이 작품 재향 군인 업무 공로 수상 (D.A.H.)에 의해 지원 되었다 그리고 건강의 국립 연구소는 부여 R01 AI 034431 (D.A.H.).

<ol>
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저자는 공개 없다.

Intraperitoneally 42 L. interrogans 돌연변이 함께 도전 하는 햄스터에 대 한 우리의 파일럿 실험 결과17제공 됩니다, 하지만 우리는 돌연변이의 더 큰 수영장 Tn이 의해 상영 될 수 있습니다 기대 Transconjugants의 주파수는 낮은 때문에 (100-200 transconjugants/짝짓기), 몇몇 matings 큰 Tn-Seq 실험에 대 한 돌연변이의 충분 한 번호를 생성할 필요가 있습니다. 액체 문화에서 돌연변이의 많은 수...

Leptospira 종 등 일부 박테리아에 대 한 독성 유전자의 유전 도구 사용할 수 있는 제한 된 수 때문에 어렵습니다. 일반적으로 사용 되는 한 가지 방법은 각 돌연변이에 삽입 사이트의 식별 및 독성 테스트 동물 모델에서 개별 transposon 돌연변이의 임의의 transposon mutagenesis에 의해 돌연변이의 컬렉션의 창조 이다. 이 방법은 소모, 비싼, 고 동물의 많은 수를 요구 한다.
무작위 mutagenesis Leptospira interrogans병원 체에 대 한 처음으로 개발, 유전자 독성에 관련 된 동물 모델1에서 개별 돌연변이 테스트 하 여 확인 되었다. 돌연변이 신호에 그들의 잠재적인 역할 또는 운동 성 또는 그들의 예상된 외부 막 또는 표면 위치 같은 기준에 따라 선정 됐다. Leptospiral의 대부분으로 유전자 인코딩 알 수 없는 함수2의 가상 단백질, 선택 돌연변이 이러한 조건 제한에 따라 소설 leptospiral 독성 유전자를 발견 하는 능력.
더 최근에, L. interrogans transposon 돌연변이의 풀 infectivity 햄스터와 마우스 모델3에 대 한 상영 했다. 각 동물 수영장 최대 10 돌연변이에 전했다. 돌연변이의 infectivity 문화의 혈액과 신장에서 얻은 PCR에 의해 검색 된 경우 긍정적으로 득점 했다. 수영장에서 각 돌연변이 대 한 개별 PCR 반응 필요 하기 때문에 PCR 테스트 하는 것은 힘 드는. 문화에 각 돌연변이의 주파수는 계량 하지, 때문에 접근 했다 높은 감쇠 돌연변이의 id 쪽으로 치우친 다.
(테네시-Seq) 기법, 독성 유전자에 대 한 화면을 보다 효율적으로 하는 전략으로 시퀀싱 하는 transposon을 설명 합니다. 대규모 병렬 시퀀싱4,,56다음 transposon mutagenesis에 의해 돌연변이의 도서관의 창조 테네시-seq에 의하여 이루어져 있었다. 간단히, transposon 돌연변이 풀링된, 동물에 주사 고 나중 다른 장기 (출력 풀)에서 복구. 출력 풀에서 DNA 추출 및 금지 효소로 소화 또는 쥡니다에 의해 전단. 2 라운드 PCR transposon 삽입 사이트의 접속을 대상으로 수행 됩니다. 이 단계는 시퀀싱에 필요한 어댑터의 추가 수 있습니다. 결과 PCR 제품의 그들의 관계 되는 풍부, 돌연변이의 수영장의 초기 구성에 비해 함께 수영장의 각 돌연변이 transposon 삽입 사이트를 식별 하려면 높은 처리량 연속 분석 된다.
이 방법의 주요 장점은 많은 동물의 작은 수로 돌연변이 동시에 화면을 기능입니다. 테네시-Seq 새로운 Leptospira발견의 기회를 증가 하는 transposon 삽입 사이트의 사전 지식이 필요 하지 않습니다-특정 유전자 독성에 더 적은 시간 및 더 큰 효율성과 관련된. 설치류 모델에 치명적인 감염 감염 조직에 leptospiral 부담에서 상대적으로 높기 때문에 (일반적으로 104 조직의 108 박테리아/g)7,,89 또한 저수지 호스트 에서 10,11, 조직 문화, 생체 외에서 성장으로 인해 편견을 줄일 필요 없이 직접 분석 될 수 있다.
테네시-Seq 날짜를 설명 하는 대부분 세균성 병원 체 연구, insertional mutagenesis의 높은 주파수 통칭 데 모든 유전자4 내 여러 긴밀 한-간격 transposon 삽입 돌연변이 포함 하는 큰 풀과 감염을 허용 ,12,,1314. 또한 테네시-Seq는 mutagenesis 주파수는 훨씬 낮은6박테리아에 대 한 개발 되었습니다. Leptospira, 함께 transposon 돌연변이의 라이브러리 활용 설명 된 대로 Slamti 외15여 의해 transposon mobilizable 플라스 미드에 도입 하 여 생성할 수 있습니다. 그러나, L. interrogans 의 transposon mutagenesis의 주파수가 낮습니다. Transconjugant 주파수만 8.5 받는 당 10-8 x L. interrogans16 라이 긴장으로 세포와 될 것입니다 알려졌다 Himar1 transposon conjugative 플라스 미드에 도입 되었다, L. interrogans의 대부분 다른 종자와 마찬가지로 가난한 사람 여기에 설명 된 프로토콜 보렐 리아 burgdorferi는 transposon insertional mutagenesis의 주파수는 또한 낮은6에 대 한 개발에 기반 하는 부분 이다.
프로토콜17우리의 파일럿 실험에 대 한 낮은 함께 피로 transposon 삽입 돌연변이 고립 시키는에 다른 그룹의 성공 때문에 L. interrogans serovar Manilae 긴장 L495와 transposon mutagenesis 실시 LD50 (치명적인 복용량) 독성1. 우리 테네시-Seq에 의해 42 돌연변이 검사 하 고 여러 돌연변이 후보 후보 adenylate 있고 유전자에서 2 삽입을 포함 한 독성에 결함을 발견. 햄스터에 두 돌연변이의 개별 테스트 그들이 결핍 독성17에 확인.

<table>
	<tbody>
		<tr>
			<td>Kanamycin sulfate from Streptomyces kanamyceticus</td>
			<td>Sigma-Aldrich</td>
			<td>K4000</td>
			<td></td>
		</tr>
		<tr>
			<td>2,6-diaminopimelic acid</td>
			<td>Sigma-Aldrich</td>
			<td>D1377</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin dihydrochloride pentahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S4014</td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Lab A1 microscope with a darkfieldcondenser</td>
			<td>Zeiss</td>
			<td>490950-001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy blood and tissue kit</td>
			<td>Qiagen</td>
			<td>69504/69506</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute PCR Purification</td>
			<td>Qiagen</td>
			<td>28004/28006</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104/28106</td>
			<td></td>
		</tr>
		<tr>
			<td>Model 505 Sonic Dismembrator</td>
			<td>Fisher Scientific</td>
			<td>FB-505</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5&#34; Cup horn</td>
			<td>Fisher Scientific</td>
			<td>FB-4625</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead Ruptor 24</td>
			<td>Omni International</td>
			<td>19-010</td>
			<td>Step 3.1.2.4</td>
		</tr>
		<tr>
			<td>Terminal deoxynucleotidyl transferase</td>
			<td>Promega</td>
			<td>M828C</td>
			<td></td>
		</tr>
		<tr>
			<td>Master mix Phusion</td>
			<td>Thermo Scientific</td>
			<td>F531</td>
			<td>Preparation of genomic libraries, step 3.4.</td>
		</tr>
		<tr>
			<td>DreamTaq Master Mix</td>
			<td>Thermo Scientific</td>
			<td>K9011/K9012</td>
			<td>Identification of the transposon insertion site, step 1.2.</td>
		</tr>
		<tr>
			<td>dCTP</td>
			<td>Thermo Scientific</td>
			<td>R0151</td>
			<td></td>
		</tr>
		<tr>
			<td>ddCTP</td>
			<td>Affymetrix/ USBProducts</td>
			<td>77112</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal cycler</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 fluorometer</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS assay kit</td>
			<td>Invitrogen</td>
			<td>Q32851/Q32854</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>Qubit assay tubes</td>
			<td>life technologies</td>
			<td>Q32856</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>PBS pH 7.2 (1X)</td>
			<td>Gibco</td>
			<td>20012-027 20012-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable scalpel No10</td>
			<td>Feather</td>
			<td>2975#10</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic K2 EDTA 2 ml tubes</td>
			<td>BD vacutainer</td>
			<td>367841</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe U-100 with 26G x &#189;&#8221; needle</td>
			<td>BD vacutainer</td>
			<td>329652</td>
			<td>IP challenge, step 2.2.1.</td>
		</tr>
		<tr>
			<td>3 mL Luer-Lok tip syringe</td>
			<td>BD vacutainer</td>
			<td>309657</td>
			<td>Cardiac puncture, step 2.4.2.</td>
		</tr>
		<tr>
			<td>25G X 5/8&#8221; needle</td>
			<td>BD vacutainer</td>
			<td>305901</td>
			<td>Cardiac puncture, step 2.4.2.</td>
		</tr>
		<tr>
			<td>25 mm fritted glass base with stopper</td>
			<td>EMD Millipore</td>
			<td>XX1002502</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>25 mm aluminum spring clamp</td>
			<td>EMD Millipore</td>
			<td>XX1002503</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>15 ml borosilcate glass funnel</td>
			<td>EMD Millipore</td>
			<td>XX1002514</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>125 ml side-arm Erlenmeyer flask</td>
			<td>EMD Millipore</td>
			<td>XX1002505</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>Acetate-cellulose filter VVPP (pore size 0.1 mm; diameter 25 mm)</td>
			<td>EMD Millipore</td>
			<td>VVLP02500</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput parallel sequencing to measure fitness of leptospira interrogans transposon insertion mutants during golden syrian hamster infection

이 원고에서 우리는 transposon 시퀀싱 (Tn-Seq) 기술을 식별 하 고 계량 Leptospira interrogans 돌연변이 피트 니스 시리아 황금 햄스터의 감염 시 변경 설명 합니다. 테네시-Seq 임의의 transposon mutagenesis 높은 처리량 시퀀싱 기술의 힘 결합합니다. 동물 뒤에 수확 혈액과 조직의 몇 일 후 식별 하 고 각 기관 (출력 풀)에 있는 돌연변이의 수를 계량 transposon 돌연변이 (입력된 풀)의 수영장 도전입니다. 출력 풀 각 돌연변이의 vivo에서 적합성을 평가 하는 입력에 비교 됩니다. 이 방법은 돌연변이 동물의 제한 된 수에서의 대형 수영장의 수 있습니다. 사소한 수정이 프로토콜 렙의 어떤 동물 모델, 저수지 호스트 모델 쥐, 햄스터, 생체 외에서 연구 등 급성 감염 모델 수행할 수 있습니다. 테네시-Seq vivo에서 그리고 생체 외에서 피트 니스 결함 돌연변이 대 한 화면에 강력한 도구를 제공합니다.

그림 1에서 보듯이 transposon 돌연변이 L. interrogans 에 활용 하 여 도서관의 창조 여과 장치를 요구 한다. 우리는 각 짝짓기에서 100-200 transconjugants를 복구.
Transposon 삽입 사이트는 transposon의 끝을 대상으로 어느 정도 임의적인 PCR에 의해 생성 된 PCR 제품을 시퀀싱으로 각 돌연변이에 식별 되 고 인접 한 ...

Watch this Scientific Journal Video about High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection at JoVE.c

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

여기를 식별 하 여 조직에서 transposon leptospiral 돌연변이 햄스터의 도전 후 계량 높은 처리량 시퀀싱 transposon mutagenesis 결합 기술을 설명 합니다. 이 프로토콜 생존 및 동물에 보급에 대 한 화면 돌연변이를 사용할 수 있습니다 하 고 생체 외에서 연구에도 적용 될 수 있습니다.

높은 처리량 측정 적합성 Leptospira interrogans Transposon 삽입 돌연변이 중 황금 시리아 햄스터 감염의 병렬 시퀀싱

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

high-throughput-parallel-sequencing-to-measure-fitness-leptospira

Research

JoVE Journal

Immunology and Infection

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

8.8K 조회수.  Veterans Affairs Greater Los Angeles Healthcare System. 여기를 식별 하 여 조직에서 transposon leptospiral 돌연변이 햄스터의 도전 후 계량 높은 처리량 시퀀싱 transposon mutagenesis 결합 기술을 설명 합니다. 이 프로토콜 생존 및 동물에 보급에 대 한 화면 돌연변이를 사용할 수 있습니다 하 고 생체 외에서 연구에도 적용 될 수 있습니다.

비디오: High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

인플루엔자 바이러스에 대한 높은 처리량 검출 방법

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

연구

면역학 및 감염병학

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 ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

높은 처리량 유전자 태그에<em&gt; 트리파노소마 (Trypanosoma) brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

높은 처리량을 지원 분석은 대식 세포 계대에 대한 약물 효능을 평가하기<em&gt; 결핵균</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

밀도 Transposon 삽입 라이브러리를 사용 하 여 세균성 활용 Enterobacterial 종자와 같은 대장균 또는 Shigella flexneri 의 창조

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

주문, 녹 농 균 스트레인 PA14 Transposon 삽입 돌연변이의 비중복 라이브러리의 복제

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

포유류 세포에서 효율 Resealing 원형질 막의 높은 처리량 측정

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

평가 하 고 호 중구 Extracellular 함정 대형 계량 높은 처리량 분석 결과

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

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 ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

혈청형 1 Streptococcus pneumoniae 균주 519/43에서 돌연변이 구성

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...