Isolation of Listeria monocytogenes mutants with high‐level in vitro expression of host cytosol‐induced gene products

Abstract

SummaryThe facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA , the protein product of which is required for cell-to-cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200-fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high-level expression of PrfAdependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild-type L. monocytogenes . These results suggest that, although constitutive activation of PrfA and PrfA-dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells.

LysPGS formation inListeria monocytogeneshas broad roles in maintaining membrane integrity beyond antimicrobial peptide resistance

“…56 Briefly, 2× BHI media containing 0.2% w/v activated charcoal was autoclaved and filter sterilized before being combined with an equal volume of 0.6% w/v media grade agar. The resulting BHI plates with 0.3% w/v agar were inoculated by stabbing a needle used to pick up isolated colonies directly into the agar; isolated colonies originated from BHI agar plates incubated for 24 h at 30 °C or 37 °C.…”

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).…”

The Virulence Regulator PrfA Promotes Biofilm Formation byListeria monocytogenes

“…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). These mutations also result in an increased level of production of the PrfA* protein via the activation of PplcA (43,47).…”