An Insertion Sequence-Dependent Plasmid Rearrangement in Aeromonas salmonicida Causes the Loss of the Type Three Secretion System

Aeromonas salmonicida, a bacterial fish pathogen, possesses a functional Type Three Secretion System (TTSS), which is essential for its virulence. The genes for this system are mainly located in a single region of the large pAsa5 plasmid. Bacteria lose the TTSS region from this plasmid through rearrangements when grown in stressful growth conditions. The A. salmonicida genome is rich in insertion sequences (ISs), which are mobile DNA elements that can cause DNA rearrangements in other bacterial species. pAsa5 possesses numerous ISs. Three IS11s from the IS256 family encircle the rearranged regions. To confirm that these IS11s are involved in pAsa5 rearrangements, 26 strains derived from strain A449 and two Canadian isolates (01-B526 and 01-B516) with a pAsa5 rearrangement were tested using a PCR approach to determine whether the rearrangements were the result of an IS11-dependent process. Nine out of the 26 strains had a positive PCR result, suggesting that the rearrangement in these strains were IS-dependent. The PCR analysis showed that all the rearrangements in the A449-derived strains were IS11-dependent process while the rearrangements in 01-B526 and 01-B516 could only be partially coupled to the action of IS11. Unidentified elements that affect IS-dependent rearrangements may be present in 01-B526 and 01-B516. Our results suggested that pAsa5 rearrangements involve IS11. This is the first study showing that ISs are involved in plasmid instability in A. salmonicida

Étude de la fonction d'Ati2, un effecteur du système de sécrétion de type trois chez Aeromonas salmonicida

La bactérie Aeromonas salmonicida utilise le système de sécrétion de type trois (SSTT) pour injecter des effecteurs à l'intérieur des cellules de l'hôte lors de l'infection. L'étude du génome d'A. salmonicida a révélé l'existence d'Ati2, un nouvel effecteur du SSTT. Ce projet avait pour but d'étudier la relation structure-fonction d'Ati2 afin de déterminer son rôle dans la virulence d'A. salmonicida. Des analyses biochimiques et en modélisation moléculaire ont permis de démontrer qu'Ati2 est une inositol polyphosphate 5-phosphatase qui hydrolyse les PtdIns(4,5)P₂ et les PtdIns(3,4,5)P₃. Divers mutants d'Ati2 ont été produits et clones dans des vecteurs d'expression chez l'amibe Dictyostelium discoideum. Les tests d'expression ont démontré qu'Ati2 est toxique pour l'amibe et que cela est lié à son activité catalytique. Ce projet de recherche a ainsi permis de démontrer l'existence d'un nouvel effecteur du SSTT et d'en spécifier la fonction dans la pathogénicité d'A. salmonicida

<i>A. salmonicida</i> strains used in this study and compilation of the various analyses done on these strains.

a<p>with 11AF and 11CR primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033725#pone-0033725-t002" target="_blank">Table 2</a>).</p>b<p>with 11B1F and 11CR primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033725#pone-0033725-t002" target="_blank">Table 2</a>).</p>c<p>see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033725#pone-0033725-g005" target="_blank">Figure 5</a>.</p>d<p>Parental strain.</p>e<p>Not applicable.</p>f<p>01-B526-R4 had lost the genes corresponding to a type 1 loss profile as well as <i>P5G011</i>, another gene 40 kb upstream from the TTSS region (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033725#pone-0033725-g001" target="_blank">Figure 1</a>).</p

IS<i>11</i>D on the chromosome is not involved in pAsa5 insertion sequence rearrangements.

<p>Unexplained rearrangements in derivative strains were assessed by PCR using the 11D1F2 and 11D2R2 primers for the regions flanking IS<i>11</i>D. <b>A.</b> The positions of these primers are illustrated in both scenarios (intact IS<i>11</i>D or following homologous recombination in a B–C rearranged pAsa5 plasmid). <b>B.</b> An agarose gel electrophoresis of the PCR products, showing a positive result for all tested strains.</p

IS-dependent rearrangement may explain loss profiles 1 and 2, and can be assessed by PCR.

<p><b>A.</b> Two ISs can lead to the excision of a region of a plasmid by homologous recombination. <b>B.</b> This event, which can generate type 1 loss profile (TTSS) and type 2 loss profile (TTSS and upstream locus) in pAsa5, can be assessed by PCR. On this map, the primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033725#pone-0033725-t002" target="_blank">Table 2</a>), shown as arrows, flank one side of their respective IS. In an intact pAsa5 plasmid, these primers are too far apart to generate an amplicon. However, both homologous recombination events involving ISs could place the primers in a suitable position to generate an amplicon. B–C rearrangements can be detected by the 11B1F and 11CR primers, and A–C rearrangements can be detected by the 11AF and 11CR primers. The position of <i>traC</i>, <i>resD</i> and <i>exsD</i> genes are also shown.</p

Primers used in this study.

<p>Primers used in this study.</p

IS<i>11</i>-dependent A–C and B–C rearrangements are involved in loss profiles 2 and 1, respectively.

<p>A449-R1 derivative strain harboring a loss profile 1 (<b>A</b>) and A449-R2 derivative strain harboring a loss profile 2 (<b>B</b>) were assessed by PCR, using <i>exsD</i>, <i>traC</i> and <i>resD</i> primers as control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033725#pone-0033725-g002" target="_blank">Figure 2B</a>). Primers 11B1F and 11CR were used to assess B–C rearrangement while primers 11AF and 11CR were used to assess A–C rearrangement.</p

Growing <i>A. salmonicida</i> in stressful conditions can lead to the loss of pAsal1.

<p><b>A and C.</b> Small plasmids (pAsa1, pAsa2, pAsa3 and pAsal1) from 01-B526 and 01-B516 derived strains were purified and digested using EcoRI leading to the linearization of the plasmids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033725#pone.0033725-Boyd1" target="_blank">[12]</a>. An agarose gel electrophoresis of the digested plasmids is shown. <b>B and D.</b> DNA lysates of the same strains were tested by PCR with pAsal1 primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033725#pone-0033725-t002" target="_blank">Table 2</a>) to confirm by a second approach the presence or the absence of pAsal1 in these strains. The presence (+) or the absence (−) of a PCR product is indicated.</p