scholarly journals The Toxoplasma gondii Cyst Wall Interactome

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Vincent Tu ◽  
Tadakimi Tomita ◽  
Tatsuki Sugi ◽  
Joshua Mayoral ◽  
Bing Han ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Interactions ◽  
Cyst Wall ◽  
Affinity Purification ◽  
Dense Granule ◽  
Hypothetical Proteins ◽  
Content Type

ABSTRACT A characteristic of the latent cyst stage of Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole. Previously, our laboratory group published a proteomic analysis of purified in vitro cyst wall fragments that identified an inventory of cyst wall components. To further refine our understanding of the composition of the cyst wall, several cyst wall proteins were tagged with a promiscuous biotin ligase (BirA*), and their interacting partners were screened by streptavidin affinity purification. Within the cyst wall pulldowns, previously described cyst wall proteins, dense granule proteins, and uncharacterized hypothetical proteins were identified. Several of the newly identified hypothetical proteins were validated to be novel components of the cyst wall and tagged with BirA* to expand the model of the cyst wall interactome. Community detection of the cyst wall interactome model revealed three distinct clusters: a dense granule, a cyst matrix, and a cyst wall cluster. Characterization of several of the identified cyst wall proteins using genetic strategies revealed that MCP3 affects in vivo cyst sizes. This study provides a model of the potential protein interactions within the cyst wall and the groundwork to understand cyst wall formation. IMPORTANCE A model of the cyst wall interactome was constructed using proteins identified through BioID. The proteins within this cyst wall interactome model encompass several proteins identified in a prior characterization of the cyst wall proteome. This model provides a more comprehensive understanding of the composition of the cyst wall and may lead to insights on how the cyst wall is formed.

scholarly journals Hammondia hammondi Harbors Functional Orthologs of the Host-Modulating Effectors GRA15 and ROP16 but Is Distinguished from Toxoplasma gondii by a Unique Transcriptional Profile

2014 ◽  
Vol 13 (12) ◽  
pp. 1507-1518 ◽  
Author(s):  
Katelyn A. Walzer ◽  
Gregory M. Wier ◽  
Rachel A. Dam ◽  
Ananth R. Srinivasan ◽  
Adair L. Borges ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Transcript Abundance ◽  
Dense Granule ◽  
Transcriptional Analysis ◽  
Content Type ◽  
Phenotypic Differences ◽  
Functional Conservation ◽  
High Transcript Abundance

ABSTRACTToxoplasma gondiiand its nearest extant relative,Hammondia hammondi, are phenotypically distinct despite their remarkable similarity in gene content, synteny, and functionality. To begin to identify genetic differences that might drive distinct infection phenotypes ofT. gondiiandH. hammondi, in the present study we (i) determined whether two known host-interacting proteins, dense granule protein 15 (GRA15) and rhoptry protein 16 (ROP16), were functionally conserved inH. hammondiand (ii) performed the first comparative transcriptional analysis ofH. hammondiandT. gondiisporulated oocysts. We found that GRA15 and ROP16 fromH. hammondi(HhGRA15 and HhROP16) modulate the host NF-κB and STAT6 pathways, respectively, when expressed heterologously inT. gondii. We also found the transcriptomes ofH. hammondiandT. gondiito be highly distinct. Consistent with the spontaneous conversion ofH. hammonditachyzoites into bradyzoites bothin vitroandin vivo,H. hammondihigh-abundance transcripts are enriched for genes that are of greater abundance inT. gondiibradyzoites. We also identified genes that are of high transcript abundance inH. hammondibut are poorly expressed in multipleT. gondiilife stages, suggesting that these genes are uniquely expressed inH. hammondi. Taken together, these data confirm the functional conservation of knownT. gondiivirulence effectors inH. hammondiand point to transcriptional differences as a potential source of the phenotypic differences between these species.

scholarly journals Identification of Tissue Cyst Wall Components by Transcriptome Analysis ofIn Vivoand In Vitro Toxoplasma gondii Bradyzoites

2011 ◽  
Vol 10 (12) ◽  
pp. 1637-1647 ◽  
Author(s):  
Kerry R. Buchholz ◽  
Heather M. Fritz ◽  
Xiucui Chen ◽  
Blythe Durbin-Johnson ◽  
David M. Rocke ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Tissue Cyst ◽  
Content Type ◽  
Repeat Domain ◽  
Developmental Form ◽  
Protein Components ◽  
New Protein

ABSTRACTTheToxoplasma gondiibradyzoite is essential to establish persistent infection, yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes ofin vitrotachyzoites 2 days postinfection,in vitrobradyzoites 4 days postinfection, andin vivobradyzoites 21 days postinfection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One hundred two putative bradyzoite-secreted effectors were identified by this approach. Two candidates, bradyzoite pseudokinase 1 and microneme adhesive repeat domain-containing protein 4, were chosen for further investigation and confirmed to be induced and secreted by bradyzoitesin vitroandin vivo. Thus, we report the first analysis of the transcriptomes ofin vitroandin vivobradyzoites and identify two new protein components of theToxoplasmatissue cyst wall.

scholarly journals Toxoplasma gondii Intravacuolar-Network-Associated Dense Granule Proteins Regulate Maturation of the Cyst Matrix and Cyst Wall

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Rebekah B. Guevara ◽  
Barbara A. Fox ◽  
Alejandra Falla ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Protein Localization ◽  
Dense Granule ◽  
Wall Material ◽  
Content Type ◽  
Wall Structures ◽  
Cyst Development ◽  
Rate Of Accumulation

ABSTRACT Little is known regarding how the chronic Toxoplasma gondii cyst develops. Here, we investigated intravacuolar-network-associated dense granule (GRA) proteins GRA1, GRA2, GRA4, GRA6, GRA9, and GRA12 during cyst development in vitro after differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 postdifferentiation, GRA1, GRA4, GRA6, GRA9, and GRA12 colocalized with Dolichos biflorus agglutinin stain at the cyst periphery. In contrast, GRA2 remained in the cyst matrix. By day 2 postdifferentiation, coinciding with localization of GRA2 to the cyst periphery, GRA1, GRA4, GRA6, and GRA9 established a continuous matrix pattern in the cyst. In contrast, GRA2 and GRA12 were colocalized in prominent cyst matrix puncta throughout cyst development. While GRA2, GRA6, and GRA12 localized in outer and inner layers of the cyst wall, GRA1, GRA4, and GRA9 localized predominantly in the inner layers of the cyst wall. GRA2 and GRA12 were colocalized in the cyst wall by day 7 postdifferentiation. However, by day 10 postdifferentiation, GRA12 was relocalized from the cyst wall to puncta in the cyst matrix. Differentiation of Δgra2 parasites revealed a defect in the ability to establish a normal cyst matrix. In addition, the deletion of any intravacuolar-network-associated GRA protein, except GRA1, reduced the rate of accumulation of cyst wall proteins at the cyst periphery relative to the cyst interior. Our findings reveal dynamic patterns of GRA protein localization during cyst development and suggest that intravacuolar-network-associated GRA proteins regulate the formation and maturation of the cyst matrix and cyst wall structures. IMPORTANCE Toxoplasma gondii establishes chronic infection in humans by forming thick-walled cysts that persist in the brain. If host immunity wanes, cysts reactivate to cause severe, and often lethal, toxoplasmic encephalitis. There is no available therapy to eliminate cysts or to prevent their reactivation. Moreover, how the vital and characteristic cyst matrix and cyst wall structures develop is poorly understood. Here, we visualized and tracked the localization of Toxoplasma intravacuolar-network-associated dense granule (GRA) proteins during cyst development in vitro. Intravacuolar-network GRAs were present within the cyst matrix and at the cyst wall in developing cysts, and genetic deletion of intravacuolar-network-associated GRAs reduced the rate of accumulation of cyst wall material at the cyst periphery. Our results show that intravacuolar-network-associated GRAs, particularly GRA2 and GRA12, play dynamic and essential roles in the development and maturation of the cyst matrix and the cyst wall structures.

scholarly journals The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis

2015 ◽  
Vol 59 (8) ◽  
pp. 4446-4452 ◽  
Author(s):  
Vadim Makarov ◽  
João Neres ◽  
Ruben C. Hartkoorn ◽  
Olga B. Ryabova ◽  
Elena Kazakova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitro Group ◽  
Covalent Bond ◽  
Antimycobacterial Activity ◽  
Content Type ◽  
Sar Studies ◽  
Covalent Bond Formation

ABSTRACT8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2′-oxidase (DprE1) and display nanomolar bactericidal activity againstMycobacterium tuberculosisin vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 μg/ml againstM. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 μM and present favorablein vitroabsorption-distribution-metabolism-excretion/toxicity (ADME/T) andin vivopharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency.

scholarly journals Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei

2015 ◽  
Vol 84 (3) ◽  
pp. 701-710 ◽  
Author(s):  
Madeleine G. Moule ◽  
Natasha Spink ◽  
Sam Willcocks ◽  
Jiali Lim ◽  
José Afonso Guerra-Assunção ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Insertion Site ◽  
Wild Type ◽  
Content Type ◽  
Growth And Survival ◽  
New Genes ◽  
Insertion Sites

Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survivalin vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuationin vivowere identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarkedbpsl2248,tex,rpiR,bpsl1728, andbpss1528deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was testedin vitroandin vivoto confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important toin vivovirulence with roles in different stages ofB. pseudomalleipathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and thetexmutant was capable of providing protective immunity against challenge with wild-typeB. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates.

scholarly journals Role of Toxoplasma gondii Chloroquine Resistance Transporter in Bradyzoite Viability and Digestive Vacuole Maintenance

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Geetha Kannan ◽  
Manlio Di Cristina ◽  
Aric J. Schultz ◽  
My-Hang Huynh ◽  
Fengrong Wang ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Chronic Infection ◽  
Chloroquine Resistance ◽  
Congenital Defects ◽  
Functional Link ◽  
Content Type ◽  
Chloroquine Resistance Transporter

ABSTRACT Toxoplasma gondii is a ubiquitous pathogen that can cause encephalitis, congenital defects, and ocular disease. T. gondii has also been implicated as a risk factor for mental illness in humans. The parasite persists in the brain as slow-growing bradyzoites contained within intracellular cysts. No treatments exist to eliminate this form of parasite. Although proteolytic degradation within the parasite lysosome-like vacuolar compartment (VAC) is critical for bradyzoite viability, whether other aspects of the VAC are important for parasite persistence remains unknown. An ortholog of Plasmodium falciparum chloroquine resistance transporter (CRT), TgCRT, has previously been identified in T. gondii. To interrogate the function of TgCRT in chronic-stage bradyzoites and its role in persistence, we knocked out TgCRT in a cystogenic strain and assessed VAC size, VAC digestion of host-derived proteins and parasite autophagosomes, and the viability of in vitro and in vivo bradyzoites. We found that whereas parasites deficient in TgCRT exhibit normal digestion within the VAC, they display a markedly distended VAC and their viability is compromised both in vitro and in vivo. Interestingly, impairing VAC proteolysis in TgCRT-deficient bradyzoites restored VAC size, consistent with a role for TgCRT as a transporter of products of digestion from the VAC. In conjunction with earlier studies, our current findings suggest a functional link between TgCRT and VAC proteolysis. This study provides further evidence of a crucial role for the VAC in bradyzoite persistence and a new potential VAC target to abate chronic Toxoplasma infection. IMPORTANCE Individuals chronically infected with the intracellular parasite Toxoplasma gondii are at risk of experiencing reactivated disease that can result in progressive loss of vision. No effective treatments exist for chronic toxoplasmosis due in part to a poor understanding of the biology underlying chronic infection and a lack of well-validated potential targets. We show here that a T. gondii transporter is functionally linked to protein digestion within the parasite lysosome-like organelle and that this transporter is necessary to sustain chronic infection in culture and in experimentally infected mice. Ablating the transporter results in severe bloating of the lysosome-like organelle. Together with earlier work, this study suggests the parasite’s lysosome-like organelle is vital for parasite survival, thus rendering it a potential target for diminishing infection and reducing the risk of reactivated disease.

scholarly journals Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Alicja M. Cygan ◽  
Terence C. Theisen ◽  
Alma G. Mendoza ◽  
Nicole D. Marino ◽  
Michael W. Panas ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Translocation ◽  
Affinity Purification ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Host Cells ◽  
Content Type ◽  
Cyclin E1 ◽  
Infected Cells

ABSTRACT Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins. IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

scholarly journals Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Jixu Li ◽  
Huanping Guo ◽  
Eloiza May Galon ◽  
Yang Gao ◽  
Seung-Hun Lee ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Targets ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Lytic Cycle ◽  
Type I ◽  
Content Type ◽  
Mechanism Of Inhibition

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

scholarly journals Characterization of Intracellular Growth RegulatoricgRby Utilizing Transcriptomics To Identify Mediators of Pathogenesis in Shigella flexneri

2013 ◽  
Vol 81 (9) ◽  
pp. 3068-3076 ◽  
Author(s):  
Carolyn R. Morris ◽  
Christen L. Grassel ◽  
Julia C. Redman ◽  
Jason W. Sahl ◽  
Eileen M. Barry ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Diarrheal Disease ◽  
Intracellular Growth ◽  
Bioinformatics Analyses ◽  
Content Type ◽  
Regulatory Pathways

ABSTRACTShigellaspecies Gram-negative bacteria which cause a diarrheal disease, known as shigellosis, by invading and destroying the colonic mucosa and inducing a robust inflammatory response. With no vaccine available, shigellosis annually kills over 600,000 children in developing countries. This study demonstrates the utility of combining high-throughput bioinformatic methods within vitroandin vivoassays to provide new insights into pathogenesis. Comparisons ofin vivoandin vitrogene expression identified genes associated with intracellular growth. Additional bioinformatics analyses identified genes that are present inS. flexneriisolates but not in the three otherShigellaspecies. Comparison of these two analyses revealed nine genes that are differentially expressed during invasion and that are specific toS. flexneri. One gene, a DeoR family transcriptional regulator with decreased expression during invasion, was further characterized and is now designatedicgR, forintracellulargrowthregulator. Deletion oficgRcaused no difference in growthin vitrobut resulted in increased intracellular replication in HCT-8 cells. Furtherin vitroandin vivostudies using high-throughput sequencing of RNA transcripts (RNA-seq) of an isogenic ΔicgRmutant identified 34 genes that were upregulated under both growth conditions. This combined informatics and functional approach has allowed the characterization of a gene and pathway previously unknown inShigellapathogenesis and provides a framework for further identification of novel virulence factors and regulatory pathways.

scholarly journals Adaptation-Based Resistance to Siderophore-Conjugated Antibacterial Agents by Pseudomonas aeruginosa

2013 ◽  
Vol 57 (9) ◽  
pp. 4197-4207 ◽  
Author(s):  
Andrew P. Tomaras ◽  
Jared L. Crandon ◽  
Craig J. McPherson ◽  
Mary Anne Banevicius ◽  
Steven M. Finegan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibacterial Agents ◽  
Iron Acquisition ◽  
Content Type ◽  
Multiple Strains ◽  
Resistance Rates ◽  
Assay Conditions

ABSTRACTMultidrug resistance in Gram-negative bacteria has become so threatening to human health that new antibacterial platforms are desperately needed to combat these deadly infections. The concept of siderophore conjugation, which facilitates compound uptake across the outer membrane by hijacking bacterial iron acquisition systems, has received significant attention in recent years. While standardin vitroMIC and resistance frequency methods demonstrate that these compounds are potent, broad-spectrum antibacterial agents whose activity should not be threatened by unacceptably high spontaneous resistance rates, recapitulation of these results in animal models can prove unreliable, partially because of the differences in iron availability in these different methods. Here, we describe the characterization of MB-1, a novel siderophore-conjugated monobactam that demonstrates excellentin vitroactivity againstPseudomonas aeruginosawhen tested using standard assay conditions. Unfortunately, thein vitrofindings did not correlate with thein vivoresults we obtained, as multiple strains were not effectively treated by MB-1 despite having low MICs. To address this, we also describe the development of newin vitroassays that were predictive of efficacy in mouse models, and we provide evidence that competition with native siderophores could contribute to the recalcitrance of someP. aeruginosaisolatesin vivo.

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