Functional and structural diversity in deubiquitinases of the Chlamydia-like bacterium Simkania negevensis.

Besides the regulation of many cellular pathways, ubiquitination is important for defense against invading pathogens. Some intracellular bacteria have evolved deubiquitinase (DUB) effector proteins, which interfere with the host ubiquitin system and help the pathogen to evade xenophagy and lysosomal degradation. Most intracellular bacteria encode one or two DUBs, which are often linkage-promiscuous or preferentially cleave K63-linked chains attached to bacteria or bacteria-containing vacuoles. By contrast, the respiratory pathogen Legionella pneumophila possesses a much larger number of DUB effectors, including a K6-specific enzyme belonging to the OTU family and an M1-specific DUB uniquely found in this bacterium. Here, we report that the opportunistic pathogen Simkania negevensis, which is unrelated to Legionella but has a similar lifestyle, encodes a similarly large number of DUBs, including M1- and K6-specific enzymes. Simkania DUBs are highly diverse and include DUB classes never before seen in bacteria. Interestingly, the M1- and K6-specific DUBs of Legionella and Simkania are unrelated, suggesting that their acquisition occurred independently. We characterize the DUB activity of eight Simkania-encoded enzymes belonging to five different DUB classes. We also provide a structural basis for the M1-specificity of a Simkania DUB, which most likely evolved from a eukaryotic otubain-like precursor.

Modulation of Host Cell Death and Lysis Are Required for the Release of Simkania negevensis.

Simkania negevensis is a Chlamydia-like bacterium and emerging pathogen of the respiratory tract. It is an obligate intracellular bacterium with a biphasic developmental cycle, which replicates in a wide range of host cells. The life cycle of S. negevensis has been shown to proceed for more than 12 days, but little is known about the mechanisms that mediate the cellular release of these bacteria. This study focuses on the investigation of host cell exit by S. negevensis and its connection to host cell death modulation. We show that Simkania-infected epithelial HeLa as well as macrophage-like THP-1 cells reduce in number during the course of infection. At the same time, the infectivity of the cell culture supernatant increases, starting at the day 3 for HeLa and day 4 for THP-1 cells and reaching maximum at day 5 post infection. This correlates with the ability of S. negevensis to block TNFα-, but not staurosporin-induced cell death up to 3 days post infection, after which cell death is boosted by the presence of bacteria. Mitochondrial permeabilization through Bax and Bak is not essential for host cell lysis and release of S. negevensis. The inhibition of caspases by Z-VAD-FMK, caspase 1 by Ac-YVAD-CMK, and proteases significantly reduces the number of released infectious particles. In addition, the inhibition of myosin II by blebbistatin also strongly affects Simkania release, pointing to a possible double mechanism of exit through host cell lysis and potentially extrusion.

Distribution and Structure of DM2 Repeat Tract Alleles in the German Population.

Autosomal dominant inherited Myotonic dystrophy type 1 and 2 (DM1 and DM2) are the most frequent muscle dystrophies in the European population and are caused by repeat expansion mutations. For Germany cumulative empiric evidence suggests an estimated prevalence of DM2 of roughly 9 in 100,000, therefore being as prevalent as DM1. In DM2, a (CCTG)n repeat tract located in the first intron of the CNBP gene is expanded. The CCTG repeat tract is part of a complex repeat structure comprising not only CCTG tetraplets but also repeated TG dinucleotides and TCTG tetraplet elements as well as NCTG interruptions. Here, we provide the distribution of normal sized alleles in the German population, which was found to be highly similar to the Slovak population. Sequencing of 34 unexpanded healthy range alleles in DM2 positive patients (heterozygous for a full expansion) revealed that the CCTG repeat tract is usually interrupted by at least three tetraplets which according to current opinion is supposed to render it stable against expansion. Interestingly, only the largest analyzed normal allele had 23 uninterrupted CCTGs and consequently could represent an instable early premutation allele. In our diagnostic history of DM2 cases, a total of 18 premutations were detected in 16 independent cases. Here, we describe two premutation families, one with an expansion from a premutation allele and the other with a contraction of a full expansion down to a premutation allele. Our diagnostic results support the general assumption that the premutation range of unstable CCTG stretches lies obviously between 25 and 75 CCTGs. However, the clinical significance of premutation alleles is still unclear. In the light of the two described families we suggest incomplete penetrance. Thus, as it was proposed for other repeat expansion diseases (e.g., Huntington's disease), a fluid transition of penetrance is more likely rather than a clear cut CCTG number threshold.