scholarly journals Evidence of Intraflagellar Transport and Apical Complex Formation in a Free-Living Relative of the Apicomplexa

2013 ◽  
Vol 13 (1) ◽  
pp. 10-20 ◽  
Author(s):  
Neil Portman ◽  
Christie Foster ◽  
Giselle Walker ◽  
Jan Šlapeta
Keyword(s):  
Close Association ◽  
Flagellar Apparatus ◽  
Intraflagellar Transport ◽  
Endomembrane System ◽  
Free Living ◽  
Content Type ◽  
Daughter Cells ◽  
Flagellar Assembly ◽  
Apical Complex ◽  
Flagellar Biogenesis

ABSTRACT Since its first description, Chromera velia has attracted keen interest as the closest free-living relative of parasitic Apicomplexa. The life cycle of this unicellular alga is complex and involves a motile biflagellate form. Flagella are thought to be formed in the cytoplasm, a rare phenomenon shared with Plasmodium in which the canonical mode of flagellar assembly, intraflagellar transport, is dispensed with. Here we demonstrate the expression of intraflagellar transport components in C. velia , answering the question of whether this organism has the potential to assemble flagella via the canonical route. We have developed and characterized a culturing protocol that favors the generation of flagellate forms. From this, we have determined a marked shift in the mode of daughter cell production from two to four daughter cells per division as a function of time after passage. We conduct an ultrastructural examination of the C. velia flagellate form by using serial TEM and show that flagellar biogenesis in C. velia occurs prior to cytokinesis. We demonstrate a close association of the flagellar apparatus with a complex system of apical structures, including a micropore, a conoid, and a complex endomembrane system reminiscent of the apical complex of parasitic apicomplexans. Recent work has begun to elucidate the possible flagellar origins of the apical complex, and we show that in C. velia these structures are contemporaneous within a single cell and share multiple connections. We propose that C. velia therefore represents a vital piece in the puzzle of the origins of the apical complex.

scholarly journals Helicobacter pylori FlhA Binds the Sensor Kinase and Flagellar Gene Regulatory Protein FlgS with High Affinity

2015 ◽  
Vol 197 (11) ◽  
pp. 1886-1892 ◽  
Author(s):  
Jennifer Tsang ◽  
Takanori Hirano ◽  
Timothy R. Hoover ◽  
Jonathan L. McMurry
Keyword(s):  
Helicobacter Pylori ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Kinase Domain ◽  
Optical Biosensing ◽  
Content Type ◽  
High Affinity ◽  
Flagellar Assembly ◽  
Flagellar Biogenesis

ABSTRACTFlagellar biogenesis is a complex process that involves multiple checkpoints to coordinate transcription of flagellar genes with the assembly of the flagellum. InHelicobacter pylori, transcription of the genes needed in the middle stage of flagellar biogenesis is governed by RpoN and the two-component system consisting of the histidine kinase FlgS and response regulator FlgR. In response to an unknown signal, FlgS autophosphorylates and transfers the phosphate to FlgR, initiating transcription from RpoN-dependent promoters. In the present study, export apparatus protein FlhA was examined as a potential signal protein. Deletion of its N-terminal cytoplasmic sequence dramatically decreased expression of two RpoN-dependent genes,flaBandflgE. Optical biosensing demonstrated a high-affinity interaction between FlgS and a peptide consisting of residues 1 to 25 of FlhA (FlhANT). TheKD(equilibrium dissociation constant) was 21 nM and was characterized by fast-on (kon= 2.9 × 104M−1s−1) and slow-off (koff= 6.2 × 10−4s−1) kinetics. FlgS did not bind peptides consisting of smaller fragments of the FlhANTsequence. Analysis of binding to purified fragments of FlgS demonstrated that the C-terminal portion of the protein containing the kinase domain binds FlhANT. FlhANTbinding did not stimulate FlgS autophosphorylationin vitro, suggesting that FlhA facilitates interactions between FlgS and other structures required to stimulate autophosphorylation.IMPORTANCEThe high-affinity binding of FlgS to FlhA characterized in this study points to an additional role for FlhA in flagellar assembly. Beyond its necessity for type III secretion, the N-terminal cytoplasmic sequence of FlhA is required for RpoN-dependent gene expression via interaction with the C-terminal kinase domain of FlgS.

scholarly journals Diversification of Campylobacter jejuni Flagellar C-Ring Composition Impacts Its Structure and Function in Motility, Flagellar Assembly, and Cellular Processes

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Louie D. Henderson ◽  
Teige R. S. Matthews-Palmer ◽  
Connor J. Gulbronson ◽  
Deborah A. Ribardo ◽  
Morgan Beeby ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Content Type ◽  
Cellular Processes ◽  
Spatial Regulation ◽  
Flagellar Motors ◽  
Core Components ◽  
Flagellar Assembly ◽  
And Function ◽  
Conserved Core ◽  
Flagellar Biogenesis

ABSTRACT Bacterial flagella are reversible rotary motors that rotate external filaments for bacterial propulsion. Some flagellar motors have diversified by recruiting additional components that influence torque and rotation, but little is known about the possible diversification and evolution of core motor components. The mechanistic core of flagella is the cytoplasmic C ring, which functions as a rotor, directional switch, and assembly platform for the flagellar type III secretion system (fT3SS) ATPase. The C ring is composed of a ring of FliG proteins and a helical ring of surface presentation of antigen (SPOA) domains from the switch proteins FliM and one of two usually mutually exclusive paralogs, FliN or FliY. We investigated the composition, architecture, and function of the C ring of Campylobacter jejuni, which encodes FliG, FliM, and both FliY and FliN by a variety of interrogative approaches. We discovered a diversified C. jejuni C ring containing FliG, FliM, and both FliY, which functions as a classical FliN-like protein for flagellar assembly, and FliN, which has neofunctionalized into a structural role. Specific protein interactions drive the formation of a more complex heterooligomeric C. jejuni C-ring structure. We discovered that this complex C ring has additional cellular functions in polarly localizing FlhG for numerical regulation of flagellar biogenesis and spatial regulation of division. Furthermore, mutation of the C. jejuni C ring revealed a T3SS that was less dependent on its ATPase complex for assembly than were other systems. Our results highlight considerable evolved flagellar diversity that impacts motor output, biogenesis, and cellular processes in different species. IMPORTANCE The conserved core of bacterial flagellar motors reflects a shared evolutionary history that preserves the mechanisms essential for flagellar assembly, rotation, and directional switching. In this work, we describe an expanded and diversified set of core components in the Campylobacter jejuni flagellar C ring, the mechanistic core of the motor. Our work provides insight into how usually conserved core components may have diversified by gene duplication, enabling a division of labor of the ancestral protein between the two new proteins, acquisition of new roles in flagellar assembly and motility, and expansion of the function of the flagellum beyond motility, including spatial regulation of cell division and numerical control of flagellar biogenesis in C. jejuni. Our results highlight that relatively small changes, such as gene duplications, can have substantial ramifications on the cellular roles of a molecular machine.

scholarly journals Partially redundant actin genes in Chlamydomonas control flagellum-directed traffic and transition zone organization

2017 ◽  
Author(s):  
Brittany Jack ◽  
David M. Mueller ◽  
Ann C. Fee ◽  
Ashley Tetlow ◽  
Prachee Avasthi
Keyword(s):  
Transition Zone ◽  
Mammalian Cells ◽  
Intraflagellar Transport ◽  
Actin Genes ◽  
Transmission Electron ◽  
Unicellular Green Alga ◽  
Absolute Requirement ◽  
Flagellar Assembly ◽  
Flagellar Proteins ◽  
Flagellar Biogenesis

ABSTRACTFlagella of the unicellular green alga Chlamydomonas reinhardtii are nearly identical to cilia of mammalian cells and provide an excellent model to study ciliogenesis. These biflagellated cells have two actin genes: one encoding a conventional actin (IDA5) and the other encoding a divergent novel actin-like protein (NAP1). Previously, we described a role for actin in the regulation of flagella-building intraflagellar transport machinery. Here, we probe how actin redundancy contributes to this process using a nap1 mutant Chlamydomonas strain. Disruption of a single actin allows normal or slower incorporation but complete flagellar assembly. However, when we disrupt both actins using Latrunculin B (LatB) treatment on the nap1 mutant background, we find flagellar growth from newly synthesized limiting flagellar proteins is actin-dependent. Upon total actin disruption during flagellar assembly, transmission electron microscopy identified an accumulation of Golgi-adjacent vesicles, suggesting impaired vesicular trafficking may be the mechanism by which actin supports flagellar growth from new flagellar proteins. We also find there is a mislocalization of a key transition zone gating and ciliopathy protein, NPHP-4. Extended (2 hour) treatment with LatB, a condition under which NAP1 is upregulated, restores NPHP-4 localization. This suggests NAP1 can perform the functions of conventional actin at the transition zone. Our experiments demonstrate that each stage of flagellar biogenesis requires redundant actin function to varying degrees, with an absolute requirement for these actins in transport of Golgi-adjacent vesicles and flagellar incorporation of newly synthesized proteins.

scholarly journals Enterobacterial Common Antigen Integrity Is a Checkpoint for Flagellar Biogenesis in Serratia marcescens

2007 ◽  
Vol 190 (1) ◽  
pp. 213-220 ◽  
Author(s):  
María E. Castelli ◽  
Griselda V. Fedrigo ◽  
Ana L. Clementín ◽  
M. Verónica Ielmini ◽  
Mario F. Feldman ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Insertional Mutagenesis ◽  
Physiological Role ◽  
Flagellar Apparatus ◽  
Common Antigen ◽  
Enterobacterial Common Antigen ◽  
Flagellar Regulon ◽  
Flagellar Assembly ◽  
New Perspective ◽  
Flagellar Biogenesis

ABSTRACT Serratia marcescens strains are ubiquitous bacteria isolated from environmental niches, such as soil, water, and air, and also constitute emergent nosocomial opportunistic pathogens. Among the numerous extracellular factors that S. marcescens is able to produce, the PhlA phospholipase is the only described exoprotein secreted by the flagellar apparatus while simultaneously being a member of the flagellar regulon. To gain insight into the regulatory mechanism that couples PhlA and flagellar expression, we conducted a generalized insertional mutagenesis and screened for PhlA-deficient strains. We found that three independent mutations in the wec cluster, which impaired the assembly of enterobacterial common antigen (ECA), provoked the inhibition of PhlA expression. Swimming and swarming assays showed that in these strains, motility was severely affected. Microscopic examination and flagellin immunodetection demonstrated that a strong defect in flagellum expression was responsible for the reduced motility in the wec mutant strains. Furthermore, we determined that in the ECA-defective strains, the transcriptional cascade that controls flagellar assembly was turned off due to the down-regulation of flhDC expression. These findings provide a new perspective on the physiological role of the ECA, providing evidence that in S. marcescens, its biosynthesis conditions the expression of the flagellar regulon.

scholarly journals Antimicrobial Photodynamic Inactivation Inhibits Candida albicans Virulence Factors and ReducesIn VivoPathogenicity

2012 ◽  
Vol 57 (1) ◽  
pp. 445-451 ◽  
Author(s):  
Ilka Tiemy Kato ◽  
Renato Araujo Prates ◽  
Caetano Padial Sabino ◽  
Beth Burgwyn Fuchs ◽  
George P. Tegos ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Virulence Factors ◽  
Sodium Dodecyl ◽  
Systemic Infection ◽  
Tube Formation ◽  
Photodynamic Inactivation ◽  
Content Type ◽  
Daughter Cells

ABSTRACTThe objective of this study was to evaluate whetherCandida albicansexhibits altered pathogenicity characteristics following sublethal antimicrobial photodynamic inactivation (APDI) and if such alterations are maintained in the daughter cells.C. albicanswas exposed to sublethal APDI by using methylene blue (MB) as a photosensitizer (0.05 mM) combined with a GaAlAs diode laser (λ 660 nm, 75 mW/cm2, 9 to 27 J/cm2).In vitro, we evaluated APDI effects onC. albicansgrowth, germ tube formation, sensitivity to oxidative and osmotic stress, cell wall integrity, and fluconazole susceptibility.In vivo, we evaluatedC. albicanspathogenicity with a mouse model of systemic infection. Animal survival was evaluated daily. Sublethal MB-mediated APDI reduced the growth rate and the ability ofC. albicansto form germ tubes compared to untreated cells (P< 0.05). Survival of mice systemically infected withC. albicanspretreated with APDI was significantly increased compared to mice infected with untreated yeast (P< 0.05). APDI increasedC. albicanssensitivity to sodium dodecyl sulfate, caffeine, and hydrogen peroxide. The MIC for fluconazole forC. albicanswas also reduced following sublethal MB-mediated APDI. However, none of those pathogenic parameters was altered in daughter cells ofC. albicanssubmitted to APDI. These data suggest that APDI may inhibit virulence factors and reducein vivopathogenicity ofC. albicans. The absence of alterations in daughter cells indicates that APDI effects are transitory. The MIC reduction for fluconazole following APDI suggests that this antifungal could be combined with APDI to treatC. albicansinfections.

scholarly journals Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori

2015 ◽  
Vol 197 (11) ◽  
pp. 1921-1930 ◽  
Author(s):  
Jennifer Tsang ◽  
Timothy R. Hoover
Keyword(s):  
Helicobacter Pylori ◽  
Basal Body ◽  
Protein Interactions ◽  
Transcript Levels ◽  
Content Type ◽  
Genes Encoding ◽  
H Pylori ◽  
Flagellar Biogenesis ◽  
Flagellar Genes

ABSTRACTFlagellar biogenesis inHelicobacter pyloriis regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ80), RpoN (σ54), and FliA (σ28), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaBandflgE) and FliA-dependent (flaA) genes. The MS ring (encoded byfliF) is one of the earliest flagellar structures assembled. Deletion offliFresulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease inflaAtranscript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions offliHand genes encoding C ring components (fliMandfliY) decreased transcript levels offlaBandflgEbut had little or no effect on transcript levels offlaA. Transcript levels offlaBandflgEwere elevated in mutants where genes encoding rod proteins (fliEandflgBC) were deleted, while transcript levels offlaAwas reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure.IMPORTANCEThe mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of theH. pyloriflagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of theH. pyloriRpoN regulon, a hypothesis that can be tested by examining protein-protein interactionsin vitro.

scholarly journals Apicortin, a Constituent of Apicomplexan Conoid/Apical Complex and Its Tentative Role in Pathogen—Host Interaction

2021 ◽  
Vol 6 (3) ◽  
pp. 118
Author(s):  
Ferenc Orosz
Keyword(s):  
Plasmodium Falciparum ◽  
Toxoplasma Gondii ◽  
Therapeutic Target ◽  
In Silico ◽  
Free Living ◽  
Binding Properties ◽  
Host Interactions ◽  
Trichoplax Adhaerens ◽  
Host Interaction ◽  
Apical Complex

In 2009, apicortin was identified in silico as a characteristic protein of apicomplexans that also occurs in the placozoa, Trichoplax adhaerens. Since then, it has been found that apicortin also occurs in free-living cousins of apicomplexans (chromerids) and in flagellated fungi. It contains a partial p25-α domain and a doublecortin (DCX) domain, both of which have tubulin/microtubule binding properties. Apicortin has been studied experimentally in two very important apicomplexan pathogens, Toxoplasma gondii and Plasmodium falciparum. It is localized in the apical complex in both parasites. In T. gondii, apicortin plays a key role in shaping the structure of a special tubulin polymer, conoid. In both parasites, its absence or downregulation has been shown to impair pathogen–host interactions. Based on these facts, it has been suggested as a therapeutic target for treatment of malaria and toxoplasmosis.

scholarly journals Highlighting the Potency of Biosurfactants Produced by Pseudomonas Strains as Anti-Legionella Agents

2018 ◽  
Vol 2018 ◽  
pp. 1-15 ◽  
Author(s):  
Clémence Loiseau ◽  
Emilie Portier ◽  
Marie-Hélène Corre ◽  
Margot Schlusselhuber ◽  
Ségolène Depayras ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Close Association ◽  
Water Systems ◽  
Antagonistic Activity ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Free Living ◽  
Legionnaires Disease ◽  
Culture Supernatants ◽  
Multispecies Biofilms

Legionella pneumophila, the causative agent of Legionnaires’ disease, is a waterborne bacterium mainly found in man-made water systems in close association with free-living amoebae and multispecies biofilms. Pseudomonas strains, originating from various environments including freshwater systems or isolated from hospitalized patients, were tested for their antagonistic activity towards L. pneumophila. A high amount of tested strains was thus found to be active. This antibacterial activity was correlated to the presence of tensioactive agents in culture supernatants. As Pseudomonas strains were known to produce biosurfactants, these compounds were specifically extracted and purified from active strains and further characterized using reverse-phase HPLC and mass spectrometry methods. Finally, all biosurfactants tested (lipopeptides and rhamnolipids) were found active and this activity was shown to be higher towards Legionella strains compared to various other bacteria. Therefore, described biosurfactants are potent anti-Legionella agents that could be used in the water treatment industry although tests are needed to evaluate how effective they would be under field conditions.

scholarly journals Complete Genome Sequence of the Arcobacter halophilus Type Strain CCUG 53805

2018 ◽  
Vol 7 (14) ◽  
Author(s):  
William G. Miller ◽  
Emma Yee ◽  
James L. Bono
Keyword(s):  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Type Strain ◽  
Marine Environments ◽  
Free Living ◽  
Content Type

Many Arcobacter spp. are free living and are routinely recovered from marine environments.

scholarly journals Serratia marcescens ShlA Pore-Forming Toxin Is Responsible for Early Induction of Autophagy in Host Cells and Is Transcriptionally Regulated by RcsB

2014 ◽  
Vol 82 (9) ◽  
pp. 3542-3554 ◽  
Author(s):  
Gisela Di Venanzio ◽  
Tatiana M. Stepanenko ◽  
Eleonora García Véscovi
Keyword(s):  
Serratia Marcescens ◽  
Promoter Region ◽  
Regulatory Mechanism ◽  
Host Cells ◽  
Binding Motif ◽  
Content Type ◽  
Life Threatening ◽  
Opportunistic Human Pathogen ◽  
Flagellar Biogenesis ◽  
Fine Tune

ABSTRACTSerratia marcescensis a Gram-negative bacterium that thrives in a wide variety of ambient niches and interacts with an ample range of hosts. As an opportunistic human pathogen, it has increased its clinical incidence in recent years, being responsible for life-threatening nosocomial infections.S. marcescensproduces numerous exoproteins with toxic effects, including the ShlA pore-forming toxin, which has been catalogued as its most potent cytotoxin. However, the regulatory mechanisms that govern ShlA expression, as well as its action toward the host, have remained unclear. We have shown thatS. marcescenselicits an autophagic response in host nonphagocytic cells. In this work, we determine that the expression of ShlA is responsible for the autophagic response that is promoted prior to bacterial internalization in epithelial cells. We show that a strain unable to express ShlA is no longer able to induce this autophagic mechanism, while heterologous expression of ShlA/ShlB suffices to confer on noninvasiveEscherichia colithe capacity to trigger autophagy. We also demonstrate thatshlBAharbors a binding motif for the RcsB regulator in its promoter region. RcsB-dependent control ofshlBAconstitutes a feed-forward regulatory mechanism that allows interplay with flagellar-biogenesis regulation. At the top of the circuit, activated RcsB downregulates expression of flagella by binding to theflhDCpromoter region, preventing FliA-activated transcription ofshlBA. Simultaneously, RcsB interaction within theshlBApromoter represses ShlA expression. This circuit offers multiple access points to fine-tune ShlA production. These findings also strengthen the case for an RcsB role in orchestrating the expression ofSerratiavirulence factors.

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