Le financement a été fourni par le NIH / NIAID octroi RO1-AI-63457 et de l&#39;ARRA subventions complément RO1-AI-63457 S1. Nous reconnaissons les contributions de tous les membres actuels et passés du groupe de laboratoires et des collaborateurs.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+of+leprosy+for+control+programmes.">Chemotherapy of leprosy for control programmes.</a> Technical Report Series. 675, 18-22 (1982).</li><li>Cole, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massive+gene+decay+in+the+leprosy+bacillus.">Massive gene decay in the leprosy bacillus.</a> Nature. 409, 1007-1011 (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leproma+Web+Server.">Leproma Web Server.</a> Leproma Web Server [Internet]. , (2004).</li><li>Groathouse, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Polymorphic+Loci+for+Molecular+Typing+of+Strains+of+Mycobacterium+leprae.">Multiple Polymorphic Loci for Molecular Typing of Strains of Mycobacterium leprae.</a> J. of Clinical Microbiology. 42, 1666-1672 (2004).</li><li>Young, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Short+Tandem+Repeat+Sequences+to+Study+Mycobacterium+leprae+in+Leprosy+Patients+in+Malawi+and+India.">Use of Short Tandem Repeat Sequences to Study Mycobacterium leprae in Leprosy Patients in Malawi and India.</a> Public Library of Science: Neglected Tropical Diseases. 2, E214-E214 (2008).</li><li>Monot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Variable-Number+Tandem+Repeats+Appropriate+for+Genotyping+Mycobacterium+leprae.">Are Variable-Number Tandem Repeats Appropriate for Genotyping Mycobacterium leprae.</a> J. of Clinical Microbiology. 46, 2291-2297 (2008).</li><li>Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Variable+Number+Tandem-Repeat+Genotyping+for+Mycobacterium+leprae+Clinical+Specimens.">Rapid Variable Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical Specimens.</a> J. of Clinical Microbiology. 47, 1757-1766 (2009).</li><li>Weng, X. i. a. o. m. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Distribution+of+Mycobacterium+leprae+Genotypes+in+a+Region+of+High+Leprosy+Prevalence+in+China:+a+3-Year+Molecular+Epidemiological+Study.">Identification and Distribution of Mycobacterium leprae Genotypes in a Region of High Leprosy Prevalence in China: a 3-Year Molecular Epidemiological Study.</a> J. of Clinical Microbiology. 45, 1728-1734 (2007).</li><li>Weng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+leprosy+in+Qiubei+County,+Yunnan,+China:+Insights+from+an+eight+year+molecular+epidemiology+investigation.">Transmission of leprosy in Qiubei County, Yunnan, China: Insights from an eight year molecular epidemiology investigation.</a> Infection, Genetics and Evolution. , (2010).</li><li>Sakamuri, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-Based+Molecular+Epidemiology+of+Leprosy+in+Cebu,+Philippines.">Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines.</a> J. of Clinical Microbiology. 47, 2844-2854 (2009).</li><li>Fontes, A. N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+diversity+of+Mycobacterium+leprae+isolates+from+Brazilian+leprosy+patients.">Genetic diversity of Mycobacterium leprae isolates from Brazilian leprosy patients.</a> Leprosy Review. 80, 302-315 (2009).</li></ol>

La collecte d&#39;échantillons de peau de patients lépreux nécessite cliniciens qualifiés ou techniciens travaillant dans des cliniques de peau. Les travailleurs de laboratoire manipulant ces échantillons doit prendre grand soin de porter des blouses de laboratoire, des gants et des lunettes de protection et de travailler dans un cabinet de bio-sécurité lors de la manipulation des échantillons de tissus humains infectés ou tatou. Désinfection des surfaces et des outils est également critique. Travailler dans un endroit propre, stérile de bio-sécurité armoire est important pour éviter la contamination des échantillons d&#39;ADN. Extraction de l&#39;ADN est devenu relativement facile grâce au développement de kits d&#39;extraction d&#39;entreprises telles que Qiagen. Des directives doivent être suivies attentivement. Tous les échantillons doivent être conservés au froid lorsqu&#39;il n&#39;est pas utilisé. Évitez répétées, des changements de température extrêmes pour les échantillons. Amorces d&#39;ADN utilisées dans ce travail peut être commandé auprès de diverses entreprises qui ont été cités dans la liste des références bibliographiques à la fin du présent document. Il faut prendre soin de travailler avec des amorces dans un environnement de l&#39;ADN libre, afin d&#39;éviter toute contamination. Primaires viennent sous forme de poudre sèche et doit être mélangé avec TE et dilué à une concentration 100 microns, puis séparés en petites quantités (figure 2a). Les solutions de travail des amorces sont encore dilués à des concentrations 10 uM. Encore une fois, ne pas utiliser des échantillons d&#39;ADN dans les zones / hottes où l&#39;on utilise des amorces et préparés. Les produits de PCR doivent également être conservés au froid lorsqu&#39;il n&#39;est pas utilisé. Un gel d&#39;agarose 3% la préparation donnera une meilleure séparation bande d&#39;ADN, mais prend plus de temps à courir. Pour des raisons purement qualitative, 2% est généralement suffisant. Solution de bromure d&#39;éthidium utilisé pour la coloration de l&#39;ADN dans les gels d&#39;agarose est une génotoxine très actif qui est absorbé par la peau. Manipuler cette solution et les gels colorés avec de l&#39;utiliser le plus grand soin et toujours en étant sûr de porter des gants. Laver soigneusement les mains après avoir manipulé des échantillons d&#39;ADN ou de matériaux de bromure d&#39;éthidium. Eliminer la solution une coloration au bromure d&#39;éthidium, les laver et les gels selon les directives institutionnelles. Les gels sont pas systématiquement requis; cette étape peut être éliminé une fois les méthodes FLA ont été établis. Il est temps et consommer réactif. La solution de formamide utilisées dans la préparation des échantillons pour FLA est également très toxique et doit être manipulé avec soin. Se laver les mains après son utilisation. Débarrassez-vous de la suite des directives institutionnelles. Lecture des résultats du FLA en utilisant le logiciel du scanner de pointe peuvent être difficiles. Un ensemble de base des appels allèle (nombre de répétitions en tandem) a été développée au CSU (tableaux 4-7). (Il convient de noter que tableaux 4-7 peut-être pas tout compris. Comme d&#39;autres souches de M. leprae sont étudiés, avec des allèles nombre de copies en dehors des plages inscrites peuvent être trouvés.) Un défi particulier consiste à &quot;bégayer&quot; pics. Ce sont des familles de pics, en particulier des déclarations de soupçon qui n&#39;impliquent que deux à trois répétitions de paires de bases, telles que (TA) 10. Parfois, la sélection des pics corriger à lire est difficile (figure 7). Dans cette figure, le pic dans le contrôle positif à 190 pb est le pic principal. Inférieure Peaks en hauteur qui sont 2, 4 ou même 6 paires de base plus grande ou plus petite que le pic principal sont appelés «pics bégaiement.&quot; A-tailing peut aussi causer de la confusion. A-tailing est décrit plus haut dans le texte et la figure 8. Enfin, si les produits de PCR sont très abondants dans les échantillons, les pics peuvent apparaître avec un pic double. Dans ce cas, lisez le centre de la forme de pointe. (Voir Figure 6, Patient 6.) La gestion des données peut être une tâche redoutable pour ce type de travail. Il est important d&#39;enregistrer toutes les informations pertinentes pour toutes les expériences telles que: la date d&#39;une tâche ou une procédure, l&#39;opérateur, bandes / plaques cartes, des ordres FLA, des conditions de PCR et les recettes, les dates des gels et des photographies, des températures de stockage, des dilutions matrice d&#39;ADN, FLA électroniques lieux de stockage de fichiers, organisation, etc Bonne gestion des données et peut sauver des heures de temps passé à chercher des éléments d&#39;information dans le futur. Le travail de laboratoire décrit ici a été en cours depuis plusieurs années à la Colorado State University et à d&#39;autres endroits dans le monde. La grande photo de ce que l&#39;ensemble des données recueillies signifie et comment elle peut être d&#39;un usage futur commence à émerger. Figures 10A et 10B montrent comment ces empreintes d&#39;ADN peuvent être utilisés pour distinguer les différentes M. leprae tensions entre les pays (10A), ou même entre les familles (10B). Cas de la famille et la communauté liée a été démontré pour mener M. leprae d&#39;similaires ou identiques types de souches VNTR. L&#39;espoir est que cela peut être possible d&#39;avoir un aperçu plus loin dans le mode (s) de transmission de la lèpre afin qu&#39;un système de détection précoce des réseaux de transmission peuvent être développés pour ces mos de personnest à risque, et que la pharmacothérapie curative peut commencer avant des dommages neurologiques permanents et dermatologiques est fait.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> Sang DNeasy et Kit de tissus </td><td> Qiagen </td><td> 69504 </td><td></td></tr><tr><td> Multiplex PCR Kit </td><td> Qiagen </td><td> 206143 </td><td></td></tr><tr><td> DNA Primers </td><td> Divers </td><td></td><td></td></tr><tr><td> Gel d&#39;agarose </td><td> Divers </td><td></td><td></td></tr><tr><td> 5x ou 6x tampon de chargement de gel </td><td> New England Biolabs </td><td> B7021S </td><td> Recettes disponibles pour rendre votre propre * </td></tr><tr><td> Solution de bromure d&#39;éthidium </td><td> Divers </td><td></td><td> Diluer du con. </td></tr><tr><td> Salut-Di Formamide Solution </td><td> Applied Biosystems </td><td> 4311320 </td><td></td></tr><tr><td> Gene scan -500 LIZ </td><td> Applied Biosystems </td><td> 4322682 </td><td></td></tr></tbody></table> <a href="http://biowww.net/buffer-reagent/6X-Gel-loading-buffer-bromophenol-blue-sucrose.html">* Http://biowww.net/buffer-reagent/6X-Gel-loading-buffer-bromophenol-blue-sucrose.html</a> <table border="1"><tbody><tr><th> Équipement </th><th> Commentaires </th></tr><tr><td> 2 Réfrigérateurs </td><td> 4 ° C: 1 pour les échantillons d&#39;ADN, une pour les amorces et les réactifs Multiplex </td></tr><tr><td> 1 four micro-ondes </td><td> Ou de façon appropriée pour le chauffage et l&#39;eau d&#39;agarose pour faire des gels </td></tr><tr><td> 1 autoclave </td><td> Autocuiseur ou pour stériliser des matériaux </td></tr><tr><td> Machine à PCR </td><td> Thermocycleur </td></tr><tr><td> 2 jeux de pipettes </td><td> 0,5 à 10 ul, 10-100 ul, 20-200 ul, 1000 ul 1 jeu pour les apprêts, 1 jeu de l&#39;ADN </td></tr><tr><td> Submarine boîte électrophorèse sur gel </td><td> Avec des formes et des peignes pour les gels préparation. </td></tr><tr><td> Alimentation d&#39;électrophorèse </td><td> Avec des câbles pour la connexion à la boîte de gel </td></tr><tr><td> Bloc de chaleur </td><td> Pour tubes Eppendorf </td></tr><tr><td> Centrifugeuse </td><td> Pour les tubes Eppendorf / bandes </td></tr><tr><td> Système d&#39;électrophorèse capillaire (analyseur génétique) </td><td> Ou l&#39;accès à une institution qui peut effectuer cette analyse </td></tr><tr><td> Plastiques </td><td> Jetable d&#39;aérosol de pipette, tubes Eppendorf, des bandes de 8 puits, des plaques de 96 puits, dont certains de qualité optique </td></tr><tr><td> Un ordinateur avec connexion Internet </td><td></td></tr></tbody></table>

dna fingerprinting of mycobacterium leprae strains using variable number tandem repeat (vntr) - fragment length analysis (fla)

L&#39;étude de la transmission de la lèpre est particulièrement difficile car l&#39;agent causal, Mycobacterium leprae, ne peuvent pas être cultivées en laboratoire. Les seules sources de la bactérie sont malades de la lèpre et de tatous infectés expérimentalement et des souris nude. Ainsi, de nombreuses méthodes utilisées en épidémiologie moderne ne sont pas disponibles pour l&#39;étude de la lèpre. Malgré un vaste programme mondial de la drogue traitement de la lèpre en œuvre par l&#39;OMS une, la lèpre reste endémique dans de nombreux pays, avec environ 250 000 nouveaux cas chaque année. 2 L&#39;ensemble de M. leprae génome a été cartographié 3,4 et de nombreux locus ont été identifiés qui ont répété les segments de deux ou plusieurs paires de bases (appelés micro et minisatellites). 5 souches cliniques de M. leprae peut varier dans le nombre de segments répétés en tandem (courtes répétitions en tandem, STR) à plusieurs de ces loci. 5,6,7 répéter en tandem en nombre variable (VNTR) 5 analyse a été utilisée pour distinguer les différentes souches du bacille de la lèpre. Certains des loci semblent être plus stables que d&#39;autres, montrant moins de variation dans le nombre le répète, tandis que d&#39;autres semblent évoluer plus rapidement, parfois chez le même patient. Alors que la variabilité de certains VNTR a soulevé des questions quant à leur aptitude à 7,8,9 typage des souches, les nouvelles données indiquent que l&#39;analyse de loci multiples, qui sont diversifiés dans leur stabilité, peut être utilisé comme un outil précieux épidémiologiques. Plusieurs locus VNTR analyse (MLVA) 10 a été utilisé pour étudier l&#39;évolution de la lèpre et la transmission dans plusieurs pays dont la Chine 11,12, au Malawi 8, les Philippines 10,13, et le Brésil 14. MLVA implique plusieurs étapes. Tout d&#39;abord, l&#39;ADN bactérien est extrait avec l&#39;ADN du tissu hôte à partir de biopsies cliniques ou frottis fente (SSS). 10 Le locus désiré sont ensuite amplifiés de l&#39;ADN extrait par réaction en chaîne de polymérase (PCR). Amorces marqués à la fluorescence pour 4-5 loci différents sont utilisés par réaction, avec 18 loci étant amplifié dans un total de quatre réactions. 10 Les produits de PCR peut être soumis à une électrophorèse sur gel d&#39;agarose pour vérifier la présence des segments d&#39;ADN désirée, puis soumis à une analyse de longueur des fragments fluorescents (FLA) par électrophorèse capillaire. ADN de bactéries de tatou passages avec un nombre connu de copies répéter pour chaque locus est utilisé comme contrôle positif. Les chromatogrammes FLA sont ensuite examinés en utilisant le logiciel du scanner Peak et de longueur des fragments est converti en nombre de copies VNTR (allèle). Enfin, les haplotypes VNTR sont analysés pour les modèles, et lorsqu&#39;ils sont combinés avec les données cliniques des patients peuvent être utilisés pour suivre la distribution des types de souches.

Watch this Scientific Journal Video about DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA at JoVE.com

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA

La lèpre, causée par<em&gt; Mycobacterium leprae</em&gt;, Est encore endémique dans de nombreux endroits. Afin d&#39;en apprendre davantage sur la propagation et le mode de transmission de la lèpre, il est important de déterminer quelle souche de<em&gt; M. leprae</em&gt; A infecté un patient. Nombre variable de répétitions en tandem (VNTR) frappe est un tel procédé.

L&#39;empreinte génétique des Mycobacterium leprae Souches à l&#39;aide variable Tandem Repeat Nombre (VNTR) - Analyse de la longueur des fragments (FLA)

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory.  ...

dna-fingerprinting-mycobacterium-leprae-strains-using-variable-number

Research

JoVE Journal

Immunology and Infection

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

26.9K vues.  Colorado State University. La lèpre, causée par Mycobacterium leprae, Est encore endémique dans de nombreux endroits. Afin d'en apprendre davantage sur la propagation et le mode de transmission de la lèpre, il est important de déterminer quelle souche de M. leprae A infecté un patient. Nombre variable de répétitions en tandem (VNTR) frappe est un tel procédé.

Vidéo: DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Séparation de l&#39;ADN simple brin, double brin de l&#39;ADN et l&#39;ARN à partir d&#39;un environnement communautaire viral en utilisant chromatographie d&#39;hydroxyapatite

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Recherche

Immunologie et infection

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

La croissance de la Mycobacterium tuberculosis Les biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Manipulation génétique<em&gt; Δku80</em&gt; Les souches pour l&#39;analyse génomique fonctionnelle de<em&gt; Toxoplasma gondii</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1nu). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Mycobacterium leprae, the causative agent of leprosy, does not grow in vitro. We describe an easy to follow protocol to prepare a bacillary suspension to ensure the maintenance of large quantities of M. leprae for a variety of applications. Protocols for propagation by mouse footpad inoculation, evaluation of viability, freezing and thawing bacillary stock are described in detail.

Protocoles optimisés pour<em&gt; Mycobacterium leprae</em&gt; Gestion de traction: Frozen Stock préservation et l&#39;entretien dans des souris nude athymiques

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Mise en place d&#39;un<em&gt; In vitro</em&gt; Système d&#39;étudier le comportement intracellulaire de<em&gt; Candida glabrata</em&gt; En droits macrophages THP-1

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Faisant preuve d&#39;une multi-résistante<em&gt; Mycobacterium tuberculosis</em&gt; Amplification de puces à ADN

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Système d&#39;efficacité et de dépistage des inhibiteurs Cytotoxicité ciblage intracellulaire<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Création d’un Dense Transposon Insertion bibliothèque à l’aide de la conjugaison bactérienne dans les entérobactéries souches telles que Escherichia Coli et Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analyse de 18FDG TEP/CT d’imagerie comme un outil pour étudier Mycobacterium tuberculosis Infection et traitement chez les Primates Non humains

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Imagerie de Mycobacterium tuberculosis chez la souris avec la journaliste Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...