Biochemical and Micrographic Evidence of Escherichia coli Membrane Damage during Incubation in Egg White under Bactericidal Conditions

Abstract

Bacterial membranes are often thought to be the main targets of the antimicrobial activity of egg white. In order to test this hypothesis, the state of the membranes of Escherichia coli K-12 cells during either bactericidal (45°C) or bacteriostatic (30°C) incubation in egg white at natural alkaline pH was studied by biochemical methods. Namely, the permeability of the outer membrane was evaluated through its ability to incorporate a hydrophobic fluorescent probe (1-N-phenylnaphthylamine), and the permeability of the cytoplasmic membrane was evaluated through the release of a specific intracellular enzyme (β-galactosidase). The bacteria were observed by atomic force microscopy in order to support the biochemical results. At 45°C, the outer membrane of E. coli K-12 incorporated the hydrophobic probe, suggesting that it was disrupted. In addition, the cytoplasmic β-galactosidase was released at this temperature. The atomic force microscopy analysis revealed the formation of spheroplasts, which provided further evidence of the cell wall disruption and a progressive release of cellular contents. At 30°C, biochemical and micrographic experiments confirmed that membrane integrity was preserved. These techniques provide a useful approach for studying the mechanisms of bacterial cell death in egg white.

Global Gene-expression Analysis of the Response of Salmonella Enteritidis to Egg White Exposure Reveals Multiple Egg White-imposed Stress Responses

“…The up-regulation of spy is consistent with the microscopy observation made on E. coli in the same conditions (Jan et al, 2013), where incubation in EWMM for 45 min at 45°C caused formation of spheroplasts. The induction of degP , encoding an endoprotease that degrades abnormal proteins, is consistent with previous studies.…”

“…Flagella-mediated motility is transcriptionally controlled in response to multiple stresses including heat-shock (RpoH dependent), pH change (CpxAR dependent), envelope stress (OmpR/EnvZ, CpxAR and PspF dependent) and low iron content (Fur dependent) (Shin and Park, 1995; Prüß et al, 2003; Lloyd et al, 2004; Jovanovic et al, 2006; Raivio et al, 2013), which are all of relevance to the conditions experienced upon EWMM exposure, as indicated by the transcriptomics data. The down-regulation of flagella/motility genes is consistent with AFM (Atomic Force Microscopy) observation in EWMM showing lack of flagella at 30 and 45°C in E. coli (Jan et al, 2013). Repression of motility genes at high pH (8.6) was also shown by Maurer et al (2005), which matches the expression effects reported here.…”

“…The probable environmental factors triggering this effect in EWMM are antimicrobial egg white components (Kohanski et al, 2008; Laubacher and Ades, 2008; Farris et al, 2010; Audrain et al, 2013; Evans et al, 2013; Raivio et al, 2013; Raivio, 2014), pmf dissipation (Weiner and Model, 1994; Kleerebezem et al, 1996; Joly et al, 2010) and alkaline pH (Danese and Silhavy, 1998; Thede et al, 2011; Tschauner et al, 2014) along with temperature (Brissette et al, 1990; Danese and Silhavy, 1998; Raivio and Silhavy, 2001; Darwin, 2005; Joly et al, 2010; Raivio, 2014). Previous work has also suggested that maintenance of cell-envelope integrity is an important facet of resistance to egg white (Gantois et al, 2008a), with cell-wall disruption and progressive cell lysis reported as the major mechanisms of egg white-mediated bactericidal action at 45°C for E. coli (Jan et al, 2013). …”

“…Appropriate balance between peptidoglycan synthesis and hydrolysis is critical for cell integrity (Meisel et al, 2003; Kumar et al, 2012). The apparent imbalance in their respective expression levels upon EWMM exposure might result in excessive peptidoglycan hydrolysis leading to loss of integrity of the peptidoglycan layer, which would be expected to promote spheroplast formation as seen for E. coli EWMM exposure (Jan et al, 2013). …”

Membrane permeabilization by conjugated oligoelectrolytes accelerates whole-cell catalysis

“…1b), we measured the release of the cytoplasmic hydrolase enzyme, b-galactosidase (b-gal), into the medium, which is commonly used as an indicator of inner membrane damage. [40][41][42][43][44] As before, E. coli cells were washed in buffer, stained with varying concentrations of DSSN+ for 5 hours and then ltered. Extracellular b-gal activity in the resulting ltrates was measured by monitoring cleavage of colorless 2-nitrophenyl b-D-galactopyranoside (ONPG) into yellow o-nitrophenol (ONP) by using absorption spectroscopy.…”