scholarly journals PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction ofLegionellainfection

2018 ◽  
Author(s):  
Catherine M. Buckley ◽  
Victoria L. Heath ◽  
Aurélie Guého ◽  
Cristina Bosmani ◽  
Paulina Knobloch ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Host Cells ◽  
Central Component ◽  
Phagosome Maturation ◽  
General Role ◽  
Phagosomal Maturation ◽  
General Importance ◽  
Wide Range ◽  
Opportunistic Human Pathogen

AbstractBy engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyteDictyostelium discoideum.PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation inDictyostelium. Disruption ofPIKfyveinhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently,PIKfyve-cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lackingPIKfyveare more susceptible to infection by the intracellular pathogenLegionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.ImportanceCells that capture or eat bacteria must swiftly kill them to prevent pathogens from surviving long enough to escape the bactericidal pathway and establish an infection. This is achieved by the rapid delivery of components that produce an antimicrobial environment in the phagosome, the compartment containing the captured microbe. This is essential both for the function of immune cells and for amoebae that feed on bacteria in their environment. Here we identify a central component of the pathway used by cells to deliver antimicrobial components to the phagosome and show that bacteria survive over three times as long within the host if this pathway is disabled. We show that this is of general importance for killing a wide range of pathogenic and non-pathogenic bacteria, and that it is physiologically important if cells are to avoid infection by the opportunistic human pathogenLegionella.

scholarly journals The Wnt/β-Catenin Signaling Pathway Controls the Inflammatory Response in Infections Caused by Pathogenic Bacteria

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Octavio Silva-García ◽  
Juan J. Valdez-Alarcón ◽  
Víctor M. Baizabal-Aguirre
Keyword(s):  
Inflammatory Response ◽  
Signaling Pathway ◽  
Bacterial Infections ◽  
Pathogenic Bacteria ◽  
Tissue Injury ◽  
Adaptive Immune Response ◽  
Host Cells ◽  
Wide Range ◽  
The Many ◽  
Canonical Wnt

Innate immunity against pathogenic bacteria is critical to protect host cells from invasion and infection as well as to develop an appropriate adaptive immune response. During bacterial infection, different signaling transduction pathways control the expression of a wide range of genes that orchestrate a number of molecular and cellular events to eliminate the invading microorganisms and regulate inflammation. The inflammatory response must be tightly regulated because uncontrolled inflammation may lead to tissue injury. Among the many signaling pathways activated, the canonical Wnt/β-catenin has been recently shown to play an important role in the expression of several inflammatory molecules during bacterial infections. Our main goal in this review is to discuss the mechanism used by several pathogenic bacteria to modulate the inflammatory response through the Wnt/β-catenin signaling pathway. We think that a deep insight into the role of Wnt/β-catenin signaling in the inflammation may open new venues for biotechnological approaches designed to control bacterial infectious diseases.

Structural basis of ubiquitin recognition by a bacterial OTU deubiquitinase LotA

2021 ◽  
Author(s):  
Norihiro Takekawa ◽  
Tomoko Kubori ◽  
Tomoya Iwai ◽  
Hiroki Nagai ◽  
Katsumi Imada
Keyword(s):  
Enzymatic Activity ◽  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Catalytic Domain ◽  
Docking Simulation ◽  
Effector Proteins ◽  
Host Cells ◽  
Structural Basis ◽  
Ubiquitin Chain ◽  
Lota Lota

Pathogenic bacteria have acquired a vast array of eukaryotic-like proteins via intimate interaction with host cells. Bacterial effector proteins that function as ubiquitin ligases and deubiquitinases (DUBs) are remarkable examples of such molecular mimicry. LotA, a Legionella pneumophila effector, belongs to the ovarian tumor (OTU) superfamily, which regulates diverse ubiquitin signals by their DUB activities. LotA harbors two OTU domains that have distinct reactivities; the first one is responsible for the cleavage of the K6-linked ubiquitin chain, and the second one shows an uncommon preference for long chains of ubiquitin. Here, we report the crystal structure of a middle domain of LotA (LotA M ), which contains the second OTU domain. LotA M consists of two distinct subdomains, a catalytic domain having high structural similarity with human OTU DUBs and an extended helical lobe (EHL) domain, which is characteristically conserved only in Legionella OTU DUBs. The docking simulation of LotA M with ubiquitin suggested that hydrophobic and electrostatic interactions between the EHL of LotA M and the C-terminal region of ubiquitin are crucial for the binding of ubiquitin to LotA M . The structure-based mutagenesis demonstrated that the acidic residue in the characteristic short helical segment termed the ‘helical arm’ is essential for the enzymatic activity of LotA M . The EHL domain of the three Legionella OTU DUBs, LotA, LotB, and LotC, share the ‘helical arm’ structure, suggesting that the EHL domain defines the Lot-OTUs as a unique class of DUBs. Importance To successfully colonize, some pathogenic bacteria hijack the host ubiquitin system. Legionella OTU-like-DUBs (Lot-DUBs) are novel bacterial deubiquitinases found in effector proteins of L. pneumophila . LotA is a member of Lot-DUBs and has two OTU domains (OTU1 and OTU2). We determined the structure of a middle fragment of LotA (LotA M ), which includes OTU2. LotA M consists of the conserved catalytic domain and the Legionella OTUs-specific EHL domain. The docking simulation with ubiquitin and the mutational analysis suggested that the acidic surface in the EHL is essential for enzymatic activity. The structure of the EHL differs from those of other Lot-DUBs, suggesting that the variation of the EHL is related to the variable cleaving specificity of each DUB.

scholarly journals Invasion of Eukaryotic Cells byLegionella Pneumophila: A Common Strategy for all Hosts?

1997 ◽  
Vol 8 (3) ◽  
pp. 139-146 ◽  
Author(s):  
Paul S Hoffman
Keyword(s):  
Legionella Pneumophila ◽  
Human Infection ◽  
Host Cells ◽  
Eukaryotic Cells ◽  
Mature Form ◽  
Cellular Processes ◽  
Wide Range ◽  
Common Strategy ◽  
Legionnaires Disease ◽  
Host Parasite

Legionella pneumophilais an environmental micro-organism capable of producing an acute lobar pneumonia, commonly referred to as Legionnaires’ disease, in susceptible humans. Legionellae are ubiquitous in aquatic environments, where they survive in biofilms or intracellularly in various protozoans. Susceptible humans become infected by breathing aerosols laden with the bacteria. The target cell for human infection is the alveolar macrophage, in which the bacteria abrogate phagolysosomal fusion. The remarkable ability ofL pneumophilato infect a wide range of eukaryotic cells suggests a common strategy that exploits very fundamental cellular processes. The bacteria enter host cells via coiling phagocytosis and quickly subvert organelle trafficking events, leading to formation of a replicative phagosome in which the bacteria multiply. Vegetative growth continues for 8 to 10 h, after which the bacteria develop into a short, highly motile form called the ‘mature form’. The mature form exhibits a thickening of the cell wall, stains red with the Gimenez stain, and is between 10 and 100 times more infectious than agar-grown bacteria. Following host cell lysis, the released bacteria infect other host cells, in which the mature form differentiates into a Gimenez-negative vegetative form, and the cycle begins anew. Virulence ofL pneumophilais considered to be multifactorial, and there is growing evidence for both stage specific and sequential gene expression. Thus,L pneumophilamay be a good model system for dissecting events associated with the host-parasite interactions.

High-throughput assays of phagocytosis, phagosome maturation, and bacterial invasion

2007 ◽  
Vol 292 (2) ◽  
pp. C945-C952 ◽  
Author(s):  
Benjamin E. Steinberg ◽  
Cameron C. Scott ◽  
Sergio Grinstein
Keyword(s):  
High Throughput ◽  
Mammalian Cells ◽  
Pathogenic Bacteria ◽  
Host Cells ◽  
Bacterial Invasion ◽  
Small Interference Rna ◽  
Phagosomal Maturation ◽  
Large Populations ◽  
Intracellular Proliferation ◽  
Efficient Screening

Ingestion of foreign particles by macrophages and neutrophils and the fate of the vacuole that contains the ingested material are generally monitored by optical microscopy. Invasion of host cells by pathogenic bacteria and their intracellular proliferation are similarly studied by microscopy or by plating assays. These labor-intensive and time-consuming methods limit the number of assays that can be performed. The effort required to test multiple reagents or conditions can be prohibitive. We describe high-throughput assays of phagocytosis and of phagosomal maturation. An automated fluorescence microscope-based platform and associated analysis software were used to study Fcγ receptor-mediated phagocytosis of IgG-opsonized particles by cultured murine macrophages. Phagosomal acidification was measured as an index of maturation. The same platform was similarly used to implement high-throughput assays of invasion of mammalian cells by pathogenic bacteria. The invasion of HeLa cells by Salmonella and the subsequent intracellular proliferation of the bacteria were measured rapidly and reliably in large populations of cells. These high-throughput methods are ideally suited for the efficient screening of chemical libraries to select potential drugs and of small interference RNA libraries to identify essential molecules involved in critical steps of the immune response.

scholarly journals Lipopolysaccharide of Legionella pneumophila shed in a liquid culture as a nonvesicular fraction arrests phagosome maturation in amoeba and monocytic host cells

2010 ◽  
Vol 307 (2) ◽  
pp. 113-119 ◽  
Author(s):  
Eva M. Seeger ◽  
Maria Thuma ◽  
Esteban Fernandez-Moreira ◽  
Enno Jacobs ◽  
Marc Schmitz ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Liquid Culture ◽  
Host Cells ◽  
Phagosome Maturation

scholarly journals The structural complexity of the Gammaproteobacteria flagellar motor is related to the type of its torque-generating stators

2018 ◽  
Author(s):  
Mohammed Kaplan ◽  
Debnath Ghosal ◽  
Poorna Subramanian ◽  
Catherine M. Oikonomou ◽  
Andreas Kjær ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Cell Envelope ◽  
Shewanella Oneidensis ◽  
Structural Complexity ◽  
High Viscosity ◽  
Flagellar Motor ◽  
Flagellar Motors ◽  
Wide Range ◽  
A Cell

AbstractThe bacterial flagellar motor is a cell-envelope-embedded macromolecular machine that functions as a propeller to move the cell. Rather than being an invariant machine, the flagellar motor exhibits significant variability between species, allowing bacteria to adapt to, and thrive in, a wide range of environments. For instance, different torque-generating stator modules allow motors to operate in conditions with different pH and sodium concentrations and some motors are adapted to drive motility in high-viscosity environments. How such diversity evolved is unknown. Here we use electron cryo-tomography to determine thein situmacromolecular structures of the flagellar motors of three Gammaproteobacteria species:Legionella pneumophila,Pseudomonas aeruginosa, andShewanella oneidensisMR-1, providing the first views of intact motors with dual stator systems. Complementing our imaging with bioinformatics analysis, we find a correlation between the stator system of the motor and its structural complexity. Motors with a single H+-driven stator system have only the core P- and L-rings in their periplasm; those with dual H+-driven stator systems have an extra component elaborating their P-ring; and motors with Na+- (or dual Na+-H+)- driven stator systems have additional rings surrounding both their P- and L-rings. Our results suggest an evolution of structural complexity that may have enabled pathogenic bacteria likeL. pneumophilaandP. aeruginosato colonize higher-viscosity environments in animal hosts.

scholarly journals Components of the Legionella pneumophila Flagellar Regulon Contribute to Multiple Virulence Traits, Including Lysosome Avoidance and Macrophage Death

2005 ◽  
Vol 73 (9) ◽  
pp. 5720-5734 ◽  
Author(s):  
A. B. Molofsky ◽  
L. M. Shetron-Rama ◽  
Michele S. Swanson
Keyword(s):  
Legionella Pneumophila ◽  
Sigma Factor ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Phagosome Maturation ◽  
Multiple Traits ◽  
Murine Bone Marrow ◽  
Flagellar Regulon ◽  
Loss Of Contact ◽  
Aquatic Pathogen

ABSTRACT Legionella pneumophila is a motile intracellular pathogen of macrophages and amoebae. When nutrients become scarce, the bacterium induces expression of transmission traits, some of which are dependent on the flagellar sigma factor FliA (σ28). To test how particular components of the L. pneumophila flagellar regulon contribute to virulence, we compared a fliA mutant with strains whose flagellar construction is disrupted at various stages. We find that L. pneumophila requires FliA to avoid lysosomal degradation in murine bone marrow-derived macrophages (BMM), to regulate production of a melanin-like pigment, and to regulate binding to the dye crystal violet, whereas motility, flagellar secretion, and external flagella or flagellin are dispensable for these activities. Thus, in addition to flagellar genes, the FliA sigma factor regulates an effector(s) or regulator(s) that contributes to other transmissive traits, notably inhibition of phagosome maturation. Whether or not the microbes produced flagellin, all nonmotile L. pneumophila mutants bound BMM less efficiently than the wild type, resulting in poor infectivity and a loss of contact-dependent death of BMM. Therefore, bacterial motility increases contact with host cells during infection, but flagellin is not an adhesin. When BMM contact by each nonmotile strain was promoted by centrifugation, all the mutants bound BMM similarly, but only those microbes that synthesized flagellin induced BMM death. Thus, the flagellar regulon equips the aquatic pathogen L. pneumophila to coordinate motility with multiple traits vital to virulence.

scholarly journals Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex

2004 ◽  
Vol 72 (10) ◽  
pp. 5983-5992 ◽  
Author(s):  
Jessica A. Sexton ◽  
Jennifer L. Miller ◽  
Aki Yoneda ◽  
Thomas E. Kehl-Fie ◽  
Joseph P. Vogel
Keyword(s):  
Cell Surface ◽  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Wide Distribution ◽  
Host Cells ◽  
Growth Defect ◽  
Intracellular Growth ◽  
Homologous Proteins ◽  
Type Iv ◽  
Cell Surface Structure

ABSTRACT Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotU ΔicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotU ΔicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

scholarly journals One-step preparation of antimicrobial silicone materials based on PDMS and salicylic acid: insights from spatially and temporally resolved techniques

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Luca Barbieri ◽  
Ioritz Sorzabal Bellido ◽  
Alison J. Beckett ◽  
Ian A. Prior ◽  
Jo Fothergill ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Laser Scanning ◽  
Pathogenic Bacteria ◽  
Pharmaceutical Products ◽  
Laser Scanning Microscopy ◽  
Wound Dressings ◽  
Confocal Laser ◽  
Vis Spectroscopy ◽  
Wide Range ◽  
One Step

AbstractIn this work, we introduce a one-step strategy that is suitable for continuous flow manufacturing of antimicrobial PDMS materials. The process is based on the intrinsic capacity of PDMS to react to certain organic solvents, which enables the incorporation of antimicrobial actives such as salicylic acid (SA), which has been approved for use in humans within pharmaceutical products. By combining different spectroscopic and imaging techniques, we show that the surface properties of PDMS remain unaffected while high doses of the SA are loaded inside the PDMS matrix. The SA can be subsequently released under physiological conditions, delivering a strong antibacterial activity. Furthermore, encapsulation of SA inside the PDMS matrix ensured a diffusion-controlled release that was tracked by spatially resolved Raman spectroscopy, Attenuated Total Reflectance IR (ATR-IR), and UV-Vis spectroscopy. The biological activity of the new material was evaluated directly at the surface and in the planktonic state against model pathogenic bacteria, combining confocal laser scanning microscopy, electron microscopy, and cell viability assays. The results showed complete planktonic inhibition for clinically relevant strains of Staphylococcus aureus and Escherichia coli, and a reduction of up to 4 orders of magnitude for viable sessile cells, demonstrating the efficacy of these surfaces in preventing the initial stages of biofilm formation. Our approach adds a new option to existing strategies for the antimicrobial functionalisation of a wide range of products such as catheters, wound dressings and in-dwelling medical devices based on PDMS.

scholarly journals A surrogate virus neutralization test to quantify antibody-mediated inhibition of SARS-CoV-2 in finger stick dried blood spot samples

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Amelia E. Sancilio ◽  
Richard T. D’Aquila ◽  
Elizabeth M. McNally ◽  
Matthew P. Velez ◽  
Michael G. Ison ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Venous Blood ◽  
Host Cells ◽  
Dried Blood Spot ◽  
Blood Collection ◽  
Widespread Application ◽  
Live Virus ◽  
Dilution Series ◽  
Wide Range ◽  
Blood Spot

AbstractThe spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample. Samples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV). Dilution series produced the expected pattern of dose–response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % surrogate neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples. Large-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying inhibition of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations.

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