PrfA and the Regulation ofVirulence Gene Expression

Virulence genes are coordinately regulated in L. monocytogenes by a trans-acting factor called PrfA (positive regulatory factor A) encoded by the prfA gene located upstream of the hly gene. PrfA, a cytoplasmic protein of 27 kDa, acts as a transcriptional activator [53] and is a member of the Crp/Fnr family of transcriptional activators. Like all members of this family it harbors a helix-turn-helix motif in its C-terminal part that binds to a 14-bp palindromic sequence called PrfA box which is present in the promoters of all PrfA-dependent genes (reviewed in Refs. [4, 54]). The essential structural and biochemical features of a functional PrfA-dependent promoter including the importance of neighboring sequences have been recently studied in detail by the use of an in vitro transcription system [55]. Aside from the well-studied promoters of the known virulence genes, a number of additional PrfA-boxes have been identified in the genome sequence of L. monocytogenes as discussed below.

The transcriptional organization of the PrfA-regulated genes is complex. Multiple transcripts of all genes of the virulence gene cluster and also of the inlAB operon have been identified [4, 54]. The full expression of the PrfA-dependent virulence genes requires the synthesis of the monocistronic prfA transcripts, which are synthesized at two PrfA-independent promoters, one of which is dependent on sigma B (SigB), while the other seems to be SigA-dependent [36, 56, 57]. It is believed that transcription of prfA via these prfA promoters results in the synthesis of a limited amount of PrfA sufficient to activate the high-affinity PrfA-de-pendent promoter located in front of plcA which initiates transcription of a bicis-tronic plcA-prfA mRNA leading to enhanced PrfA synthesis. The resulting higher level of PrfA is believed to activate the mpl and actA promoters, which seem to have a lower affinity for PrfA due to base mismatches in their palindromic PrfA boxes [54]. PrfA-dependent virulence gene expression in L. monocytogenes is also thermoregulated, and the shift from 30 °C to 37 °C results in a dramatic increase in expression of the virulence genes [58]. The low expression of virulence genes at temperatures below 30 °C coincides with the absence of PrfA protein. However, the prfA gene is still transcribed under these conditions from its own promoter, resulting in a monocistronic prfA transcript. At 37 °C prfA is transcribed from the prfA promoter and from the PrfA-dependent plcA promotor, resulting in both a monocistronic and a bicistronic plcA-prfA messenger [58]. Johansson et al. [59] were able to demonstrate that at 30 °C the monocistronic prfA messenger is not translated because the upstream untranslated leader (UTR) preceding prfA forms a secondary structure, which masks the ribosome binding region. This secondary structure is thermosensitive and hence unstable at higher temperatures (37 °C), which then allows efficient translation resulting in an about five-fold increase in PrfA levels.

Besides temperature, an increasing number of environmental signals have been shown to affect virulence gene expression in L. monocytogenes; these can be classified into either physicochemical signals (iron, readily metabolized carbohydrates taken up by phosphoenolpyruvate-dependent phosphotransferase systems such as glucose or cellobiose, higher salt concentration, low pH, activated charcoal) or stress conditions (heat shock, oxidative stress, nutritional stress). Growth inside host cells also activates PrfA, which may be caused by one or more of the above-mentioned signals [54]. The mechanisms of altered gene expression under such conditions are either unknown or only poorly understood.

An insertional mutagenesis study [60] and two in vivo expression technology (IVET) studies [61, 62] tried to identify genes preferentially expressed inside mammalian cells. However, in both studies, apart from the well-known virulence genes, only a few genes involved in nucleotide biosynthesis, an arginine transporter, and some unknown genes were identified. The complexity of the regulation of gene expression inside the host cell is thus, despite all progress, far from understood.

346 | 16 Genomics of Listeria monocytogenes 16.4

0 0

Post a comment