Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications

Abstract

High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress) proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods.

Keywords: EHEC; Escherichia coli; STEC; food preservation; high hydrostatic pressure.

FIGURE 1

Pressure effects on the outer membrane of E. coli. The outer leaflet of the outer membrane is composed of a lipopolysaccharide layer which prevents penetration of large or hydrophobic molecules to the periplasm. Lipid A molecules are cross-linked by divalent cations. Porins provide channels for small hydrophilic compounds; lipoproteins that are anchored in the peptidoglycan stabilize the outer membrane. Pressure application disrupts the electrostatic interactions between divalent cations and negatively charged LPS molecules, resulting in dissociation of lipid A from the outer membrane and the integration of phospholipids in the outer leaflet. Outer membrane proteins also dissociate from the membrane (Ritz et al., 2000). This process permits entry of hydrophobic inhibitors (Kalchayanand et al., 1992; Hauben et al., 1996; Gänzle and Vogel, 2001). The uncommon porin NmpC may contribute to pressure resistance in E. coli AW1.7 (Ruan et al., 2011), and the porin OmpX is over-expressed in E. coli during grown at elevated pressure (Nakashima et al., 1995). Lipoproteins including OsmB and NlpI contribute to structural integrity of E. coli, and mediate pressure resistance (Charoenwong et al., 2011).

FIGURE 2

Pressure effects on the cytoplasmic membrane and membrane bound proteins in E. coli. (A) High pressure decreases lateral motion and induces phase transition in the phospholipid bilayers of E. coli, and promotes gelation of the membrane lipids (Pagan and Mackey, 2000; Winter, 2002; Mañas and Mackey, 2004). Pressure resistance is influenced by membrane fluidity and fatty acid composition (Casadei et al., 2002). Exponential phase cell are more sensitive to pressure when compared to stationary phase cells (Pagan and Mackey, 2000; Casadei et al., 2002). Stationary phase cell express cfa encoding for cyclopropane fatty acyl phospholipid synthase (CFA). CFA converts unsaturated fatty acids to cyclopropane fatty acids, which contribute to acid resistance (Brown et al., 1997; Grogan and Cronan, 1997) and pressure resistance in E. coli (Charoenwong et al., 2011). (B) Sublethal pressure inactivates acid resistance in E. coli. The glutamate decarboxylase system for acid resistance is more resistant to pressure than other acid resistance mechanisms, and glutamic acid decarboxylation improved the survival of E. coli during post-pressure acid challenge (Kilimann et al., 2005). (C) The accumulation of compatible solutes including glycine-betaine, choline and sucrose, and the synthesis of trehalose protects against pressure-induced cell death (Van Opstal et al., 2003; Molina-Höppner et al., 2004; Charoenwong et al., 2011); BetT, ProP, and ProU are the major transporters for compatible solutes in E. coli. Mutants that are defective in trehalose synthesis exhibit a reduced resistance to pressure (Charoenwong et al., 2011). (D) Pressure inactivates F₀F₁-ATPase, which causes disruption of the acid efflux system (Wouters et al., 1998).

FIGURE 3

Pressure effects on cytoplasmic proteins and nucleic acids in E. coli. (A,A’) Pressure dissociates ribosomes and inhibits protein synthesis; ribosomes are stabilized by addition of divalent cations (Hauben et al., 1998; Niven et al., 1999; Gayan et al., 2013). (B,B’) Dps (DNA binding protein from starved cells) binds DNA as homo-dodecamer and protects E. coli against oxidative stress-, pressure-, and acid-induced DNA damage (Choi et al., 2000; Zhao et al., 2002; Malone et al., 2006). Deletion of the genes coding for the alternative sigma factors σ^E or σ^S increases the sensitivity of E. coli to pressure; indicating that the general stress response (σ^S) and the heat shock response (σ^E) increase pressure resistance (Robey et al., 2001; Aertsen et al., 2004, 2005; Malone et al., 2006). (C,C’) High pressure-induces oxidative stress in E. coli. Proteins that protect against peroxide and superoxide stress (thioredoxin, catalase, superoxide dismutase, and proteins that regulate their expression) also increase pressure resistance in E. coli (Aertsen et al., 2005; Malone et al., 2006; Charoenwong et al., 2011). The presence of iron and iron sulfur cluster proteins increases the lethality of pressure on E. coli (Malone et al., 2006; Yan et al., 2013), likely because free intracellular iron accumulates and catalyses the formation of reactive oxygen species. (D,D’) Pressure disassembles protein aggregates bodies in vivo, re-growth of sublethally injured cells after pressure treatment is dependent on the time required for re-assembly of protein aggregates. The presence of the locus of heat resistance which predominantly encodes genes involved in protein folding and protein turnover is generally associated with pressure resistance in E. coli and loss of the locus of heat resistance reduces the pressure resistance in E. coli AW1.7 (Garcia-Hernandez et al., 2015; Liu et al., 2015; Mercer et al., personal communication). Deletion of the inclusion body binding proteins IbpA and IbpB decreases pressure resistance (Charoenwong et al., 2011). The heat shock proteins DnaK and DnaJ contribute to assembly and segregation of protein aggregates (Aertsen et al., 2004; Govers et al., 2014), and mediate pressure resistance.