Foik, I. P. | bioRxiv Science

bioRxiv2022

Activation of a bacterial flow sensor by hypochlorous acid from stimulated neutrophils

Neutrophils kill bacteria by producing an array of lethal molecules, including hypochlorous acid (HOCl). The extent to which pathogens detect HOCl from neutrophils and defend themselves has not been understood. We report that the opportunistic pathogen Pseudomonas aeruginosa responds to activated neutrophils by upregulating the flow regulated operon (Fro), which was previously shown to be activated by fluid flow. We found that fro is upregulated in a mouse infection model where neutrophil influx occurs. This upregulation is induced in vitro by HOCl and its secondary product taurine chloramine but not by other neutrophil defense factors including LL-37, histones, or H2O2. HOCl induces the FroR-dependent upregulation of methionine sulfoxide reductases that relieve otherwise lethal oxidative stress. Fro expression is regulated by FroR's anti-sigma factor FroI, which contains the highest density of methionines and cysteines of all anti-sigma factors. The second-order rate constants of HOCl are highest with these residues, raising the possibility that the activation of fro could involve oxidation of FroI. These findings suggest a model in which flow transports oxidizing molecules that activate the fro operon, establishing an early warning system for P. aeruginosa that improves its survival against host immune defenses and persistence during infection.

microbiology↗

bioRxiv2020

Structural and molecular dynamics of Mycobacterium tuberculosis malic enzyme, a potential anti-TB drug target

Tuberculosis (TB) is the most lethal bacterial infectious disease worldwide. It is notoriously difficult to treat, requiring a cocktail of antibiotics administered over many months. The dense, waxy outer membrane of the TB-causing agent, Mycobacterium tuberculosis (Mtb), acts as a formidable barrier against uptake of antibiotics. Subsequently, enzymes involved in maintaining the integrity of the Mtb cell wall are promising drug targets. Recently, we demonstrated that Mtb lacking malic enzyme (MEZ) has altered cell wall lipid composition and attenuated uptake by macrophages. These results suggest that MEZ provides the required reducing power for lipid biosynthesis. Here, we present the X-ray crystal structure of MEZ to 3.6 [A] resolution and compare it with known structures of prokaryotic and eukaryotic malic enzymes. We use biochemical assays to determine its oligomeric state and to evaluate the effects of pH and allosteric regulators on its kinetics and thermal stability. To assess the interactions between MEZ and its substrate malate and cofactors, Mn2+ and NAD(P)+, we ran a series of molecular dynamics (MD) simulations. First, the MD analysis corroborates our empirical observations that MEZ is unusually disordered, which persists even with the addition of substrate and cofactors. Second, the MD simulations reveal that MEZ subunits alternate between open and closed states and that MEZ can stably bind its NAD(P)+ cofactor in multiple conformations, including an inactive, compact NAD+ form. Together the structure of MEZ and insights from its dynamics can be harnessed to inform the design of MEZ inhibitors that target Mtb.

biochemistry↗