Characteristics of induction of virulence factor expression by activated charcoal inListeria monocytogenes

Abstract

Activated charcoal has been previously shown to induce in vitro expression of virulence factors by Listeria monocytogenes. In trying to elucidate the nature of the charcoal action, we found that the treatment of brain heart infusion medium with activated charcoal followed by charcoal removal does not result in an increase of virulence factor expression. At the same time, the addition of fresh charcoal to the charcoal-treated medium induces expression, suggesting that the effect of activated charcoal cannot be explained only by changes in medium composition. In addition, we observed that activated charcoal induced expression of virulence factors even when L. monocytogenes was physically separated from charcoal particles by either a nitrocellulose membrane or a thin layer of agar. We propose that the interaction of charcoal with some listerial product(s) might be responsible for the effect observed.

Route of Injection Affects the Impact of InlB Internalin Domain Variants on Severity ofListeria monocytogenesInfection in Mice

“…L. monocytogenes were routinely cultivated in the Brain Heart Infusion (BHI, BD, USA) medium. To get a virulence regulon induction, L. monocytogenes was grown in BHI supplemented with 0,2% activated charcoal (Merck) [ 26 ]. E. coli used in cloning and expression procedures were cultivated in the LB medium (Sigma-Aldrich).…”

Characterization of the roles of activated charcoal and Chelex in the induction of PrfA regulon expression in complex medium

“…To test if metal ions can negate the effect of activated charcoal on the expression of hly and actA in BHI medium, GFP fluorescence levels were measured in L. monocytogenes cells expressing either P hly -eGFP or P actA -eGFP and grown in AC-BHI medium supplemented with iron, zinc, or both (S1 Fig) . While addition of zinc to AC-BHI medium marginally but significantly reduced the expression of P hly -eGFP, addition of iron or iron and zinc had no significant effect on the expression of P hly -eGFP (S1A Fig). Furthermore, addition of iron, zinc or both had no significant effect on P actA -eGFP expression in AC-BHI (S1B Fig) . However, it is important to note that the activated charcoal in AC-BHI medium may continuously chelate metal ion [36,49], negating the effect of metal ion addition, which could explain the limited effect of metal ion addition on hly and actA expression found here. Pre-treatment of BHI with activated charcoal and its subsequent removal by filtration, in a similar manner to Chelex pretreatment, was not feasible since it has been shown that pre-treatment of BHI with activated charcoal was not sufficient to induce PrfA-dependent gene expression and that activated charcoal must be present throughout the course of bacterial growth [36,37].…”

“…Furthermore, addition of iron, zinc or both had no significant effect on P actA -eGFP expression in AC-BHI (S1B Fig) . However, it is important to note that the activated charcoal in AC-BHI medium may continuously chelate metal ion [36,49], negating the effect of metal ion addition, which could explain the limited effect of metal ion addition on hly and actA expression found here. Pre-treatment of BHI with activated charcoal and its subsequent removal by filtration, in a similar manner to Chelex pretreatment, was not feasible since it has been shown that pre-treatment of BHI with activated charcoal was not sufficient to induce PrfA-dependent gene expression and that activated charcoal must be present throughout the course of bacterial growth [36,37]. To rule out a possible effect of excess metal ions in the medium on GFP fluorescence per se, we measured P hly -eGFP levels in BHI with increasing iron and zinc concentrations.…”

“…This was corroborated by the fact that the addition of iron and zinc to Chelex-treated BHI restored the lower expression levels of hly and actA. The presence of activated charcoal throughout the course of bacterial growth, which is essential for PrfA activation [36,37], will likely results in the chelation of exogenously added metal ion and GSH, thus reducing their bioavailability to L. monocytogenes cells. Activated charcoal is a complex resin that binds multiple organic and inorganic molecules.…”

“…Recently, it has been shown that Amberlite XAD induces expression of the PrfA regulon in BHI by depleting oligopeptides, which either inhibit or activate PrfA depending on their cysteine content [23]. On the other hand, the specific mechanism by which charcoal activates PrfA-dependent gene expression in BHI remains unknown [36][37][38].…”