설명이 프로젝트는 알레르기 및 전염병 국립 연구소에서 보너스 번호 5R01AI116895에 의해 지원됩니다. 내용은 전적으로 저자의 책임이며 반드시 알레르기 및 전염병 국립 연구소 또는 국립 보건원의 공식 견해를 대변하지 않습니다.

1. 생성 샘플 <ol><li> 열 C를 활성화 디피는 얼음에 30 분 및 저장을 위해 65 ° C에서 포자. 참고 : C를 앞서 설명한 바와 같이 7,9,10 디피 포자를 제조 및 정제 할 수있다. </li><li> X는 샘플의 수는 분석 중에 촬영하는 X + 1 용액에서 발아 용액을 제조 하였다. 참고 : 박테리아의 종류가 연구되고 포자에 발아 솔루션은 고유의 것입니다. C.을 분석하십시오 디피 포자 발아, 우리는 10 mM 트리스 (PH 7.5), 150 mM의 NaCl을 탈 이온수 100 mM의 글리신 및 10 mM의 타우로 콜린 산의 용액을 사용 하였다. </li><li> 분석을 시작하기 전에, 피질 조각 검출을위한 빈 역할을하는 포자없이 발아 솔루션의 1.0 ml의 샘플을 그립니다. </li><li> OD 빨리 발아 솔루션 및 소용돌이에 ~ 3.0 (600)에 포자를 추가합니다. 참고 : B. 우리의 발아 연구 내용 서브 틸리 스, 우리는 같은 OD를 사용 (6)C. 용으로 00 포자 디피. 참고 : 포자의이 금액은 C. 동안 모니터링 피질 조각 방출을 위해 잘 작동 남과 어울리지 않는 포자 발아. 이 분석에 사용 된 포자의 크기는 각각의 박테리아 균주 또는 실험적으로 결정되어야한다. </li><li> 즉시 14,000 × g에서 1 분 동안 실온에서 원심 분리 포자를 첨가 한 후 1.2 ml의 샘플을 제거한다. 이것은 제로 시점이다. </li><li> 후속 피질 단편 분석을위한 신선한 튜브 원심 분리 된 샘플로부터 상등액 1.0 ㎖에 옮긴다. 주 : 테르븀 형광 9,18,19에 기초한 분석을 이용하여 상기 용액에서 DPA의 검출을위한 별도의 100 μL 샘플을 제거 DPA가 모니터링해야해야한다. </li><li> -80 ° C에서 수집 된 샘플을 고정. </li><li> 모든 시점이 완료 될 때까지 반복하여 일정한 간격으로 1.4 및 1.5 단계를 반복합니다. 참고 : 시간의 원심 분리 및 샘플링 시점 적은 일에 필요하기 때문에2 분 떨어져 우리의 절차를 수행하는 동안 불가능했던, 그래서 우리의 샘플링 간격은 한 번에 14 분 동안 매 2 분이었다. </li><li> 0, 12.5, 25, 50, 100, 250, 500, 5000 : 설탕 검출 및 정량화를 감소시키기위한 포지티브 대조군으로서, N의 -acetylglucosamine (nmol의) 다음의 양을 포함한다. 껍질 샘플과 동시에 이들 샘플을 준비하고, 표준 곡선이 포자를 발아에 의해 생성 된 데이터와 관련되도록 껍질 샘플과 함께 실행. </li><li> 모든 시점의 샘플을 수집 한 후, 샘플을 동결 건조. 참고 : 우리는 2 ml의 스크류 캡 튜브에 우리의 샘플을 동결 건조. </li></ol> 2. 코어 텍스 조각을 측정 <ol><li> 1 %의 페놀 및 0.5 % β 메르 캅토 에탄올이 보충 된 3 N HCl을 120 μL에 재현 탁하여 동결 건조 샘플. </li><li> 2 ml의 스크류 캡 튜브에 재현 탁 샘플을 전송합니다. 참고 : 재현성를 들어, 모든 동결 건조 된 시료를 재현 탁하고 전송되어 있는지 확인합니다. </li> &lt;리&gt; 4 시간 동안 95 ° C에서 재순환 수조에서 샘플을 품어. </li><li> 그들은 멋진 때까지 배양 한 후, 얼음에 샘플을 배치합니다. </li><li> 3 M NaOH를 120 μL로 샘플을 중화. </li><li> 포화 중탄산 나트륨 용액 80 ㎕를 각 샘플 및 와류에 5 % 아세트산 무수물 용액 80 μL를 추가한다. </li><li> 실온에서 10 분 동안 샘플을 인큐베이션. </li><li> 정확히 3 분 동안 다시 95 ° C의 물을 욕조에 샘플을 전송합니다. 참고 :이 단계는 민감하고 긴 시간이 하향 신호의 개발에 영향을 미칠 수있는 시간입니다. </li><li> 수조에서 샘플을 제거하고 얼음에 냉각. </li><li> 각 튜브와 소용돌이에 6.54 % K 2 B 4 O 7 · 4H 2 O의 400 μl를 추가합니다. </li><li> 정확히 7 분 동안 95 ℃에서 재순환 수욕 샘플을 인큐베이션. 참고 :이 단계는 민감하고 긴 시간이 하향 신호의 개발에 영향을 미칠 수있는 시간입니다. </li><li> 없애다5 분 동안 얼음에 샘플 및 장소. </li></ol> 3. 색상 시약 <ol><li> 샘플을 얼음 (단계 2.12)에 있지만, 컬러 시약을 준비한다. (; 에를리히 시약 DMAB) 빙초산의 1.9 ml의에서 P는 -dimethylaminobenzaldehyde의 0.320 g을 녹여. 참고 : 시약 시간, 온도, 빛에 민감하고 사전에 할 수 없습니다. </li><li> DMAB가 완전히 용해 후 10 N 염산과 소용돌이의 100 μl를 추가합니다. </li><li> 빙초산과 소용돌이의 5 ML을 추가합니다. </li></ol> 4. 비색 반응 <ol><li> 전송 각 100 ㎕ 새로운 1.5 ml의 microcentrifuge 관에 피질 샘플을 냉각시켰다. </li><li> 각 샘플에 갓 준비 색 용액 700 μl를 추가합니다. </li><li> 즉시 37 ° C의 물을 욕조에 샘플을 전송하고 정확히 20 분 동안 품어. </li><li> 무려 96 웰 플레이트에 각 시료 200 μl를 전송합니다. </li><li> 접시에 585 nm에서 샘플을 정량화리더. 샘플은 노란색에서 시작해야하며, 긍정적 인 반응이 보라색으로 이동합니다. 더 피질에 존재하는, 깊은 보라색 색상입니다. </li></ol>

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The authors have nothing to disclose.

자극 후, 발아 과정을 거치고 포자 고분자 합성에 대한 필요없이 저항 특성을 잃는다. 포자 발아가 트리거되면 포자 코어는 물이 교환 DPA를 발표한다. 인해 휴면 포자의 높은 DPA 콘텐츠 포자 코어는 팽창이 장벽으로, 아마도 작용하여 팽윤 코어 방지 강렬한 삼투압 및 전문 펩티, 피질 중이다. 상기 방법은 보라색의 색조에 변화 측정으로 환원당의 존재를 검?...

내생 포자는 대사 세균이 불리한 환경에서 지속 할 수 있도록 박테리아의 휴면 형태이다. 많은 포자 - 형성 박테리아 포자 형성 영양 결핍에 의해 유도되지만,이 공정은 산소 또는 다른 스트레스 1 pH를 노출의 변화에 의해 제어 될 수있다. 이들 대사 휴면 포자 형태 동안 박테리아는 UV 방사선, 탈수, 고온 및이 냉동 레지스트. 포자 형성 과정에 대한 지식의 대부분 모델에서 생체 연구 고초균에서 온다. 포자 형성 과정은 DNA 복제 및 축 3,4- 필라멘트의 형성으로 시작된다. 비대칭 격막은이 불평등 크기의 구획에 셀을 분할한다. 더 큰 구획은, 머더 셀은 더 작은 구획은 forespore을 삼키기. 머더 셀과 도만을 방출 포자 결국 lyses 머더 세포 성숙의 유전자 발현을 조정 forespore 두주변 환경 1에 t 포자. 포자 구조 및 조성은 다양한 세균 종에 걸쳐 보존되어있다. 포자 코어는 식물 세포보다 낮은 수분 함량을 갖고 dipicolinic 산 (DPA) -1,2- 풍부하다. 포자 형성하는 동안, DPA 물 교환 포자 코어로 펌핑된다. 코어 주변 대부분의 포자 형성 균에, 발아 (germinants)을 자극하는 작은 분자를 인식하는 수용체 2 위치, 내부 포자 막이다. 단 포자의 내막 밖에있는 세포벽의 펩티도 글리 칸 층이다. 특수한 펩티 층 (피질) 세포벽 펩티을 둘러싸고 [N 아세틸 글루코사민 (NAG)와 N-acetylmuramic 산 (NAM)이 교대] 세포벽 펩티 같은 많은 구성 요소로 구성된다. 그러나, 피질의 NAM 잔기의 약 50 %가 무람-δ 락탐 5,6- 변환되었습니다 </s&gt;입니다. 포자 발아 동안이 무람-δ 락탐 잔기 따라서 피질 아니라 칸막이 벽 (발아 마칠 공정)을 저하 할 수 포자 피질 용균 효소 (SCLEs)에 의해 인식된다. 피질 주변은 외부 막과 코트 단백질이 여러 층이다. 발아 과정을 제어하는​​ 포자 형성 세균 매우 중요하다. 싹 트는 수용체는 해당 germinants 2와 상호 작용할 때 발아가 시작됩니다. 많은 포자 - 형성 박테리아에서 이러한 germinants 2 이의 아미노산, 당, 뉴클레오티드, 이온 또는 이들의 조합이다. C. 디피 포자 발아 특정 담즙산 [예를 들어, 타우로 콜린 산 (TA)] 및 아미노산 (예컨대, 글리신) 7-10의 조합에 의해 개시된다. C. 사이의 차이가 있지만 디피 발아 경로와에서 공부 경로 다른 포자 형성같은 B와 박테리아, 서브 틸리 모든 공통 식물 세포는 포자 발아 2,8 성장할 수 있도록 포자 외피 저하하는 절대 조건이다. 또는 SLEC (B. 서브 틸리에서 발견) (많은 클로스 트리 디아에서 발견) 코어 텍스 저하는 SCLEs CwlJ / SleB에 의해 달성 될 수있다. 피질 가수 분해 세포벽 및 포자의 제한을 감소시킨다. 이는 전체 코어 수분 보충, 세포 대사 (2)에 필요한 단백질의 대부분을 재 활성화에 필요한 단계 수 있습니다. 포자가 발아 할 때 위상이 어두운 상태로 공정 상 밝은 상태로부터 휴면 포자 변화는 600에서 광학 밀도 (OD)의 변화에​​ 의해 측정 할 수있다 나노 11. 이전의 보고서는 OD의 변화의 대부분은 포자 12 DPA의 출시로 인해 있음을 시사한다. 우리의 최근의 연구 동안, 우리는 C.의 타이밍을 비교하고자 남과 어울리지 않는 포자 발아 및 모니터링되는DPA와 피질 조각 9 릴리스. 그들은 포자를 발아가 발표되기 시작으로이 연구는 피질 조각의 방출을 모니터링하는 것이 중요했다. 여기에서 사용되는 비색 분석이 환원 말단에 당류를 검출하는 방법에 기초 하였다 Ghuysen 외. (13)을 개발 하였다. 다른 사람이 환원당 (14)의 검출을위한 프로토콜을 설명했습니다 또는 이러한 프로토콜 15을 수정 한 때문에이 주제에 대한 문헌은 혼란 스러울 수 있습니다. 여기서, C. 발아에서 해방 환원당의 비색 검출 상세히에게 단계별 방법 우리 남과 어울리지 않는 포자. 이 연구는 C.를 사용하지만 남과 어울리지 않는 포자는 다른 포자 형성 균의 포자의 발아 중에 방출 된 환원당이 프로토콜 9,16,17 검출 할 수있다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.0 mL screw cap tube</td>
			<td>USA scientific</td>
			<td>1420-3700</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p200 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p20 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100-1250uL pipet tips</td>
			<td>VWR</td>
			<td>89079-486</td>
			<td></td>
		</tr>
		<tr>
			<td>1-200uL pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-D-glucosamine</td>
			<td>Sigma</td>
			<td>A3286-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid - 10N</td>
			<td>BDH-Aristar</td>
			<td>BDH3032-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Alfa Aesar</td>
			<td>L04295</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>BDH</td>
			<td>BDH0280-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Tetraborate tetrahydrate</td>
			<td>Alfa Aesar</td>
			<td>39435</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>BDH</td>
			<td>BDH8019-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(Dimethylamino)benzaldehyde</td>
			<td>Sigma</td>
			<td>156477-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Saturated Phenol</td>
			<td>Fisher</td>
			<td>BP1750-400</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Aldrich</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M3</td>
			<td colspan="2">Molecular Devices</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid, Glacial</td>
			<td>BDH</td>
			<td>BDH3098-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated, Circulating Water Bath</td>
			<td>VWR Scientific</td>
			<td>Model 1136</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtest 96</td>
			<td>Falcon</td>
			<td>353072</td>
			<td>96 well clear tissue culture plates</td>
		</tr>
		<tr>
			<td>Culture tubes</td>
			<td>VWR</td>
			<td>89000-506</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Blocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Machine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting cortex fragments during bacterial spore germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

표 1 NAG 표준을 사용하여 통상적 인 결과를 나타낸다. 데이터는 표준 곡선을 생성하는데 사용된다. 표 2는 100 mM의 글리신 및 10mM의 TA (발아 촉진 조건) 또는 100 mM의 글리신 만 (비 발아 조건)으로 보충 발아 버퍼에서 발아 시험에서 전형적인 결과를 나타낸다. TA, C.가없는 디피 포자가 발아하지 않고, 용액의 피질 단편의 존재 하에서 거의 변?...

Watch this Scientific Journal Video about Detecting Cortex Fragments During Bacterial Spore Germination at JoVE.com

Detecting Cortex Fragments During Bacterial Spore Germination

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

세균 포자 발아 동안 코어 텍스 조각 검출

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

detecting-cortex-fragments-during-bacterial-spore-germination

Research

JoVE Journal

Immunology and Infection

9.3K 조회수.  Texas A&M University. Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

비디오: Detecting Cortex Fragments During Bacterial Spore Germination

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

전파와 녹색 형광 단백질을 표현 Maedi - visna 바이러스의 감염성 분자 클론을 검출

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

연구

면역학 및 감염병학

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 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

발광 생물 세균 이미징<em&gt; 생체 내</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

요오드 산 쉬프 염색 말초 혈액 단핵 세포에서 글리코겐을 감지

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

포유 동물 세포에 기능 세균 이펙터의 전기

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

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 ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

말라리아 감염 시 세포 외 기 기능의 평가

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

방법 감지 세포 독성 Amyloids 다음 감염의 폐 내 피 세포 녹 농 균 에 의해

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

효소와 항 원에 대 한 Nonrecombinant 디스플레이 시스템으로 포자 흡착

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

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 ...

세균 포자 발아 동안 코어 텍스 조각 검출

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