A novel homozygous missense mutation in AK7 causes multiple morphological anomalies of the flagella and oligoasthenoteratozoospermia.

PURPOSE: To identify the genetic causes of multiple morphological anomalies of the flagella (MMAF) and oligoasthenoteratozoospermia (OAT). METHODS: Whole-exome sequencing (WES) was performed on the proband to identify pathogenic mutation for infertility. Western blotting and immunofluorescence analysis detected the expression level and localization of adenylate kinase 7 (AK7). RESULTS: We identified a novel homozygous missense mutation (NM_152327: c.1846G > A; p.E616K) in AK7 in two brothers with MMAF and OAT from a consanguineous family by WES. Western blotting and immunofluorescence experiments determined that the expression level of AK7 decreased in the sperm from the proband. The proband and his wife underwent two cycles of intracytoplasmic sperm injection (ICSI) treatment but got unfavorable outcomes. CONCLUSION: This study could provide precise genetic diagnosis for the patient and expand the spectrum of AK7 mutations.

BB0326 is responsible for the formation of periplasmic flagellar collar and assembly of the stator complex in Borrelia burgdorferi.

Borrelia burgdorferi is a highly motile spirochete due to its periplasmic flagella. Unlike flagella of other bacteria, spirochetes' periplasmic flagella possess a complex structure called the collar, about which little is known in terms of function and composition. Using various approaches, we have identified a novel protein, BB0326, as a key component of the collar. We show that a peripheral portion of the collar is diminished in the Δbb0326 mutant and restored in the complemented bb0326+ cells, leading us to rename BB0326 as periplasmic flagellar collar protein A or FlcA. The ΔflcA mutant cells produced fewer, abnormally tilted and shorter flagella, as well as diminished stators, suggesting that FlcA is crucial for flagellar and stator assemblies. We provide further evidence that FlcA interacts with the stator and that this collar-stator interaction is essential for the high torque needed to power the spirochete's periplasmic flagellar motors. These observations suggest that the collar provides various important functions to the spirochete's periplasmic flagellar assembly and rotation.

Flagellar motility plays important role in Biofilm formation of Bacillus cereus and Yersinia enterocolitica.

Bacteria live either independently as planktonic cells or in organized surface associated colonies called as biofilms. Biofilms play an important role in increased pathogenesis of bacteria and it is assumed that motility is one of the contributing factors towards biofilm initiation. This study was planned to identify the role of flagella in biofilm formation by constructing flagellated (wild type) and physically disrupted variants (non-motile). Total 10 clinical bacterial strains were isolated and characterized. Morphological and biochemical study identified these strains as Enterobacter spp., Pseudomonas spp., Yersinia spp., Escherichia spp., Salmonella spp., Proteus spp., Staphylococcus spp., Streptococcus spp., Lactobacillus spp. and Bacillus spp. Among all strains, two strains including Yersinia spp and Bacillus spp. showed higher antibiotic resistance, hence studied at molecular and physiological level. Biofilm formation capacity of strains was analyzed using three methods including Congo red assay, Test tube assay and Liquid-interface coverslip assay. Afterwards, flagellar disintegration was induced by blending and centrifugation for 5, 10 and 15 minutes. 16S rRNA sequencing showed two strains as Bacillus cereus and Yersinia enterocolitica. Both strains produced significant biofilm by all three above mentioned methods. A motility test of these blended variants showed partial/diminished motility with increased blending time. The significant loss in biofilm formation after 15 minutes blending confirmed the important flagellar contribution to the initiation of biofilm formation. This biofilm defect observed in flagella paralysed/minus variants presumably may be due to defects in attachments to surface at early stages. This study indicated that flagellar motility is crucial initially for surface attachment and subsequently for biofilm formation.

The role of intrinsically disordered C-terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus.

The bacterial flagellar export switching machinery consists of a ruler protein, FliK, and an export switch protein, FlhB and switches substrate specificity of the flagellar type III export apparatus upon completion of hook assembly. An interaction between the C-terminal domain of FliK (FliKC ) and the C-terminal cytoplasmic domain of FlhB (FlhBC ) is postulated to be responsible for this switch. FliKC has a compactly folded domain termed FliKT3S4 (residues 268-352) and an intrinsically disordered region composed of the last 53 residues, FliKCT (residues 353-405). Residues 301-350 of FliKT3S4 and the last five residues of FliKCT are critical for the switching function of FliK. FliKCT is postulated to regulate the interaction of FliKT3S4 with FlhBC , but it remains unknown how. Here we report the role of FliKCT in the export switching mechanism. Systematic deletion analyses of FliKCT revealed that residues of 351-370 are responsible for efficient switching of substrate specificity of the export apparatus. Suppressor mutant analyses showed that FliKCT coordinates FliKT3S4 action with the switching. Site-directed photo-cross-linking experiments showed that Val-302 and Ile-304 in the hydrophobic core of FliKT3S4 bind to FlhBC . We propose that FliKCT may induce conformational rearrangements of FliKT3S4 to bind to FlhBC .

An in silico pan-genomic probe for the molecular traits behind Lactobacillus ruminis gut autochthony.

As an ecological niche, the mammalian intestine provides the ideal habitat for a variety of bacterial microorganisms. Purportedly, some commensal genera and species offer a beneficial mix of metabolic, protective, and structural processes that help sustain the natural digestive health of the host. Among these sort of gut inhabitants is the Gram-positive lactic acid bacterium Lactobacillus ruminis, a strict anaerobe with both pili and flagella on its cell surface, but also known for being autochthonous (indigenous) to the intestinal environment. Given that the molecular basis of gut autochthony for this species is largely unexplored and unknown, we undertook a study at the genome level to pinpoint some of the adaptive traits behind its colonization behavior. In our pan-genomic probe of L. ruminis, the genomes of nine different strains isolated from human, bovine, porcine, and equine host guts were compiled and compared for in silico analysis. For this, we conducted a geno-phenotypic assessment of protein-coding genes, with an emphasis on those products involved with cell-surface morphology and anaerobic fermentation and respiration. We also categorized and examined the core and accessory genes that define the L. ruminis species and its strains. Here, we made an attempt to identify those genes having ecologically relevant phenotypes that might support or bring about intestinal indigenousness.

Roles of RpoS in Yersinia pseudotuberculosis stress survival, motility, biofilm formation and type VI secretion system expression.

RpoS (σ(S)), the stationary phase/stress σ factor, controls the expression of a large number of genes involved in cellular responses to a variety of stresses. However, the role of RpoS appears to differ in different bacteria. While RpoS is an important regulator of flagellum biosynthesis, it is associated with biofilm development in Edwardsiella tarda. Biofilms are dense communities formed by bacteria and are important for microbe survival under unfavorable conditions. The type VI secretion system (T6SS) discovered recently is reportedly associated with several phenotypes, ranging from biofilm formation to stress sensing. For example, Vibrio anguillarum T6SS was proposed to serve as a sensor for extracytoplasmic signals and modulates RpoS expression and stress response. In this study, we investigated the physiological roles of RpoS in Yersinia pseudotuberculosis, including bacterial survival under stress conditions, flagella formation, biofilm development and T6SS expression. We found that RpoS is important in resistance to multiple stressors-including H2O2, acid, osmotic and heat shock-in Y. pseudotuberculosis. In addition, our study showed that RpoS not only modulates the expression of T6SS but also regulates flagellum formation by positively controlling the flagellar master regulatory gene flhDC, and affects the formation of biofilm on Caenorhabditis elegans by regulating the synthesis of exopolysaccharides. Taken together, these results show that RpoS plays a central role in cell fitness under several adverse conditions in Y. pseudotuberculosis.

The Hd, Hj, and Hz66 flagella variants of Salmonella enterica serovar Typhi modify host responses and cellular interactions.

Salmonella Typhi, the causative agent of typhoid fever, is a monophyletic, human-restricted bacterium that exhibits limited phenotypic variation. S. Typhi from Indonesia are a notable exception, with circulating strains expressing diverse flagella antigens including Hj, Hd and Hz66. Hypothesizing that S. Typhi flagella plays a key role during infection, we constructed an S. Typhi fliC mutant and otherwise isogenic S. Typhi strains expressing the Hj, Hd, Hz66 flagella antigens. Phenotyping revealed differences in flagellum structure, strain motility and immunogenicity, but not in the ability of flagellated isolates to induce TLR5 activity. Invasion assays using epithelial and macrophage cell lines revealed differences in the ability of these S. Typhi derivatives to invade cells or induce cellular restructuring in the form of ruffles. Notably, the Hj variant induced substantial ruffles that were not fully dependent on the GTPases that contribute to this process. These data highlight important differences in the phenotypic properties of S. Typhi flagella variation and how they impact on the pathogenesis of S. Typhi.

Brucella melitensis invades murine erythrocytes during infection.

Brucella spp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed that Brucella melitensis is able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced following Brucella vaccination. In contrast to Bartonella, the type IV secretion system and flagellar expression are not critically required for the persistence of Brucella in blood. ImageStream analysis of blood cells showed that following a brief extracellular phase, Brucella is associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated that B. melitensis is able to invade erythrocytes in vivo. The bacteria do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open up new areas for investigation and should serve in the development of novel strategies for the treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the bacterial cells from the host's immune response and hamper antibiotic treatment and suggests possible Brucella transmission by bloodsucking insects in nature.

A conserved type IV pilin signal peptide H-domain is critical for the post-translational regulation of flagella-dependent motility.

In many bacteria and archaea, type IV pili facilitate surface adhesion, the initial step in biofilm formation. Haloferax volcanii has a specific set of adhesion pilins (PilA1-A6) that, although diverse, contain an absolutely conserved signal peptide hydrophobic (H) domain. Data presented here demonstrate that these pilins (PilA1-A6) also play an important role in regulating flagella-dependent motility, which allows cells to rapidly transition between planktonic and sessile states. Cells lacking adhesion pilins exhibit a severe motility defect, however, expression of any one of the adhesion pilins in trans can rescue the motility and adhesion. Conversely, while deleting pilB3-C3, genes required for PilA pilus biosynthesis, results in cells lacking pili and having an adhesion defect, it does not affect motility, indicating that motility regulation requires the presence of pilins, but not assembled pili. Mutagenesis studies revealed that the pilin-dependent motility regulatory mechanism does not require the diverse C-terminal region of the PilA pilins but specifically involves the conserved H-domain. This novel post-translational regulatory mechanism, which employs components that promote biofilm formation to inhibit motility, can provide a rapid response to changing environmental conditions. A model for this regulatory mechanism, which may also be present in other prokaryotes, is discussed.

A conserved PapB family member, TosR, regulates expression of the uropathogenic Escherichia coli RTX nonfimbrial adhesin TosA while conserved LuxR family members TosE and TosF suppress motility.

A heterogeneous subset of extraintestinal pathogenic Escherichia coli (ExPEC) strains, referred to as uropathogenic E. coli (UPEC), causes most uncomplicated urinary tract infections. However, no core set of virulence factors exists among UPEC strains. Instead, the focus of the analysis of urovirulence has shifted to studying broad classes of virulence factors and the interactions between them. For example, the RTX nonfimbrial adhesin TosA mediates adherence to host cells derived from the upper urinary tract. The associated tos operon is well expressed in vivo but poorly expressed in vitro and encodes TosCBD, a predicted type 1 secretion system. TosR and TosEF are PapB and LuxR family transcription factors, respectively; however, no role has been assigned to these potential regulators. Thus, the focus of this study was to determine how TosR and TosEF regulate tosA and affect the reciprocal expression of adhesins and flagella. Among a collection of sequenced UPEC strains, 32% (101/317) were found to encode TosA, and nearly all strains (91% [92/101]) simultaneously carried the putative regulatory genes. Deletion of tosR alleviates tosA repression. The tos promoter was localized upstream of tosR using transcriptional fusions of putative promoter regions with lacZ. TosR binds to this region, affecting a gel shift. A 100-bp fragment 220 to 319 bp upstream of tosR inhibits binding, suggesting localization of the TosR binding site. TosEF, on the other hand, downmodulate motility when overexpressed by preventing the expression of fliC, encoding flagellin. Deletion of tosEF increased motility. Thus, we present an additional example of the reciprocal control of adherence and motility.

Lipopolysaccharide modifications of a cholera vaccine candidate based on outer membrane vesicles reduce endotoxicity and reveal the major protective antigen.

The causative agent of the life-threatening gastrointestinal infectious disease cholera is the Gram-negative, facultative human pathogen Vibrio cholerae. We recently started to investigate the potential of outer membrane vesicles (OMVs) derived from V. cholerae as an alternative approach for a vaccine candidate against cholera and successfully demonstrated the induction of a long-lasting, high-titer, protective immune response upon immunization with OMVs using the mouse model. In this study, we present immunization data using lipopolysaccharide (LPS)-modified OMVs derived from V. cholerae, which allowed us to improve and identify the major protective antigen of the vaccine candidate. Our results indicate that reduction of endotoxicity can be achieved without diminishing the immunogenic potential of the vaccine candidate by genetic modification of lipid A. Although the protective potential of anti-LPS antibodies has been suggested many times, this is the first comprehensive study that uses defined LPS mutants to characterize the LPS-directed immune response of a cholera vaccine candidate in more detail. Our results pinpoint the O antigen to be the essential immunogenic structure and provide a protective mechanism based on inhibition of motility, which prevents a successful colonization. In a detailed analysis using defined antisera, we can demonstrate that only anti-O antigen antibodies, but not antibodies directed against the major flagellar subunit FlaA or the most abundant outer membrane protein, OmpU, are capable of effectively blocking the motility by binding to the sheathed flagellum and provide protection in a passive immunization assay.

Two-dimensional optical feedback control of Euglena confined in closed-type microfluidic channels.

We examined two-dimensional (2D) optical feedback control of phototaxis flagellate Euglena cells confined in closed-type microfluidic channels (microaquariums), and demonstrated that the 2D optical feedback enables the control of the density and position of Euglena cells in microaquariums externally, flexibly, and dynamically. Using three types of feedback algorithms, the density of Euglena cells in a specified area can be controlled arbitrarily and dynamically, and more than 70% of the cells can be concentrated into a specified area. Separation of photo-sensitive/insensitive Euglena cells was also demonstrated. Moreover, Euglena-based neuro-computing has been achieved, where 16 imaginary neurons were defined as Euglena-activity levels in 16 individual areas in microaquariums. The study proves that 2D optical feedback control of photoreactive flagellate microbes is promising for microbial biology studies as well as applications such as microbe-based particle transportation in microfluidic channels or separation of photo-sensitive/insensitive microbes.

Frateuria terrea sp. nov., isolated from forest soil, and emended description of the genus Frateuria.

A bacterial strain, designated VA24(T), was isolated from forest soil of the Changbai Mountains, Heilongjiang province, China. Cells of strain VA24(T) were Gram-reaction-negative, aerobic, non-spore-forming, long rods, 0.3-0.5×2.0-3.0 µm in size and were motile by means of a subpolar flagellum. Strain VA24(T) was oxidase-positive and catalase-negative. Growth occurred at 21-36 °C, pH 5-10 and in 0-2 % (w/v) NaCl but did not occur at 37 °C. The predominant respiratory quinone was Q-8, the major polar lipids were phosphatidylethanolamine and phosphatidylmonomethylethanolamine and the major cellular fatty acids were iso-C(15 : 0) (14.9 %), iso-C(17 : 1)ω9c (14.1 %), iso-C(17 : 0) (10.8 %) and iso-C(16 : 0) (10.3 %). The DNA G+C content was 67.4 mol% (T(m)). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain VA24(T) was closely related to Frateuria aurantia IFO 13300(T) with 96.9 % sequence similarity. DNA-DNA relatedness of strain VA24(T) to F. aurantia DSM 6220(T) was 15.8 %. Based on its phenotypic and genotypic features, strain VA24(T) represents a novel species of the genus Frateuria, for which the name Frateuria terrea sp. nov. is proposed. The type strain is VA24(T) (=CGMCC 1.7053(T) =NBRC 104236(T)).

Survival of the fittest: how Brucella strains adapt to their intracellular niche in the host.

Brucella strains produce abortion and infertility in their natural hosts and a zoonotic disease in humans known as undulant fever. These bacteria do not produce classical virulence factors, and their capacity to successfully survive and replicate within a variety of host cells underlies their pathogenicity. Extensive replication of the brucellae in placental trophoblasts is associated with reproductive tract pathology in natural hosts, and prolonged persistence in macrophages leads to the chronic infections that are a hallmark of brucellosis in both natural hosts and humans. This review describes how Brucella strains have efficiently adapted to their intracellular lifestyle in the host.

Different signaling roles of two conserved residues in the cytoplasmic hairpin tip of Tsr, the Escherichia coli serine chemoreceptor.

Bacterial chemoreceptors form ternary signaling complexes with the histidine kinase CheA through the coupling protein CheW. Receptor complexes in turn cluster into cellular arrays that produce highly sensitive responses to chemical stimuli. In Escherichia coli, receptors of different types form mixed trimer-of-dimers signaling teams through the tips of their highly conserved cytoplasmic domains. To explore the possibility that the hairpin loop at the tip of the trimer contact region might promote interactions with CheA or CheW, we constructed and characterized mutant receptors with amino acid replacements at the two nearly invariant hairpin charged residues of Tsr: R388, the most tip-proximal trimer contact residue, and E391, the apex residue of the hairpin turn. Mutant receptors were subjected to in vivo tests for the assembly and function of trimers, ternary complexes, and clusters. All R388 replacements impaired or destroyed Tsr function, apparently through changes in trimer stability or geometry. Large-residue replacements locked R388 mutant ternary complexes in the kinase-off (F, H) or kinase-on (W, Y) signaling state, suggesting that R388 contributes to signaling-related conformational changes in the trimer. In contrast, most E391 mutants retained function and all formed ternary signaling complexes efficiently. Hydrophobic replacements of any size (G, A, P, V, I, L, F, W) caused a novel phenotype in which the mutant receptors produced rapid switching between kinase-on and -off states, indicating that hairpin tip flexibility plays an important role in signal state transitions. These findings demonstrate that the receptor determinants for CheA and CheW binding probably lie outside the hairpin tip of the receptor signaling domain.

The kinetoplast ultrastructural organization of endosymbiont-bearing trypanosomatids as revealed by deep-etching, cytochemical and immunocytochemical analysis.

The endosymbiont-bearing trypanosomatids present a typical kDNA arrangement, which is not well characterized. In the majority of trypanosomatids, the kinetoplast forms a bar-like structure containing tightly packed kDNA fibers. On the contrary, in trypanosomatids that harbor an endosymbiotic bacterium, the kDNA fibers are disposed in a looser arrangement that fills the kinetoplast matrix. In order to shed light on the kinetoplast structural organization in these protozoa, we used cytochemical and immunocytological approaches. Our results showed that in endosymbiont-containing species, DNA and basic proteins are distributed not only in the kDNA network, but also in the kinetoflagellar zone (KFZ), which corresponds to the region between the kDNA and the inner mitochondrial membrane nearest the flagellum. The presence of DNA in the KFZ is in accordance with the actual model of kDNA replication, whereas the detection of basic proteins in this region may be related to the basic character of the intramitochondrial filaments found in this area, which are part of the complex that connects the kDNA to the basal body. The kinetoplast structural organization of Bodo sp. was also analyzed, since this protozoan lacks the highly ordered kDNA-packaging characteristic of trypanosomatid and represents an evolutionary ancestral of the Trypanosomatidae family.

Leishmania (Leishmania) chagasi interactions with Serratia marcescens: ultrastructural studies, lysis and carbohydrate effects.

Studies on the lysis of L. chagasi caused by the bacteria Serratia marcescens were carried out. In vitro experiments demonstrated that S. marcescens variant SM 365, a prodigiosin pigment producer, lysed this species of Leishmania but variant DB11, a nonpigmented bacteria, was unable to lyse the parasite. High concentrations of d-mannose were found to protect L. chagasi markedly diminishing the lysis by S. marcescens SM 365. Promastigotes of L. chagasi bound the lectin Concanavalin A conjugated with FITC, the fluorescence was intensely found at the base of the flagellum (flagellar pocket). Scanning electron microscopy revealed that the bacteria adherence occurred mainly in the flagellar pocket. S. marcescens SM 365 formed filamentous structures, identified as biofilms, which connect the protozoan to the developing bacterial clusters, in low concentrations of bacteria after 30 min incubation time. We suggest that bacterial mannose-sensitive (MS) fimbriae are relevant to S. marcescens SM 365 in the lysis of L. chagasi.

Motility is involved in Silicibacter sp. TM1040 interaction with dinoflagellates.

Silicibacter sp. TM1040, originally isolated from a culture of the dinoflagellate Pfiesteria piscicida, senses and responds to the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) by flagella-mediated chemotaxis behaviour. In this report we show that swimming motility is important for initiating the interaction between the bacterium and dinoflagellate. Following transposon mutagenesis, three mutants defective in wild-type swimming motility (Mot-) were identified. The defects in motility were found to be in homologues of cckA and ctrA, encoding a two-component regulatory circuit, and in a novel gene, flaA, likely to function in flagellar export or biogenesis. Mutation of flaA or cckA results in the loss of flagella and non-motile cells (Fla-), while CtrA- cells possess flagella, but have reduced motility due to increased cell length. All three Mot- mutants were defective in attaching to the dinoflagellate, particularly to regions that colocalized with intracellular organelles. The growth rate of the dinoflagellates was reduced in the presence of the Fla- mutants compared with Fla+ cells. These results indicate that bacterial motility is important for the Silicibacter sp. TM1040-P. piscicida interaction.

Contribution of hemagglutinin/protease and motility to the pathogenesis of El Tor biotype cholera.

Vibrio cholerae is a highly motile organism that secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HapA). HapA has been shown to have mucinase activity and contribute to the reactogenicity of live vaccine candidates, but its role in cholera pathogenesis is not yet clear. The contribution of motility to pathogenesis is not fully understood, since conflicting results have been obtained with different strains, mutants, and animal models. The objective of this work was to determine the contribution of HapA and motility to the pathogenesis of El Tor biotype cholera. To this end we constructed isogenic motility (motY) and mucinase (hapA) single and double mutants of an El Tor biotype V. cholerae strain. Mutants were characterized for the expression of major virulence factors in vitro and in vivo. The motility mutant showed a remarkable increase in cholera toxin (CT), toxin coregulated pilus major subunit (TcpA), and HapA production in vitro. Increased TcpA and CT production could be explained by increased transcription of tcpA, ctxA, and toxT. No effect was detected on the transcription of hapA in the motility mutant. The sodium ionophore monensin diminished production of HapA in the parent but not in the motility mutant. Phenamil, a specific inhibitor of the flagellar motor, diminished CT production in the wild-type and motY strains. The hapA mutant showed increased binding to mucin. In contrast, the motY mutation diminished adherence to biotic and abiotic surfaces including mucin. Lack of HapA did not affect colonization in the suckling mouse model. The motility and mucinase defects did not prevent induction of ctxA and tcpA in the mouse intestine as measured by recombinase-based in vivo expression technology. Analysis of mutants in the rabbit ileal loop model showed that both V. cholerae motility and HapA were necessary for full expression of enterotoxicity.