scholarly journals Trypanosoma brucei Pex13.2 Is an Accessory Peroxin That Functions in the Import of Peroxisome Targeting Sequence Type 2 Proteins and Localizes to Subdomains of the Glycosome

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Logan P. Crowe ◽  
Christina L. Wilkinson ◽  
Kathleen R. Nicholson ◽  
Meredith T. Morris
Keyword(s):  
Trypanosoma Brucei ◽  
Drug Targets ◽  
Protein Import ◽  
Specific Protein ◽  
Soluble Receptors ◽  
Two Dimensional Gel Electrophoresis ◽  
Content Type ◽  
Superresolution Microscopy

ABSTRACT Kinetoplastid parasites, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, harbor unique organelles known as glycosomes, which are evolutionarily related to peroxisomes. Glycosome/peroxisome biogenesis is mediated by proteins called peroxins that facilitate organelle formation, proliferation, and degradation and import of proteins housed therein. Import of matrix proteins occurs via one of two pathways that are dictated by their peroxisome targeting sequence (PTS). In PTS1 import, a C-terminal tripeptide sequence, most commonly SKL, is recognized by the soluble receptor Pex5. In PTS2 import, a less conserved N-terminal sequence is recognized by Pex7. The soluble receptors deliver their cargo to the import channel consisting minimally of Pex13 and Pex14. While much of the import process is conserved, kinetoplastids are the only organisms to have two Pex13s, Pex13.1 and Pex13.2. It is unclear why trypanosomes require two Pex13s when one is sufficient for most eukaryotes. To interrogate the role of Pex13.2, we have employed biochemical approaches to partially resolve the composition of the Pex13/Pex14 import complexes in T. brucei and characterized glycosome morphology and protein import in Pex13.2-deficient parasites. Here, we show that Pex13.2 is an integral glycosome membrane protein that interacts with Pex13.1 and Pex14. The N terminus of Pex13.2 faces the cytoplasmic side of the membrane, where it can facilitate interactions required for protein import. Two-dimensional gel electrophoresis revealed three glycosome membrane complexes containing combinations of Pex13.1, Pex13.2, and Pex14. The silencing of Pex13.2 resulted in parasites with fewer, larger glycosomes and disrupted glycosome protein import, suggesting the protein is involved in glycosome biogenesis as well as protein import. Furthermore, superresolution microscopy demonstrated that Pex13.2 localizes to discrete foci in the glycosome periphery, indicating that the glycosome periphery is not homogenous. IMPORTANCE Trypanosoma brucei causes human African trypanosomiasis and a wasting disease called Nagana in livestock. Current treatments are expensive, toxic, and difficult to administer. Because of this, the search for new drug targets is essential. T. brucei has glycosomes that are essential to parasite survival; however, our ability to target them in drug development is hindered by our lack of understanding about how these organelles are formed and maintained. This work forwards our understanding of how the parasite-specific protein Pex13.2 functions in glycosome protein import and lays the foundation for future studies focused on blocking Pex13.2 function, which would be lethal to bloodstream-form parasites that reside in the mammalian bloodstream.

scholarly journals Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

2015 ◽  
Vol 59 (11) ◽  
pp. 6873-6881 ◽  
Author(s):  
Kathryn Winglee ◽  
Shichun Lun ◽  
Marco Pieroni ◽  
Alan Kozikowski ◽  
William Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Strong Activity ◽  
Content Type ◽  
Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

Neurofibromatosis type 2: growth stimulation of mixed acoustic schwannoma by concurrent adjacent meningioma: possible role of growth factors

1998 ◽  
Vol 89 (1) ◽  
pp. 149-154 ◽  
Author(s):  
Roberto Pallini ◽  
Angelo Tancredi ◽  
Patrizia Casalbore ◽  
Delio Mercanti ◽  
Luigi M. Larocca ◽  
...  
Keyword(s):  
Neurofibromatosis Type ◽  
Growth Stimulation ◽  
Neurofibromatosis Type 2 ◽  
Annual Growth Rate ◽  
Content Type ◽  
Acoustic Schwannoma ◽  
Epidermal Growth ◽  
The One

✓ The authors report the case of a young man suffering from neurofibromatosis type 2 (NF2) who harbored bilateral acoustic schwannomas and a parasellar meningioma. Neuroimaging studies performed during a 4-year follow-up period showed that the bilateral schwannomas had grown very little and at similar rates. However, after the meningioma had infiltrated the tentorium and approached the ipsilateral schwannoma at the incisura, both Schwann cell tumors started to grow rapidly, particularly the one adjacent to the meningioma, of which the percentage of annual growth rate increased by approximately a factor of 102. At the same time, magnetic resonance imaging showed that this tumor also changed its features. During surgery, the acoustic schwannoma was firmly adherent to both meningioma and tentorium. Histological examination revealed meningotheliomatous cells in the schwannoma adjacent to the meningioma. Antiphosphotyrosine immunoblotting of PC12 cells was compatible with the presence of an epidermal growth factor (EGF)—like molecule in the cerebrospinal fluid (CSF) of the patient. This factor was not detected in the CSF of five other NF2 patients, two of whom bore associated bilateral acoustic schwannomas and meningioma in remote locations. It is hypothesized that the meningotheliomatous cells infiltrating the schwannoma triggered an autocrine/paracrine growth—stimulatory mechanism that involved an EGF-like factor.

Cytosolic and mitochondrial Hsp90 in cytokinesis, mitochondrial DNA replication, and drug action in Trypanosoma brucei

2021 ◽  
Author(s):  
Kirsten J. Meyer ◽  
Theresa A. Shapiro
Keyword(s):  
Mitochondrial Dna ◽  
Trypanosoma Brucei ◽  
Cell Biology ◽  
Drug Targets ◽  
Heat Shock Protein 90 ◽  
Cell Cycle Regulators ◽  
Hsp90 Inhibitors ◽  
African Trypanosomes ◽  
Hsp90 Activity

Trypanosoma brucei subspecies cause African sleeping sickness in humans, an infection that is commonly fatal if not treated, and available therapies are limited. Previous studies have shown that heat shock protein 90 (Hsp90) inhibitors have potent and vivid activity against bloodstream form trypanosomes. Hsp90s are phylogenetically conserved and essential catalysts that function at the crux of cell biology, where they ensure the proper folding of proteins and their assembly into multicomponent complexes. To assess the specificity of Hsp90 inhibitors and further define the role of Hsp90s in African trypanosomes, we used RNAi to knockdown cytosolic and mitochondrial Hsp90s (HSP83 and HSP84, respectively). Loss of either protein led to cell death but the phenotypes were distinctly different. Depletion of cytosolic HSP83 closely mimicked the consequences of chemically depleting Hsp90 activity with inhibitor 17-AAG. In these cells cytokinesis was severely disrupted and segregation of the kinetoplast (the massive mitochondrial DNA structure unique to this family of eukaryotic pathogens) was impaired, leading to cells with abnormal kDNA structures. Quite differently, knockdown of mitochondrial HSP84 did not impair cytokinesis but halted the initiation of new kDNA synthesis, generating cells without kDNA. These findings highlight the central role for Hsp90s in chaperoning cell cycle regulators in trypanosomes, reveal their unique function in kinetoplast replication, and reinforce their specificity and value as drug targets.

scholarly journals Cryptococcus neoformans Evades Pulmonary Immunity by Modulating Xylose Precursor Transport

2020 ◽  
Vol 88 (8) ◽  
Author(s):  
Lucy X. Li ◽  
Camaron R. Hole ◽  
Javier Rangel-Moreno ◽  
Shabaana A. Khader ◽  
Tamara L. Doering
Keyword(s):  
Cryptococcus Neoformans ◽  
Mutant Strain ◽  
Fungal Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Helper Cell Type ◽  
Lymphoid Structures ◽  
Type Infection

ABSTRACT Cryptococcus neoformans is a fungal pathogen that kills almost 200,000 people each year and is distinguished by abundant and unique surface glycan structures that are rich in xylose. A mutant strain of C. neoformans that cannot transport xylose precursors into the secretory compartment is severely attenuated in virulence in mice yet surprisingly is not cleared. We found that this strain failed to induce the nonprotective T helper cell type 2 (Th2) responses characteristic of wild-type infection, instead promoting sustained interleukin 12p40 (IL-12p40) induction and increased IL-17A (IL-17) production. It also stimulated dendritic cells to release high levels of proinflammatory cytokines, a behavior we linked to xylose expression. We further discovered that inducible bronchus-associated lymphoid tissue (iBALT) forms in response to infection with either wild-type cryptococci or the mutant strain with reduced surface xylose; although iBALT formation is slowed in the latter case, the tissue is better organized. Finally, our temporal studies suggest that lymphoid structures in the lung restrict the spread of mutant fungi for at least 18 weeks after infection, which is in contrast to ineffective control of the pathogen after infection with wild-type cells. These studies demonstrate the role of xylose in modulation of host response to a fungal pathogen and show that cryptococcal infection triggers iBALT formation.

scholarly journals Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

2015 ◽  
Vol 59 (8) ◽  
pp. 4669-4679 ◽  
Author(s):  
Nilmar Silvio Moretti ◽  
Leonardo da Silva Augusto ◽  
Tatiana Mordente Clemente ◽  
Raysa Paes Pinto Antunes ◽  
Nobuko Yoshida ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Drug Targets ◽  
Wild Type ◽  
Content Type ◽  
Damage Repair ◽  
Lysine Deacetylases

ABSTRACTAcetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD+-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation ofTrypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion byT. cruziat concentrations that did not affect host cell viability. In addition,in vivoinfection was partially controlled upon administration of salermide. There are two sirtuins inT. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed inEscherichia coliwith a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

scholarly journals Dissection of the Role of Pili and Type 2 and 3 Secretion Systems in Adherence and Biofilm Formation of an Atypical Enteropathogenic Escherichia coli Strain

2013 ◽  
Vol 81 (10) ◽  
pp. 3793-3802 ◽  
Author(s):  
Rodrigo T. Hernandes ◽  
Miguel A. De la Cruz ◽  
Denise Yamamoto ◽  
Jorge A. Girón ◽  
Tânia A. T. Gomes
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Coli Strain ◽  
Mass Spectrometry Analysis ◽  
Secretion Systems ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Rigid Rod

ABSTRACTAtypical enteropathogenicEscherichia coli(aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2eaemutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion offimAin 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2sslEmutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a doubleeae espAmutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2eaemutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains.

scholarly journals Genetic Validation of Trypanosoma brucei Glutathione Synthetase as an Essential Enzyme

2014 ◽  
Vol 13 (5) ◽  
pp. 614-624 ◽  
Author(s):  
Chelsea Pratt ◽  
Suong Nguyen ◽  
Margaret A. Phillips
Keyword(s):  
Trypanosoma Brucei ◽  
Drug Targets ◽  
Glutathione Synthetase ◽  
Biosynthetic Enzymes ◽  
Polyamine Biosynthesis ◽  
Content Type ◽  
Stage Disease ◽  
Trypanosomatid Parasites ◽  
Vector Borne ◽  
Essential Enzyme

ABSTRACTHuman African trypanosomiasis (HAT) is a debilitating and fatal vector-borne disease. Polyamine biosynthesis is the target of one of the key drugs (eflornithine) used for the treatment of late-stage disease, suggesting that the pathway might be exploited for the identification of additional drug targets. The polyamine spermidine is required in trypanosomatid parasites for formation of a unique redox cofactor termed trypanothione, which is formed from the conjugation of glutathione to spermidine. Here we characterize recombinantTrypanosoma bruceiglutathione synthetase (TbGS) and show that depletion ofTbGS in blood-form parasites using a regulated knockout strategy leads to loss of trypanothione and to cell death as quantified by fluorescence-activated cell sorter (FACS) analysis. These data suggest that >97% depletion ofTbGS is required before trypanothione is depleted and cell growth arrest is observed. Exogenous glutathione was able to partially compensate for the loss ofTbGS, suggesting that parasites are able to transport intact glutathione. Finally, reduced expression ofTbGS leads to increased levels of upstream glutathione biosynthetic enzymes and decreased expression of polyamine biosynthetic enzymes, providing evidence that the cells cross regulate the two branches of the trypanothione biosynthetic pathway to maintain spermidine and trypanothione homeostasis.

scholarly journals A Novel, WidespreadqacAAllele Results in Reduced Chlorhexidine Susceptibility inStaphylococcus epidermidis

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Amin Addetia ◽  
Alexander L. Greninger ◽  
Amanda Adler ◽  
Shuhua Yuan ◽  
Negar Makhsous ◽  
...  
Keyword(s):  
Amino Acid ◽  
Staphylococcus Epidermidis ◽  
Sequence Data ◽  
Methicillin Resistance ◽  
Single Amino Acid ◽  
Data Sets ◽  
The Novel ◽  
Content Type

ABSTRACTChlorhexidine gluconate (CHG) is a topical antiseptic widely used in health care settings. InStaphylococcusspp., the pump QacA effluxes CHG, while the closely related QacB cannot due to a single amino acid substitution. We characterized 1,050 cutaneousStaphylococcusisolates obtained from 173 pediatric oncology patients enrolled in a multicenter CHG bathing trial. CHG susceptibility testing revealed that 63 (6%) of these isolates had elevated CHG MICs (≥4 μg/ml). Screening of all 1,050 isolates for theqacA/Bgene (the sameqacgene with A or B allele) by restriction fragment length polymorphism (RFLP) yielded 56 isolates with a novelqacA/BRFLP pattern,qacA/B273. The CHG MIC was significantly higher forqacA/B273-positive isolates (MIC50, 4 μg/ml; MIC range, 0.5 to 4 μg/ml) than for otherqacgroups:qacA-positive isolates (n = 559; MIC50, 1 μg/ml; MIC range, 0.5 to 4 μg/ml),qacB-positive isolates (n = 17; MIC50, 1 μg/ml; MIC range, 0.25 to 2 μg/ml), andqacA/B-negative isolates (n = 418, MIC50, 1 μg/ml; MIC range, 0.125 to 2 μg/ml) (P = 0.001). A high proportion of theqacA/B273-positive isolates also displayed methicillin resistance (96.4%) compared to the otherqacgroups (24.9 to 61.7%) (P = 0.001). Whole-genome sequencing revealed thatqacA/B273-positive isolates encoded a variant of QacA with 2 amino acid substitutions. This new allele, namedqacA4, was carried on the novel plasmid pAQZ1. TheqacA4-carrying isolates belonged to the highly resistantStaphylococcus epidermidissequence type 2 clone. By searching available sequence data sets, we identified 39 additionalqacA4-carryingS. epidermidisstrains from 5 countries. Curing an isolate ofqacA4resulted in a 4-fold decrease in the CHG MIC, confirming the role ofqacA4in the elevated CHG MIC. Our results highlight the importance of further studyingqacA4and its functional role in clinical staphylococci.

scholarly journals Strain-Specific Regulatory Role of Eukaryote-Like Serine/Threonine Phosphatase in Pneumococcal Adherence

2012 ◽  
Vol 80 (4) ◽  
pp. 1361-1372 ◽  
Author(s):  
Shivangi Agarwal ◽  
Shivani Agarwal ◽  
Preeti Pancholi ◽  
Vijay Pancholi
Keyword(s):  
High Efficiency ◽  
Regulatory System ◽  
Gene Encoding ◽  
Content Type ◽  
Knockout Mutants ◽  
Catalytically Active

ABSTRACTStreptococcus pneumoniaeexploits a battery of virulence factors to colonize the host. Although the eukaryote-like Ser/Thr kinase ofS. pneumoniae(StkP) has been implicated in physiology and virulence, the role of its cotranscribing phosphatase (PhpP) has remained elusive. The construction of nonpolar markerlessphpPknockout mutants (ΔphpP) in two pathogenic strains, D39 (type 2) and 6A-EF3114 (type 6A), indicated that PhpP is not indispensable for pneumococcal survival. Further, PhpP also participates in the regulation of cell wall biosynthesis/division, adherence, and biofilm formation in a strain-specific manner. Additionally, we provide hitherto-unknownin vitroandin vivoevidence of a physiologically relevant biochemical link between the StkP/PhpP-mediated cognate regulation and the two-component regulatory system TCS06 (RR06/HK06) that regulates the expression of the gene encoding an important pneumococcal surface adhesin, CbpA, which was found to be significantly upregulated in ΔphpPmutants. In particular, StkP (threonine)-phosphorylated RR06 bound to thecbpApromoter with high efficiency even in the absence of the HK06-responsive and catalytically active aspartate 51 residue. Together, our findings unravel the significant contributions of PhpP in pneumococcal physiology and adherence.

scholarly journals Rab11 Function in Trypanosoma brucei: Identification of Conserved and Novel Interaction Partners

2011 ◽  
Vol 10 (8) ◽  
pp. 1082-1094 ◽  
Author(s):  
Carme Gabernet-Castello ◽  
Kelly N. DuBois ◽  
Camus Nimmo ◽  
Mark C. Field
Keyword(s):  
Trypanosoma Brucei ◽  
Intracellular Transport ◽  
Homo Sapiens ◽  
Coiled Coil ◽  
Membrane Composition ◽  
Content Type ◽  
Evolutionarily Conserved ◽  
Interaction Partners ◽  
Hybrid Screening

ABSTRACT The Ras-like GTPase Rab11 is implicated in multiple aspects of intracellular transport, including maintenance of plasma membrane composition and cytokinesis. In metazoans, these functions are mediated in part via coiled-coil Rab11-interacting proteins (FIPs) acting as Rab11 effectors. Additional interaction between Rab11 and the exocyst subunit Sec15 connects Rab11 with exocytosis. We find that FIPs are metazoan specific, suggesting that other factors mediate Rab11 functions in nonmetazoans. We examined Rab11 interactions in Trypanosoma brucei , where endocytosis is well studied and the role of Rab11 in recycling well documented. TbSec15 and TbRab11 interact, demonstrating evolutionary conservation. By yeast two-hybrid screening, we identified additional Rab11 interaction partners. Tb927.5.1640 (designated RBP74) interacted with both Rab11 and Rab5. RBP74 shares a coiled-coil architecture with metazoan FIPs but is unrelated by sequence and appears to play a role in coordinating endocytosis and recycling. A second coiled-coil protein, Tb09.211.4830 (TbAZI1), orthologous to AZI1 in Homo sapiens , interacts exclusively with Rab11. AZI1 is restricted to taxa with motile cilia/flagella. These data suggest that Rab11 functions are mediated by evolutionarily conserved (i.e., AZI1 and Sec15) and potentially lineage-specific (RBP74) interactions essential for the integration of the endomembrane system.

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