저자 물고기 보호를위한 그림 4, Ulrike Nehrdich 및 데비 드 위트의 이미지 및 유용한 토론을 위해 다른 연구실 회원, 차오의 Cui에게 감사 층 드 Kort, 에스더의 웅크리다과 랄프 카르 발료주세요. 이 작품은 경제의 네덜란드 사역의 스마트 믹스 프로그램과 교육, 문화, 과학, 유럽위원회 일곱째 프레임 워크 프로젝트 ZF-건강 (보건-F4-2010-242048), 유럽 마리 퀴리의 교육부에 의해 지원됩니다 초기 교육 네트워크 FishForPharma (PITN-GA-2011-289209), 그리고 호주 NHMRC (637394)에 의해. 호주의 재생 의료 연구소는 빅토리아와 호주 정부의 주 정부의 보조금에 의해 지원됩니다.

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저자가 공개하는 게 없다.

이 문서에서 설명하는 감염 방식은 주로 호스트 타고난 면역 유전자 또는 세균성 독성 유전자 하나의 기능을 연구하는 데 사용됩니다. 정맥 마이크로 주입 방법 (프로토콜 5, 6.1)과 같은 공부를 가장 자주 사용됩니다. 사후 혈액 섬의 꼬리 정맥은 1 일 된 배아의 정맥 주사를위한 가장 편리한 위치입니다. 꼬리 정맥 나중 단계에서 침투하기가 더 어려워집니다로서 2-3 dpf에서 배아의 정맥 주사 사이트로 Cuvier의 덕트를 선호합니다. 모든 경우에 그것은 cfu의 계산을위한 도금 사출 inocula으로 선택되어야 박테리아의 일관성있는 번호로 배아를 주입하는 것이 중요합니다. 다음과 같은 측면 재현할 주사를 달성하기 위해 고려되어야합니다. 첫째, 이것은 고품질의 유리 microcapillary 주사 바늘을 가지고 필수적입니다. 바늘 끝이 너무 클 경우 주사가 발생하는 출혈을 위해 충분히 큰 바늘 구멍을 만들 것입니다주입된 박테리아가 혈액 순환의 돈은 절대 나오지 않습니다. 둘째, 박테리아는 혈액 순환에 직접 주입해야합니다. 바늘 끝은 난황 확장으로 분사 유체 확산 정맥 안쪽 아니면되지 않으면 배아는 실험에서 폐기되어야합니다. 셋째, M. 주입을위한 캐리어로 PVP40를 사용하여 marinum은 유리 바늘에 가라앉고에서 세균을 방지에 도움이 될 것입니다. PVP40 주사 기간에 걸쳐 더 재현할 inocula 그 결과 현탁액의 균질성 향상됩니다. PVP40 또한 S. 위해 사용될 수있다 큰 크기와 박테리아의 밝은 형광이 실험 중에 분사 inoculum 이상의 시각적 제어를 허용하기 때문에 typhimurium 주사하지만, 여기서는 덜 중요하다. 주사 연습을 위해 우리는 페놀 붉은 염료 (1 % V / V) 또는 1 μm의 다양한 색상 (Invitrogen)에서 사용 가능한 형광 분야의 사용을 권장합니다. 기술이 마스터되면, 그것은 약 30 minut 소요초밖에는 아가로 오스 플레이트에 배아를 정렬하고 세균성 사출 볼륨을 조정하는 데 필요한 시간을 포함하여 50-100 배아를 주입합니다. 혈액 섬 (프로토콜 5)로 또는 Cuvier (프로토콜 6.1)의 덕트로 정맥 주사가 가장 일반적으로 사용되고 있지만,이 비디오 문서에서 설명한 감염의 대안 노선 (프로토콜 6.2, 6.3 대식 세포 및 호중구 chemotaxis을 공부하는 데 유용 증명했습니다 및 6.4), 감쇠 박테리아 변종 (6.5 프로토콜)의 성장을 공부하고, 높은 처리량 어플 리케이션 (프로토콜 6.6) 4-8 대한 zebrafish 감염 모델을 적용할 수 있습니다. 설명 마이크로 사출 방법도 바이러스 18, 19, 곰팡이 포자 14, 20, protozoan의 기생충 (마리아 Forlenza, 개인 커뮤니케이션)의 주사에 대해 적용할 수 있습니다. 이 비디오 문서에서 설명하는 분사 절차에 유용한 첨가 zebr로 박테리아의 피하 주입에 대해 최근에 설명된 절차입니다금부어의 배아 21. 이 절차는 조직 표면에 효율적으로 빨아들이다 세균을 찾았으나되었다 neutrophils에 의한 세균의 phagocytosis를 공부하기 위해 적용되었다 macrophages과는 달리, 혈액 또는 외부로 유체가 노니는 신체 충치로 주입시 박테리아를 phagocytose 것이 거의 없었습니다. 피하 주사의 배아이 동영상에 대한 문서에서 설명한 꼬리 근육 주사 용과 비슷한 위치되지만, 바늘은 체절을 통해 박테리아를 주입하기 위해 단지 피부 아래에 삽입됩니다. 그것은 마이크로 사출은 본질적으로 감염의 인위적인 경로는 것을 고려하는 것이 중요합니다. 그러나, 초기 배아는 세균성 병원체에 대한 외부 노출에 매우 저항력이 있습니다. 실제로 태아가 세균 현탁액 목욕을하고 침지 assays는 우리 손에 22 재현할 수 없습니다. 일부 배아는 침수시 감염 될 수 있지만, 우리는 사망률 요금과 특급에 큰 변화를 관찰했습니다이러한 assays에서 개별 배아 간의 염증 마커 유전자의 이온. 이 문서에서 설명하는 마이크로 사출 방법은 재현성 감염을 달성하며 호스트 타고난 면역 세포와 세균의 상호 작용을 연구하는 데 특히 유용합니다. 각각의 배아에서 세균 감염을 계량하기위한 효율적인 접근법은 맞춤 전용 픽셀 부량 소프트웨어 17, 23에 감염된 태아의 형광 이미지를 분석하는 것입니다. 이 접근법은 감염된 태아의 도금 후 cfu 카운트의 결과와 잘 상관 관계를 보여왔다. 마찬가지로 태아 당 백혈구 수치의 상대적인 변화는 유전자 변형 백혈구의 형광단 기자 라인의 디지털 이미지 분석에 의해 계량되어,이 방법은 감염 24 호스트 leukopoietic 응답을 quantifying에 적용되는 것입니다. 호스트 타고난 면역 유전자의 기능을 설명하는 감염 모델을 결합하여 생체내에 효율적으로 조사 수morpholino를 최저로. Morpholinos 특정 RNAs을 타겟으로하고이 저널 10, 25의 다른 동영상 기사와 같이 1-세포 단계 zebrafish의 배아에 주입하면 유전자 제품의 표현을 줄이고 합성 안티 센스 oligonucleotides입니다. morpholino의 최저 연구에서는 비교되는 모든 배아 그룹이 세균 주사 이전 올바르게 개최되는 매우 중요합니다. 배아는 시간이 지남에 따라 점차 유능한되는 개발 면역 체계를 가지고 있기 때문에 이것은 특히 중요합니다. zebrafish 배아의 주요 타고난 면역 하위 집합, macrophages와 neutrophils를 구분하는 현재 사용할 수있는 여러 가지 유전자 변형 기자 라인이 있습니다. mpx 및 lyz의 발기인은 호중구 기자 노선 16, 26, 27을 생성하는 데 사용되었으며 csf1r (FMS)과 MPEG1 발기인은 대식 세포 기자에게 14, 28, 생산하는 데 사용되었다. 동안 csf1r (FMS)가 추가 xanthophores로 표현되며, MPEG1 표현이 macrophages에 독점적이다. 이 동영상을 문서에 나타난 바와 같이, 최근에 개발된 MPEG1 기자 라인 macrophages에 의해 영상 세균성 phagocytosis에 대한 매우 유용합니다.

<table border="1"><tbody><tr><td> 시약 </td><td> 공급 </td><td> 카탈로그 번호 </td></tr><tr><td> 페놀 레드 </td><td> 시그마 </td><td> P0290 </td></tr><tr><td> 글리세린 </td><td> 시그마 - 올드 리치 </td><td> 228,210 </td></tr><tr><td> Difco 미들 7H10 한천 </td><td> Becton, Dickenson 및 회사 </td><td> 262,710 </td></tr><tr><td> BBL 미들 OADC 농축 </td><td> Becton, Dickenson 및 회사 </td><td> 211,886 </td></tr><tr><td> Difco 미들 7H9 국물 </td><td> Becton, Dickenson 및 회사 </td><td> 271,310 </td></tr><tr><td> BBL 미들 ADC 농축 </td><td> Becton, Dickenson 및 회사 </td><td> 211,887 </td></tr><tr><td> 십대 초반 80 </td><td> 시그마 - 올드 리치 </td><td> P1754 </td></tr><tr><td> Polyvinylpyrrolidone (PVP40) </td><td> Calbiochem, 머크 KGaA </td><td> 529,504 </td></tr><tr><td> N &lt;/ em&gt;는-Phenylthiourea (PTU) </td><td> 시그마 - 올드 리치 </td><td> P7629 </td></tr><tr><td> 에틸 3 - aminobenzoate methanesulfonate 소금 (Tricaine) </td><td> 시그마 - 올드 리치 </td><td> A5040 </td></tr><tr><td> 메틸 셀룰로오스 </td><td> 시그마 - 올드 리치 </td><td> M0387 </td></tr><tr><td> SeaPlaque 아가로 오스 (낮은 용융 점 아가로 오스) </td><td> Lonza 주식 회사 </td><td> 50,100 </td></tr><tr><td> FluoSpheres (1.0 μm의 빨강, 형광 (605분의 580)) </td><td> Invitrogen </td><td> F8821 </td></tr><tr><td> FluoSpheres (1.0 μm의, (515분의 505) 노랑 - 녹색 형광) </td><td> Invitrogen </td><td> F8823 </td></tr></tbody></table>

infection of zebrafish embryos with intracellular bacterial pathogens

Zebrafish (Danio rerio) 배아는 점점 호스트 병원체 상호 작용 1 척추 타고난 면역 체계의 기능을 연구를위한 모델로 사용됩니다. 타고난 면역 체계의 주요 셀 유형, macrophages와 neutrophils은 사전 적응 면역 반응에 필요한 lymphocytes의 성숙에 embryogenesis의 첫 번째 일 동안 개발할 수 있습니다. 배아 다수를 획득 용이성은, 외부 개발, 배아 및 애벌레 단계의 광학 투명성으로 인해 그들의 접근은 유전자 도구, 유전자 변형 리포터 라인의 광범위한 돌연변이 자원과 콜렉션의 다양한 모든 zebrafish의 다양성에 추가 모델. 살모넬라 enterica serovar Typhimurium (S. typhimurium)와은 Mycobacterium marinum는 macrophages에 intracellularly 거주하고 자주 zebrafish 배아에서 호스트 병원체 상호 작용을 연구하는 데 사용됩니다. 이 두 가지의 감염 과정세균성 병원균은 비교하는 흥미로운 때문에 S. typhimurium 감염은 M. 반면, 급성 하나 일 이내에 치명적입니다 marinum 감염 만성이고 애벌레의 단계 2, 3까지 몇 군데하실 수 있습니다. 배아 (그림 1)로 박테리아의 마이크로 주입의 사이트는 감염이 급속도로 전신이 될 것이다 또는 초기화된 상태로 유지됩니다 여부를 결정합니다. 급속한 조직 감염에 의해 설정할 수 마이크로 주입 직접 사후 혈액 섬의 꼬리 정맥을 통해 혈액 순환에 또는 Cuvier, 트렁크 vasculature로 마음을 연결하는 난황에 대한 광범위한 유통 채널의 덕트를 통해 박테리아를. 한 dpf에서이 단계의 배아는 phagocytically 활성 macrophages을 가지고 있지만 neutrophils 아직 혈액 섬에 주입, 성숙하지 않은 경우 사용하는 것이 좋습니다. 배아도 기능적 (myeloperoxidase 생산) neutrophils를 개발 2-3 dpf,시 주사의 경우 Cuvier의 덕트는 t으로 선호그는 주사 사이트. 지역의 감염에 대한 골수양 세포의 이동 마이 그 레이션을 공부하려면, 박테리아는 꼬리 근육, 귀의 소포, 또는 hindbrain 뇌실 4-6로 주입 할 수 있습니다. 또한, notochord, 골수양 세포에 정상적으로 접근할 것 같습니다 구조, 지역 감염 7 매우 쉽습니다. 높은 처리량 어플 리케이션을위한 유용한 대안은 기름지게 8 후 첫 시간 이내에 배아의 노른자로 박테리아의 주사입니다. 형광 macrophages 또는 neutrophils과 형광 박테리아와 유전자 변형 zebrafish 라인을 결합하면 호스트 병원체 상호 작용의 멀티 컬러 이미징을위한 이상적인 환경을 만듭니다. 이 비디오 기사는 S로 zebrafish 배아의 정맥과 지역 감염에 대한 자세한 절차를 설명합니다 typhimurium 또는 M. marinum 박테리아와 타고난 면역 계통의 세포와 상호 작용의 후속 형광 이미징을위한.

Watch this Scientific Journal Video about Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens at JoVE.com

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

투명 zebrafish의 배아는 다음과 같은 시각화하는 유용한 모델 호스트와 타고난 면역 세포와 세포 세균성 병원체 간의 기능상 학습 상호 작용을 증명했습니다<em&gt; 살모넬라균 typhimurium</em&gt;과<em&gt;은 Mycobacterium marinum</em&gt;. 박테리아와 멀티 컬러 형광 이미징의 미세 주입은 zebrafish 배아 감염 모델의 응용에 관련된 필수적인 기술입니다.

세포내 세균성 병원균과 Zebrafish 태아의 감염

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

infection-of-zebrafish-embryos-with-intracellular-bacterial-pathogens

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

43.6K 조회수.  Leiden University. 투명 zebrafish의 배아는 다음과 같은 시각화하는 유용한 모델 호스트와 타고난 면역 세포와 세포 세균성 병원체 간의 기능상 학습 상호 작용을 증명했습니다 살모넬라균 typhimurium과은 Mycobacterium marinum. 박테리아와 멀티 컬러 형광 이미징의 미세 주입은 zebrafish 배아 감염 모델의 응용에 관련된 필수적인 기술입니다.

비디오: Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

카테고리의 감지 세균성 병원균에 대한 &#39;발광 생물&#39;리포터 파지

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

연구

면역학 및 감염병학

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

액포 파열의 단일 세포 측정은 세포 내 병원균에 의해 발생

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

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

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

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

Amoebal 공 배양 및 Amoebal 심화 접근에 의한 새로운 세포 내 병원균의 발견

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

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

세대와의 멀티 표현형 높은 콘텐츠 상영<em&gt; Coxiella burnetii의</em&gt; 트랜스포존 돌연변이

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

유도 발현 시스템을 적용하면 세포 내 신호와 세균 독성 요인의 간섭을 연구하기 위해

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

영상 병인과의 Cording를 해독하고<em&gt; 미코 농양</em&gt; Zebrafish의 배아에서

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

16S rRNA의 유전자 시퀀싱과 MALDI-TOF MS에 의해 희귀 세균성 병원체의 식별

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

다운스트림 단일 세포 분석에 대 한 요로 감염의 Murine 모델에서 생성 된 단일 세포내 세균성 지역 사회의 절연

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonophagocytic 살해 분석 결과 세균성 병원 체에 대 한 면역학 응답을 평가 하

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

마우스 종아리에 일관된 마사지와 같은 섭동을 적용하고 근육 내 압력 변화를 모니터링

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...