Biology subjects

Shaffer, C. L.

Publications and source records attributed to Shaffer, C. L..

4 recordsLinked to original sources

bioRxiv2025

In situ architecture of the endosymbiont Wolbachia pipientis

Hidden within host cells, the endosymbiont Wolbachia pipientis is the most prevalent bacterial infection in the animal kingdom. Scientific breakthroughs over the past century yielded fundamental mechanisms by which Wolbachia controls arthropod reproduction to shape dynamic ecological and evolutionary trajectories. However, the structure and spatial organization of symbiont machineries that underpin intracellular colonization and orchestrate maternal inheritance remain unknown. Here, we used cryo-electron tomography to directly image the nanoscale architecture of bacterial tools deployed for host manipulation and germline transmission. We discovered that Wolbachia assembles multiple structures at the host-endosymbiont interface including a filamentous ladder-like framework hypothesized to serve as a specialized motility mechanism that enables bacterial translocation to specific host cell compartments during embryogenesis and somatic tissue dissemination. In addition, we present the first in situ structure of the Rickettsiales vir homolog type IV secretion system (rvh T4SS). We provide evidence that the rvh T4SS nanomachine exhibits architectural similarities to the pED208-encoded T4SS apparatus including the biogenesis of rigid conjugative pili extending hundreds of nanometers beyond the bacterial cell surface. Coupled with integrative structural modeling, we demonstrate that in contrast to canonical T4SS architectures, the -proteobacterial T4SS outer membrane complex assembles a periplasmic baseplate structure predicted to comprise VirB9 oligomers complexed with cognate VirB10 subunits that form extended antennae projections surrounding the translocation channel pore. Collectively, these studies provide an unprecedented view into Wolbachia structural cell biology and unveil the molecular blueprints for architectural paradigms that reinforce ancient host-microbe symbioses.

microbiology↗

bioRxiv2023

Architectural asymmetry enables DNA transport through the Helicobacter pylori cag type IV secretion system

Structural asymmetry within secretion system architecture is fundamentally important for apparatus diversification and biological function. However, the mechanism by which symmetry mismatch contributes to nanomachine assembly and interkingdom effector translocation are undefined. Here, we show that architectural asymmetry orchestrates dynamic substrate selection and enables trans-kingdom DNA conjugation through the Helicobacter pylori cag type IV secretion system (cag T4SS). Structural analyses of asymmetric units within the cag T4SS periplasmic ring complex (PRC) revealed intermolecular {pi}-{pi} stacking interactions that coordinate DNA binding and license trans-kingdom conjugation without disrupting the translocation of protein and peptidoglycan effector molecules. Additionally, we identified a novel proximal translocation channel gating mechanism that regulates cargo loading and governs substrate transport across the outer membrane. We thus propose a model whereby the organization and geometry of architectural symmetry mismatch exposes {pi}-{pi} interfaces within the PRC to facilitate DNA transit through the cag T4SS translocation channel.

microbiology↗

bioRxiv2022

Helicobacter pylori provokes STING immunosurveillance via trans-kingdom conjugation

Recognition of foreign nucleic acids is an evolutionarily conserved mechanism by which the host detects microbial threats. Whereas some intracellular bacterial pathogens trigger DNA surveillance pathways following phagosomal membrane perturbation, mechanisms by which extracellular bacteria activate cytosolic nucleic acid reconnaissance systems remain unresolved. Here, we demonstrate that Helicobacter pylori exploits cag type IV secretion system (cag T4SS) activity to provoke STING signaling in gastric epithelial cells. We provide direct evidence that chromosomal fragments delivered to the host cell cytoplasm via trans-kingdom conjugation bind and activate the key DNA sensor cGMP-AMP synthase. To enable paracrine-like signal amplification, translocated H. pylori DNA is sorted into exosomes that stimulate DNA-sensing pathways in uninfected bystander cells. We show that DNA cargo is loaded into the cag T4SS apparatus in the absence of host cell contact to establish a ready-to-fire nanomachine and provide evidence that cag T4SS-dependent DNA translocation is mechanistically coupled to chromosomal replication and replichore decatenation. Collectively, these studies suggest that H. pylori evolved mechanisms to stimulate nucleic acid surveillance pathways that regulate both pro- and anti-inflammatory programs to facilitate chronic persistence in the gastric niche.

microbiology↗

bioRxiv2017

In vivo structures of the Helicobacter pylori cag type IV secretion system

The bacterial type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Using electron cryotomography, here we reveal the molecular architecture of the cancer-associated Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to previously studied T4SSs. When H. pylori encounters host cells, however, the bacterium elaborates rigid, membranous tubes perforated by lateral ports. Dense, pilus-like rod structures extending from the inner membrane were also observed. We propose that the membrane tubes assemble out of the T4SS and are the delivery system for cag T4SS cargo. These studies reveal the architecture of a dynamic molecular machine that evolved to function in the human gastric niche.

microbiology↗