Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins

2020

Preprint

Infections in many virulent bacterial strains are triggered by the release of pore forming toxins (PFTs), which form oligomeric transmembrane pore complexes on the target plasma membrane. The spatial extent of the perturbation to the surrounding lipids during pore formation is relatively unexplored. Using all-atom molecular dynamics simulations, we investigate the changes in the structure and dynamics of lipids in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer in the presence of contrasting PFTs. Cytolysin A (ClyA) an α toxin with its inserted wedge shaped bundle of inserted α helices induces significant asymmetry across the membrane leaflets in comparison with α hemolysin (AHL) a β toxin. Despite the differences in hydrophobic mismatch and uniquely different topologies of the two oligomers, perturbation to lipid order as reflected in the tilt angle and order parameters, and membrane thinning is short ranged, lying within ∼ 2.5 nm from the periphery of the either pore complex, commensurate with distances typically associated with van der Waals forces. In contrast, the spatial extent of perturbations to the lipid dynamics extend outward to at least 4 nm for both proteins, and the continuous survival probabilities reveal the presence of a tightly bound shell of lipids in this region. Displacement probability distributions show long tails and the distinctly non-Gaussian features reflect the induced dynamic heterogeneity. A detailed profiling of the protein-lipid contacts with residues tyrosine, tryptophan, lysine and arginine show increased non-polar contacts in the cytoplasmic leaflet for both PFTs, with a higher number of atomic contacts in the case of AHL in the extracellular leaflet due to the mushroom-like topology of the pore complex. The short ranged nature of the perturbations observed in this simple one component membrane suggests an inherent plasticity of membrane lipids enabling recovery of structure and membrane fluidity even in the presence of these large oligomeric trans-membrane protein assemblies. This observation has implications in membrane repair processes such as budding or vesicle fusion events used to mitigate PFT virulence, where the underlying lipid dynamics and fluidity in the vicinity of the pore complex are expected to play an important role.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Assessing the Extent of Structural and Dynamic Modulation of Membrane Lipids due to Pore Forming Toxins: Insights from Molecular Dynamics Simulations

Varadarajan¹,

Desikan²,

2020

Preprint

“…Recent studies using high resolution cryo-electron microscopy (cryo-EM) (4) and high speed atomic force microscopy (AFM) (5) of pore formation by CDCs on model membranes have revealed the kinetics of pore formation by including existence of pre-pore and pore states and the presence of a distribution of oligomeric states resembling arcs and rings (5)(6)(7). While it is known that the nature of these oligomeric states of PFTs depend on membrane fluidity (5,8,9) and, in case of CDCs, on availability of cholesterol (10), the Manuscript submitted to Biophysical Journal 1 manner in which incorporation of these states influences biomembrane dynamics is unclear. Membrane fluidity is also believed to play an important role in cellular repair processes such as exocytosis and endocytosis (11).…”

Section: Introductionmentioning

confidence: 99%

“…In case of membrane excision causing proteins that alter lipid diffusion such as antibiotics (12,13), pore-forming toxins (8,9,(14)(15)(16), and peptides (17)(18)(19), it is not clear whether existing models for integral membrane protein concentration dependent membrane dynamics will be applicable (20). A problem central to PFT attack is the manner in which lipids are either displaced or ejected during pore formation.…”

Section: Introductionmentioning

confidence: 99%

Bacterial pore-forming proteins induce non-monotonic dynamics due to lipid ejection and crowding

Ponmalar

Basu

2020

Preprint

Developing alternate strategies against pore forming toxin (PFT) mediated bacterial virulence factors require an understanding of the target cellular response to combat rising antimicrobial resistance. Membrane-bound protein complexes involving PFTs, released by virulent bacteria are known to form pores leading to host cell lysis. However, membrane disruption and related lipid mediated active repair processes during attack by PFTs remain largely unexplored. We report counter intuitive and non-monotonic variations in lipid diffusion, measured using confocal fluorescence correlation spectroscopy, due to interplay of lipid ejection and crowding by membrane bound oligomers of a prototypical cholesterol dependent cytolysin, Listeriolysin O (LLO). The observed protein concentration dependent dynamical cross-over is correlated with transitions of LLO oligomeric state populations from rings to arc-like pore complexes, predicted using a proposed two-state free area based diffusion model. At low PFT concentrations, a hitherto unexplored regime of increased lipid diffusivity is attributed to lipid ejection events due to a preponderance of ring-like pore states. At higher protein concentrations where membrane inserted arc-like pores dominate, lipid ejection is less efficient and the ensuing crowding results in a lowering of lipid diffusion. These variations in lipid dynamics are corroborated by macroscopic rheological response measurements of PFT bound vesicles. Our study correlates PFT oligomeric state transitions, membrane remodelling and mechanical property variations, providing unique insights into developing strategies to combat virulent bacterial pathogens responsible for several infectious diseases.SIGNIFICANCEDeveloping alternate strategies against pore forming toxin (PFT) mediated bacterial virulence factors requires understanding target cellular responses and cellular defence strategies to combat rising antimicrobial resistant strains. While it is well understood that PFTs exist in a wide variety of oligomeric states, the underlying membrane response to these states is unexplored. Using confocal fluorescence correlation spectroscopy and a membrane free area based model we relate non-monotonic variations in the lipid diffusivity arising from an interplay of lipid ejection events and membrane crowding due to variations in concentration of membrane bound listeriolysin O. Our observations have a direct bearing on understanding cellular defense and repair mechanisms effective during initial stages of bacterial infection and intrinsically connected to the underlying membrane fluidity.

“…To target PFTs directly, small molecules, peptides and liposome decoys that inhibit the binding of PFTs to host receptors, oligomerization of the pores on the host plasma membrane, or block already formed pores have been designed (recently reviewed in Escajadillo & Nizet, 2018) to protect against infection propagated by PFTs of Bacillus anthracis (anthrax) (Nestorovich & Bezrukov, 2014; Rai et al, 2006), Staphylococcus aureaus (pneumonia, gut and skin infections) (Ragle et al, 2010), Escherichia coli (gut and urinary tract infections) (Mandal et al, 2016), Vibrio cholerae (cholera) (Rai et al, 2006), Streptococcus pneumoniae (pneumonia) (Henry et al, 2015), and many other bacteria. While inhibitors that block fully formed pores may yield some therapeutic benefit, blocked pores can nonetheless cause other disruptive effects such as bending and deformation of the plasma membrane (Tzokov et al, 2006; Drücker et al, 2019) and alteration of the lateral organization of lipid domains (Yilmaz & Kobayashi, 2015) as well as lipid dynamics (Ponmalar et al, 2019). Further, we have also shown through experiments and modelling that not just fully formed pores but oligomeric intermediates along the pore formation pathway of the α -PFT, Cytolysin A, are also capable of spontaneously compromising membrane integrity by causing leakage (Agrawal et al, 2017; Desikan, Maiti, & Ayappa, 2017).…”

Section: Introductionmentioning

confidence: 99%

AnIn silicoAlgorithm for Identifying Amino Acids that Stabilize Oligomeric Membrane-Toxin Pores through Electrostatic Interactions

Desikan

Maiti

2019

Preprint

Self Cite

Pore forming toxins (PFTs) are a class of proteins which have specifically evolved to form unregulated pores in target plasma membranes, and represent the single largest class of bacterial virulence factors. With increasingly prevalent antibiotic-resistant bacterial strains, next generation therapies are being developed to target bacterial PFTs rather than the pathogens themselves. However, structure-based design of inhibitors that could block pore formation are hampered by a paucity of structural information about pore intermediates. On similar lines, observations of the inter-subunit interfaces in fully-formed pore complexes to identify druggable residues, whose interactions could potentially be blocked to hamper pore formation or destabilize pore assemblies, are often limited because of the presence of a large number of protein-protein interaction sites across pore inter-subunit interfaces. Narrowing down the list of plausible target residues requires a quantitative assessment of their contributions towards pore stability, which cannot be gleaned from a single, static, crystal or cryo-EM pore structure.We overcome this limitation by developing an in silico screening algorithm that employs fully atomistic molecular dynamics simulations coupled with knowledge-based screening to identify residues engaged in persistent and stabilizing electrostatic interactions across inter-subunit interfaces in membrane-inserted PFT pores. Application of this algorithm to prototypical α-PFT (cytolysin A) and β-PFT (α-hemolysin) pores yielded a small predicted subset of highly interacting residues, blocking of which could destabilize pore complexes as shown in previous mutagenesis experiments for some of these predicted residues. The algorithm also yielded a novel set of residues in both cytolysin A and α-hemolysin pores for which no mutagenesis and stability data exists to the best of our knowledge, and therefore could serve as hitherto un-CONTACT Rajat Desikan, recognised potential targets for PFT inhibitors. The algorithm worked equally well for both α and β-PFT pores, and could thus be potentially applicable to all pores with known structures to generate a database of pore-destabilizing mutations, which could then serve as a starting point for experimental validation and structure-based PFT-inhibitor design. ABBREVIATIONSAHL α-hemolysin ClyA Cytolysin A DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine PDB Protein data bank PFT(s) Pore-forming toxin(s) MD Molecular dynamics RSA Relative solvent accessibility RMSD Root mean square deviation SASA solvent accessible surface area