Ph.D Thesis Colloquium : Aditya Upasani

Membrane Interactions in Bacterial Resistance to Antimicrobial Peptides and Mammalian Susceptibility to Toxins

Membrane targeting agents such as pore-forming toxins (PFTs) and antimicrobial peptides (AMPs) specifically disrupt target  cell membranes leveraging its lipid constituents, architecture and biophysical properties. Cells respond against such attacks by modifying the membrane composition and characteristics to counteract such disruption. During co-evolution of the host and pathogens, both have learned to adapt to such modifications. In this work, I demonstrate  two systems where the cell membrane modifications  can assist or inhibit the activity of membrane disrupting agents.

First, I describe how resistance to colistin, an antimicrobial peptide, in gram-negative bacteria emerges via membrane modifications. Many colistin resistant clinical isolates have been reported though the mechanism that leads to the  emergence of drug resistance is not well understood. Using single-cell imaging combined with DNA sequencing and lipidomics, I show that a heterogeneous level of tolerance against colistin enables the E. coli bacteria to progressively adapt in high colistin concentrations by modifying their outer cell membrane to prevent colistin binding and activity. These tolerant bacteria are detectable early right after the first exposure to colistin in isogenic susceptible populations suggesting phenotypic adaptation as the precursor to emergence of resistance. These colistin tolerant bacteria display characteristic lipid A modifications including phosphoethanolamine addition leading to lowering of negative charge of the membrane albeit at the cost of membrane integrity. We take advantage of this collateral sensitivity, to show that the frequency of colistin resistance emergence can be reduced by combining colistin treatment with small membrane bioactive molecules. 

In the second half, I examine how membrane composition changes in the mammalian cell membranes affect the activity of pore forming toxins. Mammalian cells respond to pathogen attacks by disrupting the cholesterol synthesis which is known to be essential for action of bacterial pore-forming toxins. Additionally, activation of CH25H gene by bacterial components like LPS leads to oxidation of cholesterol. Oxidised sterols like 25-hydroxycholesterol tend to act as broad spectrum antimicrobials and inhibit cholesterol dependent cytolysins. In my work, I demonstrate that Cytolysin A (ClyA), a canonical alpha-toxin while being a cholesterol dependent PFT, has evolved to escape 25-HC mediated inhibition and even adapted to replace cholesterol with 25-HC for more potent activity. Single-molecule imaging on synthetic membrane systems shows that this enhancement results from the increased membrane insertion of ClyA and enhanced ClyA assembly on 25-HC membranes.

Overall, this study underscores the importance of understanding the interplay between membrane-disrupting proteins and peptides and their target cell membranes, highlighting how modifications in membrane composition are employed as a defense mechanism against these agents.