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 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 Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

2011 ◽  
Vol 80 (1) ◽  
pp. 3-13 ◽  
Author(s):  
Chen Li ◽  
Kurniyati ◽  
Bo Hu ◽  
Jiang Bian ◽  
Jianlan Sun ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Biofilm Formation ◽  
Adult Population ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Content Type ◽  
Multiple Organs ◽  
Biochemical Analyses

ABSTRACTThe oral bacteriumPorphyromonas gingivalisis a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide.P. gingivalisexhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence ofP. gingivalisremain elusive. In this report, we found thatP. gingivalisencodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed thatPG0352is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPgis an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that thePG0352deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type,in vitrostudies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement.In vivostudies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPgis an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity ofP. gingivalis, and it can potentially serve as a new target for developing therapeutic agents againstP. gingivalisinfection.

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 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 Cryptococcus neoformans Dual GDP-Mannose Transporters and Their Role in Biology and Virulence

2014 ◽  
Vol 13 (6) ◽  
pp. 832-842 ◽  
Author(s):  
Zhuo A. Wang ◽  
Cara L. Griffith ◽  
Michael L. Skowyra ◽  
Nichole Salinas ◽  
Matthew Williams ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Protein Glycosylation ◽  
Content Type ◽  
Phenotypic Differences ◽  
Major Virulence Factor ◽  
Transmembrane Transporters ◽  
Mutant Cells ◽  
Made In

ABSTRACTCryptococcus neoformansis an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed inSaccharomyces cerevisiaeshowed that both are functional, with similar kinetics and substrate specificitiesin vitro. We have now examined these proteinsin vivoand demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore,gmt1 gmt2double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis.

scholarly journals Spiroindolone That Inhibits PfATPase4 Is a Potent, Cidal Inhibitor of Toxoplasma gondii TachyzoitesIn VitroandIn Vivo

2013 ◽  
Vol 58 (3) ◽  
pp. 1789-1792 ◽  
Author(s):  
Ying Zhou ◽  
Alina Fomovska ◽  
Stephen Muench ◽  
Bo-Shiun Lai ◽  
Ernest Mui ◽  
...  
Keyword(s):  
Body Weight ◽  
Toxoplasma Gondii ◽  
Inhibitory Concentration ◽  
Parasite Burden ◽  
Content Type ◽  
Medicine Development ◽  
Lead Candidate ◽  
Oral Doses

ABSTRACTHere, we show that spiroindolone, an effective treatment for plasmodia, is also active againstToxoplasma gondiitachyzoites.In vitro, spiroindolone NITD609 is cidal for tachyzoites (50% inhibitory concentration [IC50], 1μM) and not toxic to human cells at ≥10μM. Two daily oral doses of 100 mg/kg of body weight reduced the parasite burden in mice by 90% (P= 0.002), measured 3 days after the last dose. This inhibition ofT. gondiitachyzoitesin vitroandin vivoindicates that spiroindolone is a promising lead candidate for further medicine development.

scholarly journals TolC-Dependent Secretion of an Ankyrin Repeat-Containing Protein of Rickettsia typhi

2012 ◽  
Vol 194 (18) ◽  
pp. 4920-4932 ◽  
Author(s):  
Simran J. Kaur ◽  
M. Sayeedur Rahman ◽  
Nicole C. Ammerman ◽  
Magda Beier-Sexton ◽  
Shane M. Ceraul ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Transcript Abundance ◽  
Ankyrin Repeat ◽  
Transcriptional Analysis ◽  
Insertion Mutant ◽  
Dependent Manner ◽  
Rickettsia Typhi ◽  
Content Type ◽  
E Coli

ABSTRACTRickettsia typhi, the causative agent of murine (endemic) typhus, is an obligate intracellular pathogen with a life cycle involving both vertebrate and invertebrate hosts. In this study, we characterized a gene (RT0218) encoding a C-terminal ankyrin repeat domain-containing protein, namedRickettsiaankyrinrepeatprotein 1 (RARP-1), and identified it as a secreted effector protein ofR. typhi.RT0218showed differential transcript abundance at various phases ofR. typhiintracellular growth. RARP-1 was secreted byR. typhiinto the host cytoplasm duringin vitroinfection of mammalian cells. Transcriptional analysis revealed thatRT0218was cotranscribed with adjacent genesRT0217(hypothetical protein) andRT0216(TolC) as a single polycistronic mRNA. Given one of its functions as a facilitator of extracellular protein secretion in some Gram-negative bacterial pathogens, we tested the possible role of TolC in the secretion of RARP-1. UsingEscherichia coliC600 and an isogenictolCinsertion mutant as surrogate hosts, our data demonstrate that RARP-1 is secreted in a TolC-dependent manner. Deletion of either the N-terminal signal peptide or the C-terminal ankyrin repeats abolished RARP-1 secretion by wild-typeE. coli. Importantly, expression ofR. typhitolCin theE. colitolCmutant restored the secretion of RARP-1, suggesting that TolC has a role in RARP-1 translocation across the outer membrane. This work implies that the TolC component of the putative type 1 secretion system ofR. typhiis involved in the secretion process of RARP-1.

scholarly journals New Method for the Orthogonal Labeling and Purification of Toxoplasma gondii Proteins While Inside the Host Cell

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Gregory M. Wier ◽  
Erica M. McGreevy ◽  
Mark J. Brown ◽  
Jon P. Boyle
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Click Reaction ◽  
Severe Disease ◽  
Protozoan Parasite ◽  
Trna Synthetase ◽  
Content Type ◽  
Fluorescent Tags

ABSTRACTToxoplasma gondiiis an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRSNLL) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed “click” reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRSNLL-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRSNLL-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRSNLL-expressing T. gondii. The minimal labeling of secreted proteins is likely a consequence of the selective charging of the initiator tRNA (and not the elongator methionine tRNA) by the heterologously expressed bacterial MetRS.IMPORTANCEStudying how T. gondii modifies the host cell to permit its survival is complicated by the complex protein environment of the host cell. The approach presented in this article provides the first method for specific labeling of T. gondii proteins while the parasite grows inside the host cell. We show that this approach is useful for pulse-chase labeling of parasite proteins duringin vitrogrowth. It should also be applicable duringin vivoinfections and in other apicomplexan parasites, including Plasmodium spp.

scholarly journals Transcriptional Profiling Suggests T Cells Cluster around Neurons Injected with Toxoplasma gondii Proteins

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Emily F. Merritt ◽  
Hannah J. Johnson ◽  
Zhee Sheen Wong ◽  
Adam S. Buntzman ◽  
Austin C. Conklin ◽  
...  
Keyword(s):  
T Cells ◽  
Toxoplasma Gondii ◽  
Immune Cells ◽  
Drug Targets ◽  
Transcriptional Profiling ◽  
Effector Proteins ◽  
Content Type ◽  
Symptomatic Disease

ABSTRACT Toxoplasma gondii’s tropism for and persistence in the central nervous system (CNS) underlies the symptomatic disease that T. gondii causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and which it infects in vivo. This predilection for neurons suggests that T. gondii’s persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on T. gondii-host cell interactions has been done in vitro and in nonneuronal cells. We address this gap by utilizing our T. gondii-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo. Using laser capture microdissection (LCM) and RNA sequencing using RNA-seq, we isolated and transcriptionally profiled T. gondii-injected neurons (TINs), Bystander neurons (nearby non-T. gondii-injected neurons), and neurons from uninfected mice (controls). These profiles show that TIN transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8+ T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8+ T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that T. gondii transcripts were primarily found in the TIN transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8+ T cells. IMPORTANCE Like other persistent intracellular pathogens, Toxoplasma gondii, a protozoan parasite, has evolved to evade the immune system and establish a chronic infection in specific cells and organs, including neurons in the CNS. Understanding T. gondii’s persistence in neurons holds the potential to identify novel, curative drug targets. The work presented here offers new insights into the neuron-T. gondii interaction in vivo. By transcriptionally profiling neurons manipulated by T. gondii, we unexpectedly revealed that immune cells, and specifically CD8+ T cells, appear to cluster around these neurons, suggesting that CD8+ T cells specifically recognize parasite-manipulated neurons. Such a possibility supports evidence from other labs that questions the long-standing dogma that neurons are often persistently infected because they are not directly recognized by immune cells such as CD8+ T cells. Collectively, these data suggest we reconsider the broader role of neurons in the context of infection and neuroinflammation.

scholarly journals Isolation Site Influences Virulence Phenotype of Serotype 14 Streptococcus pneumoniae Strains Belonging to Multilocus Sequence Type 15

2015 ◽  
Vol 83 (12) ◽  
pp. 4781-4790 ◽  
Author(s):  
Zarina Amin ◽  
Richard M. Harvey ◽  
Hui Wang ◽  
Catherine E. Hughes ◽  
Adrienne W. Paton ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Immune Responses ◽  
Sequence Type ◽  
Transcriptional Analysis ◽  
Innate Immune Responses ◽  
Pathogenic Potential ◽  
Content Type ◽  
Niche Adaptation

Streptococcus pneumoniaeis a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. We have previously reported thatS. pneumoniaeserotype 3 isolates belonging to the same multilocus sequence type (MLST) differed markedly inin vitroandin vivophenotypes, in accordance with the clinical site of isolation, suggesting stable niche adaptation within a clonal lineage. In the present study, we have extended our analysis to serotype 14 clinical isolates from cases of sepsis or otitis media that belong to the same MLST (ST15). In a murine intranasal challenge model, five ST15 isolates (three from blood and two from ears) colonized the nasopharynx to similar extents. However, blood and ear isolates exhibited significant differences in bacterial loads in other host niches (lungs, ear, and brain) at both 24 and 72 h postchallenge. In spite of these differences, blood and ear isolates were present in the lungs at similar levels at 6 h postchallenge, suggesting that early immune responses may underpin the distinct virulence phenotypes. Transcriptional analysis of lung tissue from mice infected for 6 h with blood isolates versus ear isolates revealed 8 differentially expressed genes. Two of these were exclusively expressed in response to infection with the ear isolate. These results suggest a link between the differential capacities to elicit early innate immune responses and the distinct virulence phenotypes of clonally relatedS. pneumoniaestrains.

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