عالية الدقة المجهر الإلكتروني ل<em> هيليكوباكتر بيلوري</em> CAG النوع الرابع إفراز نظام بيلي المنتجة في ظروف مختلفة من توافر الحديد

Summary

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.

Abstract

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population^1,2. 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, CagA^4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells^5,6. 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 networks^5-8. 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 “pilus”⁵. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface^9,10. 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.

Introduction

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’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 Fur^16,17. 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 disease^11-14. 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.

Protocol

1. ه. بيلوري النمو في مختلف الظروف من الحديد الشواغر والمشارك الثقافة مع خلايا الإنسان الظهارية المعدة حدد H. بيلوري سلالة PMSS1 لهذه الدراسات لأنه يحتوي على حالها جزيرة المساعدة النقدية المرضية ويعبر عن ن?…

Representative Results

في هذا التقرير، لقد أثبتنا أن ظروف متفاوتة توافر الحديد لديها القدرة على تعديل H. بيلوري CAG-T4SS شعرة نشوء حيوي في واجهة الممرض المضيف. عندما مثقف في المتوسط ​​وحده، H. بيلوري تشكل ما معدله 3 بيلي / الخلية. عندما H. ويزرع في بيلوري الحديد تستنزف الظروف (ب…

Discussion

الحديد هو المغذيات الدقيقة الأساسية لمعظم أشكال الحياة، بما في ذلك مسببات الأمراض البكتيرية. في محاولة للحد من قابلية غزو الكائنات الحية الدقيقة، ويستضيف الفقاريات بتنحية الحديد المغذيات في عملية تعرف باسم "مناعة الغذائية" ^(18). ردا على ذلك، تطورت مسببات ا…

Materials

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