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<front> Genetic Variation in Sialidase and Linkage to N-acetylneuraminate <lb/>Catabolism in Mycoplasma synoviae <lb/> Meghan May and Daniel R. Brown * <lb/> Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of <lb/>Florida, Gainesville, Florida 32611-0880, USA <lb/> Abstract <lb/> We explored the genetic basis for intraspecific variation in mycoplasmal sialidase activity that <lb/>correlates with virulence, and its potentially advantageous linkage to nutrient catabolism. <lb/>Polymorphism in N-acetylneuraminate scavenging and degradation genes (sialidase, N-<lb/>acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate <lb/>epimerase, N-acetylglucosamine-6-phosphate deacetylase, and glucosamine-6-phosphate <lb/>deaminase) was evident among eight strains of the avian pathogen Mycoplasma synoviae. Most <lb/>differences were single nucleotide polymorphisms, ranging from 0.34 ± 0.04 substitutions per 100 <lb/>bp for N-acetylmannosamine kinase to 0.65 ± 0.03 for the single-copy sialidase gene nanI. Missense <lb/>mutations were twice as common as silent mutations in nanI; 26% resulted in amino acids dissimilar <lb/>to consensus; and there was a 12-base deletion near the nanI promoter in strain WVU1853 T , <lb/>supporting a complex genetic basis for differences in sialidase activity. Two strains had identical <lb/>frameshifts in the N-acetylneuraminate lyase gene nanA, resulting in nonsense mutations, and both <lb/>had downstream deletions in nanA. Such genetic lesions uncouple extracellular liberation of sialic <lb/>acid from generation of fructose-6-phosphate and pyruvate via intracellular N-acetylneuraminate <lb/>degradation. Retention of nanI by such strains, but not others in the M. synoviae phylogenetic cluster, <lb/>is evidence that sialidase has an important non-nutritional role in the ecology of M. synoviae and <lb/>certain other mycoplasmas. <lb/> Keywords <lb/> Mycoplasma synoviae; polymorphism; N-acetylneuraminate catabolism; sialidase; virulence <lb/></front>
<body>1. Introduction <lb/> Mycoplasma synoviae is a major avian pathogen associated with osteoarthritis, synovitis, and <lb/>respiratory tract lesions in gallinaceous birds [1,2,3]. Infection can produce disease that ranges <lb/>from subclinical to severe, and clinical outcome can be influenced by co-infection with other <lb/>agents [4,5,6,7,8]. The majority of prior studies have focused on cytadherence and/or <lb/>hemadsorption as pathogenic mechanisms of M. synoviae, with particular attention to <lb/></body>
<front>*Corresponding author: Daniel R. Brown, Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University <lb/>of Florida, Gainesville, Florida 32611-0880, USA, Tel.: +1 352 392 2239 X3975; fax: +1 352 392 9704, Email address: <lb/>BrownD@vetmed.ufl.edu (Daniel R. Brown). <lb/></front>
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<front>NIH Public Access <lb/> Author Manuscript <lb/> Microb Pathog. Author manuscript; available in PMC 2009 July 1. <lb/> Published in final edited form as: <lb/> Microb Pathog. 2008 July ; 45(1): 38–44. <lb/> NIH-PA Author Manuscript <lb/> NIH-PA Author Manuscript <lb/> NIH-PA Author Manuscript <lb/></front>
<body>antigenically-variable hemagglutinins, although the molecular basis of M. synoviae <lb/> pathogenicity is still not well-understood [9,10]. <lb/>The recently annotated genome of M. synoviae field isolate 53 includes putative sialidase <lb/> nanI (synonymous with nanH of Gram-negative species and strain 53 GenBank accession no. <lb/> ABS50356), N-acetylneuraminate lyase nanA, N-acetylmannosamine kinase nagC, N-<lb/>acetylmannosamine-6-phosphate epimerase nanE, N-acetylglucosamine-6-phosphate <lb/>deacetylase nagA, and glucosamine-6-phosphate deaminase nagB genes in a locus comprising <lb/>a canonical sialic acid scavenging and degradation pathway (Figure 1A) [11]. This was <lb/>unexpected, although sialidase activity is common in other pathogenic bacteria [12], because <lb/>it is very rare in mycoplasmas, having been described previously only in the lethal pathogen <lb/>of alligators Mycoplasma alligatoris [13] and an extinct strain of the avian pathogen <lb/> Mycoplasma gallisepticum [14, 15, 16]. The term sialic acid is the family name covering all <lb/>derivatives of neuraminic acid [17], the aldol condensation product of D-mannosamine and <lb/>pyruvic acid, which are potential bacterial nutrients. In vertebrate animals including birds, <lb/>diverse sialic acid derivatives are involved in recognition processes, cellular connections with <lb/>extracellular matrix (ECM) components, and intercellular interactions [18]. They protect <lb/>against hydrolysis of the glycosidic or peptide bonds of oligosaccharides, glycoproteins, <lb/>glycolipids and gangliosides located on eukaryotic cell surfaces, and against degradation of <lb/>the ECM. In addition, sialylated lipopolysaccharide and polysialic acid capsules are surface <lb/>features of certain Gram-negative and Gram-positive bacteria [19]. <lb/>Exo-α-sialidases (EC 3.2.1.18) catalyze hydrolysis of α-(2–3)-, α-(2–6)-, and/or α-(2–8)-<lb/> glycosidic linkages of terminal sialic acid residues on oligosaccharides, glycoproteins, <lb/>glycolipids, colominic acid (a homopolymer of N-acetylneuraminic acid), and synthetic <lb/>substrates. Synonyms for sialidase include neuraminidase, α-neuraminidase, and N-<lb/>acylneuraminate glycohydrolase. Most bacterial sialidases preferentially cleave α-(2–3)-linked <lb/> sialic acids, and are found in species that live in close contact with vertebrate host cells as <lb/>commensals or facultative pathogens. Sialidase activity is involved in bacterial colonization <lb/>and dissemination, ECM degradation, and induced host-cell death [12,20,21,22,23]. It has also <lb/>been proposed that bacterial desialylation of host glycoconjugates could expose or form new <lb/>host antigens to play a role in autoimmune complications of infection [24,25]. <lb/>Most recently, in support of the prediction based on the genomic sequence of strain 53 that a <lb/>functional sialidase gene occurs in M. synoviae, sialidase activity was readily detected in <lb/>several additional strains [26,27]. This suggested that an ability to desialylate sialoconjugates <lb/>present in its environment is important in the ecology of M. synoviae. Strikingly, strains <lb/>originally isolated from clinically symptomatic birds had significantly more sialidase activity <lb/>than strains from asymptomatic birds [27], and certain strains lacked detectable sialidase <lb/>activity [26], suggesting substantial intraspecies genetic heterogeneity and a role for sialidase <lb/>activity in the virulence of the organism. In bacterial genomes, including that of M. synoviae <lb/> strain 53, sialidase genes are often part of a locus encoding additional enzymes that enable the <lb/>import and intracellular catabolism of free sialic acid. This pathway culminates with the <lb/>production of fructose-6-phosphate for entry to glycolysis [28], constituting a bacterial nutrient <lb/>stream that might be essential to offset any selective disadvantage of increased virulence <lb/>attributable to desialylation of host glycoconjugates [20]. In the present study, we explored the <lb/>genetic basis for the observed intraspecific variation in sialidase activity, and its potentially <lb/>advantageous linkage to nutrient catabolism, by characterizing the natural DNA sequence <lb/>diversity within the sialic acid degradation locus among eight strains of M. synoviae. <lb/>
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2. Results <lb/> 2.1. Amplification of the sialic acid degradation locus <lb/> An 8.4 kb PCR amplicon, including the 7.9 kb sialic acid degradation locus as predicted from <lb/>the genomic sequence of strain 53 [11], was amplified from M. synoviae strains K3344, <lb/>K4907A, K5395B, MS173, MS178, and WVU1853 T [27] using the flanking primers described <lb/>in section 4.2. Passages 33 and 126 of the FMT strain [3] only generated approximately 8 kb <lb/>amplicons, suggesting the deletion of a portion(s) of the locus. Homologs of nanA, nanE, nanI, <lb/>nagA, nagB, and nagC were present in each strain in the same orientations as in strain 53 (Figure <lb/>1B). The 521 bp gap between nagC and nanE, annotated for the strain 53 genome as encoding <lb/>the 151 aa hypothetical protein of unknown function MS53_0196 (GenBank accession no. <lb/> AAZ43615), was also conserved. When Southern blots of their fragmented genomic DNA <lb/>were probed to determine nanI copy number, the strains having the highest (WVU1853 T ) and <lb/>lowest (K4907A and K5395B) amounts of sialidase activity all exhibited the banding pattern <lb/>predicted from the whole-genome sequence of strain 53, showing that only a single copy of <lb/> nanI was present in those strains despite their having almost 100-fold differences in sialidase <lb/>activity per colony-forming unit (CFU) [27]. <lb/> 2.2. Nucleotide sequence variability <lb/> Numerous point mutations with respect to the consensus sequence were observed throughout <lb/>the locus in each of the eight strains. The mean (± standard error) number of substitutions per <lb/>100 bp across the six genes constituting the locus ranged from 0.34 ± 0.09 for strain MS173 <lb/>to 0.65 ± 0.08 for strain K4907A (Table 1). The mean number of substitutions per 100 bp within <lb/>each gene ranged from 0.34 ± 0.04 for the 861 bp N-acetylmannosamine kinase nagC to 0.65 <lb/>± 0.03 for the 2,817 bp sialidase nanI (MS53_0199, GenBank accession no. YP_278329). In <lb/>contrast, the number of substitutions per 100 bp within the two 16S rRNA genes of M. <lb/>synoviae was only 0.04. For comparison to another species of mycoplasma, the number of <lb/>substitutions per 100 bp in the 1,002 bp signal recognition particle receptor subunit Y gene <lb/> ftsY ranged from 0.14 in Mycoplasma hyopneumoniae strain J (GenBank accession no. <lb/> AE017243) to 0.42 in strain 232 (AE017332), and in the 1,392 bp transcriptional dual regulator <lb/> dnaA gene ranged from 0.2 in M. hyopneumoniae strains J and 232 to 0.3 in strain 7448 <lb/>(AE017244). <lb/> 2.3. Guanine+cytosine content <lb/> The guanine+cytosine content (%G+C) was calculated for the sialic acid degradation genes <lb/>from each strain, individually and for the consensus sequence of each gene (Table 2). Genes <lb/>of this locus tended to have a slightly higher %G+C than the 27% calculated for the M. <lb/>synoviae strain 53 genome as a whole [11], ranging from 27.2 ± 0.11% for N-<lb/>acetylglucosamine-6-phosphate deacetylase nagA to 33.1 ± 0.23% for N-<lb/>acetylmannosamine-6-phosphate epimerase nanE (MS53_0197, GenBank accession no. <lb/> AAZ43616). <lb/> 2.4. Insertions, deletions, frameshift, and nonsense mutations <lb/> There were no insertions, deletions, or frameshift mutations, with respect to the consensus <lb/>sequence, in the protein-coding sequences of the nanE, nanI, nagA, nagB, and nagC genes of <lb/>the eight strains examined. However, strain WVU1853 T had a 12 bp deletion in the flanking <lb/>glyceraldehyde-3-phosphate dehydrogenase gapDH-nanI intergenic region, beginning at base <lb/>minus 41 from the predicted nanI start codon (Figure 2A), that substantially altered the <lb/>predicted nucleic acid stem-loop secondary structure of the putative nanI promoter region. The <lb/>891 bp nanA gene contained a 2 bp insertion, common to strains FMT (passages 33 and 126) <lb/>and K3344, creating a frameshift with respect to the consensus sequence that resulted in seven <lb/>
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premature stop codons and effectively disabled the gene. Also, deletions of 14 and 462 bp were <lb/>present in strains K3344 and FMT (both passages), respectively, downstream of the nanA <lb/> nonsense mutations (Figure 2B). The deletion in strain FMT was bracketed by the direct repeats <lb/>5′-AAT TTC TTC A-3′, and completely overlapped the deletion in strain K3344 (Figure 2C). <lb/>The deletions accounted for the notably short PCR amplicon obtained for the locus from both <lb/>passages of strain FMT. Strain WVU1853 T had a nonsense mutation arising from a single <lb/>nucleotide substitution in nanA, predicted to result in the loss of 17 aa from the carboxyl <lb/>terminus of the 296 aa NanA of the other strains. <lb/> 2.5. Missense and silent mutations <lb/> Missense mutations with respect to the consensus sequences were approximately twice as <lb/>common as silent mutations in the nanI and nagA genes (Figure 3). The number of missense <lb/>and silent mutations was approximately equal in nanA and nagC, and nagB and nanE genes <lb/>had slightly more silent mutations. For perspective, silent mutations were approximately 2.5 <lb/>and 15 fold more common than missense mutations in the M. hyopneumoniae genes dnaA and <lb/> ftsY, respectively. <lb/>Missense mutations resulting in dissimilar amino acids were common throughout the sialic <lb/>acid degradation locus in all eight M. synoviae strains. They represented 26%, 28%, and 26% <lb/>of all mutations in NanI, NagA, and NagB, respectively (Figure 3). Approximately half of the <lb/>dissimilar amino acid substitutions in NanI were in the functional domain defined by the <lb/>Conserved Domain Database [29]. The signature Arg-Ile-Pro and two Ser-X-Asp-X-Gly-X-<lb/>Thr-Trp " Asp box " motifs [30], plus Asp box variants Thr-X-Asp-X-Gly-X-Thr-Trp and Ser-<lb/>X-Asp-X-Gly-X-Asn-Trp, were conserved in NanI. Candidate equivalents of the highly-<lb/>conserved Arg 37 , Asp 54 , Arg 56 , Asp 62 , Asp 100 , Glu 230 , Arg 245 , and Tyr 347 residues <lb/>(numbering of the Clostridium perfringens NanI; GenBank accession no. P10481) were readily <lb/>identified by inspection of local and global sequence alignments, and were conserved across <lb/>strains, but no equivalent of the Arg 312 that is strictly conserved in many other bacterial and <lb/>eukaryotic sialidases [31] could be recognized in any strain. All of the dissimilar amino acid <lb/>substitutions of NanA, NanE, NagA, NagB, and NagC were in their broadly-defined functional <lb/>domains [29]. Dissimilar amino acid changes were least common in NanA, representing just <lb/>4% of all mutations in nanA across strains. Missense mutations resulting in dissimilar amino <lb/>acids were not present in either dnaA or ftsY genes in the three strains of M. hyopneumoniae <lb/> examined. <lb/> 3. Discussion and conclusions <lb/> The recent discovery by Vasconselos et al. [11] of putative genes for sialidase and the N-<lb/>acetylneuraminate catabolism pathway in M. synoviae strain 53, when sialidase activity was <lb/>believed to be extremely rare among mycoplasmas [25,32], prompted us and others to confirm <lb/>the annotation by using assays for the enzyme to examine additional strains [26,27]. The <lb/>activity was present in most, but not all, M. synoviae strains examined. Unexpectedly, although <lb/>strain WVU1853 T had by far the most activity, there was essentially continuous variation in <lb/>the amount of sialidase activity per CFU among other strains, which also correlated positively <lb/>with the degree of strain virulence. In this study, we sought to explain the remarkable <lb/>intraspecific variation in activity of this candidate virulence factor by measuring nanI copy <lb/>number in high-and low-activity strains, and by sequencing the sialidase gene nanI and genes <lb/>constituting the sialic acid degradation locus in multiple strains. <lb/>Although the presence and relative spacing of signature motifs and active site residues are <lb/>conserved in bacterial sialidase catalytic domains, their primary amino acid sequences and <lb/>lengths are otherwise highly variable [30]. The NanI sialidase of M. synoviae has a theoretical <lb/>and observed [33] molecular weight of approximately 109 kDa. It consists of an approximately <lb/>
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420 aa N-terminal domain that includes a predicted 29 aa transmembrane region near the N-<lb/> terminus, and an approximately 520 aa C-terminal six-sheet beta-propellor catalytic domain, <lb/>Pfam 00064 [34]. NanI has been reported to be extracellular surface-localized in M. <lb/> synoviae and M. gallisepticum [15,26], consistent with most other bacterial sialidases that are <lb/>secreted to effect their actions on the surrounding environment [20]. Negligible activity was <lb/>found in M. synoviae-conditioned cell-free broth [26,27]. Since extracellular surface-<lb/>localization precludes an influence of substrate import on the enzyme's activity in situ, the <lb/>quantitative differences in sialidase activity of the magnitude observed for M. synoviae are <lb/>most simply explained by interstrain variations in the topology of the enzyme. The evidence <lb/>for a complex genetic basis for the intraspecific variation includes: 1) numerous single <lb/>nucleotide polymorphisms with respect to their consensus nanI DNA sequence; resulting in 2) <lb/>a comparatively high frequency of dissimilar missense mutations with respect to the consensus <lb/>NanI amino acid sequence; 3) presence of only a single copy of nanI regardless of sialidase <lb/>activity per CFU; and 4) strict conservation of all but one of the several residues believed to <lb/>constitute the active site [26,28,30,31]. For perspective, individual site-directed mutagenesis <lb/>of each of the active site residues other than Arg 312 reduced C. perfringens sialidase specific <lb/>activity by 100 to 10, 000 fold [31]. It was noteworthy that the sialidase activity of M. <lb/>synoviae strain FMT remained quantitatively unchanged after 93 in vitro passages [27]. Since <lb/>the 12-base deletion in the nanI promoter region in strain WVU1853 T was a singular finding <lb/>among several M. synoviae strains examined [26,27], and no distinct TATA or ribosome <lb/>binding sequences were evident in the gapDH-nanI intergenic region adjacent to the putative <lb/> nanI start codon, a causal relationship between the deletion and that strain's comparatively <lb/>high sialidase activity per CFU remains plausible but speculative. It was also remarkable that <lb/>virulent strain WVU1853 T had the least sequence variation in nanI but the highest activity per <lb/>CFU, whereas avirulent strain K4907A had the most sequence variation in nanI and nearly the <lb/>least activity per CFU of the strains with quantitated sialidase activity [27]. <lb/>From a taxonomic standpoint, the capacity to produce sialidase occurs irregularly among <lb/>bacteria, and sialidases are sometimes produced even by only a single strain within a species <lb/>[35,36]. Those findings are most readily explained by horizontal transfer of sialidase genes <lb/>[30]. The hypothetical protein MGA_0329 of M. gallisepticum strain R low (GenBank accession <lb/>no. NP_853343) shares 94.5% aa identity and 96.5% aa similarity to M. synoviae NanI <lb/>(hypothetical protein MS53_0199, GenBank accession no. YP_278329). We used the methods <lb/>described in section 4.1 [27] to confirm that M. gallisepticum strain R low does express sialidase <lb/>activity (our unpublished data). Vasconselos et al. [11] hypothesized a history of nanI homolog <lb/>transfer between M. synoviae and M. gallisepticum, even though the strain R low genome <lb/>(GenBank accession no. AE015450) lacks all of the genes of the N-acetylneuraminate <lb/>catabolism pathway [37]. The Arg 312 that is conserved in other sialidases including in M. <lb/>gallisepticum, but missing from M. synoviae, provides evidence of the direction of transfer. <lb/>The corresponding M. gallisepticum Arg 312 codon CGT was substituted in M. synoviae with <lb/>Gly codons GGT in six of eight strains, or GGC in two strains. Thus a first-position C→G <lb/>transversion mutation accounts for the loss of the Arg 312 residue in M. synoviae and indicates <lb/>that the direction of transfer was from M. gallisepticum to M. synoviae. In that case, either 1) <lb/> nanI alone was transferred from M. gallisepticum to M. synoviae or its ancestor, which <lb/>independently acquired five N-acetylneuraminate catabolism genes in a cluster precisely <lb/>adjacent to nanI in its chromosome; or, more likely 2) the entire locus was transferred to M. <lb/>synoviae from a strain of M. gallisepticum or its ancestor whose descendants later lost all of <lb/>the N-acetylneuraminate catabolism genes. The nanI %G+C, which was similar to the %G+C <lb/>of the whole genome, did not suggest any other history. Regardless of which history is correct, <lb/>the implication is that sialidase activity need not remain linked to N-acetylneuraminate <lb/>catabolism in order to persist in mycoplasmal genomes. <lb/>
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A " neuraminidase-like enzyme " was detected in an unidentified strain of M. gallisepticum <lb/> [15], and sialidase activity of M. gallisepticum strain TT was characterized in detail [14,16], <lb/>but no evidence for sialidase activity was found in M. gallisepticum strains S6 [32] or A5969 <lb/>[25]. Its genomic context suggests that sialidase activity is important in the ecology of M. <lb/>gallisepticum strain R low independently of its potentially advantageous role in nutrient <lb/>acquisition [20]. The present study provides further evidence of a separate role for extracellular <lb/>sialidase also in at least some strains of M. synoviae. Strains FMT and K3344 had identical <lb/>frameshifts in the 5′-third of nanA, resulting in multiple nonsense mutations, and both strains <lb/>also had downstream deletions in nanA. Such genetic lesions naturally uncouple extracellular <lb/>liberation of sialic acid, via sialidase, from generation of N-acetylmannosamine, pyruvate, and <lb/>eventually fructose-6-phosphate via intracellular N-acetylneuraminate catabolism. We <lb/>interpret the different deletions as subsequent decay of the gene initially disabled by frameshift <lb/>in a common ancestor, because it seems less likely that exactly the same 2 bp insertion would <lb/>occur upstream in the gene independently in two strains if the deletions had occurred first. The <lb/>deletion in strain FMT completely overlapped the deletion in strain K3344, which implies that <lb/>K3344 may be an ancestor of FMT, or at least that FMT is less similar to a common ancestor <lb/>than K3344 is at this locus. In a small sample of other species affiliated with the M. synoviae <lb/> phylogenetic cluster [38], which we screened for sialidase activity as described in section 4.1 <lb/>[27], Mycoplasma felis ATCC 23391 T , Mycoplasma leonicaptivi ATCC 49890 T , and <lb/> Mycoplasma sturni UC/MF T were negative, but 12 of 13 canine clinical isolates confirmed by <lb/>PCR-RFLP typing to be the opportunistic mammalian pathogen Mycoplasma canis [39] did <lb/>express activity (our unpublished data). <lb/>An ecological function of sialidase in strains that express activity not linked to N-<lb/>acetylneuraminate catabolism may be to modulate cytadherence. For example, M. synoviae <lb/> and M. gallisepticum both utilize sialylated glycoproteins on eukaryotic cell surface <lb/>membranes as receptors for cytadherence mediated by adhesins such as the vlhA system <lb/>hemagglutinins [40]. Since receptor desialylation reduces or abolishes cytadherence by M. <lb/>synoviae, M. gallisepticum, and M. canis [32,41,42], it is predictable that a functional balance <lb/>between the amount of sialidase activity and receptor binding affinity must be essential to <lb/>promote both colonization and transmission of these mycoplasmas. Strains with comparatively <lb/>higher sialidase activity would be expected to possess higher-affinity adhesins [43]. The <lb/> vlhA locus in the M. synoviae strain 53 genome is flanked by homologs of gapDH adjacent to <lb/>the sialic acid degradation locus. Hypervariability in the VlhA hemagglutinins expressed <lb/>within and among strains is generated by site-specific recombinations among a large <lb/>assemblage of vlhA pseudogenes constituting the 69 kb locus [44,45]. In contrast, there are <lb/>many potentially independently-transcribed vlhA genes dispersed throughout the M. <lb/>gallisepticum genome [37,46], supporting the hypothesis that, like nanI, vlhA also may have <lb/>been exchanged between these species by horizontal transfer [11,47]. Results of the present <lb/>study contribute to a foundation for further work to correlate the probably shifting balances <lb/>among host immune responses to antigenic adhesins such as VlhA, variations in receptor <lb/>binding affinity and sialidase activity, and their interplay with cytadherence and pathogenicity <lb/>in M. synoviae. <lb/> 4. Materials ands methods <lb/> 4.1. Mycoplasma synoviae strains and culture conditions <lb/> Mycoplasma synoviae strains FMT (passages 33 and 126), K3344, K4907A, K5395B, MS173, <lb/>MS178, and WVU1853 T were cultured in modified Frey's medium as previously described <lb/>[27]. The FMT strain, originally isolated from chicken trachea, induced minor respiratory <lb/>lesions following experimental infection [3]; stocks FMT33 and FMT126 were derived from <lb/>serial in vitro passages of FMT. Strain K3344 was isolated during an outbreak of apparent <lb/>
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reproductive disease in a breeder flock in 1992, but was demonstrated to produce respiratory <lb/>lesions during experimental infections [48]. Strains MS173 and MS178 were isolated during <lb/>an outbreak of severe synovitis in Argentina [49]. Lesions from infected birds involved <lb/>synovial membranes, bursa of Fabricius, liver, kidney, and the lower respiratory tract in <lb/>breeders [50]. Strain WVU1853 T has been most commonly reported to cause airsacculitis and <lb/>synovitis [2], however, experimental infection studies indicated that this strain is capable of <lb/>systemic spread and the generation of lesions in multiple tissues [3,51]. Strains K4907A and <lb/>K5395B were isolated from clinically normal chickens, and are not suspected to cause <lb/>significant lesions. A quantitative analysis using using the fluorogenic substrate 2′-(4-<lb/>methylumbelliferyl)-α-D-N-acetylneuraminic acid and the sialidase inhibitor 2-deoxy-2,3-<lb/>didehydro-N-acetylneuraminic acid [27] showed that the units (U) of sialidase activity per CFU <lb/>varied as much as 65 fold (ANOVA P < 0.0001) among these strains. The highest (Fisher's <lb/>Protected Least Significant Difference test P < 0.001) activity observed was for strain <lb/>WVU1853 T (1.3 × 10 −7 U/CFU), intermediate amounts were observed for FMT33, FMT126, <lb/>K3344, and MS178 (1.3–3.9 × 10 −8 U/CFU), and low amounts were observed for strains <lb/>K4907A, K5395B, and MS173 (2.7–6.0 × 10 −9 U/CFU). The M. synoviae field isolate 53 was <lb/>not readily available for phenotypic analysis, and neither clinical data nor sialidase activity <lb/>have been reported for that strain. <lb/> 4.2. PCR amplification of the sialic acid degradation locus <lb/> Genomic DNA was extracted using Easy DNA reagents (Invitrogen, Carlsbad, California) <lb/>according to the manufacturer's instructions. The sialic acid degradation locus (Figure 1B) was <lb/>amplified from strains FMT33, FMT126, K3344, K4907A, K5395B, MS173, MS178, and <lb/>WVU1853 T using PCR primers designed to anneal in the flanking gapDH (5′-TGT TGA ATC <lb/>AAA AGA CGG AAG A-3′) and hypothetical gene MS53_0192 (5′-TCA TCG CTT AAT <lb/>ACT GGG CTT T-3′) open reading frames of strain 53. Amplification reactions were carried <lb/>out as follows, using the Expand High Fidelity PCR System (Roche Applied Sciences, <lb/>Indianapolis, Indiana): initial denaturation at 94°C for 2 min, followed by 30 cycles of template <lb/>denaturation at 94°C for 20 sec, primer annealing at 50°C for 30 sec, and extension at 68°C <lb/>for 9 min, completed by a final extension at 68°C for 10 min. The expected length of the product <lb/>was approximately 8.4 kb. <lb/> 4.3. Nucleotide sequencing and sequence analyses of the sialic acid degradation locus <lb/> Nucleotide sequencing for the sialic acid degradation locus of each strain, from the gapDH-<lb/>nanI intergenic region to the nagB-MS53_0192 junction, was achieved by primer walking <lb/>using four-dye fluorescent dideoxy labeling methods and the Model 3130 capillary system <lb/>(Applied Biosystems, Foster City, California). The uncloned amplified DNA described in <lb/>section 4.2 served as the cycle-sequencing templates. For each strain, 31 reads were required <lb/>to assemble a contig of reconciled double-stranded sequences using Sequencher version 4.7 <lb/>software (Gene Codes, Ann Arbor, Michigan). Open reading frames were identified by BLAST <lb/>alignments with homologs from the M. synoviae strain 53 genome (GenBank accession no. <lb/> AE017245). The nucleotide sequence of each gene was translated using the ExPASy Translate <lb/>Tool [52]. The secondary structure of the putative nanI promoter region and structural features <lb/>of the NanI protein were investigated using tools of the European Molecular Biology Open <lb/>Software Suite [53,54]. Nucleotide and amino acid substitutions among strains were mapped <lb/>using ClustalW alignments [55]. As a benchmark, 1,240 bases of each of the two M. <lb/>synoviae 16S rRNA genes were sequenced from strains WVU1853 T , K3344, MS173, and <lb/>MS178 using internal primers previously described [56], and compared similarly to the <lb/>corresponding sequences from strain 53. For another perspective, intraspecies heterogeneity <lb/>among M. hyopneumoniae strains 232, 7448, and J (GenBank accession nos. AE017332, <lb/>AE017244, and AE017243) in the sequences of housekeeping genes dnaA and ftsY was also <lb/>measured. <lb/>
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4.4. Southern blotting to determine nanI copy number <lb/> To compare the nanI copy number among strains by Southern blotting, a probe consisting of <lb/>the 3′ 2.8 kb of nanI was amplified from WVU1853 T genomic DNA using PCR primers 5′-<lb/>TCT CTT CCT TTT TGA GGG CTA-3′ and 5′-GCA AAT CAT CTT AAG AAA AGT CAT <lb/>T-3′. Amplification conditions, using GoTaq reagents (Promega, Madison, Wisconsin), were <lb/>as described in section 4.2, with the exception of extension steps of 3 min at 72°C. The <lb/>amplicons were labeled with digoxygenin (DIG Hi prime, Roche Applied Sciences) according <lb/>to the manufacturer's instructions. Genomic DNA from the strains with the highest <lb/>(WVU1853 T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with <lb/>endonuclease VspI (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% <lb/>agarose gel. The DNA fragments were electrotransferred to nitrocellulose, then cross-linked <lb/>with shortwave ultraviolet light, using standard methods. Hybridization of the nanI probe and <lb/>detection of the digoxygenin label were carried out using the DIG EasyHyb system (Roche <lb/>Applied Sciences) according to the manufacturer's instructions. <lb/>
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<div type="acknowledgement"> Acknowledgements <lb/> The M. synoviae strains FMT (passages 33 and 126), K3344, K4907A, and K5395B were a gift from Dr. Stanley H. <lb/>Kleven, University of Georgia, Athens, Georgia, USA. Strain K5395B was referred to as K5599A in a preliminary <lb/>report [27]. Strains MS173 and MS178 were a gift from Dr. Raul Cerda, La Plata University, Buenos Aires, Argentina. <lb/>The M. gallisepticum strain R low was a gift from Dr. Steven J. Geary, University of Connecticut, Storrs, Connecticut, <lb/>USA. The canine clinical isolates were a gift from Dr. Mary B. Brown, University of Florida, Gainesville, Florida, <lb/>USA. Cycle sequencing reactions were performed by the Interdisciplinary Center for Biotechnology Research DNA <lb/>Sequencing Core Laboratory at the University of Florida. This work was supported by Public Health Service grant <lb/>1R01GM076584-01A1 from the National Institute of General Medical Sciences (DRB). <lb/></div>
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34. Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths-Jones S, et al. The Pfam protein families <lb/>database. Nucleic Acids Res 2004;32:D138–41. [PubMed: 14681378] <lb/>35. Popoff MR, Dodin A. Survey of neuraminidase production by Clostridium butyricum, Clostridium <lb/>beijerinckii, and Clostridium difficile strains from clinical and nonclinical sources. J Clin Microbiol <lb/>1985;22:873–6. [PubMed: 4056013] <lb/>36. Hoyer LL, Hamilton AC, Steenbergen SM, Vimr ER. Cloning, sequencing and distribution of the <lb/> Salmonella typhimurium LT2 sialidase gene, nanH, provides evidence for interspecies gene transfer. <lb/>Mol Microbiol 1992;6:873–84. [PubMed: 1602967] <lb/>37. Papazisi L, Frasca S Jr, Gladd M, Liao X, Yogev D, Geary SJ. The complete genome sequence of <lb/>the avian pathogen Mycoplasma gallisepticum strain R(low). Microbiology 2003;149:2307–16. <lb/>[PubMed: 12949158] <lb/>38. Johansson, K-E.; Pettersson, B. Taxonomy of Mollicutes. In: Razin, S.; Herrmann, R., editors. <lb/>Molecular biology and pathogenicity of mycoplasmas. London: Kluwer Academic Press; 2002. p. <lb/>1-29. <lb/>39. Spergser J, Rosengarten R. Identification and differentiation of canine Mycoplasma isolates by <lb/>16S-23S rDNA PCR-RFLP. Vet Microbiol 2007;125:170–4. [PubMed: 17544231] <lb/>40. Allen JL, Noormohammadi AH, Browning GF. The vlhA loci of Mycoplasma synoviae are confined <lb/>to a restricted region of the genome. Microbiology 2005;151:935–40. [PubMed: 15758238] <lb/>41. Gesner B, Thomas L. Sialic acid binding sites: role in hemagglutination by Mycoplasma <lb/>gallisepticum. Science 1965;151:590–1. [PubMed: 5903587] <lb/>42. Manchee RJ, Taylor-Robinson D. Utilization of neuraminic acid receptors by mycoplasmas. J <lb/>Bacteriol 1969;98:914–9. [PubMed: 5788718] <lb/>43. Wagner R, Matrosovich M, Klenk H-D. Functional balance between haemagglutinin and <lb/>neuraminidase in influenza virus infections. Rev Med Virol 2002;12:159–66. [PubMed: 11987141] <lb/>44. Noormohammadi AH, Markham PF, Kanci A, Whithear KG, Browning GF. A novel mechanism for <lb/>control of antigenic variation in the haemagglutinin gene family of Mycoplasma synoviae. Mol <lb/>Microbiol 2000;35:911–23. [PubMed: 10692167] <lb/>45. Hong Y, Garcia M, Leiting V, Bencina D, Dufour-Zavala L, Zavala G, et al. Specific detection and <lb/>typing of Mycoplasma synoviae strains in poultry with PCR and DNA sequence analysis targeting <lb/>the hemagglutinin encoding gene vlhA. Avian Dis 2004;48:606–16. [PubMed: 15529983] <lb/>46. Glew MD, Browning GF, Markham PF, Walker ID. pMGA phenotypic variation in Mycoplasma <lb/>gallisepticum occurs in vivo and is mediated by trinucleotide repeat length variation. Infect Immun <lb/>2000;68:6027–33. [PubMed: 10992515] <lb/>47. Markham PF, Duffy MF, Glew MD, Browning GF. A gene family in Mycoplasma imitans closely <lb/>related to the pMGA family of Mycoplasma gallisepticum. Microbiology 1999;145:2095–2103. <lb/>[PubMed: 10463176] <lb/>48. Ewing ML, Cookson KC, Phillips RA, Turner KR, Kleven SH. Experimental infection and <lb/>transmissibility of Mycoplasma synoviae with delayed serologic response in chickens. Avian Dis <lb/>1998;42:230–8. [PubMed: 9645313] <lb/>49. Cerda RO, Giacoboni GI, Xavier JA, Sansalone PL, Landoni MF. In vitro antibiotic susceptibility of <lb/>field isolates of Mycoplasma synoviae in Argentina. Avian Dis 2002;46:215–8. [PubMed: 11922338] <lb/>50. Cerda RO, Xavier JA, Petrucelli MA, Everrigaray ME. Aislamento de Mycoplasma Synoviae de <lb/>pollos parrilleros y gallinas reproducturas primera comunicacion an la Republica Argentina. Analect <lb/>Vet 1998;18:41–6. <lb/>51. Hinz KH, Blome C, Ryll M. Virulence of Mycoplasma synoviae strains in experimentally infected <lb/>broiler chickens. Berl Munch Tierarztl Wochenschr 2003;116:59–66. [PubMed: 12592932] <lb/>52. Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A. ExPASy: the proteomics server <lb/>for in-depth protein knowledge and analysis. Nucleic Acids Res 2003;31:3784–8. [PubMed: <lb/>12824418] <lb/>53. Rice P, Longden I, Bleasby A. EMBOSS: The European Molecular Biology Open Software Suite. <lb/>Trends Genet 2000;16:276–7. [PubMed: 10827456] <lb/>54. SantaLucia J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor <lb/>thermodynamics. Proc Natl Acad Sci USA 1998;95:1460–5. [PubMed: 9465037] <lb/></listBibl>
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<listBibl> 55. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive <lb/>multiple sequence alignment through sequence weighting, position-specific gap penalties and weight <lb/>matrix choice. Nucleic Acids Res 1994;22:4673–80. [PubMed: 7984417] <lb/>56. Brown DR, Farley JM, Zacher LA, Carlton JM, Clippinger TL, Tully JG, et al. Mycoplasma <lb/> alligatoris sp. nov from American alligators. Int J Syst Evol Microbiol 2001;51:419–24. [PubMed: <lb/>11321088] <lb/></listBibl>
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<body> Figure 1. <lb/> (A) The canonical sialic acid degradation pathway [28]. The horizontal line represents the <lb/>interface between extracellular and intracellular processes. (B) Organization of the 7.9 kb sialic <lb/>acid degradation locus in the M. synoviae strain 53 genome [11]. <lb/>
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Figure 2. <lb/> Deletion mutations in the sialic acid degradation locus. The M. synoviae strains examined are <lb/>listed (left). Nucleotide numbering is from the M. synoviae strain 53 genome (GenBank <lb/>accession no. NC_007294) [11]. (A) Nucleotides 224131–224155. A 12 bp deletion occurred <lb/>in the intergenic sequence immediately upstream of the sialidase gene nanI in strain <lb/>WVU1853 T . (B) Nucleotides 220678–221153. Slashes represent 446 intervening bases. A 462 <lb/>base deletion occurred in the 891 bp N-acetylneuraminate lyase gene nanA of strain FMT. A <lb/>direct repeat bracketing the deletion is boxed. (C) Nucleotides 220953–220976. A 14 bp <lb/>deletion, that was overlapped by the larger deletion in strain FMT, occurred in the nanA gene <lb/>of strain K3344. <lb/>
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Figure 3. <lb/> Composition of substitution mutations with respect to consensus sequences. Percentages <lb/>represent the mean across eight M. synoviae strains for nanI, nagA, nanA, nagC, nanE, and <lb/> nagB genes of the sialidase locus, and three M. hyopneumoniae strains for housekeeping genes <lb/> dnaA and ftsY. <lb/>
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Table 1 <lb/> Genetic heterogeneity in the Mycoplasma synoviae sialic acid degradation locus a <lb/> . <lb/> Strain <lb/> K4907A <lb/> K5395B <lb/> WVU1853 T <lb/> K3344 <lb/> FMT <lb/> 53 <lb/> MS178 <lb/> MS173 <lb/> Mean ± S.E. <lb/> Gene b <lb/> nanI <lb/> 0.78 <lb/> 0.61 <lb/> 0.57 <lb/> 0.61 <lb/> 0.61 <lb/> 0.82 <lb/> 0.61 <lb/> 0.61 <lb/> 0.65 ± 0.03 <lb/> nanA <lb/> 0.92 <lb/> 0.61 <lb/> 0.58 <lb/> 0.38 <lb/> 0.49 <lb/> 0.58 <lb/> 0.46 <lb/> 0.46 <lb/> 0.56 ± 0.06 <lb/> nagA <lb/> 0.64 <lb/> 0.55 <lb/> 0.55 <lb/> 0.73 <lb/> 0.55 <lb/> 0.55 <lb/> 0.46 <lb/> 0.46 <lb/> 0.56 ± 0.03 <lb/> nanE <lb/> 0.7 <lb/> 0.99 <lb/> 0.7 <lb/> 0.56 <lb/> 0.56 <lb/> 0.56 <lb/> 0 <lb/> 0 <lb/> 0.51 ± 0.12 <lb/> nagB <lb/> 0.42 <lb/> 0.42 <lb/> 0.56 <lb/> 0.56 <lb/> 0.71 <lb/> 0.28 <lb/> 0.14 <lb/> 0.14 <lb/> 0.40 ± 0.07 <lb/> nagC <lb/> 0.46 <lb/> 0.23 <lb/> 0.34 <lb/> 0.46 <lb/> 0.34 <lb/> 0.11 <lb/> 0.46 <lb/> 0.34 <lb/> 0.34 ± 0.04 <lb/> Mean ± S.E. <lb/> 0.65 ± 0.08 <lb/> 0.57 ± 0.10 <lb/> 0.55 ± 0.05 <lb/> 0.55 ± 0.05 <lb/> 0.54 ± 0.05 <lb/> 0.48 ± 0.10 <lb/> 0.36 ± 0.10 <lb/> 0.34 ± 0.09 <lb/> 16S rDNA c <lb/> N/D d <lb/> N/D d <lb/> 0.04 <lb/> 0.04 <lb/> N/D d <lb/> 0.04 <lb/> 0.04 <lb/> 0.04 <lb/> 0.04 <lb/> a <lb/> Nucleotide substitutions per 100 bases of consensus sequence. <lb/> b <lb/> nanI = sialidase MS53_0199 (synonymous with nanH of GenBank accession no. ABS50356); nanA = N-acetylneuraminate lyase; nagA = N-acetylglucosamine-6-phosphate deacetylase; nanE = N-<lb/> acetylmannosamine-6-phosphate epimerase MS53_0197 (GenBank accession no. AAZ43616); nagB = glucosamine-6-phosphate deaminase; nagC = N-acetylmannosamine kinase. <lb/> c <lb/> Both copies. <lb/> d <lb/> N/D = not determined. <lb/>
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Table 2 <lb/> Guanine+cytosine content of genes in the Mycoplasma synoviae sialic acid degradation locus. <lb/> Strain <lb/> K4907A <lb/> K5395B <lb/> WVU1853 T <lb/> K3344 <lb/> FMT <lb/> 53 <lb/> MS178 <lb/> MS173 <lb/> Mean ± S.E. <lb/> Consensus <lb/> Gene a <lb/> nanI <lb/> 29.3 <lb/> 29.2 <lb/> 29.1 <lb/> 29.0 <lb/> 29.0 <lb/> 29.1 <lb/> 29.1 <lb/> 29.1 <lb/> 29.1 ± 0.04 <lb/> 29.3 <lb/> nanA <lb/> 32.4 <lb/> 32.4 <lb/> 32.6 <lb/> 31.9 <lb/> 31.9 <lb/> 33.2 <lb/> 32.8 <lb/> 32.8 <lb/> 32.5 ± 0.05 <lb/> 32.4 <lb/> nagA <lb/> 27.2 <lb/> 27.2 <lb/> 27.0 <lb/> 27.3 <lb/> 27.3 <lb/> 27.1 <lb/> 27.3 <lb/> 27.3 <lb/> 27.2 ± 0.11 <lb/> 27.4 <lb/> nanE <lb/> 33.1 <lb/> 32.6 <lb/> 32.9 <lb/> 33.2 <lb/> 33.1 <lb/> 33.3 <lb/> 33.2 <lb/> 33.2 <lb/> 33.1 ± 0.23 <lb/> 33.2 <lb/> nagB <lb/> 30.4 <lb/> 30.2 <lb/> 30.1 <lb/> 30.2 <lb/> 30.2 <lb/> 30.2 <lb/> 30.6 <lb/> 30.6 <lb/> 30.3 ± 0.20 <lb/> 30.2 <lb/> nagC <lb/> 30.7 <lb/> 30.9 <lb/> 30.8 <lb/> 30.9 <lb/> 31.0 <lb/> 31.0 <lb/> 30.9 <lb/> 31.0 <lb/> 30.9 ± 0.04 <lb/> 31.1 <lb/> a <lb/> nanI = sialidase MS53_0199 (synonymous with nanH of GenBank accession ABS50356); nanA = N-acetylneuraminate lyase; nagA = N-acetylglucosamine-6-phosphate deacetylase; nanE = N-<lb/> acetylmannosamine-6-phosphate epimerase MS53_0197 (GenBank accession no. AAZ43616); nagB = glucosamine-6-phosphate deaminase; nagC = N-acetylmannosamine kinase. The calculated %G <lb/> +C for the M. synoviae strain 53 genome as a whole was 27% [11]. <lb/></body>
<note place="footnote">Microb Pathog. Author manuscript; available in PMC 2009 July 1.</note>
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