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Demonstration of neuraminidase activity in Mycoplasma neurolyticum and of neuraminidase proteins in three canine Mycoplasma species
Veterinary Microbiology 155 (2012) 425–429
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Demonstration of neuraminidase activity in Mycoplasma neurolyticum and of neuraminidase proteins in three canine Mycoplasma species Rebeka Lucijana Bercˇicˇ a, Ivanka Cizelj a, Mateja Bencˇina a, Mojca Narat a, Janet M. Bradbury b, Peter Dovcˇ a, Dusˇan Bencˇina a,* a b
Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domzˇale, Slovenia University of Liverpool, School of Veterinary Science, Leahurst Campus, Leahurst, Neston CH64 7TE, United Kingdom
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 March 2011 Received in revised form 8 August 2011 Accepted 25 August 2011
Neuraminidases are virulence factors in many pathogenic microorganisms. They are present also in some Mycoplasma species that cause disease in birds, dogs and alligators. Thirty-seven Mycoplasma species have been examined previously for neuraminidase (sialidase) activity, whereas many of the species causing disease in man, ruminants, pigs, rodents and other animals have not. In this study neuraminidase enzymatic activity (NEAC) was examined in 45 previously untested Mycoplasma species, including those causing diseases in man, farm animals and laboratory animals. The only species in which NEAC was found was Mycoplasma neurolyticum, specifically, its type strain (Type AT) which is capable of inducing neurologic signs in inoculated young mice and rats. The NEAC of washed cells was relatively weak, but it differed even more than 10-fold among cells of cultures derived from individual colonies of M. neurolyticum. A weak NEAC was also detected in the supernatant of the M. neurolyticum broth culture. Canine Mycoplasma spp. with high sialidase activity reported previously, Mycoplasma canis, Mycoplasma cynos and Mycoplasma molare had 100-fold more NEAC than M. neurolyticum, but apparent differences in NEAC levels existed among strains of M. canis and of M. cynos. Zymograms using neuraminidase-specific chromogenic substrate were used to show proteins having NEAC. In M. canis (a field isolate Larissa and the type strain PG14T), M. cynos (isolate 896) and M. molare (type strain H542T) proteins with NEAC had molecular masses of 130 kDa, 105 kDa and 110 kDa, respectively. Identification of these neuraminidases could provide the basis for their molecular characterization. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Mycoplasmas Neuraminidase activity Zymograms
1. Introduction Mycoplasmas are distinguished from other bacteria by their total lack of a cell wall and possession of a minimal genome (Razin et al., 1998). The genus Mycoplasma includes over 110 recognized Mycoplasma species. Among them are species that cause diseases in humans, ruminants, pigs, dogs, rodents, birds and other animals.
* Corresponding author. Tel.: +386 13203809; fax: +386 17241005. E-mail address: [email protected] (D. Bencˇina). 0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.08.026
Factors associated with pathogenicity of individual Mycoplasma species are generally undetermined. However, recent studies indicated that sialidases (neuraminidases) might be involved in the pathogenicity of certain Mycoplasma spp. that cause diseases in birds, dogs and alligators (Bercˇicˇ et al., 2008b; Brown et al., 2004; May and Brown, 2009). Systematic surveys for neuraminidase (or sialidase) activity have been done so far for avian Mycoplasma spp. (Bercˇicˇ et al., 2008b) and Mycoplasma spp. frequently or first isolated from dogs (May and Brown, 2009). In addition, it has been reported that Mycoplasma alligatoris has sialidase activity, whereas such activity was
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not detected in Mycoplasma crocodyli (Brown et al., 2004) or in a human pathogen, Mycoplasma pneumoniae (Kahane et al., 1990). In all, 37 recognized Mycoplasma species were tested and sialidase activity, termed also neuraminidase enzymatic activity (NEAC), was found in 12 species, including eight species isolated from birds (Bercˇicˇ et al., 2008b) and three canine species, Mycoplasma canis, Mycoplasma cynos and Mycoplasma molare (May and Brown, 2009). In this study we investigated NEAC in 45 Mycoplasma species that previously have not been examined for this activity and one species (M. pneumoniae) that has been examined before (Kahane et al., 1990). In addition, we used zymograms to demonstrate the proteins with NEAC in M. canis, M. cynos and M. molare, information that could lead to the identification and sequencing of the coding genes. 2. Materials and methods 2.1. Mycoplasma isolates and their cultivation The type strains of Mycoplasma species were mainly used. They were from Mycoplasma spp. culture collections at the University of Liverpool or at the University of Ljubljana. They were M. adleri (G145T), M. agalactiae (PG2T), M. amphoriforme (A39T), M. arthritidis (PG 6T), M. auris (U1AT), M. bovirhinis (PG43T), M. bovoculi (M165/69T), M. bovis type strain (DonettaT) and nine field isolates, M. buccale (CH20247T), M. californicum (ST-6T), M. canadense (275 CT), M. capricolum subsp. capricolum (Calif. KidT), M. citelli (RG-2 CT), M. collis (58 BT), M. conjunctivae (HRC 581T), M. cottewii (VIST), M. elephantis (E42T), M. equigenitalium (T37T), M. equirhinis (M 432/72T), M. fermentans (PG18T), M. flocculare (MS42T), M. genitalium (G 37T and strain 7), M. hominis (PG21T and two clinical isolates (Ja and Ho), M. hyopharyngis, (H3-6BFT), M. hyopneumoniae (JT and strain Pi), M. hyorhinis (BTS-7T and strain 108), M. hyosynoviae (S16T), M. mobile (163 KT), M. mustelae (MX9T), M. mycoides subsp. capri (PG3T), Mycoplasma neurolyticum (Type AT), M. orale (CH 19299T), M. ovipneumoniae (Y98T), M. oxoniensis (128T), M. penetrans (GTU-54T), M. phocidae (105T), M. phocicerebrale (1049T), M. phocirhinis (852T), M. pneumoniae (FHT, reference strain M129 and clinical isolates ULB 932, ULB 933, ULB 934 and ULB 935A), M. primatum (HRC 292T), M. pulmonis (PG34T), M. salivarium (PG20T), M. sualvi (Mayfield BT), M. subdolum (TBT), M. testudinis (O1008T) and M. yeatsii (GIHT). Three canine Mycoplasma species with high sialidase activity reported previously (May and Brown, 2009) were also tested for NEAC, including by zymograms. These were M. canis type strain PG14T and clinical isolates Larissa and Luna, isolated from bitches (vaginal swab) in Slovenia, M. cynos type strain H831T and clinical isolates (strains 105, 896 and 2297) isolated from dogs in Austria (Zeugswetter et al., 2007) and the type strain of M. molare (H542T). For most Mycoplasma species we used Mycoplasma broth and agar media containing either 10% horse serum or 10–15% porcine serum (Bradbury, 1977; Bercˇicˇ et al., 2008b). Depending on the species being grown the broth
contained glucose (1% w/v) or L-arginine (0.2% w/v). For Mycoplasma spp. with more demanding requirements other media were used. Thus SP-4 medium was used to grow M. genitalium (Whitcomb, 1983), and Friis medium was used for M. hyopneumoniae, M. flocculare and M. hyosynoviae (Freundt, 1983). The M. neurolyticum type strain (Sabin’s Type A) was used in this study. Its identity was confirmed by indirect immunofluorescence using reference (rabbit) antiserum (Bradbury et al., 1993) and it was grown in broth containing Bacto PPLO broth base (BD, US), 10% porcine serum (Invitrogen, US), 1% glucose, 0.25% BME vitamins and 0.003% phenol red (Sigma, Germany). Strains of M. canis, M. cynos, Mycoplasma corogypsi and M. molare also were grown in media with this composition. The M. neurolyticum log-phase broth culture was filtered-cloned using filtration through 0.45 mm-pore membrane (Minisart, Sartorius Stedim, Germany) and seeding (25 ml) onto mycoplasma agar in plastic dishes (diameter 60 mm). Individual colonies were transferred to mycoplasma broth to obtain M. neurolyticum clones. All Mycoplasma spp. cultures were incubated at 37– 38 8C under aerobic conditions. Colony forming units (CFU) were determined by standard procedures (Rodwell and Whitcomb, 1983). 2.2. Measurement of neuraminidase enzymatic activity (NEAC) NEAC of washed mycoplasma cells was assayed using the chromogenic substrate 5-bromo-4-chloro-3-indol-aD-N-acetylneuraminic acid sodium salt (B4666, Sigma, St. Louis, MO) hereafter referred to as BIN. Mycoplasma cells were harvested in log-growth phase (Bercˇicˇ et al., 2008b). Samples with cells (10 ml of cell suspension corresponding to 109 CFU) were mixed with 10 ml phosphate buffered saline (PBS), pH 7.4 containing 5 mg of BIN in small microcentrifuge tubes with caps. Samples were incubated at room temperature and the time at which the positive reaction occurred, i.e. the colourless sample became indigo blue, was recorded. For the first hour of incubation samples were observed every 10 min and then at intervals of 1 hour (except overnight) for at least three days. Negative controls contained BIN and PBS alone. The positive control of NEAC was neuraminidase of Clostridium perfringens (type V, N2876, Sigma) 0.1 mg/sample. Since putative secreted sialidases have been reported in supernatants of the above canine Mycoplasma spp. (May and Brown, 2009) we investigated NEAC of M. neurolyticum supernatant in late log-phase broth cultures, after the cells were pelleted by centrifugation (25,000 g for 20 min). Ten ml of the supernatant broth was mixed with 10 ml of PBS containing 5 ml of BIN. The negative control used here was an aliquot of mycoplasma broth, and the positive control was C. perfringens neuraminidase (0.1 mg) in mycoplasma broth. NEAC of M. neurolyticum clones with high or low activity was compared by titration of samples with similar numbers of mycoplasmas (109 CFU). They were diluted in PBS by serial doubling dilutions. Assays were conducted
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with samples in triplicate. When samples with low NEAC became positive, we recorded the dilution at which samples with high NEAC gave a comparable level of staining. Reactions were then monitored for the next two to three days. The neuraminidase inhibitor, 2-deoxy-2,3-didehydroN-acetylneuraminic acid (DANA; D9050, Sigma, St. Louis, MO) was used to confirm the specificity of NEAC observed with M. neurolyticum cells. Thirty mg of DANA was included in the sample with 109 CFU of mycoplasma cells and BIN, whereas the positive control was without DANA. 2.3. NEAC of mycoplasma colonies Examination of NEAC was conducted either with intact colonies of M. neurolyticum and M. canis ‘‘in situ’’ on agar blocks or with colonies transferred from agar onto nitrocellulose membrane (Sartorius) by careful lifting of colonies with a piece of the membrane (1 cm 1 cm). In both cases colonies were then incubated with a solution (PBS, pH 7.4) containing 0.5 mg BIN/ml in a humid atmosphere at room temperature at least for 1 h. Staining of colonies ‘‘in situ’’ was observed by microscope and images were recorded.
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Fig. 1. Putative neuraminidases of M. canis, M. cynos and M. molare demonstrated by zymogram using BIN. Proteins of Mycoplasma species: lane 1, M. corogypsi (strain BV1, 105 CFU); lane 2, M. molare (H542, 108 CFU); lane 3, M. canis (Luna, 108 CFU); lane 4, M. canis (Larissa, 108 CFU), lane 5, M. cynos (896, 108 CFU) and lane 6, prestained protein, molecular mass marker (Fermentas). Note: M. molare 110-kDa protein, M. canis (Larissa) 130-kDa protein and M. cynos 100-kDa protein showed NEAC following overnight incubation of the membrane with BIN (0.5 mg/ml). Proteins were separated in PhastGel Gradient (8–25) and blotted to the membrane at 45 8C for 20 min.
2.6. Statistical analysis The difference in NEAC among M. neurolyticum clones was evaluated by Fisher’s exact test. P values <0.05 were considered significant.
2.4. Identification of proteins with NEAC by zymograms Potent NEAC of M. corogypsi enabled identification of a protein of about 110 kDa with this activity by the use of SDS-PAGE and zymogram with BIN (Bercˇicˇ et al., 2008b). This approach was used in an attempt to identify proteins with NEAC in M. neurolyticum, M. canis (strains PG14T, Larissa and Luna), M. cynos (896) and M. molare (H 542T). Thus, cells with strong NEAC, were harvested, washed in PBS pH 7.4 and lysed by 5% sodium dodecyl sulphate (SDS). Proteins were separated with the PhastSystem (Pharmacia) in PhastGel Gradient 8-25 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Samples (1 ml) containing 1 mg of protein (108 CFU) separated by SDSPAGE, were transferred from the gel to an Immobilon P membrane (Millipore) using diffusion blot at 45 8C for 25 min. Proteins remaining in the gel were then stained by Coomassie blue (PhastGelTM BlueR). The Immobilon P membrane with transferred proteins was incubated in PBS (pH 7.2) for at least 2 h or overnight. Then, the membrane was put into a sterile plastic petri dish and carefully overlaid with PBS (pH 7.2) containing 0.5 mg BIN per ml. Incubation took place in a humid container at room temperature. Reactions were monitored every hour. The membrane showing staining of proteins was scanned and images are presented in Fig. 1 and Supplementary Figs. S3 and S4. 2.5. N-terminal sequencing of M. canis protein(s) M. canis (Larissa) proteins were separated in a large gel (10 cm 15 cm) and transferred to the Immobilon P membrane to permit subsequent sequencing. Following staining with Coomassie blue a protein (of 130 kDa) with NEAC was excised and submitted for sequencing (Bercˇicˇ et al., 2008a).
3. Results 3.1. NEAC in previously untested Mycoplasma species Forty-five previously untested Mycoplasma species were examined for NEAC (see Section 2). Among them were species that cause economically important diseases in ruminants and pigs or diseases in rodents or in humans. Forty-four species lacked detectable NEAC. Among them were species whose genomes do not have gene(s) for neuraminidase (see Section 4). As reported previously M. pneumoniae showed no NEAC activity. M. neurolyticum (Type AT) was, thus, the only species of this collection that showed NEAC. 3.2. Different levels of NEAC in M. neurolyticum cultures NEAC was detected in washed cells of all M. neurolyticum Type AT cultures that were tested. Most cultures reached 109 CFU/ml within 30–40 h. Their supernatants had weak, but detectable NEAC. However when M. neurolyticum broth cultures became acidic (pH < 7), further incubation resulted in decreasing NEAC and finally its total disappearance. Native M. neurolyticum colonies ‘‘in situ’’ and colonies transferred to nitrocellulose membranes had weak but detectable NEAC when they were ‘‘overlaid’’ with BIN. However, some colonies showed more intense staining than others, indicating a higher level of NEAC. Therefore, 12 broth cultures derived from individual M. neurolyticum colonies were established and tested for NEAC. At a similar density of cells (109 CFU/sample) there were differences in the times at which they showed positive NEAC. Four cloned cultures (cultures B, C, E and F) gave a positive NEAC reaction in 45–50 min, five after 2–3 h and three (including
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cultures A and D) only after overnight incubation (Fig. S1 and not shown). Thus, there was a considerable variation in NEAC among cells of cloned M. neurolyticum cultures derived from the same parental culture (Fig. S1). For instance, after 10-h incubation, 16-fold diluted samples (containing 6.3 107 CFU) of culture B had NEAC comparable to undiluted samples of culture A which contained 1.1 109 CFU. This difference is statistically significant (P < 0.05). DANA strongly inhibited NEAC. In samples giving NEAC within 60 min, the addition of DANA (30 mg) prevented positive reactions for at least 48 h, indicating that the M. neurolyticum neuraminidase was involved in NEAC (Fig. S1). The activity did not change if PBS with pH 6, 7, 8 or 9 was added to M. neurolyticum cells, but pH 10 prevented the positive NEAC reaction (data not shown). Attempts to identify M. neurolyticum neuraminidase by zymography using BIN were not successful. There was no positive reaction after 72-h incubation. 3.3. Putative neuraminidases of M. canis, M. cynos and M. molare Native colonies of M. canis strains Larissa, Luna and PG14T ‘‘in situ’’ on agar reacted with BIN (Fig. S2 and not shown). Such reactions indicated that neuraminidase was exposed at the surface of M. canis colonies. Zymograms were used to detect M. canis protein with NEAC. In the field strain Larissa, whose cells had strong NEAC (the sample with 106 CFU was positive in 10 min) a 130 kDa protein became indigo blue following incubation with BIN (Fig. 1 and Supplementary Fig. S3). Strain PG14T, which had about 5-fold lower NEAC than Larissa, also expressed a 130 kDa protein with NEAC (not shown). However such a protein was not detected in Luna strain (Fig. 1, lane 3). Sequencing of the 130 kDa protein of Larissa yielded two N-terminal amino acid sequences, MNPLVEPAPN and EKAQFSIVK. There was no match for these sequences in protein databases (NCBI, Blast protein). Cells of four M. cynos strains were assayed for NEAC. A lung isolate, 896 from a puppy with fatal bronchopneumonia, had the highest NEAC. Samples containing 106 CFU showed positive NEAC in 5–10 min. Field isolates 105 and 2297 revealed 4- and 10-fold lower NEAC than 896. The type strain H831T had approximately 5-fold lower NEAC than strain 896. The zymogram showed that a 105 kDa protein of strain 896 had strong NEAC (Fig. 1 and Fig. S3). Cells of the type strain of M. molare (H542T) showed higher NEAC than most strains of M. canis and M. cynos apart from strains Larissa and 896. In the zymogram a 110 kDa protein of M. molare revealed an apparent NEAC (Fig. 1 and Fig. S4). In the protein profile of M. molare a 110kDa protein was the most abundant protein in the region containing proteins from 90 to 130 kDa (Fig. S4). 4. Discussion Our study shows that NEAC is relatively rare among Mycoplasma species. Indeed, of 45 species examined for the first time in this study, only M. neurolyticum revealed apparent NEAC. However it is important to note that, for
most species only the type strain was examined. Previous surveys have shown that individual strains of certain species, e.g. Mycoplasma synoviae and M. canis, were without detectable NEAC (Bercˇicˇ et al., 2008b; May and Brown, 2009). So it is possible that strains of some species may possess NEAC, although a strain representing such species was found NEAC-negative in this survey. Among Mycoplasma species whose genomes lack genes encoding neuraminidases (sialidases) are the human pathogens M. pneumoniae, M. genitalium, M. fermentans, M. penetrans, M. hominis, the species causing disease in pigs, M. hyopneumoniae and M. hyorhinis, the species pathogenic for ruminants M. agalactiae, M. bovis, M. capricolum, M. mycoides supbsp. capri and the rodent pathogens M. arthritidis and M. pulmonis. So far, 96 Mycoplasma spp. have been assayed for NEAC and activity was found in only 13 species including M. neurolyticum (Bercˇicˇ et al., 2008b; May and Brown, 2009; D.R. Brown, personal communication). M. neurolyticum produces a putative neurotoxin causing ‘‘rolling’’ disease in young mice and rats (Gabridge et al., 1985; Thomas, 1967; Tully, 1983). Although there are some ‘‘common’’ properties of the neurotoxin and the protein(s) involved in NEAC, such as their inactivation by heating at 45–50 8C (Thomas, 1967; our unpublished data), their appearance in supernatant of broth culture and their disappearance in older M. neurolyticum cultures, both remain to be identified and molecularly characterized. Using zymograms we failed to demonstrate a M. neurolyticum protein with NEAC. It is likely that it is a minor protein or it has much lower specific activity than neuraminidases of M. canis, M. cynos, M. molare and M. corogypsi which were identified by zymograms. A putative M. neurolyticum neurotoxin with Mw 300 kDa (Tully, 1983) is probably a minor protein, because we did not see such a protein in the protein profile of 10 M. neurolyticum cultures (data not shown). Sialidase of M. alligatoris can induce apoptosis of host’s cells (Hunt and Brown, 2007), whereas the M. synoviae neuraminidase NanH can desialylate chicken glycoproteins, including heavy chain of IgG (Bercˇicˇ et al., 2011). Whether the neuraminidases of M. neurolyticum, M. canis, M. cynos and M. molare can induce apoptosis or desialylation of host glycoproteins remains to be investigated. So far only the neuraminidase (sialidase) genes of M. alligatoris, Mycoplasma gallisepticum and M. synoviae have been sequenced. Attempts to sequence such genes in species with high NEAC (M. canis, M. corogypsi, M. cynos and M. molare) were not successful, because degenerate primers did not work in PCR (May and Brown, 2009; Bercˇicˇ et al., unpublished data). However, demonstration of proteins with NEAC (Fig. 1 and Figs. S3 and S4) in these species may help to identify and then to sequence the genes encoding neuraminidases. One way to do this would be to determine their partial amino acid sequences and then synthesise the corresponding oligo-nucleotides (primers and/or probes) that would bind to the DNA of their neuraminidase genes. 5. Conclusions The M. neurolyticum type strain has NEAC, but levels can vary among cultures. In M. canis, M. cynos and M. molare
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their putative neuraminidases, demonstrated by zymograms, had molecular masses of 130, 105 and 110 kDa, respectively. Strong NEAC of these three Mycoplasma species may be important in interactions with sialylated molecules of their hosts, but this remains to be assessed by further studies. Acknowledgements This work was supported by Slovenian Research Agency (ARRS) (Grant P4 – 0220). We thank Drs. Brigita Slavec, Natasˇa Tozon, Irena Oven and also Daliborka Dusˇanic´ and Teja Zakrajsˇek for help and acknowledge the technical assistance of Ana Jakopicˇ. We thank Drs. Renate Rosengarten, Christine Citti, Anne Gautier-Bouchardon and Joachim Spergser for Mycoplasma cultures. We also thank Drs. Meghan May and Daniel R. Brown for the permission to use their unpublished data.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vetmic.2011. 08.026. References Bercˇicˇ, R.L., Slavec, B., Lavricˇ, M., Narat, M., Bidovec, A., Dovcˇ, P., Bencˇina, D., 2008a. Identification of major immunogenic proteins of Mycoplasma synoviae isolates. Vet. Microbiol. 127, 147–154. Bercˇicˇ, R.L., Slavec, B., Lavricˇ, M., Narat, M., Zorman-Rojs, O., Dovcˇ, P., Bencˇina, D., 2008b. A survey of avian Mycoplasma species for neuraminidase enzymatic activity. Vet. Microbiol. 130, 391–397.
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Bercˇicˇ, R.L., Cizelj, I., Dusˇanic´, D., Narat, M., Zorman-Rojs, O., Dovcˇ, P., Bencˇina, D., 2011. Neuraminidase of Mycoplasma synoviae desialylates heavy chain of the chicken immunoglobulin G and glycoproteins of chicken tracheal mucus. Avian Pathol. 40, 298–308. Bradbury, J.M., 1977. Rapid biochemical tests for characterization of the Mycoplasmatales. J. Clin. Microbiol. 5, 531–534. Bradbury, J.M., Abdul-Wahab, O.M., Yavari, C.A., Dupiellet, J.P., Bove´, J.M., 1993. Mycoplasma imitans sp. nov. is related to Mycoplasma gallisepticum and found in birds. Int. J. Syst. Bacteriol. 43, 721–728. Brown, D.R., Zacher, L.A., Farmerie, W.G., 2004. Spreading factors of Mycoplasma alligatoris, a flesh-eating mycoplasma. J. Bacteriol. 186, 3922–3927. Freundt, E.A., 1983. Culture media for classic mycoplasma. In: Razin, S., Tully, J.B. (Eds.), Methods in Mycoplasmology. I. Mycoplasma Characterization. Academic Press, New York, pp. 127–135. Gabridge, M.G., Chandler, D.K.F., Daniels, M.J., 1985. Pathogenicity factors in Mycoplasmas and Spiroplasmas. In: Razin, S., Barile, M.F. (Eds.), The Mycoplasmas. IV. Mycoplasma Pathogenicity. Academic Press, Orlando, pp. 313–351. Hunt, M.E., Brown, D.R., 2007. Role of sialidase in Mycoplasma alligatorisinduced pulmonary fibroblast apoptosis. Vet. Microbiol. 121, 73–82. Kahane, I., Reisch-Saada, A., Almagor, M., Abeliuck, P., Yatziv, S., 1990. Glycosidase activities of Mycoplasmas. Zentralbl. Bakteriol. 273, 200–205. May, M., Brown, D.R., 2009. Secreted sialidase activity of canine mycoplasmas. Vet. Microbiol. 137, 380–383. Razin, S., Yogev, D., Naot, Y., 1998. Molecular biology and pathogenicity of Mycoplasmas. Microbiol. Mol. Biol. Rev. 62, 1094–1156. Rodwell, A.W., Whitcomb, R.F., 1983. Methods for direct and indirect measurement of mycoplasma growth. In: Razin, S., Tully, J.G. (Eds.), Methods in Mycoplasmology. I. Mycoplasma Characterization. Academic Press, New York, pp. 185–196. Thomas, L., 1967. The neurotoxins of M. neurolyticum and M. gallisepticum. Ann. N. Y. Acad. Sci. 143, 218–224. Tully, J.G., 1983. Tests for Mycoplasma neurolyticum neurotoxin. In: Razin, S., Tully, J.G. (Eds.), Methods in Mycoplasmology. II. Diagnostic Mycoplasmology. Academic Press, New York, pp. 311–314. Whitcomb, R.F., 1983. Culture media for spiroplasmas. Culture media for spiroplasmas. In: Razin, S., Tully, J.G. (Eds.), Methods in Mycoplasmology. I. Mycoplasma Characterization. Academic Press, New York, pp. 147–158. Zeugswetter, F., Weissenbock, H., Shibly, S., Hassan, J., Spergser, J., 2007. Lethal bronchopneumonia caused by Mycoplasma cynos in a litter of golden retriever puppies. Vet. Rec. 161, 626–627.
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Demonstration of neuraminidase activity in Mycoplasma neurolyticum and of neuraminidase proteins in three canine Mycoplasma species Rebeka Lucijana Bercˇicˇ a, Ivanka Cizelj a, Mateja Bencˇina a, Mojca Narat a, Janet M. Bradbury b, Peter Dovcˇ a, Dusˇan Bencˇina a,* a b
Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domzˇale, Slovenia University of Liverpool, School of Veterinary Science, Leahurst Campus, Leahurst, Neston CH64 7TE, United Kingdom
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 March 2011 Received in revised form 8 August 2011 Accepted 25 August 2011
Neuraminidases are virulence factors in many pathogenic microorganisms. They are present also in some Mycoplasma species that cause disease in birds, dogs and alligators. Thirty-seven Mycoplasma species have been examined previously for neuraminidase (sialidase) activity, whereas many of the species causing disease in man, ruminants, pigs, rodents and other animals have not. In this study neuraminidase enzymatic activity (NEAC) was examined in 45 previously untested Mycoplasma species, including those causing diseases in man, farm animals and laboratory animals. The only species in which NEAC was found was Mycoplasma neurolyticum, specifically, its type strain (Type AT) which is capable of inducing neurologic signs in inoculated young mice and rats. The NEAC of washed cells was relatively weak, but it differed even more than 10-fold among cells of cultures derived from individual colonies of M. neurolyticum. A weak NEAC was also detected in the supernatant of the M. neurolyticum broth culture. Canine Mycoplasma spp. with high sialidase activity reported previously, Mycoplasma canis, Mycoplasma cynos and Mycoplasma molare had 100-fold more NEAC than M. neurolyticum, but apparent differences in NEAC levels existed among strains of M. canis and of M. cynos. Zymograms using neuraminidase-specific chromogenic substrate were used to show proteins having NEAC. In M. canis (a field isolate Larissa and the type strain PG14T), M. cynos (isolate 896) and M. molare (type strain H542T) proteins with NEAC had molecular masses of 130 kDa, 105 kDa and 110 kDa, respectively. Identification of these neuraminidases could provide the basis for their molecular characterization. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Mycoplasmas Neuraminidase activity Zymograms
1. Introduction Mycoplasmas are distinguished from other bacteria by their total lack of a cell wall and possession of a minimal genome (Razin et al., 1998). The genus Mycoplasma includes over 110 recognized Mycoplasma species. Among them are species that cause diseases in humans, ruminants, pigs, dogs, rodents, birds and other animals.
* Corresponding author. Tel.: +386 13203809; fax: +386 17241005. E-mail address: [email protected] (D. Bencˇina). 0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.08.026
Factors associated with pathogenicity of individual Mycoplasma species are generally undetermined. However, recent studies indicated that sialidases (neuraminidases) might be involved in the pathogenicity of certain Mycoplasma spp. that cause diseases in birds, dogs and alligators (Bercˇicˇ et al., 2008b; Brown et al., 2004; May and Brown, 2009). Systematic surveys for neuraminidase (or sialidase) activity have been done so far for avian Mycoplasma spp. (Bercˇicˇ et al., 2008b) and Mycoplasma spp. frequently or first isolated from dogs (May and Brown, 2009). In addition, it has been reported that Mycoplasma alligatoris has sialidase activity, whereas such activity was
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not detected in Mycoplasma crocodyli (Brown et al., 2004) or in a human pathogen, Mycoplasma pneumoniae (Kahane et al., 1990). In all, 37 recognized Mycoplasma species were tested and sialidase activity, termed also neuraminidase enzymatic activity (NEAC), was found in 12 species, including eight species isolated from birds (Bercˇicˇ et al., 2008b) and three canine species, Mycoplasma canis, Mycoplasma cynos and Mycoplasma molare (May and Brown, 2009). In this study we investigated NEAC in 45 Mycoplasma species that previously have not been examined for this activity and one species (M. pneumoniae) that has been examined before (Kahane et al., 1990). In addition, we used zymograms to demonstrate the proteins with NEAC in M. canis, M. cynos and M. molare, information that could lead to the identification and sequencing of the coding genes. 2. Materials and methods 2.1. Mycoplasma isolates and their cultivation The type strains of Mycoplasma species were mainly used. They were from Mycoplasma spp. culture collections at the University of Liverpool or at the University of Ljubljana. They were M. adleri (G145T), M. agalactiae (PG2T), M. amphoriforme (A39T), M. arthritidis (PG 6T), M. auris (U1AT), M. bovirhinis (PG43T), M. bovoculi (M165/69T), M. bovis type strain (DonettaT) and nine field isolates, M. buccale (CH20247T), M. californicum (ST-6T), M. canadense (275 CT), M. capricolum subsp. capricolum (Calif. KidT), M. citelli (RG-2 CT), M. collis (58 BT), M. conjunctivae (HRC 581T), M. cottewii (VIST), M. elephantis (E42T), M. equigenitalium (T37T), M. equirhinis (M 432/72T), M. fermentans (PG18T), M. flocculare (MS42T), M. genitalium (G 37T and strain 7), M. hominis (PG21T and two clinical isolates (Ja and Ho), M. hyopharyngis, (H3-6BFT), M. hyopneumoniae (JT and strain Pi), M. hyorhinis (BTS-7T and strain 108), M. hyosynoviae (S16T), M. mobile (163 KT), M. mustelae (MX9T), M. mycoides subsp. capri (PG3T), Mycoplasma neurolyticum (Type AT), M. orale (CH 19299T), M. ovipneumoniae (Y98T), M. oxoniensis (128T), M. penetrans (GTU-54T), M. phocidae (105T), M. phocicerebrale (1049T), M. phocirhinis (852T), M. pneumoniae (FHT, reference strain M129 and clinical isolates ULB 932, ULB 933, ULB 934 and ULB 935A), M. primatum (HRC 292T), M. pulmonis (PG34T), M. salivarium (PG20T), M. sualvi (Mayfield BT), M. subdolum (TBT), M. testudinis (O1008T) and M. yeatsii (GIHT). Three canine Mycoplasma species with high sialidase activity reported previously (May and Brown, 2009) were also tested for NEAC, including by zymograms. These were M. canis type strain PG14T and clinical isolates Larissa and Luna, isolated from bitches (vaginal swab) in Slovenia, M. cynos type strain H831T and clinical isolates (strains 105, 896 and 2297) isolated from dogs in Austria (Zeugswetter et al., 2007) and the type strain of M. molare (H542T). For most Mycoplasma species we used Mycoplasma broth and agar media containing either 10% horse serum or 10–15% porcine serum (Bradbury, 1977; Bercˇicˇ et al., 2008b). Depending on the species being grown the broth
contained glucose (1% w/v) or L-arginine (0.2% w/v). For Mycoplasma spp. with more demanding requirements other media were used. Thus SP-4 medium was used to grow M. genitalium (Whitcomb, 1983), and Friis medium was used for M. hyopneumoniae, M. flocculare and M. hyosynoviae (Freundt, 1983). The M. neurolyticum type strain (Sabin’s Type A) was used in this study. Its identity was confirmed by indirect immunofluorescence using reference (rabbit) antiserum (Bradbury et al., 1993) and it was grown in broth containing Bacto PPLO broth base (BD, US), 10% porcine serum (Invitrogen, US), 1% glucose, 0.25% BME vitamins and 0.003% phenol red (Sigma, Germany). Strains of M. canis, M. cynos, Mycoplasma corogypsi and M. molare also were grown in media with this composition. The M. neurolyticum log-phase broth culture was filtered-cloned using filtration through 0.45 mm-pore membrane (Minisart, Sartorius Stedim, Germany) and seeding (25 ml) onto mycoplasma agar in plastic dishes (diameter 60 mm). Individual colonies were transferred to mycoplasma broth to obtain M. neurolyticum clones. All Mycoplasma spp. cultures were incubated at 37– 38 8C under aerobic conditions. Colony forming units (CFU) were determined by standard procedures (Rodwell and Whitcomb, 1983). 2.2. Measurement of neuraminidase enzymatic activity (NEAC) NEAC of washed mycoplasma cells was assayed using the chromogenic substrate 5-bromo-4-chloro-3-indol-aD-N-acetylneuraminic acid sodium salt (B4666, Sigma, St. Louis, MO) hereafter referred to as BIN. Mycoplasma cells were harvested in log-growth phase (Bercˇicˇ et al., 2008b). Samples with cells (10 ml of cell suspension corresponding to 109 CFU) were mixed with 10 ml phosphate buffered saline (PBS), pH 7.4 containing 5 mg of BIN in small microcentrifuge tubes with caps. Samples were incubated at room temperature and the time at which the positive reaction occurred, i.e. the colourless sample became indigo blue, was recorded. For the first hour of incubation samples were observed every 10 min and then at intervals of 1 hour (except overnight) for at least three days. Negative controls contained BIN and PBS alone. The positive control of NEAC was neuraminidase of Clostridium perfringens (type V, N2876, Sigma) 0.1 mg/sample. Since putative secreted sialidases have been reported in supernatants of the above canine Mycoplasma spp. (May and Brown, 2009) we investigated NEAC of M. neurolyticum supernatant in late log-phase broth cultures, after the cells were pelleted by centrifugation (25,000 g for 20 min). Ten ml of the supernatant broth was mixed with 10 ml of PBS containing 5 ml of BIN. The negative control used here was an aliquot of mycoplasma broth, and the positive control was C. perfringens neuraminidase (0.1 mg) in mycoplasma broth. NEAC of M. neurolyticum clones with high or low activity was compared by titration of samples with similar numbers of mycoplasmas (109 CFU). They were diluted in PBS by serial doubling dilutions. Assays were conducted
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with samples in triplicate. When samples with low NEAC became positive, we recorded the dilution at which samples with high NEAC gave a comparable level of staining. Reactions were then monitored for the next two to three days. The neuraminidase inhibitor, 2-deoxy-2,3-didehydroN-acetylneuraminic acid (DANA; D9050, Sigma, St. Louis, MO) was used to confirm the specificity of NEAC observed with M. neurolyticum cells. Thirty mg of DANA was included in the sample with 109 CFU of mycoplasma cells and BIN, whereas the positive control was without DANA. 2.3. NEAC of mycoplasma colonies Examination of NEAC was conducted either with intact colonies of M. neurolyticum and M. canis ‘‘in situ’’ on agar blocks or with colonies transferred from agar onto nitrocellulose membrane (Sartorius) by careful lifting of colonies with a piece of the membrane (1 cm 1 cm). In both cases colonies were then incubated with a solution (PBS, pH 7.4) containing 0.5 mg BIN/ml in a humid atmosphere at room temperature at least for 1 h. Staining of colonies ‘‘in situ’’ was observed by microscope and images were recorded.
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Fig. 1. Putative neuraminidases of M. canis, M. cynos and M. molare demonstrated by zymogram using BIN. Proteins of Mycoplasma species: lane 1, M. corogypsi (strain BV1, 105 CFU); lane 2, M. molare (H542, 108 CFU); lane 3, M. canis (Luna, 108 CFU); lane 4, M. canis (Larissa, 108 CFU), lane 5, M. cynos (896, 108 CFU) and lane 6, prestained protein, molecular mass marker (Fermentas). Note: M. molare 110-kDa protein, M. canis (Larissa) 130-kDa protein and M. cynos 100-kDa protein showed NEAC following overnight incubation of the membrane with BIN (0.5 mg/ml). Proteins were separated in PhastGel Gradient (8–25) and blotted to the membrane at 45 8C for 20 min.
2.6. Statistical analysis The difference in NEAC among M. neurolyticum clones was evaluated by Fisher’s exact test. P values <0.05 were considered significant.
2.4. Identification of proteins with NEAC by zymograms Potent NEAC of M. corogypsi enabled identification of a protein of about 110 kDa with this activity by the use of SDS-PAGE and zymogram with BIN (Bercˇicˇ et al., 2008b). This approach was used in an attempt to identify proteins with NEAC in M. neurolyticum, M. canis (strains PG14T, Larissa and Luna), M. cynos (896) and M. molare (H 542T). Thus, cells with strong NEAC, were harvested, washed in PBS pH 7.4 and lysed by 5% sodium dodecyl sulphate (SDS). Proteins were separated with the PhastSystem (Pharmacia) in PhastGel Gradient 8-25 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Samples (1 ml) containing 1 mg of protein (108 CFU) separated by SDSPAGE, were transferred from the gel to an Immobilon P membrane (Millipore) using diffusion blot at 45 8C for 25 min. Proteins remaining in the gel were then stained by Coomassie blue (PhastGelTM BlueR). The Immobilon P membrane with transferred proteins was incubated in PBS (pH 7.2) for at least 2 h or overnight. Then, the membrane was put into a sterile plastic petri dish and carefully overlaid with PBS (pH 7.2) containing 0.5 mg BIN per ml. Incubation took place in a humid container at room temperature. Reactions were monitored every hour. The membrane showing staining of proteins was scanned and images are presented in Fig. 1 and Supplementary Figs. S3 and S4. 2.5. N-terminal sequencing of M. canis protein(s) M. canis (Larissa) proteins were separated in a large gel (10 cm 15 cm) and transferred to the Immobilon P membrane to permit subsequent sequencing. Following staining with Coomassie blue a protein (of 130 kDa) with NEAC was excised and submitted for sequencing (Bercˇicˇ et al., 2008a).
3. Results 3.1. NEAC in previously untested Mycoplasma species Forty-five previously untested Mycoplasma species were examined for NEAC (see Section 2). Among them were species that cause economically important diseases in ruminants and pigs or diseases in rodents or in humans. Forty-four species lacked detectable NEAC. Among them were species whose genomes do not have gene(s) for neuraminidase (see Section 4). As reported previously M. pneumoniae showed no NEAC activity. M. neurolyticum (Type AT) was, thus, the only species of this collection that showed NEAC. 3.2. Different levels of NEAC in M. neurolyticum cultures NEAC was detected in washed cells of all M. neurolyticum Type AT cultures that were tested. Most cultures reached 109 CFU/ml within 30–40 h. Their supernatants had weak, but detectable NEAC. However when M. neurolyticum broth cultures became acidic (pH < 7), further incubation resulted in decreasing NEAC and finally its total disappearance. Native M. neurolyticum colonies ‘‘in situ’’ and colonies transferred to nitrocellulose membranes had weak but detectable NEAC when they were ‘‘overlaid’’ with BIN. However, some colonies showed more intense staining than others, indicating a higher level of NEAC. Therefore, 12 broth cultures derived from individual M. neurolyticum colonies were established and tested for NEAC. At a similar density of cells (109 CFU/sample) there were differences in the times at which they showed positive NEAC. Four cloned cultures (cultures B, C, E and F) gave a positive NEAC reaction in 45–50 min, five after 2–3 h and three (including
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cultures A and D) only after overnight incubation (Fig. S1 and not shown). Thus, there was a considerable variation in NEAC among cells of cloned M. neurolyticum cultures derived from the same parental culture (Fig. S1). For instance, after 10-h incubation, 16-fold diluted samples (containing 6.3 107 CFU) of culture B had NEAC comparable to undiluted samples of culture A which contained 1.1 109 CFU. This difference is statistically significant (P < 0.05). DANA strongly inhibited NEAC. In samples giving NEAC within 60 min, the addition of DANA (30 mg) prevented positive reactions for at least 48 h, indicating that the M. neurolyticum neuraminidase was involved in NEAC (Fig. S1). The activity did not change if PBS with pH 6, 7, 8 or 9 was added to M. neurolyticum cells, but pH 10 prevented the positive NEAC reaction (data not shown). Attempts to identify M. neurolyticum neuraminidase by zymography using BIN were not successful. There was no positive reaction after 72-h incubation. 3.3. Putative neuraminidases of M. canis, M. cynos and M. molare Native colonies of M. canis strains Larissa, Luna and PG14T ‘‘in situ’’ on agar reacted with BIN (Fig. S2 and not shown). Such reactions indicated that neuraminidase was exposed at the surface of M. canis colonies. Zymograms were used to detect M. canis protein with NEAC. In the field strain Larissa, whose cells had strong NEAC (the sample with 106 CFU was positive in 10 min) a 130 kDa protein became indigo blue following incubation with BIN (Fig. 1 and Supplementary Fig. S3). Strain PG14T, which had about 5-fold lower NEAC than Larissa, also expressed a 130 kDa protein with NEAC (not shown). However such a protein was not detected in Luna strain (Fig. 1, lane 3). Sequencing of the 130 kDa protein of Larissa yielded two N-terminal amino acid sequences, MNPLVEPAPN and EKAQFSIVK. There was no match for these sequences in protein databases (NCBI, Blast protein). Cells of four M. cynos strains were assayed for NEAC. A lung isolate, 896 from a puppy with fatal bronchopneumonia, had the highest NEAC. Samples containing 106 CFU showed positive NEAC in 5–10 min. Field isolates 105 and 2297 revealed 4- and 10-fold lower NEAC than 896. The type strain H831T had approximately 5-fold lower NEAC than strain 896. The zymogram showed that a 105 kDa protein of strain 896 had strong NEAC (Fig. 1 and Fig. S3). Cells of the type strain of M. molare (H542T) showed higher NEAC than most strains of M. canis and M. cynos apart from strains Larissa and 896. In the zymogram a 110 kDa protein of M. molare revealed an apparent NEAC (Fig. 1 and Fig. S4). In the protein profile of M. molare a 110kDa protein was the most abundant protein in the region containing proteins from 90 to 130 kDa (Fig. S4). 4. Discussion Our study shows that NEAC is relatively rare among Mycoplasma species. Indeed, of 45 species examined for the first time in this study, only M. neurolyticum revealed apparent NEAC. However it is important to note that, for
most species only the type strain was examined. Previous surveys have shown that individual strains of certain species, e.g. Mycoplasma synoviae and M. canis, were without detectable NEAC (Bercˇicˇ et al., 2008b; May and Brown, 2009). So it is possible that strains of some species may possess NEAC, although a strain representing such species was found NEAC-negative in this survey. Among Mycoplasma species whose genomes lack genes encoding neuraminidases (sialidases) are the human pathogens M. pneumoniae, M. genitalium, M. fermentans, M. penetrans, M. hominis, the species causing disease in pigs, M. hyopneumoniae and M. hyorhinis, the species pathogenic for ruminants M. agalactiae, M. bovis, M. capricolum, M. mycoides supbsp. capri and the rodent pathogens M. arthritidis and M. pulmonis. So far, 96 Mycoplasma spp. have been assayed for NEAC and activity was found in only 13 species including M. neurolyticum (Bercˇicˇ et al., 2008b; May and Brown, 2009; D.R. Brown, personal communication). M. neurolyticum produces a putative neurotoxin causing ‘‘rolling’’ disease in young mice and rats (Gabridge et al., 1985; Thomas, 1967; Tully, 1983). Although there are some ‘‘common’’ properties of the neurotoxin and the protein(s) involved in NEAC, such as their inactivation by heating at 45–50 8C (Thomas, 1967; our unpublished data), their appearance in supernatant of broth culture and their disappearance in older M. neurolyticum cultures, both remain to be identified and molecularly characterized. Using zymograms we failed to demonstrate a M. neurolyticum protein with NEAC. It is likely that it is a minor protein or it has much lower specific activity than neuraminidases of M. canis, M. cynos, M. molare and M. corogypsi which were identified by zymograms. A putative M. neurolyticum neurotoxin with Mw 300 kDa (Tully, 1983) is probably a minor protein, because we did not see such a protein in the protein profile of 10 M. neurolyticum cultures (data not shown). Sialidase of M. alligatoris can induce apoptosis of host’s cells (Hunt and Brown, 2007), whereas the M. synoviae neuraminidase NanH can desialylate chicken glycoproteins, including heavy chain of IgG (Bercˇicˇ et al., 2011). Whether the neuraminidases of M. neurolyticum, M. canis, M. cynos and M. molare can induce apoptosis or desialylation of host glycoproteins remains to be investigated. So far only the neuraminidase (sialidase) genes of M. alligatoris, Mycoplasma gallisepticum and M. synoviae have been sequenced. Attempts to sequence such genes in species with high NEAC (M. canis, M. corogypsi, M. cynos and M. molare) were not successful, because degenerate primers did not work in PCR (May and Brown, 2009; Bercˇicˇ et al., unpublished data). However, demonstration of proteins with NEAC (Fig. 1 and Figs. S3 and S4) in these species may help to identify and then to sequence the genes encoding neuraminidases. One way to do this would be to determine their partial amino acid sequences and then synthesise the corresponding oligo-nucleotides (primers and/or probes) that would bind to the DNA of their neuraminidase genes. 5. Conclusions The M. neurolyticum type strain has NEAC, but levels can vary among cultures. In M. canis, M. cynos and M. molare
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their putative neuraminidases, demonstrated by zymograms, had molecular masses of 130, 105 and 110 kDa, respectively. Strong NEAC of these three Mycoplasma species may be important in interactions with sialylated molecules of their hosts, but this remains to be assessed by further studies. Acknowledgements This work was supported by Slovenian Research Agency (ARRS) (Grant P4 – 0220). We thank Drs. Brigita Slavec, Natasˇa Tozon, Irena Oven and also Daliborka Dusˇanic´ and Teja Zakrajsˇek for help and acknowledge the technical assistance of Ana Jakopicˇ. We thank Drs. Renate Rosengarten, Christine Citti, Anne Gautier-Bouchardon and Joachim Spergser for Mycoplasma cultures. We also thank Drs. Meghan May and Daniel R. Brown for the permission to use their unpublished data.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vetmic.2011. 08.026. References Bercˇicˇ, R.L., Slavec, B., Lavricˇ, M., Narat, M., Bidovec, A., Dovcˇ, P., Bencˇina, D., 2008a. Identification of major immunogenic proteins of Mycoplasma synoviae isolates. Vet. Microbiol. 127, 147–154. Bercˇicˇ, R.L., Slavec, B., Lavricˇ, M., Narat, M., Zorman-Rojs, O., Dovcˇ, P., Bencˇina, D., 2008b. A survey of avian Mycoplasma species for neuraminidase enzymatic activity. Vet. Microbiol. 130, 391–397.
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