Flagellar Protein Research Articles

Flexible fitting of AlphaFold2-Predicted models to Cryo-EM density maps using elastic network models: a methodical affirmation

Abstract Motivation This study investigates the flexible refinement of AlphaFold2 models against corresponding cryo-electron microscopy (cryo-EM) maps using normal modes derived from elastic network models (ENMs) as basis functions for displacement. AlphaFold2 generally predicts highly accurate structures, but 18 of the 137 models of isolated chains exhibit a TM-score below 0.80. We achieved a significant improvement in four of these deviating structures and used them to systematically optimize the parameters of the ENM motion model. Results We successfully refined four AlphaFold2 models with notable discrepancies: lipid-preserved respiratory supercomplex (TM-score increased from 0.52 to 0.69), flagellar L-ring protein (TM-score increased from 0.53 to 0.64), cation diffusion facilitator YiiP (TM-score increased from 0.76 to 0.83), and Sulfolobus islandicus pilus (TM-score increased from 0.77 to 0.85). We explored the effect of three different mode ranges (modes 1–9, 7–9, and 1–12), masked or box-cropped density maps, numerical optimization methods, and two similarity measures (Pearson correlation and inner product). The best results were achieved for the widest mode range (modes 1–12), masked maps, inner product, and local Powell optimization. These optimal parameters were implemented in the flexible fitting utility elforge.py in version 1.4 of our Python-based ModeHunter package. Availability https://modehunter.biomachina.org.

Novel variants in DNAH9 are present in two infertile patients with severe asthenospermia.

Asthenospermia is a type of sperm that has malformed sperm with movement disorders that lead to male infertility. DNAH9 is a member of the dynein family and a central part of the outer dynein arm of cilia and flagella. DNAH9 gene defects are associated with primary ciliary dyskinesia and ultrastructural abnormalities in ciliary axial ultrastructure. However, the role of DNAH9 in sperm motility remains unclear, prompting us to investigate its function in spermatozoa. Familial Sanger sequencing showed that sterile males carried homozygous DNAH9 variants (c. 12218A>C, p. N4073T) and compound heterozygous variants (c.8617G>A, p.V2873M; c.11742A>T, p.E3914D), respectively. Transmission electron microscopy revealed these variants resulted in a significant lack of outer dynein arms in the cross-sectional view of the axoneme in both patients. Immunofluorescence results showed that these variants can lead to decline in DNAH9 protein expression, which led to the dysfunction of flagellar ultrastructure-related proteins, including DNAI1, DNAH1 and DNAH10. In conclusion, we identified novel biallelic variants in DNAH9 that likely bring about sharply decreased motility of spermatozoa in the two patients with asthenospermia. Our findings will widen the variant spectrum of known DNAH9 variants involving asthenospermia and further offer more proofs for genetic counseling and diagnosis.

Uncovering bacterial-mammalian cell interactions via single-cell tracking

BackgroundThe interactions between bacterial pathogens and host cells are characterized by a multitude of complexities, leading to a wide range of heterogeneous outcomes. Despite extensive research, we still have a limited understanding of how bacterial motility in complex environments impacts their ability to tolerate antibiotics and adhere to mammalian cell surfaces. The challenge lies in unraveling the complexity of these interactions and developing quantitative microscopy approaches to predict the behavior of bacterial populations.ResultsTo address this challenge, we directed our efforts towards Pseudomonas aeruginosa, a pathogenic bacterium known for producing thick films in the lungs of cystic fibrosis patients, and Escherichia coli, used as a proof of concept to develop and demonstrate our single-cell tracking approaches. Our results revealed that P. aeruginosa exhibits diverse and complex interactions on mammalian cell surfaces, such as adhesion, rotational motion, and swimming, unlike the less interactive behavior of Escherichia coli. Our analysis indicated that P. aeruginosa demonstrated lower mean-squared displacement (MSD) values and greater adherence to mammalian cells compared to E. coli, which showed higher MSD slopes and less frequent adherence. Genetic mutations in membrane proteins of P. aeruginosa resulted in altered displacement patterns and reduced adhesion, with the ΔfliD mutant displaying a more Gaussian displacement distribution and significantly less adherence to mammalian cells. Adhesion and tolerance mechanisms are diverse and complex, potentially involving distinct pathways; however, our findings highlight the therapeutic potential of targeting the fliD gene (encoding a critical flagellum protein), as its deletion not only reduced adherence but also antibiotic tolerance.ConclusionsOverall, our findings underscore the importance of single cell tracking in accurately assessing bacterial behavior over short time periods and highlight its significant potential in guiding effective intervention strategies.

DNAH3 deficiency causes flagellar inner dynein arm loss and male infertility in humans and mice

Axonemal protein complexes, including the outer and inner dynein arms (ODA/IDA), are highly ordered structures of the sperm flagella that drive sperm motility. Deficiencies in several axonemal proteins have been associated with male infertility, which is characterized by asthenozoospermia or asthenoteratozoospermia. Dynein axonemal heavy chain 3 (DNAH3) resides in the IDA and is highly expressed in the testis. However, the relationship between DNAH3 and male infertility is still unclear. Herein, we identified biallelic variants of DNAH3 in four unrelated Han Chinese infertile men with asthenoteratozoospermia through whole-exome sequencing (WES). These variants contributed to deficient DNAH3 expression in the patients’ sperm flagella. Importantly, the patients represented the anomalous sperm flagellar morphology, and the flagellar ultrastructure was severely disrupted. Intriguingly, Dnah3 knockout (KO) male mice were also infertile, especially showing the severe reduction in sperm movement with the abnormal IDA and mitochondrion structure. Mechanically, nonfunctional DNAH3 expression resulted in decreased expression of IDA-associated proteins in the spermatozoa flagella of patients and KO mice, including DNAH1, DNAH6, and DNALI1, the deletion of which has been involved in disruption of sperm motility. Moreover, the infertility of patients with DNAH3 variants and Dnah3 KO mice could be rescued by intracytoplasmic sperm injection (ICSI) treatment. Our findings indicated that DNAH3 is a novel pathogenic gene for asthenoteratozoospermia and may further contribute to the diagnosis, genetic counseling, and prognosis of male infertility.

DNAH3 deficiency causes flagellar inner dynein arm loss and male infertility in humans and mice.

Axonemal protein complexes, including the outer and inner dynein arms (ODA/IDA), are highly ordered structures of the sperm flagella that drive sperm motility. Deficiencies in several axonemal proteins have been associated with male infertility, which is characterized by asthenozoospermia or asthenoteratozoospermia. Dynein axonemal heavy chain 3 (DNAH3) resides in the IDA and is highly expressed in the testis. However, the relationship between DNAH3 and male infertility is still unclear. Herein, we identified biallelic variants of DNAH3 in four unrelated Han Chinese infertile men with asthenoteratozoospermia through whole-exome sequencing (WES). These variants contributed to deficient DNAH3 expression in the patients' sperm flagella. Importantly, the patients represented the anomalous sperm flagellar morphology, and the flagellar ultrastructure was severely disrupted. Intriguingly, Dnah3 knockout (KO) male mice were also infertile, especially showing the severe reduction in sperm movement with the abnormal IDA and mitochondrion structure. Mechanically, nonfunctional DNAH3 expression resulted in decreased expression of IDA-associated proteins in the spermatozoa flagella of patients and KO mice, including DNAH1, DNAH6, and DNALI1, the deletion of which has been involved in disruption of sperm motility. Moreover, the infertility of patients with DNAH3 variants and Dnah3 KO mice could be rescued by intracytoplasmic sperm injection (ICSI) treatment. Our findings indicated that DNAH3 is a novel pathogenic gene for asthenoteratozoospermia and may further contribute to the diagnosis, genetic counseling, and prognosis of male infertility.

A flagellar accessory protein links chemotaxis to surface sensing.

Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact sensing by the flagellum plays a central role in activating colonization programs. Caulobacter crescentus adheres to surfaces using a polysaccharide adhesin called the holdfast. In C. crescentus, disruption of the flagellum through interactions with a surface or mutation of flagellar genes increases holdfast production. Our group previously identified several C. crescentus genes involved in flagellar surface sensing. One of these, fssF, codes for a protein with homology to the flagellar C-ring protein FliN. We show here that a fluorescently tagged FssF protein localizes to the flagellated pole of the cell and requires all components of the flagellar C-ring for proper localization, supporting the model that FssF associates with the C-ring. Deleting fssF results in a severe motility defect, which we show is due to a disruption of chemotaxis. Epistasis experiments demonstrate that fssF promotes adhesion through a stator-dependent pathway when late-stage flagellar mutants are disrupted. Separately, we find that disruption of chemotaxis through deletion of fssF or other chemotaxis genes results in a hyperadhesion phenotype. Key genes in the surface sensing network (pleD, motB, and dgcB) contribute to both ∆flgH-dependent and ∆fssF-dependent hyperadhesion, but these genes affect adhesion differently in the two hyperadhesive backgrounds. Our results support a model in which the stator subunits of the flagella incorporate both mechanical and chemical signals to regulate adhesion.IMPORTANCEBacterial biofilms pose a threat in clinical and industrial settings. Surface sensing is one of the first steps in biofilm formation. Studying surface sensing can improve our understanding of biofilm formation and develop preventative strategies. In this study, we use the freshwater bacterium Caulobacter crescentus to study surface sensing and the regulation of surface attachment. We characterize a previously unstudied gene, fssF, and find that it localizes to the cell pole in the presence of three proteins that make up a component of the flagellum called the C-ring. Additionally, we find that fssF is required for chemotaxis behavior but dispensable for swimming motility. Lastly, our results indicate that deletion of fssF and other genes required for chemotaxis results in a hyperadhesive phenotype. These results support that surface sensing requires chemotaxis for a robust response to a surface.

Proteomics and EPS Compositional Analysis Reveals Desulfovibrio bisertensis SY-1 Induced Corrosion on Q235 Steel by Biofilm Formation

Microorganisms that exist in the seawater form microbial biofilms on materials used in marine construction, especially on metal surfaces submerged in seawater, where they form biofilms and cause severe corrosion. Biofilms are mainly composed of bacteria and their secreted polymeric substances. In order to understand how biofilms promote metal corrosion, planktonic and biofilm cells of Desulfovibrio bizertensis SY-1 (D. bizertensis) from Q235 steel were collected and analyzed as to their intracellular proteome and extracellular polymeric substances (EPS). The intracellular proteome analysis showed that the cellular proteins were strongly regulated in biofilm cells compared to planktonic cells, e.g., along with flagellar proteins, signaling-related proteins were significantly increased, whereas energy production and conversion proteins and DNA replication proteins were significantly regulated. The up-and-down regulation of proteins revealed that biofilm formation by bacteria on metal surfaces is affected by flagellar and signaling proteins. A significant decrease in DNA replication proteins indicated that DNA is no longer replicated and transcribed in mature biofilms, thus reducing energy consumption. Quantitative analysis and lectin staining of the biofilm on the metal’s surface revealed that the bacteria secreted a substantial amount of EPS when they began to attach to the surface, and proteins dominated the main components of EPS. Further, the infrared analysis showed that the secondary structure of the proteins in the EPS of the biofilm was mainly dominated by β-sheet and 3-turn helix, which may help to enhance the adhesion of EPS. The functional groups of EPS analyzed using XPS showed that the C element of EPS in the biofilm mainly existed in the form of combinations with N. Furthermore, the hydroxyl structure in the EPS extracted from the biofilm had a stronger hydrogen bonding effect, which could maintain the stability of the EPS structure and biofilm. The study results revealed that D. bizertensis regulates the metabolic pathways and their secreted EPS structure to affect biofilm formation and cause metal corrosion, which has a certain reference significance for the study of the microbially influenced corrosion (MIC) mechanism.

Recombinant Lactococcus lactis secreting FliC protein nanobodies for resistance against Salmonella enteritidis invasion in the intestinal tract

Salmonella Enteritidis is a major foodborne pathogen throughout the world and the increase in antibiotic resistance of Salmonella poses a significant threat to public safety. Natural nanobodies exhibit high affinity, thermal stability, ease of production, and notably higher diversity, making them widely applicable for the treatment of viral and bacterial infections. Recombinant expression using Lactococcus lactis leverages both acid resistance and mucosal colonization properties of these bacteria, allowing the effective expression of exogenous proteins for therapeutic effects. In this study, nine specific nanobodies against the flagellar protein FliC were identified and expressed. In vitro experiments demonstrated that FliC-Nb-76 effectively inhibited the motility of S. Enteritidis and inhibited its adhesion to and invasion of HIEC-6, RAW264.7, and chicken intestinal epithelial cells. Additionally, a recombinant L. lactis strain secreting the nanobody, L. lactis-Nb76, was obtained. Animal experiments confirmed that it could significantly reduce the mortality rates of chickens infected with S. Enteritidis, together with alleviating the inflammatory response caused by the pathogen. These results provide a novel strategy for the treatment of antibiotic-resistant S. Enteritidis infection in the intestinal tract.Graphical

The ABCD_AF291 antibody gives strong and specific signal in ultra-expansion microscopy analysis of HA tagged proteins in African trypanosomes

African trypanosomes expressing a flagellar central pair protein tagged with a triple hemagglutinin (HA) epitope in tandem repeat were analyzed by ultra-expansion microscopy (U-ExM) using the recombinant anti-HA antibody ABCD_AF291. ABCD_AF291 gave a strong and specific signal, demonstrating that it can be used to localize tagged proteins in U-ExM experiments.

Unveiling Allium sativum Phytocompounds as New Antileptospiral Agents via a Structural‐Based Virtual Screening Approach

AbstractLeptospirosis, a disease of zoonotic origin, is transmitted to humans via the Leptospira bacteria, which infected rats and domestic animals carry. This disease is prevalent in certain regions of South India and North‐East India. As per the Leptospirosis Burden Epidemiology Group (WHO), there are approximately 873,000 reported cases and 48,600 deaths annually. Interestingly, we have identified specific targets that are vital to metabolic processes, lipid synthesis, the flagellar motor protein system, and bacterial chemotaxis. By developing new drugs that target these areas, we could significantly mitigate the risks associated with severe Leptospirosis. This study aims to create a new antileptospiral drug using data from the IMPPAT database. We performed virtual screening with Schrodinger, molecular dynamic simulation, and free energy investigation using GROMACS throughout 50 ns. The primary objective was to identify eight potential therapeutic targets in Leptospira for drug development. The screening process successfully identified promising drug candidates for the treatment of Leptospirosis, specifically IMPHY015116, IMPHY004388, IMPHY004619, IMPHY014919, IMPHY007598, and IMPHY001246. These phytochemical compounds exhibit higher binding energy than conventional drug molecules and show stability in dynamic environments. However, additional studies are required to validate their effect on disease progression and pathogenicity through in vitro studies.

Temperature-dependent alterations in the proteome of the emergent fish pathogen Edwardsiella piscicida.

Edwardsiella piscicida is an emerging bacterial pathogen and the aetiological agent of edwardsiellosis among cultured and wild fish species globally. The increased frequency of outbreaks of this Gram-negative, facultative intracellular pathogen pose not only a threat to the aquaculture industry but also a possible foodborne/waterborne public health risk due to the ill-defined zoonotic potential. Thus, understanding the role of temperature on the virulence of this emerging pathogen is essential for comprehending the pathogenesis of piscine edwardsiellosis in the context of current warming trends associated with climate change, as well as providing insight into its zoonotic potential. In this study, significant temperature-dependent alterations in bacterial growth patterns were observed, with bacterial isolates grown at 17°C displaying higher peak growth sizes, extended lag times, and slower maximal growth rates than isolates grown at 27or 37°C. When E. piscicida isolates were grown at 37°C compared to 27 and 17°C, mass spectrometry analysis of the E. piscicida proteome revealed significant downregulation of crucial virulence proteins, such as Type VI secretion system proteins and flagellar proteins. Although invivo models of infection are warranted, this invitro data suggests possible temperature-associated alterations in the virulence and pathogenic potential of E. piscicida in poikilotherms and homeotherms.

Flagellar point mutation causes social aggregation in laboratory-adapted Bacillus subtilis under conditions that promote swimming.

Motility allows microbes to explore and maximize success in their environment; however, many laboratory bacterial strains have a reduced or altered capacity for motility. Swimming motility in Bacillus subtilis depends on peritrichous flagella and is carried out individually as cells move by biased random walks toward attractants. Previously, we adapted Bacillus subtilis strain 3610 to the laboratory for 300 generations in lysogeny broth (LB) batch culture and isolated lab-adapted strains. Strain SH2 is motility-defective and in broth culture forms large, frequently spherical aggregates of cells. A single point mutation in the flagellin gene hag that causes amino acid 259 to switch from A to T is necessary and sufficient to cause these social cell aggregates, and aggregation occurs between flagellated cells bearing this point mutation regardless of the strain background. Cells associate when bearing this mutation, but flagellar rotation is needed to pull associating cells into spherical aggregates. Using electron microscopy, we are able to show that the SH2 flagellar filament has limited polymorphism when compared to other flagellar structures. This limited polymorphism hinders the flagellum's ability to function as a motility apparatus but appears to alter its function to that of cell aggregation/adhesion. We speculate that the genotype-specific aggregation of cells producing HagA259T flagella could have increased representation in a batch-culture experiment by allowing similar cells to go through a transfer together and also that this mutation could serve as an early step to evolve sociality in the natural world.IMPORTANCEThe first life forms on this planet were prokaryotic, and the earliest environments were aquatic, and from these relatively simple starting conditions, complex communities of microbes and ultimately multicellular organisms were able to evolve. Usually, motile cells in aqueous environments swim as individuals but become social by giving up motility and secreting extracellular substances to become a biofilm. Here, we identify a single point mutation in the flagellum that is sufficient to allow cells containing this mutation to specifically form large, suspended groups of cells. The specific change in the flagellar filament protein subunits causes a unique change in the flagellar structure. This could represent a distinct way for closely related cells to associate as an early precursor to sociality.

Diametral influence of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) on the growth of Campylobacter jejuni with consequences on the bacterial transcriptome

BackgroundDeoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly found in cereals and grains worldwide. The presence of this fungal secondary-metabolite raises public-health concerns at both the agriculture and food industry level. Recently, we have shown that DON has a negative impact on gut integrity, a feature also noticed for Campylobacter (C.) jejuni. We further demonstrated that DON increased the load of C. jejuni in the gut and inner organs. In contrast, feeding the less toxic DON metabolite deepoxy-deoxynivalenol (DOM-1) to broilers reduced the Campylobacter load in vivo. Consequently, it can be hypothesized that DON and DOM-1 have a direct effect on the growth profile of C. jejuni. The aim of the present study was to further resolve the nature of this interaction in vitro by co-incubation and RNA-sequencing.ResultsThe co-incubation of C. jejuni with DON resulted in significantly higher bacterial growth rates from 30 h of incubation onwards. On the contrary, the co-incubation of C. jejuni with DOM-1 reduced the CFU counts, indicating that this DON metabolite might contribute to reduce the burden of C. jejuni in birds, altogether confirming in vivo data. Furthermore, the transcriptomic profile of C. jejuni following incubation with either DON or DOM-1 differed. Co-incubation of C. jejuni with DON significantly increased the expression of multiple genes which are critical for Campylobacter growth, particularly members of the Flagella gene family, frr (ribosome-recycling factor), PBP2 futA-like (Fe3+ periplasmic binding family) and PotA (ATP-binding subunit). Flagella are responsible for motility, biofilm formation and host colonization, which may explain the high Campylobacter load in the gut of DON-fed broiler chickens. On the contrary, DOM-1 downregulated the Flagella gene family and upregulated ribosomal proteins.ConclusionThe results highlight the adaptive mechanisms involved in the transcriptional response of C. jejuni to DON and its metabolite DOM-1, based on the following effects: (a) ribosomal proteins; (b) flagellar proteins; (c) engagement of different metabolic pathways. The results provide insight into the response of an important intestinal microbial pathogen against DON and lead to a better understanding of the luminal or environmental acclimation mechanisms in chickens.

Flagellar protein FliL: A many-splendored thing.

FliL is a bacterial flagellar protein demonstrated to associate with, and regulate ion flow through, the stator complex in a diverse array of bacterial species. FliL is also implicated in additional functions such as stabilizing the flagellar rod, modulating rotor bias, sensing the surface, and regulating gene expression. How can one protein do so many things? Its location is paramount to understanding its numerous functions. This review will look at the evidence, attempt to resolve some conflicting findings, and offer new thoughts on FliL.

Polar confinement of a macromolecular machine by an SRP-type GTPase

The basal structure of the bacterial flagellum includes a membrane embedded MS-ring (formed by multiple copies of FliF) and a cytoplasmic C-ring (composed of proteins FliG, FliM and FliN). The SRP-type GTPase FlhF is required for directing the initial flagellar protein FliF to the cell pole, but the mechanisms are unclear. Here, we show that FlhF anchors developing flagellar structures to the polar landmark protein HubP/FimV, thereby restricting their formation to the cell pole. Specifically, the GTPase domain of FlhF interacts with HubP, while a structured domain at the N-terminus of FlhF binds to FliG. FlhF-bound FliG subsequently engages with the MS-ring protein FliF. Thus, the interaction of FlhF with HubP and FliG recruits a FliF-FliG complex to the cell pole. In addition, the modulation of FlhF activity by the MinD-type ATPase FlhG controls the interaction of FliG with FliM-FliN, thereby regulating the progression of flagellar assembly at the pole.

A flagellar accessory protein links chemotaxis to surface sensing.

Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact sensing by the flagellum plays a central role in activating colonization programs. Caulobacter crescentus adheres to surfaces using a polysaccharide adhesin called the holdfast. In C. crescentus, disruption of the flagellum through interactions with a surface or mutation of flagellar genes increases holdfast production. Our group previously identified several C. crescentus genes involved in flagellar surface sensing. One of these, called fssF, codes for a protein with homology to the flagellar C-ring protein FliN. We show here that a fluorescently tagged FssF protein localizes to the flagellated pole of the cell and requires all components of the flagellar C-ring for proper localization, supporting the model that FssF associates with the C-ring. Deleting fssF results in a severe motility defect that we show is due to a disruption of chemotaxis. Epistasis experiments demonstrate that fssF promotes adhesion through a stator-dependent pathway when late-stage flagellar mutants are disrupted. Separately, we find that disruption of chemotaxis through deletion of fssF or other chemotaxis genes results in a hyperadhesion phenotype. Key genes in the surface sensing network (pleD, motB, and dgcB) contribute to both ∆flgH-dependent and ∆fssF-dependent hyperadhesion, but these genes affect adhesion differently in the two hyperadhesive backgrounds. Our results support a model in which the stator subunits of the flagella incorporate both mechanical and chemical signals to regulate adhesion.

Shotgun proteomics of detergent-solubilized proteins from Trypanosoma evansi

Trypanosoma evansi, the causative agent of surra, is the most prevalent pathogenic salivarian trypanosome and affects the majority of domesticated and wild animals in endemic regions. This work aimed to analyze detergent-solubilized T. evansi proteins and identify potential diagnostic biomarkers for surra. Triton X-114-extracted membrane-enriched proteins (MEP) of T. evansi bloodstream forms were analyzed using a gel-free technique (LC-ESI-MS/MS). 247 proteins were identified following the MS analysis of three biological and technical replicates. Two of these proteins were predicted to have a GPI-anchor, 100 (40%) were predicted to have transmembrane domains, and 166 (67%) were predicted to be membrane-bound based on at least one of six features: location (WolfPSORT, DeepLoc-2.0, Protcomp-9.0), transmembrane, GPI, and gene ontology. It was predicted that 76 (30%) of proteins had membrane evidence. Typical membrane proteins for each organelle were identified, among them ISG families (64, 65, and 75 kDa), flagellar calcium-binding protein, 24 kDa calflagin, syntaxins and oligosaccharyltransferase some of which had previously been studied in other trypanosomatids. T. evansi lacks singletons and exclusive orthologous groups, whereas three distinct epitopes have been identified. Data are available via ProteomeXchange with identifier PXD040594. SignificanceTrypanosoma evansi is a highly prevalent parasite that induces a pathological condition known as “surra” in various species of ungulates across five continents. The infection gives rise to symptoms that are not pathognomonic, thereby posing challenges in its diagnosis and leading to substantial economic losses in the livestock industry. A significant challenge arises from the absence of a diagnostic test capable of distinguishing between Trypanosoma equiperdum and T. evansi, both of which are implicated in equine diseases. Therefore, there is a pressing need to conduct research on the biochemistry of the parasite in order to identify proteins that could potentially serve as targets for differential diagnosis or therapeutic interventions.

Integrative omics reveals clues as to why Cynoglossus semilaevis pseudomales produce no W sperm

Cynoglossus semilaevis (half-smooth tongue sole) is an important aquaculture species that has a ZW/ZZ sex-determination system (females, ZW; males, ZZ). Some genotypic females may develop phenotypic males, and are known as pseudomales (ZW). These pseudomales can develop mature testes and produce sperm. However, previous studies have shown that pseudomales cannot produce sperms bearing W chromosome (W sperm). In this study, transcriptomic, proteomic, and ubiquitinated proteomic analyses were employed simultaneously to investigate the differences in gonads between pseudomales and normal males. Functional enrichment analysis of the differentially expressed genes/proteins showed that non-coding RNA metabolic processes and RNA degradation were more active in the gonads of pseudomales, and they showed higher levels of ubiquitination compared to males. In addition, most genes/proteins related to sperm morphogenesis expressed downwards in pseudomales. These genes included genes located exclusively on the Z chromosome, such as sperm flagellar protein 2 (spef2), doublesex and mab-3 related transcription factor 1 (dmrt1), doublesex and mab-3 related transcription factor 3 (dmrt3), and cilia- and flagella-associated protein 157 (cfap157). These findings characterized the transcriptional and proteomic profiles of the gonads of pseudomales and males, and suggested that W sperm were absent in pseudomales, possibly due to the disruption of sperm morphogenesis, resulting from W chromosome lacking spermatogenesis-related genes.

Genetic Causes of Qualitative Sperm Defects: A Narrative Review of Clinical Evidence.

Several genes are implicated in spermatogenesis and fertility regulation, and these genes are presently being analysed in clinical practice due to their involvement in male factor infertility (MFI). However, there are still few genetic analyses that are currently recommended for use in clinical practice. In this manuscript, we reviewed the genetic causes of qualitative sperm defects. We distinguished between alterations causing reduced sperm motility (asthenozoospermia) and alterations causing changes in the typical morphology of sperm (teratozoospermia). In detail, the genetic causes of reduced sperm motility may be found in the alteration of genes associated with sperm mitochondrial DNA, mitochondrial proteins, ion transport and channels, and flagellar proteins. On the other hand, the genetic causes of changes in typical sperm morphology are related to conditions with a strong genetic basis, such as macrozoospermia, globozoospermia, and acephalic spermatozoa syndrome. We tried to distinguish alterations approved for routine clinical application from those still unsupported by adequate clinical studies. The most important aspect of the study was related to the correct identification of subjects to be tested and the correct application of genetic tests based on clear clinical data. The correct application of available genetic tests in a scenario where reduced sperm motility and changes in sperm morphology have been observed enables the delivery of a defined diagnosis and plays an important role in clinical decision-making. Finally, clarifying the genetic causes of MFI might, in future, contribute to reducing the proportion of so-called idiopathic MFI, which might indeed be defined as a subtype of MFI whose cause has not yet been revealed.

Overexpression of the flagellar motor protein MotB sensitizes Bacillus subtilis to aminoglycosides in a motility-independent manner.

The flagellar motor proteins, MotA and MotB, form a complex that rotates the flagella by utilizing the proton motive force (PMF) at the bacterial cell membrane. Although PMF affects the susceptibility to aminoglycosides, the effect of flagellar motor proteins on the susceptibility to aminoglycosides has not been investigated. Here, we found that MotB overexpression increased susceptibility to aminoglycosides, such as kanamycin and gentamicin, in Bacillus subtilis without affecting swimming motility. MotB overexpression did not affect susceptibility to ribosome-targeting antibiotics other than aminoglycosides, cell wall-targeting antibiotics, DNA synthesis-inhibiting antibiotics, or antibiotics inhibiting RNA synthesis. Meanwhile, MotB overexpression increased the susceptibility to aminoglycosides even in the motA-deletion mutant, which lacks swimming motility. Overexpression of the MotB mutant protein carrying an amino acid substitution at the proton-binding site (D24A) resulted in the loss of the enhanced aminoglycoside-sensitive phenotype. These results suggested that MotB overexpression sensitizes B. subtilis to aminoglycosides in a motility-independent manner. Notably, the aminoglycoside-sensitive phenotype induced by MotB requires the proton-binding site but not the MotA/MotB complex formation.