scholarly journals Probing the Biology of Giardia intestinalis Mitosomes UsingIn VivoEnzymatic Tagging

2015 ◽  
Vol 35 (16) ◽  
pp. 2864-2874 ◽  
Author(s):  
Eva Martincová ◽  
Luboš Voleman ◽  
Jan Pyrih ◽  
Vojtěch Žárský ◽  
Pavlína Vondráčková ◽  
...  
Keyword(s):  
Protein Import ◽  
Single Step ◽  
Giardia Intestinalis ◽  
Homology Detection ◽  
Content Type ◽  
Unique Character ◽  
Unfolded State ◽  
Specific Proteins ◽  
Species Specific

Giardia intestinalisparasites contain mitosomes, one of the simplest mitochondrion-related organelles. Strategies to identify the functions of mitosomes have been limited mainly to homology detection, which is not suitable for identifying species-specific proteins and their functions. Anin vivoenzymatic tagging technique based on theEscherichia colibiotin ligase (BirA) has been introduced toG. intestinalis; this method allows for the compartment-specific biotinylation of a protein of interest. Known proteins involved in the mitosomal protein import werein vivotagged, cross-linked, and used to copurify complexes from the outer and inner mitosomal membranes in a single step. New proteins were then identified by mass spectrometry. This approach enabled the identification of highly diverged mitosomal Tim44 (GiTim44), the first known component of the mitosomal inner membrane translocase (TIM). In addition, our subsequent bioinformatics searches returned novel diverged Tim44 paralogs, which mediate the translation and mitosomal insertion of mitochondrially encoded proteins in other eukaryotes. However, most of the identified proteins are specific toG. intestinalisand even absent from the related diplomonad parasiteSpironucleus salmonicida, thus reflecting the unique character of the mitosomal metabolism. Thein vivoenzymatic tagging also showed that proteins enter the mitosome posttranslationally in an unfolded state and without vesicular transport.

scholarly journals Ex VivoMaturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites

2016 ◽  
Vol 60 (11) ◽  
pp. 6859-6866 ◽  
Author(s):  
Zi Wei Chang ◽  
Benoit Malleret ◽  
Bruce Russell ◽  
Laurent Rénia ◽  
Carla Claser
Keyword(s):  
Ex Vivo ◽  
Single Step ◽  
Parasite Development ◽  
Ring Stage ◽  
Malaria Parasites ◽  
Rodent Malaria ◽  
Content Type ◽  
Parasite Biology

ABSTRACTEx vivoassay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-termin vitroculture andex vivoantimalarial susceptibility assays are relatively cumbersome, relying onin vivopassage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stagePlasmodium berghei,P. yoelii, andP. vinckei vinckeiusing a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.

scholarly journals Signaling through Syk or CARD9 Mediates Species-Specific Anti- Candida Protection in Bone Marrow Chimeric Mice

mBio ◽  
2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Erik Zajta ◽  
Katalin Csonka ◽  
Adél Tóth ◽  
Laszló Tiszlavicz ◽  
Tamás Németh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chimeric Mice ◽  
Emerging Pathogen ◽  
Content Type ◽  
Clinically Significant ◽  
Species Specific

While C. albicans remains the most clinically significant Candida species, C. parapsilosis is an emerging pathogen with increased affinity to neonates. Syk/CARD9 signaling is crucial in immunity to C. albicans , but its role in in vivo responses to other pathogenic Candida species is largely unexplored.

scholarly journals Evidence ofIn VivoProphage Induction during Clostridium difficile Infection

2012 ◽  
Vol 78 (21) ◽  
pp. 7662-7670 ◽  
Author(s):  
Mathieu Meessen-Pinard ◽  
Ognjen Sekulovic ◽  
Louis-Charles Fortier
Keyword(s):  
Clostridium Difficile ◽  
Bioinformatics Analyses ◽  
Content Type ◽  
Unique Character ◽  
Viral Particles ◽  
Potential Impact ◽  
First Time ◽  
Culture Supernatants

ABSTRACTProphages contribute to the evolution and virulence of most bacterial pathogens, but their role inClostridium difficileis unclear. Here we describe the isolation of fourMyoviridaephages, ϕMMP01, ϕMMP02, ϕMMP03, and ϕMMP04, that were recovered as free viral particles in the filter-sterilized stool supernatants of patients suffering fromC. difficileinfection (CDI). Furthermore, identical prophages were found in the chromosomes ofC. difficileisolated from the corresponding fecal samples. We therefore provide, for the first time, evidence ofin vivoprophage induction during CDI. We completely sequenced the genomes of ϕMMP02 and ϕMMP04, and bioinformatics analyses did not reveal the presence of virulence factors but underlined the unique character of ϕMMP04. We also studied the mobility of ϕMMP02 and ϕMMP04 prophagesin vitro. Both prophages were spontaneously induced, with 4 to 5 log PFU/ml detected in the culture supernatants of the corresponding lysogens. When lysogens were grown in the presence of subinhibitory concentrations of ciprofloxacin, moxifloxacin, levofloxacin, or mitomycin C, the phage titers further increased, reaching 8 to 9 log PFU/ml in the case of ϕMMP04. In summary, our study highlights the extensive genetic diversity and mobility ofC. difficileprophages. Moreover, antibiotics known to represent risk factors for CDI, such as quinolones, can stimulate prophage mobilityin vitroand probablyin vivoas well, which underscores their potential impact on phage-mediated horizontal gene transfer events and the evolution ofC. difficile.

scholarly journals ModifiedmarinerTransposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

2011 ◽  
Vol 78 (3) ◽  
pp. 778-785 ◽  
Author(s):  
Eric R. Pozsgai ◽  
Kris M. Blair ◽  
Daniel B. Kearns
Keyword(s):  
Bacillus Subtilis ◽  
Insertional Mutagenesis ◽  
Inducible Promoter ◽  
Insertion Sequences ◽  
Content Type ◽  
Transcriptional Reporter ◽  
Species Specific ◽  
Genetically Manipulated

ABSTRACTTransposons are mobile genetic elements bounded by insertion sequences that are recognized by a specific mobilizing transposase enzyme. The transposase may mobilize not only the insertion sequences but also intervening DNA.marineris a particularly efficient transposon for the random chromosomal integration of genes and insertional mutagenesis. Here, we modify an existingmarinertransposon, TnYLB, such that it can easily be genetically manipulated and introduced intoBacillus subtilis. We generate a series of three newmarinerderivatives that mobilize spectinomycin, chloramphenicol, and kanamycin antibiotic resistance cassettes. Furthermore, we generate a series of transposons with a strong, outward-oriented, optionally isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter for the random overexpression of neighboring genes and a series of transposons with a promoterlesslacZgene for the random generation of transcriptional reporter fusions. We note that the modification of the base transposon is not restricted toB. subtilisand should be applicable to anymariner-compatible host organism, provided thatin vitromutagenesis or anin vivospecies-specific delivery vector is employed.

scholarly journals Amphotericin B- and Voriconazole-Echinocandin Combinations against Aspergillus spp.: Effect of Serum on Inhibitory and Fungicidal Interactions

2013 ◽  
Vol 57 (10) ◽  
pp. 4656-4663 ◽  
Author(s):  
Antigoni Elefanti ◽  
Johan W. Mouton ◽  
Paul E. Verweij ◽  
Athanassios Tsakris ◽  
Loukia Zerva ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Colorimetric Method ◽  
Antifungal Drugs ◽  
Content Type ◽  
Synergistic Interactions ◽  
Inhibitory Activities ◽  
Additive Interactions ◽  
Species Specific

ABSTRACTAntifungal combination therapy with voriconazole or amphotericin B and an echinocandin is often employed as primary or salvage therapy for management particularly of refractory aspergillosis. The pharmacodynamic interactions of amphotericin B- and voriconazole-based combinations with the three echinocandins caspofungin, micafungin, and anidulafungin in the presence of serum were tested against 15Aspergillus fumigatuscomplex,A. flavuscomplex, andA. terreuscomplex isolates to assess both their growth-inhibitory and fungicidal activities. Thein vitroactivity of each drug alone and in combination at a 1:1 fixed concentration ratio was tested with a broth microdilution colorimetric method, and interactions were assessed by isobolographic analysis. Synergy was found for all amphotericin B- and voriconazole-based combinations, with amphotericin B-based combinations showing strong inhibitory synergistic interactions (interaction indices of 0.20 to 0.52) and with voriconazole-based combinations demonstrating strong fungicidal synergistic interactions (interaction indices of 0.10 to 0.29) (P< 0.001). Drug- and species-specific differences were found, with caspofungin and theA. fumigatuscomplex exhibiting the weakest synergistic interactions. In the presence of serum, the synergistic interactions were reduced in the order (from largest to smallest decrease) micafungin > anidulafungin > caspofungin, andA. flavuscomplex >A. fumigatuscomplex >A. terreuscomplex, resulting in additive interactions, particularly for inhibitory activities of amphotericin B-echinocandin combinations and fungicidal activities of voriconazole-echinocandin combinations. Drug- and species-specific differences were found in the presence of serum for inhibitory activities of antifungal drugs, with the lowest interaction indices being observed for amphotericin B-caspofungin (median, 0.77) and for theA. terreuscomplex (median, 0.56). The presentin vitrodata showed that serum had a major impact on synergistic interactions of amphotericin B-echinocandin and voriconazole-echinocandin combinations, resulting in additive interactions and explaining the indifferent outcomes usually observedin vivo.

scholarly journals Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages

mSphere ◽  
2016 ◽  
Vol 1 (5) ◽  
Author(s):  
Jennifer M. Willingham-Lane ◽  
Londa J. Berghaus ◽  
Steeve Giguère ◽  
Mary K. Hondalus
Keyword(s):  
Host Species ◽  
Opportunistic Pathogen ◽  
Rhodococcus Equi ◽  
Replication Capacity ◽  
Virulence Plasmid ◽  
Intracellular Replication ◽  
Content Type ◽  
Disease Agent ◽  
Species Specific

ABSTRACT This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a conjugative virulence plasmid, pVAPA1037, that expresses Vap proteins, including VapA, essential for intramacrophage replication and virulence in vivo. The understudied R. equi isolates from pigs carry a related but different plasmid, pVAPB, expressing distinct Vap proteins, including VapB. In this work, we document for the first time that R. equi isolates carrying pVAPB-type plasmids are capable of intramacrophage replication. Moreover, we show that R. equi isolates carrying either plasmid type can replicate in both equine and swine macrophages, indicating that host species tropism is not due to species-specific intramacrophage replication capabilities defined by plasmid type. Furthermore, plasmid swapping between equine and swine strains did not alter intracellular replication capacity, indicating that coevolution of the plasmid and chromosome is not essential for intracellular growth. The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a multihost, facultative intracellular pathogen of macrophages. When inhaled by susceptible foals, it causes severe bronchopneumonia. It is also a pathogen of pigs, which may develop submaxillary lymphadenitis upon exposure. R. equi isolates obtained from foals and pigs possess conjugative plasmids housing a pathogenicity island (PAI) containing a novel family of genes of unknown function called the virulence-associated protein or vap family. The PAI regions of the equine and swine plasmids differ in vap gene composition, with equine isolates possessing six vap genes, including the major virulence determinant vapA, while the PAIs of swine isolates house vapB and five other unique vap genes. Possession of the pVAPA-type virulence plasmid by equine isolates bestows the capacity for intramacrophage replication essential for disease development in vivo. Swine isolates of R. equi are largely unstudied. Here, we show that R. equi isolates from pigs, carrying pVAPB-type plasmids, are able to replicate in a plasmid-dependent manner in macrophages obtained from a variety of species (murine, swine, and equine) and anatomical locations. Similarly, equine isolates carrying pVAPA-type plasmids are capable of replication in swine macrophages. Plasmid swapping between equine and swine strains through conjugation did not alter the intracellular replication capacity of the parental strain, indicating that coevolution of the plasmid and chromosome is not crucial for this attribute. These results demonstrate that while distinct plasmid types exist among R. equi isolates obtained from equine and swine sources, this tropism is not determined by host species-specific intramacrophage replication capabilities. IMPORTANCE This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a conjugative virulence plasmid, pVAPA1037, that expresses Vap proteins, including VapA, essential for intramacrophage replication and virulence in vivo. The understudied R. equi isolates from pigs carry a related but different plasmid, pVAPB, expressing distinct Vap proteins, including VapB. In this work, we document for the first time that R. equi isolates carrying pVAPB-type plasmids are capable of intramacrophage replication. Moreover, we show that R. equi isolates carrying either plasmid type can replicate in both equine and swine macrophages, indicating that host species tropism is not due to species-specific intramacrophage replication capabilities defined by plasmid type. Furthermore, plasmid swapping between equine and swine strains did not alter intracellular replication capacity, indicating that coevolution of the plasmid and chromosome is not essential for intracellular growth.

scholarly journals Characterization ofIn VitroResistance Development to the Novel Echinocandin CD101 in Candida Species

2016 ◽  
Vol 60 (10) ◽  
pp. 6100-6107 ◽  
Author(s):  
Jeffrey B. Locke ◽  
Amanda L. Almaguer ◽  
Douglas E. Zuill ◽  
Ken Bartizal
Keyword(s):  
Drug Exposure ◽  
Hot Spot ◽  
Serial Passage ◽  
Single Step ◽  
Cross Resistance ◽  
Time Curve ◽  
Resistance Development ◽  
Content Type ◽  
Development Of Resistance

ABSTRACTCD101 is a novel echinocandin with a long half-life undergoing clinical development for treatment of candidemia/invasive candidiasis and vulvovaginal candidiasis. The potential for and mechanisms underlying the development of resistance to CD101 inCandidaspecies were investigated by using spontaneous resistance and serial passage selection methodologies. FourCandidaspp. (C. albicans,C. glabrata,C. parapsilosis, andC. krusei) were chosen for resistance characterization with CD101, anidulafungin, and caspofungin. The frequency of spontaneous, single-step mutations conferring reduced susceptibility to CD101 at 1× the agar growth inhibition concentration was low across all species, with median frequencies ranging from 1.35 × 10−8to 3.86 × 10−9, similar to ranges generated for anidulafungin and caspofungin. Serial passage ofCandidaspp. on agar plates containing drug gradients demonstrated a low potential for resistance development, with passage 20 CD101-selected strains possessing increases in MICs equivalent to or lower than those for the majority of strains generated under selection with anidulafungin and caspofungin. A total of 12fks“hot spot” mutations were identified, typically in strains with the highest MIC shifts. Cross-resistance was broadly observed among the 3 echinocandins evaluated, with no CD101-selected mutants (with or withoutfkshot spot mutations) exhibiting reduced susceptibility to CD101 but not also to anidulafungin and/or caspofungin. Consistent with currently approved echinocandins, CD101 demonstrates a low potential for resistance development, which could be further enhancedin vivoby the high maximum concentration of drug in serum (Cmax)/area under the concentration-time curve (AUC) plasma drug exposure achieved with once-weekly dosing of CD101.

scholarly journals Proximity Staining Using Enzymatic Protein Tagging in Diplomonads

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Ásgeir Ástvaldsson ◽  
Kjell Hultenby ◽  
Staffan G. Svärd ◽  
Jon Jerlström-Hultqvist
Keyword(s):  
Ascorbate Peroxidase ◽  
Cell Biology ◽  
Ultrastructural Localization ◽  
Giardia Intestinalis ◽  
Model Systems ◽  
Human Pathogen ◽  
Content Type ◽  
Superresolution Microscopy ◽  
Cellular Compartments

ABSTRACT The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis. Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM) to enable the study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags require light-induced activation (1, 2) or are inactive in many cellular compartments (3), while ascorbate peroxidase has not been shown to have those limitations. Here, we have optimized the in vivo activities of two versions of pea ascorbate peroxidase (APXW41F and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis. We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3′-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida. Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with superresolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM, and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalis. IMPORTANCE The function of many proteins is intrinsically related to their cellular location. Novel methods for ascertainment of the ultrastructural location of proteins have been introduced in recent years, but their implementation in protists has so far not been readily realized. Here, we present an optimized proximity labeling protocol using the APEX system in the salmon pathogen Spironucleus salmonicida. This protocol was also applicable to the human pathogen Giardia intestinalis. Both organisms required extraneous addition of hemin to the growth medium to enable detectable peroxidase activity. Further, we saw no inherent limitation in labeling efficiency coupled to the cellular compartment, as evident with some other proximity labeling systems. We anticipate that the APEX proximity labeling system might offer a great resource to establish the ultrastructural localization of proteins across genetically tractable protists but might require organism-specific labeling conditions.

scholarly journals Distribution of the SELMA Translocon in Secondary Plastids of Red Algal Origin and Predicted Uncoupling of Ubiquitin-Dependent Translocation from Degradation

2012 ◽  
Vol 11 (12) ◽  
pp. 1472-1481 ◽  
Author(s):  
Simone Stork ◽  
Daniel Moog ◽  
Jude M. Przyborski ◽  
Ilka Wilhelmi ◽  
Stefan Zauner ◽  
...  
Keyword(s):  
Phaeodactylum Tricornutum ◽  
Protein Import ◽  
Content Type ◽  
Evolutionary Origins ◽  
Red Algal ◽  
Multistep Process ◽  
Core Components ◽  
Secondary Plastids ◽  
Periplastidal Compartment

ABSTRACT Protein import into complex plastids of red algal origin is a multistep process including translocons of different evolutionary origins. The s ymbiont-derived E RAD- l ike ma chinery (SELMA), shown to be of red algal origin, is proposed to be the transport system for preprotein import across the periplastidal membrane of heterokontophytes, haptophytes, cryptophytes, and apicomplexans. In contrast to the canonical endoplasmic reticulum-associated degradation (ERAD) system, SELMA translocation is suggested to be uncoupled from proteasomal degradation. We investigated the distribution of known and newly identified SELMA components in organisms with complex plastids of red algal origin by intensive data mining, thereby defining a set of core components present in all examined organisms. These include putative pore-forming components, a ubiquitylation machinery, as well as a Cdc48 complex. Furthermore, the set of known 20S proteasomal components in the periplastidal compartment (PPC) of diatoms was expanded. These newly identified putative SELMA components, as well as proteasomal subunits, were in vivo localized as PPC proteins in the diatom Phaeodactylum tricornutum . The presented data allow us to speculate about the specific features of SELMA translocation in contrast to the canonical ERAD system, especially the uncoupling of translocation from degradation.

scholarly journals An Improved Recombination-BasedIn VivoExpression Technology-Like Reporter System Reveals DifferentialcyaAGene Activation in Bordetella Species

2013 ◽  
Vol 81 (4) ◽  
pp. 1295-1305 ◽  
Author(s):  
Matthew S. Byrd ◽  
Eliza Mason ◽  
Michael W. Henderson ◽  
Erich V. Scheller ◽  
Peggy A. Cotter
Keyword(s):  
Virulence Factors ◽  
Gene Activation ◽  
Regulatory System ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Genes Encoding ◽  
Species Specific

ABSTRACTBordetella pertussisandBordetella bronchisepticarely on the global two-component regulatory system BvgAS to control expression of distinct phenotypic phases. In the Bvg−phase, expression ofvrggenes, including those required for motility inB. bronchiseptica, is activated and genes encoding virulence factors are not expressed. Conversely, in the Bvg+phase, genes encoding virulence factors are highly expressed while genes necessary for motility are repressed. Although several genetic analyses have demonstrated the importance of the Bvg+phase during respiratory infection, Bvg-regulated gene activation inB. bronchisepticahas not been investigatedin vivo. To address this, we developed a plasmid, pGFLIP, that encodes a sensitive Flp recombinase-based fluorescent reporter system able to document gene activation bothin vitroandin vivo. Using pGFLIP, we demonstrated thatcyaA, considered to be a “late” Bvg+phase gene, is activated substantially earlier inB. bronchisepticathanB. pertussisfollowing a switch from Bvg−to Bvg+phase conditions. We show that the altered activation ofcyaAis not due to differences in thecyaApromoter or in thebvgASalleles ofB. bronchisepticacompared toB. pertussis, but appears to be species specific. Finally, we used pGFLIP to show thatflaAremains repressed during infection, confirming thatB. bronchisepticadoes not modulate to the Bvg−phasein vivo.

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