This work has been supported by Elizabeth E. Kennedy Research Award.

생산자 균주의 1 준비 <ol><li> 킬 폐렴 구균 균주는 -80 ° C 냉동고 주식에서 5 % 양 혈액 트립 틱 소이 한천 접시에 박테리오신 생산을위한 시험한다. NOTE : 억제를 판정하는 균주의 초기 성장을위한 혈액 플레이트의 사용은 크게 분석의 재현성을 증가시킨다. 성장 조건은 특정 유기체에 대해 수정해야 할 수도 있지만, 또한, 박테리오신을 생산하는 다른 비 - 폐렴 구균 균주를 사용할 수있다. 우리는 성공적으로 오버레이 분석에서 연쇄상 구균 mitis 및 락토 코커스 락 티스의를 사용하고 있습니다. </li><li> 5 % CO 2와 37 ° C에서 밤새 플레이트 (O / N)를 품어. </li><li> 생산자 균주의 배양 후, TSA 25ml를 함유 플레이트에 카탈라제 3,000 4,000 단위를 피펫 팅하여 트립신 콩 한천 (TSA) 판을 제조하고 멸균 유리 비드를 이용하여 용액을 카탈라아제 확산. 약 10 분 아래에 접시를 건조 시키생물 안전 캐비닛. </li><li> 피펫 팁에 박테리아의 가시적 양을 수집합니다. 건조 TSA 플레이트에 피펫 팁을 찔러. 참고 : 하나의 생산 균주 이상은 하나의 TSA 플레이트에 찔 수 있습니다. 결과 후광이 겹치지 않도록 그것은 찢고 밖으로 공간으로 중요합니다. <ol><li> 가능한 경우, 양성 대조군 및 음성 대조군으로서 오버레이에 사용되는 균주로서 프로듀서 박테리오신 알려진 것을 포함한다. 참고 : 양성 및 음성 컨트롤은 이전에 게시 된 아르 요청 10시 사용할 수 있습니다. </li></ol></li><li> 6 시간 동안 5 % CO 2와 37 ° C에 여러 찌르기를 포함하는 TSA 플레이트를 품어. </li></ol> 오버레이 스트레인의 2 만들기 글리세롤 주식 <ol><li> 오버레이 분석의 일 이전에 (글리세롤의 추가 후 중간 지수 단계로 성장하고 -80 ° C에서 보관 문화) 글리세롤 주식을 준비합니다. 폐렴 구균 STR 5-10 식민지를 접종아인은 0.5 % 효모 추출물 (THY)와 토드 휴잇 매체 감도 또는 페로몬 분비 검사를한다. 참고 : BlpC 유도 기자의 건설 이전에 2를 설명하고 균주의 요청에 따라 사용할 수 있습니다. </li><li> 흔들림없이 37 ° C의 물을 욕조에 단단히 밀폐 뚜껑 THY의 5 ㎖의 유리 튜브에 5 ~ 10 식민지를 품어. </li><li> 그들은 0.5 OD에게 0.3의 620에 도달 할 때까지 37 ° C에서 문화를 품다. 참고 :이 사용되는 균주에 따라 약 4-5 시간이 소요됩니다. <ol><li> 부드럽게 튜브에게 OD를 측정하기 전에 몇 번 반전. </li></ol></li><li> 실험 당일 수행 될 수 없거나 균주는 글리세롤을 보유하고 나중에 실험을 재개, 향후 여러 번 사용할 경우. 글리세롤 축적량 들어, 20 %의 최종 농도로 배양하여 글리세롤을 추가한다. -80 ° C에서 1.5 ML의 microcentrifuge 튜브 및 저장소로 나누어지는 문화. 참고 : 샘플의 할 수있다-80 ° C에서 달 tored. 글리세롤 주식을하지 않는 경우, 오버레이 분석을 위해 직접 샘플을 사용합니다. </li></ol> 3 오버레이 분석 <ol><li> 직전, 응용 프로그램을 오버레이 오버레이 구성 요소를 혼합한다. 15 ML 원뿔 관에서 THY 5 ㎖, 카탈라아제의 3,000 ~ 4,000 U, 그리고 그 중 하나 일 또는 실온에서 해동 된 문화의 글리세롤 주식을 성장시켰다 오버레이 균주 배양액의 200 μl를 추가합니다. 기자 균주를 사용하는 경우, 오버레이 혼합물에 X 갤런의 50 μL (40 ㎎ / ㎖)를 추가합니다. 접시 당 하나의 원뿔 튜브를 사용합니다. </li><li> 이전 용융 TSA의 추가로 실온에서 오버레이 혼합 평형. </li><li> 인큐베이터에서 칼에 찔린 균주 TSA 플레이트를 제거합니다. 전자 레인지에 고체 TSA 녹여 사용할 준비가 될 때까지 55 ° C의 물을 욕조에 보관하십시오. 1.5 % 한천로 용융 TSA 3 ㎖를 추가 혼합물을 오버레이 55 ° C로 냉각. <ol><li> 피펫 직전 오버레이 혼합물을 제조하고, 혼합물은 아주 고화 시작신속. 참고 : 오버레이 도금하기 전에 응고되기 시작하면, 그 결과는 해석 할 수없는 것입니다. </li></ol></li><li> 피펫 팅에 의해 및 5 ㎖의 피펫이 혼합물에 거품을 소개하지 않도록주의 여러 번 아래로 오버레이 혼합물을 섞는다. 천천히 칼에 찔린 TSA 플레이트에 직접 오버레이 혼합물을 피펫. 한천이 굳어 할 수 있도록 몇 분 동안 벤치 탑에있는 플레이트에 보관하십시오. <ol><li> 오버레이 혼합물을 배포 할 수있는 판을 돌리지 마십시오,이 오버레이에 찔 문화에서 성장을 끌어. </li></ol></li><li> 조심스럽게 5 % CO 2 O / N (약 16 ~ 18 시간)을 포함 37 ° C 배양기에 오버레이 TSA 플레이트를 배치합니다. </li><li> 하룻밤 성장 후 번호판을 관​​찰한다. 판 억제 또는 페로몬 신호가 발생한 지역을 제외하고 오버레이 균주의 성장 불투명 나타납니다. 이들은 각각 찔 변형 주위를 청소 또는 청색의 영역으로 표시됩니다. 참고 : 비록 지표 성과청색 또는 정화 직경 ements은 정량적 평가를 위해 결정될 수있다 직경의 차이는 또한 접시에 찔린 박테리아의 양에 따라 달라질 수있다. 오버레이 분석은 정량적 평가는 신중하게 해석되어야한다, 질적 분석이다. 음성 대조군에 비해 눈에 띄는 개간 활동으로 간주된다. </li></ol>

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The authors have nothing to disclose.

이 오버레이 분석은 박테리오신 생산자 활동의 범위를 결정하고 폐렴 구균 균주 사이에서 발생할 수있는 경쟁적인 상호 작용을 보여주기 위해 빠른 방법이다. 오버레이 분석은 하나의 플레이트에 대한 여러 박테리오신 생산 균주 선별을 위해 사용하도록 구성 될 수있다. 우리는 성공적으로 96 웰 플레이트의 절반에서 SBA 판을 접종 48 핀 복제기를 사용하여이 분석에 큰 부담 모음을 상영했다​​. 밤새 배양 한 후, 성장 복제기를 사용 리플리케이터의 핀의 접시 한천 표면 피어싱 TSA 플레이트에 전달된다. 란티 바이오 틱은 같은 농도 의존적으로 조절되는 기타 세균 분비하는 항균 펩타이드는 오버레이 방식을 사용 7,12,13 검사 할 수있다. 이 박테리오신 또는 lantibiotic의 정화가 어려운 경우에 특히 유용합니다. 상청액 항균 시험을 할 수 있지만활성은 배양액에서 BLP 유래 박테리오신의 발현은 (데이터는 도시하지 않음) 매우 제한적이거나 존재하지 않는 것이다. 오버레이 분석은 생물 액체에서 재배 할 때 달성되지 않는 높은 밀도로 성장 할 수 있습니다. 고체 배지에서의 성장도 더 밀접하게 인두의 생체 성장 조건을 반영 할 수 있습니다. 오버레이 분석은 또한 특정 시그널링 박테리오신 페로몬 분비 찔 균주의 능력을 검사하기 위해 사용될 수있다. 이것은 페로몬 수용체 단백질을 발현하고있는 페로몬 반응 촉진제의 lacZ 유전자의 상류에 배치된다 리포터 균주의 구축을 요구한다. 찔 균주 오버레이 변형을 저해하지 않는 경우 찔 균주에 의한 페로몬 분비 절상 BLP 궤적 활동 판독로서 특히 유용하다. 오버레이 변형을 억제 할 수 없다는 등의 같은 비 기능적 BLP 궤적 (의 결과가 될 수수송 돌연변이로 변형) 또는 분비 박테리오신 테스트 특정 오버레이 변형에 대한 활성화되지 때문이다. 기자 스트레인 오버레이는 생산자의 궤적이 페로몬의 상향 조절하고 분비 수 있도록 완벽하게 두 구성 요소 시스템 기능 및 BlpA 수송을 가지고 추가 정보를 제공합니다. 이것은 어떤 인코딩 박테리오신도 분비 것을 의미한다. 이 분석에 몇 가지 제한이 적용됩니다. 저해 활성 또는 상이한 균주 페로몬 분비 사이의 비교가 깊게 이루어져야 할 필요가 있으므로 오버레이 분석은 정성 분석이다. 분석의 결과는 오버레이 및 찔 균주의 성장 속도에 크게 의존하기 때문에 다른 균주를 비교했을 때의 성장 속도에 관한 조건이 고려되어야한다. 플레이트 및 오버레이 모두 카탈라아제의 첨가는 2 O 2 생산 폐렴의 H의 억제 효과를 제거하기 위해 중요오버레이 균주의 성장은, 특히 오버레이 카탈라아제 네거티브 유기체를 사용하는 경우. 폐렴 인구 게놈 컨텐츠의 다양성 공지 주어이 분석 함께 볼 어떤 저해 활성이 자동 분석 결실없이 BLP 궤적에 기인 할 수 없다.

폐렴 구균, 비 인두의 polymicrobial 지역 사회의 일반적인 식민지 개척자는, 세균 생성 된 항균 펩타이드 (박테리오신)의 생산을 통해 다른 폐렴 구균과 밀접하게 관련 종과 경쟁 할 수있다. 현재까지 조사마다 완전히 순서가 폐렴 구균 균주는 B의 acteriocin- 리터의 이케 페이지의 eptide 궤적, BLP의 버전이 포함되어 있습니다. 폐렴 구균에 의하여 생산 박테리오신은 양의 시험 관내 결과 및 생체 내 경쟁 1-4에 중요한 것으로 입증되었다. 경쟁적인 역학 특정 펩티드 페로몬에 응답하여 동족 내성 단백질과 함께 다양한 박테리오신의 발현에 의해 영향을 받는다. BLP 궤적 유도 펩티드 페로몬, BlpC는, 센서 키나아제, BlpH에 결합되는 정족수 인식 시스템에 의해 제어하고, 그 결과 신호 캐스케이드를 개시한다BLP 궤적 5,6의 전사. BlpC의 사 대립 유전자 변형이 있습니다 자사의 동족 BlpH 6 각 바인드. 셀 자체 박테리오신의 효과 살아남 들어 동족 내성 단백질을 인코딩하는 유전자는 유전자 박테리오신 각각 같은 오페론에 전사된다. 변형 방지에 필요한 특정 내성 유전자를 결여하는 경우 경쟁자 균주는 외인성 페로몬에 응답 BLP 궤적을 상향 조절 할 수없는 경우, 또는 죽이는 발생한다. 수송 복합체, BlpAB이 분비 및 처리 펩타이드 페로몬과 박테리오신 펩티드 2,4 필요합니다. 최근에는 폐렴 구균 균주의 상당한 부분들이없는 그들과 박테리오신 1,2 페로몬 분비 렌더링 blpA 유전자의 보존 된 돌연변이를 의미하는 &quot;사기꾼&quot;가 있다고 밝혀졌다. 이 균주는 울음 소리에 의해 분비되는 외인성 BlpC 2에 응답 할 수 있습니다면역 단백질의 생산을 허용 보링 균주. 상청액으로부터 박테리오신의 조질 제제는 순도를 달성하는 생화학 방법의 단계를 사용하여 제조자 균주로부터 제조 될 수 있지만,이 방법은 균주의 큰 컬렉션을 스크리닝 유용한 아니며 박테리오신 발현 수준은 국물 성장 배양 낮은 경우 2,7- 9. 오버레이 분석은 시험 관내에 존재할 수있는 균주간에 경쟁 역학 조사 신속한 방법으로 사용된다. 그렇게하려면, 폐렴 구균 균주는 미리 접종 한천 플레이트 상과 BLP 궤적의 유도에 충분한 로컬 박테리아 농도를 달성하기 위해 증가시킨다. 두 번째 변형은 다음 다음 감도를 테스트 할 수있는 사전 접종 균주에 걸쳐 적용되는 용융 부드러운 한천 솔루션에 접종한다. (억제 활성 무관) BLP 궤적 행위 평가하기 위해 균주 번째 일련의 오버레이 할 수이전에 설명 BlpC 기자 균주를 REE. 이 균주는 폐렴 구균에 의해 분비되는 가장 일반적인 세 BlpC 페로몬에 반응 세 가지 blpH 대립 유전자 중 하나를 포함하도록 구성되었다. 리포터 균주 BlpH 의존 프로모터의 제어하에 있고 자기 자극을 방지 10,11 blpC 유전자에서 삭제를 수행하는 통합의 lacZ 유전자를 가지고있다.

<table><tbody><tr><td>Trypticase Soy Agar with 5% Sheep Blood</td><td>Fisher</td><td>B21261X</td><td>For growth from freezer cultures</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C100-500mg</td><td>4,741 Units per plate</td></tr><tr><td>Todd Hewitt Broth + Yeast Extract (THY)</td><td>Fisher</td><td>DF0492-17-6&amp;nbsp;</td><td>30 g of THB, 5 g yeast extract, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>Trypticase Soy Broth + Agar plates (TSA)</td><td>Fisher</td><td>B11768&amp;nbsp;</td><td>30 g of TS, 15 g agar, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>5-bromo-4-chloro-3-indolyl &amp;beta;-D-galactopyranoside (X-gal)</td><td>Sigma</td><td>B4252-50MG</td><td>Dissolve in dimethylformamide</td></tr><tr><td>Petri dishes</td><td>Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Spectrophometer</td><td></td><td></td><td>Measures the OD&lt;sub&gt;620&lt;/sub&gt; of cultures</td></tr><tr><td>37 &amp;deg;C water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C incubator with 5% CO2</td><td></td><td></td><td></td></tr><tr><td>15 ml conical tube</td><td></td><td></td><td></td></tr></tbody></table>

using the overlay assay to qualitatively measure bacterial production of and sensitivity to pneumococcal bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

오버레이 분석에서 두 가지 결과가 있습니다. 하룻밤 배양 한 후 칼에 찔린 균주의 성장을 할 수 있어야한다. 그림 1A에서 오버레이 변형 박테리오신이 생산되고에 민감하다. 통관의 영역은 칼에 부담을 둘러싼 시각화 할 수 있습니다. 또한, 오버레이 혼합물에 찔 균주의 소용돌이가 가능하며,도 1a에서 볼 수있다. 그림 1B에서 오버레이 변형은 자상을 둘러싼 후광의 부재에 의해 언급 된 바와 같이 분비되는 박테리오신 영향을받지 않는다. 이 찔 균주 박테리오신을 분비하지 않은 것 또한 가능하다. 신호 분석의 경우, 두 개의 서로 다른 결과들이 가능하다. 찔 균주는도 2a에 도시 된 바와 같이 X-GAL의 분해를 통해 본, 오버레이 균주의 히스티딘 키나아제와 상호 작용 페로몬을 분비 할 수있다. 페로몬의 전사를 활성화하지 않는 경우찔 균주 페로몬 분비되지 않는 경우도 2b에 도시 된 바와 같이 리포터 균주 BLP 궤적, 또는 X-GAL의 파괴가 발생하지 않을 것이다. <img alt="그림 1" fo:content-width="5in" src="/files/ftp_upload/51876/51876fig1highres.jpg" width="500" /> 오버레이 분석에서 BLP 매개 억제 및 내성의 1 외관 그림. (A) P133 균주, 박테리오신 제조자는 TSA 플레이트로 아군과 6 시간 동안 성장시켰다. 균주 P250, 박테리오신 네거티브 균주 오버레이에 접종 하였다. 플레이트는 O / N 배양 후 촬영 하였다. 통관의 영역은 화살표로 표시됩니다. (B) 스트레인 P133은 TSA 플레이트로 아군과 6 시간 동안 배양 하였다. blpA에 틀 이동 돌연변이를 함유하는 변형 (130)는, 오버레이에 접종 하였다. O / N의 배양 후, 플레이트를 촬영했다. 그림 2 페로몬은 분석 신호에 BLP 궤적의 활성화를 매개. (A) 스트레인 P133, BlpC 유형 R6의 secretor은, TSA 플레이트로 아군과 6 시간 동안 배양 하였다. 스트레인 P981, BlpC에서 삭제되는 BlpC 유형 R6 기자, 오버레이에 접종 하였다. 사진은 O / N 배양 후 촬영 하였다. (B) P133은 TSA 플레이트에 타서. 6 시간 동안 배양 한 후, P845, BlpC에서 삭제되는 BlpC 형 P164 기자는 오버레이에 접종 하였다. 사진은 O / N 성장 후 촬영 하였다.

Watch this Scientific Journal Video about Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins at JoVE.com

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

오버레이 분석을 사용하여 성적으로 폐렴 구균 박테리오신에 대한 세균의 생산 및 감도를 측정하는

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

using-overlay-assay-to-qualitatively-measure-bacterial-production

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.4K 조회수.  University of Michigan. Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.  Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. 

비디오: Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

박테리아에서 단백질 RNA 결합을 정량화하기위한 분석

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

연구

유전학

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS로 인한 세균 경쟁을 모니터링 할 비주얼 분석

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

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 ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

막 극성, 멤브레인 무결성 및 세포 내 이온 농도의 모니터링 변경<em&gt; 폐렴 구균</em&gt; 형광 염료를 사용

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

를 사용하여 폐렴 구균 식민지에서 생균제의 효과를 조사<em&gt; 체외</em&gt; 준수의 분석

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

박테리아 - 호스트 상호 작용의 특성을 생체 모방 재료

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

소프트 한천 오버레이 기법의 사용은 세균 생산 억제 화합물에 대한 화면으로

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

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 ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

미생물 상호 작용의 조사를 위한 높은 처리량 공동 문화 애세서

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. 아루기노사 감염된 3D 기관상피세포 및 대식세포의 공동 배양

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

오버레이 분석을 사용하여 성적으로 폐렴 구균 박테리오신에 대한 세균의 생산 및 감도를 측정하는

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