Flagellar Function Research Articles

Achievements in bacterial flagellar research with focus on Vibrio species.

In 1980s, the most genes involved in the bacterial flagellar function and formation had been isolated, although many of their functions or roles were not clarified. Bacterial flagella are the primary locomotive organ and are not necessary for growing in vitro but are probably essential for living in natural condition and are involved in the pathogenicity. In vitro, the flagella-deficient strains can grow at rates similar to wild-type strains. More than 50 genes are responsible for flagellar function, and the flagellum is constructed by more than 20 structural proteins. The maintenance cost of flagellum is high as several genes are required for its development. The fact that it evolved as a motor organ even with such high cost shows that the motility is indispensable to survive under the harsh environment of Earth. In this review, we focus on flagella-related research conducted by the authors for about 40 years and flagellar research focused on Vibrio spp.

Novel DNAH1 Mutation Loci Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Literature Review.

The protein encoded by dynein axonemal heavy chain 1 (DNAH1) is a part of dynein, which regulates the function of cilia and sperm flagella. The mutant of DNAH1 causes the deletion of inner dynein arm 3 in the flagellum, leading to multiple morphological abnormalities of the sperm flagella (MMAF) and severe asthenozoospermia. However, instead of asthenozoospermia and MMAF, the result caused by the mutation of DNAH1 remains unknown. Here we report a male infertility patient with severe asthenozoospermia and teratozoospermia. We found two heterozygous mutations in DNAH1 (c.6912C>A and c.7076G>T) and which were reported to be associated with MMAF for the first time. We next collected and analyzed 65 cases of DNAH1 mutation and found that the proportion of short flagella is the largest, while the bent flagella account for the smallest, and the incidence of head deformity is not high in the sperm of these patients. Finally, we also analyzed 31 DNAH1 mutation patients who were treated with intracytoplasmic sperm injection (ICSI) and achieved beneficial outcomes. We hope our research will be helpful in the diagnosis and treatment of male infertility caused by DNAH1 mutation.

Bridging the N-terminal and middle domains in FliG of the flagellar rotor.

Flagella are necessary for bacterial movement and contribute to various aspects of virulence. They are complex cylindrical structures built of multiple molecular rings with self-assembly properties. The flagellar rotor is composed of the MS-ring and the C-ring. The FliG protein of the C-ring is central to flagellar assembly and function due to its roles in linking the C-ring with the MS-ring and in torque transmission from stator to rotor. No high-resolution structure of an assembled C-ring has been resolved to date, and the conformation adopted by FliG within the ring is unclear due to variations in available crystallographic data. Here, we use molecular dynamics (MD) simulations to study the conformation and dynamics of FliG in different states of assembly, including both in physiologically relevant and crystallographic lattice environments. We conclude that the linker between the FliG N-terminal and middle domain likely adopts an extended helical conformation in vivo, in contrast with the contracted conformation observed in some previous X-ray studies. We further support our findings with integrative model building of full-length FliG and a FliG ring model that is compatible with cryo-electron tomography (cryo-ET) and electron microscopy (EM) densities of the C-ring. Collectively, our study contributes to a better mechanistic understanding of the flagellar rotor assembly and its function.

Bhalin, an Essential Cytoskeleton-Associated Protein of Trypanosoma brucei Linking TbBILBO1 of the Flagellar Pocket Collar with the Hook Complex.

Background: In most trypanosomes, endo and exocytosis only occur at a unique organelle called the flagellar pocket (FP) and the flagellum exits the cell via the FP. Investigations of essential cytoskeleton-associated structures located at this site have revealed a number of essential proteins. The protein TbBILBO1 is located at the neck of the FP in a structure called the flagellar pocket collar (FPC) and is essential for biogenesis of the FPC and parasite survival. TbMORN1 is a protein that is present on a closely linked structure called the hook complex (HC) and is located anterior to and overlapping the collar. TbMORN1 is essential in the bloodstream form of T. brucei. We now describe the location and function of BHALIN, an essential, new FPC-HC protein. Methodology/Principal Findings: Here, we show that a newly characterised protein, BHALIN (BILBO1 Hook Associated LINker protein), is localised to both the FPC and HC and has a TbBILBO1 binding domain, which was confirmed in vitro. Knockdown of BHALIN by RNAi in the bloodstream form parasites led to cell death, indicating an essential role in cell viability. Conclusions/Significance: Our results demonstrate the essential role of a newly characterised hook complex protein, BHALIN, that influences flagellar pocket organisation and function in bloodstream form T. brucei parasites.

BB0259 Encompasses a Peptidoglycan Lytic Enzyme Function for Proper Assembly of Periplasmic Flagella in Borrelia burgdorferi.

Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most β- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb.

Molecular Determinants of Filament Capping Proteins Required for the Formation of Functional Flagella in Gram-Negative Bacteria.

Bacterial flagella are cell surface protein appendages that are critical for motility and pathogenesis. Flagellar filaments are tubular structures constructed from thousands of copies of the protein flagellin, or FliC, arranged in helical fashion. Individual unfolded FliC subunits traverse the filament pore and are folded and sorted into place with the assistance of the flagellar capping protein complex, an oligomer of the FliD protein. The FliD filament cap is a stool-like structure, with its D2 and D3 domains forming a flat head region, and its D1 domain leg-like structures extending perpendicularly from the head towards the inner core of the filament. Here, using an approach combining bacterial genetics, motility assays, electron microscopy and molecular modeling, we define, in numerous Gram-negative bacteria, which regions of FliD are critical for interaction with FliC subunits and result in the formation of functional flagella. Our data indicate that the D1 domain of FliD is its sole functionally important domain, and that its flexible coiled coil region comprised of helices at its extreme N- and C-termini controls compatibility with the FliC filament. FliD sequences from different bacterial species in the head region are well tolerated. Additionally, head domains can be replaced by small peptides and larger head domains from different species and still produce functional flagella.

CONCURRENT ULTRASTRUCTURAL ANALYSIS AND GENOMIC ASSESSMENT OF SPERMATOZOA TO ACCURATELY DIAGNOSE RARE AND SEVERE FORMS OF MALE INFERTILITY

In men with ART failure, sperm DNA sequencing with ultrastructural analysis can identify specific sperm defects and guide ART treatment. In the past 2 years, 22 consenting men who failed IVF cycles due to severe astheno-/teratozoospermia were recruited; specimens from 3 fertile men were used as controls. To assess ultrastructure by transmission electron microscopy (TEM), pelleted spermatozoa were fixed, embedded, sectioned to 100-nm slides by ultramicrotome, and viewed with an electron microscope (JEOL-1400) at 300,000X magnification. Sperm head morphology, fibrous sheath intactness, and axonemal/periaxonemal structure were examined on at least 100 spermatozoa to diagnose specific sperm organelle defects. DNA was extracted and amplified from at least 500 spermatozoa for next-generation sequencing (NGS) to identify mutations contributing to the ultrastructural abnormalities. Four types of sperm ultrastructural defects were identified: globozoospermia, dysplasia of fibrous sheath (DFS), proximal centriole defect, and primary ciliary dyskinesia (PCD). One case of combined globozoospermia and DFS and one case of combined DFS and proximal centriole defect were identified. In 13 globozoospermic cases, 97–100% of the spermatozoa displayed complete spherical heads with the absence of acrosomes, perinuclear theca deformities, and dispersed chromatin. Microtubular arrangement of centrosome and flagellum was conserved. NGS identified deletions (DPY19L, PICK1, SPATA16) directly responsible for the globozoospermic phenotype. Based on the findings, assisted oocyte activation was performed for 7 couples with an oocyte-activating agent, boosting fertilization from 5.7% to 40.2% (p<0.00001), resulting in 5 pregnancies and 4 deliveries. In patients with DFS (n=6), flagellar deformities were observed in 90–100% of spermatozoa with mitochondria disorganization, disarranged microtubules, and cytoplasmic remnant enveloping coiled flagella, indicating incomplete late spermiogenesis. Normal acrosomes and nuclei were seen. NGS identified deletions (AKAP4, SPAG16, CATSPER1) involved in flagellar development and function. For patients with proximal centriole defects (n=2), the sperm nuclei and fibrous sheath appeared normal. Approximately 90% of proximal centrioles exhibited microtubular disorganization, confirmed by an ODF2 mutation identified by NGS. For a patient with PCD, chaotic flagellar microtubular arrangement and the absence of outer dynein arms prevailed in 90% of the axonemal cross-sections examined, confirmed by a DNAH5 gene deletion. Nonetheless, a few motile spermatozoa were identified and used for ICSI, yielding 80% fertilization and the delivery of a healthy twin. Concurrent ultrastructural and genomic analysis can pinpoint sperm organelle disruption and lead to successful ART cycles.

Bi-allelic mutations in DNAH7 cause asthenozoospermia by impairing the integrality of axoneme structure.

Asthenozoospermia is the most common cause of male infertility. Dynein protein arms play a crucial role in the motility of both the cilia and flagella, and defects in these proteins generally impair the axoneme structure and cause primary ciliary dyskinesia. But relatively little is known about the influence of dynein protein arm defects on sperm flagella function. Here, we recruited 85 infertile patients with idiopathic asthenozoospermia and identified bi-allelic mutations in DNAH7 (NM_018897.3) from three patients using whole-exome sequencing. These variants are rare, highly pathogenic, and very conserved. The spermatozoa from the patients with DNAH7 bi-allelic mutations showed specific losses in the inner dynein arms. The expression of DNAH7 in the spermatozoa from the DNAH7-defective patients was significantly decreased, but these patients were able to have their children via intra-cytoplasmic sperm injection treatment. Our study is the first to demonstrate that bi-allelic mutations in DNAH7 may impair the integrality of axoneme structure, affect sperm motility, and cause asthenozoospermia in humans. These findings may extend the spectrum of etiological genes and provide new clues for the diagnosis and treatment of patients with asthenozoospermia.

Bi-allelic truncating variants in CFAP206 cause male infertility in human and mouse.

Spermatozoa are polarized cells with a head and a flagellum joined together by the connecting piece. Flagellum integrity is critical for normal sperm function, and flagellum defects consistently lead to male infertility. Multiple morphological abnormalities of the flagella (MMAF) is a distinct sperm phenotype consistently leading to male infertility due to a reduced or absent sperm motility associated with severe morphological and ultrastructural flagellum defects. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analyzed remain unresolved, suggesting that many yet uncharacterized gene defects account for this phenotype. By performing a retrospective exome analysis of the unsolved cases from our initial cohort of 167 infertile men with a MMAF phenotype, we identified one individual carrying a homozygous frameshift variant in CFAP206, a gene encoding a microtubule-docking adapter for radial spoke and inner dynein arm. Immunostaining experiments in the patient's sperm cells demonstrated the absence of WDR66 and RSPH1 proteins suggesting severe radial spokes and calmodulin and spoke-associated complex defects. Using the CRISPR-Cas9 technique, we generated homozygous Cfap206 knockout (KO) mice which presented with male infertility due to functional, structural and ultrastructural sperm flagellum defects associated with a very low rate of embryo development using ICSI. Overall, we showed that CFAP206 is essential for normal sperm flagellum structure and function in human and mouse and that bi-allelic mutations in CFAP206 cause male infertility in man and mouse by inducing morphological and functional defects of the sperm flagellum that may also cause ICSI failures.

Molecular chaperone Hsp90 protects KCBP from degradation by proteasome in Dunaliella salina cells.

Kinesin-like calmodulin-binding protein (KCBP) is a unique kinesin with half kinesin and half myosin, with kinesin motor domain at C-terminus and myosin tail homology region 4 (MyTH4) and band 4.1, ezrin, radixin, moesin (FERM) domains at N-terminus. The special structure endows KCBP multi-intracellular functions, including cell division, trichome morphogenesis in plants, and flagellar function in algae. However, little is known about the molecular mechanism underlying these functions. Here, we identified a molecular chaperone Hsp90 as a novel binding partner with KCBP in Dunaliella salina using a yeast two-hybrid screen. Further analysis showed that Hsp90 interacted with both the N-terminal and C-terminal of DsKCBP. Since Hsp90 was involved in the stability and proteolytic turnover of numerous proteins, whether Hsp90 regulated the degradation of DsKCBP was investigated. Our results showed that both Hsp90 and DsKCBP presented in the purified proteasome, and the interaction of DsKCBP-Hsp90 was inhibited upon Hsp90 inhibitor geldanamycin treatment. The level of DsKCBP proteins was diminished remarkably indicating that the disassociation of DsKCBP from Hsp90 accelerated the degradation of the former. Furthermore, immunofluorescence results showed that the localization of DsKCBP at basal body and flagella was disappeared by Hsp90 inhibition. The increased mRNA level of DsKCBP during flagellar assembly was not obvious by geldanamycin treatment. These data provided evidence that Hsp90 protected DsKCBP from degradation by proteasome and was involved in the role of DsKCBP in flagellar assembly.

Flagellum Malfunctions Trigger Metaboly as an Escape Strategy in Euglena gracilis.

Euglenoids present the ability to alter the shape of their bodies, a process referred to as metaboly. Metaboly is usually used by phagotrophic cells to engulf their prey. However, Euglena gracilis is osmotrophic and photosynthetic. Though metaboly was discovered centuries ago, it remains unclear why E.gracilis undergo metaboly and what causes them to deform, and some consider metaboly to be a functionless ancestral vestige. Here, we discover that flagellum malfunctions trigger metaboly and metaboly is a smart escape strategy adopted by E. gracilis when the proper rotation and beating of the flagellum are hindered by restrictions including surface obstruction, sticking, resistance, or limited space. Metaboly facilitates escape in five ways: (i) detaching the body from the surface; (ii) enlarging the space between flagellum and the restricting surface which restores beating and rotation of the flagellum; (iii) decreasing the torque of viscous resistance for rotation of the body; (iv) decreasing the length of the body; and (v) crawling backwards on a surface or swimming backwards if the flagellum completely malfunctions or has broken off. Our findings suggest that metaboly plays a key role in enabling E.gracilis to escape from harmful conditions when flagellar functions are impaired or absent.

Motility, Adhesion and c-di-GMP Influence the Endophytic Colonization of Rice by Azoarcus sp. CIB.

Proficient crop production is needed to ensure the feeding of a growing global population. The association of bacteria with plants plays an important role in the health state of the plants contributing to the increase of agricultural production. Endophytic bacteria are ubiquitous in most plant species providing, in most cases, plant promotion properties. However, the knowledge on the genetic determinants involved in the colonization of plants by endophytic bacteria is still poorly understood. In this work we have used a genetic approach based on the construction of fliM, pilX and eps knockout mutants to show that the motility mediated by a functional flagellum and the pili type IV, and the adhesion modulated by exopolysaccarides are required for the efficient colonization of rice roots by the endophyte Azoarcus sp. CIB. Moreover, we have demonstrated that expression of an exogenous diguanylate cyclase or phophodiesterase, which causes either an increase or decrease of the intracellular levels of the second messenger cyclic di-GMP (c-di-GMP), respectively, leads to a reduction of the ability of Azoarcus sp. CIB to colonize rice plants. Here we present results demonstrating the unprecedented role of the universal second messenger cyclic-di-GMP in plant colonization by an endophytic bacterium, Azoarcus sp. CIB. These studies pave the way to further strategies to modulate the interaction of endophytes with their target plant hosts.

Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility.

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.

Asymmetric random walks reveal that the chemotaxis network modulates flagellar rotational bias in Helicobacter pylori.

The canonical chemotaxis network modulates the bias for a particular direction of rotation in the bacterial flagellar motor to help the cell migrate toward favorable chemical environments. How the chemotaxis network in Helicobacter pylori modulates flagellar functions is unknown, which limits our understanding of chemotaxis in this species. Here, we determined that H. pylori swim faster (slower) whenever their flagella rotate counterclockwise (clockwise) by analyzing their hydrodynamic interactions with bounding surfaces. This asymmetry in swimming helped quantify the rotational bias. Upon exposure to a chemo-attractant, the bias decreased and the cells tended to swim exclusively in the faster mode. In the absence of a key chemotaxis protein, CheY, the bias was zero. The relationship between the reversal frequency and the rotational bias was unimodal. Thus, H. pylori's chemotaxis network appears to modulate the probability of clockwise rotation in otherwise counterclockwise-rotating flagella, similar to the canonical network.

The Effect of Antimicrobial Resistance Plasmids Carrying blaCMY-2 on Biofilm Formation by Escherichia coli from the Broiler Production Chain.

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCR E. coli) with plasmids carrying the blaCMY-2 resistance gene have been isolated from the Norwegian broiler production chain through the Norwegian monitoring program for antimicrobial resistance in animals, food and feed, NORM-VET. The aim of the present study was to investigate the biofilm forming abilities of these strains, and in particular to see whether these might be influenced by the carriage of blaCMY-2 plasmids. The ESCR E. coli from the broiler production chain displayed relatively low biofilm forming abilities in the crystal violet biofilm assay as compared to quinolone-resistant E. coli (QREC) from the same population (mean ± SD = 0.686 ± 0.686 vs. 1.439 ± 0.933, respectively). Acquisition of two different blaCMY-2 plasmids by QREC strains reduced their biofilm production in microtiter plates, but not their biofilm production on Congo Red agar plates. Furthermore, motility was reduced, but not planktonic growth. We hypothesize that genes carried by these plasmids may have caused the observed reduction in biofilm formation, possibly mediated through changes in flagellar expression or function. Furthermore, this may help explain the different biofilm forming abilities observed between ESCR E. coli and QREC. The results also indicate that the risk of biofilm reservoirs of antimicrobial resistant E. coli on in the broiler production is lower for ESCR E. coli than for QREC.

Experimental Evolution of Campylobacter jejuni Leads to Loss of Motility, rpoN (σ54) Deletion and Genome Reduction.

Evolution experiments in the laboratory have focused heavily on model organisms, often to the exclusion of clinically relevant pathogens. The foodborne bacterial pathogen Campylobacter jejuni belongs to a genus whose genomes are small compared to those of its closest genomic relative, the free-living genus Sulfurospirillum, suggesting genome reduction during the course of evolution to host association. In an in vitro experiment, C. jejuni serially passaged in rich medium in the laboratory exhibited loss of flagellar motility–an essential function for host colonization. At early time points the motility defect was often reversible, but after 35 days of serial culture, motility was irreversibly lost in most cells in 5 independently evolved populations. Population re-sequencing revealed disruptive mutations to genes in the flagellar transcriptional cascade, rpoN (σ54)—therefore disrupting the expression of the genes σ54 regulates—coupled with deletion of rpoN in all evolved lines. Additional mutations were detected in virulence-related loci. In separate in vivo experiments, we demonstrate that a phase variable (reversible) motility mutant carrying an adenine deletion within a homopolymeric tract resulting in truncation of the flagellar biosynthesis gene fliR was deficient for colonization in a C57BL/6 IL-10–/– mouse disease model. Re-insertion of an adenine residue partially restored motility and ability to colonize mice. Thus, a pathogenic C. jejuni strain was rapidly attenuated by experimental laboratory evolution and demonstrated genomic instability during this evolutionary process. The changes observed suggest C. jejuni is able to evolve in a novel environment through genome reduction as well as transition, transversion, and slip-strand mutations.

Restoration of wild-type motility to flagellin-knockout Escherichia coli by varying promoter, copy number and induction strength in plasmid-based expression of flagellin.

Flagellin is the major constituent of the flagellar filament and faithful restoration of wild-type motility to flagellin mutants may be beneficial for studies of flagellar biology and biotechnological exploitation of the flagellar system. However, gene complementation studies often fail to report whether true wild-type motility was restored by expressing flagellin from a plasmid. Therefore, we explored the restoration of motility by flagellin expressed from a variety of combinations of promoter, plasmid copy number and induction strength. Motility was only partially (~50%) restored using the tightly regulated rhamnose promoter due to weak flagellin gene expression, but wild-type motility was regained with the T5 promoter, which, although leaky, allowed titration of induction strength. The endogenous E. coli flagellin promoter also restored wild-type motility. However, flagellin gene transcription levels increased 3.1–27.9-fold when wild-type motility was restored, indicating disturbances in the flagellar regulatory mechanisms. Motility was little affected by plasmid copy number when dependent on inducible promoters. However, plasmid copy number was important when expression was controlled by the native E. coli flagellin promoter. Motility was poorly correlated with flagellin transcription levels, but strongly correlated with the amount of flagellin associated with the flagellar filament, suggesting that excess monomers are either not exported or not assembled into filaments. This study provides a useful reference for further studies of flagellar function and a simple blueprint for similar studies with other proteins.

Structural Conservation and Adaptation of the Bacterial Flagella Motor.

Many bacteria require flagella for the ability to move, survive, and cause infection. The flagellum is a complex nanomachine that has evolved to increase the fitness of each bacterium to diverse environments. Over several decades, molecular, biochemical, and structural insights into the flagella have led to a comprehensive understanding of the structure and function of this fascinating nanomachine. Notably, X-ray crystallography, cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) have elucidated the flagella and their components to unprecedented resolution, gleaning insights into their structural conservation and adaptation. In this review, we focus on recent structural studies that have led to a mechanistic understanding of flagellar assembly, function, and evolution.

Variants in RABL2A causing male infertility and ciliopathy.

Approximately 7% of men worldwide suffer from infertility, with sperm abnormalities being the most common defect. Though genetic causes are thought to underlie a substantial fraction of idiopathic cases, the actual molecular bases are usually undetermined. Because the consequences of most genetic variants in populations are unknown, this complicates genetic diagnosis even after genome sequencing of patients. Some patients with ciliopathies, including primary ciliary dyskinesia and Bardet-Biedl syndrome, also suffer from infertility because cilia and sperm flagella share several characteristics. Here, we identified two deleterious alleles of RABL2A, a gene essential for normal function of cilia and flagella. Our in silico predictions and in vitro assays suggest that both alleles destabilize the protein. We constructed and analyzed mice homozygous for these two single-nucleotide polymorphisms, Rabl2L119F (rs80006029) and Rabl2V158F (rs200121688), and found that they exhibit ciliopathy-associated disorders including male infertility, early growth retardation, excessive weight gain in adulthood, heterotaxia, pre-axial polydactyly, neural tube defects and hydrocephalus. Our study provides a paradigm for triaging candidate infertility variants in the population for in vivo functional validation, using computational, in vitro and in vivo approaches.

A structure-function study of C-terminal residues predicted to line the export channel in Salmonella Flagellin

BackgroundStructural studies of a Salmonella Typhimurium flagellin protein indicated that four polar or charged C-terminal amino acid residues line the inner channel of the flagellum. The hydrophilic character of these putative channel-lining residues was predicted to be essential to facilitate the transport of unfolded flagellin monomers during flagellar assembly. The structure-function relationship of these putative channel-lining residues was investigated by site-directed mutagenesis to examine effects of side chain polarity and size on flagella assembly and function. MethodsChannel-lining residue variants were generated using site-directed mutagenesis to substitute alanine and other residues to examine the effects of altered side-chain polarity on export and assembly. The export, in vivo motility function, and flagellar structure of variants was characterized by agar motility, video microscopy, immunofluorescence, and SDS-PAGE. ResultsAlanine substitution yielded decreased motility and flagellar assembly for three of the four residues. However, alanine substitution of residue Arg 494 did not alter export, although substitution with negatively charged glutamate decreased motility and flagellar filament length. Furthermore, many of the C-terminal mutations affected flagellar filament morphology and stability, often resulting in more tightly coiled and/or more brittle flagella than the wild type. ConclusionsThe four channel-lining C-terminal residues may facilitate monomer protein transport but also have structural roles in determining the stability and morphology of the flagellum. General significanceThese results provide further insight into the complex process of bacterial flagellin export and flagellar assembly and provide evidence of previously unknown structural functions for the four putative channel-lining residues.