Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958

Helicobacter pylori is a gastric pathogen which infects ~50% of the global population and can lead to the development of gastritis, gastric and duodenal ulcers and carcinoma. Genome sequencing of H. pylori revealed high levels of genetic variability; this pathogen is known for its adaptability due to mechanisms including phase variation, recombination and horizontal gene transfer. Motility is essential for efficient colonisation by H. pylori. The flagellum is a complex nanomachine which has been studied in detail in E. coli and Salmonella. In H. pylori, key differences have been identified in the regulation of flagellum biogenesis, warranting further investigation. In this study, the genomes of two H. pylori strains (CCUG 17874 and P79) were sequenced and published as draft genome sequences. Comparative studies identified the potential role of restriction modification systems and the comB locus in transformation efficiency differences between these strains. Core genome analysis of 43 H. pylori strains including 17874 and P79 defined a more refined core genome for the species than previously published. Comparative analysis of the genome sequences of strains isolated from individuals suffering from H. pylori related diseases resulted in the identification of “disease-specific” genes. Structure-function analysis of the essential motility protein HP0958 was performed to elucidate its role during flagellum assembly in H. pylori. The previously reported HP0958-FliH interaction could not be substantiated in this study and appears to be a false positive. Site-directed mutagenesis confirmed that the coiled-coil domain of HP0958 is involved in the interaction with RpoN (74-284), while the Zn-finger domain is required for direct interaction with the full length flaA mRNA transcript. Complementation of a non-motile hp0958-null derivative strain of P79 with site-directed mutant alleles of hp0958 resulted in cells producing flagellar-type extrusions from non-polar positions. Thus, HP0958 may have a novel function in spatial localisation of flagella in H. pylori

Three motAB stator gene products in Bdellovibrio bacteriovorus contribute to motility of a single flagellum during predatory and prey-independent growth

The predatory bacterium Bdellovibrio bacteriovorus uses flagellar motility to locate regions rich in Gram-negative prey bacteria, colliding and attaching to prey and then ceasing flagellar motility. Prey are then invaded to form a "bdelloplast" in a type IV pilus-dependent process, and prey contents are digested, allowing Bdellovibrio growth and septation. After septation, Bdellovibrio flagellar motility resumes inside the prey bdelloplast prior to its lysis and escape of Bdellovibrio progeny. Bdellovibrio can also grow slowly outside prey as long flagellate host-independent (HI) cells, cultured on peptone-rich media. The B. bacteriovorus HD100 genome encodes three pairs of MotAB flagellar motor proteins, each of which could potentially form an inner membrane ion channel, interact with the FliG flagellar rotor ring, and produce flagellar rotation. In 2004, Flannagan and coworkers (R. S. Flannagan, M. A. Valvano, and S. F. Koval, Microbiology 150:649-656, 2004) used antisense RNA and green fluorescent protein (GFP) expression to downregulate a single Bdellovibrio motA gene and reported slowed release from the bdelloplast and altered motility of the progeny. Here we inactivated each pair of motAB genes and found that each pair contributes to motility, both predatorily, inside the bdelloplast and during HI growth; however, each pair was dispensable, and deletion of no pair abolished motility totally. Driving-ion studies with phenamil, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and different pH and sodium conditions indicated that all Mot pairs are proton driven, although the sequence similarities of each Mot pair suggests that some may originate from halophilic species. Thus, Bdellovibrio is a "dedicated motorist," retaining and expressing three pairs of mot genes.

The efficiency of propulsion by a rotating flagellum

[At very low Reynolds number, the regime in which fluid dynamics is governed by Stokes equations, a helix that translates along its axis under an external force but without an external torque will necessarily rotate. By the linearity of the Stokes equations, the same helix that is caused to rotate due to an external torque will necessarily translate. This is the physics that underlies the mechanism of flagellar propulsion employed by many microorganisms. Here, I examine the linear relationships between forces and torques and translational and angular velocities of helical objects to understand the nature of flagellar propulsion.]

Short-rib polydactyly and jeune syndromes are caused by mutations in WDR60

Short-rib polydactyly syndromes (SRPS I-V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis.

Kertooko luonnon järjestys suunnittelusta? : Michael Behe älykkään suunnittelun ajatuksen puolustajana

This study presents a systematical analysis of biochemist Michael Behe's thinking. Behe is a prominent defender of the Intelligent Design Movement which has gaines influence particularly in the United States, but also in elsewhere. At the core of his thinking is the idea of intelligent design, according to which the order of the cosmos and of living things is the handiwork of a non-human intelligence. This "design argument" had previously been popular in the tradition of natural theology. Behe attempts to base his argument on the findings of 20th century biology, however. It has been revealed by biochemistry that cells, formerly thought to be simple, in fact contain complex structures, for instance the bacterial flagellum, which are reminiscent of the machines built by humans. According to Behe these can be believably explained only by referring to intelligent design, not by invoking darwinian natural laws. My analysis aims to understand Behe's thought on intelligent design, to bring forward its connections to intellectual history and worldviews, and to study whether Behe has formulated his argument so as to avoid common criticisms directed against design arguments. I use a large amount literature and refer to diverse writers participating in the intelligent design debate. The results of the analysis are as follows. Behe manages to avoid a large amount of classical criticisms against the design argument, and new criticisms have to be developed to meet his argument. Secondly, positions on intelligent design appear to be linked to larger philosophical and religious worldviews.vaan myös maailmankuvat ja uskonnolliset näkemykset.

Inhibition of Tyrosine Phosphorylation of Sperm Flagellar Proteins, Outer Dense Fiber Protein-2 and Tektin-2, Is Associated With Impaired Motility During Capacitation of Hamster Spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.

Observation of Enhanced Diffusivity in Magnetically Powered Reciprocal Swimmers

We report on the development of a system of micron-sized reciprocal swimmers that can be powered with small homogeneous magnetic fields, and whose motion resembles that of a helical flagellum moving back and forth. We have measured the diffusivities of the swimmers to be higher compared to nonactuated objects of identical dimensions at long time scales, in accordance with the theoretical predictions made by Lauga Phys. Rev. Lett. 106, 178101 (2011)]. Randomness in the reciprocity of the actuation strokes was found to have a strong influence on the enhancement of the diffusivity, which has been investigated with numerical calculations.

Development of an ex vivo organ culture model using human gastro-intestinal tissue and Campylobacter jejuni.

Campylobacter jejuni is an important food-borne pathogen. However, relatively little is understood regarding its pathogenesis, and research is hampered by the lack of a suitable model. Recently, a number of groups have developed assays to study the pathogenic mechanisms of C. jejuni using cell culture models. Here, we report the development of an ex vivo organ culture model, allowing for the maintenance of intestinal mucosal tissue, to permit more complex host-bacterium interactions to be studied. Ex vivo organ culture highlights the propensity for C. jejuni to adhere to mucosal tissue via the flagellum, either as discrete colonies or as multicellular units.

The Voltage-Gated Sodium Channel Na(v)1.8 Is Expressed in Human Sperm

The role of Na+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC a subunit Na(v)1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na-v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na-v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na-v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 mu M), the Na-v1.8 antagonist A-803467, or a specific Na-v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca2+-containing or Ca2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na+, [Na+](i), and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na+ channel Na-v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.

Efeito da 1,10-fenantrolina e seus derivados complexados em metal na atividade proteolítica de Leishmania braziliensis

Leishmanioses são um grupo de doenças com um largo espectro de manifestações clínicas, as quais variam desde lesões cutâneas até o envolvimento visceral severo, podendo levar ao óbito. A leishmaniose é, ainda hoje, uma doença negligenciada, estando entre os agravos prioritários do programa de pesquisa sobre doenças da pobreza da Organização Mundial da Saúde (OMS). Além de não haver vacinas disponíveis, a terapia é baseada em medicamentos injetáveis que causam sérios efeitos colaterais, tornando o tratamento inviável para muitos países endêmicos. Drogas derivadas de metal representam um novo arsenal terapêutico antimicrobiano e anti-câncer. Os inibidores de peptidase/agentes quelantes tais como 1,10-fenantrolina e seus derivados, no estado livre de metal ou como ligantes com metais de transição, interferem com a função de vários sistemas biológicos. Em trabalhos anteriores, nosso grupo descreveu que o parasito L. braziliensis produziu moléculas gp63 sensíveis a 1,10-fenantrolina. No presente trabalho, demonstramos a distribuição celular da molécula gp63 em uma cepa virulenta de L. braziliensis por meio de análises bioquímicas e imuno-histoquímica. Depois disso, relatamos os efeitos inibitórios de três compostos derivados da 1,10-fenantrolina, 1,10-fenantrolina-5,6-diona (phendio), [Cu(phendio)2] e [Ag(phendio)2], nas atividades metalopeptidases celulares e extracelulares produzidas por promastigotas de L. braziliensis, bem como as suas ações sobre a viabilidade do parasita e na interação com as células de macrófagos murinos. As moléculas gp63 foram detectadas em compartimentos de parasitos, incluindo membrana citoplasmatica e bolsa flagelar. O tratamento de promastigotas de L. braziliensis durante 1 hora com 1,10-fenantrolina e seus derivados resultou numa inibição significativa da viabilidade celular e mostrou um mecanismo de ação irreversível. Estes inibidores de metalopeptidases induziram apoptose em promastigotas de L. braziliensis, demonstrada através da marcação com anexina/iodeto de propídio e ensaio TUNEL. O pré-tratamento de promastigotas com os inibidores de metalopeptidases induziram uma diminuição na expressão de moléculas de superfície gp63, assim como uma redução significativa no índice de associação com macrófagos. Em paralelo, macrófagos infectados com L. braziliensis e tratados com 1,10-fenantrolina e seus derivados promoveram uma potente redução sobre o número de amastigotas intracelulares. O tratamento de macrófagos com 1,10-fenantrolina e seus derivados não induziram o aumento de óxido nítrico. A ação combinatória sobre a capacidade de crescimento entre os compostos derivados da 1,10-fenantrolina e Glucantime, quando ambos foram utilizados em concentracões sub-inibidoras, também foi observada. In vivo os compostos derivados da 1,10-fenantrolina e seus drivados foram capazes de controlar o tamanho das lesões a partir da terceira semana de tratamento em relação ao controle não tratado em hamsters infectados quando administrado por via intraperitoneal. Os animais tratados com os compostos apresentaram maior resposta intradérmica (DTH) aos antígenos de L. braziliensis. Coletivamente, a 1,10-fenantrolina e seus derivados metálicos apresentam uma nova perspectiva de estudos para o desenvolvimento de novos fármacos anti-L. braziliensis

Atividade in vitro da pterocarpanoquinona LQB-118 sobre o Trypanosoma cruzi

A doença de Chagas é endêmica na América Latina sendo considerada uma doença negligenciada com grande impacto socioeconômico. A infecção é causada pelo protozoário Trypanosoma cruzi que é transmitido pela forma vetorial, entre outros mecanismos. O tratamento consiste basicamente no uso de dois fármacos, o benznidazol e o Nifurtimox que apresentam uma série de efeitos colaterais e atuam muito pouco nas formas amastigotas intracelulares o que faz com que o tratamento atual seja restrito e insatisfatório.Várias atividades farmacológicas foram atribuídas ao lapachol e a pterocarpanos, tais como atividade antitumoral e antiparasitária. Devido a esse potencial foi sintetizado uma molécula híbrida, a pterocarpanoquinona LQB-118, e algumas moléculas derivadas. A LQB-118 mostrou anteriormente atividade antitumoral e anti-Leishmania. O objetivo do presente trabalho foi investigar a atividade in vitro da LQB-118 e suas moléculas derivadas sobre o Trypanosoma cruzi clone Dm28c. Para avaliação inicial do efeito anti-parasitário das moléculas, amastigotas intracelulares, tripomastigotas metacíclicos e epimastigotas foram incubados com 20 M das LQBs 118, 168, 187, 182 e 236. A LQB-118 demonstrou atividade antiparasitária nas três formas evolutivas (90% na forma amastigota, 44% na forma tripomastigota e 70% na forma epimastigota) do parasito, enquanto as moléculas derivadas não mostraram atividade significativa. Sendo assim os estudos foram continuados com a molécula LQB-118. A ação da LQB-118 sobre as amastigotas intracelulares foi dose dependente, com redução do índice de infecção em 81% e 88% nas concentrações de 20 e 30 M respectivamente. Já sobre tripomastigotas, a LQB-118 foi menos ativa reduzindo a mobilidade dessas formas em até 45% a 30 M. Sobre a forma epimastigota a ação foi dose-dependente chegando a inibir 96% o crescimento dos parasitos a 20 M, com alterações da morfologia tais como arrendondamento do corpo celular e perda do flagelo. A dose capaz de inibir 50% foi de 4,2 M para amastigota intracelular e 38,1 M para tripomastigotas. Para macrófagos, a LC50 ficou em 40 M, uma concentração quase dez vezes maior que a IC50 para amastigotas. A capacidade das formas amastigotas intracelulares se diferenciarem em tripomatigotas e lisar os macrófagos foi avaliada após o tratamento com a LQB-118 por 72h. Observou-se um atraso do ciclo intracelular do parasito de modo dose-dependente, onde na concentração de 30 M o surgimento de tripomastigota foi no 9 dia enquanto nos controles foi no 5 dia de cultura. Para delinear o mecanismo de ação, foi avaliado o efeito direto sobre o parasito como a indução da fragmentação de DNA. A análise de indução da fragmentação do DNA feita pela marcação pelo TUNEL mostrou que o tratamento com a LQB-118 induziu seletivamente a fragmentação do núcleo das amastigotas enquanto o núcleo dos macrófagos se mantiveram íntegros. Macrófagos peritoneais pré-tratados com LQB-118 por 24 horas foram capazes de reduzir o número de amastigotas após 72h de cultivo na ausência da molécula, mas sem alteração na produção de óxido nítrico. Esses resultados mostram que a LQB-118 é ativa contra o T. cruzi, principalmente sobre a forma amastigota intracelular, que é a forma presente na fase crônica da infecção. O mecanismo de ação sugere que a LQB-118 é capaz de ser seletivamente tóxica para o parasito e também ativar os mecanismos microbicidas dos macrófagos de modo independente da produção de óxido nítrico.

Morphometric differentiation in small juveniles of the pink spotted shrimp (Farfantepenaeus brasiliensis) and the southern pink shrimp (F. notialis) in the Yucatan Peninsula, Mexico

The morphometric and morphological characters of the rostrum have been widely used to identify penaeid shrimp species (Heales et al., 1985; Dall et al., 1990; Pendrey et al., 1999). In this setting, one of the constraints in studies of penaeid shrimp populations has been the uncertainty in the identification of early life history stages, especially in coastal nursery habitats, where recruits and juveniles dominate the population (Dall et al., 1990; Pérez-Castañeda and Defeo, 2001). In the western Atlantic Ocean, Pérez-Farfante (1969, 1970, 1971a) described diagnostic characters of the genus Farfantepenaeus that allowed identification of individuals in the range of 8−20 mm CL (carapace length) on the basis of the following morphological features: 1) changes in the structure of the petasma and thelycum; 2) absence or presence of distomarginal spines in the ventral costa of the petasma; 3) the ratio between the keel height and the sulcus width of the sixth abdominal somite; 4) the shape and position of the rostrum with respect to the segments and flagellum of the antennule; and 5) the ratio between rostrum length (RL) and carapace length (RL/CL). In addition, she classified Farfantepenaeus into two groups according to the shape and position of the rostrum with respect to the segments and flagellum of the antennule and the ratio RL/CL: 1) F. duorarum and F. notialis: short rostrum, straight distally, and the proximodorsal margin convex, usually extending anteriorly to the end of distal antennular segment, sometimes reaching to proximal one-fourth of broadened portion of lateral antennular flagellum, with RL/CL <0.75; and 2) F. aztecus, F. brasiliensis, F. paulensis, and F. subtilis: long rostrum, usually almost straight along the entire length, extending anteriorly beyond the distal antennular segment, sometimes reaching to the distal one-third of broadened portion of lateral antennular flagellum, with RL/CL >0.80. Pérez-Farfante stressed that, for the recognition to species level of juveniles <10 mm CL, all the characters listed above should be considered because occasionally one alone may not prove to be diagnostic. However, the only characters that could be distinguished for small juveniles in the range 4−8 mm CL are those defined on the rostrum. Therefore, it has been almost impossible to identify and separate small specimens of Farfantepenaeus (Pérez-Farfante, 1970, 1971a; Pérez-Farfante and Kensley, 1997).

Hydrodynamic entrapment of bacteria swimming near a solid surface.

The near-surface motility of bacteria is important in the initial formation of biofilms and in many biomedical applications. The swimming motion of Escherichia coli near a solid surface is investigated both numerically and experimentally. A boundary element method is used to predict the hydrodynamic entrapment of E. coli bacteria, their trajectories, and the minimum separation of the cell from the surface. The numerical results show the existence of a stable swimming distance from the boundary that depends only on the shape of the cell body and the flagellum. The experimental validation of the numerical approach allows one to use the numerical method as a predictive tool to estimate with reasonable accuracy the near-wall motility of swimming bacteria of known geometry. The analysis of the numerical database demonstrated the existence of a correlation between the radius of curvature of the near-wall circular trajectory and the separation gap. Such correlation allows an indirect estimation of either of the two quantities by a direct measure of the other without prior knowledge of the cell geometry. This result may prove extremely important in those biomedical and technical applications in which the near-wall behavior of bacteria is of fundamental importance.