Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme

International audienceTo our knowledge, the phylodistribution of M. hominis clinical strains associated with various pathological conditions of the urogenital tract has not been explored hitherto. Here we analyzed the genetic diversity and phylogenetic relationships among 59 M. hominis Tunisian clinical isolates, categorized as gynecological infections-or infertility-associated pathotypes. For this purpose, we developed an expanded multilocus sequence typing (eMLST) scheme, combining the previously reported multilocus sequence typing (MLST) loci (gyrB, tuf, ftsY, uvrA, gap) with a new selected set of putative virulence genes (p120', vaa, lmp1, lmp3, p60), referred herein to as multi-virulence-locus sequence typing (MVLST) loci. In doing so, M. hominis population was segregated into two distinct genetic lineages, which were differentially associated with each pathotype. Such a clear dichotomy was supported by several phylogenetic and population genetic analysis tools. Recombination was found to take place, but not sufficient enough to break down the overall clonal population structure of M. hominis, most likely as a result of purifying selection, which accommodated the most fit clones. In sum, and owing to the eMLST scheme described herein, we provide insightful data on the phylogenetics of M. hominis, arguing for the existence of genetically differentiable urogenital pathotypes. Mycoplasma hominis, which belongs to the Mycoplasmataceae family, in the Mollicutes class, was the first myco-plasma species isolated from humans in 1937 1. It resides, as a commensal, in the lower urogenital tract of healthy persons. Under certain circumstances, M. hominis can cause a variety of genital infections such as bacterial vag-inosis, pelvic inflammatory disease, and cervicitis 2. This microorganism seems to be associated with pregnancy complications and neonatal diseases 3. In addition, several studies reported the pathogenic role of M. hominis in infertility 4,5. More interestingly, this species has been linked to a wide range of extragenital infections (septic arthritis, endocarditis, brain abscess), especially in immunocompromised patients 6-8. To better understand the epidemiology and the mode of spread of M. hominis, several molecular typing systems have been developed. These include Pulse-Field Gel Electrophoresis (PFGE), Restriction Fragment Length Polymorphism (RFLP) analysis, Amplified Fragment Length Polymorphism (AFLP), and Random Amplified Polymorphic DNA (RADP). All these methods have revealed a high degree of both genetic and antigenic het-erogeneity among M. hominis strains 9-12. Although informative, these approaches proved to be quite difficult t

Genome Sequences of Two Tunisian Field Strains of Avian <I>Mycoplasma, M. meleagridis<I> and <I>M. gallinarum<I>

International audienceMycoplasma meleagridis and Mycoplasma gallinarum are bacteria that affect birds, but little is known about the genetic basis of their interaction with chickens and other poultry. Here, we sequenced the genomes of M. meleagridis strain MM_26B8_IPT and M. gallinarum strain Mgn_IPT, both isolated from chickens showing respiratory symptoms, poor growth, reduction in hatchability, and loss of production

Genetic variability of the P120' surface protein gene of Mycoplasma hominis isolates recovered from Tunisian patients with uro-genital and infertility disorders

<p>Abstract</p> <p>Background</p> <p>Among the surface antigens of <it>Mycoplasma hominis</it>, the P120' protein was previously shown to elicit a subtle antibody response and appears to be relatively conserved. To get better insight into the evolution of this protein, we analysed the genetic variability of its surface exposed region in 27 <it>M. hominis </it>isolates recovered from the genital tract of Tunisian patients with infertility disorders.</p> <p>Methods</p> <p>All specimens were processed for culture and PCR amplification of the N-terminal surface exposed region of p120' gene. PCR products were sequenced to evaluate the genetic variability, to test for adaptive selection, and to infer the phylogenetic relationship of the <it>M. hominis </it>isolates.</p> <p>Results</p> <p>Sequence analysis showed a total of 25 single nucleotide polymorphisms distributed through 23 polymorphic sites, yielding 13 haplotypes. All but one mutation were confined within three distinct regions. Analysis of the amino acid-based phylogenetic tree showed a predominant group of 17 closely related isolates while the remaining appear to have significantly diverged.</p> <p>Conclusion</p> <p>By analysing a larger sample of <it>M. hominis </it>recovered from patients with urogenital infections, we show here that the P120' protein undergoes substantial level of genetic variability at its surface exposed region.</p

Nuclease activity of GST-Mm19 fusion protein.

<p><b>(A)</b> Effect of divalent cations on nuclease activity of purified GST-Mm19 against pasmid pCR2.1 DNA. Nuclease activity was analyzed in the absence or presence of 2 mM of different divalent cations (indicated under each panel). Molecular weight markers, 1 kb DNA Plus Ladder (Thermoscientific), are shown on the left margin of each panel. Lanes C1 and C2 in each panel are plasmid DNA untreated or treated with GST protein, respectively. Purified GST-Mm19 fusion protein was incubated with pasmid pCR2.1 DNA. Incubation times in minutes are indicated above each lane. All reactions were performed at 37°C. <b>(B)</b> Substrate specificity of nuclease activity of purified GST-Mm19. Nuclease activity of GST-Mm19 was assessed against Vero cell double-stranded DNA (left panel), M13 phage single-stranded DNA (middle panel), and <i>E</i>. <i>coli</i> strain BL21 total RNA (right panel). The positions of 1 kb DNA ladder markers are indicated as MW on the left of each panel. Lanes C1 and C2 in each panel indicate end point reactions of the undigested nucleic acid controls after incubation in the nuclease buffer alone or supplemented with GST protein, respectively. Reactions were stopped at the times indicated above each lane.</p

<i>In silico</i> characterization of Mm19 nuclease.

<p><b>(A)</b> Schematic illustration of RE_<i>Alw</i>I conserved domain as determined by BLASTP analysis of Mm19 amino acid sequence. The deduced 300-residue sequence of the RE_<i>Alw</i>I superfamily domain mapped to the C-terminal region of Mm19, from residues 307 to 607. <b>(B)</b> Genomic location of <i>M</i>. <i>meleagridis Mm19</i> nuclease ORF and <i>methyltransferase</i> sequences. The two genes encoding these enzymes are located next to each other with an intergenic region of 130 bp and opposing orientations; <i>Mm19</i> is on the positive strand, while the <i>methyltransferase</i> gene is encoded on the negative strand. A “TATAAT box” (in red) was detected upstream of Mm19 ORF, at position -77. In the same orientation and approximately 10 kb upstream of the <i>Mm19</i> gene sequence, genes coding for an ABC transporter ATP-binding protein and a transport system permease protein were detected. Genomic coordinates of coding sequences are indicated. All genomic data were extracted from the MolliGen database.</p

Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme

Abstract To our knowledge, the phylodistribution of M. hominis clinical strains associated with various pathological conditions of the urogenital tract has not been explored hitherto. Here we analyzed the genetic diversity and phylogenetic relationships among 59 M. hominis Tunisian clinical isolates, categorized as gynecological infections- or infertility-associated pathotypes. For this purpose, we developed an expanded multilocus sequence typing (eMLST) scheme, combining the previously reported multilocus sequence typing (MLST) loci (gyrB, tuf, ftsY, uvrA, gap) with a new selected set of putative virulence genes (p120’, vaa, lmp1, lmp3, p60), referred herein to as multi-virulence-locus sequence typing (MVLST) loci. In doing so, M. hominis population was segregated into two distinct genetic lineages, which were differentially associated with each pathotype. Such a clear dichotomy was supported by several phylogenetic and population genetic analysis tools. Recombination was found to take place, but not sufficient enough to break down the overall clonal population structure of M. hominis, most likely as a result of purifying selection, which accommodated the most fit clones. In sum, and owing to the eMLST scheme described herein, we provide insightful data on the phylogenetics of M. hominis, arguing for the existence of genetically differentiable urogenital pathotypes

Mm19, a <i>Mycoplasma meleagridis</i> Major Surface Nuclease that Is Related to the RE_<i>Alw</i>I Superfamily of Endonucleases

<div><p><i>Mycoplasma meleagridis</i> infection is widespread in turkeys, causing poor growth and feathering, airsacculitis, osteodystrophy, and reduction in hatchability. Like most mycoplasma species, <i>M</i>. <i>meleagridis</i> is characterized by its inability to synthesize purine and pyrimidine nucleotides <i>de novo</i>. Consistent with this intrinsic deficiency, we here report the cloning, expression, and characterization of a <i>M</i>. <i>meleagridis</i> gene sequence encoding a major surface nuclease, referred to as Mm19. Mm19 consists of a 1941- bp ORF encoding a 646-amino-acid polypeptide with a predicted molecular mass of 74,825 kDa. BLASTP analysis revealed a significant match with the catalytic/dimerization domain of type II restriction enzymes of the RE_<i>Alw</i>I superfamily. This finding is consistent with the genomic location of Mm19 sequence, which dispalys characteristics of a typical type II restriction-modification locus. Like intact <i>M</i>. <i>meleagridis</i> cells, the <i>E</i>. <i>coli-</i>expressed Mm19 fusion product was found to exhibit a nuclease activity against plasmid DNA, double-stranded DNA, single-stranded DNA, and RNA. The Mm19-associated nuclease activity was consistently enhanced with Mg²⁺ divalent cations, a hallmark of type II restriction enzymes. A rabbit hyperimmune antiserum raised against the bacterially expressed Mm19 strongly reacted with <i>M</i>. <i>meleagridis</i> intact cells and fully neutralized the surface-bound nuclease activity. Collectively, the results show that <i>M</i>. <i>meleagridis</i> expresses a strong surface-bound nuclease activity, which is the product of a single gene sequence that is related to the RE_<i>Alw</i>I superfamily of endonucleases.</p></div

Hypothetical and experimental evidence of Mm19 surface exposure.

<p><b>(A)</b> Schematic representation of the secondary structure of Mm19 protein as predicted by PSIPRED based on PSI-BLAST. Using MEMSAT-SVM, a segment of 464 amino acids, including the N-terminal region of the Mm19 nuclease protein, was predicted to be located in the cytoplasm. The C-terminal 167 residues were predicted to be exposed to the extracellular milieu, while 15 amino acid residues were embedded within the membrane. A similar topology was predicted by MEMSAT3 but with a few differences in the segments lengths (cytoplasmic segment, 581 aa; transmembrane segment, 18 aa; extracellular segment, 47 aa). <b>(B)</b> Surface expression of <i>M</i>. <i>meleagridis</i> Mm19 as revealed by colony blotting. <i>M</i>. <i>meleagridis</i> colonies growing on agar plates were left untreated or subjected to trypsin digestion then imprinted onto nitrocellulose discs. The anti-GST (panel 1) and the specific polyclonal anti-<i>M</i>. <i>meleagridis</i> (panel 2) sera were used as negative and positive controls, respectively. The reactivity of anti-GST-Mm19 serum against non- and trypsin-treated <i>M</i>. <i>meleagridis</i> colonies is shown in panels 3 and 4, respectively. Colored imprints of colonies were observed under a microscope at 100 x magnification.</p

Neutralization of <i>M</i>. <i>meleagridis</i> membrane-associated endonuclease activity.

<p>Nuclease activity neutralization was tested with three controls (lanes C1, C2, and C3). Lane C1, plasmid pCR2.1 DNA incubated in reaction buffer only; Lane C2, plasmid pCR2.1 DNA incubated with untreated <i>M</i>. <i>meleagridis</i> cells; Lane C3, plasmid pCR2.1 DNA incubated with intact <i>M</i>. <i>meleagridis</i> cells that had been treated with rabbit antiserum against GST. Lanes 1–5, plasmid pCR2.1 DNA incubated with <i>M</i>. <i>meleagridis</i> cells treated with rabbit anti-GST-Mm19 antiserum diluted 1/10, 1/100, 1/1000, 1/2000, or 1/4000, respectively. Lane MW, 1 kb DNA ladder (Amersham).</p