Molecular characterisation of XvRG6 and XvRF17 genes isolated from the resurrection plant Xeropyta viscosa

Includes bibliographical references (leaves 59-71).A number of genes up-regulated in response to abiotic stress have been isolated from X. viscosa by various methods, one of which includes differential screening of cDNA libraries. Of these genes, a large number are undescribed, with no identity to known plant genes. Two such genes, XvRG6 and XvRF17, isolated from a X. viscosa cDNA library have been partially characterised. Southern blot analysis confirmed the presence of both XvRG6 and XvRF17 genes in the X. viscosa genome. In silico analyses predicted XvRG6 to be proline-rich protein, since it possesses many of the features common to PRP such as a signal peptide, proline repeats, a cysteine residue and possible phosphorylation sites. It also possesses a tyrosine residue present in some of the repeats, and this residue is belivied to play a role in protecting the plant against environmental stresses. The XvRF17 protein was predicted by in silico analyses to belong to the family of metallothioneins, a family of metal-binding proteins. Anaylis of the XvRG6 mRNA transcript showed that the gene was not dehydration stress inducible, but was induced by ethylene, endogenous ABA, SA and JA application. In contrast, the XvRF17 mRNA transcript was shown by RT-PCR to be induced by dehydration stress, endogenous ABA, SA and JA application but not by ethylene treatment. Interestingly, western blot analyses revealed that XvRG6 protein levels increased only during dehydration and not during any other imposed stress. This study has shown that both XvRG6 and XvRF17 and responsive to stress. Since there are no known orthologues of these genes, their respective roles in X. viscosa remains speculative

Acanthamoeba polyphaga-Enhanced Growth of Mycobacterium smegmatis

Background: Mycobacterium smegmatis is a rapidly-growing mycobacterium causing rare opportunistic infections in human patients. It is present in soil and water environments where free-living amoeba also reside, but data regarding M. smegmatis-amoeba relationships have been contradictory from mycobacteria destruction to mycobacteria survival. Methodology/Principal Findings: Using optic and electron microscopy and culture-based microbial enumeration we investigated the ability of M. smegmatis mc 2 155, M. smegmatis ATCC 19420 T and M. smegmatis ATCC 27204 organisms to survive into Acanthamoeba polyphaga trophozoites and cysts. We observed that M. smegmatis mycobacteria penetrated and survived in A. polyphaga trophozoites over five-day co-culture resulting in amoeba lysis and the release of viable M. smegmatis mycobacteria without amoebal cyst formation. We further observed that amoeba-co-culture, and lysed amoeba and supernatant and pellet, significantly increased five-day growth of the three tested M. smegmatis strains, including a four-fold increase in intra-amoebal growth. Conclusions/Significance: Amoebal co-culture increases the growth of M. smegmatis resulting in amoeba killing by replicating M. smegmatis mycobacteria. This amoeba-M. smegmatis co-culture system illustrates an unusual paradigm in the mycobacteria-amoeba interactions as mycobacteria have been mainly regarded as amoeba-resistant organisms. Using these model organisms, this co-culture system could be used as a simple and rapid model to probe mycobacterial factors implicated in the intracellular growth of mycobacteria

Mycobacterium tuberculosis Complex Mycobacteria as Amoeba-Resistant Organisms

International audienceBackground: Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii.Methodology/Principal Findings: Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10−6) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34×104 CFU/mL) than M. tuberculosis (42×101 CFU/mL) and M. bovis (35×101 CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde.Conclusions/Significance: These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle

Le rôle des cellulases dans les interactions entre les mycobactéries du complexe Mycobacterium tuberculosis et les amibes libres

Le génome de Mycobacterium tuberculosis, l’agent causal de la tuberculose, code pour une protéine ayant la capacité de se fixer sur la cellulose (Rv1987), une cellulase potentielle (Rv1090), et une cellulase pleinement active (Rv0062). Cette observation est surprenante, car la cellulose est un composant majeur des parois des cellules végétales, tandis que M. tuberculosis est un pathogène humain sans contact connu avec des plantes. Nous avons émis l’hypothèse que ces protéines pourraient jouer un rôle dans les interactions entre les mycobactéries du complexe M. tuberculosis avec les kystes d’amibes libres, dont la paroi contient également de la cellulose. Dans notre travail de thèse, nous avons cherché par une analyse in silico la présence de ces trois gènes chez toutes les bactéries ayant un génome complètement séquencé présentes dans la base de données CAZy (accessible en ligne à l’adresse www.cazy.org). Cette étude a montré que seulement 2,5% des bactéries codent pour les trois gènes simultanément. Parmi ces bacteries, nous avons ensuite confirmé expérimentalement par PCR et séquençage la présence des gènes Rv0062, Rv1090 et Rv1987 chez les mycobactéries du complexe M. tuberculosis. Nous avons ensuite vérifié la transcription de ces trois gènes chez la souche de référence M. tuberculosis H37Rv, puis produit dans Escherichia coli des protéines de fusion Rv1090 et Rv1987 et montré qu'elles étaient capables d'hydrolyser la cellulose (Rv1090) et de s’y fixer (Rv1987). De plus, nous avons mis en place un model expérimental d’interaction entre les mycobactéries du complexe M. tuberculosis et les amibes libres dans le but de comprendre le rôle des gènes Rv0062, Rv1090 et Rv1987. Dans un premier temps nous avons montré que M. tuberculosis, Mycobacterium bovis, Mycobacterium canettii ainsi que Mycobacterium avium utilisé ici comme un controle positif étaient capables de survivre dans le cytoplasme des amibes libres telles que Acanthamoeba polyphaga. Ensuite, nous avons montré que M. tuberculosis et M. bovis mais pas M. canettii étaient capables de survivre à l’intérieur des kystes d’amibes. Enfin nous avons montré que M. tuberculosis, M. bovis et M. canettii étaient capables de survivre dans le sol pendant au moins 6 mois. Les données établies dans cette thèse soutiennent le rôle des cellulases dans la survie environnementale des mycobactéries du complexe M. tuberculosis, et ouvrent la voie à l’étude de cette phase méconnue dans le cycle de ces organismesThe genome of Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes a protein with the ability to bind to cellulose (Rv1987), one potential cellulase (Rv1090), and one fully active cellulase (Rv0062). This observation is puzzling, because cellulose is a major component of plant cell walls, whereas M. tuberculosis is a human pathogen without known contact with plants. We hypothesized that these genes could play a role in the interactions between M. tuberculosis complex organisms and amoebal cysts, whose wall contains cellulose.In our thesis work, we have searched by in silico analysis for the presence of these three genes in all bacteria with complete sequenced genomes present in the CAZy database (available online at www.cazy. org). This study showed that only 2.5% of bacteria encode the three genes simultaneously. Among these bacteria we have confirmed experimentally by PCR and sequencing the presence of Rv0062, Rv1090 and Rv1987 in the M. tuberculosis complex organisms. We have checked the transcript of the three genes in the reference strain M. tuberculosis H37Rv and we subsequently produced Rv1090 and Rv1987 fusion proteins in Escherichia coli and demonstrated that they were indeed able to hydrolyze (Rv1090) and to bind (Rv1987) cellulose. In addition, we have developed an experimental model of interaction between M. tuberculosis organisms and the free-living amoebae in order to understand the role of Rv0062, Rv1090 and Rv1987 genes. Initially we have shown that M. tuberculosis, Mycobacterium bovis, Mycobacterium canettii and Mycobacterium avium used here as a positive control were able to survive in the cytoplasm of the free-living amoeba such as Acanthamoeba polyphaga. We have further shown that M. tuberculosis and M. bovis but not M. canettii were able to survive within the amoebal cysts. Finally we have shown that M. tuberculosis, M. bovis and M. canettii were able to survive in soil for at least 6 months. The data obtained in this thesis support the role of cellulase in the survival of M. tuberculosis complex organisms in the environment and pave the way for the study of this unknown phase in the cycle of these organisms

Growth of <i>M. smegmatis</i> in the presence of amoeba lysis.

<p>Three <i>M. smegmatis</i> organisms were tested: (A) <i>M. smegmatis</i> mc² 155, (B) <i>M. smegmatis</i> ATCC 19420^T and (C) <i>M. smegmatis</i> ATCC 27204. <i>M. smegmatis</i> strains cultured with amoeba lysis pellet (white bar) and supernatant (black bar). PAS medium was used as negative control (gray bar). Each bar represents the mean of triplicate wells, and the standard errors are represented by error bars.</p

Amoeba increases the growth of <i>M. smegmatis</i>.

<p>Counting of amoeba alive with and without <i>M. smegmatis</i> mc² 155 (A), <i>M. smegmatis</i> ATCC 19420^T (B) and <i>M. smegmatis</i> ATCC 27204 (C) in PAS. Asterix represent significant variation (p≤0.05). Each bar represents the mean of triplicate wells, and the standard errors are represented by error bars.</p

Transmission electron-microscopy observation of <i>A. polyphaga</i> cysts.

<p>(A) The mature form of cyst. <i>M. smegmatis</i> mc² 155 (▸) exit from <i>A. polyphaga</i> pre-cyst (B) and present in the outside of pre-cyst (C); <b>n:</b> nucleus, <b>m:</b> mitochondria. Scale bar: 5 µm (A, C) and 2 µm (B).</p

Different forms of amoeba-mycobacteria interactions.

<p>Different forms of amoeba-mycobacteria interactions.</p

Described interactions of rapid and slow-growing mycobacteria with FLA.

<p>IC, intracellular; IK, intracyst; Ap, <i>Acanthamoeba polyphaga</i>; Ac, <i>Acanthamoeba castellanii</i>; Tp, <i>Tetrahymena pyriformis</i>.</p