scholarly journals How bacterial effectors hijack small GTPases to divert membrane traffic

2014 ◽  
Vol 70 (a1) ◽  
pp. C802-C802
Author(s):  
Jacqueline Cherfils ◽  
Marcia Folly-Klan ◽  
Valérie Campanacci
Keyword(s):  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Membrane Traffic ◽  
Severe Pneumonia ◽  
Small Gtpases ◽  
Covalent Attachment ◽  
Rab Gtpase ◽  
Postal Service ◽  
Nucleotide Exchange ◽  
Vesicular Traffic

Membrane traffic, which is the "cellular postal service" that shuttles biomolecules around the cell and organizes the structure of organelles, is among the primary targets of effectors injected by intracellular pathogenic bacteria to invade their host and avoid from being destroyed. I will present our recent structural and biochemical studies of effectors from Legionella pneumophila (the bacteria that causes the legionnaire's disease, a severe pneumonia) that divert membrane traffic to generate a membrane-bound vacuole where the pathogen hides and replicates. One of these effectors, AnkX, is a FIC domain-containing toxin that alters the functions of a Rab GTPase involved in vesicular traffic at the endoplasmic reticulum, by covalent attachment of a phosphocholine molecule. The other one, RalF, functions as an illegitimate guanine nucleotide exchange factor to activate an Arf GTPase on the vacuole. Our studies showed how AnkX binds and processes CDP-choline to transfer phosphocholine onto Rab1 [1], and uncover a novel membrane sensor in RalF that controls its localization and activity [2]

scholarly journals More than 18,000 effectors in theLegionellagenus genome provide multiple, independent combinations for replication in human cells

2019 ◽  
Vol 116 (6) ◽  
pp. 2265-2273 ◽  
Author(s):  
Laura Gomez-Valero ◽  
Christophe Rusniok ◽  
Danielle Carson ◽  
Sonia Mondino ◽  
Ana Elena Pérez-Cobas ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Chromatin Modification ◽  
Severe Pneumonia ◽  
Rab Gtpase ◽  
Functional Screening ◽  
Evolutionary Analysis ◽  
Human Pathogen ◽  
Human Pathogens ◽  
Intracellular Replication ◽  
Type Iv

The genusLegionellacomprises 65 species, among whichLegionella pneumophilais a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80Legionellagenomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes andLegionella. Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted toL. pneumophilaand seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens.

scholarly journals Exploitation of Phosphoinositides by the Intracellular Pathogen, Legionella pneumophila

2020 ◽  
Author(s):  
Colleen M. Pike ◽  
Rebecca R. Noll ◽  
M. Ramona Neunuebel
Keyword(s):  
Membrane Trafficking ◽  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Severe Pneumonia ◽  
Bacterial Proteins ◽  
Specific Host ◽  
Spatiotemporal Regulation ◽  
Host Membrane ◽  
Membrane Compartments ◽  
Legionnaires Disease

Manipulation of host phosphoinositide lipids has emerged as a key survival strategy utilized by pathogenic bacteria to establish and maintain a replication-permissive compartment within eukaryotic host cells. The human pathogen, Legionella pneumophila, infects and proliferates within the lung’s innate immune cells causing severe pneumonia termed Legionnaires’ disease. This pathogen has evolved strategies to manipulate specific host components to construct its intracellular niche termed the Legionella-containing vacuole (LCV). Paramount to LCV biogenesis and maintenance is the spatiotemporal regulation of phosphoinositides, important eukaryotic lipids involved in cell signaling and membrane trafficking. Through a specialized secretion system, L. pneumophila translocates multiple proteins that target phosphoinositides in order to escape endolysosomal degradation. By specifically binding phosphoinositides, these proteins can anchor to the cytosolic surface of the LCV or onto specific host membrane compartments, to ultimately stimulate or inhibit encounters with host organelles. Here, we describe the bacterial proteins involved in binding and/or altering host phosphoinositide dynamics to support intracellular survival of L. pneumophila.

scholarly journals Dot/Icm Effector Translocation by Legionella longbeachae Creates a Replicative Vacuole Similar to That of Legionella pneumophila despite Translocation of Distinct Effector Repertoires

2015 ◽  
Vol 83 (10) ◽  
pp. 4081-4092 ◽  
Author(s):  
Rebecca E. Wood ◽  
Patrice Newton ◽  
Eleanor A. Latomanski ◽  
Hayley J. Newton
Keyword(s):  
Legionella Pneumophila ◽  
A549 Cells ◽  
Severe Pneumonia ◽  
Rab Gtpase ◽  
Human Pathogens ◽  
Immunofluorescence Analysis ◽  
Content Type ◽  
Extensive Analysis ◽  
Legionnaires Disease ◽  
Environmental Bacteria

Legionellaorganisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed theLegionella-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis ofLegionella pneumophila. Here, we have characterized theLegionella longbeachaereplicative vacuole (longbeachae-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV ofL. pneumophila(pneumophila-LCV). We constructed a Dot/Icm-deficient strain by deletingdotBand demonstrated the inability of this mutant to replicate inside THP-1 cells.L. longbeachaedoes not enter THP-1 cells as efficiently asL. pneumophila, and this is reflected in the observation that translocation of BlaM-RalFLLO(where RalFLLOis theL. longbeachaehomologue of RalF) into THP-1 cells by theL. longbeachaeDot/Icm system is less efficient than that byL. pneumophila. This difference is negated in A549 cells whereL. longbeachaeandL. pneumophilainfect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, theRab-likeeffector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on thelongbeachae-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to thelongbeachae-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of theL. longbeachaereplicative vacuole, highlighting phenotypic similarities to the vacuole ofL. pneumophilaas well as unique aspects of LCV biology.

scholarly journals Early Detection of Legionella pneumophila and Aspergillus by mNGS in a Critically Ill Patient With Legionella Pneumonia After Extracorporeal Membrane Oxygenation Treatment: Case Report and Literature Review

2021 ◽  
Vol 8 ◽  
Author(s):  
Ruiming Yue ◽  
Xiaoxiao Wu ◽  
Tianlong Li ◽  
Li Chang ◽  
Xiaobo Huang ◽  
...  
Keyword(s):  
Extracorporeal Membrane Oxygenation ◽  
Antibiotic Treatment ◽  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Early Stage ◽  
Clinical Manifestations ◽  
Severe Pneumonia ◽  
Detection Methods ◽  
Critically Ill Patient ◽  
Membrane Oxygenation

Legionella pneumophila can cause pneumonia, leading to severe acute respiratory distress syndrome (ARDS). Because of its harsh growth requirements, limited detection methods, and non-specific clinical manifestations, diagnosing Legionella pneumonia remains still challenging. Metagenomic next-generation sequencing (mNGS) technology has increased the rate of detection of Legionella. This study describes a patient who rapidly progressed to severe ARDS during the early stage of infection and was treated with extracorporeal membrane oxygenation (ECMO). Although his bronchoalveolar lavage fluid (BALF) was negative for infection and his serum was negative for anti-Legionella antibody, mNGS of his BALF and blood showed only the presence of Legionella pneumophila (blood mNGS reads 229, BALF reads 656). After antibiotic treatment and weaning from ECMO, however, he developed a secondary Aspergillus and Klebsiella pneumoniae infection as shown by mNGS. Mechanical ventilation and antibiotic treatment were effective. A search of PubMed showed few reports of secondary Aspergillus infections after Legionella infection. Severe pneumonia caused by any type of pathogenic bacteria may be followed by Aspergillus infection, sometimes during extremely early stages of infection. Patients with severe pneumonia caused by Legionella infection should undergo early screening for secondary infections using methods such as mNGS, enabling early and precise treatment, thereby simplifying the use of antibiotics and improving patient prognosis.

scholarly journals Stochastic activation and bistability in a Rab GTPase regulatory network

2020 ◽  
Vol 117 (12) ◽  
pp. 6540-6549
Author(s):  
Urban Bezeljak ◽  
Hrushikesh Loya ◽  
Beata Kaczmarek ◽  
Timothy E. Saunders ◽  
Martin Loose
Keyword(s):  
Regulatory Network ◽  
Regulatory Networks ◽  
Membrane Surface ◽  
Activity Patterns ◽  
Small Gtpases ◽  
Signaling Networks ◽  
Rab Gtpase ◽  
Endomembrane System ◽  
In Vitro Reconstitution

The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell.

scholarly journals TBC-2 Regulates RAB-5/RAB-7-mediated Endosomal Trafficking inCaenorhabditis elegans

2010 ◽  
Vol 21 (13) ◽  
pp. 2285-2296 ◽  
Author(s):  
Laëtitia Chotard ◽  
Ashwini K. Mishra ◽  
Marc-André Sylvain ◽  
Simon Tuck ◽  
David G. Lambright ◽  
...  
Keyword(s):  
Rab Gtpase ◽  
Endosomal Trafficking ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Late Endosomes ◽  
Arginine Finger ◽  
Endosome Maturation ◽  
Hops Complex

During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7–positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(−) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(−) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.

scholarly journals The DHR1 Domain of DOCK180 Binds to SNX5 and Regulates Cation-independent Mannose 6-phosphate Receptor Transport

2008 ◽  
Vol 19 (9) ◽  
pp. 3823-3835 ◽  
Author(s):  
Shigeo Hara ◽  
Etsuko Kiyokawa ◽  
Shun-ichiro Iemura ◽  
Tohru Natsume ◽  
Thomas Wassmer ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Retrograde Transport ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Sorting Nexins ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Mannose 6 Phosphate

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

scholarly journals Hijacking Components of the Cellular Secretory Pathway for Replication of Poliovirus RNA

2006 ◽  
Vol 81 (2) ◽  
pp. 558-567 ◽  
Author(s):  
George A. Belov ◽  
Nihal Altan-Bonnet ◽  
Gennadiy Kovtunovych ◽  
Catherine L. Jackson ◽  
Jennifer Lippincott-Schwartz ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Brefeldin A ◽  
Rna Replication ◽  
Bound State ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Virus Growth ◽  
Guanine Nucleotide Exchange ◽  
Membrane Reorganization

ABSTRACT Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase ∼4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.

scholarly journals CYK-4 regulates Rac, but not Rho, during cytokinesis

2017 ◽  
Vol 28 (9) ◽  
pp. 1258-1270 ◽  
Author(s):  
Yelena Zhuravlev ◽  
Sophia M. Hirsch ◽  
Shawn N. Jordan ◽  
Julien Dumont ◽  
Mimi Shirasu-Hiza ◽  
...  
Keyword(s):  
Alternative Model ◽  
Single Cell Analysis ◽  
Myosin Ii ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Filamentous Actin ◽  
Contractile Ring ◽  
Division Plane ◽  
Ring Constriction

Cytokinesis is driven by constriction of an actomyosin contractile ring that is controlled by Rho-family small GTPases. Rho, activated by the guanine-nucleotide exchange factor ECT-2, is upstream of both myosin-II activation and diaphanous formin-mediated filamentous actin (f-actin) assembly, which drive ring constriction. The role for Rac and its regulators is more controversial, but, based on the finding that Rac inactivation can rescue cytokinesis failure when the GTPase-activating protein (GAP) CYK-4 is disrupted, Rac activity was proposed to be inhibitory to contractile ring constriction and thus specifically inactivated by CYK-4 at the division plane. An alternative model proposes that Rac inactivation generally rescues cytokinesis failure by reducing cortical tension, thus making it easier for the cell to divide when ring constriction is compromised. In this alternative model, CYK-4 was instead proposed to activate Rho by binding ECT-2. Using a combination of time-lapse in vivo single-cell analysis and Caenorhabditis elegans genetics, our evidence does not support this alternative model. First, we found that Rac disruption does not generally rescue cytokinesis failure: inhibition of Rac specifically rescues cytokinesis failure due to disruption of CYK-4 or ECT-2 but does not rescue cytokinesis failure due to disruption of two other contractile ring components, the Rho effectors diaphanous formin and myosin-II. Second, if CYK-4 regulates cytokinesis through Rho rather than Rac, then CYK-4 inhibition should decrease levels of downstream targets of Rho. Inconsistent with this, we found no change in the levels of f-actin or myosin-II at the division plane when CYK-4 GAP activity was reduced, suggesting that CYK-4 is not upstream of ECT-2/Rho activation. Instead, we found that the rescue of cytokinesis in CYK-4 mutants by Rac inactivation was Cdc42 dependent. Together our data suggest that CYK-4 GAP activity opposes Rac (and perhaps Cdc42) during cytokinesis.

scholarly journals Bacterial Guanine Nucleotide Exchange Factors SopE-Like and WxxxE Effectors

2010 ◽  
Vol 78 (4) ◽  
pp. 1417-1425 ◽  
Author(s):  
Richard Bulgin ◽  
Benoit Raymond ◽  
James A. Garnett ◽  
Gad Frankel ◽  
Valerie F. Crepin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rho Gtpases ◽  
Shigella Flexneri ◽  
Small Gtpases ◽  
Effector Proteins ◽  
Actin Dynamics ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
T3ss Effector

ABSTRACT Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce “the trigger mechanism of cell entry.” Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection.

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