Name: detection of enterohemorrhagic escherichia coli colonization in murine host by non-invasive in vivo bioluminescence system
Uploaded: 2018-04-09T12:30:00
Description: Watch this Scientific Journal Video about Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System at JoVE.com

Vi erkänner Chi-Chung Chen från Institutionen för medicinsk forskning, Chi Mei Medical Center (Tainan, Taiwan) för hjälpen i mus infektion och stöd från stadens laboratorium djur National Cheng Kung University. Detta arbete stöds av den Minister av vetenskap och teknik (de flesta) bidrag (mest 104-2321-B-006-019, 105-2321-B-006-011, and106-2321-B-006-005) till CC.

Varning: EHEC O157: H7 är en biosäkerhet nivå 2 (BSL-2) patogener enligt Centers for Disease Control and Prevention (CDC) biosäkerhet instruktion (https://www.cdc.gov/). Därför måste alla experimentella procedurer som involverar EHEC utföras i en BSL-2 anläggning. Använd lab rockar och skyddshandskar när de utför experimentet. Arbeta i en certifierad biosäkerhet skåp (BSC). Desinficera experimentella bänken före och efter det experimentella förfarandet med 70% etanol. Alla instrument eller utrustning att...

<ol>
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Det har rapporterats att EHEC omvandlas med luciferas plasmid har utnyttjats för att undersöka dess lokalisering i värdar eller gen uttryck i vivo8,11,12. Murina modellen visat här har också rapporterats att upptäcka EHEC koloniserade timing och lokalisering i murina värd8. Dock ger vi protokollet detalj att administrera EHEC inympning till möss intragastrically och hur noggrant förbered...

EHEC O157: H7 är en patogen som orsakar diarré1, HS2, HUS3och även akut njursvikt4 genom förorenat vatten eller mat. EHEC är en patogena enterobacterium och colonizes till mag-tarmkanalen människor1. När EHEC följer först värd intestinal epitel, injicera de colonization faktorerna i värdceller genom typ III-sekretionssystemet (T3SS) som fungerar som en molekylär spruta förmå en fästa och utplåna (A/E) lesion därefter att genomdriva adhesion (kolonisation)5. Dessa gener som är involverade i A/E lesion formation kodas av locus av enterocyter utplåning (LEE) patogenicitet island5.
Mareld är en ljus-producerande kemisk reaktion, i vilka luciferas katalyserar dess substrat luciferin för att generera synliga ljus6. Detta enzymatisk process kräver ofta närvaro av syre eller adenosintrifosfat (ATP)6. Mareld imaging (BLI) tillåter forskare visualisering och kvantisering av värd-patogen interaktioner i levande djur7. BLI kan karakterisera bakterieinfektion cykeln i levande djur genom att följa de självlysande bakterierna som de migrerar till och invadera olika vävnader7; Detta avslöjar en dynamisk progression av infektionen. Dessutom är bakteriehalten i djur relaterad till den självlysande signal8; Därför är det en bekväm indikator att uppskatta de sjukliga tillstånd försöksdjur på ett enkelt och direkt sätt.
Plasmiden används här innehöll luciferas operon, luxCDABE, som är från bakterien Photorhabdus hjälp som kodar egna luciferas substrat7,9. Genom att omvandla denna luciferas-uttryckande plasmid till bakterier, kan colonization och infektion processerna övervakas genom att observera dessa självlysande bakterier i levande djur. Sammantaget tillåter BLI och Mareld-märkt bakterier forskare att övervaka bakteriell siffrorna och läge, bakteriell bärkraft med antibiotika/terapi behandling och bakteriell genuttryck i infektion/kolonisering6, 7. många patogena bakterier har rapporterats att uttrycka den luxCDABE operon att undersöka deras infektion cykel och/eller gen uttryck i infektion. Dessa bakterier, inklusive uropathogenic E. coli10, EHEC8,11,12,13, enteropathogenic E. coli (EPEC)8, Citrobakter rodentium14,15, Salmonella typhimurium16, Listeria monocytogenes17, Yersinia enterocolitica18,19, och Vibriocholerae20, har dokumenterats.
Flera experimentella modeller har utvecklats för att underlätta studiet av EHEC colonization in vitro- och in-vivo21,22,23. Det finns dock en brist på lämplig djurmodeller studera den EHEC koloniseringen i vivo, och därmed en resulterande bristen på Detaljer. För att underlätta studiet av den EHEC colonization mekanism i vivo, är det värdefullt att bygga djurmodeller för att observera och kvantifiera EHEC kolonisationen i levande djur i en icke-invasiv metod.
Detta manuskript beskriver en mus-EHEC colonization modell som använder en självlysande uttrycker system för att övervaka EHEC colonization över tid i levande värdar. Möss är intragastrically inokuleras med Mareld-märkt EHEC och den självlysande signalen detekteras hos möss med en icke-invasiv i vivo imaging system13. Möss infekterade med Mareld-märkt EHEC visade betydande självlysande signaler i deras tarmen efter 2 dagar efter infektion, som föreslog att dessa bakterier koloniserade i tarmen värd efter 2 dagar efter infektion. Ex vivo bilddata visade att denna kolonisering är specifikt i blindtarmen och tjocktarmen av möss. Genom att använda denna mus-EHEC modell, kan självlysande EHEC koloniseringen upptäckas i den levande mottagande av en in-vivo imaging system, för att studera de detaljerade mekanismerna av enteriska bakterier kolonisering, som kan främja ytterligare förståelse i EHEC-inducerad fysiologiska och patologiska förändringar.

<table border="0" cellpadding="0" cellspacing="0" width="721">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Shaker incubator</td>
			<td style="width:175px;">YIH DER</td>
			<td style="width:143px;">LM-570R</td>
			<td style="width:167px;">bacteria incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Orbital shaking incubator</td>
			<td style="width:175px;">FIRSTEK</td>
			<td style="width:143px;">S300</td>
			<td>bacteria incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">pBSL180</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>source of nptII gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pAKlux2</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>source of luxCDABE operon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T&#38;A Cloning Kit</td>
			<td>Yeastern Biotech</td>
			<td style="width:143px;">FYC001-20P</td>
			<td>use for TA cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nsi I</td>
			<td style="width:175px;">NEB</td>
			<td>R0127S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sca I</td>
			<td style="width:175px;">NEB</td>
			<td>R0122S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spe I-HF</td>
			<td style="width:175px;">NEB</td>
			<td>R0133S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sma I&#160;</td>
			<td style="width:175px;">NEB</td>
			<td>R0141S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">T4 ligase</td>
			<td style="width:175px;">NEB</td>
			<td style="width:143px;">M0202S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ex Taq</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">10X Ex Taq Buffer</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP Mixture&#160;</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">PCR machine</td>
			<td style="width:175px;">applied Biosystem&#160;</td>
			<td style="width:143px;">2720 thermal cycler&#160;</td>
			<td>&#160;for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glycerol</td>
			<td style="width:175px;">SIGMA</td>
			<td style="width:143px;">G5516-1L</td>
			<td>use for bacteria stocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">NaCl</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">31434-5KG-R</td>
			<td>chemical for making LB medium, 10 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Tryptone</td>
			<td style="width:175px;">CONDA pronadisa</td>
			<td style="width:143px;">Cat 1612.00</td>
			<td>chemical for making LB medium, 10 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Yeast Extract powder</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:143px;">23547-1 KG</td>
			<td>chemical for making LB medium, 5 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Agar</td>
			<td style="width:175px;">CONDA pronadisa</td>
			<td style="width:143px;">Cat 1802.00</td>
			<td>chemical for making LB agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">kanamycin&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">K4000-5G</td>
			<td>antibiotics, use for seleciton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">streptomycin&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">S6501-100G</td>
			<td>antibiotics, eliminate the microbiota in mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">EDL933 competent cell</td>
			<td style="width:175px;">Homemade</td>
			<td style="width:143px;"></td>
			<td>method is on supplemental document&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Electroporator</td>
			<td style="width:175px;">MicroPulser</td>
			<td style="width:143px;"></td>
			<td>for electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Electroporation Cuvettes</td>
			<td style="width:175px;">Gene Pulser/MicroPulser</td>
			<td style="width:143px;">1652086</td>
			<td>for electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">High-speed centrifuge</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">Avanti, J-26S XP</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Fixed-Angle Rotor</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">JA25.5</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Fixed-Angle Rotor</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">JLA10.5</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge bottles</td>
			<td>Beckman Coulter</td>
			<td>REF357003</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge bottles</td>
			<td style="width:175px;">Thermo Fisher scientific</td>
			<td style="width:143px;">3141-0500</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">eppendorf biophotometer plus&#160;</td>
			<td>eppendorf</td>
			<td>AG 22331 hamburg</td>
			<td>for measuring the OD600 value of bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">C57BL/6 mice&#160;</td>
			<td colspan="2">Laboratory Animal Center of NCKU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">lab coat, gloves</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>for personnel protection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isoflurane&#160;</td>
			<td>Panion &#38; BF Biotech Inc.</td>
			<td style="width:143px;">G-8669</td>
			<td>for mice anesthesia, pharmaceutical grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1ml syringe&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>use for oral gavage of mice</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Reusable 22 G ball-tipped feeding needle</td>
			<td style="width:143px;">&#966;0.9 mm X L 50 mm</td>
			<td>use for oral gavage of mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">surgical&#160; scissors&#160;</td>
			<td></td>
			<td style="width:143px;"></td>
			<td>use for mice experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Xenogen IVIS 200 imaging system</td>
			<td>Perkin Elmer</td>
			<td>IVIS spectrum</td>
			<td>use for bioluminescent image capture&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Living Image Software</td>
			<td>Perkin Elmer</td>
			<td style="width:143px;">version 4.1</td>
			<td>use for quantifying the image data</td>
		</tr>
	</tbody>
</table>

detection of enterohemorrhagic escherichia coli colonization in murine host by non-invasive in vivo bioluminescence system

Entrohemoragiska E. coli (EHEC) O157: H7, som är en livsmedelsburna patogener som causesdiarrhea, hemorragisk kolit (HS), och Hemolytiskt uremiskt syndrom (HUS), kolonisera till tarmkanalen av människor. För att studera detaljerade mekanismen av EHEC colonization in vivo är det viktigt att ha djurmodeller att övervaka och kvantifiera EHEC colonization. Vi visar här en mus-EHEC colonization modell genom att omvandla självlysande uttrycker plasmiden att EHEC att övervaka och kvantifiera EHEC kolonisationen i levande värdar. Djur som inokulerats med Mareld-märkt EHEC Visa intensiva självlysande signaler hos möss genom detektion med en icke-invasiv i vivo imaging system. Efter 1 och 2 dagar efter infektion, kunde självlysande signaler fortfarande upptäckas hos infekterade djur, vilket antyder att EHEC kolonisera i värdar för minst 2 dagar. Vi visar också att dessa självlysande EHEC lokalisera till mus tarmen, speciellt i blindtarmen och tjocktarmen, från ex vivo bilder. Denna mus-EHEC colonization modell kan fungera som ett verktyg att avancera den aktuella kunskapen om EHEC colonization mekanismen.

Vi administrerade Mareld-märkt EHEC (~ 109 bakterieceller) till 6 - vecka-gammal kvinnlig C57BL/6 möss av oral sondmatning. Efter oral inokulering av EHEC till möss inom 1 h undersöktes djuren för självlysande signal av i vivo imaging systemet som visas i figur 7. Resultaten visade en stark självlysande signal i sondmatning möss med Mareld-märkt EHEC. Vi undersökte signalerna på 2 dagar efter infektion. Som visas i <strong class...

Watch this Scientific Journal Video about Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System at JoVE.com

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Ett detaljerat protokoll av en musmodell för entrohemoragiska E. coli (EHEC) koloniseringen med hjälp av Mareld-märkt bakterier presenteras. Påvisande av dessa självlysande bakterier av en icke-invasiv i vivo imaging system med levande djur kan avancera vår nuvarande förståelse av EHEC colonization.

Påvisande av entrohemoragiska Escherichia Coli koloniseringen i murina mottagande av icke-invasiva In Vivo Mareld System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Research

JoVE Journal

Immunology and Infection

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Icke-invasiv avbildning av Leukocyt Homing och migration In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Icke-invasiv avbildning av disseminerad candidiasis i zebrafisk Larver

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Icke-invasiv Modell av Neuropathogenic<em&gt; Escherichia coli</em&gt; Infektion i neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Enstegs Negativ kromatografisk rening av<em&gt; Helicobacter pylori</em&gt; Neutrofilaktiverande Protein överuttryckt i<em&gt; Escherichia coli</em&gt; I batch-läge

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

A Murine Model of Group B Streptococcus Vaginal Colonization

A Murine Model of Group B Streptococcus Vaginal Colonization

En musmodell av grupp B<em&gt; Streptococcus</em&gt; Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Icke-invasiv<em&gt; In vivo</em&gt; Fluorescence optisk avbildning av inflammatorisk MMP aktivitet Använda en aktiverbar Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overuttryck och rening av människa<em&gt; Cis</em&gt; -prenyltransferas i<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Ett lättköpt protokoll att generera anläggningsvis acetylerade proteiner i Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Återkommande Escherichia coli urinvägsinfektion utlöst av Gardnerella vaginalis urinblåsans exponering hos möss

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Kvantifiering i realtid av reaktiva syrearter i neutrofiler infekterade med meningitisk escherichia coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...