Chemotaxis Pathway Research Articles

Chemotactic network responses to live bacteria show independence of phagocytosis from chemoreceptor sensing.

Aspects of innate immunity derive from characteristics inherent to phagocytes, including chemotaxis toward and engulfment of unicellular organisms or cell debris. Ligand chemotaxis has been biochemically investigated using mammalian and model systems, but precision of chemotaxis towards ligands being actively secreted by live bacteria is not well studied, nor has there been systematic analyses of interrelationships between chemotaxis and phagocytosis. The genetic/molecular model Dictyostelium and mammalian phagocytes share mechanistic pathways for chemotaxis and phagocytosis; Dictyostelium chemotax toward bacteria and phagocytose them as food sources. We quantified Dictyostelium chemotaxis towards live gram positive and gram negative bacteria and demonstrate high sensitivity to multiple bacterially-secreted chemoattractants. Additive/competitive assays indicate that intracellular signaling-networks for multiple ligands utilize independent upstream adaptive mechanisms, but common downstream targets, thus amplifying detection at low signal propagation, but strengthening discrimination of multiple inputs. Finally, analyses of signaling-networks for chemotaxis and phagocytosis indicate that chemoattractant receptor-signaling is not essential for bacterial phagocytosis.

Recent advances and future prospects in bacterial and archaeal locomotion and signal transduction.

Unraveling the structure and function of two-component and chemotactic signaling along with different aspects related to motility of bacteria and archaea are key research areas in modern microbiology. Escherichia coli is the traditional model organism to study chemotaxis signaling and motility. However, the recent study of a wide range of bacteria and even some archaea with different lifestyles has provided new insight into the eco-physiology of chemotaxis, which is essential for the host establishment of different pathogens or beneficial bacteria. The expanded range of model organisms has also permitted the study of chemosensory pathways unrelated to chemotaxis, multiple chemotaxis pathways within an organism, and new types of chemoreceptors. This research has greatly benefitted from technical advances in the field of cryo-microscopy that continues to reveal with increasing resolution the complexity and diversity of large protein complexes like the flagellar motor or chemoreceptor arrays. In addition, sensitive instruments now allow for an increasing number of experiments to be conducted at the single-cell level, thereby revealing information that is beginning to bridge the gap between individual cells and population behavior. Evidence has also accumulated showing that bacteria have evolved different mechanisms for surface sensing, which appears to be mediated by flagella and possibly type IV pili, and that the downstream signaling involves chemosensory pathways and two-component system based processes. Herein we summarize the recent advances and research tendencies in this field as presented at the latest Bacterial Locomotion and Signal Transduction (BLAST XIV) conference.

Insights into the Mechanism of Proliferation on the Special Microbes Mediated by Phenolic Acids in the Radix pseudostellariae Rhizosphere under Continuous Monoculture Regimes.

As potent allelochemicals, phenolic acids are believed to be associated with replanting disease and cause microflora shift and structural disorder in the rhizosphere soil of continuously monocultured Radix pseudostellariae. The transcriptome sequencing was used to reveal the mechanisms underlying the differential response of pathogenic bacterium Kosakonia sacchari and beneficial bacterium Bacillus pumilus on their interactions with phenolic acids, the main allelochemicals in root exudates of R. pseudostellariae in the monoculture system. The microbes were inoculated in the pots containing soil and the medicinal plant in this study. The results showed that the addition of beneficial B. pumilus to the 2-year planted soil significantly decreased the activity of soil urease, catalase, sucrase, and cellulase and increased the activity of chitinase compared with those in the 2nd-year monocropping rhizosphere soil without any treatment. However, opposite results were obtained when K. sacchari was added. Transcriptome analysis showed that vanillin enhanced glycolysis/gluconeogenesis, fatty acid biosynthesis, pentose phosphate, bacterial chemotaxis, flagellar assembly, and phosphotransferase system pathway in K. sacchari. However, protocatechuic acid, a metabolite produced by K. sacchari from vanillin, had negative effects on the citrate cycle and biosynthesis of novobiocin, phenylalanine, tyrosine, and tryptophan in B. pumilus. Concurrently, the protocatechuic acid decreased the biofilm formation of B. pumilus. These results unveiled the mechanisms how phenolic acids differentially mediate the shifts of microbial flora in rhizosphere soil, leading to the proliferation of pathogenic bacteria (i.e., K. sacchari) and the attenuation of beneficial bacteria (i.e., B. pumilus) under the monocropping system of R. pseudostellariae.

3-Amino 1,8-naphthalimide, a structural analog of the anti-cholera drug virstatin inhibits chemically-biased swimming and swarming motility in vibrios

A screen for inhibitors of Vibrio cholerae motility identified the compound 3-amino 1,8-naphthalimide (3-A18NI), a structural analog of the cholera drug virstatin. Similar to virstatin, 3-A18NI diminished cholera toxin production. In contrast, 3-A18NI impeded swimming and/or swarming motility of V. cholerae and V. parahemolyticus suggesting that it could target the chemotaxis pathway shared by the polar and lateral flagellar system of vibrios. 3-A18NI did not inhibit the expression of V. cholerae major flagellin FlaA or the assembly of its polar flagellum. Finally, 3-A18NI enhanced V. cholerae colonization mimicking the phenotype of chemotaxis mutants that exhibit counterclockwise-biased flagellum rotation.

Involvement of the α/β Isoform of p38 MAP Kinase in Chemotactic Responses of Human Eosinophils to Eotaxin (CCL11) and RANTES (CCL5)

Eosinophils are the principal effector cells for allergic inflammation in a variety of diseases, in which they contribute to tissue damage and remodelling processes via the secretion of cytotoxic granular proteins and cytokines. The intracellular mechanisms that control the activation, recruitment and survival of eosinophils are fundamental in understanding these disease processes. Phosphoinositide 3-kinase (PI3K) has been shown previously to be essential for eosinophil chemotactic responses to some stimuli but not others. Human blood neutrophils have been shown to utilize two antagonistic signalling pathways for chemotaxis: PI3K and p38 mitogen-activated protein kinase (p38 MAPK). In the present study, the role of p38 MAPK in chemotactic responses of an eosinophil-differentiated myeloid leukaemia cell line (EOL-1) and human peripheral blood eosinophils to a range of stimuli - platelet-activating factor (PAF), eotaxin 1 (CCL11), RANTES (CCL5), interleukin 8 (IL8, CXCL8) and IL16 - was explored through the use of the p38 MAPK α/β isoform inhibitor, SB 203580. SB 203580 caused significant inhibition of chemotactic responses of both EOL-1 cells and blood eosinophils to eotaxin 1 and RANTES (≥75% inhibition at 1 μM SB 203580, p<0.01) but had no effect on the migration induced by PAF and IL16 (<25%) and little or no effect on responses to IL8. Responses to PAF - but not eotaxin - have been shown previously to be suppressed by PI3K inhibition. The complementary pattern of inhibition observed in the present study provides evidence that distinct PI3K-dependent and p38 MAPK-dependent chemoattractants may also exist for eosinophils.

Carbon Monoxide–Releasing Molecule-401 Suppresses Polymorphonuclear Leukocyte Migratory Potential by Modulating F-Actin Dynamics

PMN co-cultures were perfused for additional 15 minutes with PMN-free medium containing CORM-401/inactive CORM-401. The experiments were videorecorded (phase-contrast microscopy), and PMN adhesion/migration were assessed off-line. In parallel, CORM-401-dependent modulation of PMN chemotaxis, F-actin expression/distribution, and actin-regulating pathways [eg, p21-activated protein kinases (PAK1/2) and extracellular signal-regulated kinase (ERK)/C-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK)] were assessed in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. Pretreating PMNwith CORM-401 did not suppress PMN adhesion to HUVEC, but significantly reduced PMN transendothelial migration (P<0.0001) and fMLP-induced PMN chemotaxis (ie, migration directionality and velocity). These changes were associated with CORM-401-dependent suppression of F-actin levels/cellular distributionand fMLP-induced phosphorylation of PAK1/2 and ERK/JNK MAPK (P<0.05). CORM-401 had no effect on p38 MAPK activation. In summary, this study demonstrates, for the first time, CORM-401-dependent suppression of neutrophilmigratory potential associated with modulation of PAK1/2 and ERK/JNK MAPK signaling and F-actin dynamics.

Subtyping Chronic Obstructive Pulmonary Disease Using Peripheral Blood Proteomics.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disorder. COPD patients may have different clinical features, imaging characteristics and natural history. Multiple studies have investigated heterogeneity using statistical methods such as unsupervised clustering to define different subgroups of COPD based largely on clinical phenotypes. Some studies have performed clustering using genetic data or limited numbers of blood biomarkers. Our primary goal was to use proteomic data to find subtypes of COPD within clinically similar individuals. In the Treatment of Emphysema with a gamma-Selective Retinoid Agonist (TESRA) study, multiplex biomarker panels were run in serum samples collected prior to randomization. After implementing an algorithm to minimize missing values, the dataset included 396 COPD individuals and 87 biomarkers. Using hierarchical clustering, we identified 3 COPD subgroups, containing 267 (67.4%), 104 (26.3%), and 25 (6.3%) individuals, respectively. The third cluster had less emphysema on quantitative analysis of chest computed tomography scans (p=0.03) and worse disease-related quality of life based on the St. George's Respiratory Questionnaire (total score cluster 1: 45.6, cluster 2: 45.4, cluster 3: 56.6; p=0.01), despite similar levels of lung function impairment (forced expiratory volume in 1 second (49.2%, 49.2%, 48.2 % predicted, respectively). Enrichment analysis showed the biomarkers distinguishing cluster 3 mapped to platelet alpha granule and cell chemotaxis pathways. Thus, we identified a subgroup which has less emphysema but may have greater inflammation, which could be potentially targeted with anti-inflammatory therapies.

Comparative genomics of Pseudomonas syringae pathovar tomato reveals novel chemotaxis pathways associated with motility and plant pathogenicity.

The majority of bacterial foliar plant pathogens must invade the apoplast of host plants through points of ingress, such as stomata or wounds, to replicate to high population density and cause disease. How pathogens navigate plant surfaces to locate invasion sites remains poorly understood. Many bacteria use chemical-directed regulation of flagellar rotation, a process known as chemotaxis, to move towards favorable environmental conditions. Chemotactic sensing of the plant surface is a potential mechanism through which foliar plant pathogens home in on wounds or stomata, but chemotactic systems in foliar plant pathogens are not well characterized. Comparative genomics of the plant pathogen Pseudomonas syringae pathovar tomato (Pto) implicated annotated chemotaxis genes in the recent adaptations of one Pto lineage. We therefore characterized the chemosensory system of Pto. The Pto genome contains two primary chemotaxis gene clusters, che1 and che2. The che2 cluster is flanked by flagellar biosynthesis genes and similar to the canonical chemotaxis gene clusters of other bacteria based on sequence and synteny. Disruption of the primary phosphorelay kinase gene of the che2 cluster, cheA2, eliminated all swimming and surface motility at 21 °C but not 28 °C for Pto. The che1 cluster is located next to Type IV pili biosynthesis genes but disruption of cheA1 has no observable effect on twitching motility for Pto. Disruption of cheA2 also alters in planta fitness of the pathogen with strains lacking functional cheA2 being less fit in host plants but more fit in a non-host interaction.

Characterization of Inflammatory Response in Acute-on-Chronic Liver Failure and Relationship with Prognosis.

ACLF is characterized by a systemic inflammatory response, but the cytokines involved in this process have not been well studied. The aim of this study was to characterize the systemic inflammatory response in patients with cirrhosis and ACLF and its relationship with prognosis. Fifty-five patients with cirrhosis, 26 with ACLF, were studied prospectively. Systemic inflammatory response was analyzed by measuring a large array of plasma cytokines by using a multiplex kit. A principal component analysis show noticeable differences between ACLF and decompensated cirrhosis without ACLF. Patients with ACLF had significant abnormal levels of 12 cytokines compared to those without ACLF, including: VCAM-1, VEGF-A, Fractalkine, MIP-1α, Eotaxin, IP-10, RANTES, GM-CSF, IL-1β, IL-2, ICAM-1, and MCP-1. Cytokines showing the most marked relationship with ACLF were VCAM-1 and VEGF-A (AUCROC 0.77; p = 0.001). There was a significant relationship between some of inflammatory mediators and 3-month mortality, particularly VCAM-1, ICAM-1, and GM-CSF (AUCROC>0.7; p < 0.05). Functional Enrichment Analysis showed that inflammatory markers differentially expressed in ACLF patients were enriched in leukocyte migration, particularly monocytes and macrophages, and chemotaxis pathways. In conclusion, ACLF is characterized by a marked inflammatory reaction with activation of mediators of adhesion and migration of leukocytes. The intensity of the inflammatory reaction correlates with prognosis.

Sugar Influx Sensing by the Phosphotransferase System of Escherichia coli.

The phosphotransferase system (PTS) plays a pivotal role in the uptake of multiple sugars in Escherichia coli and many other bacteria. In the cell, individual sugar-specific PTS branches are interconnected through a series of phosphotransfer reactions, thus creating a global network that not only phosphorylates incoming sugars but also regulates a number of cellular processes. Despite the apparent importance of the PTS network in bacterial physiology, the holistic function of the network in the cell remains unclear. Here we used Förster resonance energy transfer (FRET) to investigate the PTS network in E. coli, including the dynamics of protein interactions and the processing of different stimuli and their transmission to the chemotaxis pathway. Our results demonstrate that despite the seeming complexity of the cellular PTS network, its core part operates in a strikingly simple way, sensing the overall influx of PTS sugars irrespective of the sugar identity and distributing this information equally through all studied branches of the network. Moreover, it also integrates several other specific metabolic inputs. The integrated output of the PTS network is then transmitted linearly to the chemotaxis pathway, in stark contrast to the amplification of conventional chemotactic stimuli. Finally, we observe that default uptake through the uninduced PTS network correlates well with the quality of the carbon source, apparently representing an optimal regulatory strategy.

Bacterial pathogenesis of plants: future challenges from a microbial perspective: Challenges in Bacterial Molecular Plant Pathology.

Summary Future Challenges in Plant Pathology Plant infection is a complicated process. On encountering a plant, pathogenic microorganisms must first adapt to life on the epiphytic surface, and survive long enough to initiate an infection. Responsiveness to the environment is critical throughout infection, with intracellular and community‐level signal transduction pathways integrating environmental signals and triggering appropriate responses in the bacterial population. Ultimately, phytopathogens must migrate from the epiphytic surface into the plant tissue using motility and chemotaxis pathways. This migration is coupled with overcoming the physical and chemical barriers to entry into the plant apoplast. Once inside the plant, bacteria use an array of secretion systems to release phytotoxins and protein effectors that fulfil diverse pathogenic functions (Fig. 1) (Melotto and Kunkel, 2013; Phan Tran et al., 2011).As our understanding of the pathways and mechanisms underpinning plant pathogenicity increases, a number of central research challenges are emerging that will profoundly shape the direction of research in the future. We need to understand the bacterial phenotypes that promote epiphytic survival and surface adaptation in pathogenic bacteria. How do these pathways function in the context of the plant‐associated microbiome, and what impact does this complex microbial community have on the onset and severity of plant infections?The huge importance of bacterial signal transduction to every stage of plant infection is becoming increasingly clear. However, there is a great deal to learn about how these signalling pathways function in phytopathogenic bacteria, and the contribution they make to various aspects of plant pathogenicity.We are increasingly able to explore the structural and functional diversity of small‐molecule natural products from plant pathogens. We need to acquire a much better understanding of the production, deployment, functional redundancy and physiological roles of these molecules. Type III secretion systems (T3SSs) are important and well‐studied contributors to bacterial disease. Several key unanswered questions will shape future investigations of these systems. We need to define the mechanism of hierarchical and temporal control of effector secretion.For successful infection, effectors need to interact with host components to exert their function. Advanced biochemical, proteomic and cell biological techniques will enable us to study the function of effectors inside the host cell in more detail and on a broader scale.Population genomics analyses provide insight into evolutionary adaptation processes of phytopathogens. The determination of the diversity and distribution of type III effectors (T3Es) and other virulence genes within and across pathogenic species, pathovars and strains will allow us to understand how pathogens adapt to specific hosts, the evolutionary pathways available to them, and the possible future directions of the evolutionary arms race between effectors and molecular plant targets. Although pathogenic bacteria employ a host of different virulence and proliferation strategies, as a result of the space constraints, this review focuses mainly on the hemibiotrophic pathogens. We discuss the process of plant infection from the perspective of these important phytopathogens, and highlight new approaches to address the outstanding challenges in this important and fast‐moving field.

(1)H, (13)C and (15)N resonance assignments for the response regulator CheY3 from Rhodobacter sphaeroides.

Rhodobacter sphaeroides has emerged as a model system for studies of the complex chemotaxis pathways that are a hallmark of many non-enteric bacteria. The genome of R. sphaeroides encodes two sets of flagellar genes, fla1 and fla2, that are controlled by three different operons. Each operon encodes homologues of most of the proteins required for the well-studied E. coli chemotaxis pathway. R. sphaeroides has six homologues of the response regulator CheY that are localized to and are regulated by different clusters of chemosensory proteins in the cell and have different effects on chemotaxis. CheY6 is the major CheY stopping the fla1 flagellar motor and associated with a cytoplasmically localised chemosensory pathway. CheY3 and CheY4 are associated with a membrane localised polar chemosensory cluster, and can bind to but not stop the motor. CheY6 and either CheY3 or CheY4 are required for chemotaxis. We are using NMR spectroscopy to characterise and compare the structure and dynamics of CheY3 and CheY6 in solution. We are interested in defining the conformational changes that occur upon activation of these two proteins and to identify differences in their properties that can explain the different functions they play in chemotaxis in R. sphaeroides. Here we present the 1H, 13C and 15N assignments for CheY3 in its active, inactive and Mg2+-free apo form. These assignments provide the starting point for detailed investigations of the structure and function of CheY3.

The Role of Adaptation in Bacterial Speed Races.

Evolution of biological sensory systems is driven by the need for efficient responses to environmental stimuli. A paradigm among prokaryotes is the chemotaxis system, which allows bacteria to navigate gradients of chemoattractants by biasing their run-and-tumble motion. A notable feature of chemotaxis is adaptation: after the application of a step stimulus, the bacterial running time relaxes to its pre-stimulus level. The response to the amino acid aspartate is precisely adapted whilst the response to serine is not, in spite of the same pathway processing the signals preferentially sensed by the two receptors Tar and Tsr, respectively. While the chemotaxis pathway in E. coli is well characterized, the role of adaptation, its functional significance and the ecological conditions where chemotaxis is selected, are largely unknown. Here, we investigate the role of adaptation in the climbing of gradients by E. coli. We first present theoretical arguments that highlight the mechanisms that control the efficiency of the chemotactic up-gradient motion. We discuss then the limitations of linear response theory, which motivate our subsequent experimental investigation of E. coli speed races in gradients of aspartate, serine and combinations thereof. By using microfluidic techniques, we engineer controlled gradients and demonstrate that bacterial fronts progress faster in equal-magnitude gradients of serine than aspartate. The effect is observed over an extended range of concentrations and is not due to differences in swimming velocities. We then show that adding a constant background of serine to gradients of aspartate breaks the adaptation to aspartate, which results in a sped-up progression of the fronts and directly illustrate the role of adaptation in chemotactic gradient-climbing.

A Gα-Stimulated RapGEF Is a Receptor-Proximal Regulator of Dictyostelium Chemotaxis

Chemotaxis, or directional movement toward extracellular chemical gradients, is an important property of cells that is mediated through G-protein-coupled receptors (GPCRs). Although many chemotaxis pathways downstream of Gβγ have been identified, few Gα effectors are known. Gα effectors are of particular importance because they allow the cell to distinguish signals downstream of distinct chemoattractant GPCRs. Here we identify GflB, a Gα2 binding partner that directly couples the Dictyostelium cyclic AMP GPCR to Rap1. GflB localizes to the leading edge and functions as a Gα-stimulated, Rap1-specific guanine nucleotide exchange factor required to balance Ras and Rap signaling. The kinetics of GflB translocation are fine-tuned by GSK-3 phosphorylation. Cells lacking GflB display impaired Rap1/Ras signaling and actin and myosin dynamics, resulting in defective chemotaxis. Our observations demonstrate that GflB is an essential upstream regulator of chemoattractant-mediated cell polarity and cytoskeletal reorganization functioning to directly link Gα activation to monomeric G-protein signaling.

Azospirillum brasilense Chemotaxis Depends on Two Signaling Pathways Regulating Distinct Motility Parameters.

The genomes of most motile bacteria encode two or more chemotaxis (Che) systems, but their functions have been characterized in only a few model systems. Azospirillum brasilense is a motile soil alphaproteobacterium able to colonize the rhizosphere of cereals. In response to an attractant, motile A. brasilense cells transiently increase swimming speed and suppress reversals. The Che1 chemotaxis pathway was previously shown to regulate changes in the swimming speed, but it has a minor role in chemotaxis and root surface colonization. Here, we show that a second chemotaxis system, named Che4, regulates the probability of swimming reversals and is the major signaling pathway for chemotaxis and wheat root surface colonization. Experimental evidence indicates that Che1 and Che4 are functionally linked to coordinate changes in the swimming motility pattern in response to attractants. The effect of Che1 on swimming speed is shown to enhance the aerotactic response of A. brasilense in gradients, likely providing the cells with a competitive advantage in the rhizosphere. Together, the results illustrate a novel mechanism by which motile bacteria utilize two chemotaxis pathways regulating distinct motility parameters to alter movement in gradients and enhance the chemotactic advantage. Chemotaxis provides motile bacteria with a competitive advantage in the colonization of diverse niches and is a function enriched in rhizosphere bacterial communities, with most species possessing at least two chemotaxis systems. Here, we identify the mechanism by which cells may derive a significant chemotactic advantage using two chemotaxis pathways that ultimately regulate distinct motility parameters.

Role of eosinophils and apoptosis in PDIMs/PGLs deficient mycobacterium elimination in adult zebrafish

The cell wall lipids phthiocerol dimycocerosates (PDIMs) and its structurally-related compound, phenolic glycolipids (PGLs) are major virulence factors of mycobacterium, as shown by the reduced growth of PDIMs/PGLs deficient mutants in various animal models. PDIMs/PGLs play active roles in modulating host immune responses. However, the cellular and molecular mechanisms of how PDIMs/PGLs deficient mutant was eliminated in vivo are still elusive. Our aim was to investigate what host immune responses have effect on mycobacterium elimination in vivo. Using microarray, we find PDIMs/PGLs modulate divergent host responses, including chemotaxis and focal adhesion's downstream pathway and apoptosis. We examine these two host responses by Diff-Quik stain, coupled with transmission electron microscopy and TUNEL stain respectively. The ultrastructure observation showed that eosinophils appeared in WT-infected zebrafish at day 1, however eosinophils arrived was delayed to day 7 in PDIMs/PGLs-deficient mutant-infected animals. More intriguingly, apoptosis was markedly increased in PDIMs/PGLs-mutant infected zebrafish at day 1 after infection, compared to WT-infected fishes at this time. However, apoptosis trend was fully reversed by day 7, with increased apoptosis were detected in WT-infected zebrafish compared with the PDIMs/PGLs-deficient mutant, especially more apoptosis within the granuloma. This study shows that the anti-apoptotic effects of PDIMs/PGLs and the recruitment of eosinophils in tissue during the early infection in zebrafish might promote bacterium growth in vivo.

Evolutionary Genomics Suggests That CheV Is an Additional Adaptor for Accommodating Specific Chemoreceptors within the Chemotaxis Signaling Complex

Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a “phosphate sink” possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Overall, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.

Megadock 4.0. An Ultra-High-Performance Protein-Protein Docking Software for Heterogeneous Supercomputers

Protein-protein interaction (PPI) plays a core role in cellular functions. In recent years, PPI prediction methods based on protein docking have been developed and have been applied for large-scale PPI network prediction based on tertiary structures. However, such network prediction requires much computing resources, and a faster PPI prediction method is eagerly demanded. We have developed an ultra-high-throughput PPI prediction system based on rigid-body protein docking, “MEGADOCK 4.0”. MEGADOCK 4.0 can perform faster docking based on its original scoring function and implementation for heterogeneous supercomputers. MEGADOCK 4.0 was implemented by two parallelization techniques for massively parallel supercomputing environment; the MPI and OpenMP hybrid parallelization and a CUDA-based implementation on GPUs. MEGADOCK 4.0 achieved an excellent scalability on supercomputing environments, such as K computer and TSUBAME 2.5. We also present newly implemented MEGADOCK for many integrated core architecture like Intel Xeon Phi co-processor. In addition, we have already applied MEGADOCK system to a number of interactome analyses such as (a) bacterial chemotaxis pathway consisted of 101 PDB structures and required 10,201 pairs of protein dockings, (b) human apoptosis pathway consisted of 158 PDB structures and required 24,964 pairs of protein dockings, and (c) human epidermal growth factor receptor related pathway consisted of 1,921 PDB structures and required 3,690,241 pairs of protein dockings. We present a new protein-protein docking engine aimed at exhaustive docking of millions of protein pairs. The system was shown to be scalable when running on thousands of nodes and multiple accelerators.

Function and Regulation of Heterotrimeric G Proteins during Chemotaxis.

Chemotaxis, or directional movement towards an extracellular gradient of chemicals, is necessary for processes as diverse as finding nutrients, the immune response, metastasis and wound healing. Activation of G-protein coupled receptors (GPCRs) is at the very base of the chemotactic signaling pathway. Chemotaxis starts with binding of the chemoattractant to GPCRs at the cell-surface, which finally leads to major changes in the cytoskeleton and directional cell movement towards the chemoattractant. Many chemotaxis pathways that are directly regulated by Gβγ have been identified and studied extensively; however, whether Gα is just a handle that regulates the release of Gβγ or whether Gα has its own set of distinct chemotactic effectors, is only beginning to be understood. In this review, we will discuss the different levels of regulation in GPCR signaling and the downstream pathways that are essential for proper chemotaxis.

Modulation of B-cell receptor and microenvironment signaling by a guanine exchange factor in B-cell malignancies.

Objective: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells over-express a guanine exchange factor (GEF), Rasgrf-1. This GEF increases active Ras as it catalyzes the removal of GDP from Ras so that GTP can bind and activate Ras. This study aims to study the mechanism of action of Rasgrf-1 in B-cell malignancies. Methods: N-terminus truncated Rasgrf-1 variants have a higher GEF activity as compared to the full-length transcript therefore a MCL cell line with stable over-expression of truncated Rasgrf-1 was established. The B-cell receptor (BCR) and chemokine signaling pathways were compared in the Rasgrf-1 over-expressing and a control transfected cell line. Results: Cells over-expressing truncated form of Rasgrf-1 have a higher proliferative rate as compared to control transfected cells. BCR was activated by lower concentrations of anti-IgM antibody in Rasgrf-1 over-expressing cells as compared to control cells indicating that these cells are more sensitive to BCR signaling. BCR signaling also phosphorylates Rasgrf-1 that further increases its GEF function and amplifies BCR signaling. This activation of Rasgrf-1 in over-expressing cells resulted in a higher expression of phospho-ERK, AKT, BTK and PKC-alpha as compared to control cells. Besides BCR, Rasgrf-1 over-expressing cells were also more sensitive to microenvironment stimuli as determined by resistance to apoptosis, chemotaxis and ERK pathway activation. Conclusions: This GEF protein sensitizes B-cells to BCR and chemokine mediated signaling and also upregulates a number of other signaling pathways which promotes growth and survival of these cells.