Name: high resolution electron microscopy of the helicobacter pylori cag type iv secretion system pili produced in varying conditions of iron availability
Uploaded: 2014-11-21T14:00:00.000+00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability at JoVE.c

This research was supported by the Department of Veterans Affairs Career Development Award 1IK2BX001701 and the CTSA award UL1TR000445 from the National Center for Advancing Translational Sciences. Its contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health. Scanning electron microscopy experiments were performed in part through the use of the VUMC Cell Imaging Shared Resource, supported by NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126.

1. H. 인간의 위 상피 세포와 철 가용성 및 공동 문화의 다양한 조건에서 균 성장 <ol><li> H.를 선택 이러한 연구에 대한 파이로리 스트레인 PMSS1 그것이 그대로 CAG 병원성 아일랜드를 가지며 작동 타입 IV 분비 시스템을 표현하기 때문이다. 또한 음성 대조군으로 동질 케이지 돌연변이 (PMSSI Δ 케이지)를 사용합니다. 5 % CO 2 존재하에 37 ° C에서 24 시간 동안 5 % 양 혈액 (아가 플레이트)로 보충 TSA 플레이트상에서 세균 성장. 참고 : 시약을 준비하고 재료 및 장비 표에 요약 된 자료를 조립한다. 각 시약에 대한 최종 농도는 괄호 안에 표시됩니다. </li><li> 멸균 면봉을 사용하여 판에서 박테리아를 긁고 구성 요소에서 준비 수정 브루셀라 배지에 접종 (재료 및 장비 표 참조) 콜레스테롤 보충. 37에서 하룻밤 문화를 품다분 수 (rpm)에 200 회전에서 진탕 5 % CO 2로 보충 된 실내 공기를 ° C. NOTE : 콜레스테롤 인해 혈청이 복잡하다는 사실에 소 태아 혈청 대신에 사용된다; 이러한 결과에 변화가 발생 헴 (heme) 등의 영양소 철의 다양한 소스를 포함. </li><li> 세균 배양의 날, AGS 인간 위 상피 세포를 시드 (ATCC CRL-1739) 12 웰 플레이트 (웰 당 약 1 × 105 세포)의 폴리 D 라이신 처리 된 커버 슬립 상. </li><li> 다음 날, 수정 브루셀라 국물 혼자 또는 100 μM 50ml을, 200 μM의 피리 딜 (합성 철 킬레이트), 또는 200 μM의 피리 딜 플러스 250 μM 50ml을 보충 0.3의 OD600에 박테리아 문화를 희석. 200 rpm으로 진탕하면서 5 % CO 2로 보충 된 실내 공기에서 37 ° C에서 4 시간 동안 배양이 부화. </li><li> 1,000 XG에 원심 분리기 박테리아는 세포를 수집하고 난을 재 부유하기 전에 상층 액을 제거n은 신선한 수정 된 브루셀라 브로스 동량 콜레스테롤 보충. 문화 (OD600 = 1.0 = 5.5 × 10 8 세포 / ml)의 측정 OD600 감염 20 (MOI)의 다수의 상피 세포에 박테리아를 추가 : 1. 박테리아 세포 생존력을 평가하기 위해 혈액 아가 플레이트 상에 연속 희석 접시 균체를 수행한다. </li><li> H.을 품어 주사 전자 현미경 (SEM) 시료 전처리를 수행하기 전에 정적 인 상태에서 5 % CO 2 존재 하 37 ° C에서 4 시간 동안 파이로리 AGS 인간 위 상피 세포 공동 배양. </li></ol> 2. SEM H.을 시각화하는 샘플 준비 pylori의 CAG-T4SS 필리 <ol><li> H. 제거 균 및 인큐베이터와 캔트 배양 상층 액에서 AGS 공동 문화 (IL-8 ELISA에 대한 저장). 0.05 M 나트륨 cacodylate 완충액 (pH 7.4)으로 가볍게 세 번 씻으십시오. , 2.0 % 파라 포름 알데히드 용액에 2.5을 실온에서 2~4시간에 대한 샘플을 수정% 글루 테르 알데히드, 0.05 M 나트륨 cacodylate 버퍼입니다. 차 고정 후, 샘플을 0.05 M 나트륨 cacodylate 버퍼로 3 회 씻는다. </li><li> 두 연속 10 분 정착 단계에서 0.05 M 소듐 cacodylate 버퍼에 0.1 % 사 산화 오스뮴을 이용하여 차 고정을 수행한다. 차 고정 후, 샘플을 0.05 M 나트륨 cacodylate 버퍼로 3 회 씻는다. </li><li> 기본 및 보조 고정 후, 표 1에 따라 에탄올의 농도가 증가함에 따라 순차적으로 세척하여 샘플을 탈수. </li><li> 에탄올 탈수 후 액체 이산화탄소 세 순차 세척을​​ 수행한다. 1073 PSI의 31.1 ºC의 온도 및 압력에서 임계점 건조 기계를 사용 임계점이어서 건조한 샘플. </li><li> 마운트는 알루미늄 SEM 샘플 스텁에 된 커버하고 적절한 접지를 달성하고 SEM 이미징 동안 충전을 방지하기 위해 샘플 가장자리에 콜로이드 실버의 얇은 선으로 페인트. </li><li> 코트 샘플과는 5nm샘플 2 차 전자 신호 및 에지 효과를 증가시키기 위해 스퍼터 코팅 기계를 사용하여 금 - 팔라듐. </li></ol> 고해상도 현미경 분석 3. 이미징 매개 변수 <ol><li> 이 필드 방출 총 스캐닝 전자 현미경 (FEG-SEM)으로보기 샘플. </li><li> 5~10mm의 작동 거리에서 고진공 모드에서 이미지. </li><li> 5 kV의에 가속 전압을 설정합니다. </li><li> 2-2.5에 자리 크기를 설정합니다. </li><li> 호스트 인터페이스 병원체의 더 나은 관점을 달성하기 위해 15 내지 25도 사이의 샘플을 기울인다. </li><li> 호스트 병원체 상호 작용의 이들 영역은 필리 - 형성 박테리아 농후 한, 고배율 관찰 용 상피 세포의 가장자리에 부착 된 세균을 이미징 우선 순위. </li></ol> 4. 통계 분석은 필리 정량화를 평가하기 <ol><li> H. 분석 ImageJ에 소프트웨어를 사용하여 파이로리 CAG-T4SS의 필리는 piliated 프 전시 세포의 필리 / 세포의 수뿐만 아니라 퍼센트를 정량화하기아래의 지침에 따라 표현형. </li><li> ImageJ에 소프트웨어의 열기 현미경 사진 파일. 균일 한 폭 (10 ~ 13 ㎚)과 길이 (60-150 나노 미터)와 박테리아 세포와 숙주 세포 사이에 형성되는 구조로 필리을 확인합니다. NOTE : pilus 구조 주요 ATP 아제 힘 타입 IV 분비 pilus 조립체를 코딩하는 유전자에 불활 항구 이러한 Δ 케이지 변이주로서 동질 균주에 WT 균주에 존재하지만, 존재하지 않는다. </li><li> &quot;분석&quot;탭을 클릭 한 다음 직선 도구를 사용하여 선 그리기에 의해 측정 도구를 보정합니다. 드롭 다운 메뉴에서 &quot;설정 규모&quot;를 선택하고 상자 표시 &quot;알려진 거리&quot;로 확대 바 값을 입력하고 적절한 설정 (㎚)로 길이의 단위를 설정합니다. &quot;확인&quot;을 클릭합니다. </li><li> pilus의 길이나 폭에 걸쳐 그려 직선 도구를 사용하여 CAG-T4SS의 필리를​​ 측정하고 각 pilus를 측정하기 위해 &quot;Ctrl 키를-M&quot;을 눌러. 측정 값과의 대화 상자를 관찰합니다. 이 값을 기록하거나 복사하여 스프레드 시트 프로그램에 붙여 넣습니다. </li><li> 길이를 사용하여 폭 매개 변수는 이전에 CAG-T4SS의 프로필에 맞지 않는 24 무시 기능을 설립했다. 그런 다음 10 ~ 13 nm의 폭 및 60-150 nm의 길이 컷오프 내에있는 값에 기초 필리의 수를 정량화. </li><li> 그래프 패드 프리즘 소프트웨어를 사용하여 두 개의 꼬리 학생의 T 시험에 의해 통계적으로 필리의 정량을 분석 할 수 있습니다. </li></ol> 5. 선택 사항 : IL-8 분비의 평가 Pilus 생산과의 상관 관계를 분석하기 <ol><li> 2.1 단계에서 상층 액을 사용하여, 키트 제조 업체의 지침 (재료 및 장비 표 참조) 당 IL-8 ELISA를 수행합니다. </li><li> IL-8 숙주 세포에 의해 분비를 분석 단독 배지에서 성장 WT PMSS1에 대해 퍼센트 IL-8 유도를 계산한다. 그래서 그래프 패드 프리즘을 사용하여 두 꼬리 학생의 T 테스트를 사용하여 통계 분석을 수행ftware. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+host+iron+utilization+mechanisms+of+Helicobacter+pylori+revealed+with+iron-deficient+chemically+defined+media.">Unique host iron utilization mechanisms of Helicobacter pylori revealed with iron-deficient chemically defined media.</a> Infect. Immun. 78 (5), 1841-1849 (2010).</li><li>Shaffer, C. L., 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=Helicobacter+pylori+exploits+a+unique+repertoire+of+type+IV+secretion+system+components+for+pilus+assembly+at+the+bacteria-host+cell+interface.">Helicobacter pylori exploits a unique repertoire of type IV secretion system components for pilus assembly at the bacteria-host cell interface.</a> PLoS Pathog. 7 (9), (2011).</li><li>Barrozo, R. M., 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=Functional+plasticity+in+the+type+IV+secretion+system+of+Helicobacter+pylori.">Functional plasticity in the type IV secretion system of Helicobacter pylori.</a> PLoS Pathog. 9 (2), (2013).</li></ol>

The authors declare that they have no competing financial interests.

철 박테리아 병원균을 포함한 삶의 대부분의 형태에 필수적인 미량 영양소이다. 미생물 침입의 가능성을 제한하기위한 노력으로, 척추 호스트는 &quot;영양 면역&quot;(18)로 알려진 과정에서 영양소 철을 격리시키는. 이에 응답하여, 박테리아 병원균은 주변 환경을 감지하고 철 수집 시스템, 독소 및 독소 분비 기계 (19, 20)으로 병원성 기능 정교화 조절하는 전역 신호 분자로서 철을 사용하도록 진화했다. H. 파이로리는 철, 아연, 및 마그네슘 (22)을 성장 및 헴 (heme), 헤모글로빈, 트랜스페린 등의 대체 철 공급원을 사용할 수 있으며, 락토페린 (23) 등의 미량 영양소를 필요로한다. H.을 파이로리는 가장 성공적인 병원균처럼, 호스트 (21)로부터 영양 철을 청소할 수 있도록 독성 요인의 넓은 레퍼토리를 진화했다. 한 독성 인자와 cagA, 이펙터 molecuCAG-T4SS의 제작은, 그것이 용이 영양소 철 공급원으로서 균체가 사용하는 15 및 복제를 촉진 할 수있는 선 단면에 편광 셀 측저 측으로부터 트랜스페린 경로 전환에 관여한다. 따라서, 낮은 철 가용성의 조건에서와 cagA의 분비를 촉진 할 수있는 직관적 인 진화 전략을 보인다. H. 파일로리 CAG-T4SS 인해 그것이 유도하는 염증 숙주 세포 반응에 중요한 세균성 소기관이다. CAG-T4SS의 효과는 잘 공동 배양 감염의 동물 모델 모두에서 연구되어왔다. 그러나,이 독소 분비 시스템의 고분자 구조와 구성의 연구는 고해상도 이미징 프로토콜의 부족에 의해 방해되어왔다. CAG-T4SS을 공부하고 우리의 이전에 게시 된 보고서는 고해상도 전자 현미경으로 박테리아 세포에 네 CAG-T4SS의 필리 평균 (24)를 분석보고했다. 우리의 개선 된 방법을 제공강력한 이미징 기술은 박테리아 세포의 표면에서이 기능의 생성을 촉진하는 배양 기술을 이용하여 CAG-T4SS를 시각화한다. 고분해능 전계 방출 총 주사 전자 현미경과 함께 배양 기술의 이용이 촬상 프로토콜이 중요하다. CAG-T4SS 표면 기능의 두 배 농축은 면역 또는 면역 기술이 더 CAG-T4SS의 세포 내 위치 나 구성을 특징으로 면역 조직 화학 염색을 수행에 관심이있는 연구자에 대한 상당한 증가는 분석이다. CAG-T4SS의 고분해능 전자 현미경 촬상가 마스터되면, 그것은 CAG-T4SS의 pilus (25)에 대해 박테리아 단백질의 위치를 결정하기 위해, 예컨대 immunogold 표지와 같은 다른 응용 프로그램을 개발하는 것도 가능하다. 우리의 기술의 한 가지 제한은 조사자가 여전히 USI로 제한된다는 사실이다CAG-T4SS의 시각화를위한 공동 문화 시스템 겨. H.의 단일 필드에서 하위 집합 만 파이로리 정상 배양 조건은 이러한 구조를 형성 하에서 (50-70 퍼센트도 3 패널 B 참조). 그래서 필드에있는 수많은 세포가 아닌 piliated 될 것입니다. 이러한 하위 집단이 동일한 위치에 존재하는 이유는 불명하지만, 이들 세포는 항상 표면에 필리와 필리 / 셀 및 셀들의 퍼센트 정량화 모두 내 차지된다. CAG-T4SS 할수있는 숙주 박테리아 계면에서 관측되기 때문에, SEM 샘플 제제 내의 일차 및 이차 정착 단계는 단백질 구조와 진핵 및 원핵 세포의 막 모두의 보존에 중요하다. 고정 단계 이상 수행 설명, 또는 솔루션은 갓 준비하지 않으면하는 경우에는 오버 고정가 발생할 수 있습니다. 오버 고정이 발생하면 SEM에서 볼 수 있고 CAG-T4SS이에 의해 마스크 될 때, 샘플은 돌의 모양을해야합니다. troubl하려면e 촬영 덜 농축 고정 제 및 / 또는 고정 제의 존재 하에서 배양 시간이 적게 사용 SEM 샘플 제제를 반복한다. 공동 배양 시스템은 또한 호스트 인한 단백질의 대량의 오염 생화학 (질량 분광 분석 등) 특성화 성가신를 렌더링한다. 그러나,이 박테리아 분비 시스템의 생산을 조절하는 분자 적 메커니즘을 이해하는 것은 궁극적으로 숙주 세포의 부재하에 CAG-T4SS pilus의 형성을 트리거 새로운 기술로 이어질 수있다. 이것은 크게 생화학 적 분석에 대한 분리 및 정제 기술을 개선하는 것이다. 결론적으로, 우리는보고 그 H.의 생산 증가에 제한 철 가용성 결과의 조건 고해상도 주사 전자 현미경 기법에 의해 시각화 될 수 파이로리 CAG-T4SS. 또한 호스트 병원체 interfa에서이 표면 특징의 철 - 의존성 억제를보고CE. 이 분석은 최적화 된 철 - 조절 독소 분비물을 형성 호스트 미생물 상호 작용의 다양한 광범위하게 적용 가능하다 수많은 세균 병원균에 이용 될 수있다.

H. pylori infection is a significant risk factor for gastric cancer1. However, disease outcomes vary and depend on numerous factors such as host genetics, genetic diversity of H. pylori strains, and environmental elements such as host diet11. Previous reports have established that a correlation exists between H. pylori infection, iron deficiency (as measured by decreased blood ferritin and hemoglobin concentrations), and increased proinflammatory cytokine production, including IL-8 secretion, which ultimately leads to increased gastric disease progression12. Acute H. pylori infection is also associated with hypochlorhydria which impairs the host&#8217;s ability to absorb nutrient iron, and ultimately leads to changes in iron homeostasis13. These clinical findings suggest that iron availability within the gastic niche could be an important factor in disease outcome. In fact, animal models of H. pylori infection have demonstrated that low dietary iron consumption exacerbates gastric disease14. The reduced iron levels in these animals necessitate that H. pylori induce an iron-acquisition response in order to obtain the iron needed for bacterial replication. H. pylori has the capacity to perturb iron trafficking within host cells to facilitate bacterial replication in a CagA-dependent fashion15. Interestingly, the cag-pathogenicity island has been shown to be regulated by the iron-responsive transcription factor Fur16,17. Furthermore, Cag+ strains are associated with increased inflammation and gastric diseases such as cancer1. These findings support a model whereby H. pylori alters Cag-T4SS expression in an effort to obtain iron from host cells that reside in an iron deplete environment resulting in exacerbated disease outcomes. 
Two factors that increase inflammation and morbidity are Cag expression and low dietary iron intake. These facts support the hypothesis that reduced iron availability increases the production of Cag-T4SS pili at the host pathogen interface resulting in worse gastric disease11-14. The goal of the method provided in this manuscript is to establish the role of the micronutrient iron in the regulation of the Cag-T4SS pilus biogenesis. In previous work, we utilized two approaches to observe an iron-dependent increase in Cag-T4SS expression. First, output strains from animals maintained on high and low iron diets were analyzed and revealed that low-iron diet output strains produced more Cag-T4SS pili than high-iron diet strains14. Second, growing the H. pylori 7.13 strain in vitro in iron replete conditions resulted in reduced pili formation while cells grown in the presence of an iron chelator produced significantly more pili. 
We have continued to investigate the iron-dependent regulation of Cag-T4SS pili phenotype and offer the following optimized protocol and representative results performed with an additional Helicobacter pylori strain, PMSS1. The rationale behind the development of this technique was to correlate increased Cag-T4SS activity in conditions of iron-limitation with increased Cag-T4SS pilus formation. The broader implication and use of this technique will provide optimized culture conditions that result in elevated production of the Cag-T4SS pili. This assay will be useful to researchers seeking to determine the composition and architecture of the Cag-T4SS by enriching for this important bacterial surface feature. The sample preparation and visualization by field-emission gun electron microscopy has numerous advantages over alternative techniques such as light-microscopy methods to visualize the Cag-T4SS and will be appropriate to investigators interested in studying the regulation of this organelle10.

<table><tbody><tr><td>Modified brucella broth</td><td></td><td></td><td></td></tr><tr><td>Peptone from casein (10 g/L)</td><td>Sigma</td><td>70172</td><td></td></tr><tr><td>Peptic digest of animal tissue (10 g/L)</td><td>Sigma</td><td>70174</td><td></td></tr><tr><td>Yeast extract (2 g/L)</td><td>Sigma</td><td>92144</td><td></td></tr><tr><td>Dextrose (1 g/L)</td><td>Sigma</td><td>D9434</td><td></td></tr><tr><td>Sodium chloride (5 g/L)</td><td>Thermo Fisher</td><td>S271-10</td><td></td></tr><tr><td>Cholesterol (250X) (4 ml/L)</td><td>Life Technologies</td><td>12531018</td><td></td></tr><tr><td>Ferric chloride (100 or 250 uM)</td><td>Sigma</td><td>157740-100G</td><td></td></tr><tr><td>Dipyridyl (200 uM)</td><td>Sigma</td><td>D216305-100G</td><td></td></tr><tr><td>Modified RPMI</td><td></td><td></td><td></td></tr><tr><td>RPMI+HEPES (1X)</td><td>Life Technologies</td><td>22400-121</td><td></td></tr><tr><td>Fetal bovine serum (100 ml/L)</td><td>Life Technologies</td><td>10438-026</td><td></td></tr><tr><td>Electron Microscopy Preparation</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde (2.0% aqueous)</td><td>Electron Microscopy Sciences</td><td>15713</td><td></td></tr><tr><td>Gluteraldehyde (2.5% aqueous)</td><td>Electron Microscopy Sciences</td><td>16220</td><td></td></tr><tr><td>Sodium cacodylate (0.05 M)</td><td>Electron Microscopy Sciences</td><td>12300</td><td></td></tr><tr><td>Osmium tetroxide (0.1% aqueous)</td><td>Electron Microscopy Sciences</td><td>19150</td><td></td></tr><tr><td>Ethanol (absolute)</td><td>Sigma</td><td>E7023</td><td></td></tr><tr><td>Colloidal silver paint</td><td>Electron Microscopy Sciences</td><td>12630</td><td></td></tr><tr><td>SEM sample stubs</td><td>Electron Microscopy Sciences</td><td>75220</td><td></td></tr><tr><td>Coverslips</td><td>Thermo Fisher</td><td>08-774-383</td><td></td></tr><tr><td>IL-8 Secretion Evaluation</td><td></td><td></td><td></td></tr><tr><td>Quantikine IL-8 ELISA kit</td><td>R&amp;amp;D Systems</td><td>D8000C</td><td></td></tr></tbody></table>

high resolution electron microscopy of the helicobacter pylori cag type iv secretion system pili produced in varying conditions of iron availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

이 보고서에서 우리는 철의 가용성을 변화의 조건이 H.을 조절하는 능력을 가지고 있음을 증명하고있다 호스트 병원체 인터페이스에서 파이로리 CAG-T4SS의 pilus의 생합성. , H. 단독 배지에서 배양하면 파일로리 3 필리 / 셀의 평균을 형성한다. 때 H. 파일로리 철에서 성장 고갈 세균 성장 (도 1)은 하위 억제이다 (합성 킬레이트 피리 딜 사용) 조건, 박테리아 생성 수많은 CAG-T4SS 필리 (~ 7 필리 / 셀) 때 인간 위암 세포와 공 배양 (그림 2). 영양소 철 외인성 소스가 존재하면 반대로, CAG-T4SS의 필리의 형성이 억제된다 (FE 샘플 1 개 미만 pilus / 셀, 1 미만 pilus / FE +의 DIP 시료 중의 세포) (도 2). 호스트 병원체 인터페이스에서 필리의 정량화 밝혀 그 철 제한, H.의 조건 파이로리는 CAG-T4SS의 필리 (P에서 2 배 증가 &lt;0.000 전시<html> 1) 혼자 배지에서 성장 세포에 비해 11 % (P &lt;0.05)에 의한 비율 piliated 세포 증가 (그림 3). 흥미롭게도, 호스트 IL-8 분비에 의해 측정 CAG-T4SS의 활성은, 철 제한 조건 하에서 107% 향상 및 배지 단독 (p = 0.03, 및 P에 비해 과량의 철 가용성의 조건 하에서 49 %를 억압 &lt; SEM 데이터를 지원 0.001) 결과. 그러나 필리 치수 (그림 4) 철 가용성의 모든 조건 사이에서 일관성을 유지. 또한, 철 가용성 케이지 돌연변이의 표면에 필리의 생합성을 변경하지 않으며, 사실 증명 구조임을 가설을지지하는, 숙주 세포에서 IL-8 반응을 유도하기 위해이 동질 유도체의 능력을 변경 않는다 CAG-T4SS 아닌 추가 관련이없는 표면 기능입니다. N &quot;SRC =&quot;/ 파일 / ftp_upload / 52122 / 52122fig1highres.jpg &quot;폭 =&quot;500 &quot;/&gt; 그림 1 :. 철 가용성의 다양한 조건에서 결정 세균 생존 H. 파이로리는 (중간 형) 만 수정 브루셀라 배지에서 배양, 또는 100 μM 50ml을 (FE), 200 μM의 피리 딜 (DIP), 또는 200 μM의 피리 딜 플러스 250 μM 50ml을 (FE + 딥)로 보충했다. 박테리아는 5 % CO 2 존재하에 37 ° C에서 2 일 동안 배양하기 전에 세균 매체 상에 순차적으로 희석하고, 플레이 팅 하였다. 콜로니를 카운트 함과 콜로니 형성 단위는 / ㎖ (CFU / ml)을 계산 하였다. 바는 3 개의 독립적 인 실험의 의미 나타냅니다 +/- SEM을. DIP, FE, 또는 FE + 딥 치료는 크게 박테리아의 생존 능력을 변경하지 않습니다. <img alt="그림 2" fo:content-width="5in" src="/files/ftp_upload/52122/52122fig2highres.jpg" width="500" /> 그림 2 : 높은 Resolution 주사 전자 현미경 분석 H. pylori의 CAG-T4SS의 필리. 박테리아가 혼자 매체)에서 성장했다, B) 매체 100 μM 50ml을 보충, C) 매체는 200 μM의 피리 딜 보충 200 μM의 피리 딜, 또는 D) 매체로 보충 플러스 250 μM 50ml을 이전 AGS 인체와 공동으로 문화 위 세포. 화살표는 호스트 병원체 인터페이스에 형성 CAG-T4SS의 필리을 나타냅니다. 샘플 CAG-T4SS의 생합성을 평가 고해상도 주사 전자 현미경으로 분석 하였다. 초과 가능한 영양소 철의 조건이 CAG-T4SS의 pilus 형성을 억제하면서 낮은 철 가용성의 조건, CAG-T4SS의 pilus 형성의 유병률을 증가시킨다. <img alt="그림 3" fo:content-width="5in" src="/files/ftp_upload/52122/52122fig3highres.jpg" width="500" /> 그림 3 : 다양한 배양 필리 / 셀 및 퍼센트의 정량화 piliated 세포철 가용성의 조건. 박테리아는 혼자 (중간 형) 수정 브루셀라 배지에서 성장했다, 또는 매체는 100 μM 50ml을 (FE), 200 μM의 피리 딜 (DIP), 또는 200 μM의 피리 딜 플러스 250 μM 50ml을 (FE +의 DIP) 공동 - 이전에 보충 인간의 위 상피 세포와 문화. 시료는 고해상도 주 사형 전자 현미경 및 필리은 ImageJ에 소프트웨어를 사용하여 분석 하였다 열거 하였다. 세포 (* P &lt;0.0001 혼자 매체에 비해 당 필리의) 산점도) 및 조건 (3) 별도의에서 파생 된 문화 당 60-112 세포에서 파생 된 piliated 세포 (* P &lt;0.05 형 매체에 비해)의 B) 막대 그래프 묘사 퍼센트 생물학적 실험. <img alt="그림 4" fo:content-width="5in" src="/files/ftp_upload/52122/52122fig4highres.jpg" width="500" /> 그림 4 : 철 가용성의 다양한 조건에서 배양 된 세포에서 측정 Pilus 치수. BACTeria 형 (보통 형) 수정 브루셀라 배지에서 재배, 또는 매체는 100 μM 50ml을 (FE), 200 μM의 피리 딜 (DIP), 또는 200 μM의 피리 딜 플러스 250 μM 50ml을 (FE +의 DIP) 공동 - 이전에 보충되었다 인간의 위 상피 세포와 문화. 샘플은 고해상도 주사 전자 현미경으로 분석하고, 필리 치수 ImageJ에 소프트웨어를 사용하여 측정 하였다. A) Pilus 폭은 모든 조건 중 13.1 +/- 2.4 nm의 평균입니다. B) Pilus 길이는 모든 조건 중 75.8 +/- 16.0 nm의 평균입니다. pilus 크기에 유의 한 차이는 철 가용성의 다양한 조건에서 볼 수 없습니다. 보충 그림 1 : 고해상도 주사 전자 현미경은 H. 분석 철 가용성의 다양한 조건에서 균 케이지 돌연변이. 박테리아 50ml을 100 μM 보충) 배지에서 성장하고, B) 매체 D 200 μM의 피리 딜 (보충IP) 또는 C) 매체는 200 μM의 피리 딜 (DIP) 플러스 AGS 인체 위암 세포를 250 μM 50ml을 이전하는 공동 문화 보충. 샘플 CAG-T4SS의 생합성을 평가 고해상도 주사 전자 현미경으로 분석 하였다. 낮거나 높은 철의 조건은 그림 2에서 시각화 구조 CAG-T4SS의 필리 있다는 가설을 지원, 어떤 CAG 독립적 pilus의 생합성을 조절하지 않습니다. 보충 그림 2 : 철 가용성의 조건을 변화에서 자란 박테리아에 대응 호스트 IL-8 분비의 ELISA 분석. 야생형 (흰색 막대) 또는 케이지 돌연변이 (회색 막대) 박테리아는 혼자 (중간 형) 수정 브루셀라 배지에서 성장했다, 또는 매체는 100 μM 50ml을 (FE), 200 μM의 피리 딜 (DIP), 또는 200 μM의 피리 딜 보충 플러스 250 μM의 50ml을 (FE +의 DIP) 인간의 위 상피 세포와 이전하는 공동 문화. 증가 IL 철 제한 결과-8, CAG-T4SS 활동에 의존 호스트 응답. 바 3 별도의 생물학적 실험에서 파생 된, 평균 +/- SEM을 나타냅니다. (* p &lt;0.05 단독 배지에서 성장 PMSS1 비교).

Watch this Scientific Journal Video about High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability at JoVE.c

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

고해상도 전자 현미경<em&gt; 헬리코박터 파이로리</em철 가용성의 다양한 조건에서 생산&gt; CAG 유형 IV 분비 시스템 필리

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

high-resolution-electron-microscopy-helicobacter-pylori-cag-type-iv

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

15.0K 조회수.  Vanderbilt University School of Medicine. Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

비디오: High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE를 사용한 전자 저온 현미경 이미지의 고해상도 단일 입자 분석

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

연구

생화학

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

단일 합성 Curli 오페론을 생성하여 공학 점착성의 박테리아를

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

생체공학

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

철 소스로 인간 혈색소를 사용하여 황색 포도상 구균 성장

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

면역학 및 감염병학

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.
X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented.

그램 음성 박테리아에 필수 금속 통풍 관: 형광, Radioisotopes, x 선 및 세포 분별 법

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

화학

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

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

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.

숙주 세포와 혐기성 세균의 상호 작용을 평가하기위한 모델 생물로 포피 진지 발리

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

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility provides X. fastidiosa a means for long-distance intra-plant movement and colonization, contributing toward pathogenicity in X. fastidiosa. The twitching motility of X. fastidiosa is operated by type IV pili. Type IV pili of Xylella fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon encoding proteins that are involved with signal transduction pathways. To elucidate the roles of pilG in the twitching motility of X. fastidiosa, a pilG-deficient mutant Xf&#916;pilG and its complementary strain Xf&#916;pilG-C containing native pilG were developed. A microfluidic chambers integrated with a time-lapse image recording system was used to observe twitching motility in Xf&#916;pilG, Xf&#916;pilG-C and its wild type strain. Using this recording system, it permits long-term spatial and temporal observations of aggregation, migration of individual cells and populations of bacteria via twitching motility. X. fastidiosa wild type and complementary Xf&#916;pilG-C strain showed typical twitching motility characteristics directly observed in the microfluidic flow chambers, whereas mutant Xf&#916;pliG exhibited the twitching deficient phenotype. This study demonstrates that pilG contributes to the twitching motility of X. fastidiosa. The microfluidic flow chamber is used as a means for observing twitching motility.

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce&#39;s disease in grapevine.

의 역할의 꿈틀 운동성의 시각화 및 특성<em&gt; PilG</em&gt;에서<em&gt; Xylella의 fastidiosa</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

레지오넬라 폐렴 구균 도트/Icm 분비 시스템의 시공간적 특징을 해결하기 위해 라이브 세포 이미징 및 극저온 단층 촬영 적용

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein structure. Metal sequestration by hosts often leads to metal limitation for the bacterium. This limitation induces bacterial gene expression whose protein products allow bacteria to overcome their metal-limited environment. Characterization of such genes is challenging. Bacteria must be grown in meticulously prepared media that allows sufficient access to nutritional metals to permit bacterial growth while maintaining a metal profile conducive to achieving expression of the aforementioned genes. As such, a delicate balance must be established for the concentrations of these metals. Growing a nutritionally fastidious organism such as Neisseria gonorrhoeae, which has evolved to survive only in the human host, adds an additional level of complexity. Here, we describe the preparation of a defined metal-limited medium sufficient to allow gonococcal growth and the desired gene expression. This method allows the investigator to chelate iron and zinc from undesired sources while supplementing the media with defined sources of iron or zinc, whose preparation is also described. Finally, we outline three experiments that utilize this media to help characterize the protein products of metal-regulated gonococcal genes.

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

We describe here a method for growth of Neisseria gonorrhoeae in metal-restricted liquid medium to facilitate the expression of genes for metal uptake. We also outline downstream experiments to characterize the phenotype of gonococci grown in these conditions. These methods may be adapted to be suitable for characterization of metal-responsive genes in other bacteria.

호스트 리간드에서 금속 반응 유전자 및 금속 취득의 특성화를 위한 Neisseria 임질의 금속 제한 성장

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein ...

T3SS Activity in Yersinia pseudotuberculosis Under Iron Depletion & Anaerobic Stress

Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or immunomodulatory effector proteins into host cells. The T3SS is a target for antimicrobial discovery campaigns since it is accessible extracellularly and largely absent from non-pathogenic bacteria. Recent studies demonstrated that the T3SS of Yersinia and Salmonella are regulated by factors responsive to iron and oxygen, which are important niche-specific signals encountered during mammalian infection. Described here is a method for iron starvation of Yersinia pseudotuberculosis, with subsequent optional supplementation of inorganic iron. To assess the impact of oxygen availability, this iron starvation process is demonstrated under both aerobic and anaerobic conditions. Finally, incubating the cultures at the mammalian host temperature of 37 &#176;C induces T3SS expression and allows quantification of Yersinia T3SS activity by visualizing effector proteins released into the supernatant. The steps detailed here offer an advantage over the use of iron chelators in the absence of iron starvation, which is insufficient for inducing robust iron starvation, presumably due to efficient Yersinia iron uptake and scavenging systems. Likewise, acid-washing laboratory glassware is detailed to ensure the removal of residual iron, which is essential for inducing robust iron starvation. Additionally, using a chelating agent is described to remove residual iron from media, and culturing the bacteria for several generations in the absence of iron to deplete bacterial iron stores. By incorporating standard protocols of trichloroacetic acid-induced protein precipitation, SDS-PAGE, and silver staining, this procedure demonstrates accessible ways to measure T3SS activity. While this procedure is optimized for Y. pseudotuberculosis, it offers a framework for studies in pathogens with similar robust iron uptake systems. In the age of antibiotic resistance, these methods can be expanded to assess the efficacy of antimicrobial compounds targeting the T3SS under host-relevant conditions.

Bacteria colonize host tissues that vary in oxygen and iron bioavailability, yet most approaches to studying bacteria use aerated, rich media. This protocol describes culturing the human pathogen Yersinia pseudotuberculosis under varying iron concentrations and oxygen tension, and quantifying activity of the Yersinia type III secretion system, which is an important virulence factor.

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or ...

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host cells. Upon entry, the injected effector proteins cooperatively modulate eukaryotic signaling pathways and reprogram cellular functions, enabling bacterial entry and survival. Monitoring and localizing these secreted effector proteins in the context of infections provides a footprint for defining the dynamic interface of host-pathogen interactions. However, labeling and imaging bacterial proteins in host cells without disrupting their structure/function is technically challenging. 
Constructing fluorescent fusion proteins does not resolve this problem, because the fusion proteins jam the secretory apparatus and thus are not secreted. To overcome these obstacles, we recently employed a method for site-specific fluorescent labeling of bacterial secreted effectors, as well as other difficult-to-label proteins, using genetic code expansion (GCE). This paper provides a complete step-by-step protocol to label Salmonella secreted effectors using GCE site-specifically, followed by directions for imaging the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM) 
Recent findings suggest that the incorporation of non-canonical amino acids (ncAAs) via GCE, followed by bio-orthogonal labeling with tetrazine-containing dyes, is a viable technique for selective labeling and visualization of bacterial secreted proteins and subsequent image analysis in the host. The goal of this article is to provide a straightforward and clear protocol that can be employed by investigators interested in conducting super-resolution imaging using GCE to study various biological processes in bacteria and viruses, as well as host-pathogen interactions.

This article provides a straightforward and clear protocol to label Salmonella secreted effectors using genetic code expansion (GCE) site-specifically and image the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)

유전자 코드 확장을 이용한 박테리아 분비 단백질의 초고해상도 이미징

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host ...

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