Modulation of PrfA activity in Listeria monocytogenes upon growth in different culture media

Abstract

PrfA is the major transcriptional activator of most virulence genes of Listeria monocytogenes. Its activity is modulated by a variety of culture conditions. Here, we studied the PrfA activity in the L. monocytogenes wild-type strain EGD and an isogenic prfA deletion mutant (EGDDprfA) carrying multiple copies of the wild-type prfA or the mutant prfA* gene (strains EGDDprfApPrfA and EGDDprfApPrfA*) in response to growth in brain heart infusion (BHI), Luria-Bertani broth (LB) or a defined minimal medium (MM) supplemented with one of the three phosphotransferase system (PTS) carbohydrates, glucose, mannose and cellobiose, or the non-PTS carbon source glycerol. Low PrfA activity was observed in the wild-type strain in BHI and LB with all of these carbon sources, while PrfA activity was high in minimal medium in the presence of glycerol. EGDDprfApPrfA*, expressing a large amount of PrfA* protein, showed high PrfA activity under all growth conditions. In contrast, strain EGDDprfApPrfA, expressing an equally high amount of PrfA protein, showed high PrfA activity only when cultured in BHI, and not in LB or MM (in the presence of any of the carbon sources). A ptsH mutant (lacking a functional HPr) was able to grow in BHI but not in LB or MM, regardless of which of the four carbon sources was added, suggesting that in LB and MM the uptake of the used PTS carbohydrates and the catabolism of glycerol are fully dependent on the functional common PTS pathway. The BHI culture medium, in contrast, apparently contains carbon sources (supporting listerial growth) which are taken up and metabolized by L. monocytogenes independently of the common PTS pathway. The growth rates of L. monocytogenes were strongly reduced in the presence of large amounts of PrfA (or PrfA*) protein when growing in MM, but were less reduced in LB and only slightly reduced in BHI. The expression of the genes encoding the PTS permeases of L. monocytogenes was determined in the listerial strains under the applied growth conditions. The data obtained further support the hypothesis that PrfA activity correlates with the expression level and the phosphorylation state of specific PTS permeases.

The major PEP-phosphotransferase systems (PTSs) for glucose, mannose and cellobiose of Listeria monocytogenes, and their significance for extra- and intracellular growth

“…Recent reports Stoll et al, 2008) indicate a close link between the glucose-, mannose-and cellobiose-specific PTS permeases and the modulation of the PrfA activity in L. monocytogenes. Since PrfA is the central transcriptional activator of the major listerial virulence genes, a deeper knowledge of these PTS permeases is therefore of utmost interest for the understanding of the regulation of listerial virulence.…”

“…Even a ptsH insertion mutant, lacking functional HPr and EI, still grows in BHI (Mertins et al, 2007;Stoll et al, 2008). The following studies instead used well-defined media supplemented with glucose, mannose or cellobiose as sole carbon source (Premaratne et al, 1991;Stoll et al, 2008).…”

“…Transcriptome analyses were performed using special PTS microarrays (oligonucleotides from Operon were spotted on epoxy-coated glass slides from BF-BIOlabs Schneider und Zeltz), as described in Stoll et al (2008).…”

Association of ActA to Peptidoglycan Revealed by Cell Wall Proteomics of Intracellular Listeria monocytogenes

“…Of interest, differences in the type of virulence-related proteins synthesized by L. monocytogenes have been reported in BHI-rich medium compared with a minimal medium containing distinct fermentable or nonfermentable carbohydrates (19). For instance, the activity of the master virulence regulator of L. monocytogenes PrfA, which controls expression of functions involved in virulence such as the listeriolysin LLO and the phospholipases PlcA and PlcB (15,20,21), differs in these distinct growth conditions (19). PrfA also modulates the expression of surface proteins, including proteins bearing an LPXTG-sorting motif, GW-rich modules, and the membrane protein ActA, which induces host actin assembly (15,20,21).…”

Glucose-Dependent Activation ofBacillus anthracisToxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA

“…Investigations into bacterial pathogenesis have revealed an intuitive connection between basic metabolic processes of adaptation and virulence. For example, it has been reported that bacteria alter the transcription of carbohydrate utilization genes and virulence factor-encoding genes in response to the sugar availability encountered in the environment, in particular, in the infected host (44,65,67,69,79). Consequently, regulatory mechanisms that connect carbohydrate metabolism to virulence factor production must have evolved.…”