Die Finanzierung wurde durch die NIH / NIAID gewähren RO1-AI-63457 und ARRA gewähren ergänzen RO1-AI-63457 S1 zur Verfügung gestellt. Wir erkennen die Beiträge aller aktuellen und ehemaligen Mitgliedern des Labors Gruppe und Mitarbeiter.

<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>

Die Sammlung von Hautproben von Lepra-Patienten erfordert qualifizierte Ärzte oder Techniker arbeiten an Hautkliniken. Labormitarbeiter Umgang mit diesen Proben müssen mit großer Sorgfalt zu Laborkittel, Schutzhandschuhe und Schutzbrille tragen zu tragen und in einem Bio-Sicherheitsschrank Arbeit beim Umgang mit infizierten Proben menschlichen oder Gürteltier Gewebe. Desinfektion von Oberflächen und Tools ist ebenfalls entscheidend. Arbeiten in einem sauberen, sterilen biologisch sichere Schrank ist zur Vermeidung von Kontamination von DNA-Proben wichtig. DNA-Extraktion hat sich relativ einfach durch die Entwicklung der Extraktion Kits von Firmen wie Qiagen. Anfahrt müssen unbedingt beachtet werden. Alle Proben sollten kühl gehalten werden, wenn nicht in Gebrauch ist. Vermeiden Sie wiederholte, extreme Temperaturschwankungen für die Proben. DNA-Primer in dieser Arbeit verwendet werden kann aus verschiedenen Unternehmen, die in der Liste der Literaturangaben wurden am Ende dieses Beitrags zitiert bestellt werden. Es ist darauf zu mit Primern in einem DNA-freien Umgebung zu arbeiten, um eine Kontamination zu vermeiden. Primer kommen als trockenes Pulver und muss mit TE gemischt und verdünnt, um eine 100 &amp;mgr; Konzentration, dann in kleineren Mengen (Abbildung 2a) getrennt. Working-Lösungen von Primer sind weiter auf 10 um Konzentrationen verdünnt. Auch hier verwenden Sie keine DNA-Proben in den Bereichen / Hauben wo Primer verwendet und sind bereit. PCR-Produkte sollten auch gehalten, wenn nicht in Gebrauch ist kalt. Eine 3% Agarosegel Vorbereitung wird eine bessere DNA-Bande Trennung, dauert aber länger zu laufen. Für rein qualitative Zwecke, 2% in der Regel ausreichend. Ethidiumbromidlösung zur Färbung der DNA in Agarosegelen verwendet ist ein hochaktiver genotoxische Wirkung, die durch die Haut absorbiert wird. Handle dieser Lösung und Gele mit ihm mit großer Sorgfalt und stets sicher, dass das Tragen von Handschuhen gefärbt. Waschen Sie Ihre Hände nach dem Umgang mit allen DNA-Proben oder Ethidiumbromid Materialien. Entsorgen Ethidiumbromidfärbung Lösung, waschen und Gele nach den Richtlinien des Instituts. Gele werden nicht routinemäßig erforderlich, kann dieser Schritt entfallen, sobald FLA-Methoden festgelegt wurden. Es ist Zeit und Reagenzien verbraucht. Die Formamidlösung in Vorbereitung der Proben für die FLA verwendet wird, auch sehr giftig und sollte mit Sorgfalt behandelt werden. Waschen Sie Ihre Hände nach dem Gebrauch. Entsorgen Sie folgende Richtlinien des Instituts. Beim Lesen der Ergebnisse der FLA mit Peak-Scanner-Software kann eine Herausforderung sein. Eine Reihe grundlegender Allel Anrufe (Anzahl der Tandem-Repeats) hat der CSU (Tabellen 4-7) entwickelt worden. (Anzumerken ist, dass Tabellen 4-7 nicht all inclusive sein. Wie auch andere Stämme von M. leprae studiert werden, Allele mit Kopie Zahlen außerhalb der Bereiche aufgeführt gefunden werden kann.) Eine besondere Herausforderung besteht darin, &#39;stottern&#39; Gipfel. Dies sind Familien von Gipfeln, vor allem von Verdachtsanzeigen, die nur um 2-3 Basenpaare wiederholt, wie (TA) 10. Manchmal Auswahl der richtigen Höhepunkt zu lesen ist schwierig (Abbildung 7). In dieser Abbildung ist der Höhepunkt in der positiven Kontrolle bei 190 bp der Hauptpeak. Peaks geringer Höhe, die 2 sind, 4 oder sogar 6 Basenpaare größer oder kleiner als der Hauptgipfel heißen &quot;stottern Gipfel.&quot; A-Tailing kann auch zu Verwechslungen führen. A-Tailing ist bereits im Text und in Abbildung 8 beschrieben. Schließlich, wenn PCR-Produkte sehr häufig in den Proben sind, kann Peaks mit einer Doppel-Spitze erscheinen. In diesem Fall lesen Sie die Mitte des Peaks zu bilden. (Siehe Abbildung 6, Patient 6). Datenmanagement kann eine gewaltige Aufgabe für diese Art von Arbeit sein. Es ist wichtig, alle relevanten Informationen für alle Experimente wie Rekord: Datum einer Aufgabe oder eines Verfahrens, Betreiber, Streifen / Platte Karten, FLA Bestellungen, PCR-Bedingungen und Rezepte, Termine von Gelen und Fotografien, Lagertemperaturen, DNA-Template Verdünnungen, FLA elektronische Datei Lagerorte, etc. Eine gute Organisation und Datenverwaltung, kann Ihnen Stunden Zeit damit verbracht, auf der Suche nach spezifischen Informationen in die Zukunft. Das Labor hier beschriebenen Arbeiten hat schon seit mehreren Jahren an der Colorado State University und an anderen Orten auf der ganzen Welt. Das große Bild von dem, was all der gesammelten Daten bedeutet und wie es bezüglich der künftigen Nutzung ist im Entstehen begriffen. 10A und 10B zeigen, wie diese DNA-Fingerprints verwendet werden, um verschiedene M. zu unterscheiden leprae Stämme zwischen den Ländern (10A) oder auch zwischen Familien (10B). Familie und Gemeinschaft verbunden Fällen hat sich gezeigt, M. tragen leprae von ähnlichen oder identischen VNTR Belastung Typen. Die Hoffnung ist, dass es möglich sein, weitere Einblicke in die Wirkungsweise (n) der Lepra Übertragung zu gewinnen, so dass eine Früherkennung von Übertragungsnetzen für jene Leute mos entwickelt werden könnent in Gefahr, und das heilende medikamentöse Therapie beginnen kann, bevor bleibende neurologische und dermatologische Schaden entsteht.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> DNeasy Blood and Tissue Kit </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-Primer </td><td> Verschiedene </td><td></td><td></td></tr><tr><td> Agarose Gel </td><td> Verschiedene </td><td></td><td></td></tr><tr><td> 5x oder 6x Gel-Ladepuffer </td><td> New England Biolabs </td><td> B7021S </td><td> Rezepte zur Verfügung, um Ihre eigenen * machen </td></tr><tr><td> Ethidiumbromid-Lösung </td><td> Verschiedene </td><td></td><td> Verdünnen von con. </td></tr><tr><td> Hallo-Di Formamidlösung </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> Ausstattung </th><th> Kommentare </th></tr><tr><td> 2 Kühlschränke </td><td> 4 ° C: 1 für die DNA-Proben, 1 für Grundierungen und Multiplex-Reagenzien </td></tr><tr><td> 1 Mikrowellenherd </td><td> Oder geeigneter Weise für Heizung und Wasser Agarose Gele machen </td></tr><tr><td> 1-Autoklaven </td><td> Oder Dampfkochtopf für die Sterilisation Materialien </td></tr><tr><td> PCR-Maschine </td><td> Thermocycler </td></tr><tr><td> 2 Sätze von Pipetten </td><td> 0,5-10 ul, 10-100 ul, 20-200 ul, 1000 ul 1 Satz für Primer, 1 Satz für DNA </td></tr><tr><td> Submarine Gelelektrophorese box </td><td> Mit Formen und Kämme für Herstellung von Gelen. </td></tr><tr><td> Elektrophorese-Netzteil </td><td> Mit Kabel für den Anschluss an Gel-Box </td></tr><tr><td> Heizblock </td><td> Für Eppendorf-Röhrchen </td></tr><tr><td> Zentrifuge </td><td> Für Eppendorf-Röhrchen / Streifen </td></tr><tr><td> Kapillarelektrophorese-System (Genetic Analyzer) </td><td> Oder um eine Institution, die diese Analyse durchzuführen zugreifen können </td></tr><tr><td> Plastics </td><td> Einweg-Aerosol-Pipettenspitzen, Eppendorf-Röhrchen, 8-Well-Streifen, 96-Well-Platten, einige optische Qualität </td></tr><tr><td> Computer mit Internet-Anschluss </td><td></td></tr></tbody></table>

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

Das Studium der Übertragung von Lepra ist besonders schwierig, da der Erreger, Mycobacterium leprae, kann nicht im Labor kultiviert werden. Die einzige Quelle für die Bakterien sind Lepra-Patienten und experimentell infizierten Gürteltiere und Nacktmäuse. So sind viele der Methoden in der modernen Epidemiologie verwendet werden, nicht für das Studium der Lepra zur Verfügung. Trotz einer umfangreichen weltweiten medikamentösen Behandlung Programm für die Lepra durch die WHO 1 implementiert, bleibt Lepra endemisch in vielen Ländern mit rund 250.000 Neuerkrankungen pro Jahr. 2 Die gesamte M. leprae Genom wurde 3,4 abgebildet und viele Loci identifiziert worden, die haben Segmenten von 2 oder mehr Basenpaare wiederholt (so genannte Mikro-und Minisatelliten). 5 Klinische Stämme von M. leprae kann in der Anzahl der Tandem wiederholte Segmente (short tandem repeats, STR) auf vielen dieser Loci variieren. 5,6,7 Variable Anzahl Tandem-Repeat (VNTR) 5 Analyse verwendet wurde, um verschiedene Stämme des Lepra-Bazillen zu unterscheiden. Einige der loci scheinen stabiler zu sein als andere, zeigen weniger Schwankungen in wiederholen Zahlen, während andere sich schneller ändern, manchmal in den gleichen Patienten zu sein scheinen. Während die Variabilität bestimmter VNTRs hat Fragen hinsichtlich ihrer Eignung für Stammtypisierung 7,8,9 brachte, legen die Schwellenländer Daten, die Analyse mehrerer Loci, die vielfältig in ihrer Stabilität sind, können als wertvolle epidemiologische Instrument verwendet werden. Multiple locus VNTR-Analyse (MLVA) 10 wurde verwendet, um die Lepra Entwicklung und Übertragung in mehreren Ländern, darunter China 11,12, Malawi 8, auf den Philippinen 10,13 und Brasilien 14 studierst. MLVA umfasst mehrere Schritte. Erstens ist die bakterielle DNA zusammen mit Wirtsgewebe DNA aus klinischen Biopsien oder Schlitz Haut schmiert (SSS) extrahiert. 10 Die gewünschte loci dann aus der extrahierten DNA mittels Polymerase-Kettenreaktion (PCR) amplifiziert. Fluoreszenzmarkierten Primer für 4-5 verschiedene Loci pro Reaktion eingesetzt, mit 18 Loci in insgesamt vier Reaktionen verstärkt. 10 Die PCR-Produkte auf Agarose-Gelelektrophorese unterworfen werden können, um das Vorhandensein des gewünschten DNA-Abschnitte zu überprüfen, und dann eingereicht für Leuchtstofflampen-Fragment-Längen-Analyse (FLA) mittels Kapillarelektrophorese. DNA aus Gürteltier passagiert Bakterien mit einer bekannten Anzahl von wiederholten Kopien für jeden Locus wird als positive Kontrolle verwendet. Die FLA-Chromatogramme werden dann unter Verwendung Peak-Scanner-Software-und Fragment-Länge ist auf die Anzahl der VNTR Kopien (Allel) umgewandelt. Schließlich werden die VNTR Haplotypen nach Mustern analysiert und, wenn sie mit Patienten klinische Daten kombiniert werden verwendet, um Verteilung der Belastung Arten zu verfolgen.

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

Lepra, verursacht durch<em&gt; Mycobacterium leprae</em&gt;, Ist immer noch in vielen Orten endemisch. Um über die Verbreitung und Art der Übertragung der Lepra zu lernen, ist es wichtig zu ermitteln, welche Belastung der<em&gt; M. leprae</em&gt; Hat ein Patient infiziert. Variable Anzahl von Tandem Repeats (VNTR-Typisierung) ist eine solche Methode.

DNA Fingerprinting von Mycobacterium leprae Die Stämme mit variablen Anzahl Tandem Repeat (VNTR) - Fragment Length Analysis (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)

27.0K Aufrufe.  Colorado State University. Lepra, verursacht durch Mycobacterium leprae, Ist immer noch in vielen Orten endemisch. Um über die Verbreitung und Art der Übertragung der Lepra zu lernen, ist es wichtig zu ermitteln, welche Belastung der M. leprae Hat ein Patient infiziert. Variable Anzahl von Tandem Repeats (VNTR-Typisierung) ist eine solche Methode.

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

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Probenvorbereitung von<em&gt; Mycobacterium tuberculosis</em&gt; Extrakte für Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Forschung

Immunologie und Infektion

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.

Wachstum von Mycobacterium tuberculosis Biofilme

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.

Genetische Manipulation in<em&gt; Δku80</em&gt; Die Stämme für Funktionelle Genomanalyse<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.

Optimierte Protokolle für<em&gt; Mycobacterium leprae</em&gt; Dehnungs Management: Gefrorene Lizenz Erhaltung und Wartung in Nacktmaus

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.

Einrichtung einer<em&gt; In-vitro-</em&gt; System zur intrazelluläre Verhalten der Studie<em&gt; Candida glabrata</em&gt; Menschen THP-1-Makrophagen

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

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 der 18FDG PET/CT Imaging als Werkzeug für Studium Mycobacterium-Tuberkulose -Infektion und Behandlung bei nicht-menschlichen Primaten

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.

Mycobacterium Tuberculosis in Mäusen mit Reporter Enzym Fluoreszenz Imaging

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

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

Identifikation der Virulenz Marker von Mycobacterium Abscessus für die intrazelluläre Replikation in Phagozyten

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Erkennung von niedrigen Kopie Nummer integrierte virale DNA gebildet durch In-vitro- Hepatitis B-Infektion

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of tuberculosis (TB). We have established an experimental protocol to follow immune polarization of myeloid-derived cells into M1 (classically activated) or M2 (alternatively activated) macrophage-like cells through assessment with a 10-color flow cytometry panel that allows visualization and deep-characterization of green-fluorescent-protein (GFP)-labeled Mtb in diverse macrophages subsets. Monocytes obtained from healthy blood donors were polarized into M1 or M2 cells using differentiation with granulocyte macrophage-colony-stimulating factor (GM-CSF) or macrophage-colony stimulating factor (M-CSF) followed by polarization with IFN-&#947; and lipopolysaccharide (LPS) or IL-4, respectively. Fully polarized M1 and M2 cells were infected with Mtb-GFP for 4 hours before detached Mtb-infected macrophages were stained with flow cytometry at 4- or 24-hours post-infection. Sample acquisition was performed with flow cytometry and the data was analyzed using a flow cytometry analysis software. Manual gating as well as dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP) and phenograph analysis was performed. This protocol resulted in effective M1/M2 polarization characterized by elevated levels of CD64, CD86, TLR2, HLA-DR and CCR7 on uninfected M1 cells, while uninfected M2 cells exhibited a strong up-regulation of the M2 phenotype markers CD163, CD200R, CD206 and CD80. M1-polarized cells typically contained fewer bacteria compared to M2-polarized cells. Several M1/M2 markers were downregulated after Mtb infection, which suggests that Mtb can modulate macrophage polarization. In addition, 24 different cell clusters of different sizes were found to be uniquely distributed among the M1 and M2 uninfected and Mtb-infected cells at 24-hours post-infection. This M1/M2 flow cytometry protocol could be used as a backbone in Mtb-macrophage research and be adopted for special needs in different areas of research.

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

This protocol provides a method to study Mycobacterium tuberculosis infection in human M1- or M2-polarized macrophages based on differentiation of peripheral-blood-monocytes to macrophage-like cells that are infected with the GFP-labeled virulent strain H37Rv, and analyzed with flow cytometry using a 10-color panel including expression of selected M1/M2 markers.

Polarisation von M1 und M2 Human Monocyte-Derived Cells und Analyse mit Durchflusszytometrie bei Mycobacterium tuberculosis Infection

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of ...