We would like to thank Dr. Hiroshi Miyazaki, Dr. Scott Henderson, Dr. Todd Kitten, Dr. Justin Hutcherson, Dr. Catherine Jauregui, and Collin R. Berry. This work was supported by NIH NIDCR grants R01DE016124, R01DE018039, and R01DE023304 to J.P. Lewis.
Microscopy was performed at the VCU Department of Anatomy and Neurobiology Microscopy Facility, supported, in part, with funding from NIH-NINDS Center core grant (5P30NS047463).

Authors have nothing to disclose.

上記のすべての方法は、真核細胞で嫌気性細菌の相互作用を評価するために、特定のアッセイを設計することができます。しかし、成功した実験を行うには、いくつかの考慮事項があります。最初の研究で使用される菌株です。 それは彼らが同じような成長段階にあり、上記の違いと同様の細胞濃度を達成侵入効率13に影響を与えることができること生存アッセ?...

嫌気性細菌は、人間の体のほぼすべての面にコロニーを形成します。酸素濃度が低い腸管および尿生殖路の細菌叢において優勢が、それらはまた、皮膚、口、鼻、喉1上に高レベルで存在します。嫌気性細菌は、内因性感染症の一般的な原因であり、病気のサイトから頻繁に分離されています。しかし、その気難しい性質上、嫌気性菌が分離し、培養することは困難です。嫌気性細菌が関与する研究が制限された条件の下で行われなければなりません。現代嫌気培養技術は、研究者は、多くの嫌気性実験室株、あるいは臨床分離2,3を研究するために必要な嫌気性の設定を模倣することができます。 病原性嫌気性細菌は、それらが存在する宿主細胞とのダイナミックな関係と共進化を開発しました。ほとんどの嫌気性菌はinfectiに到達する前に宿主免疫応答による殺傷に影響されやすいです組織単位（OU）レベル。しかし、いくつかの病原性細菌から逃れるか、宿主の免疫応答を破壊するメカニズムを開発しました。彼らは、 ポルフィロモナス・ジンジバリス 、両方の経口に関与し、グラム陰性嫌気性菌。4に信号を送るような免疫認識の回避、免疫メディエーターの中和、細胞性免疫の変化、宿主細胞の浸潤、および免疫の変更などのメカニズムを通じてこの目標を達成し、口外疾患、 ホスト5-7の病原性の変化を引き起こすことができる非常に適合細菌性病原体の一例です。 歯と歯肉粘膜組織との間に形成された深い裂け目に計上バイオフィルムプラークのポケットには、大気中の酸素8から保護されている嫌気性細菌を抱くことができます。これらの歯周ポケットは、P.など、様々な嫌気性病原体のためのニッチとして機能ジンジバリス 9。P.ジンジバリス改造が可能であるキーストン病原体であります歯周病10の発症と進行を促進する方法で、口腔微生物のコミュニティをる。これは、宿主タンパク質の広いスペクトルに対して活性である毒性因子を大量に生成し、宿主防御の回避11ためのメカニズムを提供します。また、上皮細胞、内皮細胞、線維芽細胞、 および in vitro および in vivo 12-14 15 で歯根膜細胞に侵入することが可能です。効果的に宿主細胞に侵入することにより、P。ジンジバリスは、宿主の免疫を逃れることができます。宿主細胞の効果的な侵入だけでなく、細菌が宿主防御をエスケープすることを可能にするだけでなく、将来の再感染するためのリザーバとして働くだけでなく、宿主細胞を変化させます。宿主細胞が細菌の接着および内在化に関与する分子機構の研究が必要とされています。いくつかの研究室での研究は 、Pの内部に関連する分子事象を理解することに焦点を当てています宿主細胞によってジンジバリス同様に抑制し、免疫応答をハイジャックし、敵対的な宿主防御機構を生き残るために使用されるメカニズムとして。 宿主細胞に侵入することができる病原体を同定し、特徴付けることが可能な多くのアッセイがあります。これは、酸素の不存在下でかさばる器具に依存して研究を行うことが困難であるためしかし、嫌気性病原体によるインビトロ研究は、主に研究者のために多くの実験的な問題を引き起こします。これは、真核細胞が増殖するために酸素を必要とし、従って、組織培養インキュベーター中で別途用意しなければならないという事実によって悪化します。このような障害物を回避する一つの方法は、大気中の酸素の下で研究を行うことであろうが、それは、嫌気性細菌の増殖を不可能にするであろう。別の方法は、感染して宿主細胞の相互作用を研究するために加熱殺菌細菌を使用することであろう。しかし、違いは、ホスト病原体interactiの関連性を低下させる熱殺菌し、生菌の間に存在します16に。これは、宿主細胞との相互作用変更のない表現で生菌を研究するために中央です。 Pを培養するため、方法嫌気性環境におけるジンジバリスが与えられます 。また、2つの簡単なコスト効率の高いプロトコルがPの能力を評価するための実証されヒト臍帯静脈内皮細胞（HUVEC）によって内在するジンジバリス 。最初のプロトコルは、人気のある抗生物質保護アッセイに基づいています。アッセイは簡単であるが、嫌気性微生物を用いた検討事項が挙げられます。第二プロトコルは、相互作用可視化する蛍光顕微鏡の使用を必要とし、Pを内在化ジンジバリス 。各アッセイは、制限および嫌気性細菌の侵襲性を研究するための研究者に概要を提供するために説明される利点を有します。現在の原稿は 、P を研究しているがジンジバリス及びHUVECを、これらのプロトコルは、他の多くの嫌気性細菌のために使用することができます宿主細胞の他のタイプのように。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Vinyl Anaerobic Chamber-Type B</td>
			<td>Coy Laboratory Products</td>
			<td></td>
			<td>Model 2000 incubator&#160;</td>
		</tr>
		<tr>
			<td>TSA II Trypticase Soy Agar w/5% Sheep Blood</td>
			<td>BBL</td>
			<td>221261</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells 10-donor Pool</td>
			<td>LifeLine Technology</td>
			<td>FC-0044</td>
			<td></td>
		</tr>
		<tr>
			<td>VascuLife VEGF Medium Complete Kit</td>
			<td>LifeLine Technology</td>
			<td>LL-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypKit</td>
			<td>LifeLine</td>
			<td>LL-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Riedel-de Haen</td>
			<td>16109</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin Sulfate Salt</td>
			<td>Sigma-Aldrich</td>
			<td>G-1264</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole</td>
			<td>Sigma-Aldrich</td>
			<td>M-3761</td>
			<td></td>
		</tr>
		<tr>
			<td>BCECF-AM</td>
			<td>LifeTechnologies</td>
			<td>B1150</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC Phalloidin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm Circular Coverslips</td>
			<td>Electron Microscopy Sciences</td>
			<td>72222-01</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaShield Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

porphyromonas gingivalis as a model organism for assessing interaction of anaerobic bacteria with host cells

嫌気性細菌は、これまで、このような腸、口、膣などの多くのヒトのニッチで好気性菌数を上回ります。また、嫌気性感染は一般的であり、しばしば先住民起源のものです。ヒトの細胞に侵入するためにいくつかの嫌気性病原体の能力は、彼らに先天性免疫を逃れるためだけでなく、宿主細胞の挙動を調節するために、適応策を提供します。しかし、嫌気性細菌は、イベントの実験的調査中に生きていることを確保することが課題を提起することができる。 ポルフィロモナス・ジンジバリス 、グラム陰性嫌気性菌は、真核生物の非食細胞の多様な侵入が可能です。この記事では、成功した文化とは、Pの能力を評価する方法について説明ジンジバリスは、ヒト臍帯静脈内皮細胞（HUVEC）を浸潤します。宿主細胞と相互作用する細菌を可視化するために成功裏に侵入し、宿主内で生存することができる細菌を測定するためのものを、その他：2つのプロトコルが開発されました。これらの技術はanaeの使用を必要とP.を供給する robic室最適な成長のための嫌気性環境でジンジバリス 。 第一のプロトコルは、主に細菌に​​よる宿主細胞の浸潤を研究するために使用される抗生物質保護アッセイに基づいています。しかし、抗生物質保護アッセイは限られています。抗生物質処置および宿主細胞の溶解後に培養可能である唯一の細胞内細菌が測定されます。生と死の両方の宿主細胞と相互作用するすべての細菌を評価するために、我々は、宿主 - 病原体相互作用を調べるために、蛍光顕微鏡を使用するプロトコルを開発しました。細菌は蛍光2 &#39;、7&#39;-ビス - （2-カルボキシエチル）-5-（および-6） - カルボキシフルオレセインアセトキシメチルエステル（BCECF-AM）で標識し、嫌気性条件下で真核細胞を感染させるために使用されます。 0.2％トリトンX-100で透過処理し、パラホルムアルデヒドで固定した後、宿主細胞は、それぞれ、細胞骨格と細胞核を標識するためのTRITCファロイジンおよびDAPIで標識されます。複数のIMA異なる焦点（Zスタック）で撮影されたGESは、細菌の時空間視覚化のために得られます。本研究で使用した方法は、任意の培養可能嫌気性菌及び任意の真核細胞型に適用することができます。

上記で概説したプロトコルはPとの間の宿主-病原体相互作用を研究に使用しましたジンジバリスおよび内皮細胞。P.ジンジバリス W83 と P. PG0228の欠失を保有するジンジバリス V3150を研究に使用しました。 PG0228は、最終的にPの相互作用に影響を与える可能性があるRNAおよびタンパク質のレベルを変化させることができるタンパク質をコードすることが予?...

Watch this Scientific Journal Video about Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells at JoVE.com

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

宿主細胞と嫌気性菌の相互作用を評価するためのモデル生物としてポルフィロモナス・ジンジバリス

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

porphyromonas-gingivalis-as-model-organism-for-assessing-interaction

Research

JoVE Journal

Immunology and Infection

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

26.1K ビュー.  Virginia Commonwealth University. This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type. 

ビデオ: Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

モデルヘルペスウイルスの溶菌レプリケーションにおける解剖ホストウイルス作用

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

免疫・感染学

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

長期宿主 - 病原体相互作用を特徴付けるためのEpiAirwayモデルの利用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

宿主細胞に関連して病原性真菌の定量化のための画像サイトメトリーの使用

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;ハチノスツヅリガ</em&gt;モデル生物として研究する<em&gt;レジオネラ·ニューモフィラ</em&gt;感染症

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

微生物群集を学びます<em&gt;インビボ</em&gt;：ホスト間の媒介の相互作用のモデル<em&gt;カンジダ</em&gt;と<em&gt;緑膿菌</em&gt;航空で

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

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.

細菌 - 宿主相互作用を特徴づけるために、バイオミメティック材料

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 ...

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.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

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.

と実験感染<em&gt;リステリア菌</em&gt;ホストインターフェロンγ応答を研究するためのモデルとして、

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 ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

グループBのマウスモデル<em&gt;連鎖球菌</em&gt;膣植民地化

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

アルボ ウイルス感染症ウイルス免疫調節剤およびホスト抗ウイルス因子の同定のためのスクリーニング ツールとして

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...