thus preventing the gene's transcrip-tion, or the translation of the mRNA. Alternatively, thegene may be deleted.

Isolates of M. fermentans are very di¤cult to obtain.However, if more strains could be tested, it is possiblethat a link would be revealed between the expression of5PN and source of the isolate.

Fig. 3. The e¡ect of putative inhibitors on the phosphatase activity ofM. arthritidis. b AMPCP, S L-glycerophosphate. The points shown arethe mean of duplicates for the type strain only.

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3.7. Mycoplasmas and arthritis

Several species of mycoplasma have been stronglylinked to naturally or experimentally induced arthritis inanimals with severe symptoms similar to RA in humans.Notably, Mycoplasma bovis and Mycoplasma capricolumin ruminants, Mycoplasma hyosynoviae in pigs, Mycoplas-ma synoviae in poultry and M. pulmonis and M. arthritidisin rodents [11]. We have shown that the two rodent strainscan both hydrolyse AMP at pH 8.5, using di¡erent en-zymes.

U. urealyticum, which has a non-5PN AMP phospha-tase, has been isolated from the arthritic joints of hypo-gammaglobulinaemic patients [7].

We were unable to detect 5PN or any phosphatase activeon 5P-AMP at pH 8.5 on M. genitaliumT, M. hominisT,M. pneumoniaeT and M. salivariumT, but these specieshave been occasionally detected in arthritic joints [7^10].

3.8. The value of 5PN to a mycoplasma in the joint

M. fermentans is detected only in in£ammatory arthri-tides, not in osteoarthritis [4]. For the mycoplasmal 5PNenzyme to be of any value to the organism, the nucleosidemonophosphate substrates must be available. This hap-pens only in in£ammatory arthritis, where the joints areinvaded by activated neutrophils, which release AMP [31].In the mouse, M. pulmonis and M. arthritidis could alsoboth utilise the AMP provided by the in£ammatory neu-trophils.

3.9. The cause of the raised 5PN detected in the joints ofRA patients

The 5PN is raised on both the synovial lining cells and inthe synovial £uid of RA patients. The raised 5PN observedon the synovial lining cells could have been induced by a5PN positive M. fermentans strain similar to M106 whereasthat in the synovial £uid of the RA patients may either bemycoplasma-derived, or human 5PN released by activatedmacrophage-like synoviocytes [3], or a combination ofboth sources.

3.10. In£ammatory factor induced by M. fermentans

M. fermentans possesses a potent immunomodulatorproduct named macrophage activating lipopeptide 2(MALP-2) which activates macrophages to release cyto-kines [32,33], which may in turn increase the neutrophilicin¢ltrate into the joint. It is possible that MALP-2 couldalso trigger the release of macrophage-derived 5PN. Thusboth endogenous and mycoplasma-derived 5PN may occurin joints in excessive amounts when M. fermentans ispresent, and account for the raised 5PN observed in theRA patients.

3.11. Ecto 5PN on other bacteria

There are few reports of ecto 5PN on any bacteria, but amembrane-bound 5P-nucleotidase has been reported andsequenced on Vibrio parahaemolyticus [34]. This maywell prove to be important in identifying the 5PN geneon M. pulmonis, since, at the time of writing, the sequenc-ing of the genome of this species is underway at the In-stitut Pasteur.

Acknowledgements

Thanks are due to P.C.T. Hannan and R.H. Leach whodonated strains, and Professor Mark Fisher for readingthe paper. S.J. wishes to thank St. George's Hospital Spe-cial Trustees for ¢nancial support.

References

[1] Johnson, S.M., Patel, S.J., Bruckner, F.E. and Collins, D.A. (1999)5P-Nucleotidase as a marker of both general and local in£ammationin rheumatoid arthritis patients. Rheumatology 38, 391^396.

[2] Henderson, B., Johnstone, J.J. and Chayen, J. (1980) 5P-Nucleotidaseactivity in the human synovial lining in rheumatoid arthritis. Ann.Rheum. Dis. 39, 248^252.

[3] Karnovsky, M.L. and Lazdins, J.K. (1978) Biochemical criteria foractivated macrophages. J. Immunol. 121, 809^813.

[4] Johnson, S.M., Sidebottom, D., Bruckner, F. and Collins, D. (2000)Identi¢cation of Mycoplasma fermentans in the synovial £uid fromarthritis patients with in£ammatory disease. J. Clin. Microbiol. 38,90^93.

[5] Schaeverbeke, T., Gilroy, C.B., Bebear, C., Dehais, J. and Taylor-Robinson, D. (1996) Mycoplasma fermentans in joints of patientswith rheumatoid arthritis and other joint disorders. Lancet 347, 1418.

[6] Williams, M.H., Brosto¡, J. and Roitt, I.M. (1970) Possible role ofMycoplasma fermentans in the pathogenesis of rheumatoid arthritis.Lancet ii, 277^280.

[7] Furr, P.M., Taylor-Robinson, D. and Webster, A.D. (1994) Myco-plasmas and Ureaplasmas in patients with hypogammaglobulinaemiaand their role in arthritis : microbiological observations over 20 years.Ann. Rheum. Dis. 53, 183^187.

[8] Luttrell, L.M., Kanj, S.S., Corey, G.R., Lins, R.E., Spinner, R.J.,Mallon, W.J. and Sexton, D.J. (1994) Mycoplasma hominis septicarthritis : two case reports and a review. Clin. Infect. Dis. 19, 1067^1070.

[9] Taylor-Robinson, D., Gilroy, C.B., Horowitz, S. and Horowitz, J.(1994) Mycoplasma genitalium in the joints of two patients with ar-thritis. Eur. J. Clin. Microbiol. Infect. Dis. 13, 1066^1069.

[10] Tully, J.G., Rose, D.L., Baseman, J.B., Dallo, S.F., Lazzell, A.L. andDavis, C.P. (1995) Mycoplasma pneumoniae and Mycoplasma genita-lium mixture in synovial £uid isolate. J. Clin. Microbiol. 33, 1851^1855.

[11] Simecka, J.W., Davis, J.K., Davidson, M.K., Ross, S.E., Sta«dtlander,C.T. and Cassell, G.H. (1992) Mycoplasma diseases of animals. In:Mycoplasmas, Molecular Biology and Pathogenesis (Manilo¡, J.,McElhaney, R.N., Finch, L.R. and Baseman, J.B., Eds.), pp. 391^415. American Society for Microbiology, Washington, DC.

[12] Bu«rger, R.M. and Lowenstein, J.M. (1975) 5P-Nucleotidase fromsmooth muscle of small intestine and brain. Inhibition by nucleotides.Biochemistry 14, 2362^2367.

FEMSLE 9630 12-10-00

S. Johnson, D. Pitcher / FEMS Microbiology Letters 192 (2000) 59^6564

[13] Zimmermann, H. (1992) 5P-Nucleotidase : molecular structure andfunctional aspects. Biochem. J. 285, 345^365.

[14] Reiter, M. and Wentholt, H.M. (1952) The occurrence of a pleuro-pneumonia-like organism in fuso-spirillary infections of the humangenital mucosa. J. Invest. Dermatol. 18, 313^325.

[15] McGarrity, G.J., Kotani, H. and Butler, G.H. (1992) Mycoplasmasand tissue culture cells. In: Mycoplasmas, Molecular Biology andPathogenesis (Manilo¡, J., McElhaney, R.N., Finch, L.R. and Base-man, J.B., Eds.), pp. 445^454. American Society for Microbiology,Washington, DC.

[16] Ma®rdh, P.A., Nilsson, F.J. and Bjelle, A. (1973) Mycoplasmas andbacteria in synovial £uid from patients with rheumatoid arthritis.Ann. Rheum. Dis. 32, 319^325.

[17] Wang, R.Y.-H., Hu, W.S., Dawson, M.S., Shih, J.W.-K. and Lo,S.-C. (1992) Selective identi¢cation of Mycoplasma fermentans bypolymerase chain reaction and by using a nucleotide sequence withinthe insertion-like element. J. Clin. Microbiol. 30, 245^248.

[18] Ditty, S.E., Connolly, M.A., Li, B.J. and Lo, S.-C. (1999) Mycoplas-ma orale has a sequence similar to the insertion-like sequence of M.fermentans. Mol. Cell Probes 13, 183^189.

[19] Pitcher, D.G. and Hilbocus, J. (1998) Variability in the distributionand composition of insertion sequence-like elements in strains ofMycoplasma fermentans. FEMS Microbiol. Lett. 160, 101^109.

[20] Tully, J.G. (1995) Culture medium formulation for primary isolationand maintenance of mollicutes. In: Molecular and Diagnostic Proce-dures in Mycoplasmology (Razin, S. and Tully, J.G., Eds.), pp. 33^39. Academic Press, London.

[21] Rowe, M., de Gast, G.C., Platts-Mills, T.A.E., Asherson, G.L., Web-ster, A.D.B. and Johnson, S.M. (1979) 5P-nucleotidase of B and Tlymphocytes isolated from human peripheral blood. Clin. Exp. Im-munol. 36, 97^101.

[22] McGarrity, G.J. and Kotani, H. (1986) Detection of cell culturemycoplasmas by a genetic probe. Exp. Cell. Res. 163, 273^278.

[23] Bel¢eld, A. and Goldberg, D.M. (1968) Inhibition of the nucleotidasee¡ect of alkaline phosphatase by L-glycerophosphate. Nature 219,73^75.

[24] Black, F.T. (1973) Phosphatase activity in T-mycoplasmas. Int. J.Syst. Bacteriol. 23, 65^66.

[25] Shibata, K.-I., Totsuka, M. and Watanabe, T. (1986) Phosphataseactivity as a criterion for di¡erentiation of oral mycoplasmas. J. Clin.Microbiol. 23, 970^972.

[26] Aluotto, B.B., Wittler, R.G., Williams, C.O. and Faber, J.E. (1970)Standardised bacteriologic techniques for the characterisation of My-coplasma species. Int. J. Syst. Bacteriol. 20, 35^58.

[27] Van Belle, H. (1976) Alkaline phosphatase. 1. Kinetics and inhibitionby levamisole of puri¢ed isoenzymes from humans. Clin. Chem. 22,972^976.

[28] Goldstein, I.J., Hollerman, C.E. and Smith, E.E. (1965) Protein-car-bohydrate interaction. II. Inhibition studies on the interaction ofconcanavalin A with polysaccharides. Biochemistry 4, 876^883.

[29] Tryon, V.V. and Pollack, D. (1984) Purine metabolism in Acholeplas-ma laidlawii B: a novel PPi-dependent nucleoside kinase activity.J. Bacteriol. 159, 265^270.

[30] Noda, M., Shibata, K., Sawa, Y., Shimokoube, H. and Watanabe, T.(1994) Puri¢cation and characterization of an acid phosphatase fromMycoplasma fermentans. Microbiol. Immunol. 38, 103^107.

[31] Madara, J.L., Patapo¡, T.W., Gillece-Castro, B., Colgan, S.P., Par-kos, C., Delp, C. and Mrsny, R.J. (1993) 5P-Adenosine monophos-phate is the neutrophil-derived paracrine factor that elicits chloridesecretion from T84 intestinal epithelial cell monolayers. J. Clin. In-vest. 91, 2320.

[32] Deiters, U. and Mu«hlradt, P.F. (1999) Mycoplasmal lipopeptideMALP-2 induces the chemoattractant proteins macrophage in£am-matory protein 1K (MIP-1K), monocyte chemoattractant protein 1,and MIP-2 and promotes leukocyte in¢tration in mice. Infect. Im-mun. 67, 3390^3398.

[33] Calcutt, M.J., Kim, M.F., Karpas, A.B., Mu«hlradt, P.F. and Wise,K.S. (1999) Di¡erential posttranslational processing confers intraspe-cies variation of a major surface lipoprotein and a macrophage-acti-vating lipopeptide of Mycoplasma fermentans. Infect. Immun. 67,760^771.

[34] Tamao, Y., Noguchi, K., Sakai-Tomita, Y., Hama, H., Shimamoto,T., Kanazawa, H., Tsuda, M. and Tsuchiya, T. (1991) Sequenceanalysis of nutA gene encoding membrane-bound Cl3-dependent5P-nucleotidase of Vibrio parahaemolyticus. J. Biochem. 109, 24^29.

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