Name: forward genetic approaches in chlamydia trachomatis
Uploaded: 2013-10-23T18:30:00
Description: Watch this Scientific Journal Video about Forward Genetic Approaches in Chlamydia trachomatis at JoVE.com

1.. Kemisk mutagenese Bemærk: Vi fandt, at den replikative RB formen er mere modtagelig for kemisk mutagenese end EB formularen. Midt i cyklus (mellem 18.-20 HPI) er RB på det største antal forud for RB-EB overgang. Fordi Chlamydia trachomatis er en obligat intracellulær patogen, kan virkningerne af mutagen på værten sundhed begrænse bakteriel genopretning. Vero-celler blev fundet at være mere modstandsdygtige over for de negative virkninger af høje niveauer af EMS end andre ...

<ol>
	<li>Haggerty, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+sequelae+after+Chlamydia+trachomatis+genital+infection+in+women.">Risk of sequelae after Chlamydia trachomatis genital infection in women.</a> J Infect Dis. 201, S134-S155 (2010).</li><li>Dautry-Varsat, A., Subtil, A., Hackstadt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+the+mechanisms+of+Chlamydia+entry.">Recent insights into the mechanisms of Chlamydia entry.</a> Cell Microbiol. 7 (12), 1714-1722 (2005).</li><li>Hybiske, K., Stephens, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Chlamydia+trachomatis+entry+into+nonphagocytic+cells.">Mechanisms of Chlamydia trachomatis entry into nonphagocytic cells.</a> Infect Immun. 75 (8), 3925-3934 (2007).</li><li>Heuer, D., Kneip, C., Maurer, A. P., Meyer, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tackling+the+intractable+-+approaching+the+genetics+of+Chlamydiales.">Tackling the intractable - approaching the genetics of Chlamydiales.</a> Int J Med Microbiol. 297 (7-8), 569-576 (2007).</li><li>Stephens, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+an+obligate+intracellular+pathogen+of+humans,+Chlamydia+trachomatis.">Genome sequence of an obligate intracellular pathogen of humans, Chlamydia trachomatis.</a> Science. 282 (5389), 754-759 (1998).</li><li>Thomson, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydia+trachomatis,+genome+sequence+analysis+of+lymphogranuloma+venereum+isolates.">Chlamydia trachomatis, genome sequence analysis of lymphogranuloma venereum isolates.</a> Genome Res. 18 (1), 161-171 (2008).</li><li>Harris, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-genome+analysis+of+diverse+Chlamydia+trachomatis+strains+identifies+phylogenetic+relationships+masked+by+current+clinical+typing.">Whole-genome analysis of diverse Chlamydia trachomatis strains identifies phylogenetic relationships masked by current clinical typing.</a> Nat Genet. 44 (4), 413-419 (2012).</li><li>Somboonna, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypervirulent+Chlamydia+trachomatis+clinical+strain+is+a+recombinant+between+lymphogranuloma+venereum+(L(2))+and+D+lineages.">Hypervirulent Chlamydia trachomatis clinical strain is a recombinant between lymphogranuloma venereum (L(2)) and D lineages.</a> MBio. 2 (3), e00045-00011 (2011).</li><li>Voigt, A., Schofl, G., Saluz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chlamydia+psittaci+genome,+a+comparative+analysis+of+intracellular+pathogens.">The Chlamydia psittaci genome, a comparative analysis of intracellular pathogens.</a> PLoS One. 7 (4), e35097 (2012).</li><li>Jeffrey, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequencing+of+recent+clinical+Chlamydia+trachomatis+strains+identifies+loci+associated+with+tissue+tropism+and+regions+of+apparent+recombination.">Genome sequencing of recent clinical Chlamydia trachomatis strains identifies loci associated with tissue tropism and regions of apparent recombination.</a> Infect Immun. 78 (6), 2544-2553 (2010).</li><li>Gomes, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Chlamydia+trachomatis+diversity+occurs+by+widespread+interstrain+recombination+involving+hotspots.">Evolution of Chlamydia trachomatis diversity occurs by widespread interstrain recombination involving hotspots.</a> Genome Res. 17 (1), 50-60 (2007).</li><li>DeMars, R., Weinfurter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstrain+gene+transfer+in+Chlamydia+trachomatis+in+vitro,+mechanism+and+significance.">Interstrain gene transfer in Chlamydia trachomatis in vitro, mechanism and significance.</a> J Bacteriol. 190 (5), 1605-1614 (2008).</li><li>Demars, R., Weinfurter, J., Guex, E., Lin, J., Potucek, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+gene+transfer+in+vitro+in+the+intracellular+pathogen+Chlamydia+trachomatis.">Lateral gene transfer in vitro in the intracellular pathogen Chlamydia trachomatis.</a> J Bacteriol. 189 (3), 991-1003 (2007).</li><li>Nguyen, B. D., Valdivia, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+determinants+in+the+obligate+intracellular+pathogen+Chlamydia+trachomatis+revealed+by+forward+genetic+approaches.">Virulence determinants in the obligate intracellular pathogen Chlamydia trachomatis revealed by forward genetic approaches.</a> Proc Natl Acad Sci U S A. 109 (4), 1263-1268 (2012).</li><li>Tipples, G., McClarty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+initial+characterization+of+a+series+of+Chlamydia+trachomatis+isolates+selected+for+hydroxyurea+resistance+by+a+stepwise+procedure.">Isolation and initial characterization of a series of Chlamydia trachomatis isolates selected for hydroxyurea resistance by a stepwise procedure.</a> J Bacteriol. 173 (16), 4932-4940 (1991).</li><li>Wang, L. L., Henson, E., McClarty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+trimethoprim-+and+sulphisoxazole-resistant+Chlamydia+trachomatis.">Characterization of trimethoprim- and sulphisoxazole-resistant Chlamydia trachomatis.</a> Mol Microbiol. 14 (2), 271-281 (1994).</li><li>Scidmore, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+and+Laboratory+Maintenance+of+Chlamydia+trachomatis.">Cultivation and Laboratory Maintenance of Chlamydia trachomatis.</a> Curr Protoc Microbiol. Chapter 11, Unit 11A 11 (2005).</li><li>Li, H., Ruan, J., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+short+DNA+sequencing+reads+and+calling+variants+using+mapping+quality+scores.">Mapping short DNA sequencing reads and calling variants using mapping quality scores.</a> Genome Res. 18 (11), 1851-1858 (2008).</li><li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+transform.">Fast and accurate short read alignment with Burrows-Wheeler transform.</a> Bioinformatics. 25 (14), 1754-1760 (2009).</li><li>Suchland, R. J., Sandoz, K. M., Jeffrey, B. M., Stamm, W. E., Rockey, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+transfer+of+tetracycline+resistance+among+Chlamydia+spp.+in+vitro.">Horizontal transfer of tetracycline resistance among Chlamydia spp. in vitro.</a> Antimicrob Agents Chemother. 53 (11), 4604-4611 (2009).</li><li>Sladek, F. M., Melian, A., Howard-Flanders, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incision+by+UvrABC+excinuclease+is+a+step+in+the+path+to+mutagenesis+by+psoralen+crosslinks+in+Escherichia+coli.">Incision by UvrABC excinuclease is a step in the path to mutagenesis by psoralen crosslinks in Escherichia coli.</a> Proc Natl Acad Sci U S A. 86 (11), 3982-3986 (1989).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+transformation+system+for+Chlamydia+trachomatis,+restoration+of+glycogen+biosynthesis+by+acquisition+of+a+plasmid+shuttle+vector.">Development of a transformation system for Chlamydia trachomatis, restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector.</a> PLoS Pathog. 7 (9), e1002258 (2011).</li></ol>

Denne metode opfylder de grundlæggende krav til genetisk analyse da det etablerer forbindelsen mellem genotyper og fænotyper. Vigtigere er, opnås dette uden hjælp af konventionelle molekylære værktøjer til rekombinant DNA-transformation og insertionel inaktivering af gener i bakterier, som ofte er et hastighedsbegrænsende trin i analysen af ​​genfunktion i ikke-model mikrober. Et kritisk trin er at sikre klonalitet af plak-oprensede mutanter. Krydskontaminering med vildtype eller...

De obligate intracellulære bakterien Chlamydia trachomatis regnskab for en anslået 2,8 millioner genitale infektioner om året i USA (Center for Disease Control) med tilhørende følgesygdomme såsom underlivsbetændelse, graviditet udenfor livmoderen, og infertilitet (1). Chlamydia-arter har en unik fysiologi med en bifasisk udviklingsmæssige cyklus bestående af to former: den infektiøse, men ikke-replikerende elementær krop (EB) og infektiøse men replikative reticulate legeme (RB). Infektion begynder med fastgørelsen af ​​EBS til epitelceller efterfulgt af endoctyotosis (2). Inden for en membran-bundet vacuole betegnet en optagelse, EBS differentiere i RB formular, som så replikater ved binær fission. Midt i cyklus, RBS overgange tilbage i EBS, som derefter bortvist i det ekstracellulære rum til at starte en ny omgang infektion, når værten celle lyserer (3). C. trachomatis errefraktær over for rutinemæssig manipulation med standard molekylærgenetiske redskaber, såsom målrettet genudskiftning, transposoner og transducerende fager, som har været centralt for de fleste studier i bakterielle genetik, Det er uklart, i hvilket omfang de enkelte Chlamydia gener bidrager til unddragelse af medfødte immunitet , næringsstof erhvervelse, udviklingsmæssige overgange, eller andre processer der er vigtige for patogenet overlevelse i en eukaryot vært (4). Følgelig dette patogen stadig dårligt karakteriseret trods sin klinisk betydning. Genomer af Chlamydia spp. er relativt lille (~ 1 Mb) (5) med flere arter og biovarer sekventeret bruge næste generation sekventering teknologier. Sammenlignende genom analyse WGS har givet enestående indblik i udviklingen af ​​chlamydia arter og deres tilpasning til mennesker (6-8), og til en vis grad har givet nogle oplysninger, som den potentielle funktion virulensfaktorer (9, 10). THan genetiske mangfoldighed vises af kliniske isolater giver ikke beslutningen forpligtet til systematisk at kortlægge funktionen af ​​de fleste virulensfaktorer, formentlig fordi mutationer i sådanne gener ville have været let vælges imod. Uden forstyrrende effekter fra naturlig udvælgelse, mutagen-induceret gen variation, kombineret med definerede analyser, der måler fejl i virulens, kan udvide det spektrum af mutationer, der kan adspurgte. Kemiske mutagener i særdeleshed, er nyttige, da de kan generere null, betinget hypomorphic (nedsat funktion) og hypermorphic (gevinst på funktion) alleler. Med ankomsten af ​​robuste næste generations genom-sekventering teknologier, kan sådanne mutationer let identificeres og kortlægges. På denne måde kan stærke associationer foretages mellem mutationer i et gen eller genetisk vej og en fælles fænotype, der muliggør anvendelse af forward genetiske metoder. Genom-sekvenser af kliniske stammer afslørede mosaicisme Between serovarer og loci for hyppig rekombination (11). Empiriske beviser for rekombination blev demonstreret gennem co-infektion af to forskellige antibiotikaresistente stammer og valg af dobbelt resistente rekombinant afkom, som blev åbenbaret for at have genetiske bidrag fra begge stammer (12, 13). Således genetisk udveksling mellem vildtype og mutantstammer i en co-infektion indstilling giver adskillelse af kemisk inducerede mutationer til at lokalisere genet påvirkede, der fører til den observerede fænotype. Her beskriver vi en metode til at udføre genetisk analyse i Chlamydia baseret på kemisk mutagenese, WGS, og et system til DNA udveksling i inficerede celler (14) (figur 1).

<table border="0" cellspacing="0" cols="4">
	<colgroup width="291">
	</colgroup>
	<colgroup width="123">
	</colgroup>
	<colgroup width="144">
	</colgroup>
	<colgroup width="84">
	</colgroup>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl methanesulfonate (EMS)</td>
			<td>Sigma</td>
			<td>M0880</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclohexamide</td>
			<td>Sigma</td>
			<td>C4859-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) solution with 0.493 mM MgCl2 and 0.901 mM CaCl2 (PBS+MgCl2/CaCl2)</td>
			<td>Life Technologies</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5x SPG buffer (1.25 M sucrose, 50 mM sodium phosphate, 25 mM glutamic acid)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SPG buffer (0.25 M sucrose, 10 mM sodium phosphate, 5 mM glutamic acid)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water (sterile, tissue culture grade)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x DMEM (prepared from powder, buffered with 7.4 g/L Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>D7777</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids (NEAA)</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2% GTG Agarose , autoclaved</td>
			<td>Lonzo</td>
			<td>50070</td>
			<td></td>
		</tr>
		<tr>
			<td>Genomic DNA purification kits</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAzol</td>
			<td>Life Technologies</td>
			<td>10503-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (molecular biology grade)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8 mM NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1 M HEPES ( 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 cm2 tissue culture flasks (T25 flasks)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical safety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#62;Centrifuge and adaptors for spinning tissue plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#62;Dissection microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer (Qubit)</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td></td>
		</tr>
		<tr>
			<td>Adaptive Focused Acoustics S220 instrument</td>
			<td>Covaris</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

forward genetic approaches in chlamydia trachomatis

Chlamydia trachomatis, den ætiologiske agens af seksuelt overførte sygdomme og øjeninfektioner, forbliver dårligt karakteriseret på grund af sin intractability til eksperimentel transformation med rekombinant DNA. Vi udviklede en metode til at udføre genetisk analyse i C. trachomatis trods manglen på molekylærgenetiske værktøjer. Vores fremgangsmåde involverer: i) kemisk mutagenese til hurtigt at generere omfattende biblioteker af genetisk definerede mutanter med forskellige fænotyper ii) hel-genom sekventering (WGS) at kortlægge de underliggende genetiske læsioner og finde sammenhænge mellem muterede gen (r) og.. en fælles fænotype,. iii) generation af rekombinante stammer gennem co-infektion af pattedyrceller med mutant og vildtype bakterier. Derfor var vi i stand til at etablere årsagssammenhænge genotyper og fænotyper. Koblingen af ​​kemisk-induceret gen variation og WGS at etablere korrelationsmaalinger genotype-fænotype foreninger Should være bredt anvendelig til den store liste over lægeuddannede og miljømæssigt vigtige mikroorganismer i øjeblikket uløselige til genetisk analyse.

Udsættelse for mutagen fører til indeslutninger, der vises uden bakterier, formentlig på grund af bakteriel celledød. Typisk vil serovar LGV-L2 helt lysere inficerede celler inden for 48 timer efter infektion, men behandling med mutagener kan udvide denne cyklus&gt; 90 timer. Omkring 10% af indeslutninger ventes at komme sig. I vores eksperimenter, behandlede inficerede Vero celler med 20 mg / ml EMS førte til et 99% fald i inddrivelse af infektiøse afkom i forhold til ubehandlede kontroller. Mutagen behandling f?...

Watch this Scientific Journal Video about Forward Genetic Approaches in Chlamydia trachomatis at JoVE.com

Forward Genetic Approaches in Chlamydia trachomatis

Vi beskriver en metode til at udføre genetisk analyse i Chlamydia baseret på kemisk mutagenese og hele genomet sekventering. Desuden er et system til DNA-udveksling inden inficerede celler beskrevet, som kan anvendes til genetisk kortlægning. Denne metode kan være bredt anvendelig til mikrobielle systemer mangler transformation systemer og molekylærgenetiske værktøjer.

Forward Genetiske tilgange i<em&gt; Chlamydia trachomatis</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

forward-genetic-approaches-in-chlamydia-trachomatis

Research

JoVE Journal

Immunology and Infection

Forward Genetic Approaches in Chlamydia trachomatis

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

En genetisk skærm for at isolere Toxoplasma gondii Host-celle afstigningstrin Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

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.

Genetisk Manipulation i<em&gt; Δku80</em&gt; Stammer til Functional Genomisk Analyse af<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 ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

Opdagelsen af ​​nye intracellulære patogener af Amoebal cokultur og Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Brug fluorescerende proteiner til at visualisere og kvantificere<em&gt; Chlamydia</em&gt; Vakuole Vækst Dynamics i levende celler

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&amp;#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

Forward Genetik skærme med makrofager til at identificere<em&gt; Toxoplasma gondii</em&gt; Gener Vigtigt for Modstand mod IFN-γ-Dependent Cell Autonome Immunitet

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

Udnytte den Antigen capsidet-Indbygning strategi for udvikling af adenovirusserotype 5-vektoriserede Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Fag Phenomics: Fysiologiske tilgange til Karakterisere Novel virale proteiner

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

En simpel Red Blood Cell Lysis metode til fastsættelse af B lymfoblastoidcellelinier

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

En effektiv og enkel metode til at etablere NK og T-cellelinier fra patienter med kronisk aktiv Epstein - Barr virusinfektion

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...