Intrabody-Induced Cell Death by Targeting the T. brucei Cytoskeletal Protein Tb BILBO1

Trypanosoma brucei belongs to a group of important zoonotic parasites. We investigated how these organisms develop their cytoskeleton (the internal skeleton that controls cell shape) and focused on an essential protein (BILBO1) first described in T. brucei .

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TolC-Dependent Modulation of Host Cell Death by the Francisella tularensis Live Vaccine Strain

Infection and Immunity ◽

10.1128/iai.00044-14 ◽

2014 ◽

Vol 82 (5) ◽

pp. 2068-2078 ◽

Cited By ~ 16

Author(s):

Christopher R. Doyle ◽

Ji-An Pan ◽

Patricio Mena ◽

Wei-Xing Zong ◽

David G. Thanassi

Keyword(s):

Cell Death ◽

Immune Responses ◽

Host Cell ◽

Francisella Tularensis ◽

Vaccine Strain ◽

Live Vaccine ◽

Live Vaccine Strain ◽

Type I ◽

Content Type ◽

Host Cell Death

ABSTRACTFrancisella tularensisis a facultative intracellular, Gram-negative pathogen and the causative agent of tularemia. We previously identified TolC as a virulence factor of theF. tularensislive vaccine strain (LVS) and demonstrated that a ΔtolCmutant exhibits increased cytotoxicity toward host cells and elicits increased proinflammatory responses compared to those of the wild-type (WT) strain. TolC is the outer membrane channel component used by the type I secretion pathway to export toxins and other bacterial virulence factors. Here, we show that the LVS delays activation of the intrinsic apoptotic pathway in a TolC-dependent manner, both during infection of primary macrophages and during organ colonization in mice. The TolC-dependent delay in host cell death is required forF. tularensisto preserve its intracellular replicative niche. We demonstrate that TolC-mediated inhibition of apoptosis is an active process and not due to defects in the structural integrity of the ΔtolCmutant. These findings support a model wherein the immunomodulatory capacity ofF. tularensisrelies, at least in part, on TolC-secreted effectors. Finally, mice vaccinated with the ΔtolCLVS are protected from lethal challenge and clear challenge doses faster than WT-vaccinated mice, demonstrating that the altered host responses to primary infection with the ΔtolCmutant led to altered adaptive immune responses. Taken together, our data demonstrate that TolC is required for temporal modulation of host cell death during infection byF. tularensisand highlight how shifts in the magnitude and timing of host innate immune responses may lead to dramatic changes in the outcome of infection.

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Antioxidants promote establishment of trypanosome infections in tsetse

Parasitology ◽

10.1017/s0031182007002247 ◽

2007 ◽

Vol 134 (6) ◽

pp. 827-831 ◽

Cited By ~ 72

Author(s):

E. T. MacLEOD ◽

I. MAUDLIN ◽

A. C. DARBY ◽

S. C. WELBURN

Keyword(s):

Reactive Oxygen Species ◽

Cell Death ◽

Trypanosoma Brucei ◽

Sub Saharan Africa ◽

Tsetse Flies ◽

Oxygen Species ◽

Infection Rates ◽

Trypanosoma Brucei Brucei ◽

Sub Saharan ◽

Cyclical Transmission

SUMMARYEfficient, cyclical transmission of trypanosomes through tsetse flies is central to maintenance of human sleeping sickness and nagana across sub-Saharan Africa. Infection rates in tsetse are normally very low as most parasites ingested with the fly bloodmeal die in the fly gut, displaying the characteristics of apoptotic cells. Here we show that a range of antioxidants (glutathione, cysteine, N-acetyl-cysteine, ascorbic acid and uric acid), when added to the insect bloodmeal, can dramatically inhibit cell death of Trypanosoma brucei brucei in tsetse. Both L- and D-cysteine invoked similar effects suggesting that inhibition of trypanosome death is not dependent on protein synthesis. The present work suggests that antioxidants reduce the midgut environment protecting trypanosomes from cell death induced by reactive oxygen species.

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Asc Modulates the Function of NLRC4 in Response to Infection of Macrophages by Legionella pneumophila

mBio ◽

10.1128/mbio.00117-11 ◽

2011 ◽

Vol 2 (4) ◽

Cited By ~ 36

Author(s):

Christopher L. Case ◽

Craig R. Roy

Keyword(s):

Cell Death ◽

Legionella Pneumophila ◽

Fluorescent Protein ◽

Microbial Infection ◽

Content Type ◽

Caspase 1 ◽

Cell Death Pathway ◽

Dependent Cell ◽

Green Fluorescent ◽

In Cells

ABSTRACTNucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) activate caspase-1 in response to a variety of bacterium-derived signals in macrophages. NLR-mediated activation of caspase-1 byLegionella pneumophilaoccurs through both an NLRC4/NAIP5-dependent pathway and a pathway requiring the adapter protein Asc. Both pathways are needed for maximal activation of caspase-1 and for the release of the cytokines interleukin-1β (IL-1β) and IL-18. Asc is not required for caspase-1-dependent pore formation and cell death induced upon infection of macrophages byL. pneumophila. Here, temporal and spatial localization of caspase-1-dependent processes was examined to better define the roles of Asc and NLRC4 during infection. Imaging studies revealed that caspase-1 localized to a single punctate structure in infected cells containing Asc but not in cells lacking this adapter. Both endogenous Asc and ectopically produced NLRC4 tagged with green fluorescent protein (GFP) were found to localize to caspase-1 puncta followingL. pneumophilainfection, suggesting that NLRC4 and Asc coordinate signaling through this complex during caspase-1 activation. Formation of caspase-1-containing puncta correlated with caspase-1 processing, suggesting a role for the Asc/NLRC4/caspase-1 complex in caspase-1 cleavage. In cells deficient for Asc, NLRC4 did not assemble into discrete puncta, and pyroptosis occurred at an accelerated rate. These data indicate that Asc mediates integration of NLR components into caspase-1 processing platforms and that recruitment of NLR components into an Asc complex can dampen pyroptotic responses. Thus, a negative feedback role of complexes containing Asc may be important for regulating caspase-1-mediated responses during microbial infection.IMPORTANCECaspase-1 is a protease activated during infection that is central to the regulation of several innate immune pathways. Studies examining the macromolecular complexes containing this protein, known as inflammasomes, have provided insight into the regulation of this protease. This work demonstrates that the intracellular bacteriumLegionella pneumophilainduces formation of complexes containing caspase-1 by multiple mechanisms and illustrates that an adapter molecule called Asc integrates signals from multiple independent upstream caspase-1 activators in order to assemble a spatially distinct complex in the macrophage. There were caspase-1-associated activities such as cytokine processing and secretion that were controlled by Asc. Importantly, this work uncovered a new role for Asc in dampening a caspase-1-dependent cell death pathway called pyroptosis. These findings suggest that Asc plays a central role in controlling a distinct subset of caspase-1-dependent activities by both assembling complexes that are important for cytokine processing and suppressing processes that mediate pyroptosis.

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Growth Retardation, Reduced Invasiveness, and Impaired Colistin-Mediated Cell Death Associated with Colistin Resistance Development in Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01439-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 828-832 ◽

Cited By ~ 54

Author(s):

Spyros Pournaras ◽

Aggeliki Poulou ◽

Konstantina Dafopoulou ◽

Yassine Nait Chabane ◽

Ioulia Kristo ◽

...

Keyword(s):

Cell Death ◽

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Bloodstream Infections ◽

Single Amino Acid ◽

Colistin Resistance ◽

Resistance Development ◽

Content Type ◽

Alkyl Hydroperoxide ◽

Severe Soft Tissue

ABSTRACTTwo colistin-susceptible/colistin-resistant (Cols/Colr) pairs ofAcinetobacter baumanniistrains assigned to international clone 2, which is prevalent worldwide, were sequentially recovered from two patients after prolonged colistin administration. Compared with the respective Colsisolates (Ab248 and Ab299, both having a colistin MIC of 0.5 μg/ml), both Colrisolates (Ab249 and Ab347, with colistin MICs of 128 and 32 μg/ml, respectively) significantly overexpressedpmrCABgenes, had single-amino-acid shifts in the PmrB protein, and exhibited significantly slower growth. The Colrisolate Ab347, tested by proteomic analysis in comparison with its Colscounterpart Ab299, underexpressed the proteins CsuA/B and C from thecsuoperon (which is necessary for biofilm formation). This isolate also underexpressed aconitase B and different enzymes involved in the oxidative stress response (KatE catalase, superoxide dismutase, and alkyl hydroperoxide reductase), suggesting a reduced response to reactive oxygen species (ROS) and, consequently, impaired colistin-mediated cell death through hydroxyl radical production. Colsisolates that were indistinguishable by macrorestriction analysis from Ab299 caused six sequential bloodstream infections, and isolates indistinguishable from Ab248 caused severe soft tissue infection, while Colrisolates indistinguishable from Ab347 and Ab249 were mainly colonizers. In particular, a Colsisolate identical to Ab299 was still invading the bloodstream 90 days after the colonization of this patient by Colrisolates. These observations indicate considerably lower invasiveness ofA. baumanniiclinical isolates following the development of colistin resistance.

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The Nuclear Export Receptors TbMex67 and TbMtr2 Are Required for Ribosome Biogenesis in Trypanosoma brucei

mSphere ◽

10.1128/msphere.00343-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 3

Author(s):

Constance Rink ◽

Martin Ciganda ◽

Noreen Williams

Keyword(s):

Trypanosoma Brucei ◽

Nuclear Export ◽

Ribosome Biogenesis ◽

Biological Process ◽

Large Subunit ◽

Critical Role ◽

Ribosomal Subunits ◽

Content Type ◽

Protein Components ◽

Export Receptors

ABSTRACT Ribosomal maturation is a complex and highly conserved biological process involving migration of a continuously changing RNP across multiple cellular compartments. A critical point in this process is the translocation of individual ribosomal subunits (60S and 40S) from the nucleus to the cytoplasm, and a number of export factors participate in this process. In this study, we characterize the functional role of the auxiliary export receptors TbMex67 and TbMtr2 in ribosome biogenesis in the parasite Trypanosoma brucei. We demonstrate that depletion of each of these proteins dramatically impacts the steady-state levels of other proteins involved in ribosome biogenesis, including the trypanosome-specific factors P34 and P37. In addition, we observe that the loss of TbMex67 or TbMtr2 leads to aberrant ribosome formation, rRNA processing, and polysome formation. Although the TbMex67-TbMtr2 heterodimer is structurally distinct from Mex67-Mtr2 complexes previously studied, our data show that they retain a conserved function in ribosome biogenesis. IMPORTANCE The nuclear export of ribosomal subunits (60S and 40S) depends in part on the activity of the essential auxiliary export receptors TbMtr2 and TbMex67. When these proteins are individually depleted from the medically and agriculturally significant parasite Trypanosoma brucei, distinct alterations in the processing of the rRNAs of the large subunit (60S) are observed as well as aberrations in the assembly of functional ribosomes (polysomes). We also established that TbMex67 and TbMtr2 interact directly or indirectly with the protein components of the 5S RNP, including the trypanosome-specific P34/P37. The critical role that TbMex67 and TbMtr2 play in this essential biological process together with their parasite-specific interactions may provide new therapeutic targets against this important parasite.

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Toxoplasma gondii Extends the Life Span of Infected Human Neutrophils by Inducing Cytosolic PCNA and Blocking Activation of Apoptotic Caspases

mBio ◽

10.1128/mbio.02031-20 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Tatiane S. Lima ◽

Sharmila Mallya ◽

Allen Jankeel ◽

Ilhem Messaoudi ◽

Melissa B. Lodoen

Keyword(s):

Cell Death ◽

Toxoplasma Gondii ◽

Life Span ◽

Peripheral Blood ◽

Productive Infection ◽

Human Neutrophils ◽

Serum Starvation ◽

Rna Seq ◽

Content Type ◽

Blood Neutrophils

ABSTRACT Toxoplasma gondii is an intracellular protozoan parasite that has the remarkable ability to infect and replicate in neutrophils, immune cells with an arsenal of antimicrobial effector mechanisms. We report that T. gondii infection extends the life span of primary human peripheral blood neutrophils by delaying spontaneous apoptosis, serum starvation-induced apoptosis, and tumor necrosis alpha (TNF-α)-mediated apoptosis. T. gondii blockade of apoptosis was associated with an inhibition of processing and activation of the apoptotic caspases caspase-8 and -3, decreased phosphatidylserine exposure on the plasma membrane, and reduced cell death. We performed a global transcriptome analysis of T. gondii-infected peripheral blood neutrophils using RNA sequencing (RNA-Seq) and identified gene expression changes associated with DNA replication and DNA repair pathways, which in mature neutrophils are indicative of changes in regulators of cell survival. Consistent with the RNA-Seq data, T. gondii infection upregulated transcript and protein expression of PCNA, which is found in the cytosol of human neutrophils, where it functions as a key inhibitor of apoptotic pro-caspases. Infection of neutrophils resulted in increased interaction of PCNA with pro-caspase-3. Inhibition of this interaction with an AlkB homologue 2 PCNA-interacting motif (APIM) peptide reversed the infection-induced delay in cell death. Taken together, these findings indicate a novel strategy by which T. gondii manipulates cell life span in primary human neutrophils, which may allow the parasite to maintain an intracellular replicative niche and avoid immune clearance. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that can cause life-threatening disease in immunocompromised individuals and in the developing fetus. Interestingly, T. gondii has evolved strategies to successfully manipulate the host immune system to establish a productive infection and evade host defense mechanisms. Although it is well documented that neutrophils are mobilized during acute T. gondii infection and infiltrate the site of infection, these cells can also be actively infected by T. gondii and serve as a replicative niche for the parasite. However, there is a limited understanding of the molecular processes occurring within T. gondii-infected neutrophils. This study reveals that T. gondii extends the life span of human neutrophils by inducing the expression of PCNA, which prevents activation of apoptotic caspases, thus delaying apoptosis. This strategy may allow the parasite to preserve its replicative intracellular niche.

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The lysosomotropic drug LeuLeu-OMe induces lysosome disruption and autophagy-independent cell death in Trypanosoma brucei

Microbial Cell ◽

10.15698/mic2015.08.217 ◽

2015 ◽

Vol 2 (8) ◽

pp. 288-298 ◽

Cited By ~ 3

Author(s):

Hazel Koh ◽

Htay Aye ◽

Kevin Tan ◽

Cynthia He

Keyword(s):

Cell Death ◽

Trypanosoma Brucei

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Pumilacidin-Like Lipopeptides Derived from Marine Bacterium Bacillus sp. Strain 176 Suppress the Motility of Vibrio alginolyticus

Applied and Environmental Microbiology ◽

10.1128/aem.00450-17 ◽

2017 ◽

Vol 83 (12) ◽

Cited By ~ 21

Author(s):

Pengyuan Xiu ◽

Rui Liu ◽

Dechao Zhang ◽

Chaomin Sun

Keyword(s):

Cell Death ◽

Antibiotic Resistance ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Marine Bacterium ◽

Vibrio Alginolyticus ◽

Bacterial Motility ◽

Great Promise ◽

Content Type ◽

Cyclic Lipopeptides

ABSTRACT Bacterial motility is a crucial factor during the invasion and colonization processes of pathogens, which makes it an attractive therapeutic drug target. Here, we isolated a marine bacterium (Vibrio alginolyticus strain 178) from a seamount in the tropical West Pacific that exhibits vigorous motility on agar plates and severe pathogenicity to zebrafish. We found that V. alginolyticus 178 motility was significantly suppressed by another marine bacterium, Bacillus sp. strain 176, isolated from the same niche. We isolated, purified, and characterized two different cyclic lipopeptides (CLPs) from Bacillus sp. 176 using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The two related CLPs have a pumilacidin-like structure and were both effective inhibitors of V. alginolyticus 178 motility. The CLPs differ by only one methylene group in their fatty acid chains. In addition to motility suppression, the CLPs also induced cell aggregation in the medium and reduced adherence of V. alginolyticus 178 to glass substrates. Notably, upon CLP treatment, the expression levels of two V. alginolyticus flagellar assembly genes (flgA and flgP) dropped dramatically. Moreover, the CLPs inhibited biofilm formation in several other strains of pathogenic bacteria without inducing cell death. This study indicates that CLPs from Bacillus sp. 176 show promise as antimicrobial lead compounds targeting bacterial motility and biofilm formation with a low potential for eliciting antibiotic resistance. IMPORTANCE Pathogenic bacteria often require motility to establish infections and subsequently spread within host organisms. Thus, motility is an attractive therapeutic target for the development of novel antibiotics. We found that cyclic lipopeptides (CLPs) produced by marine bacterium Bacillus sp. strain 176 dramatically suppress the motility of the pathogenic bacterium Vibrio alginolyticus strain 178, reduce biofilm formation, and promote cellular aggregation without inducing cell death. These findings suggest that CLPs hold great promise as potential drug candidates targeting bacterial motility and biofilm formation with a low overall potential for triggering antibiotic resistance.

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YejM Modulates Activity of the YciM/FtsH Protease Complex To Prevent Lethal Accumulation of Lipopolysaccharide

mBio ◽

10.1128/mbio.00598-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 10

Author(s):

Randi L. Guest ◽

Daniel Samé Guerra ◽

Maria Wissler ◽

Jacqueline Grimm ◽

Thomas J. Silhavy

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Content Type ◽

Essential Protein ◽

Ftsh Protease ◽

Acyl Chains ◽

Lipid Balance

ABSTRACT Lipopolysaccharide (LPS) is an essential glycolipid present in the outer membrane (OM) of many Gram-negative bacteria. Balanced biosynthesis of LPS is critical for cell viability; too little LPS weakens the OM, while too much LPS is lethal. In Escherichia coli, this balance is maintained by the YciM/FtsH protease complex, which adjusts LPS levels by degrading the LPS biosynthesis enzyme LpxC. Here, we provide evidence that activity of the YciM/FtsH protease complex is inhibited by the essential protein YejM. Using strains in which LpxC activity is reduced, we show that yciM is epistatic to yejM, demonstrating that YejM acts upstream of YciM to prevent toxic overproduction of LPS. Previous studies have shown that this toxicity can be suppressed by deleting lpp, which codes for a highly abundant OM lipoprotein. It was assumed that deletion of lpp restores lipid balance by increasing the number of acyl chains available for glycerophospholipid biosynthesis. We show that this is not the case. Rather, our data suggest that preventing attachment of lpp to the peptidoglycan sacculus allows excess LPS to be shed in vesicles. We propose that this loss of OM material allows continued transport of LPS to the OM, thus preventing lethal accumulation of LPS within the inner membrane. Overall, our data justify the commitment of three essential inner membrane proteins to avoid toxic over- or underproduction of LPS. IMPORTANCE Gram-negative bacteria are encapsulated by an outer membrane (OM) that is impermeable to large and hydrophobic molecules. As such, these bacteria are intrinsically resistant to several clinically relevant antibiotics. To better understand how the OM is established or maintained, we sought to clarify the function of the essential protein YejM in Escherichia coli. Here, we show that YejM inhibits activity of the YciM/FtsH protease complex, which regulates synthesis of the essential OM glycolipid lipopolysaccharide (LPS). Our data suggest that disrupting proper communication between LPS synthesis and transport to the OM leads to accumulation of LPS within the inner membrane (IM). The lethality associated with this event can be suppressed by increasing OM vesiculation. Our research has identified a completely novel signaling pathway that we propose coordinates LPS synthesis and transport.

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Basal Body Protein TbSAF1 Is Required for Microtubule Quartet Anchorage to the Basal Bodies in Trypanosoma brucei

mBio ◽

10.1128/mbio.00668-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 2

Author(s):

Xiaoduo Dong ◽

Teck Kwang Lim ◽

Qingsong Lin ◽

Cynthia Y. He

Keyword(s):

Trypanosoma Brucei ◽

Basal Body ◽

Spatial Organization ◽

Screening Method ◽

Interaction Network ◽

General Mechanism ◽

Basal Bodies ◽

Body Protein ◽

Content Type ◽

Molecular Description

ABSTRACT Sperm flagellar protein 1 (Spef1, also known as CLAMP) is a microtubule-associated protein involved in various microtubule-related functions from ciliary motility to polarized cell movement and planar cell polarity. In Trypanosoma brucei, the causative agent of trypanosomiasis, a single Spef1 ortholog (TbSpef1) is associated with a microtubule quartet (MtQ), which is in close association with several single-copied organelles and is required for their coordinated biogenesis during the cell cycle. Here, we investigated the interaction network of TbSpef1 using BioID, a proximity-dependent protein-protein interaction screening method. Characterization of selected candidates provided a molecular description of TbSpef1-MtQ interactions with nearby cytoskeletal structures. Of particular interest, we identified a new basal body protein TbSAF1, which is required for TbSpef1-MtQ anchorage to the basal bodies. The results demonstrate that MtQ-basal body anchorage is critical for the spatial organization of cytoskeletal organelles, as well as the morphology of the membrane-bound flagellar pocket where endocytosis takes place in this parasite. IMPORTANCE Trypanosoma brucei contains a large array of single-copied organelles and structures. Through extensive interorganelle connections, these structures replicate and divide following a strict temporal and spatial order. A microtubule quartet (MtQ) originates from the basal bodies and extends toward the anterior end of the cell, stringing several cytoskeletal structures together along its path. In this study, we examined the interaction network of TbSpef1, the only protein specifically located to the MtQ. We identified an interaction between TbSpef1 and a basal body protein TbSAF1, which is required for MtQ anchorage to the basal bodies. This study thus provides the first molecular description of MtQ association with the basal bodies, since the discovery of this association ∼30 years ago. The results also reveal a general mechanism of the evolutionarily conserved Spef1/CLAMP, which achieves specific cellular functions via their conserved microtubule functions and their diverse molecular interaction networks.

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