New Listeria monocytogenes prfA* mutants, transcriptional properties of PrfA* proteins and structure–function of the virulence regulator PrfA

Abstract

SummaryPrfA, a transcription factor structurally related to Crp/ Fnr, activates Listeria monocytogenes virulence genes during intracellular infection. We report two new PrfA* mutations causing the constitutive overexpression of the PrfA regulon. Leu-140Phe lies in a a a a D adjacent to the DNA-binding motif in the C-terminal domain, like a previously characterized PrfA* mutation (Gly-145Ser). Ile-45Ser, in contrast, maps to the N-terminal b b b b -roll, a structure similar to that of the Crp cAMP binding site. The in vitro transcriptional properties of recombinant PrfA* I45S and PrfA* G145S were compared to those of PrfA WT at two differentially regulated PrfA-dependent promoters, P plcA and P actA . The two PrfA* mutations increased the affinity for the target DNA to a different extent, and the differences in DNA binding (PrfA* G145S > PrfA* I45S >>> PrfA WT ) correlated with proportional differences in transcriptional activity. The use of the PrfA* proteins revealed that P plcA had a greater affinity for, and was more sensitive to, PrfA than P actA . RNA polymerase (RNAP) initiated transcription independently of PrfA at P plcA , but not at P actA , consistent with bandshift experiments suggesting that P plcA has a greater affinity for RNAP than P actA . Thus, differences in affinity for both PrfA and RNAP appear to determine the different expression pattern of PrfA-regulated promoters. Modelling of the PrfA* mutations in the crystal structure of PrfA and comparison with structure-function analyses of Crp, in which similar mutations lead to constitutively active (cAMPindependent) Crp* proteins, suggested that PrfA shares with Crp an analogous mechanism of cofactormediated allosteric shift. Our data support a regulatory model in which changes in PrfA-dependent gene expression are primarily accounted for by changes in PrfA activity.

The Virulence Regulator PrfA Promotes Biofilm Formation byListeria monocytogenes

“…Small-molecule cofactors regulate the protein activity state of many family members (19,39). While no such cofactor has been identified yet for PrfA, a variety of point mutations in prfA (prfA* mutants) that result in variant PrfA proteins whose function is locked in the intracellular activity state support the theory that PrfA activity is allosterically regulated (16,28,37,40,44,47). These prfA* mu-tants constitutively express levels of the actin-nucleating protein ActA comparable to those observed when bacteria are in the host cytosol (40).…”

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

“…The interaction of PrfA with cofactors that modulate its activity is suggested by the existence of mutants generated by single amino acid exchanges at different positions in the PrfA protein which lead to permanently active PrfA proteins (termed PrfA*) that are no longer subject to modulation by environmental conditions (Mueller & Freitag, 2005;Ripio et al, 1997;Shetron-Rama et al, 2003;Vega et al, 1998Vega et al, , 2004Wong & Freitag, 2004). Based on this finding and the structural similarity between PrfA and Crp (Lampidis et al, 1994), it has been postulated that wild-type PrfA may be activated by (a) component(s) in a way similar to that by which the PrfA-related Crp (or CAP) factor of E. coli is activated by cAMP (Eiting et al, 2005;Kreft & Vázquez-Boland, 2001).…”

Activation of PrfA results in overexpression of virulence factors but does not rescue the pathogenicity of Listeria monocytogenes M7

“…4). These results indicate apparent activation of plcB and hly by the M7 PrfA due to G145S substitution (Ripio et al, 1997;Vega et al, 2004;Wong & Freitag, 2004). At similar inoculum levels in mice simultaneously infected with the two competition strains, the M7 load in liver samples was about 19.1 and 0.2 % of the total bacterial counts at 48 and 72 h pi, respectively, far less than the EGDe strain (Pv0.01) (Fig.…”

“…(Bruno & Freitag, 2010). However, the relationship between virulence and PrfA activation remains unclear (Lauer et al, 2008;Ripio et al, 1997;Vega et al, 2004;Wong & Freitag, 2004). Glomski et al (2003) demonstrated that mutants failing to compartmentalize LLO activities were cytotoxic and of low virulence.…”

“…Sequence analysis has shown that PrfA of the serovar 4a strain M7 from milk has three amino acids (Ser145, Ala165 and Asn197) different from those of EGDe (Gly145, Thr165 and Lys197) (Chen et al, 2011b), indicating that the PrfA of M7 could be constitutively active for transcriptional regulation, seen as strong phospholipase and haemolytic activities (Ripio et al, 1997;Vega et al, 2004;Wong & Freitag, 2004). Transcriptional analysis of PrfA-regulated genes in BHI medium and whole sheep blood showed that M7 did have a significantly higher expression than EGDe (Pv0.01) (Fig.…”