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

Benjamin J Koestler,Ahmed Gaballa,Tyler Bechtel,Etienne Doll,Sriya Sunil,Martin Wiedmann,Sarah I Murphy,Veronica Guariglia-Oropeza

doi:10.1371/journal.pone.0250989

Abstract

The foodborne pathogen Listeria monocytogenes is able to survive across a wide range of intra- and extra-host environments by appropriately modulating gene expression patterns in response to different stimuli. Positive Regulatory Factor A (PrfA) is the major transcriptional regulator of virulence gene expression in L. monocytogenes. It has long been known that activated charcoal is required to induce the expression of PrfA-regulated genes in complex media, such as Brain Heart Infusion (BHI), but not in chemically defined media. In this study, we show that the expression of the PrfA-regulated hly, which encodes listeriolysin O, is induced 5- and 8-fold in L. monocytogenes cells grown in Chelex-treated BHI (Ch-BHI) and in the presence of activated charcoal (AC-BHI), respectively, relative to cells grown in BHI medium. Specifically, we show that metal ions present in BHI broth plays a role in the reduced expression of the PrfA regulon. In addition, we show that expression of hly is induced when the levels of bioavailable extra- or intercellular iron are reduced. L. monocytogenes cells grown Ch-BHI and AC-BHI media showed similar levels of resistance to the iron-activated antibiotic, streptonigrin, indicating that activated charcoal reduces the intracellular labile iron pool. Metal depletion and exogenously added glutathione contributed synergistically to PrfA-regulated gene expression since glutathione further increased hly expression in metal-depleted BHI but not in BHI medium. Analyses of transcriptional reporter fusion expression patterns revealed that genes in the PrfA regulon are differentially expressed in response to metal depletion, metal excess and exogenous glutathione. Our results suggest that metal ion abundance plays a role in modulating expression of PrfA-regulated virulence genes in L. monocytogenes.

Highlights

Listeria monocytogenes is a Gram-positive foodborne pathogen that can survive across a wide range of extra- and intra-host environments [1]
To further determine the mechanisms by which activated charcoal and Chelex pre-treatment induce expression of the Positive Regulatory Factor A (PrfA) regulon, we constructed an L. monocytogenes strain that expresses a Phly-eGFP transcriptional fusion. This strain was used to assess whether activated charcoal prenst in Brain Heart Infusion (BHI) (“AC-BHI”) or Chelex pre-treatment of BHI (Ch-BHI) induces the expression of Phly-eGFP in L. monconcytgenes grown in these media
On the other hand and as expected, Phly-eGFP was constitutively and highly expressed in BHI in a L. monocytogenes strain that encodes a constitutively active PrfA (PrfA ), which is locked in the active form (34,161± 539.9 eGFP fluorescence/OD600)

Summary

Introduction

Listeria monocytogenes is a Gram-positive foodborne pathogen that can survive across a wide range of extra- and intra-host environments [1]. Regulation of virulence gene expression in L. monocytogenes environment, L. monocytogenes can survive in the presence of multiple bacterial growth deterrents such as low temperature, low pH, variable nutrient availabilities, and the presence of some anti-microbial compounds [3,4]. L. monocytogenes’ ability to survive under a wide-range of environmental conditions is supported by coordinated expression of elaborate networks of genes that are involved in virulence and stress response [5,6,7,8]. The majority of genes involved in virulence and stress response are regulated, respectively, by the transcriptional activator Positive Regulatory Factor A (PrfA) and the alternative sigma factor σB [5,9,10,11,12]

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Discussion

Conclusion