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Highly dynamic genomic loci drive the synthesis of two types of capsular or secreted 1
polysaccharides within the “Mycoplasma mycoides” cluster. 2
3
4
5
Running title: Mycoplasma mycoides polysaccharides 6
7
8
Clothilde Bertin1,2
, Corinne Pau-Roblot3, Josiane Courtois
3, Lucía Manso-Silván
1,2, Florence 9
Tardy4,5
, François Poumarat4,5
, Christine Citti6,7
, Pascal Sirand-Pugnet8,9
, Patrice Gaurivaud 10
4,5,
François Thiaucourt
#1,2 11
12
1 CIRAD, UMR CMAEE, F-34398 Montpellier, France. 2 INRA, UMR1309 CMAEE, F-13
34398 Montpellier, France. 3 Unité Biologie des Plantes et Innovation, EA 3900, Université 14
de Picardie Jules Verne, Amiens, France, 4 Agence Nationale de Sécurité Sanitaire, 15
Laboratoire de Lyon, UMR Mycoplasmoses des Ruminants, Lyon, France, 5 Université de 16
Lyon, VetAgro Sup, UMR Mycoplasmoses des Ruminants, Marcy-L’étoile, France, 6
INRA, 17
UMR 1225, IHAP, 31076 Toulouse, France, 7 Université de Toulouse, INP, ENVT, 18
UMR1225, IHAP, 31076 Toulouse, France, 8
INRA, UMR 1332 de Biologie du Fruit et 19
Pathologie, F-33140 Villenave d’Ornon, France, 9
Univ. Bordeaux, UMR 1332 de Biologie du 20
Fruit et Pathologie, F-33140 Villenave d’Ornon, France. 21
22
AEM Accepts, published online ahead of print on 14 November 2014Appl. Environ. Microbiol. doi:10.1128/AEM.02892-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract 23
Mycoplasmas of the “Mycoplasma mycoides” cluster are all ruminant pathogens. 24
Mycoplasma mycoides subsp. mycoides (MmmSC) is responsible for contagious bovine 25
pleuropneumonia and is known to produce capsular polysaccharide (CPS) and 26
exopolysaccharide (EPS). Previous studies have strongly suggested a role for MmmSC 27
polysaccharides in pathogenicity. MmmSC-secreted EPS was recently characterized as a -(1-28
6)- galactofuranose homopolymer (galactan) identical to the capsular product. Here, we 29
extended the characterization of secreted polysaccharides to all other members of the “M. 30
mycoides” cluster: M. capricolum subsp capripneumoniae (Mccp) M. capricolum subsp 31
capricolum (Mcc), M. leachii and M. mycoides subsp capri (Mmc, including the “LC” and 32
“capri” serovars). Extracted EPS were characterized by nuclear magnetic resonance, resulting 33
in the identification of a homopolymer of -(1-2)-glucopyranose (glucan) in Mccp and M. 34
leachii. Monoclonal antibodies specific for this glucan and for the MmmSC-secreted galactan, 35
were used to detect the two polysaccharides. While Mmc strains of the “LC” serovar produced 36
only capsular galactan, no polysaccharide could be detected in strains of the “capri” serovar. 37
All strains of Mccp and M. leachii produced glucan CPS and EPS, whereas glucan production 38
and localization varied among Mcc strains. Genes associated with polysaccharide synthesis, 39
and forming a biosynthetic pathway, were predicted in all cluster members. These genes were 40
organized in clusters within two loci representing genetic variability hotspots. Phylogenetic 41
analysis showed that some of these genes, notably galE and glf, were acquired via horizontal 42
gene transfer. These findings call for a reassessment of the specificity of the serological tests 43
based on mycoplasma polysaccharides. 44
45
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Introduction 46
47
Within the Class Mollicutes, the so-called “Mycoplasma mycoides” cluster (MMC) (1) is 48
unique: all the members of the MMC are pathogenic for ruminants yet, from a phylogenetic 49
point of view, this cluster belongs to the "Spiroplasma" clade, which includes a number of 50
species isolated from plants or insects (2). The taxonomy of the MMC was modified recently 51
to reflect more precisely the phylogeny of this group. Because all the cluster members are 52
closely related, their 16S rDNA sequences did not provide sufficient resolution to 53
discriminate them accurately (3). A multi-locus sequence typing approach was used to obtain 54
a more precise phylogeny (4), which lead to a simplification of the taxonomy (5) (Table 1). 55
The cluster currently comprises five species or subspecies: Mycoplasma mycoides subsp. 56
mycoides, which was known formerly as the “Small Colony” biotype (1) (MmmSC), M. 57
mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum (Mcc), M. capricolum subsp. 58
capripneumoniae (Mccp) and M. leachii. In addition, Mmc is subdivided in two serovars: the 59
“Large Colony” serovar (Mmc“LC”) and the “capri” serovar (Mmc“capri”). Among the 60
MMC, two subspecies are particularly important, Mccp and MmmSC, which are, 61
respectively, the etiologic agents of contagious caprine pleuropneumonia (CCPP) and 62
contagious bovine pleuropneumonia (CBPP). These two diseases are characterized by 63
unilateral pleuropneumonia in goats and cattle respectively. Because they are absent or have 64
been eradicated from many regions or continents they are notifiable to the world organization 65
for animal health (OIE) and their detection leads to a ban on live animal trade. By 66
comparison, the other members of the MMC have a worldwide distribution and induce lesions 67
affecting various organs: mastitis, arthritis, keratitis, pneumonia, septicemia etc. 68
Due to the importance of CBPP for international trade, many studies have been performed to 69
try and unravel the virulence factors of MmmSC. The release of the whole genome sequence 70
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of MmmSC, in 2004 (6) did not allow for the identification of any known virulence factors 71
such as genes coding for toxins, adhesins or secretion systems described in other pathogenic 72
bacteria. Putative phase variation of surface lipoproteins was identified (7) that could play a 73
role in escaping the host immune response but this phenomenon was certainly not as 74
pronounced in MmmSC as in other mycoplasmas (8). Finally, the close association to host 75
cells and the release of H2O2 and other reactive oxygen species through glycerol metabolism 76
was proposed as a virulence mechanism (9) in a similar way to what had been shown in M. 77
pneumoniae (10). However, some MmmSC vaccine strains, such as T1/44, KH3J and V5, 78
possess the ability to metabolize glycerol and produce significant quantities of H2O2 while 79
being attenuated (11). Hence, H2O2 production alone is not the sole virulence mechanism and 80
other factors may be important. One, that certainly deserves attention, is the synthesis and 81
secretion of polysaccharides. We will refer here to polysaccharides that are found in tight 82
association with the mycoplasma membrane forming a capsule as capsular polysaccharide 83
(CPS), whereas free, secreted polysaccharides or those only loosely adherent to the membrane 84
will be referred to as exopolysaccharide (EPS). This polysaccharide secretion was 85
documented long ago and EPS was detected as a soluble product in the blood of MmmSC-86
infected cattle at an early stage of the infection with simple techniques such as the 87
"Ouchterlony" immune-precipitation in agar (12). Polysaccharides were soon regarded as 88
virulence candidates for MmmSC that could be involved notably in adhesion, resistance to 89
desiccation, resistance to complement activity or direct stimulation of inflammation. As a 90
consequence, immunological reactions to intravenous or subcutaneous inoculations were 91
studied (13) and many efforts were made to characterize these products. The composition of 92
MmmSC-CPS was determined from concentrated washed mycoplasmas (14) but the 93
characterization of the EPS was more problematic. The main difficulty resulted from the use 94
of complex medium for the growth of MmmSC, which hampered the extraction of pure EPS 95
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devoid of medium contaminants. More recently, a procedure that included the incubation of 96
washed mycoplasma cells into a defined medium, devoid of extraneous polysaccharide 97
components, was a key factor for the characterization of MmmSC’s EPS (15). Results showed 98
notably that the composition of this EPS matched exactly that of the CPS (i.e. a 99
galactofuranose homopolymer) and that the alternate secretion of CPS or EPS was correlated 100
with the variation of expression of an active glucose permease belonging to a 101
phosphotransferase system (PTS-G). Owing to the importance of the MMC members as 102
pathogens for small and large ruminants worldwide, as well the possible use of the 103
polysaccharides as diagnostic or vaccine antigens, we decided to characterize these secreted 104
polysaccharides in all mycoplasmas of the MMC. In addition, the recent publication of at least 105
one full genome for each of the MMC members opened new avenues for comparative and 106
evolutionary genomic studies. We searched for genes that could be part of the metabolic 107
pathways for CPS or EPS production to understand why all these species differ in terms of 108
polysaccharide production and secretion while being so closely related from a phylogenetic 109
point of view. 110
111
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Materials and methods 112
Mycoplasma strains and culture conditions 113
Mycoplasma strains used in this study are listed in table 2. They were selected to reflect the 114
broad geographical distribution of the species. For biochemical analyses, mycoplasma strains 115
with sequenced and annotated genomes and, whenever possible, few in vitro passages, were 116
selected. Mycoplasmas were cultured in PPLO medium according to Hayflick with few 117
modifications [PPLO base (Difco, 21g/L), enriched with fresh yeast extract (10%), horse 118
serum decomplemented for 1h at 56°C (15%), glucose (1g/L) and sodium pyruvate (2g/L)] at 119
37°C, 5% CO2. 120
121
EPS production and extraction 122
EPS were produced and extracted as previously described (15). Briefly, mycoplasmas were 123
harvested in the late exponential phase of growth (14 000×g, 30 min, 4°C) and washed once 124
in phosphate buffered saline (PBS) before being resuspended in CMRL-1066 medium. After 3 125
days of incubation at 37°C, mycoplasmas were pelleted by centrifugation at 14 000×g for 1h 126
at 4°C. Then, tri-chloroacetic acid (TCA) was added to the supernatant (10% w/v) and the 127
mixture was incubated at 4°C for 2h. The proteinaceous precipitate was removed after 128
centrifugation (14 000×g, 1h, 4°C). Nine volumes of cold acetone were added to the final 129
supernatant and the mixture was incubated at least 24h at -20°C to allow precipitation of 130
polysaccharides. After centrifugation (14 000×g, 1h, 4°C) and drying, the EPS pellets were 131
dissolved in ultrapure sterile water. 132
133
Chemical analyses 134
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135
The monosaccharide components were determined by high-performance anion exchange 136
chromatography (HPAEC) with a pulsed amperometric detector (Dionex ICS 3000 system) 137
and polysaccharide structure by NMR as described previously (15). 138
Briefly, for HPAEX analysis, the EPS were hydrolysed, and anionic compounds separated 139
through CarboPac PA1 columns with a multistep gradient elution procedure. Peak analysis 140
was performed using Chromeleon software, version 7.0. 141
For the NMR analysis, the 1H NMR spectra were recorded, at 80 °C, on a Bruker Avance 500 142
spectrometer equipped with a 5 mm BBI probe and Topspin 1.3 software. 1H NMR spectra 143
were accumulated using a 30° pulse angle, a recycle time of 1 s and an acquisition time of 2 s 144
for a spectral width of 3 000 Hz for 32 K data points with a pre-saturation of the HOD signal 145
using a pre-saturation sequence provided by Bruker. 13
C NMR experiments were conducted 146
on the same spectrometer operating at 125.48 MHz with 2 s as relaxation delay. 147
The 2D 1H/
1H COSY,
1H/
1H TOCSY,
1H/
1H NOESY,
1H/
13C HSQC and
1H/
13C HMBC 148
spectra were acquired with standard pulse sequences delivered by Bruker. 149
150
Monoclonal antibody production and characterization 151
Balb/c mice were immunized twice intraperitonally with MmmSC-EPS extracts mixed with 152
incomplete Freund adjuvant and once intravenously with EPS (50 µg) suspended in water. 153
Three days later, dilacerated spleen cells and SP2/O cells were fused according to standard 154
procedures (16). The protocol (ref: CIRAD-12CBP001) was approved by the regional ethical 155
committee. The hybridoma culture supernatants were screened using MmmSC-EPS extract as 156
a positive control and a sham extract from non-inoculated PPLO medium as negative control 157
antigens. The reactivity of the MAbs was tested according to Harlow and Lane (16). Briefly, 158
these EPS extracts were spotted on nitrocellulose, incubated 30 min with the supernatants, 159
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washed and incubated with anti-mouse HRP conjugate (P0260 DAKO). After the final 160
washing the spots were revealed with diaminobenzidine (DAB) -Nickel chloride-H2O2 161
substrate. Another hybridoma cell line, "4.83" kept frozen in liquid nitrogen, was retrieved 162
from a previous fusion for the production of Mccp-recognizing MAbs (17). It was selected as 163
it recognized a soluble product with a distinct smear in dot-blot experiments. 164
The reactivity of the selected Mabs was monitored by western blotting using antigens 165
harvested after the incubation into CMRL-1066 medium: concentrated and washed 166
mycoplasma pellets, crude CMRL-1066 supernatant and concentrated EPS. These samples 167
were analyzed on denaturing polyacrylamide gel gradient 4 to 15% (Biorad 456-1083EDU) in 168
1x SDS, Tris, Glycine running buffer (Biorad 161-0732). After electrophoresis, protein and 169
polysaccharidic detection was performed with Coomassie Brilliant Blue and Periodic acid-170
Schiff (PAS) methods respectively. Immunodetection by western-blotting was performed 171
after transfer to nitrocellulose membranes at 10V during 1h15. Membranes were probed with 172
MAbs 4.83 and 2.1.31, followed by horseradish peroxidase-conjugated secondary antibodies 173
and detection with DAB-Nickel-H2O2 substrate. Dot blotting was used to detect the 174
polysaccharides in various mycoplasma cultures. Concentrated and PBS washed mycoplasma 175
pellet was used to detect CPS and culture supernatant was used as antigen to detect EPS. Dot 176
blotting was performed as described for western blotting except that samples (1 to 5 µl) were 177
directly spotted onto nitrocellulose. 178
179
In silico analyses 180
Genes potentially involved in polysaccharide biosynthetic pathways in MMC members were 181
identified and retrieved by BLASTP similarity search (18) using the MolliGen database 182
(19)(http://molligen.org) with genes already identified within MmmSC genomes (15) and a e-183
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Value threshold of 10-8
. Additional genes were identified by queries on annotated genes 184
within the MolliGen database or through the Carbohydrate-Active enZymes database (CAZy, 185
http://www.cazy.org/). The evolutionary history of biosynthetic pathways in individual 186
subspecies was inferred from phylogenetic analyses. Protein sequences were retrieved and 187
aligned by MUSCLE (20) using the MolliGen database. Protein phylogenies were inferred by 188
the maximum likelihood method in the MEGA6 molecular evolutionary genetic tool package 189
(21). The best fitting model of evolution was evaluated with ProtTest (22) for each of the 190
alignments and the “LG with frequencies (+F)” model was finally chosen. All positions 191
containing gaps and missing data were eliminated (complete deletion option) and bootstrap 192
analysis was carried out with 500 replicates. When supported by significant bootstrap values, 193
incongruence between protein and species phylogenies was considered indicative of a 194
potential HGT. A tree with the housekeeping gene fusA was built as a reference tree reflecting 195
the species phylogeny. 196
197
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Results 198
199
Chemical characterization of EPS from “Mycoplasma mycoides” cluster members by HPAEC 200
and NMR. 201
EPS was extracted from six mycoplasma cultures representing the MMC as described in 202
material and methods. Despite several attempts, no detectable amount of EPS was obtained 203
with strains 95010 (Mmc”LC”), PG3T
(Mmc”capri”) and C.kidT (Mcc). By contrast, EPS 204
were obtained with strains 8740-Rita (MmmSC), 95043 (Mccp) and PG50T (M. leachii) and 205
they were subjected to total acid hydrolysis with 4 M trifluoroacetic acid. Sugar analysis 206
revealed the presence of galactose as the only sugar for MmmSC strain 8740-Rita, as 207
previously described for MmmSC strain Afadé. The 1H NMR and 2D NMR spectra of EPS 208
for MmmSC 8740-Rita was identical to that of MmmSC strain Afadé (data not shown) and 209
characteristic of (1→6)-galactofuranose polymers. 210
The acid hydrolysate and 1H NMR spectra of EPS from Mccp 95043 and M. leachii PG50
T 211
showed that these polysaccharides were composed only by glucose. Based on correlations 212
observed in 2D NMR COSY spectrum (Figure 1A) various chemical shifts to proton of 213
glucosyl residues could be attributed: 4.873 (H-1), 3.575 (H-2), 3.793 (H-3), 3.477 (H-4), 214
3.503 (H-5), 3.774 (H-6) and 3.946 (H-6’) ppm. On the 2D NMR HSQC spectrum (Figure 215
1B), the connectivities observed between H-1 and C-1 (103.26 ppm), H-2/C-2 (83.91 ppm), 216
H-3/C-3 (76.82 ppm), H-4/C-4 (70.17 ppm), H-5/C-5 (77.47 ppm) and H-6, H-6’/C-6 (62.17 217
ppm) were characteristic of a glucan polymer. The H-1 signal at 4.873 ppm (1,2
J = 7.83 Hz) 218
and C-1 at 103.26 ppm were typical of D-glucose residues with pyranoside configuration. 219
H-2 chemical values indicated the presence of a linkage on the C-2 in glucose residues. This 220
linkage was confirmed by the presence of connectivities observed in 1H/
1H NOESY between 221
H-1 of glucose (δ 4.873 ppm) and signal at δ 3.575 ppm belonging to H-2 of glucose. The 222
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connectivities were also observed in 1H/
13C HMBC spectrum. These results showed that EPS 223
of Mccp 95043 and M.leachii PG50T were identical and composed of a (1→2)-224
glucopyranose polymer. Only one signal for C-2 (83.91 ppm) was present in the 13
C spectrum. 225
This was an indication that the polymer was linear as a cyclic (1→2)-glucan polymer would 226
have yielded multiple signals (23). 227
In summary, EPS was successfully extracted and characterized in 3 out of 6 strains from the 228
MMC. EPS from 8740-Rita (MmmSC) was made of (1→6)-galactofuranose polymer 229
(galactan) whereas EPS from 95043 (Mccp) and PG50T (M. leachii) was made of (1→2)-230
glucopyranose polymer (glucan). 231
Monoclonal antibody characterization and polysaccharide detection. 232
Monoclonal antibodies (MAbs) specific for polysaccharides were produced to allow the 233
detection of these products. More specifically, two MAbs 2.1.31 (IgG1) and 4.83 (IgM) were 234
selected by dot blot analyses, as they bound to the homologous extracted EPS and PPLO 235
culture supernatants of MmmSC 8740-Rita and Mccp 95043 respectively but not to PPLO 236
medium control or to the other EPS (Figure S1). The Coomassie blue staining of the SDS-237
PAGE gel allowed the detection of protein bands only in the concentrated mycoplasma pellet 238
(Figure 2A), an indication that the EPS extracts did not contain proteins or peptides at 239
detectable concentrations by this technique, although low levels of protein contamination may 240
have been needed for the migration of the neutral polysaccharide component. The PAS 241
staining, which binds to polysaccharide compounds, revealed a clear difference between the 242
concentrated mycoplasma pellets and the EPS extracts (Figure 2B). All preparations from 243
both mycoplasmas showed a positive smear at high molecular weight but the concentrated 244
mycoplasma pellets contained an additional conspicuous spot of low molecular weight, very 245
probably a glycolipid, which was not present in the EPS extracts. The western blot performed 246
with the two MAbs yielded a positive smear of high molecular weight, similar to the PAS 247
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positive smear (Figure 2C). This ruled out the possibility of recognition by the MAb of any 248
immunoreactive contaminating product that may have been present in the antigen used to 249
immunize the mice. The MAbs 2.1.31 and 4.83 were therefore confirmed to recognize a 250
(1→6)-galactofuranose polymer (galactan) and a (1→2)-glucopyranose polymer (glucan), 251
repectively. Owing to the lack of cross reactivity of each of the two MAbs with the other 252
polysaccharide, we could use these MAbs to detect galactan and glucan in various 253
mycoplasma species using concentrated washed mycoplasma pellets to detect CPS and culture 254
supernatant to detect EPS (Figure 3). 255
As expected, galactan, recognized by MAb 2.1.31, was detected as CPS and EPS in MmmSC 256
strains Afadé and 8740 (Figure 3A). Galactan was also clearly detected as CPS in Mmc”LC” 257
strains YGR and 95010 but not in Mmc”capri” strain PG3
T, which yielded a very faint 258
reaction. Dots with faint positivity were observed in the supernatants of Mmc”LC” strains 259
YGR and 95010 suggesting a weak secretion of EPS by these strains or the presence of 260
cellular debris in the supernatant. With MAb 2.1.31, very faint or no reactions were observed 261
with Mcc (C.kidT, 7714), Mccp (Abomsa, 95043) and M. leachii (PG50
T, 06049-C3). Results 262
obtained with 4 additional Mmc”LC” strains were quite homogeneous, with galactan being 263
detected as CPS in the cell pellet and a faint positivity as EPS in the supernatant (Figure 264
S2A). No positivity was detected with any of the 4 additional Mmc”capri” strains tested (data 265
not shown). 266
Glucan, recognized by MAb 4.83, was detected in Mccp strains as well as in M. leachii strains 267
as CPS and EPS (Figure 3B). Results were more variable with Mcc strains. No glucan was 268
detected with the reference strain C.kidT while glucan CPS and EPS was detected for strain 269
7714. Very faint or no reactivity was observed with MAb 4.83 with Mmc and MmmSC 270
strains. Results obtained with 5 additional Mcc strains differed from one strain to another, 271
with glucan being detected as CPS and EPS clearly in some strains (07046, 7714, 02053, 272
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10074), while it was only faintly detected as CPS in two other strains (IPX, 13051) (Figure 273
S2B). The ability to synthesize and secrete glucan varies greatly among Mcc strains under 274
these conditions. 275
276
In silico analysis of EPS biosynthetic pathways in the ”Mycoplasma mycoides” cluster 277
We compared the galactan predicted biosynthetic pathway that was described for MmmSC 278
(15) with those found in the other MMC members by performing BLASTP searches as well as 279
data mining using the complete genomes and databases that are now available. Two predicted 280
biosynthetic pathways, possibly leading to the production of either galactofuranose or 281
glucopyranose homopolymers, were evidenced and are presented in Figure 4. The first steps 282
leading to the formation of UDP-glucose are similar in the two pathways. They involve 283
transport systems predicted to import and phosphorylate glucose (PTS permeases, Glk), a 284
phosphoglucomutase, (ManB) to isomerize Glucose-6-P into Glucose-1-P, and UTP-glucose-285
1-phosphate uridylyltransferases (GalU) to obtain UDP-Glucose. The first pathway, leading to 286
the production of galactan, includes an UDP-glucose 4-epimerase (GalE) and an UDP-287
galactofuranose mutase (Glf) that are involved in the isomerization of UDP-Glc into UDP- 288
galactopyranose and the conformational change of the pyranose ring into UDP-289
galactofuranose, respectively. Multiple glycosyl transferases were identified in this pathway. 290
Two of them were identified as belonging to the GT2 family, but were clearly segregated into 291
two sub-families based on similarity comparisons. Another glycosyl transferase could not be 292
classified according to CAZY. This first pathway was observed in MmmSC and Mmc”LC” 293
genomes. For sake of clarity we will refer hereafter to the genes and locus tags observed in 294
the Mmc”LC” genome. The second pathway, which was found in all the other MMC 295
members, differs from the first by the absence of GalE and Glf. Nevertheless, it contains 296
genes leading to the formation of UDP-glucose residues and those coding for 297
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glycosyltransferases, which is consistent with the synthesis of a glucan homopolymer. An 298
additional subfamily of glycosyl transferase, GT2-3, was identified in this second pathway. 299
Interestingly, Mmc serovar ”capri” differed with serovar “LC”, which possesses both galE 300
and glf genes. 301
The genes that differentiate the two pathways (galU, galE and glf-GT2) are organized in two 302
clusters positioned at two loci that show a remarkable degree of polymorphism within the 303
MMC (Figure 5). A single and well conserved locus exists among Mcc, Mccp and M. leachii 304
genomes with the glycosyl transferase and galU genes being flanked by the pepQ-ribCF and 305
rplK clusters of genes. However, the two loci in the Mycoplasma mycoides genomes are 306
highly variable. They are hotspots for the insertion of mobile elements such as insertion 307
sequences (IS1296, IS1634, ISMmy1, ISMmy2, notably in MmmSC and Mmc”LC”) and a 308
restriction modification system (hsdR/hsdM) paired with a phage family integrase (int) in 309
Mmc”capri”. They are also hotspots for horizontal gene transfer. Notably in locus two, the 310
phylogenetic analysis indicated that the pair of genes galE/glf, involved in galactan synthesis, 311
has been exchanged between ancestors of the MMC and M. bovigenitalium (Figure 6A and 312
B), as the phylogenetic trees differed markedly from that of manB (Figure 6C) and that of the 313
housekeeping gene fusA (Figure S3A). Within Mmc, significant genome re-arrangements 314
have occurred with highly different structures of the loci in PG3T compared to 95010 and 315
GM12. The genome of strain PG3T has retained the structure of the loci observed in M. 316
capricolum-related genomes, while a recombination seems to have occurred between the two 317
loci in the Mmc”LC” genomes, with the downstream ilvA cluster instead of rplK cluster. In 318
addition, a 40 kbp inversion between two IS sequences has occurred just upstream of the 319
galE/glf pair in GM12 locus 2 (Figure 5). In MmmSC genomes, the general organization of 320
the two loci is similar to that of Mmc”LC” genomes but with an increased complexity due to 321
the duplication of the genes related to polysaccharide synthesis. Interestingly, the number of 322
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duplications varies from one strain to another, as shown by comparison of the genomes from 323
strains PG1T and Gladysdale. 324
325
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DISCUSSION 326
Monosaccharide composition and glycosidic linkages of polysaccharides synthesized by 327
mycoplasmas have been determined for a limited number of species. In M. pulmonis, EPS-I 328
contains equimolar amounts of glucose and galactose, with undetermined linkage (24) while 329
EPS-II, involved in biofilm formation, and another polysaccharide of M. pneumoniae contain 330
also N-acetylglucosamine (25). Members of the MMC differ from M. pneumoniae and M. 331
pulmonis and appear diverse in terms of polysaccharide production and secretion in spite of 332
their very close phylogenetic relationship. Two different polysaccharide products were 333
detected and strains differed in their ability to produce and/or secrete these compounds. 334
Galactan, a galactofuranose homopolymer, was detected as CPS and EPS in MmmSC strains. 335
The same compound was detected with the galactan-specific MAb in all Mmc serovar “LC” 336
strains as CPS, in the mycoplasma cell pellets. However, only faint signals were observed in 337
the supernatants, indicating that EPS secretion was very limited or that the positivity was due 338
to cellular debris. No galactan was detected in Mmc serovar “capri” strains. Our results 339
showing that MmmSC and Mmc“LC” share the same type of CPS may explain why cross-340
reactions were observed between these two mycoplasmas (previously classed as different 341
biotypes of the same subspecies) in the growth inhibition technique, which was formerly 342
considered a reference technique to identify species (26). Strains of Mmc serovar “capri”, 343
which do not produce galactan but are phylogenetically very closely related to Mmc’LC”, do 344
not produce the same cross-reactions with MmmSC. The absence of galactan in Mmc“capri” 345
strains had already been noticed when the chemical composition of the nucleic acids and 346
macromolecular components was studied by Jones (27). In this previous study, the only 347
polysaccharide detected in the type strain PG3T was composed of glucose in very small 348
amount (0.5% of the dry weight) compared to galactan, which represented 10% of the dry 349
weight of MmmSC. The potential ability of Mmc”capri” strains to produce a polysaccharide, 350
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as suggested by its gene repertoire, remains to be studied, as well as the conditions that would 351
favor such a production. 352
A linear (1→2)-glucopyranose homopolymer was detected as CPS and EPS in Mccp and M. 353
leachii. This composition differed from what had been already described for Mccp strains 354
(28). In that study, the monosaccharide components of the Mccp-EPS had been identified as 355
glucose, galactose, mannose, fucose, galactosamine and glucosamine. The difference with our 356
results may be attributed to the crude preparations used in 1987 to precipitate the 357
polysaccharide from the culture supernatant. These preparations were probably contaminated 358
by polysaccharides present in the very rich medium needed to grow these fastidious 359
mycoplasma strains. Serum and yeast extract are often the source of contamination (29) and 360
may have biased some earlier studies. Our concentration procedure ensured that the 361
precipitated product was pure mycoplasma polysaccharide. The linear (1→2)-glucopyranose 362
homopolymer identified in Mccp and M. leachii is rarely found in bacteria. Very short linear 363
-D-(1,2)-glucans were evidenced in the periplasm of E. coli and tropical Rhizobium species 364
(30, 31) and cyclic -D-(1→2)-glucans found for instance in Mesorhizobium species (23, 32) 365
or Brucella (33) are more frequent. 366
Diverse polysaccharide profiles were observed with Mcc strains when tested with the MAb 367
4.83. No polysaccharide was detected in the type strain C. kidT, while some other Mcc strains 368
yielded positive results in the pellet only and others in the pellet and supernatant. The genetic 369
basis of this phenotypic difference has not been unraveled yet. The stock of strain C. kidT 370
analyzed could well be a variant that does not produce polysaccharide. Mycoplasma strains, 371
notably those kept as reference cultures, have been passaged and cloned many times to ensure 372
their purity. This cloning procedure may have been responsible for the selection of variants 373
that differ from the original stock. 374
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Within the MMC, there are three species or subspecies that were shown to secrete EPS: 375
MmmSC, M. leachii, Mccp and one species with varying abilities to secrete EPS, Mcc. We 376
could not identify any specific transport system in their genomes that would distinguish them 377
from their non-secreting relatives of the MMC. However, we observed a common 378
characteristic to the three secreting species: the disruption of the putative lipoprotein gene 379
located immediately upstream of a glycosyltransferase (homologous to MLC_7190) that was 380
shown to be specific to the Spiroplasma clade (Figure S4). The disruption of the lipoprotein 381
gene was not a strain-specific event, as it was also disrupted in all the Mccp, M. leachii and 382
MmmSC strains examined. This glycosyltransferase is also flanked downstream by a 383
conserved hypothetical protein (MLC_7180) with four predicted transmembrane segments 384
and an RDD motif that may be associated with a transport activity. This cluster of genes 385
(MLC_7200-7190-7180) may therefore code for a polysaccharide export complex in the 386
MMC. If confirmed, this would imply that polysaccharide secretion in these three species may 387
be altered, a consequence of genome decay, with a reduced functionality, preventing the 388
linkage of the polysaccharide moiety to the membrane-anchored moiety to form a CPS. A 389
previous study had already shown that the levels of CPS/EPS production in MmmSC was 390
modulated by the expression of a PTS-G gene (15) that depended on phase variations. It is 391
highly probable that CPS/EPS production in the members of the “Mycoplasma mycoides” 392
cluster is tightly regulated considering the importance of these products for bacterial survival 393
in various ecological niches. In many bacteria, CPS/EPS regulation may depend on promoters 394
(34) and external factors such as oxygen availability (35) or quorum sensing (36). Similar 395
studies would be needed to decipher the regulatory mechanisms at stake in mycoplasmas. The 396
polysaccharide specific MAbs could be used in capture ELISA tests to monitor the level of 397
polysaccharide secretion in vitro or in vivo and evaluate how these levels are correlated with 398
strain virulence. They could also be used in electron microscopic assays to evaluate CPS 399
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thickness. Parallel studies, with in vitro cellular models, could be performed with purified 400
EPS to evaluate their impact on host gene transcription. 401
The phenotypic variability that we observed in the MMC, as regards to polysaccharide 402
production, was paralleled with a marked genetic polymorphism. The candidate genes 403
involved in polysaccharide production are grouped in clusters and positioned within two loci 404
which are hotspots of genetic variability. This variability is highlighted by the presence of 405
insertion sequences of various types, recombination events, gene duplication, (with duplicated 406
genes being often truncated by frameshift mutations) and by the presence of genes that have 407
been shown to be exchanged by horizontal gene transfer (HGT). The importance of HGT has 408
been recognized only recently in mycoplasmas (37, 38). Genes involved in polysaccharide 409
synthesis must be added to the list of genes that can be exchanged by HGT in mycoplasmas. 410
This is no exception to other prokaryotes, as genes for polysaccharide synthesis are often 411
carried by mobile genetic elements such as plasmids in lactococci (39) or are closely 412
associated to insertion sequences, as in Desulfovibrio (40), Lactococcus (41) or 413
Crocosphaera (42). Moreover, the genes identified in the MMC often do not have any 414
homologue in other mycoplasma genomes (Figure S3B). The other mycoplasmas may not 415
produce any polysaccharide or have completely different polysaccharide gene repertoires, 416
leading to the production of a variety of products. Further work will be needed to elucidate 417
their exact composition and their role in host-pathogen interactions. At the onset of infection, 418
polysaccharides are involved in the resistance to the innate immune responses (43, 44) but 419
further investigations are needed to evaluate the respective role of CPS and EPS in the 420
induction of the exacerbated lung inflammation which is the hallmark of CBPP and CCPP. 421
Polysaccharides present as soluble products are T-independent antigens, while CPS, 422
polysaccharide coupled probably to diglycosyldiacylglycerol residues (45), will trigger a T-423
dependent immune response (46) and a strong memory response. This type of response, which 424
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is directed to a mycoplasma surface-exposed antigen, may be important for protection and 425
may help designing more potent vaccines in the future. 426
Meanwhile, our results may have rapid practical applications for diagnostic tests. The 427
complement fixation test (prescribed by the OIE) and the latex agglutination test for CCPP are 428
based on polysaccharide antigen (47, 48). Their specificity may be jeopardized by the 429
frequent infections of goats with MMC species, and notably Mcc strains, which are likely to 430
induce false positive results. Mcc strains represented 26% of the French mycoplasma strains 431
isolated from goats between 2003 and 2008 (49). The specificity of these tests has to be re-432
evaluated. 433
434
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Figure captions 435
436
437
Figure 1: 2D 1H/
1H COSY NMR spectrum (A) and 2D
1H/
13C HSQC NMR spectrum (B) of 438
EPS produced by M. leachii strain PG50T. The H-1 signal at 4.873 ppm (
1,2J = 7.83 Hz) and 439
C-1 at 103.26 ppm were typical of D-glucose residues with pyranoside configuration (dark 440
arrow). The single signal for C-2 (83.91 ppm) was present in the 13
C spectrum (light arrow) 441
was indicative of a linear polymer. This spectrum is typical of a linear (1→2)-glucopyranose 442
polymer. Identical spectra were obtained with Mccp EPS. 443
444
445
Figure 2: Staining of MmmSC 8740-Rita and Mccp 95043 profiles after polyacrylamide gel 446
electrophoresis: (A) Coomassie blue, (B) and Schiff’s reagent, and (C) immunoblotting of 447
their extracted EPS by MAbs 448
Three samples were analyzed for strain 8740-Rita (MmmSC) and 95043 (Mccp) : washed 449
mycoplasmas obtained after incubation during 80h in CMRL supplemented with 3g/L of 450
glucose (lanes 1), CMRL supernatant after 80h of incubation (lanes 2) and extracted EPS 451
from these supernatants (lanes 3). In Western immunoblot analysis, EPS from MmmSC and 452
Mccp were labeled respectively with MAbs 2.1.31 and 4.83. 453
454
455
Figure 3: Dot blot analysis of polysaccharides produced by representative strains of the 456
“Mycoplasma mycoides” cluster with MAb 2.1.31 (A) and MAb 4.83 (B). 457
P: Pelleted mycoplasma cells grown from PPLO medium to detect CPS; S: supernatants from 458
PPLO culture to detect EPS. 459
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MAb 2.1.31 yielded positive results with pelleted MmmSC strains (Afadé and 8740) and 460
Mmc”LC” (95010 and YGR). MmmSC supernatants yielded clearly positive dots while 461
Mmc”LC” supernatants gave only faint positive dots. No product was detected by this MAb 462
with M. leachii or M. capricolum (Mcc and Mccp) strains, nor with the reference Mmc”capri” 463
strain PG3T. 464
MAb 4.83 yielded positive results with pelleted cells and supernatant from M. leachii (PG50T) 465
and Mccp (95043). A discrepancy of results was observed with Mcc reference strain C.kidT 466
that does not yield any positivity, while positive dots are observed for Mcc strain 7714, both 467
in the pellet and supernatant. No positivity is observed with Mmc and MmmSC strains with 468
this MAb. 469
470
471
Figure 4: Overview of the polysaccharide biosynthetic pathways in the “Mycoplasma 472
mycoides” cluster (MMC). 473
The various genes involved in these pathways are represented on the left side for M. mycoides 474
(MmmSC and Mmc”LC”) and on the right side for M. capricolum (Mcc and Mccp) and M. 475
leachii. Locus tag numbers are mentioned for a representative genome for each of the species. 476
Genes highlighted in blue were detected in all members of the MMC. Others were found in a 477
limited number of species. Highlighted genes in green, orange and yellow correspond to genes 478
found in clusters. Genes that show the highest similarity level within the MMC were 479
positioned on the same line. Two galU families were identified as well as three glycosyl 480
transferase type 2 sub-families. Pseudogenes are represented with vertical stripes in the 481
background. galE, glf and some glycosyl transferase-coding genes were notably absent from 482
Mmc”capri”, M. capricolum and M. leachii. 483
ND: not detected; CPS: capsular polysaccharide; EPS: secreted exopolysaccharide. 484
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485
Figure 5: Comparison of genome regions including galU, galE, glf and gt2 genes in the 486
“Mycoplasma mycoides” cluster. 487
The two loci involved are represented. Only locus 1 is found in Mcc with highly similar 488
organisation in Mccp and M. leachii. The two loci are flanked by conserved clusters of genes, 489
named pepQ, ilvA, opp and rplK clusters and used in the schematic representation as 490
milestones. Homologous genes are represented as boxes with the same color. Pseudogenes are 491
represented with vertical stripes in the background. The locus tag of one Mmc”LC” gene is 492
indicated as a reference for each locus, to facilitate association with the text and other figures. 493
494
495
496
Figure 6: Phylogeny of some genes involved in polysaccharide biosynthetic pathways. 497
A and B: Phylogenetic tree obtained with GalE and Glf respectively. The trees were generated 498
by the Maximum Likelyhood method and bootstrap values are indicated at each node of the 499
tree. These genes are found only in a few number of Mollicutes and the tree topology differs 500
from the species tree. In the “Mycoplasma mycoides” cluster, galE and glf are found only in 501
Mmc”LC” genomes as well as in MmmSC strains. Their closest homologues are found in the 502
M. bovigenitalium genome or in M. fermentans genome (for galE). This topology and the 503
observation that galE and glf are not found in the other members of the Spiroplasma clade 504
strongly suggests that these genes were acquired by Mmc”LC” and MmmSC by horizontal 505
gene transfer from an M. bovigenitalium ancestor. 506
C: Phylogenetic tree obtained with manB homologs. The topology of this tree is similar to that 507
of the species tree (Fig S2A), except that some branches are missing. Notably, manB is found 508
in all members of the Spiroplasma clade. The tree topology is indicative of a vertical 509
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transmission of this gene in all the Mollicutes where it is found. It may have been deleted 510
from the other genomes. 511
512
Figure S1: Dot blot analysis of exopolysaccharides extracted from representative strains of 513
the “Mycoplasma mycoides” cluster detected by MAb 2.1.31 (A) and MAb 4.83 (B). 514
The reactivity of the two selected MAbs was assessed by dot-blot with extracted EPS. MAb 515
2.1.31 reacted only with MmmSC EPS (strains Afadé and 8740). MAb 4.83 reacted with EPS 516
extracted from Mccp (Abomsa and 95043) and M. leachii (PG50, 06049-C3) strains. There 517
was no evidence of any cross-reaction with the two MAbs. An absence of positive reaction 518
signs either an absence of EPS or the presence of an EPS which is not detected by the MAb. 519
520
521
522
Figure S2: Dot blot analysis of polysaccharides detected in Mmc serovar “LC” strains with 523
MAb 2.1.31 (A) and Mcc strains with MAb 4.83 (B). 524
The two MAbs were used to detect polysaccharides in mycoplasma cells grown in PPLO 525
medium and washed once in PBS (pellets, P) and corresponding culture supernatants (S). 526
MmmSC 8740 and Mccp 95043 were used as positive controls for each of the MAbs. 527
All Mmc”LC” strain pellets yielded a positive result with MAb 2.1.31 while a faint positivity 528
existed with the supernatants. The detection of Mcc strains with MAb 4.83 was more variable, 529
as the intensity varied among pellets although all cultures were very turbid at the time of 530
harvest. In addition this MAb yielded a positive result for some supernatants. 531
532
Figure S3: Distribution of polysaccharide-associated genes in the “Mycoplasma mycoides” 533
cluster (MMC) and their homologues in Mollicutes. 534
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A: Phylogenetic tree of the Mollicutes based on a conserved housekeeping gene, fusA. The 535
tree was generated by the Maximum Likelyhood method; bootstrap values are indicated for 536
each node. Phylogenetic groups are indicated: S, spiroplasma (boxed in blue); MMC (boxed 537
in green); H, hominis; P, pneumoniae; AP, Acholeplasma/Phytoplasma. 538
B: Distribution of the polysaccharide-associated genes in Mollicutes. Species names and locus 539
tags are mentioned on the left side of the table. Mnemonics for the genes identified in the 540
MMC are indicated on top of the table, using the genome of Mmc”LC” strain 95010 as a 541
reference. Colored dots are indicative of a BLASTP significant result, evalue e-8
, for the 542
respective Mollicutes species. The blue color was retained for genes found in all MMC 543
members (manB and a glycosyltransferase MLC_7190). Orthologs to MLC_7190 were 544
notably absent from all other Mollicutes genomes. Orange, yellow and green colors were used 545
for clusters of genes found only in some species of the MMC as defined in figure 4. 546
547
548
Figure S4: Synteny of the gene cluster surrounding a type 2 glycosyltransferase in the 549
“Mycoplasma mycoides” cluster. 550
The cluster of genes found in the genome of Mmc serovar"LC" strain 95010 was chosen as 551
reference. Gene names and AA numbers are indicated on the top. The species and names of 552
strains are indicated on the left and underlined names correspond to strains for which EPS was 553
detected. The figures under the genes indicate similarity values to the reference Mmc 554
serovar"LC" genes. 555
The lipoprotein with a DUF285 motif found upstream of the glycosyltransferase is duplicated 556
in M. capricolum subsp. capricolum C. kidT, as well as in M. capricolum subsp. 557
capripneumoniae 9231-Abomsa. In the latter genome, the two copies are interrupted by 558
frameshift mutations, and are considered pseudogenes. There is a strict correlation between 559
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EPS-secreting strains and the presence of a pseudogenized lipoprotein upstream from the 560
glycosyl transferase. The conserved hypothetical protein downstream from the glycosyl 561
transferase possesses an RDD domain that may be associated with transport activity. 562
563
564
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Acknowledgements 565
This work was supported by a PhD grand awarded jointly by ANSES and CIRAD to 566
Clothilde Bertin. We wish to thank the LABGeM team within the “France Genomics” 567
infrastructure, Evry France, for their help in the genome annotation and analysis through the 568
Microscope platform as well as the CBiB in Bordeaux, France, for their help in the annotation 569
of the Mmc”capri” PG3T genome within the EVOLMYCO project. 570
Finally we would like to thank L. Lakhdar for her technical help in mice immunization at the 571
“Animal experimentation platform” in Lyon as well as M. Dejean for the extraction of the 572
polysaccharides at CIRAD, Montpellier. 573
574
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References 575
576
577
1. Cottew GS, Breard A, DaMassa AJ, Erno H, Leach RH, Lefevre PC, Rodwell AW, 578
Smith GR. 1987. Taxonomy of the Mycoplasma mycoides cluster. Isr. J. Med. Sci. 23:632-579
635. 580
2. Gasparich GE, Whitcomb RF, Dodge D, French FE, Glass J, Williamson DL. 2004. The 581
genus Spiroplasma and its non-helical descendants: phylogenetic classification, correlation 582
with phenotype and roots of the Mycoplasma mycoides clade. Int. J. Syst. Evol. Micr. 54:893-583
918. 584
3. Weisburg WG, Tully JG, Rose DL, Petzel JP, Oyaizu H, Yang D, Mandelco L, Sechrest 585
J, Lawrence TG, Van Etten J, et al. 1989. A phylogenetic analysis of the mycoplasmas: 586
basis for their classification. J. Bacteriol. 171:6455-6467. 587
4. Manso-Silvan L, Perrier X, Thiaucourt F. 2007. Phylogeny of the Mycoplasma mycoides 588
cluster based on analysis of five conserved protein-coding sequences and possible implications 589
for the taxonomy of the group. Int. J. Syst. Evol. Micr. 57:2247-2258. 590
5. Manso-Silvan L, Vilei EM, Sachse K, Djordjevic SP, Thiaucourt F, Frey J. 2009. 591
Proposal to assign Mycoplasma leachii sp. nov. as a new species designation for Mycoplasma 592
sp. bovine group 7 of Leach, and to reclassify Mycoplasma mycoides subsp. mycoides LC as a 593
serovar of Mycoplasma mycoides subsp. capri Int. J. Syst. Evol. Micr. 59:1353-1358. 594
6. Westberg J, Persson A, Holmberg A, Goesmann A, Lundeberg J, Johansson KE, 595
Pettersson B, Uhlen M. 2004. The genome sequence of Mycoplasma mycoides subsp. 596
mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia 597
(CBPP). Genome Res. 14:221-227. 598
7. Persson A, Jacobsson K, Frykberg L, Johansson KE, Poumarat F. 2002. Variable surface 599
protein Vmm of Mycoplasma mycoides subsp. mycoides small colony type. J. Bacteriol. 600
184:3712-3722. 601
on June 9, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
Page 29
29
8. Glew MD, Marenda M, Rosengarten R, Citti C. 2002. Surface diversity in Mycoplasma 602
agalactiae is driven by site-specific DNA inversions within the vpma multigene locus. J. 603
Bacteriol. 184:5987-5998. 604
9. Pilo P, Frey J, Vilei EM. 2007. Molecular mechanisms of pathogenicity of Mycoplasma 605
mycoides subsp. mycoides SC. Vet. J. 174:513-521. 606
10. Hames C, Halbedel S, Hoppert M, Frey J, Stülke J. 2009. Glycerol metabolism is 607
important for cytotoxicity of Mycoplasma pneumoniae. J. Bacteriol. 191:747-753. 608
11. Bischof DF, Janis C, Vilei EM, Bertoni G, Frey J. 2008. Cytotoxicity of Mycoplasma 609
mycoides subsp. mycoides small colony type to bovine epithelial cells. Infect. Immun. 76:263-610
269. 611
12. Kurotchkin TJ. 1937. Specific carbohydrate from Asterococcus mycoides for serological 612
tests of bovine pleuropneumonia. Proc. Soc. Exp. Biol. Med. 37:21-22. 613
13. Shifrine M, Gourlay RN. 1965. The immediate type allergic skin reaction in contagious 614
bovine pleuropneumonia. J. Comp. Pathol. 75:381-385. 615
14. Buttery SH, Plackett P. 1960. A specific polysaccharide from Mycoplasma mycoides. J. Gen. 616
Microbiol. 23:357-368. 617
15. Bertin C, Pau-Roblot C, Courtois J, Manso-Silván L, Thiaucourt F, Tardy F, Le Grand 618
D, Poumarat F, Gaurivaud P. 2013. Characterization of Free Exopolysaccharides Secreted 619
by Mycoplasma mycoides Subsp. mycoides. PLoS ONE 8:e68373. 620
16. Harlow E, Lane D. 1988. Antibodies, a laboratory manual. Cold Spring Harbor Laboratory, 621
New York. 622
17. Thiaucourt F, Bolske G, Libeau G, Le Goff C, Lefevre PC. 1994. The use of monoclonal 623
antibodies in the diagnosis of contagious caprine pleuropneumonia (CCPP). Vet. Microbiol. 624
41:191-203. 625
18. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. 626
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. 627
Nucleic Acids Res. 25:3389-3402. 628
on June 9, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
Page 30
30
19. Barré A, de Daruvar A, Blanchard A. 2004. MolliGen, a database dedicated to the 629
comparative genomics of Mollicutes. Nucleic Acids Res. 32:D307-D310. 630
20. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high 631
throughput. Nucleic Acids Res. 32:1792-1797. 632
21. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: 633
Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, 634
and maximum parsimony methods. Mol. Biol. Evol. 28:2731-2739. 635
22. Abascal F, Zardoya R, Posada D. 2005. ProtTest: selection of best-fit models of protein 636
evolution. Bioinformatics 21:2104-2105. 637
23. Choma A, Komaniecka I. 2003. Characterisation of Mesorhizobium huakuii cyclic beta-638
glucan. Acta biochim. Pol. 50:1273-1281. 639
24. Daubenspeck JM, Bolland JR, Luo W, Simmons WL, Dybvig K. 2009. Identification of 640
exopolysaccharide-deficient mutants of Mycoplasma pulmonis. Mol. Microbiol. 72:1235-641
1245. 642
25. Simmons WL, Daubenspeck JM, Osborne JD, Balish MF, Waites KB, Dybvig K. 2013. 643
Type 1 and type 2 strains of Mycoplasma pneumoniae form different biofilms. Microbiology 644
159:737-747. 645
26. Al-Aubaidi JM, Dardiri AH, Fabricant J. 1972. Biochemical characterization and antigenic 646
relationship of Mycoplasma mycoides subsp. mycoides, Freundt and Mycoplasma mycoides 647
subsp. capri (Edward) Freundt. Int. J. Syst. Bacteriol. 22:155-164. 648
27. Jones AS, Tittensor JR, Walker RT. 1965. The chemical composition of the nucleic acids 649
and other macromolecular constituents of Mycoplasma mycoides var. capri. J. Gen. Microbiol. 650
40:405-411. 651
28. Rurangirwa FR, McGuire TC, Magnuson NS, Kibor A, Chema S. 1987. Composition of a 652
polysaccharide from mycoplasma (F-38) recognised by antibodies from goats with contagious 653
pleuropneumonia. Res. Vet. Sci. 42:175-178. 654
on June 9, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
Page 31
31
29. Daubenspeck JM, Jordan DS, Dybvig K. 2014. The glycocalyx of Mollicutes, p. 131–147. 655
In Browning GF, Citti C (ed.), Mollicutes: Molecular Biology and Pathogenesis. Caister 656
Academic Press, Norfolk. 657
30. Geiger O, Weissborn AC, Kennedy EP. 1991. Biosynthesis and excretion of cyclic glucans 658
by Rhizobium meliloti 1021. J. Bacteriol. 173:3021-3024. 659
31. Weissborn AC, Rumley MK, Kennedy EP. 1992. Isolation and characterization of 660
Escherichia coli mutants blocked in production of membrane-derived oligosaccharides. J. 661
Bacteriol. 174:4856-4859. 662
32. Kawaharada Y, Kiyota H, Eda S, Minamisawa K, Mitsui H. 2008. Structural 663
characterization of neutral and anionic glucans from Mesorhizobium loti. Carbohyd. Res. 664
343:2422-2427. 665
33. Guidolin LS, Ciocchini AE, Iñón de Iannino N, Ugalde RA. 2009. Functional mapping of 666
Brucella abortus cyclic β-1,2-glucan synthase: Identification of the protein domain required 667
for cyclization. J. Bacteriol. 191:1230-1238. 668
34. Shainheit MG, Mulé M, Camilli A. 2014. The core promoter of the capsule operon of 669
Streptococcus pneumoniae is necessary for colonization and invasive disease. Infect. Immun. 670
82:694-705. 671
35. Geno KA, Hauser JR, Gupta K, Yother J. 2014. Streptococcus pneumoniae 672
phosphotyrosine phosphatase CpsB and alterations in capsule production resulting from 673
changes in oxygen availability. J. Bacteriol. 196:1992-2003. 674
36. Lee K-J, Kim J-A, Hwang W, Park S-J, Lee K-H. 2013. Role of capsular polysaccharide 675
(CPS) in biofilm formation and regulation of CPS production by quorum-sensing in Vibrio 676
vulnificus. Mol. Microbiol. 90:841-857. 677
37. Sirand-Pugnet P, Lartigue C, Marenda M, Jacob D, Barre A, Barbe V, Schenowitz C, 678
Mangenot S, Couloux A, Segurens B, de Daruvar A, Blanchard A, Citti C. 2007. Being 679
pathogenic, plastic, and sexual while living with a nearly minimal bacterial genome. PLoS 680
Genet 3:e75. 681
on June 9, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
Page 32
32
38. Beven L, Charenton C, Dautant A, Bouyssou G, Labroussaa F, Skollermo A, Persson A, 682
Blanchard A, Sirand-Pugnet P. 2012. Specific evolution of F1-like ATPases in 683
mycoplasmas. PLoS One 7:e38793. 684
39. Forde A, Fitzgerald GF. 2003. Molecular organization of exopolysaccharide (EPS) encoding 685
genes on the lactococcal bacteriophage adsorption blocking plasmid, pCI658. Plasmid 49:130-686
142. 687
40. Walker CB, Stolyar S, Chivian D, Pinel N, Gabster JA, Dehal PS, He Z, Yang ZK, Yen 688
H-CB, Zhou J, Wall JD, Hazen TC, Arkin AP, Stahl DA. 2009. Contribution of mobile 689
genetic elements to Desulfovibrio vulgaris genome plasticity. Environ. Microbiol. 11:2244-690
2252. 691
41. Dabour N, LaPointe G. 2005. Identification and molecular characterization of the 692
chromosomal exopolysaccharide biosynthesis gene cluster from Lactococcus lactis subsp. 693
cremoris SMQ-461. Appl. Environ. Microbiol. 71:7414-7425. 694
42. Bench SR, Ilikchyan IN, Tripp HJ, Zehr JP. 2011. Two strains of Crocosphaera watsonii 695
with highly conserved genomes are distinguished by strain-specific features. Front. Microbiol. 696
2:261. 697
43. Shaw BM, Daubenspeck JM, Simmons WL, Dybvig K. 2013. EPS-I polysaccharide 698
protects Mycoplasma pulmonis from phagocytosis. FEMS Microbiol. Lett. 338:155-160. 699
44. Gaurivaud P, Lakhdar L, Grand DL, Poumarat F, Tardy F. 2014. Comparison of in vivo 700
and in vitro properties of capsulated and non-capsulated variants of Mycoplasma mycoides 701
subsp. mycoides strain Afadé: a potential new insight into the biology of contagious bovine 702
pleuropneumonia. FEMS Microbiol. Lett. Aug 14. doi: 10.1111/1574-6968.12579 703
45. Andres E, Martinez N, Planas A. 2011. Expression and characterization of a Mycoplasma 704
genitalium glycosyltransferase in membrane glycolipid biosynthesis: potential target against 705
mycoplasma infections. J. Biol. Chem. 286:35367-35379. 706
46. Snapper CM. 2012. Mechanisms underlying in vivo polysaccharide-specific immunoglobulin 707
responses to intact extracellular bacteria. Ann. NY Acad. Sci. 1253:92-101. 708
on June 9, 2019 by guesthttp://aem
.asm.org/
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Page 33
33
47. March JB, Gammack C, Nicholas R. 2000. Rapid detection of contagious caprine 709
pleuropneumonia using a Mycoplasma capricolum subsp. capripneumoniae capsular 710
polysaccharide-specific antigen detection latex agglutination test. J. Clin. Microbiol. 38:4152-711
4159. 712
48. Rurangirwa FR, McGuire TC, Kibor A, Chema S. 1987. A latex agglutination test for field 713
diagnosis of contagious caprine pleuropneumonia. Vet. Rec. 121:191-193. 714
49. Chazel M, Tardy F, Le Grand D, Calavas D, Poumarat F. 2010. Mycoplasmoses of 715
ruminants in France: recent data from the national surveillance network. BMC Vet. Res. 6:32. 716
717
718
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H-3/H-4
H-1/H2
H-2/H3
H-3/H4
H-5/H6’ H-6/H-6’
H-3/H4
H-2/H3
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kDa
250
150
100
75
50
37
25
20
15
10
MmmSC Mccp 1 2 3 1 2 3
(A)
kDa
250
150
100
75
50
37
25
20
15
10
MmmSC Mccp 1 2 3 1 2 3
(B) kDa
250
150
100
75
50
37
25
20
2.1.31 4.83 MmmSC Mccp
(C)
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A
B
Afadé 8740
PG3T YGR 95010
Abomsa 95043 C.kidT
7714 PG50T 06049-C3
Afadé 8740
PG3T YGR 95010
Abomsa 95043 C.kidT
7714 PG50T 06049-C3
P S P S P S
P S P S P S
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Glucose
Glc-6-P
Glc-1-P
UDP-Glc
UDP-Galp
UDP-Galf
Free galactan
Glucose
Glk 2.7.1.2
ManB 5.4.2.2
GalU 2.7.7.9
GalE 5.1.3.2
Glf 5.4.99.9
Glycosyl Transferases GT2 family
Membrane bound
galactan Lack
of
coupling
MmmSC Mmc «LC » Mmc «capri » Mcc Mccp M. leachii
PG1T 95010 PG3T C.kidT Abomsa PG50T
MSC_0054 ND MMC_8150 MCAP_0844 MCCP01_0964 ND
MSC_0873 MLC_7890 MMC_0400 MCAP_0094 MCCP01_0117 MSB_A0139
MSC_0860
MSC_0874 MLC_7900 MMC_0390 MCAP_0093 MCCP01_0116 MSB_A0138
MSC_0861
MSC_0875 MLC_7910 MMC_0380 MCAP_0092 MCCP01_0115 MSB_A0137
MSC_0863
MSC_0829 MLC_7500 MMC_0730 MCAP_0756 MCCP01_0854 MSB_A0787
MSC_0110 MLC_1070 MMC_6600 ND ND ND
MSC_0990
ND MLC_8170 ND MCAP_0064 MCCP01_0082 MSB_A0105
MSC_0971 MLC_8200 ND ND ND ND
MSC_0978
MSC_0985
MSC_0970
MSC_0977 MLC_8210 ND ND ND ND
MSC_0984
MSC_0108 MLC_8190 ND ND ND ND
MSC_0973 MLC_1040 ND ND ND ND
MSC_0980
MSC_0987
MSC_0993
MSC_0109 MLC_1060 ND ND ND ND
MSC_0974
MSC_0982
MSC_0988
ND ND MMC_0120 MCAP_0063 MCCP01_0081 MSB_A0104
MMC_6610
MSC_0771 MLC_7190 MMC_1050 MCAP_0719 MCCP01_0815 MSB_A0745
CPS EPS
PTS permease
Glucose
Glc-6-P
Glc-1-P
UDP-Glc
Free glucan
Glucose
Glk 2.7.1.2
ManB 5.4.2.2
GalU 2.7.7.9
Membrane bound glucan
Lack
of
coupling
CPS EPS
PTS permease
Pathway I Pathway II
Glycosyl Transferases
1
2
GT2-1
GT2-2
GT2-3
Glycosyl Transferases (not classified)
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pepQ cluster ilvA cluster
ribCF cluster
rplK cluster pepQ cluster
pepQ cluster ilvA cluster
3 imperfect repeats in Gladysdale)
Mcc (Mccp) (M. leachii)
pepQ cluster rplK cluster
MmmSC PG1
Locus 1
gt2-3 galU-2
hsdM hsdR gt2-3
gt2-1 ISMmy2
gt2-2 galU-1
gt2-1
gt2-2
IS1634
IS1634
pepQ cluster ilvA cluster gt2-1
gt2-2
MCAP_0064
MMC_0120
MLC_1070
MMCAP2_0115
MSC_0110
Mmc 95010
PG3
GM12
int
galU-1
galU-1
MLC_1120
MLC_1060
MLC_1040
MLC_1020
MLC_0880
ribCF cluster
ribCF cluster
ribCF cluster
opp cluster rplK cluster
glf galE
gt2-1
opp cluster ilvA cluster
gt2
MmmSC PG1 opp cluster rplK cluster
3 imperfect repeats in PG1 and Gladysdale
Locus 2
HP
ISMmy1
IS1634 glf galE
gt2-1
gt2-2 IS1634
gt2-1
uvr cluster rplK cluster
glf galE
gt2-1
IS1296
40 kbp inversion
MMCAP2_0811
MMC_6600
MLC_8170
MSC_0990
galU-2
galU-2
galU-1
galU-1
Mmc 95010
PG3
GM12
opp cluster ilvA cluster
Mcc (Mccp) (M. leachii)
MLC_8160 MLC_8190
MLC_8200
MLC_8210
MLC_8220
ribCF cluster
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Spiroplasma clade
MMC
C
A
B
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TABLE1 Taxonomy of the "Mycoplasma mycoides cluster" "Mycoplasma mycoides cluster" _________________________________________________________________________________________________________________________ Mycoides sub-cluster Capricolum sub-cluster ____________________________________________________________ _________________________________________________________ 1987 taxonomy a M. m. capri M. m. mycoides LC M. m. mycoides SC M. sp. Group 7 M.c. capricolum M.c. capripneumoniae Current taxonomy b M. m. capri M. m. mycoides M. leachii M.c. capricolum M.c. capripneumoniae Abbreviation Mmc MmmSC Mcc Mccp Serovars Mmc "capri" Mmc "LC" Type/reference strain PG3T YGR PG1T PG50T C. kidT F38T a based on Cottew et al. 1987 b based on Manso-Silvan et al. 2009 M. m.: Mycoplasma mycoides subsp. M. c.: Mycoplasma capricolum subsp.
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Table 2: List of Mycoplasma strains or genome sequences studied _______________________________________________________________________________________________ Species Strain designation Country Supplier/reference Genome Acc Number ____________________________________ ________________ ______________ ______________ __________________ M. capricolum subsp. capricolum California kidT USA Cordy, 1955 NC_007633.1
(Mcc) 7714 France CIRAD
13051 (14232) France ANSES-Lyon
10074 Tajikistan SVIS
2007-046 France ANSES-Niort
IPX France CIRAD
2002-053 (VP28L) India IVRI
90122 (C1547) Ivory Coast LPA
M. capricolum subsp. capripneumoniae 9231-Abomsa Ethiopia NVI-E LM995445
(Mccp) 95043 Niger CIRAD
M1601 China Chu et al. 2011 NZ_CM001150.1
99108 Eritrea SVS JMJI01000000
M. leachii PG50T Australia Simmons & Johnston, 1963 NC_014751.1
06049-C3 Nigeria NVRI-N
M. mycoides subsp. capri serovar capri PG3T Turkey Tully, 1974 JFAE00000000
(Mmc “capri”) 2003-045 India CIRG
9139 Turkey NVI-ET
N108 Nigeria CIRAD
L France CIRAD
M. mycoides subsp. capri serovar LC Y-goatR Australia Laws, 1956
(Mmc “LC”) 95010-C1 France CIRAD NC_015431.1
Kombolcha Ethiopia NVI-ET
99045 (55507-1) Germany IVRI
99111 India TiHo
2007-073 France ANSES-Niort
GM12 USA NZ_CP001621.1
M. mycoides subsp. mycoides Afadé Cameroon CIRAD
(MmmSC) 8740-Rita Cameroon LANAVET
PG1T Not known NC_005364.2
Gladysdale Australia NC_021025.1
_______________________________________________________________________________________________
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ANSES: Agence Nationale de Sécurité Sanitaire, Lyon, France, Dr. Poumarat and Niort, France, Dr. Mercier CIRAD : Centre de coopération Internationale en Recherche Agronomique pour le Développement, Montpellier, France,
Dr Thiaucourt CIRG: Central Institute for Research in Goats, Mathura, India, Dr. Rana IVRI : Indian Veterinary Institute, Izatnagar, India, Dr. Singh LANAVET : Laboratoire National Vétérinaire, Garoua, Cameroon, Dr .Yaya LPA : Laboratoire Central de Pathologie Animale, Bingerville, Ivory Coast, Dr. Domenech NVI-ET : National Veterinary Institute, Debre-Zeit, Ethiopia, Dr. Laikemariam NVRI-N: National Veterinary Research Institute, Vom, Nigeria, Dr. Nwankpa SVIS: State veterinary Inspection Services, Dushanbe, Tajikistan, Dr. Amirbekov SVS: Senhit Veterinary Service, Asmara, Eritrea, Dr. Tesfaalem Teklegiorgis TiHo: Tierärztliche Hochschule, Hannover, Germany, Dr. Schmidt
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