The Virulence Regulator PrfA Promotes Biofilm Formation by Listeria monocytogenes

Listeria monocytogenes is an opportunistic food-borne pathogen that can be isolated from a variety of environments (5, 14). This Gram-positive bacterium can contaminate foods by persisting on food-processing surfaces, and it has been hypothesized that this persistence is at least partly due to the formation of biofilms, which can be difficult to eradicate (29). Once ingested, L. monocytogenes causes life-threatening infections in fetuses, newborns, and immunocompromised people (9, 23). The high case fatality rate of listeriosis (∼20%) makes L. monocytogenes food contamination, and, thus, biofilms, an important health issue (24). In contrast to its pathogenesis, little is known about L. monocytogenes biofilm development. L. monocytogenes has three distinct life-styles: (i) an intracellular pathogen that utilizes actin-based motility for cytoplasmic movement and cell-to-cell spread; (ii) an extracellular, free-living, flagellum-propelled bacterium in the environment; and (iii) an extracellular bacterial member of a multicellular biofilm community. Biofilm formation and intracellular invasion appear to be mutually exclusive life-styles. The critical virulence transcription factor PrfA regulates the switch from extracellular, flagellum-propelled bacterium to intracellular pathogen (reviewed in references 10, 13, and 39). PrfA binds to a specific DNA sequence (the PrfA box) and positively regulates the expression of the canonical L. monocytogenes virulence genes, including itself. During intracellular invasion, the PrfA-dependent transcription of virulence genes increases first in the phagocytic vacuole. After vacuolar escape into the host cytosol, a subset of PrfA-dependent virulence genes is induced to an even greater extent. PrfA production is regulated at transcriptional, translational, and protein activity levels (13, 39). Three promoters drive prfA transcription (Fig. ​(Fig.1).1). Two of these promoters, PprfAP1 and PprfAP2, control the expression of a monocistronic prfA transcript (11, 12). PprfAP1 is dependent on the housekeeping sigma factor, σA. PprfAP2 can be driven by either σA or the stress response sigma factor, σB (30, 36, 38). In addition, the promoter of the upstream gene plcA, PplcA, contains a canonical PrfA box and is positively autoregulated by PrfA, resulting in the transcription of a bicistronic message containing plcA and prfA (4, 26). The resulting increased level of PrfA expression from PplcA is necessary for wild-type virulence (4, 12). The transcript from PprfAP1 contains a 5′ untranslated region (UTR) that acts as a thermosensor; the translation of this transcript is efficient at the host body temperature but inefficient at 30°C and below (18). In contrast to PplcA, transcription from PprfAP2 is PrfA independent, and in contrast to PprfAP1, the translation of this transcript is temperature independent. It has been postulated that this transcript is important for PrfA production outside mammalian hosts; we provide evidence here to support this hypothesis. FIG. 1. Schematic that shows the three promoters that drive the expression of prfA. PplcA is positively autoregulated by PrfA, which binds to the PrfA box (black box), and produces a bicistronic message. PprfAP1 is σA dependent, and its transcript contains ... PrfA is a member of the cyclic AMP receptor protein-fumarate and nitrate reduction regulator (CRP-FNR) family of transcriptional regulators (20). 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* mutants constitutively express levels of the actin-nucleating protein ActA comparable to those observed when bacteria are in the host cytosol (40). These mutations also result in an increased level of production of the PrfA* protein via the activation of PplcA (43, 47). Another recently isolated mutation in prfA (Y154C) results in a PrfA protein that cannot fully transition from the extracellular activity state to the intracellular activity state (27). The prfA(Y154C) mutant demonstrates a 4-fold increase in actA expression levels in brain heart infusion (BHI) medium compared to the wild type but fails to increase actA expression levels to wild-type levels (100-fold less than the wild type) during intracellular infection (27). It effectively enters the host cytosol but is defective in cell-to-cell spread and is attenuated for virulence (150-fold reduction in a mouse model) (27). Thus, there are PrfA protein variants that are locked in the intracellular activity state (PrfA*) and one [PrfA(Y154C)] that is unable to fully transition to the intracellular activity state. L. monocytogenes has never been observed to form biofilms in host cells; intracellular bacteria are single cells that move through the cytosol using actin-based motility (34). Extracellularly, L. monocytogenes motility is mediated via flagella. Flagellum-mediated motility appears to be important for initial epithelial cell attachment, and flagellum-minus mutants show an early host invasion defect in a competitive oral infection model (32). Similarly, flagellum-mediated motility is critical for initial abiotic surface attachment, and flagellum-minus mutants are defective in biofilm formation (22, 41). This common need for flagellum-mediated motility to transition from an existence as a single, extracellular cell to either environmental biofilm formation or intracellular invasion led us to hypothesize that there might be regulatory cross talk between PrfA and biofilm formation. Indeed, here we report that prfA mutants are defective in biofilm formation at a stage after initial surface attachment. The regulatory connection between PrfA and biofilm formation demonstrates that PrfA has a global role in regulating the life-style of L. monocytogenes.