Name: assessing bacterial invasion of cardiac cells in culture and heart colonization in infected mice using listeria monocytogenes
Uploaded: 2015-05-27T14:00:00.000+00:00
Duration: 9 min 18 s
Description: Watch this Scientific Journal Video about Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes at JoVE.com

이 작업은 공중 보건 서비스 보조금 AI41816 및 AI099339 (N.E.F.) 및 NiAID의 F31AI094886-01 (P.D.M)에 의해 지원되었습니다. 내용은 전적으로 저자의 책임이며 반드시 자금 출처의 공식 견해를 나타내는 것은 아닙니다.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes:+silage,+sandwiches+and+science.">Listeria monocytogenes: silage, sandwiches and science.</a> Anim Health Res Rev. 6, 211-217 (2005).</li><li>Grundler, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surface+proteins+InlA+and+InlB+are+interdependently+required+for+polar+basolateral+invasion+by+Listeria+monocytogenes+in+a+human+model+of+the+blood-cerebrospinal+fluid+barrier.">The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood-cerebrospinal fluid barrier.</a> Microbes Infect. 15, 291-301 (2013).</li><li>Zeldovich, V. B., Robbins, J. R., Kapidzic, M., Lauer, P., Bakardjiev, A. 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저자는 경쟁 적인 재정적 이익을 보고하지 않습니다.

L. 단세포 유전자는 광범위하고 잘 특징인 인간 병원체, 다른 질병 표현의 다수를 일으키는 원인이 될 수 있는15. 박테리아는 이전에 중앙 신경계에 도달하고 식민지화하고 태아를 개발하기 위하여 혈액 두뇌 장벽 및 태반 태아 장벽과 같은 장벽을 가로질러 배분하는 기능에 대해 설명되었습니다. 이 조직을 식민지화하는 유기체의 생체 내 능력은 종종 표적 장기를 구성하는 ?...

리스테리아 단세포 유전자는 노인, 임산부, HIV/AIDS를 가진 사람들 및 화학요법을 받는 사람을 포함하여 취약한 인구에 있는 가혹한 질병을 일으키는 원인이 될 수 있는 그람 양성 세포내병원체입니다 1. 이 인구에 있는 감염은 수시로 오염한 식품을 섭취의 결과이고, 대부분의 감염은 대규모 식품 매개 발발과 연관됩니다2,3. 인간과 다른 포유동물에서, L. monocytogenes는 소장의 상피 경계를 가로질러 배설할 수 있고, 그 후에 간4,5로이송된다. 동물 모델은 섭취한 박테리아가 장 내의 담악과 포털 정맥을 통해 간으로 이동하거나 혈류로 막대한 림프절을 통해 퍼지며 간 및 비장에 혈액 선서를 통해 혈액 류로 확산되는 것을 제안한다6,7. 간과 비장에서 박테리아는 전문 식세포뿐만 아니라 상주 한창 세포로 섭취를 중재 할 수 있으며 이러한 장기 내에서 신속하게 감염을 확립합니다. 세균성 하중이 증가함에 따라, 수많은 박테리아는 혈액으로 다시 분산되어 중추 신경계와 태반 (현재)을 포함하여 취약한 조직을 더 식민지화 할 수 있습니다. 이 사이트의 식민지는 뇌막염, 뇌염 및 태아 낙태2를포함하여 인간에서 리스테리오증의 일반적인 표현을 배제합니다.
L. monocytogenes의 선택된 하위 인구는 최근에 심장 조직8내에서 침략하고 복제하는 향상된 능력을 갖는 것으로 나타났습니다. 심장 참여의 증상은 다양하며 내막염과 복막염에서 부터 전도 이상으로 완성된 충만성 심근염에 이르기까지다양하다. L. monocytogenes 심장 케이스의 전반적인 수는 1 년에 낮습니다 그러나 감염의 이 면이 일반적으로 잘 인식되지 않기 때문에 추정될 수 있습니다. 병원체에 의한 심장의 식민지화는 종종 기존의 용기 손상 또는 인공 심장 판막과 같은 호스트 소인이 필요합니다. 그러나, 어떤 심장 손상 및/또는 이상이 없는 경우에 감염된 동물의 심혼을 식민지화하는 그들의 능력에 대해 주목할 만한 확인된 L. monocytogenes의 격리가있습니다 8.
본명에서 조직 배양뿐만 아니라 살아있는 동물 감염에 침입 적인 소심을 이용하여 감염된 동물 내의 심장 조직의 세균성 식민지화를 평가하기 위한 체외 및 생체 내 방법으로 기재된다. 이러한 방법은 감염된 동물의 심장 조직을 식민지화 할 수있는 잠재력을 가진 균주를 식별하는 데 유용할뿐만 아니라 심장 세포 침입을 강화하고 심장 병리학의 변화를 초래하는 세균 성 유전자 제품의 식별에 유용해야합니다. 이러한 방법은 여러 균주 사이의 심부전의 비교를 용이하게. 여기서 설명된 방법에 대해, L. monocytogenes 10403S는 비심혈성 균주를 잘 연구한 대표자로 사용되며 임상 분리 07PF0776은 심장영양균의 대표적인 예로 사용된다. 이 두 균주는 생체 내에서 감염된 마우스의 심장의 체외 및 식민지에서 심장 세포의 세균성 침입에대한 비교를 제공하기 위해 선택되었다. 분리07PF0776은 HIV+ 환자8에서치명적인 부정맥을 일으킨 간 농양에서 회복된 임상 격리이다. L. monocytogenes 분리그들의 독성 잠재력에 다를 수 있습니다., 그리고 리스테리아 면역 억제와 임산부를 감염 하는 성향을 감안할 때, 이러한 인구 내의 사람들은 다른 임상 격리를 평가 하는 동안 주의 행사 해야.

<table><tbody><tr><td>Difco</td><td>BHI Broth Base</td><td>237200</td><td>2 kg of Brain Heart Infusion powder (without agar)</td></tr><tr><td>Difco</td><td>Agar</td><td>214510</td><td>2 kg of Granulated Agar</td></tr><tr><td>Difco</td><td>BHI Agar</td><td>241810</td><td>2 kg of Brain Heart Infusion Powder with 7.5% Agar</td></tr><tr><td>in vitrogen</td><td>LB Broth Base</td><td>12975-084</td><td>2 kg of Luria Broth Base Powder (without agar)</td></tr><tr><td>Fisher</td><td>Glass Coverslips</td><td>12-545-80</td><td>12 mm glass coverslips</td></tr><tr><td>Falcon</td><td>24-well Tissue Culture Plate</td><td>35-3047</td><td>24 well, Plasma-treated Tissue Culture Plate</td></tr><tr><td>Falcon</td><td>14mL Polystyrene tube</td><td>352051</td><td>14 ml Polystyrene tubes</td></tr><tr><td>Falcon</td><td>96-well U-bottom plate</td><td>35-3077</td><td>Non-tissue culture treated 96-well plate</td></tr><tr><td>Corning</td><td>0.05% Trypsin, 0.53 mM EDTA (1X)</td><td>25-052-CI</td><td>Stock solution of Trypsin EDTA for tissue culture</td></tr><tr><td>Corning</td><td>DMEM (High glucose, high pyruvate)</td><td>15-013-CU</td><td>DMEM media without FBS, glutamine, or antibiotics</td></tr><tr><td>Corning</td><td>Pen/Strep/Glut Solution</td><td>30-009-CI</td><td>Stock mixture of penicillin, streptomycin, and L-glutamine for tissue culture</td></tr><tr><td>Corning</td><td>Gentamicin</td><td>30-005-CR</td><td>50 mg/ml Stock solution of Gentamicin for tissue culture</td></tr><tr><td>Cellgro (Corning)</td><td>L-Glutamine</td><td>25005197</td><td>Stock L-glutamine solution without antibiotics</td></tr><tr><td>Denville</td><td>75cm^2 Flask</td><td>T1225</td><td>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture treated flask</td></tr><tr><td>Denville</td><td>1.5mL microcentrifuge tubes</td><td>2013004</td><td>1.5 ml microcentrifuge tubes</td></tr><tr><td>ATCC</td><td>H9c2 Cardiac Myoblasts</td><td>CRL-1446</td><td>Rat cardiac myoblasts</td></tr><tr><td>Hyclone</td><td>Fetal Bovine Serum</td><td>SH30070.63</td><td>Fetal Bovine Serum</td></tr></tbody></table>

assessing bacterial invasion of cardiac cells in culture and heart colonization in infected mice using listeria monocytogenes

리스테리아 단세포 유전자는 면역 손상 환자, 노인 및 임산부에서 심각한 침습적 감염을 일으킬 수있는 그램 양성 성 세포 내 병원균입니다. 인간에 있는 listeriosis의 일반적인 표현은 뇌막염, 뇌염 및 태아 낙태를 포함합니다. 침략적인 L. monocytogenes 감염의 중요하지만 훨씬 적게 문서화된 속편은 심혼을 관련시킵니다. 심장 병에서 죽음 비율은 처리에도 불구하고 최대 35%까지 일 수 있습니다, 그러나 아주 거의 심장 조직 및 그것의 결과병학의 L. monocytogenes 식민지에 관하여 알려져 있습니다. 또한, 최근 L. 단세포 유전자의 하위 인구가 심장 조직 내에서 침입하고 성장할 수 있는 향상된 용량을 가지고 있다는 것이 명백해졌습니다. 이 프로토콜은 L. monocytogenes 분리의 심폐소생학을 평가하는 데 사용할 수있는 생체 외 및 생체 내 방법에 대해 자세히 설명합니다. 방법은 H9c2 쥐 심장 근막의 감염뿐만 아니라 감염된 마우스의 심장의 세균 식민지의 결정에 대해 제시된다. 이러한 방법은 감염된 동물의 심장 조직을 식민지화 할 수있는 잠재력을 가진 균주를 식별하는 데 유용할뿐만 아니라 심장 세포 침입및 / 또는 심장 병리학의 변화를 촉진하는 역할을하는 세균 성 유전자 제품의 식별을 용이하게 할 수 있습니다. 이러한 방법은 또한 다중 L. monocytogenes 균주 사이 심혈 성 부속의 직접적인 비교를 제공.

L. monocytogenes의 선택된 분리는 세포 배양및 감염의 마우스 모형에서 심장 세포의 강화한 침략을 전시합니다. 도 1은 한천 매체에서 현탁액의 현탁액을 반점 도금 한 다음 세균성 식민지가 어떻게 나타날 수 있는지를 보여주는 예입니다. 이 방법을 사용하면 많은 수의 천 미디어 플레이트를 사용하지 않고 샘플 내에서 CFO를 정확하게 평가할 수 있습니다. 도 2는 긴장0...

Watch this Scientific Journal Video about Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes at JoVE.com

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

리스테리아 단세포 유전자는 취약한 인구에 있는 임산부및 뇌막염에 있는 태아 감염을 일으키는 원인이 됩니다. 박테리아의 하위 인구는 심장 조직을 식민지화 할 수 있습니다, 환자와 실험실 동물에 심근염을 일으키는. 여기에서 우리는 감염된 동물에 있는 체외 및 심장 식민지에 있는 L. monocytogenes 심장 세포 침략을 평가하는 방법을 설명하는 프로토콜을 제시합니다.

리스테리아 단세포 유전자를 사용하여 감염된 마우스에 있는 문화와 심혼 식민지에 있는 심장 세포의 세균성 침략 평가

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

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JoVE Journal

Immunology and Infection

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

5.4K 조회수.  College of Medicine, University of Illinois at Chicago. 리스테리아 단세포 유전자는 취약한 인구에 있는 임산부및 뇌막염에 있는 태아 감염을 일으키는 원인이 됩니다. 박테리아의 하위 인구는 심장 조직을 식민지화 할 수 있습니다, 환자와 실험실 동물에 심근염을 일으키는. 여기에서 우리는 감염된 동물에 있는 체외 및 심장 식민지에 있는 L. monocytogenes 심장 세포 침략을 평가하는 방법을 설...

비디오: Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

감염된 마우스에 세균 하중과 면역 응답 측정 리스테리아 monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

연구

면역학 및 감염병학

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

경구 전송<em&gt; 리스테리아</em&gt; 오염 된 음식의 섭취를 통해 마우스의

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

세균성 병원체에 의해 세포 감염을 조사하기 위해 InlC 분비 이미징<em&gt; 리스테리아</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

와 실험 감염<em&gt; 리스테리아</em&gt; 호스트 인터페론-γ 응답 유학을위한 모델로

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin &#38; eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

시각화<em&gt; 폐렴 구균</em&gt; 심장 Microlesions 및 후속 심장 리모델링 내

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

의학

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

Listeria monocytogenes 뇌의 감염

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

셀룰러 및 Organismic 수준 호스트 응답 세균성 병원 체 프로브를 사용 하 여

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

A Technique to Establish a Bacterial Infection Model in an Ex Vivo Organ Culture

Source: Rizzuto, G. A., et al. <a href="https://app.jove.com/v/54237">Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface.</a> J. Vis. Exp. (2016)
This video demonstrates a method to establish an infection model in placental tissues to study infection propagation. An ex vivo placental organ culture is infected with Listeria monocytogenes, a facultative intracellular parasite. The bacteria invade extravillous trophoblasts (EVTs) to enter the placental tissue and then propagate to other cells via cell-to-cell spread.

생체 외 장기 배양에서 박테리아 감염 모델을 확립하는 기술

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

In Vitro Models

Quantification of Bacterial Infection in Host Cells by the Colony-Forming Unit Assay

Source: Gayle, P. et al., <a href="https://app.jove.com/v/58775">Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.</a>&#160;J. Vis. Exp.&#160; (2019).
This video demonstrates an in vitro quantification technique to determine Listeria monocytogenes infection of macrophages by the colony-forming unit assay. The bacterial colonies on the agar plate represent the intracellular bacterial population that escapes the host-defense mechanism and survives within the macrophages.

Colony-Forming Unit Assay에 의한 숙주 세포의 박테리아 감염 정량화

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.&#160;J. Vis. Exp.&#160; (2019).
This video ...

Immune Response

Host-Pathogen Interaction

Flow Cytometry Analysis of Intracellular and Emergent Extracellular Listeria monocytogenes In Vitro

Source: Gayle, P. et al., <a href="https://app.jove.com/v/58775">Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.</a> J. Vis. Exp. (2019)
In this video, we quantify intracellular and extracellular Listeria monocytogenes, an intracellular pathogenic bacteria, following their infection of cultured macrophages in vitro using flow cytometry.

In vitro에서 Intracellular and Emergent Extracellular Listeria monocytogenes의 유세포 분석

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses. J. Vis. Exp. (2019)
In this video, we ...

리스테리아 단세포 유전자를 사용하여 감염된 마우스에 있는 문화와 심혼 식민지에 있는 심장 세포의 세균성 침략 평가

요약

더 많은 비디오 탐색

이 비디오의 챕터

감염된 마우스에 세균 하중과 면역 응답 측정 리스테리아 monocytogenes

경구 전송

세균성 병원체에 의해 세포 감염을 조사하기 위해 InlC 분비 이미징

와 실험 감염

시각화

Listeria monocytogenes 뇌의 감염

셀룰러 및 Organismic 수준 호스트 응답 세균성 병원 체 프로브를 사용 하 여

생체 외 장기 배양에서 박테리아 감염 모델을 확립하는 기술

Colony-Forming Unit Assay에 의한 숙주 세포의 박테리아 감염 정량화

In vitro에서 Intracellular and Emergent Extracellular Listeria monocytogenes의 유세포 분석