scholarly journals Attenuated Endocytosis and Toxicity of a Mutant Cholera Toxin with Decreased Ability To Cluster Ganglioside GM1 Molecules

2008 ◽  
Vol 76 (4) ◽  
pp. 1476-1484 ◽  
Author(s):  
Anne A. Wolf ◽  
Michael G. Jobling ◽  
David E. Saslowsky ◽  
Eli Kern ◽  
Kimberly R. Drake ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Host Cells ◽  
Wild Type ◽  
Intact Cells ◽  
B Subunit ◽  
Binding Pockets ◽  
Detergent Resistant Membranes ◽  
Toxin Uptake ◽  
Chimeric Toxins

ABSTRACT Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM1. The homopentomeric B-subunit of the toxin can bind up to five GM1 molecules at once. Here, we examined the role of polyvalent binding of GM1 in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM1. The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM1 when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM1 molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM1 molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.

Role of the β1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia

2002 ◽  
Vol 115 (13) ◽  
pp. 2669-2678 ◽  
Author(s):  
Anna Gustavsson ◽  
Annika Armulik ◽  
Cord Brakebusch ◽  
Reinhard Fässler ◽  
Staffan Johansson ◽  
...  
Keyword(s):  
Marked Reduction ◽  
Yersinia Pseudotuberculosis ◽  
Host Cells ◽  
Β1 Integrin ◽  
Complex Structures ◽  
Wild Type ◽  
Similar Reduction ◽  
Focal Complex ◽  
Β1 Integrins

Invasin of Yersinia pseudotuberculosis binds to β1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the β1-integrin-mediated internalization of Yersinia, a β1-integrin-deficient cell line, GD25, transfected with wild-type β1A, β1B or different mutants of the β1A subunit was used. Both β1A and β1B bound to invasin-expressing bacteria, but only β1A was able to mediate internalization of the bacteria. The cytoplasmic region of β1A, differing from β1B, contains two NPXY motifs surrounding a double threonine site. Exchanging the tyrosines of the two NPXYs to phenylalanines did not inhibit the uptake, whereas a marked reduction was seen when the first tyrosine (Y783) was exchanged to alanine. A similar reduction was seen when the two nearby threonines (TT788-9) were exchanged with alanines. It was also noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex structures was unable to mediate uptake of invasin-expressing bacteria.

scholarly journals Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Susmita Ghosh ◽  
Elizabeth A. Ruelke ◽  
Joshua C. Ferrell ◽  
Maria D. Bodero ◽  
Kenneth A. Fields ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Host Cells ◽  
Actin Binding ◽  
Wild Type ◽  
Content Type ◽  
Allelic Exchange ◽  
Binding Domains

ABSTRACT The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

OmpD but not OmpC is involved in adherence ofSalmonella entericaserovar Typhimurium to human cells

2004 ◽  
Vol 50 (9) ◽  
pp. 719-727 ◽  
Author(s):  
Bochiwe Hara-Kaonga ◽  
Thomas G Pistole
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Human Cells ◽  
Host Cells ◽  
Intestinal Epithelial ◽  
Wild Type ◽  
Human Macrophages ◽  
Significant Difference ◽  
Serovar Typhimurium

Conflicting reports exist regarding the role of porins OmpC and OmpD in infections due to Salmonella enterica serovar Typhimurium. This study investigated the role of these porins in bacterial adherence to human macrophages and intestinal epithelial cells. ompC and ompD mutant strains were created by transposon mutagenesis using P22-mediated transduction of Tn10 and Tn5 insertions, respectively, into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with host cells at various bacteria to cell ratios for 1 h at 37 °C and analyzed by flow cytometry. The mean fluorescence intensity of cells with associated wild-type and mutant bacteria was used to estimate the number of bacteria bound per host cell. Adherence was also measured by fluorescence microscopy. Neither assay showed a significant difference in binding of the ompC mutant and wild-type strains to the human cells. In contrast, the ompD mutant exhibited lowered binding to both cell types. Our findings suggest that OmpD but not OmpC is involved in the recognition of Salmonella serovar Typhimurium by human macrophages and intestinal epithelial cells.Key words: Salmonella, adherence, porins, intestinal epithelial cells, macrophage.

scholarly journals Role of Adhesin Release for Mucosal Colonization by a Bacterial Pathogen

2003 ◽  
Vol 197 (6) ◽  
pp. 735-742 ◽  
Author(s):  
Loïc Coutte ◽  
Sylvie Alonso ◽  
Nathalie Reveneau ◽  
Eve Willery ◽  
Brigitte Quatannens ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Wild Type ◽  
Early Step ◽  
Bacterial Surface ◽  
Extracellular Milieu ◽  
Wild Type Parent

Pathogen attachment is a crucial early step in mucosal infections. This step is mediated by important virulence factors called adhesins. To exert these functions, adhesins are typically surface-exposed, although, surprisingly, some are also released into the extracellular milieu, the relevance of which has previously not been studied. To address the role of adhesin release in pathogenesis, we used Bordetella pertussis as a model, since its major adhesin, filamentous hemagglutinin (FHA), partitions between the bacterial surface and the extracellular milieu. FHA release depends on its maturation by the specific B. pertussis protease SphB1. We constructed SphB1-deficient mutants and found that they were strongly affected in their ability to colonize the mouse respiratory tract, although they adhered even better to host cells in vitro than their wild-type parent strain. The defect in colonization could be overcome by prior nasal instillation of purified FHA or by coinfection with FHA-releasing B. pertussis strains, but not with SphB1-producing FHA-deficient strains, ruling out a nonspecific effect of SphB1. These results indicate that the release of FHA is important for colonization, as it may facilitate the dispersal of bacteria from microcolonies and the binding to new sites in the respiratory tract.

scholarly journals Differential Biological and Adjuvant Activities of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin Hybrids

2001 ◽  
Vol 69 (3) ◽  
pp. 1528-1535 ◽  
Author(s):  
Christal C. Bowman ◽  
John D. Clements
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Cholera Toxin ◽  
The Other ◽  
Toxin Gene ◽  
Wild Type ◽  
B Subunit ◽  
Heat Labile ◽  
A Subunit ◽  
Ifn Γ

ABSTRACT Two bacterial products that have been demonstrated to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique. The goal of this study was to determine the basis for these biological differences by constructing and characterizing chimeric CT-LT molecules. Toxin gene fragments were subcloned to create two constructs, each expressing the enzymatically active A subunit of one toxin and the receptor binding B subunit of the other toxin. These hybrid toxins were purified, and the composition and assembly of CT A subunit (CT-A)-LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confirmed. Hybrids were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxin was assessed in a patent-mouse assay. The results demonstrated that LT-A–CT-B induces the accumulation of lower levels of cyclic AMP and has less enterotoxicity than either wild-type toxin or the other hybrid. Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with tetanus toxoid for intranasal immunization of BALB/c mice. Importantly, the ability of LT to induce a type 1 cytokine response was found to be a function of LT-A. Specifically, LT-A–CT-B was able to augment the levels of antigen-specific gamma interferon (IFN-γ) and interleukin 5 to levels comparable to those achieved with native LT, while CT-A–LT-B and native CT both produced lower levels of antigen-specific IFN-γ. Thus, these toxin hybrids possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.

scholarly journals Legionella pneumophila Entry GenertxA Is Involved in Virulence

2001 ◽  
Vol 69 (1) ◽  
pp. 508-517 ◽  
Author(s):  
Suat L. G. Cirillo ◽  
Luiz E. Bermudez ◽  
Sahar H. El-Etr ◽  
Gerald E. Duhamel ◽  
Jeffrey D. Cirillo
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Integrin Receptors ◽  
Legionnaires Disease ◽  
Cell Spread ◽  
Gain Access

ABSTRACT Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogen Legionella pneumophila. Currently, the mechanisms used by L. pneumophila to gain access to the intracellular environment are not well understood. We have recently isolated three loci, designated enh1,enh2, and enh3, that are involved in the ability of L. pneumophila to enter host cells. One of the genes present in the enh1 locus, rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to β2 integrin receptors. In the current study, we demonstrate that the L. pneumophila rtxA gene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, an rtxA mutant does not replicate as well as wild-type L. pneumophila in monocytes and is less virulent in mice. Thus, we conclude that the entry genertxA is an important virulence determinant in L. pneumophila and is likely to be critical for the production of Legionnaires' disease in humans.

scholarly journals Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity.

1992 ◽  
Vol 119 (5) ◽  
pp. 1297-1307 ◽  
Author(s):  
M J Levis ◽  
H R Bourne
Keyword(s):  
Adenylyl Cyclase ◽  
Cholera Toxin ◽  
Soluble Fraction ◽  
Covalent Modification ◽  
Alpha Subunit ◽  
Wild Type ◽  
Hormonal Stimulation ◽  
Intact Cells ◽  
Cell Extracts ◽  
Membrane Bound

Binding of GTP induces alpha subunits of heterotrimeric G proteins to take on an active conformation, capable of regulating effector molecules. We expressed epitope-tagged versions of the alpha subunit (alpha s) of Gs in genetically alpha s-deficient S49 cyc- cells. Addition of a hemagglutinin (HA) epitope did not alter the ability of wild type alpha s to mediate hormonal stimulation of adenylyl cyclase or to attach to cell membranes. The HA epitope did, however, allow a mAb to immunoprecipitate the recombinant protein (HA-alpha s) quantitatively from cell extracts. We activated the epitope-tagged alpha s in intact cells by: (a) exposure of cells to cholera toxin, which activates alpha s by covalent modification; (b) mutational replacement of arginine-201 in HA-alpha s by a cysteine residue, to create HA-alpha s-R201C; like the cholera toxin-catalyzed modification, this mutation activates alpha s by slowing its intrinsic GTPase activity; and (c) treatment of cells with the beta-adrenoceptor agonist, isoproterenol, which promotes binding of GTP to alpha s, thereby activating adenylyl cyclase. Both cholera toxin and the R201C mutation accelerated the rate of degradation of alpha s (0.03 h-1) by three- to fourfold and induced a partial shift of the protein from a membrane bound to a soluble compartment. At steady state, 80% of HA-alpha s- R201C was found in the soluble fraction, as compared to 10% of wild type HA-alpha s. Isoproterenol rapidly (in < 2 min) caused 20% of HA-alpha s to shift from the membrane-bound to the soluble compartment. Cholera toxin induced a 3.5-fold increase in the rate of degradation of a second mutant, HA-alpha s-G226A, but did not cause it to move into the soluble fraction; this observation shows that loss of membrane attachment is not responsible for the accelerated degradation of alpha s in response to activation. Taken together, these findings show that activation of alpha s induces a conformational change that loosens its attachment to membranes and increases its degradation rate.

scholarly journals Revisiting the role of Toxoplasma gondii ERK7 in the maintenance and stability of the apical complex

2021 ◽  
Author(s):  
Dominique Soldati-Favre ◽  
Nicolas Dos Santos Pacheco ◽  
Nicolò Tosetti ◽  
Aarti Krishnan ◽  
Romuald Haase
Keyword(s):  
Toxoplasma Gondii ◽  
Comparative Proteomics ◽  
Host Cells ◽  
Wild Type ◽  
Proteomics Analysis ◽  
Polar Ring ◽  
Intracellular Parasites ◽  
Apical Complex ◽  
Microneme Proteins

Toxoplasma gondii ERK7 is known to contribute to the integrity of the apical complex and to be involved only in the final step of the conoid biogenesis. In the absence of ERK7, mature parasites lose their conoid complex and are unable to glide, invade or egress from host cells. In contrast to a previous report, we show here that depletion of ERK7 phenocopies the depletion of the apical cap proteins AC9 or AC10. The absence of ERK7 leads to the loss of the apical polar ring, the disorganization of the basket of subpellicular microtubules and an impairment in micronemes secretion. Ultra-expansion microscopy (U-ExM) coupled to NHS-Ester staining on intracellular parasites offers an unprecedented level of resolution and highlights the disorganization of the rhoptries as well as the dilated plasma membrane at the apical pole in the absence of ERK7. Comparative proteomics analysis of wild-type and ERK7 or AC9 depleted parasites led to the disappearance of known, predicted, as well as putative novel components of the apical complex. In contrast, the absence of ERK7 led to an accumulation of microneme proteins, resulting from the defect in exocytosis of the organelles.

scholarly journals Role of Aspergillus fumigatus DvrA in Host Cell Interactions and Virulence

2010 ◽  
Vol 9 (10) ◽  
pp. 1432-1440 ◽  
Author(s):  
Daniele E. Ejzykowicz ◽  
Norma V. Solis ◽  
Fabrice N. Gravelat ◽  
Josee Chabot ◽  
Xuexian Li ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Type Strain ◽  
Wild Type Strain ◽  
Host Cells ◽  
Epithelial Cell Line ◽  
Wild Type ◽  
Content Type ◽  
Pulmonary Epithelial Cell

ABSTRACT The transcription factors that regulate Aspergillus fumigatus interactions with host cells and virulence are incompletely defined. We investigated the role of the putative C2H2 transcription factor DvrA in governing these processes. Although DvrA was identified by its limited homology to Candida albicans Bcr1, a ΔdvrA mutant strain of A. fumigatus had wild-type adherence to host constituents in vitro. However, it had increased capacity to damage both endothelial cells and a pulmonary epithelial cell line compared to the ability of the wild-type strain and a ΔdvrA::dvrA-complemented strain. This increase in damage required direct contact between the mutant and host cells. The ΔdvrA mutant also stimulated greater CCL20, interleukin-8, and tumor necrosis factor mRNA expression in a pulmonary epithelial cell line compared to levels induced by the control strains. Also, it was resistant to nikkomycin Z, suggesting an altered cell wall composition. As predicted by these in vitro results, the ΔdvrA mutant had increased virulence and stimulated a greater pulmonary inflammatory response than the wild-type strain and ΔdvrA::dvrA-complemented strains in the nonneutropenic mouse model of invasive pulmonary aspergillosis. These results indicate that DvrA influences A. fumigatus virulence as well as its capacity to damage host cells and stimulate a proinflammatory response.

scholarly journals Activation of GPR37 in macrophages confers protection against infection-induced sepsis and pain-like behaviour in mice

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sangsu Bang ◽  
Christopher R. Donnelly ◽  
Xin Luo ◽  
Maria Toro-Moreno ◽  
Xueshu Tao ◽  
...  
Keyword(s):  
Plasmodium Berghei ◽  
Bacterial Infections ◽  
Protective Role ◽  
Host Cells ◽  
Biological Functions ◽  
Wild Type ◽  
Neuroprotectin D1 ◽  
Protective Actions ◽  
Protection Against Infection

AbstractGPR37 was discovered more than two decades ago, but its biological functions remain poorly understood. Here we report a protective role of GPR37 in multiple models of infection and sepsis. Mice lacking Gpr37 exhibited increased death and/or hypothermia following challenge by lipopolysaccharide (LPS), Listeria bacteria, and the mouse malaria parasite Plasmodium berghei. Sepsis induced by LPS and Listeria in wild-type mice is protected by artesunate (ARU) and neuroprotectin D1 (NPD1), but the protective actions of these agents are lost in Gpr37−/− mice. Notably, we found that ARU binds to GPR37 in macrophages and promotes phagocytosis and clearance of pathogens. Moreover, ablation of macrophages potentiated infection, sepsis, and their sequelae, whereas adoptive transfer of NPD1- or ARU-primed macrophages reduced infection, sepsis, and pain-like behaviors. Our findings reveal physiological actions of ARU in host cells by activating macrophages and suggest that GPR37 agonists may help to treat sepsis, bacterial infections, and malaria.

Sign in / Sign up

Export Citation Format

Share Document