Bioenergetic Mechanism for Nisin Resistance, Induced by the Acid Tolerance Response of Listeria monocytogenes

Listeria monocytogenes causes ∼2,500 cases/year of listeriosis out of an estimated 76 million cases/year of food-borne disease in the United States (23). The pathogen targets mainly newborn, pregnant, elderly, and immunocompromised individuals, and it is associated with mortality rates of up to 37%. Control of L. monocytogenes is difficult, due to its widespread presence in nature, intrinsic physiologic resistance, adaptation capacity, and ability to grow at low temperatures (37). The responses of L. monocytogenes to acid, osmotic, and thermal stresses increase its resistance and virulence (16, 28). The acid tolerance response (ATR) is the abnormal resistance to lethal acid after an exposure to mild acidic conditions (21). The regulation of stress response proteins changes during induction of the ATR (29, 31). These proteins include chaperones, transcriptional regulators (13), the glutamic acid decarboxylase system, and the FoF1 ATPase enzyme complex (10, 31). The ATR also increases virulence and cross-protects listeriae from other stressors, such as elevated temperatures (16) and antimicrobials (28). The specific acids affect the pH range at which the ATR is induced and the range within which the pH becomes lethal; lactic acid is a stronger inducer than HCl (2). The ATR also confers resistance to the bacteriocin nisin, an antimicrobial peptide that is approved for food use in >40 countries (6). ATR-induced L. monocytogenes cells (ATR+) but not the control cells (ATR−) survive for at least 30 days at 4°C in a model fermented system where Lactococcus lactis produced lactic acid (pH 5.7) and nisin (50 μg/ml) (2). The mechanism by which the ATR protects L. monocytogenes against nisin is uncertain. Analysis of membrane lipids of constitutively nisin-resistant listeriae shows that their membrane is more rigid, due to changes in the proportion of fatty acids (11, 24, 25). Similar temperature-induced changes in membrane composition cause measurable changes in membrane fluidity as demonstrated by fluorescence anisotropy (22). However, these changes in membrane lipid composition do not fully explain the increased nisin resistance of ATR+ listeriae (38). Cell membranes have low permeability to protons, which are subjected to specific transport mechanisms such as FoF1 ATPase, Na+/H+ antiporters, and electron transport systems (31). This enables living cells to build a potential across their membranes, which is essential for energy transduction (41). The peptide nisin targets energized cell membranes, and its insertion is activated by the difference in free available energy across the membrane (12). Nisin molecules insert cooperatively into the cell membrane, which is disrupted by transient pore formation (4). Destruction of the membrane integrity collapses the proton motive force (PMF), causing cell death. The PMF-dependent action of nisin suggested a bioenergetic contribution to nisin resistance in ATR+ listeriae. We hypothesized that decreased PMF contributes to the increased nisin resistance of ATR+ L. monocytogenes. In L. monocytogenes, the PMF is generated primarily by the membrane-associated FoF1 ATPase, which builds a PMF using energy derived from ATP hydrolysis (8). We present surprising evidence that ATR+ listeriae have significantly higher intracellular ATP (ATPi) concentrations than ATR− cells. This vital ATP sparing in ATR+ L. monocytogenes is correlated to the downregulation of the FoF1 ATPase c subunit.