우리 연구실의 연구는 맥스 폰 Pettenkofer 연구소, 루드비히 Maximilians 대학 뮌헨, 독일 연구 재단 (DFG, HI 1511/3-1)와 Bundesministerium FÜR Bildung 깨물어 Forschung (BMBF) &quot;의료 감염 지노 믹스&quot;이니셔티브에 의해 지원되었다 ( 0315834C).

1. LCV 절연을위한 준비 <ol><li> Legionella pneumophila는 LCV 절연 전에 5 μg / ML chloramphenicol (캠) 4 일 동안 CYE 한천 플레이트에 글리세롤 주식에서 DsRed-Express에서 13,14 생산 밖으로 행진. 37 세균을 품어 ° C. </li><li> 1 × 10 7 종자 디 GFP-calnexin 또는 75cm 두 조직 문화에 RAW264.7 murine macrophages를 생산 discoideum 언젠가 전에 감염을 flasks. 20 μg / 디위한 ML G418처럼 10 ML HL5 매체를 사용하여 23 ° C.에 discoideum과 amoebae을 품어 RAW264.7 macrophages 10 % FCS (열 inactivated)과 1 %의 글루타민과 함께 보충 10 ML RPMI 1640 배지를 사용하고 37에서 세포 성장을 위해 ° C에서 5 % CO 2.에게 감염 및 샘플마다 적어도 세 75cm 2 flasks를 (최소 6 X 10 7 세포)를 사용합니다. </li><li> L.과 하룻밤 문화의 예방 CYE 판에서 pneumophila. 예 중간 세 ML로 15 ML 테스트 튜브를 타고5 μg / ML의 캠. 0.1의 OD 600nm를 얻을 수있는 세균 현탁액을 100 μl와 예방. 21-22 시간 동안 37 ° C에서 오버헤드 회전 바퀴에서 하룻밤 문화를 품다. </li></ol> 2. LCV 절연 <ol><li> D.의 매체를 변경 discoideum 세포는 다음과 같은 감염을 방해하는 것이 항생제를 제거합니다. </li><li> 하룻밤 문화의 OD를 측정합니다. 박테리아는 2 X 10 9 박테리아 / ML에 해당 ≥ 3의 OD 600nm, 자신들의 절정 감염에 도달해야합니다. </li><li> L.의 약 500 μl를 첨가하여 세포를 감염 10 ML HL5 매체 (D. discoideum) 또는 10 ML RPMI 1640 (macrophages)에 성장 세포에 pneumophila 하룻밤 문화. 이것은 50 감염의 다수 (방어​​)에 해당합니다. 그 후에는 감염이 10 분, 500 × g에서 세포 고정 박테리아의 원심 분리에 의해 동기화됩니다. 원심 분리는 다음에 있습니다D.의 배양에 의한 25 ° C와 ° C 5 % CO 2, 각각 37 RAW264.7 macrophages에서 discoideum 세포. 1 시간의 감염 시간은 원심 분리 단계가 포함됩니다. </li><li> 감염 후 매체를 제거하고 세포 밖 박테리아를 제거하는 한 세포를 씻는다. D. 위해 얼음처럼 차가운 SorC 버퍼를 사용 discoideum 및 RAW264.7 macrophages를위한 얼음처럼 차가운 PBS. 각각의 플라스크에 단백 분해 효소 억제제 (로슈)로 보충 3 ML HS 버퍼를 추가하고 셀 스크레이퍼를 사용하여 세포를 수집합니다. 15 ML 테스트 튜브에 풀 대응하는 샘플. </li><li> 샘플 사용 3 ML 플라스틱 Luer-잠금 주사기와 스테인리스 스틸 볼을 균 질기의 균질하십시오. 당신이 얼음에서 작동하는지 확인하십시오. 시작하기 전에, 어떤 세제 오염을 방지하고 기포를 제거하기 위해 얼음 추위 HS 버퍼로 플러시, 증류수로 볼 균 질기 씻는다. 8 μm의 통관 공을 사용합니다. </li><li> 주사기 깨물어으로 처음 3 ML을 작성하는 일에 마운트전자 균 질기. 균 질기를 통해 앞뒤로 9 번 샘플을 누르십시오. - 균질하지 물질과 오염을 피하기 위해 나중에 주사기를 교환. 모아 15 ML 테스트 튜브에 균질 견본을 수영장과 미세한 분석을 위해 150 μl 채취. 다른 예제를 진행하기 전에 해체와 공 균 질기 씻는다. </li><li> 4에서 얼음 뿌리에 30 분 또는 오버헤드-물레 (10-20 RPM)에 2퍼센트 CS 또는 FCS와 homogenate를 차단 ° C. </li><li> 차단하기 SidC (희석 1:3000)에 반대하거나 LCV 막에 독점적으로 바인딩 기타 세균성 마커에 대한 친화력 - 정화 일차 항체를 사용하면. homogenate에 추가하기 전에 소용돌이 항체 솔루션입니다. 4 ° C.에 얼음 뿌리에서 1 시간 동안이나 오버헤드-물레 (10-20 RPM)에서 샘플을 품어 </li><li> 그동안 Histodenz 기울기를 준비합니다. 15 ML 기울기 당 시험관 하나의 샘플 세 75cm 2 flasks을 사용합니다. 전나무일, 튜브에 35 % Histodenz의 5.75 ML을 추가하고, 둘째, 조심스럽게 상위 10 % Histodenz 솔루션의 5.75 ML을 추가합니다. 이후 신중하게 1 시간 동안 다운 수평으로 튜브를 바칩니다. </li><li> 차단 시약 및 일차 항체, 4 펠렛에 ° C 시료에서 15 분, 600 × g에서 원심 분리기와 homogenate의 부화 후. 뜨는을 무시하고 1.5 ML HS 버퍼에 펠렛을 resuspend. 신선한 15 ML 테스트 튜브에 샘플을 전송하고 나중에 미세한 분석을 위해 40 μl 샘플을 채취한다. </li><li> 이차 항체와 펠렛을 품어 - 맥 염소 항 토끼 IgG 마이크로 비즈 (희석 1시 25분) - 흔드는 30 분을 4 ° C.에 그러는 동안, 맥 자석 홀더에 열을 올리고 0.5 ML HS 버퍼 그들을 평형. </li><li> 그 후, 컬럼에 샘플을 적용합니다. 이후 현미경 분석을 통해 유동-150 μl를 수집합니다. 0.5 ML HS 버퍼로 열 세 번 씻는다. </li><li> t에서 열 제거squirting 즉각 0.5 ML HS 버퍼와 그 자석과 eluate LCVs. 현미경 분석을 위해 20 μl 샘플을 가져가라. immuno-자성 분리에 의한 정화 단계는 1 인당 얻을 것으로 예상된다 × 10 7 D. 감염 discoideum 전지 약 1 × 10 6 그대로 LCVs에 대한 칠배로 흐름을 통해 13에 비해 eluate에 풍부합니다. </li><li> 수직 위치로 Histodenz 기울기 튜브를 다시 넣어 조심스럽게 위에 eluate를로드합니다. 4에서 1 시간 동안 3,350 × g에서 튜브를 원심 ° C. </li><li> 원심 분리 후 긴 유리 파스퇴르 피펫을 사용하여 시험관의 바닥부터 1.5 ML 분수 가져가라. 여덟 개의 분수, 튜브의 바닥에서 각 수집합니다. 아무 보이는 레이어는 지속 Histodenz 기울기에서 관찰되지 않습니다. LCVs의 높은 수율은 대개 ~ 2의 비율로 분수 4 예상되고 1 회 × 10 5 손상 LCVs × 10 7amoebae 13 fected. LCVs의 마이너 수량이 각각 분수 3과 5도 존재한다. 따라서 정화 LCVs의 전체 수확량은 ~ 1 × 10 6 × 10 7 당 6 그대로 LCVs는 감염이 디 discoideum 세포 13. LCVs의 복구는 실온 (RT)에서 수행하고, LCVs는 몇 시간 동안 안정적으로 나타날 수 있습니다. </li></ol> 3. 수집된 샘플의 현미경 분석 <ol><li> 폴리-L-라이신 코팅 coverslips으로 24도 평면 바닥 조직 배양 플레이트를 준비합니다. </li><li> LCV 정화 플러스 24 잘 플레이트에 각 밀도 기울기 분율 150 μl 중에 촬영한 모든 시료를 피펫. 높은 Histodenz 농도를 희석하기 위해 각도에 0.5 ML HS 버퍼를 추가합니다. 그 후, 4에서 10 분 동안 600 × g에서 번호판을 원심 분리기 ° C. 조심스럽게 뜨는을 제거하고 RT 20 분 4 % paraformaldehyde (PFA)로 샘플을 채운다. 고정 후 샘플 두번 씻는다. 우리D.위한 e SorC discoideum 및 RAW264.7 macrophages를위한 PBS. </li><li><ol class="lalpha"><li> D.을 표현 GFP-calnexin에서 샘플을 탑재 직접 예 Vectashield를 사용하여 유리 슬라이드에 LCV 절연, 동안 여러 단계에서 찍은 discoideum. </li><li> RT 10 분 차단 용액 (1 % PBS에서 BSA)과 함께 LCV 분리하는 동안 여러 단계에서 찍은 RAW264.7 macrophages에서 샘플을 품어. (; 버퍼를 차단에서 1:1000 친화력 - 정화 토끼 반 SidC) LCVs를 착색하고 RT에 젖은 챔버에서 1 시간 동안 부화 방지 SidC 일차 항체를 사용합니다. 그 후, PBS로 샘플을 세 번 씻는다. RT에서 습식 및 어두운 실내에서 30 분, 이차 항체 (솔루션을 차단에서 1:200 예 : 안티 토끼 Cy5)로 품어. 마지막으로, PBS와 함께 샘플을 세 번 씻고 예 Vectashield를 사용하여 유리 슬라이드에 그들을 탑재합니다. </li></ol></li><li> GFP-calnexin으로부터 격리 LCVs의 형태, 무결성 및 수량 표현 디 말요coideum 또는 RAW264.7 macrophages에서는 적절한 필터가 장착된 형광 현미경으로 분석된다. </li></ol> 4. 대표 결과 immuno-친화성 분리 및 밀도 기울기 원심 분리에 의한 LCV 정화의 품질과 수율은 형광 현미경 (그림 1) 뒤에는 수 있습니다. 샘플 개요 디에서 LCV 격리 수집된 discoideu m 또는 macrophages는 (그림 2) 표시됩니다. L.의 homogenate pneumophila에 감염된 phagocytes 그대로 LCVs를 보여줍니다뿐만 아니라, 세포 파편 및 세포외 박테리아의 많은. 샘플을 pelleting 후 이미지가 가끔 비트 밀도 homogenate와 비슷합니다. 그대로 LCVs이 칼럼에 충실해야한다고 이후 흐름을 통해 주로 세포외 박테리아와 세포 부스러기가 포함되어 있습니다. 칼럼에서 샘플을 eluting 후 eluate는 대량의 손상 LCVs가 포함되어 있습니다. 우리 손에서 LCV purificati<html> 에 디에 대한 더 효과적인 것 같습니다 macrophages에 비해 discoideum. D. 년 discoideum 병원체 vacuoles은 둥글게 만드는 표시 및 격리된 LCVs의 수율은 macrophages (그림 3)와 비교하여 10 회 이상 높다. 그대로 macrophages의 여러 항체 물들일 LVCs도 더욱 불규칙 모양의 나타납니다. <img alt="그림 1" src="/files/ftp_upload/4118/4118fig1.jpg" /> LCV 절연의 그림 1. 도식 개요. LCV 막에 독점적으로 현지화, 세균성 효과기 단백질 SidC 대한 친화력 - 정제 항체를 이용한 immuno-자성 분리에 의한 LCVs의) 분리. 이차 맥에서 마이크로 비드 결합 항체와 자석은 세포 파편으로부터 LCVs를 분리하는 데 사용됩니다. B) immuno-자성 분리 LCVs 후 추가 Histodenz 밀도 기울기 원심 분리에 의해 정화된다. LCVs은 분획 4에서 풍부합니다. upload/4118/4118fig2.jpg &quot;/&gt; 그림 2. 샘플 LCV 정화 수집된. homogenate, 펠렛에서 이미지 플로우 스루와 D로부터 eluate discoideum 또는 macrophages가 그려져있다. 샘플 L. 표시 pneumophila 디의 DsRed-익스프레스 (빨간색)를 생산 GFP-calnexin 신호 (녹색, 위쪽 패널) 또는 방지 SidC 항체 (시안, 낮은 패널) 물들일 macrophages의 생산 discoideum. 바, 10 μm의. <img alt="그림 3" src="/files/ftp_upload/4118/4118fig3.jpg" /> 그림 3. D.부터 LCVs 투석을 discoideum 또는 macrophages. 샘플은 L.의 Histodenz 밀도 기울기 원심 분리 후 분획 4를 표시 pneumophila는 (A) 라에서 DsRed-익스프레스 (빨간색)를 생산 GFP-calnexin 신호 (녹색)를 제작 discoideum 또는 (b) 항 SidC 항체 (시안) 물들일 macrophages 인치 바, 10 μm의.

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Chapter 3, Unit 3 (2010).</li><li>Urwyler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Legionella+vacuoles+purified+by+magnetic+immunoseparation+reveals+secretory+and+endosomal+GTPases.">Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases.</a> Traffic. 10, 76-87 (2009).</li><li>Mampel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planktonic+replication+is+essential+for+biofilm+formation+by+Legionella+pneumophila+in+a+complex+medium+under+static+and+dynamic+flow+conditions.">Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions.</a> Appl. Environ. Microbiol. 72, 2885-2895 (2006).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

이전에 다음 ID로 출판 메소드와는 달리,이 프로토콜은 두 단계 먼저 immuno-마그네틱 방식에 의한 LCVs의 분리, 둘째, 밀도 기울기 원심 분리에 의한 LCVs의 정화를 기준으로합니다. LCV 절연 쉽게 fluorescently 분류 박테리아와 GFP 생산 세포 또는 항체 염색법 중 하나를 사용하는 형광 현미경, 다음에 할 수 있습니다. 여기에서 설명한 프로토콜은 높은 순도와 LCVs의 농축에 결과 단순 스트레이트 포워드 방...

purification of pathogen vacuoles from legionella-infected phagocytes

기회 병원체 Legionella pneumophila도 따라서 중증 폐렴 &#39;Legionnaires&#39;병 &quot;1 원인이 폐포 macrophages에 복제 아메바에 강한 세균이다. protozoan과 포유류의 phagocytes에서, L. pneumophila는 구체적이고 복제 허용 구획, &quot;Legionella 함유 공포를&quot;(LCV) 결성 보존 메커니즘을 고용합니다. LCV 형성은 호스트 세포에 많은 275으로 &quot;이펙터&quot;단백질 translocates 세균성 ICM / 도트 타입 IV 분비 시스템 (T4SS)를 필요로합니다. effectors는 호스트 단백질뿐만 아니라 lipids를 조작하고 분비, endosomal 및 mitochondrial organelles 2-4와 통신합니다. LCVs의 형성은 reductionist의 방식으로 이해하기 어려운 복잡한 강력하고 중복 프로세스를 나타냅니다. 통합 접근 방식은 종합적 경로의 세계적인 분석을 포함하여 LCV 형성을 이해하는 데 필요한ogen - 호스트 요소의 상호 작용과 그 시간적 및 공간적 역학. 이러한 목표를 향한 첫 단계로서 그대로 LCVs이 정화되고 proteomics와 lipidomics으로 분석했다. 조성과 병원균 함유 vacuoles 형성은 액체 크로마 토그래피 또는 대량 분석법에 결합하여 2-D 겔 전기 영동을 사용 proteomic 분석에 의해 조사되었다. 사회적 토양 아메바 Dictyostelium의 discoideum이나 포유 동물 phagocytes 중 격리 Vacuoles는 Leishmania 5, 리스테리아 6,은 Mycobacterium 7, Rhodococcus 8, 살모넬라균 9 Legionella SPP을 숨겨두고. 10. 그들은 예를 들어 전자 현미경은 LCV의 형태, 무결성 및 순도를 분석할 필요로하지만, 이러한 연구들에서 사용된 정화 프로토콜은 시간이 오래 걸릴 지루한입니다. 또한 이러한 프로토콜은 실내의 병원균 공포의 특정 기능을 이용하지richment. 여기에 제시된 방법은 시각을 채용하여 이러한 한계를 극복 형광 LCV 마커를 생산 discoideum 및 세균성 효과기 단백질 SidC을 대상으로하여 어떤 선택 (PtdIns (4) P) 4 - 인산 phosphatidylinositol에 3,11을 바인딩하여 LCV 막에 앵커. LCVs는 고전 Histodenz 밀도 기울기 원심 분리 12,13 (그림 1)에 의해 두 번째 단계에 따라 SidC와 자기 구슬로 결합된 이차 항체, 대한 친화력 - 정화 일차 항체를 이용한 immuno-자성 분리에 의한 첫 번째 단계에서 풍부하고 . D.으로부터 격리된 LCVs의 프로테옴 연구 discoideum 13 전에 LCV 형성에 연루되지 않았 phagocytic vesicles, mitochondria, 응급실 및 잠돌군 Zz뿐만 아니라 여러 GTPases와 관련된 단백질을 포함한 560 개 이상의 숙주 세포 단백질을 발표했다. LCVs이 풍부하고 함께 정화프로토콜은 더욱 현미경 (immunofluorescence, 전자 현미경), 생화 학적 방법 (서양 얼룩) 및 proteomic이나 lipidomic 접근하여 분석할 수있다 여기서 설명했다.

Watch this Scientific Journal Video about Purification of Pathogen Vacuoles from Legionella-infected Phagocytes at JoVE.com

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

이 문서 그대로의 고립과 정화하는 방법을 설명합니다<em&gt; Legionella</em아메바와 macrophages에서&gt; 함유 vacuoles (LCVs). 두 단계 프로토콜은 밀도 기울기 원심 분리에 의한 세균성 LCV 표시 나아가 정화에 대한 항체를 이용한 immuno-자성 분리함으로써 LCV 농축 구성되어있다.

병원체 Vacuoles의 정제에서<em&gt; Legionella</em&gt; - 감염된 Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

purification-of-pathogen-vacuoles-from-legionella-infected-phagocytes

Research

JoVE Journal

Immunology and Infection

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

12.4K 조회수.  Ludwig-Maximilians-Universität. 이 문서 그대로의 고립과 정화하는 방법을 설명합니다 Legionella 함유 vacuoles (LCVs). 두 단계 프로토콜은 밀도 기울기 원심 분리에 의한 세균성 LCV 표시 나아가 정화에 대한 항체를 이용한 immuno-자성 분리함으로써 LCV 농축 구성되어있다.

비디오: Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

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 ...

연구

면역학 및 감염병학

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

혈액 배양에서 세균 병원체 탐지 및 항균 저항 테스트를위한 하루 워크플로 계획

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

감염된 세포의 염색질에서 인플루엔자 바이러스 Ribonucleoprotein 컴플렉스의 동질 정제

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

곰팡이 병원체 Phagocytosis의 라이브 셀 비디오 현미경

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

의 종 특이 검출을위한 루프 - 매개 등온 증폭 (LAMP) 분석 실험<em&gt; 에이 메리아</em&gt; 닭을 감염이

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

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. ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

인간 호중구 유래 거 식세포의 소설 모집단의 개발 및 식별<em&gt; 체외</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

효모 두 하이브리드 화면을 사용하여 비 경작 식물 병원체로부터 세균 이펙터의 기능을 해결했습니다

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

절연, 특성화 및 비만 관련 염증에 의해 영향을 받는 조직에서 세포의 정화

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

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

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

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

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

식 세포에서 세포내 복제에 대 한의 진 균 abscessus 독성 마커의 식별

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