Novel axonemal protein ZMYND12 interacts with TTC29 and DNAH1, and is required for male fertility and flagellum function.

Male infertility is common and complex, presenting a wide range of heterogeneous phenotypes. Although about 50% of cases are estimated to have a genetic component, the underlying cause often remains undetermined. Here, from whole-exome sequencing on samples from 168 infertile men with asthenoteratozoospermia due to severe sperm flagellum, we identified homozygous ZMYND12 variants in four unrelated patients. In sperm cells from these individuals, immunofluorescence revealed altered localization of DNAH1, DNALI1, WDR66, and TTC29. Axonemal localization of ZMYND12 ortholog TbTAX-1 was confirmed using the Trypanosoma brucei model. RNAi knock-down of TbTAX-1 dramatically affected flagellar motility, with a phenotype similar to the sperm from men bearing homozygous ZMYND12 variants. Co-immunoprecipitation and ultrastructure expansion microscopy in T. brucei revealed TbTAX-1 to form a complex with TTC29. Comparative proteomics with samples from Trypanosoma and Ttc29 KO mice identified a third member of this complex: DNAH1. The data presented revealed that ZMYND12 is part of the same axonemal complex as TTC29 and DNAH1, which is critical for flagellum function and assembly in humans, and Trypanosoma. ZMYND12 is thus a new asthenoteratozoospermia-associated gene, bi-allelic variants of which cause severe flagellum malformations and primary male infertility.

A Homozygous Ancestral SVA-Insertion-Mediated Deletion in WDR66 Induces Multiple Morphological Abnormalities of the Sperm Flagellum and Male Infertility.

Multiple morphological abnormalities of the sperm flagellum (MMAF) is a severe form of male infertility defined by the presence of a mosaic of anomalies, including short, bent, curled, thick, or absent flagella, resulting from a severe disorganization of the axoneme and of the peri-axonemal structures. Mutations in DNAH1, CFAP43, and CFAP44, three genes encoding axoneme-related proteins, have been described to account for approximately 30% of the MMAF cases reported so far. Here, we searched for pathological copy-number variants in whole-exome sequencing data from a cohort of 78 MMAF-affected subjects to identify additional genes associated with MMAF. In 7 of 78 affected individuals, we identified a homozygous deletion that removes the two penultimate exons of WDR66 (also named CFAP251), a gene coding for an axonemal protein preferentially localized in the testis and described to localize to the calmodulin- and spoke-associated complex at the base of radial spoke 3. Sequence analysis of the breakpoint region revealed in all deleted subjects the presence of a single chimeric SVA (SINE-VNTR-Alu) at the breakpoint site, suggesting that the initial deletion event was potentially mediated by an SVA insertion-recombination mechanism. Study of Trypanosoma WDR66's ortholog (TbWDR66) highlighted high sequence and structural analogy with the human protein and confirmed axonemal localization of the protein. Reproduction of the human deletion in TbWDR66 impaired flagellar movement, thus confirming WDR66 as a gene associated with the MMAF phenotype and highlighting the importance of the WDR66 C-terminal region.

A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells.

Inside the adenovirus virion, the genome forms a chromatin-like structure with viral basic core proteins. Core protein VII is the major DNA binding protein and was shown to remain associated with viral genomes upon virus entry even after nuclear delivery. It has been suggested that protein VII plays a regulatory role in viral gene expression and is a functional component of viral chromatin complexes in host cells. As such, protein VII could be used as a maker to track adenoviral chromatin complexes in vivo. In this study, we characterize a new monoclonal antibody against protein VII that stains incoming viral chromatin complexes following nuclear import. Furthermore, we describe the development of a novel imaging system that uses Template Activating Factor-I (TAF-I/SET), a cellular chromatin protein tightly bound to protein VII upon infection. This setup allows us not only to rapidly visualize protein VII foci in fixed cells but also to monitor their movement in living cells. These powerful tools can provide novel insights into the spatio-temporal regulation of incoming adenoviral chromatin complexes.

BILBO1 is a scaffold protein of the flagellar pocket collar in the pathogen Trypanosoma brucei.

The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC.

The amphipathic helix of adenovirus capsid protein VI contributes to penton release and postentry sorting.

UNLABELLED: Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry. IMPORTANCE: In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle.

Activation of the Pseudomonas aeruginosa type III secretion system requires an intact pyruvate dehydrogenase aceAB operon.

Pseudomonas aeruginosa clinical cystic fibrosis isolate CHA was mutagenized with Tn5Tc to identify new genes involved in type III secretion system (TTSS)-dependent cytotoxicity toward human polymorphonuclear neutrophils. Among 25 mutants affected in TTSS function, 14 contained the insertion at different positions in the aceAB operon encoding the PDH-E1 and -E2 subunits of pyruvate dehydrogenase. In PDH mutants, no transcriptional activation of TTSS genes in response to calcium depletion occurred. Expression in trans of ExsA restored TTSS function and cytotoxicity.