Characterization of DNA rearrangements within the vpma multigene locus of Mycoplasma agalactiae

Mycoplasma agalactiae, ein Vertreter der taxonomischen Klasse Mollicutes, stellt als Erreger der Infektiösen Agalaktie der Schafe und Ziegen ein wichtiges pathogenes Bakterium dar. Die klinische Symptomatik der Krankheit ist charakterisiert prädominant durch Mastitis, Arthritis und Konjunktivitis. Innerhalb des M. agalactiae-Genoms konnte ein einer Pathogenitätsinsel ähnlicher Genlokus identifiziert werden, welcher die wichtigsten immunodominanten Oberflächenproteine, die sogenannten Vpmas (variable proteins of M. agalactiae) kodiert. Durch ortsspezifische DNA-Umlagerungen, die durch das Xer1-Protein katalysiert werden, welches in direkter Nachbarschaft zu den vpma-Genen kodiert wird, wird die Expression der Vpma-Proteine mit einer ungewöhnlich hohen Frequenz variiert. Durch gezielte Disruption des xer1-Gens konnte gezeigt werden, dass die Xer1-Rekombinase den essentiellen Faktor für ortsspezifische DNA-Inversionen innerhalb des vpma-Genlokus darstellt. In diesen sogenannten xer1-phase-locked mutants (PLMs) finden daher keine spezifischen DNA-Umlagerungen mehr statt, da die Zellen in einer bestimmten Vpma-Expressionsphase arretiert bleiben. Die vorliegende Arbeit beinhaltet neue Daten bezüglich dieses Antigenvariationssystems von M. agalactiae. Rekombinationsexperimente in Escherichia coli zeigten, dass vorausgesagte Rekombinationsstellen (RS) von 21 bp, welche sich in der 5’ UTR aller sechs vpma-Gene befinden, tatsächlich in Xer1-katalysierten Rekombinationsereignissen involviert sind. Angrenzende Nukleotide konnten die Rekombinationsraten positiv beeinflussen. Zusätzlich konnte gezeigt werden, dass die Anordnung dieser RS für das Ergebnis der Rekombinationsereignisse eine essentielle Rolle spielt. Während invertiert angeordnete RS zu Inversionen führten, kam es an RS in direkter Abfolge zu Exzisionsereignissen. Zusätzlich konnten ortsspezifische Exzisionen im Genom von M. agalactiae mithilfe eines neu entwickelten lacZ-Reportersystems nachgewiesen werden. Neben ortsspezifischen DNA-Umlagerungen konnten neue molekulare Mechanismen der Vpma-Antigenvariabilität von M. agalactiae gezeigt werden, die speziell in der Abwesenheit der Xer1- Rekombinase stattfinden. Dazu wurden PLMs, die nur ein bestimmtes Vpma-Genprodukt exprimieren, im Tierversuchsmodell verwendet. Überraschenderweise zeigten jene PLMs, die aus den Tieren reisoliert wurden, einen neuen Vpma-Phenotyp. Die molekulare Analyse durch DNA-Sequenzierung zeigte, dass komplexe DNA-Umlagerungen, wie beispielsweise die Entstehung chimärer vpma-Gene bzw. Genduplikation und -deletionen, durch den Selektionsdruck des Immunsystems in den Reisolaten stattgefunden haben. Ähnliche Ergebnisse wurden auch in einem neu entwickelten in vitro-Experiment unter Selektionsdruck in Gegenwart von Vpma-spezifischen Antikörpern erhalten. Diese neuen Daten unterstreichen die Bedeutung der Antigenvariation als wichtige Überlebensstrategie des Erregers M. agalactiae in seinem Wirt und repräsentieren eine wichtige Grundlage für die Untersuchung ähnlicher Systeme in anderen pathogenen Mykoplasmenarten.Mycoplasma agalactiae, a cell wall-less bacterium belonging to the class Mollicutes, is considered the main etiological agent of the syndrome Contagious Agalactia, which primarily causes mastitis, arthritis and conjunctivitis in sheep and goats. Antigenic diversity in M. agalactiae is governed by DNA rearrangements within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. Six vpma genes were shown to undergo high-frequency phase variation by site-specific DNA inversions mediated by a site-specific recombinase (Xer1) encoded adjacent to the vpma genes. Disruption of the xer1 recombinase gene has demonstrated that the Xer1 recombinase is essential for site-specific recombination. In the so-called phase-locked xer1 mutants (PLMs) further vpma switching by site-specific DNA inversions was abolished and cells were locked in an expression phase where only one vpma gene is steadily expressed. In this study, recombination experiments in Escherichia coli demonstrated that a putative 21-bp recombination site (RS) located in the 5’ UTR of all six vpma genes is indeed involved in Xer1-mediated recombination. Furthermore, adjacent nucleotides flanking this conserved 21-bp region were shown to positively influence the recombination reaction. The orientation of the RS was crucial for the outcome of the recombination reaction. Recombination between inverted repeated RS led to inversions, whereas direct repeated alignment of the RS resulted in excisions. Excision events were also successfully demonstrated under native conditions in M. agalactiae, using a newly developed assay based on the lacZ reporter gene, which was the first application of this reporter system in M. agalactiae. Beside site-specific Xer1-mediated recombination, novel molecular mechanisms resulting in Vpma surface variation in the absence of the Xer1 recombinase were demonstrated. In another ongoing project PLMs lacking the xer1 gene, and therefore steadily expressing only a single Vpma product, were used in animal infection studies. Interestingly, PLMs isolated from the animals displayed a new Vpma phenotype. Molecular characterization of selected PLM switchover clones revealed that complex DNA rearrangements including generation of hybrid vpma genes, as well as gene duplications and deletions were triggered in the vpma locus, most likely due to the host immune pressure. Similar Xer1-independent Vpma variation could be demonstrated during in vitro immune pressure assays using Vpma-specific antibodies. These results clearly demonstrate that phase variation of Vpma surface proteins in M. agalactiae is necessary to survive in the immunocompetent host and provide important data for studying similar antigenic variation systems in other pathogenic Mycoplasma species

Quantitative analysis of mutant subclones in chronic myeloid leukemia : comparison of different methodological approaches

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points

Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points