Peter Agre, Mélanie Bonhivers, Mario J. Borgnia

Spermatogenesis defects concern millions of men worldwide, yet the vast majority remains undiagnosed. Here we report men with primary infertility due to multiple morphological abnormalities of the sperm flagella with severe disorganization of the sperm axoneme, a microtubule-based structure highly conserved throughout evolution. Whole-exome sequencing was performed on 78 patients allowing the identification of 22 men with bi-allelic mutations in DNAH1 (n = 6), CFAP43 (n = 10), and CFAP44 (n = 6). CRISPR/Cas9 created homozygous CFAP43/44 male mice that were infertile and presented severe flagellar defects confirming the human genetic results. Immunoelectron and stimulated-emission-depletion microscopy performed on CFAP43 and CFAP44 orthologs in Trypanosoma brucei evidenced that both proteins are located between the doublet microtubules 5 and 6 and the paraflagellar rod. Overall, we demonstrate that CFAP43 and CFAP44 have a similar structure with a unique axonemal localization and are necessary to produce functional flagella in species ranging from Trypanosoma to human.

Trypanosoma brucei is a protozoan parasite that is used as a model organism to study such biological phenomena as gene expression, protein trafficking, and cytoskeletal biogenesis. In T. brucei, endocytosis and exocytosis occur exclusively through a sequestered organelle called the flagellar pocket (FP), an invagination of the pellicular membrane. The pocket is the sole site for specific receptors thus maintaining them inaccessible to components of the innate immune system of the mammalian host. The FP is also responsible for the sorting of protective parasite glycoproteins targeted to, or recycling from, the pellicular membrane, and for the removal of host antibodies from the cell surface. Here, we describe the first characterisation of a flagellar pocket cytoskeletal protein, BILBO1. BILBO1 functions to form a cytoskeleton framework upon which the FP is made and which is also required and essential for FP biogenesis and cell survival. Remarkably, RNA interference (RNAi)-mediated ablation of BILBO1 in insect procyclic-form parasites prevents FP biogenesis and induces vesicle accumulation, Golgi swelling, the aberrant repositioning of the new flagellum, and cell death. Cultured bloodstream-form parasites are also nonviable when subjected to BILBO1 RNAi. These results provide the first molecular evidence for cytoskeletally mediated FP biogenesis. Trypanosomes are ubiquitous unicellular parasites that infect humans, animals, insects, and plants. African, Asian, and some South American trypanosomes have evolved the amazing ability to change their surface coat proteins, an essential strategy for their survival. The surface coat proteins are recycled and targeted to the surface of the parasite via an endocytic and exocytotic organelle called the flagellar pocket, which is sequestered in the trypanosome cell's cytoplasm. The flagellar pocket is also used to remove host-derived antibodies that are bound to the surface of the parasite, making this organelle critical for the parasite's evasion of the host immune system. We describe a novel protein, “BILBO1,” which was identified from the insect-form parasite of the African trypanosome Trypanosoma brucei. We show that BILBO1 is part of a ring or horseshoe-like cytoskeletal structure that is located in a region of the flagellar pocket called the collar. When BILBO1 transcripts were knocked down with inducible RNA interference, trypanosome cells became arrested in a post-mitotic cell-cycle stage. Induced cells lost the normal flagellum-to-cell-body attachment, were unable to regulate endocytosis and exocytosis, and most importantly, were unable to construct a new flagellar pocket. These results provide molecular evidence for the idea that flagellar pocket biogenesis is cytoskeletally mediated.

The NIMA-related kinase 2 (NEK 2) has important cell cycle functions related to centriole integrity and splitting. Trypanosoma brucei does not possess centrioles, however, cytokinesis is coupled to basal body separation events. Here we report the first functional characterisation of a T. brucei basal body-cytoskeletal NIMA-related kinase (NRK) protein, TbNRKC. The TbNRKC kinase domain has high amino acid identity with the human NEK1 kinase domain (50%) but also shares 42% identity with human NEK2. TbNRKC is expressed in bloodstream and procyclic cells and functions as a bona fide kinase in vitro. Remarkably, RNAi knockdown of TbNRKC and overexpression of kinase-dead TbNRKC in procyclic forms induces the accumulation of cells with four basal bodies, whereas overexpression of active protein produces supernumary basal bodies and blocks cytokinesis. TbNRKC is located on mature and immature basal bodies and is the first T. brucei NRK to be found associated with the basal body cytokinesis pathway.

Can whole-exome sequencing (WES) of infertile patients identify new genes responsible for multiple morphological abnormalities of the sperm flagella (MMAF)? WES analysis of 78 infertile men with a MMAF phenotype permitted the identification of four homozygous mutations in the fibrous sheath (FS) interacting protein 2 (FSIP2) gene in four unrelated individuals. The use of high-throughput sequencing techniques revealed that mutations in the dynein axonemal heavy chain 1 (DNAH1) gene, and in the cilia and flagella associated protein 43 (CFAP43) and 44 (CFAP44) genes account for approximately one-third of MMAF cases thus indicating that other relevant genes await identification. This was a retrospective genetics study of 78 patients presenting a MMAF phenotype who were recruited in three fertility clinics between 2008 and 2015. Control sperm samples were obtained from normospermic donors. Allelic frequency for control subjects was derived from large public databases. WES was performed for all 78 subjects. All identified variants were confirmed by Sanger sequencing. Relative mRNA expression levels for the selected candidate gene (FSIP2) was assessed by quantitative RT-PCR in a panel of normal human and mouse tissues. To characterize the structural and ultrastructural anomalies present in patients' sperm, immunofluorescence (IF) was performed on sperm samples from two subjects with a mutation and one control and transmission electron microscopy (TEM) analyses was performed on sperm samples from one subject with a mutation and one control. We identified four unrelated patients (4/78, 5.1%) with homozygous loss of function mutations in the FSIP2 gene, which encodes a protein of the sperm FS and is specifically expressed in human and mouse testis. None of these mutations were reported in control sequence databases. TEM analyses showed a complete disorganization of the FS associated with axonemal defects. IF analyses confirmed that the central-pair microtubules and the inner and outer dynein arms of the axoneme were abnormal in all four patients carrying FSIP2 mutations. Importantly, and in contrast to what was observed in patients with MMAF and mutations in other MMAF-related genes (DNAH1, CFAP43 and CFAP44), mutations in FSIP2 led to the absence of A-kinase anchoring protein 4 (AKAP4). The low number of biological samples and the absence of a reliable anti-FSIP2 antibody prevented the formal demonstration that the FSIP2 protein was absent in sperm from subjects with a FSIP2 mutation. Our findings indicate that FSIP2 is one of the main genes involved in MMAF syndrome. In humans, genes previously associated with a MMAF phenotype encoded axonemal-associated proteins (DNAH1, CFAP43 and CFAP44). We show here that FSIP2, a protein of the sperm FS, is also logically associated with MMAF syndrome as we showed that it is necessary for FS assembly and for the overall axonemal and flagellar biogenesis. As was suggested before in mouse and man, our results also suggest that defects in AKAP4, one of the main proteins interacting with FSIP2, would induce a MMAF phenotype. Finally, this work reinforces the demonstration that WES sequencing is a good strategy to reach a genetic diagnosis for patients with severe male infertility phenotypes. This work was supported by the following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 (14-CE15) and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.