Extracellular Bacterial Pathogen Induces Host Cell Surface Reorganization to Resist Shear Stress

Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow–generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream

Tightening the knot in phytochrome by single molecule atomic force microscopy

A growing number of proteins have been shown to adopt knotted folds. Yet the biological roles and biophysical properties of these knots remain poorly understood. We have used protein engineering and atomic force microscopy to explore single-molecule mechanics of the figure-of-eight knot in the chromophore-binding domain of the red/far red photoreceptor, phytochrome. Under load, apo phytochrome unfolds at forces of ~47 pN, while phytochrome carrying its covalently bound tetrapyrrole chromophore unfolds at ~73 pN. These forces are among the lowest measured in mechanical protein unfolding, hence the presence of the knot does not automatically indicate a super-stable protein. Our experiments reveal a stable intermediate along the mechanical unfolding pathway, reflecting sequential unfolding of two distinct subdomains in phytochrome, potentially the GAF and PAS domains. For the first time, our experiments allow direct determination of knot size under load. In the unfolded chain, the tightened knot is reduced to 17 amino acids, resulting in apparent shortening of the polypeptide chain by 6.2 nm. Steered molecular dynamics simulations corroborate this number. Finally, we found that covalent phytochrome dimers created for these experiments retain characteristic photoreversibility, unexpectedly arguing against dramatic rearrangement of the native GAF dimer interface upon photoconversion.Comment: 12 pages plus five figures; has been submitted to Biophysical J. Replacement on 9/16 is ONLY to correct a typo in the meta data; the uploaded file is identical to first versio

The Bacterial Fimbrial Tip Acts as a Mechanical Force Sensor

The subunits that constitute the bacterial adhesive complex located at the tip of the fimbria form a hook-chain that acts as a rapid force-sensitive anchor at high flow

Type IV pili: dynamics, biophysics and functional consequences

The surfaces of many bacteria are decorated with long, exquisitely thin appendages called type IV pili (T4P), dynamic filaments that are rapidly polymerized and depolymerized from a pool of pilin subunits. Cycles of pilus extension, binding and retraction enable T4P to perform a phenomenally diverse array of functions, including twitching motility, DNA uptake and microcolony formation. On the basis of recent developments, a comprehensive understanding is emerging of the molecular architecture of the T4P machinery and the filament it builds, providing mechanistic insights into the assembly and retraction processes. Combined microbiological and biophysical approaches have revealed how T4P dynamics influence self-organization of bacteria, how bacteria respond to external stimuli to regulate T4P activity for directed movement, and the role of T4P retraction in surface sensing. In this Review, we discuss the T4P machine architecture and filament structure and present current molecular models for T4P dynamics, with a particular focus on recent insights into T4P retraction. We also discuss the functional consequences of T4P dynamics, which have important implications for bacterial lifestyle and pathogenesis

Aquifex aeolicus PilT, Homologue of a Surface Motility Protein, Is a Thermostable Oligomeric NTPase

Bacterial surface motility works by retraction of surface-attached type IV pili. This retraction requires the PilT protein, a member of a large family of putative NTPases from type II and IV secretion systems. In this study, the PilT homologue from the thermophilic eubacterium Aquifex aeolicus was cloned, overexpressed, and purified. A. aeolicus PilT was shown to be a thermostable ATPase with a specific activity of 15.7 nmol of ATP hydrolyzed/min/mg of protein. This activity was abolished when a conserved lysine in the nucleotide-binding motif was altered. The substrate specificity was low; UTP, CTP, ATP, GTP, dATP, and dGTP served as substrates, UTP having the highest activity of these in vitro. Based on sedimentation equilibrium and size exclusion chromatography, PilT was identified as a ≈5- to 6-subunit oligomer. Potential implications of the NTPase activity of PilT in pilus retraction are discussed