Abstract
The type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Here, using electron cryotomography, we reveal the molecular architecture of the Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to other T4SSs. Our images revealed that, when H. pylori encounters host cells, the bacterium elaborates membranous tubes perforated by lateral ports. Sub-tomogram averaging of the cag T4SS machinery revealed periplasmic densities associated with the outer membrane, a central stalk, and peripheral wing-like densities. Additionally, we resolved pilus-like rod structures extending from the cag T4SS into the inner membrane, as well as densities within the cytoplasmic apparatus corresponding to a short central barrel surrounded by four longer barrels. Collectively, these studies reveal the structure of a dynamic molecular machine that evolved to function in the human gastric niche.
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🧪 Sample Preparation
🔬 Cell Lines
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🏛️ Research Organizations (ROR)
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📋 Methods
Bacterial Strains and Growth Conditions
H. pylori strain 26695 and corresponding mutants ( Shaffer et al., 2011 ) were routinely maintained on Trypticase soy agar supplemented with 5% sheep blood (BD Biosciences) under microaerobic conditions. For all experiments, H. pylori cells were seeded into Brucella broth supplemented with 10% fetal bovine serum (FBS) and were grown overnight in shaking culture at 37°C, 5% CO 2 . Overnight bacterial cultures were normalized to an optical density at 600 nm (OD 600 ) to ~ 0.3 in fresh Brucella broth supplemented with 10% FBS, and cells were grown to mid-log phase at 37°C, 5% CO 2 , prior to generating samples for microscopy analysis.
Human Cell Culture
The gastric adenocarcinoma cell line AGS (ATCC CRL-1739) was maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 10 mM HEPES buffer. Cells were grown at 37°C in 5% CO 2 . Electron Crytomography Detailed methods of electron cryotomography sample preparation, data collection, and data processing are outlined in the Supplemental Experimental Procedures .
Statistical Analysis
The statistical analysis was undertaken in GraphPad Prism 7 by one-way ANOVA, followed by Dunnett’s multiple comparisons test.
Supplementary Material 1 2
📊 Figures
Figure 1
H. pylori Assembles Membranous Tubes When Proximal to Gastric Epithelial Cells
(A) Low-magnification view of a gastric epithelial cell grown on an electron microscopy grid and infected with H. pylori . (B) Low-magnification view of H. pylori interacting with a gastric epithelial...
Figure 2
In Vivo Structure of the cag T4SS
(A and B) Distal (A) and proximal (B) tomographic slices of the same region of the bacterial envelope identifying a cag T4SS particle (white arrow) adjacent to a tube-like appendage (black arrow). Das...
Figure 3
Pilus-like Rods Emerging from the cag T4SS
(A) cag T4SS particle exhibiting a typical cytoplasmic structure (arrow). (B) cag T4SS particle with a pilus-like rod density surrounded by an IM invagination (white arrow). The gastric epithelial cel...
Figure 4
Comparison of Diverse T4SS Machinery Structures
(A and B) Side (A) and top (B) views of the purified E. coli R388 conjugation system ( Low et al., 2014 ). (C and D) Side (C) and top (D) views of immunopurified cag T4SS core complex particles (adapt...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
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