Il finanziamento è stato fornito dal NIH / NIAID concedere RO1-AI-63457 e ARRA concedere supplemento RO1-AI-63457 S1. Riconosciamo il contributo di tutti i membri attuali e passati del gruppo di laboratorio e collaboratori.

<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>

Nessun conflitto di interessi dichiarati.

La raccolta di campioni di pelle di malati di lebbra richiede medici specializzati o tecnici che lavorano nelle cliniche della pelle. Personale di laboratorio maneggiare questi campioni devono prestare particolare attenzione ad indossare camici, guanti e occhiali protettivi e di lavorare in un bio-sicurezza gabinetto durante la manipolazione di campioni di tessuti umani infetti o armadillo. Disinfezione delle superfici e degli strumenti è anche critico. Lavorare in un ambiente pulito, sterile bio-safe mobile è importante per evitare la contaminazione dei campioni di DNA. L&#39;estrazione del DNA è diventato relativamente facile grazie allo sviluppo di kit di estrazione da aziende come Qiagen. Indicazioni devono essere seguite attentamente. Tutti i campioni devono essere conservati al freddo, quando non in uso. Evitare il ripetersi, sbalzi di temperatura per i campioni. Primer DNA usato in questo lavoro può essere ordinato presso diverse aziende che sono state citate nella lista dei riferimenti bibliografici alla fine di questo documento. Si deve prestare attenzione a lavorare con primer in un ambiente DNA libero al fine di evitare la contaminazione. Primer vengono come polvere e deve essere miscelato con TE e diluito ad una concentrazione 100μM, poi separati in piccole quantità (Figura 2a). Soluzioni di lavoro di primer sono ulteriormente diluito a concentrazioni 10μM. Anche in questo caso, non utilizzare campioni di DNA in aree / cappe in cui vengono utilizzati primer e preparati. I prodotti di PCR devono essere conservati al freddo, quando non in uso. Un 3% di agarosio preparazione gel darà una migliore separazione banda del DNA, ma richiede più tempo per l&#39;esecuzione. Per scopi puramente qualitativi, 2% è solitamente sufficiente. Soluzione di bromuro di etidio utilizzato per la colorazione del DNA in gel di agarosio è un genotoxin altamente attivo che viene assorbito attraverso la pelle. Gestire questa soluzione e gel colorati con essa utilizzando grande attenzione e sempre con la certezza di indossare i guanti. Lavarsi accuratamente le mani dopo aver toccato qualsiasi DNA campioni o materiali di bromuro di etidio. Smaltire soluzione colorante etidio bromuro, lavare e gel secondo le linee guida istituzionali. Gel non sono normalmente necessarie; questo passo può essere eliminata una volta metodi FLA sono state stabilite. E &#39;tempo e consumare reagenti. La soluzione formamide utilizzati nella preparazione dei campioni per la FLA è anche altamente tossico e deve essere maneggiato con cura. Lavarsi le mani dopo l&#39;uso. Disporre di esso le linee guida istituzionali. Lettura dei risultati di FLA utilizzando il software di picco Scanner può essere impegnativo. Un insieme di base delle chiamate allele (numero di ripetizioni in tandem) è stato sviluppato a CSU (Tabelle 4-7). (Va notato che le tabelle 4-7 non può essere tutto compreso. Come altri ceppi di M. leprae sono studiati, alleli con numero di copie di fuori degli intervalli elencati possono essere trovati.) Una sfida particolare consiste &#39;balbettare&#39; picchi. Si tratta di famiglie di picchi, in particolare di segnalazioni di operazioni sospette che coinvolgono solo 2-3 ripete coppia di basi, come ad esempio (TA) 10. Volte la scelta della punta giusta per leggere è difficile (Figura 7). In questa figura, il picco nel controllo positivo a 190 bp è la vetta principale. Picchi inferiori in altezza che sono 2, 4 o anche 6 coppie di basi più grande o più piccola della vetta principale si chiamano &#39;picchi balbettare.&#39; A-tailing può anche causare confusione. A-tailing è descritto in precedenza nel testo e nella Figura 8. Infine, se i prodotti di PCR sono molto abbondanti nei campioni, i picchi possono apparire con un picco doppio. In questo caso, leggere il centro della forma di picco. (Vedi Figura 6, paziente 6.) La gestione dei dati può essere un compito arduo per questo tipo di lavoro. E &#39;importante registrare tutte le informazioni pertinenti per tutti gli esperimenti, quali: data di un compito o di procedura, l&#39;operatore, striscia / piastra mappe, ordini FLA, condizioni di PCR e le ricette, le date di gel e fotografie, temperature di conservazione, diluizioni modello di DNA, FLA percorsi di memorizzazione di file elettronici, organizzazione ecc Buono e gestione dei dati consente di risparmiare ore di tempo dedicato alla ricerca di informazioni specifiche per il futuro. Il lavoro di laboratorio qui descritta è andata avanti per diversi anni presso la Colorado State University e in altri luoghi in tutto il mondo. L&#39;immagine grande di quello che tutti i dati raccolti mezzi e come può essere di uso futuro sta iniziando ad emergere. Figure 10A e 10B dimostrare come questi le impronte digitali del DNA può essere utilizzato per distinguere differenti M. leprae tensioni tra paesi (10A) o anche tra le famiglie (10B). In materia di famiglia e di comunità collegate hanno dimostrato di portare M. leprae di tipo VNTR simili o identici ceppo. La speranza è che possa essere possibile ottenere una visione più completa la modalità (s) di trasmissione di lebbra in modo che un sistema di individuazione precoce delle reti di trasmissione possono essere sviluppate per quelle persone most a rischio, e che la terapia farmacologica curativa può iniziare prima di danni neurologici permanenti e dermatologici è fatto.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Sangue DNeasy e Kit Tissue </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> Vari </td><td></td><td></td></tr><tr><td> Gel di agarosio </td><td> Vari </td><td></td><td></td></tr><tr><td> 5x o 6x Gel Loading Buffer </td><td> New England Biolabs </td><td> B7021S </td><td> Ricette a disposizione per rendere il vostro proprio * </td></tr><tr><td> Bromuro di etidio soluzione </td><td> Vari </td><td></td><td> Diluire da truffa. </td></tr><tr><td> Hi-Di Formammide 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> Attrezzatura </th><th> Commenti </th></tr><tr><td> 2 frigoriferi </td><td> 4 ° C: 1 per i campioni di DNA, 1 per primer e reagenti Multiplex </td></tr><tr><td> 1 forno a microonde </td><td> O modo adatto per il riscaldamento agarosio e acqua per fare gel </td></tr><tr><td> 1 autoclave </td><td> O pentola a pressione per la sterilizzazione dei materiali </td></tr><tr><td> PCR macchina </td><td> Termociclatore </td></tr><tr><td> 2 set di pipette </td><td> 0,5-10 microlitri, 10-100 microlitri, 20-200 microlitri, 1000 ml 1 set per primer, 1 set di DNA </td></tr><tr><td> Sottomarino gel elettroforesi scatola </td><td> Con forme e pettini per la preparazione gel. </td></tr><tr><td> Elettroforesi alimentazione </td><td> Con cavi per il collegamento alla casella gel </td></tr><tr><td> Calore blocco </td><td> Per tubi Eppendorf </td></tr><tr><td> Centrifuga </td><td> Per tubi Eppendorf / strisce </td></tr><tr><td> Sistema di elettroforesi capillare (analisi genetiche) </td><td> O l&#39;accesso a un istituto in grado di eseguire questa analisi </td></tr><tr><td> Plastica </td><td> Aerosol monouso puntali, provette Eppendorf, strisce da 8 pozzetti, piastre a 96 pozzetti, alcune delle qualità ottica </td></tr><tr><td> Computer con collegamento Internet </td><td></td></tr></tbody></table>

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

Lo studio della trasmissione della lebbra è particolarmente difficile dato che l&#39;agente eziologico, Mycobacterium leprae, non può essere coltivato in laboratorio. Le uniche fonti dei batteri sono malati di lebbra, e armadilli sperimentalmente infetti e topi nudi. Così, molti dei metodi utilizzati in epidemiologia moderna non sono disponibili per lo studio della lebbra. Nonostante un ampio programma globale di trattamento farmacologico per la lebbra attuato dalla WHO 1, la lebbra rimane endemica in molti paesi con circa 250.000 nuovi casi ogni anno. 2 L&#39;intera M. leprae genoma è stata mappata 3,4 e molti loci sono stati identificati che si sono ripetuti segmenti di 2 o più paia di basi (chiamate micro-e minisatelliti). 5 ceppi clinici di M. leprae possono variare nel numero di segmenti ripetuti in tandem (ripetizioni in tandem corto, STR) a molti di questi loci. 5,6,7 numero variabile di ripetizione in tandem (VNTR) 5 analisi è stato utilizzato per distinguere i diversi ceppi di bacilli della lebbra. Alcuni dei loci sembrano essere più stabili di altri, mostrando meno variazioni di numeri si ripetono, mentre altri sembrano cambiare più rapidamente, a volte nello stesso paziente. Mentre la variabilità di alcuni VNTRs ha portato fino domande riguardanti la loro idoneità per la tipizzazione ceppo 7,8,9, i dati emersi suggeriscono che loci multipli, che sono diversi nella loro stabilità, può essere utilizzato come un valido strumento epidemiologico. Multiple locus VNTR analisi (MLVA) 10 è stato usato per studiare l&#39;evoluzione lebbra e la trasmissione in diversi paesi tra cui Cina 11,12, Malawi 8, le Filippine 10,13, e in Brasile 14. MLVA prevede più passaggi. In primo luogo, DNA batterico viene estratto insieme al DNA tessuto ospite da biopsie clinici o macchie della pelle fessura (SSS). 10 I loci desiderati sono poi amplificati dal DNA estratto tramite reazione a catena della polimerasi (PCR). Primers marcati con fluorescenza per 4-5 loci diversi sono usati per reazione, con 18 loci essere amplificato in un totale di quattro reazioni. 10 I prodotti di PCR può essere sottoposto a elettroforesi su gel di agarosio per verificare la presenza di segmenti di DNA desiderato, quindi sottoposti ad analisi a fluorescenza della lunghezza dei frammenti (FLA) usando elettroforesi capillare. DNA da batteri armadillo diversi passaggi con un numero definito di copie ripetere per ogni locus viene utilizzato come controllo positivo. I cromatogrammi FLA vengono poi esaminati usando il software dello scanner e di picco della lunghezza dei frammenti viene convertito in numero di copie VNTR (allele). Infine, gli aplotipi VNTR sono analizzati per i modelli, e se combinata con i dati clinici del paziente possono essere utilizzati per monitorare la distribuzione dei tipi di deformazione.

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 lebbra, causata da<em&gt; Mycobacterium leprae</em&gt;, È ancora endemica in molti luoghi. Al fine di conoscere la diffusione e la modalità di trasmissione della lebbra, è importante per determinare quale ceppo di<em&gt; M. leprae</em&gt; Ha infettato un paziente. Un numero variabile di ripetizioni tandem (VNTR) digitazione è uno di questi metodi.

DNA fingerprinting di Mycobacterium leprae Ceppi Utilizzando variabile Tandem Repeat Number (VNTR) - Lunghezza Analisi Fragment (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 Views.  Colorado State University. La lebbra, causata da Mycobacterium leprae, È ancora endemica in molti luoghi. Al fine di conoscere la diffusione e la modalità di trasmissione della lebbra, è importante per determinare quale ceppo di M. leprae Ha infettato un paziente. Un numero variabile di ripetizioni tandem (VNTR) digitazione è uno di questi metodi.

Video: 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.

Preparazione del campione di<em&gt; Mycobacterium tuberculosis</em&gt; Estratti di studi di risonanza magnetica nucleare metabolomica

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

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Immunologia e infezioni

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.

Crescita Mycobacterium tuberculosis I biofilm

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.

La manipolazione genetica in<em&gt; Δku80</em&gt; Ceppi per analisi genomica funzionale di<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.

Protocolli ottimizzati per<em&gt; Mycobacterium leprae</em&gt; Strain Management: congelata Stock Conservazione e Manutenzione in topi nudi atimici

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.

Istituzione di un<em&gt; In vitro</em&gt; Sistema per studiare intracellulare Comportamento<em&gt; Candida glabrata</em&gt; In umani THP-1 macrofagi

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.

Analisi di 18FDG PET/CT Imaging come strumento per studiare l'infezione di tubercolosi del micobatterio e trattamento in primati Non umani

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.

Tubercolosi del micobatterio in topi con fluorescenza enzima Reporter di 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.

Identificazione di fattori di virulenza di Mycobacterium abscessus per replica intracellulare nei fagociti

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.

Rilevamento di copia basso numero di DNA virale integrato formato da In Vitro l'infezione di epatite B

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.

Polarizzazione delle cellule derivate da monociti umani M1 e M2 e analisi con citometria a flusso sull'infezione da tubercolosi del micobatterio

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