저자는 Goodrich-블레어 연구실 과거 회원 감사 할 : 사만다 오차드 (Orchard), 킴벌리 Cowles, 에린 허버트 - 트란, 그렉 리차드, 메건 Menard, 그리고 Youngjin 공원이 프로토콜의 발전에 기여. 이 작품은 국립 과학 재단 (National Science Foundation) 기금 IOS-0950873 및 건강 NRSA 교제 FAI084441Z의 국립 연구소에 의해 자금을 지원했다.

1. 곤충 달걀 살균 및 양육 <ol><li> 900-1,000 ML H 2 O.에서 제공되는 한천의 첫 autoclaving 15g으로 음식을 준비 즉시 잘 실험실 믹서기에서 166g의 밀 배아식이 요법과 조화를 이루고있는 autoclaving, 혼합. 4에 알루미늄 호일에 음식을 전송 한 후 냉각 할 수있는 요리 (또는 요리)에 넣어, 단단히 포장 및 저장 ° C. </li><li> 도착시, M. 멸균을 때때로 교반 90mm 필터 종이 유리 필터 홀더와 진공 플라스크 장치에서 2-3 분에 0.6 %의 표백제 솔루션 250 ML과 sexta 달걀. </li><li> 세탁 당 250 ML 멸균 증류수 H 2 O로 표백 솔루션과 세척 계란 3-4 회를 배출하는 진공을 사용합니다. </li><li> 페트리 접시 뚜껑에 배치 신선한 90mm 필터 종이에 계란을 전송하고 건조 할 수 있도록 할 때까지 함께 그들은 더 이상 스틱 (20-30 분 정도). </li><li> 각 용기에 약 40 알을 배치, 곤충 음식을 플라스틱 용기의 바닥에 달걀을 전송(그림 1A). 달걀은 습기 유발 곰팡이 오염을 방지하기 위해식이 요법과 구분됩니다. 8 시간 짙은의 photoperiod : 16 시간의 빛으로 26 알 ° C를 유지합니다. 계란은 부화하기 전에 노란색 - 흰색으로 밝게합니다. </li><li> 부화가 완료되면 조심스럽게 아래 (그림 1B)에 다이어트를 통해 새로운 용기로 약 25 애벌레를 각각 전송합니다. 그것은 자신의 약한 초기 개발 단계에서 그들에게 손상을주지 않도록 집게를 사용하여 긴 검은 뿔 각 유충을 데리러하는 것이 중요합니다. 위와 같이 2 일 품다. 애벌레의 죽음이이 단계에서 특이한이지만, 죽은, 또는 가능성, 발달 저하 애벌레가 관찰 될 수 있습니다. 이러한 곤충들은 더 연구에서 제외하고 냉동 희생해야합니다. </li><li> 사람을 잡아 먹는 행동, 음식의 작은 조각 (그림 1C)과 개별 용기에 전송 애벌레를 피 이상으로 품다합니다. 곤충 개발을 모니터링, 음식을 교체 및 청소 배설물은 으세요매일 컨테이너의 t가 세 번째 애벌레의의 털을 갈다 (보통 6-9일 부화 후) 받아야까지. 이 곤충 몸 (그림 1D)를 따라 줄무늬의 각 proleg 및 증가 부각에 검은 색 훅 같은 crochets의 모습을 특징으로, 4 일 instar 애벌레의 단계입니다. 애벌레의 크기는 상당히 다양하지만, 일반적으로 4 번째 instar 단계를 입력시 0.15-0.4 g 무게 수 있습니다. 유사한 크기의 애벌레가 분사에 대한 선택해야합니다. </li></ol> 2. 사출에 대한 박테리아의 작성 <ol><li> 당신은 테스트하고자하는 성장 단계에 표준 절차에 따라 테스트 할 수있는 박테리아 변종 (들)을 성장. </li><li> 각 박테리아 스트레인의 펠렛 500 μl은 실온에서 13,000 rpm으로 microcentrifuge에서 2 분 동안 회전하여 테스트 할 수 있습니다. </li><li> 한 ML 멸균 1X 인산염의 Resuspend 세포가 위 다시 생리 버퍼 (PBS) 및 펠릿. </li><li> 0.5 ML 멸균 1X PBS 및 MEA에 Resuspend 세포정지 있는지 광학 밀도 (OD). 필요한 경우, 동일한 OD에 대한 모든 정지를 희석. 원하는 경우 멸균 성장 매체는 또한 1X PBS 대신에 세포 resuspension 및 희석에 사용할 수 있습니다. </li></ol> 3. 제 4 회 Instar의 애벌레의 주입 <ol><li> 각 희석에 대한 새로운 피펫 팁을 사용하여 멸균 1X PBS에서 96 잘 microtiter 플레이트 6 우물에서 처음으로 세균 변형의 일련 10 배 dilutions (그림 2A)를 준비합니다. 각 잘에 세포 현탁액의 최종 볼륨이 분사 (곤충 당 10 μl) 플러스 판 20 μl inoculum (단계 3.2 3.7 참조​​)에 필요한 볼륨을 초과해야합니다. </li><li> 적절한 성장 매체를 포함하는 페트리 접시 상단의 5 dilutions (10 -6까지 10 -2)에 대한 현장 10 μl 각. 장소는 중앙 (그림 2B)으로 확산되도록 판을 기울입니다. </li><li> 에 얼음 용기에 곤충식이 요법과 배설물 및 장소 곤충을 제거약 5 분 거리에 있습니다. </li><li> 3 회 70-100% 에탄올의 2 튜브의 각, 멸균 H 2 O (그림 2C) 중 하나 튜브 다음을 린스하여 주사기 바늘을 소독하다. 액체의 볼륨이 잠기 바늘​​에 충분해야합니다. </li><li> hemocytometer를 사용하여 현미경으로 희석 세포 현탁액을 Quantitate. 원하는 inoculum에 따라 잘 맞는 microtiter (그림 2D)에서 10 μl를 그립니다. </li><li> 견본이 곤충 95 % 에탄올로 표면, 그리고 복부 prolegs 중 하나 뒤에 (이하 45 °) 각도 곤충에 10 ML의 세포 현탁액을 주사. 펑크 용기를 내서 않도록주의를 타고 그냥 표피 층 (그림 2E) 아래의 내용을 삽입. hemolymph의 일부 손실 (일반, 녹색 파란색 액체)이 정상이지만, 사출 사이트에서 새로운 미립자과 노란색 액체가 내장 주사를 나타내는 수 있습니다. 이 경우 곤충를 희생하고 다시 할 수있는 새로운 애벌레를 얻을를 넣습니다. </li><li> 첫 번째 주입 그룹의 각 남아있는 곤충에 대한 단계 3.6을 반복합니다. 니들 살균은 오염이 발생하지 않는 한 박테리아의 동일한 희석과 변형을 주입 각각의 곤충 사이에 필요하지 않습니다. 또한, 반복 디스펜서는 샘플 사이에 주입 볼륨에서 더 일관성에 사용할 수 있습니다. </li><li> 박테리아의 첫 번째 변형 각 곤충을 주입 한 후,이 시간이 접시의 중앙에 세포 현탁액을 찾는하고 아래 방향으로 그 흐름 (그림 2 층) 주셔서 단계 3.2를 반복합니다. 적절한 온도에서 번호판을 배양하고 inoculum를 열거하는 식민지를 계산합니다. 이 두 번째 도금 단계는이 문서의 첫 번째와 두 번째 platings 사이의 서로 다른 식민지 번호 될 것 같은 샘플은, 주입 과정에서 오염되지 않은 것을 확인하는 데 사용됩니다. </li><li> 반복 실험에서 박테리아의 각 변형에 대해 3.8으로 3.1 단계를 반복합니다. 마지막으로, 동생이야를 사용하여 멸균 1X PBS로 3-5 벌레를 삽입부정적인 제어 그룹으로 구출 바늘. 8 시간 짙은의 photoperiod : 16 시간의 빛으로 26 ° C에서 곤충을 품다. </li><li> 주사 후 시간이 지남에 따라 곤충 생존을 모니터링합니다. 곤충 죽음을 자극시 자원 봉사 운동의 부족으로 특징입니다. 곤충은 종종 죽기 전에 감염 동안 식욕 감소, 설사 (물, 노란색 배설물), 및 / 또는 물 손실을 ( &quot;땀&quot;) 전시, 이러한 특성은뿐만 아니라 지적 가치가있을 수 있습니다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

M.의 직접 분사 여기를 설명 entomopathogenic 박테리아와 sexta의 유충은 박테리아 독성을 분석 할 수있는 간단하고 효과적인 수단으로 제공하고 있습니다. 방법은 다른 실험 과목 및 / 또는 조건에 맞게도 매우 적응합니다. 박테리아는 주입하기 전에 여러 가지 준비 할 수 있습니다. X의 경우 nematophila은 중간 로그 단계로 영양이 풍부한 Luria-Bertani (LB) 배지에서 자란 야생 형 ?...

<table border="1"><tbody><tr><td> 시약 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 90mm 필터 종이 </td><td> 워트 먼지 </td><td> 1001 090 </td><td></td></tr><tr><td> 유리 필터 홀더 </td><td> Millipore </td><td> XX1004700 </td><td></td></tr><tr><td> Manduca sexta 계란 </td><td> 캐롤라이나 생물학 공급 </td><td> 143880 </td><td></td></tr><tr><td> 집시 나방의 다이어트 + 한천 </td><td> MP의 Biomedicals </td><td> 0296029301 </td><td></td></tr><tr><td> 5.5 온스 플라스틱 용기 및 뚜껑 </td><td> 솔로 컵 회사 </td><td> URC55-0090 Pl4-0090 </td><td></td></tr><tr><td> 1온스 플라스틱 용기 및 뚜껑 </td><td> DART 컨테이너 주식회사 </td><td> 100PC 100PCL25 </td><td></td></tr><tr><td> 1X PBS </td><td></td&gt; <td></td><td> 137mm NaCl, 2.7 MM KCl, 8 MM이 그렇단 HPO 4, 1.46 MM KH 2 PO 4, pH를 7.4 </td></tr><tr><td> 주사기 </td><td> 해밀턴 </td><td> 80,208 </td><td> 30 게이지, 0.375 &quot;길이, 포인트 스타일이 </td></tr></tbody></table>

rearing and injection of manduca sexta larvae to assess bacterial virulence

일반적으로 담배 hornworm으로 알려진 Manduca sexta는, 담배, 토마토 등의 solanaceous 식물에 공급, 상당한 농업 해충으로 간주됩니다. M.의 민감도 entomopathogenic 박테리아 종 1-5,뿐만 아니라 곤충의 면역 체계 6-8에 대한 사용 가능한 정보의 부의 다양한 sexta의 유충, 그리고 보류중인 게놈 시퀀스 9는 호스트 미생물의 상호 작용을 연구에 사용에 대한 좋은 모델 생물 확인 pathogenesis 중. 또한, M. sexta의 애벌레는 다른 민감한 곤충 종의 실험실 상대적으로 조작 및 유지 관리가 비교적 크고 용이합니다. 그들의 대형도 감염에 대한 호스트 응답의 분석을위한 효율적인 조직 / hemolymph 추출을 용이하게한다. 여기에 제시된 방법은 M.의 hemocoel에 박테리아의 직접 분사 (혈액 캐비티)에 대해 설명합니다 sexta의 애벌레. 이 방법단순히 주사 후 곤충 죽음에 시간을 모니터링하여 다양한 세균 종, 변종, 또는 돌연변이의 독성 특성을 분석하고 비교하는 데 사용할 수 있습니다. 이 방법은 일반적으로 곤충에 항목을 얻을 수있는 수단으로 선충류 벡터와 연결 Xenorhabdus 및 Photorhabdus 종의 pathogenicity을 연구하기 위해 개발되었습니다. Entomopathogenic의 nematodes은 일반적으로 자연 소화 나 호흡 구멍을 통해 애벌레를 감염, 그리고 곤충 hemolymph (혈액) 즉시 (10) 내로의 공생 박테리아 내용을 놓습니다. 여기에 설명 된 분사 방법은 따라서 곤충에 박테리아와 선충류의 효과를 uncoupling, 선충류 벡터의 필요성을 무시합니다. 이 방법은 nicking 11 구두 독성 assays 12을 포함 entomopathogenesis을 분석하는 기존의 다른 방법을 사용 불가능 inoculum, 내 전염성 물질의 정확한 열거 (전지 또는 단백질)을 할 수 있습니다 <e직접 분사 방식은 전체 셀 inocula의 독성을 해결 반면, m&gt;는. 또한, 경구 독성 assays는 유충의 소화 시스템에 도입 분비 독소의 독성을 해결합니다. 여기에 설명 된대로 직접 분사 방식의 유틸리티는 곤충 사망률을 모니터링하여 세균 pathogenesis를 분석하는 것입니다. 그러나,이 방법은 쉽게 M.에 감염의 효과를 연구에 사용하기 위해 확장 할 수 있습니다 sexta 면역 시스템입니다. 벌레가 체액과 세포 반응 모두를 통해 감염에 응답합니다. 체액 반응은 박테리아 - 관련 패턴 인식 등 다양한 항균 펩티드의 후속 생산 7이 포함되며, 이러한 펩티드를 인코딩 유전자의 표현은 RNA 추출 및 정량 PCR 13을 통해 직접 감염 이후에 모니터링 할 수 있습니다. 감염에 대한 세포 반응은 유괴, 캡슐화, 그리고 hemocytes 6 전염성 대리인의 phagocytosis를 포함 </sup&gt;. 이 응답을 분석하기 위해 주입 곤충 해부와 현미경 13, 14으로 시각화 할 수 있습니다.

곤충 사망률 분석의 대표 예는 그림 3에 그려져 있습니다. 이 실험에서, 곤충이 중 야생 유형 (ATCC19061) 또는 중순 로그 단계 (변형 당 n은 = 6 곤충)로 성장 Xenorhabdus nematophila의 감쇠 돌연변이 스트레인 (lrp 13)의 약 50 식민지 성형 단위 (CFU)를 주입했다. 곤충은 약 72 시간에 관찰, 각 timepoint에서 아직 살아 주입 곤충의 %가 기록되었습니다. 이 경우, 감쇠 변형은 곤충 ?...

Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

방법은의 hemocoel에 entomopathogenic 박테리아의 직접 분사을 이용하여 여기에서 설명한<em&gt; Manduca sexta</em&gt; 곤충 애벌레.<em&gt; M. sexta</em&gt; 상업적으로 사용할 수 있고 잘 공부 벌레입니다. 따라서,이 방법은 하나 또는 두 파트너의 관점에서 호스트 박테리아의 상호 작용을 분석에 대한 간단한 접근 방식을 나타냅니다.

의 양육 및 분사<em&gt; Manduca sexta</em박테리아 독성을 평가하기&gt; 애벌레

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

rearing-injection-manduca-sexta-larvae-to-assess-bacterial

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JoVE Journal

Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

12.6K 조회수.  University of Wisconsin-Madison. 방법은의 hemocoel에 entomopathogenic 박테리아의 직접 분사을 이용하여 여기에서 설명한 Manduca sexta 곤충 애벌레. M. sexta 상업적으로 사용할 수 있고 잘 공부 벌레입니다. 따라서,이 방법은 하나 또는 두 파트너의 관점에서 호스트 박테리아의 상호 작용을 분석에 대한 간단한 접근 방식을 나타냅니다.

비디오: Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

연구

면역학 및 감염병학

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

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 ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

마다 가스 카 르 독성, 병 인, 및 약물 효능을 조사 하기 위해 대체 비 포유류 동물 모델로 서 치찰음의 바퀴벌레

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

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 ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

꼬마 선 충 모델에서 Aeromonas 감염의 병 인 및 독성 평가

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

마우스 모델을 평가 타고 난 면역 반응 포도 상 구 균 감염

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

높은 처리량 Shigella-특정 살 균 시험

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

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 ...