P. Cossart 실험실에서 연구는 파스퇴르 연구소, 문화원 국립 드 라 상테 외 드 라 공들인 Médicale, 문화원 내셔널 드 라 공들인 Agronomique, ERC 고급 그랜트 (233348), 직원은 국립 드 라 공들인 (부여 MIE-에서 지원 SignRupVac), 루이 Jeantet 재단과 매그 르 로크 레 무 스케 타이. AK는 파스퇴르 파리 대학 국제 박사 과정 / 문화원 카르노 만성 질환 Infectieuses에서 장학금을받는 사람입니다. 우리는 세포의 형질 전환 프로토콜을 최적화 제이슨 머서 감사합니다.

1. 세포의 준비 및 세균 배양, 형질 도구 및 차 항체 <ol><li> 개인 L.를 분리 신선한 한천 플레이트를 준비 C. -80 °로 유지 세균 글리세롤 (세균의 액체 배양 물을 포화 glycerol/50 % 50 %)에서 식민지 모노 사이토 겐 <ol><li> 뇌 심장 주입 (BHI) 한천 플레이트에 고정 된 세균 글리세롤, 줄무늬 박테리아를 전송 (-80 ° C 유지) 알루미늄 랙을 사용. </li><li> 48 시간 (또는 개별 박테리아의 식민지가 고립 될 수있을 때까지) 동안 37 ° C에서 접시를 품어. </li><li> (액체의 문화를 시드하는 데 사용됩니다) 1 개월 최대 시간 동안 4 ° C에서, 그 후이 작업 판을 보관하십시오. </li><li> 우리의 프로토콜에서는, 우리는 L.를 사용 혈청보기 1/2A EGDe.PrfA * 균주 모노 사이토 겐 만도 1에 예시 된 바와 같이 L. P14.PrfA 4B monocytogenes의 혈청 형은 * 변형은 유사한 결과를 제공합니다. </li></ol></li><li> 그라 ATCC CCL2의 셀은 항생제의 부재 하에서 10 %의 소 태아 혈청 (FBS)이 보충 된 둘 베코 변형 이글 중간 (DMEM)에서 성장된다. </li><li> 스크램블 (제어) 및 안티 메트로 된 siRNA는 블랙 384도 현미경 세포 배양 접시에로드 된 독립 풀. <ol><li> RNase가없는 물 500 μL에 siRNA를 160 pmol의 희석 잘 당이 솔루션의 5 μL (최종 농도 1.6 pmol의)를 추가합니다. </li><li> 1 년을 최대 -20 ° C에서이 판을 보관하십시오. </li></ol></li><li> 이전 25 설명한대로 InlC-GST 재조합 단백질을 사용하여 예방 접종에 의해 얻어졌다 InlC 세균의 단백질에 대한 다 클론 토끼 항체. </li></ol> 2. siRNA를 세포 형질 반전 <ol><li> 감염 전에 72 시간 실온 아니라 각각의 RNase없는 물 5 μL에서 1.6 pmol의 siRNA에 포함 된 블랙 384도 현미경 세포 배양 접시에 가져다. </li><li> 300에서 3 분 동안 접시를 원심 분리기우물의 벽에 입금 된 수의 siRNA를 가지고 실온에서 RCF. </li><li> 0.4 % 리포 펙 타민 RNAiMAX 보충 실내 온도 DMEM을 준비합니다. </li><li> (siRNA를 추가하기 전에 더 이상 20 분 이상 형질 매체를 배양하지 않는다) 각 웰에 DMEM / RNAiMAX 형질 매체의 25 μl를 추가합니다. </li><li> 형질 감염 용액으로 siRNA를 혼합 앞뒤로 플레이트를 놓은 후 siRNA를 리포 펙 타민 - 복합체 형성하도록 실온에서 1 시간 동안 플레이트를 유지한다. </li><li> 한 10 ㎖ 37 ° C 미리 예열 PBS와 합류 또는 하위 합류 세포 배양 플라스크에서 세척 HeLa 세포. </li><li> 1 ㎖ 37 °를 추가하여 HeLa 세포를 분리 C는 세포 배양 플라스크에 트립신을 데워진 37 ℃에서 3 ~ 5 분 동안 품어 </li><li> 10 ㎖ 37 ° C에서 다시 정지 세포는 16 % FBS와 보충 DMEM을 데워진. </li><li> 세포를 계산하고 준비 DMEM의 ML 당 12,000 세포의 세포 현탁액은 보충16 % FBS. </li><li> 384 - 웰 플레이트의 각 웰에 세포 현탁액의 50 μl를 추가합니다. </li><li> 세포를 배포하고 세포는 실온에서 10 분 동안 정착하게 앞뒤로 빠르게 판을 이동합니다. </li><li> 파라 필름과 플레이트를 밀봉하고 37 ℃에서 가습 5 % CO 2 함유 분위기에서 72 시간 동안 보관 </li></ol> 3. 세포 감염 및 염색 <ol><li> 감염 전날, L.의 단일 식민지을 BHI 한천 플레이트에서의 monocytogenes 및 15 ml의 폴리스티렌 튜브에서 액체 BHI 배지 5 ㎖에 재현 탁. </li><li> 박테리아의 성장을 허용 동거 장치에서 37 ° C에서 밤새 품어. </li><li> 감염의 날, 하룻밤 L. 1 ㎖를 씻어 테이블 탑 원심 분리기에서 10,600 RCF에서 2 분 원심 분리하여 문화 모노 사이토 겐. </li><li> (분비 세포 독소의리스 테리 O를 포함) 상층 액을 버리고 PBS 1 ㎖ (반복 하오의 펠렛을 다시 일시 중지) 세척 단계 3 더 많은 시간을 t. </li><li> 600 nm에서 세균의 광학 밀도를 읽고 박테리아의 수를 (OD = 1 1E9 박테리아 / ㎖에 해당)으로 추정된다. </li><li> 적절한 L.에게 준비 1 % FBS와 보충 DMEM에 희석 모노 사이토 겐 : EGDe.PrfA *와 같은 고도의 침략 균주를 사용하여, 우리는 잘 당 매체의 30 μL에 5E4 박테리아의 사용을 (감염의 다중성이 2,000 셀 (25)로 추정된다)하는 것이 좋습니다. </li><li> 각 웰의 세포 배양 배지 (80 μL)를 제거하고 L.의 30 μl를 추가하여 교체 매체 모노 사이토 함유. </li><li> 감염 프로세스를 동기화하기 위해, 실온에서 5 분 동안 200 RCF로 플레이트를 원심 분리기. </li><li> 데워진 알루미늄 블록에 37 ° C에서 가습 5 % CO 2 함유 분위기에서 1 시간 동안 접시를 품어. </li><li> L.에게 제거 각 웰로부터 배지를 모노 사이토 겐을 함유하고 10 % FBS 보충 된 DMEM 데워진의 30 μL를 추가40 ㎍ / ㎖의 겐타 마이신 세포 L.를 죽일 모노 사이토 제네스. </li><li> 데워진 금속 블록에 37 ° C에서 5 % CO 2 함유 분위기에서 4 시간 동안 배양한다. </li><li> 8 %의 포름 알데히드 (오히려 파라 포름 알데히드 폴리머보다 포름 알데히드 단량체가 사용되도록이 솔루션은 신선한 준비를해야합니다)와 보충 PBS의 솔루션을 준비합니다. </li><li> 세포 배양 배지를 폐기하지 않고, 8 % 포름 알데히드 (최종 농도 4 %)으로 보충 된 PBS 30 μL를 추가하고 실온에서 15 분 동안 배양한다. </li><li> 정착액을 제거하고 (물론 당 PBS 80 μL의 최종 볼륨에있는 세포를 유지) 잘 당 PBS 80 μL와 세포를 세 번 씻는다. </li><li> 준비 1:250는 PBS의 토끼 안티 InlC 혈청의 희석은 0.2 % 사포닌. </li><li> 잘 PBS를 제거한 후 각각 차 항체 용액 10 ㎕를 추가하고 실온에서 30 분 동안 배양한다. </li><li> 차를 폐기항체 용액과 잘 당 PBS 40 μL로 4 회 세척한다. </li><li> PBS에서 보조 알렉사 형석 546 커플 안티 - 토끼 항체 (1:250), DAPI 용액 (1:1,500) 및 phalloidin도 - Dy647 (1:150)을 희석하여 0.2 %의 사포닌. </li><li> 각 웰에이 보조 염색 용액 10 μL를 추가하고 실온에서 30 분 동안 배양한다. </li><li> 차 얼룩 솔루션을 취소하고 (물론 각 40 μL의 최종 볼륨을두고 판을 밀봉) 잘 당 PBS 40 μL로 4 회 세척한다. </li><li> 플레이트는 바로 몇 군데 있습니다 또는 후속 분석을 위해 빛으로부터 보호 4 ° C (알루미늄 호일로 커버)에 저장할 수 있습니다. </li></ol> 4. 이미지 수집 및 분석 <ol><li> 자동화 된 현미경에 장착 된 10 배 목표를 (물론 당 우선적으로 9 이미지를 얻기)를 사용하여 세 개의 서로 다른 채널 (350 nm의, 546 ㎚, 647 nm의) 이미지를 획득. </li><li> InlC 신호는 화상을 이용하여 측정 할 수있다각각 DAPI와 phalloidin도 염색을 이용하여 핵 및 세포 기관의 자동 분할 할 수 CellProfiler 같은 분석 소프트웨어. </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Gly145Ser+substitution+in+the+transcriptional+activator+PrfA+causes+constitutive+overexpression+of+virulence+factors+in+Listeria+monocytogenes.">A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes.</a> Journal of Bacteriology. 179, 1533-1540 (1997).</li><li>Rajabian, T., 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=The+bacterial+virulence+factor+InlC+perturbs+apical+cell+junctions+and+promotes+cell-to-cell+spread+of+Listeria.">The bacterial virulence factor InlC perturbs apical cell junctions and promotes cell-to-cell spread of Listeria.</a> Nature Cell Biology. 11, 1212-1218 (2009).</li><li>Gouin, E., 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=The+Listeria+monocytogenes+InlC+protein+interferes+with+innate+immune+responses+by+targeting+the+I&#954;B+kinase+subinit+IKK&#945;.">The Listeria monocytogenes InlC protein interferes with innate immune responses by targeting the I&#954;B kinase subinit IKK&#945;.</a> Proceedings of the National Academy of Sciences USA. 107, 17333-17338 (2010).</li><li>Pizarro-Cerd&#225;, J., Lecuit, M., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+and+analysing+invasion+of+mammalian+cells+by+bacterial+pathogens:+the+Listeria+monocytogenes+system.">Measuring and analysing invasion of mammalian cells by bacterial pathogens: the Listeria monocytogenes system.</a> Methods in Molecular Microbiology. 31, 161-177 (2002).</li><li>Snijder, B., Sacher, R., R&#228;m&#246;, P., Damm, E. M., Liberali, P., Pelkmans, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population+context+determines+cell-to-cell+variability+in+endocytosis+and+virus+infection.">Population context determines cell-to-cell variability in endocytosis and virus infection.</a> Nature. 461, 520-523 (2009).</li></ol>

우리는 공개 아무것도 없어.

몇개의 파라미터는 InlC 신호의 명확한 검출을 허용하기 위해 충분히 큰 세포질 표시 건강한 세포주의 사용을 포함하는 트립 InlC 검출 프로토콜의 성공에 중요하다. 분석에서 우리는 우리가 인해 세포질 공간의 확장에 대한 우리의 분석에 특히 적합하다 헬라 CCL2 세포의 사용을 제안하고이 문서에서 제시 등의 헬라 교토 세포와 같은 다른 헬라 클론 작은 세포질을 표시하지만, 사용할 수 있습니다 ...

A correction was made to <a href="http://www.jove.com/video/51043">Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes</a>. Three authors were omitted from the article at the time of publication. The acknowledgments section was also updated. The author list has been updated from:
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute, 2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020
to
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Jason Mercer4, Mario Emmenlauer5, Christoph Dehio5, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute,2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020, 4Institute of Biochemistry, ETH Z&#252;rich 5Focal Area Infection Biology, Biozentrum, University of Basel
The acknowledgments where update from:
Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We thank Jason Mercer for optimizing the cellular transfection protocol.
to
Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We acknowledge support by grant 51RT 0_126008 for the Research and Technology Development (RTD) project InfectX in the frame of SystemsX.ch, the Swiss Initiative for Systems Biology (to C.D.).

A correction to author list has been made for the article Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes. Three authors were omitted from the article at the time of publication.

Erratum: Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

그람 양성 세균 리스테리아 균은 숙주 세포에 침입의 국제화 공포를 방해하고 숙주 세포 1의 세포질에서 복제 식품 매개 병원체이다. L. 세포 및 동물 모델에서 관찰 된 세균의 독성 특성의 지속성과 관련된 실험실 컨텍스트 (급속한 성장, 건강한 개인에 대한 낮은 독성)의 조작의 용이성 모노 사이토 겐 (용혈성 활동, 백혈구가)의 주요 모델로 1960 년에 처음 사용 허용 세포 내 기생의 감염 2에 대한 세포 성 면역의 이론적 기초의 설립을위한 연구. 1980 년대 후반과 1990 년대 초반, 세균의 세포주기 3뿐만 아니라 가장 중요한 세균의 독성의 분자 특성의 해부는 4-7 L.의 사용을 선호하는 요인 조작 및 스터드의 핵심 분자 도구로 모노 사이토 제네스숙주 세포 기능의 Y. 리스테리아 속에서 무독성의 존재 (L.의 innocua)과 악성 (L. monocytogenes에) 종 비교 게놈 연구 8 도로를 포장하는, 함께 완전한 L.의 최근 설립과 함께 모노 사이토는 L.의 진화에 대한 우리의 이해를 증가, 9 사체 인간 병원체 등의 감염을위한 모델 시스템으로 모노 사이토는 10 연구. L. monocytogenes에 자신의 숙주 세포 수용체 각각 E-cadherin의와 메트로, 11 ~ 12과 박테리아 표면 단백질 InlA 및 InlB의 상호 작용에 따라 숙주 세포로의 내면화를 유도한다. 초기 후보 기반 연구의 InlB - 중요한 음향과 InlA 침략의 통로 (13)와 phosphoinositide 3 - 키나제 (PI 3-K)의 중요한 구성 요소로서 α / β의 catenins - 굴지 링크의 식별에지도 의존 침공 casca데 14 ~ 15. 프로테오믹스 및 기능 기반의 분석 이후에 허용 된 새로운 골격 요소 (16)와 숙주 세포의 침공에 필요한 지질 두 번째 메신저 (17)의 식별. 전사 연구 (18)와 질량 분석 기반의 정량 프로테오믹스 (19)는 최근 호스트 신호 폭포의 활성화와 L. 동안 면역 반응의 억제에 관한 새로운 빛을 발산했다 감염 모노 사이토 겐. 시스템 생물학은 작은 간섭 RNA에 의한 유전자 (kinomes, 전체 게놈)의 큰 세트의 불 활성화에 기반 접근 (siRNA의) 침묵은 최근 식균 작용을 포함한 특정 세포 기능의 컨텍스트에서 폭포 신호 글로벌 호스트의 분석을위한 새로운 길을 열고있다 병원체 내면화 20. 게놈 전체의 siRNA 화면은 이전에 L.의 감염에 필요한 세포의 계곡을 조사하기 위해 수행 된 식세포 초파리 모노 사이토 제네스 </em&gt; S2 세포를 21 ~ 22 만, 이러한 유형의 분석은 비 식세포로 수행되지 않은, 생체 내에서 감염을위한 중요한 표적을 나타낸다. 우리는 L.에 의해 감염의 후반 단계의 현미경 검출을위한 프로토콜을 최적화 한 상피 세포 내 세균 항목의 높은 처리량의 siRNA 연구에 적합 모노 사이토 제네스. 우리의 분석은 매우 침습적 L. 활용 PRFA, L.의 주요 전사 조절에 점 돌연변이를 제시 변형 모노 사이토 겐 독성이 6 요인 리스테리아 모노 사이토 겐 (PRFA * 이름)이 돌연변이는 23 PRFA는 구조적으로 활성 렌더링 때문에, 그렇지 않으면 제대로 감염된 비 식세포의 세균 항목을 호의를 침공 단백질 InlA 및 InlB의 발현 증가로 연결됩니다. 감염에 대한 우리의 판독은 분비 된 세균의 단백질 InlC의 세포질 축적의 검출을 기반으로이 분자는내 세포질 L.에 의해 우선적으로 발현되는 형질 발현 이펙터 모노 사이토 9 참여는 박테리아 세포에 세포 24를 전파뿐만 아니라, 호스트 면역 반응 25을 변조하는뿐만 아니라. 세포 내 세균에 의해 InlC 분비의 형광 표시가 명확하게 감염되지 않은 세포에서 감염과 구별 할 수 있지만, 또한 엔드 포인트 이후의 여러 단계에서 감염을 해부하는 데 사용할 수있는 판독 나타냅니다뿐만 아니라 : 항목, 액포 탈출, 세포 내 세균을 증식과 세포 간 확산. 이 현미경 기반 프로토콜 따라서 L. 의한 숙주 세포의 감염에 관련된 세포 경로를 연구하는 siRNA를 스크린에 연결될 수있다 모노 사이토 제네스.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		 </tr>
		 <tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>BD</td>
			<td>237500</td>
			<td>For liquid BHI preparation</td>
		 </tr>
		 <tr>
			<td>Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>Supplement to liquid BHI for BHI agar plates </td>
		 </tr>
		 <tr>
			<td>Heat-Inactivated Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>51830-500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>61965-026</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamicin</td>
			<td>Sigma </td>
			<td>G1397-10ML</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Formaldehyde (16%)</td>
			<td>EMS</td>
			<td>15710</td>
			<td>Prepare fresh before each experiment</td>
		 </tr>
		 <tr>
			<td>Anti-Rabbit Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A-11035</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D-1306</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phalloidin Dy647</td>
			<td>Dyomics</td>
			<td>647-33</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Scramble</td>
			<td>Dharmacon</td>
			<td>D-001810-10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Met</td>
			<td>Dharmacon</td>
			<td>L-003156-00-0005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Black 384-well microscopy cell culture plate</td>
			<td>Corning</td>
			<td>3985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>AxioObserver Z1 microscope</td>
			<td>Zeiss</td>
			<td>431007 9901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>Neo</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Metamorph analysis software</td>
			<td>Molecular Devices</td>
			<td>4000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CellProfiler analysis software</td>
			<td>Broad Institute</td>
			<td>Public software available at <a href="http://www.cellprofiler.org/" target="_blank">http://www.cellprofiler.org/</a></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

imaging inlc secretion to investigate cellular infection by the bacterial pathogen listeria monocytogenes

세균의 세포 내 병원균 절묘하게 조작 및 호스트 대상 조직의 감염에 필요한 세포의 기능을 파괴하는 그들의 능력에 의한 세포 신호 폭포를 해부하는 분자 도구로 생각 될 수있다. 이러한 세균성 병원균 가운데, 리스테리아 균은 세포 면역 반응의 특성에 세포 내 기생에 대한 패러다임으로 사용 된 그람 양성 미생물이며, 세포 골격과 막 인신 매매의 역 동성을 제어하는 분자 경로의 발견에서 쓸모있는 역할을했다한다. 이 문서에서는, 우리는 L. 늦은 세포 감염 단계의 검출을위한 강력한 현미경 분석을 설명 모노 사이토는 InlC, 감염된 세포의 세포질에 축적 박테리아 분비 단백질의 형광 표지에 근거,이 분석은 숯불하기 위해 자동화 고 처리량 소 간섭 RNA 스크린에 연결될 수있다감염의 상향 또는 하향 조절에 관여 세포 신호 전달 경로를 acterize.

세포질 InlC의 형광 표지는 L.에 의해 세포 감염에 대한 강력한 판독을 제공합니다 모노 사이토는,도 1에 도시 된 바와 같이 : 현미경의 중앙 셀은 높게 P14.PrfA는 * 23 균주에 의한 감염에는 위상 콘트라스트 화상 (화살촉,도 1a)에서 관찰 될 수 있으며, 이러한 DAPI 신호에 의해 확인 된 것처럼 여기서 개별 박테리아 깨끗이 (도 1b)를 구?...

Watch this Scientific Journal Video about Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes at JoVE.com

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

리스테리아 균은 자주 세포 내 기생의 연구를위한 주요 모델로 사용되는 그람 양성 세균 병원체이다. 후반 L. 이미징 소 간섭 RNA 화면의 문맥 내 감염 단계 모노 사이토 겐은 표적 숙주 세포의 세균 감염에 필요한 세포 경로의 글로벌 학습을 허용한다.

세균성 병원체에 의해 세포 감염을 조사하기 위해 InlC 분비 이미징<em&gt; 리스테리아</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

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Immunology and Infection

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

9.8K 조회수.  Pasteur Institute.  리스테리아 균은 자주 세포 내 기생의 연구를위한 주요 모델로 사용되는 그람 양성 세균 병원체이다. 후반 L. 이미징 소 간섭 RNA 화면의 문맥 내 감염 단계 모노 사이토 겐은 표적 숙주 세포의 세균 감염에 필요한 세포 경로의 글로벌 학습을 허용한다. 

비디오: Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

사이 Synergism을 맡은 이후로 발광 생물 이미징을 사용하여 연쇄상 구균 pneumoniae</em유아 마우스에서>와 인플루엔자 바이러스

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

연구

면역학 및 감염병학

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

세균성 병원체에 의해 인간의 세포의 침략

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

감염된 마우스에 세균 하중과 면역 응답 측정 리스테리아 monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

곤충<em&gt; 갤러리아 mellonella</em&gt; 세균 Pathogenesis를 조사 할 수있는 강력한 감염 모델로

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

경구 전송<em&gt; 리스테리아</em&gt; 오염 된 음식의 섭취를 통해 마우스의

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

의 자동 분리<em&gt; C. 엘레</em&gt; 가변 세균성 병원체에 의해 식민지

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

리스테리아 단세포 유전자를 사용하여 감염된 마우스에 있는 문화와 심혼 식민지에 있는 심장 세포의 세균성 침략 평가

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA 정제는 세포 내에서 재배<em&gt; 리스테리아</em&gt; 식세포 세포에

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

와 실험 감염<em&gt; 리스테리아</em&gt; 호스트 인터페론-γ 응답 유학을위한 모델로

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.