Optimizing the balance between host and environmental survival skills: lessons learned from Listeria monocytogenes

Abstract

Environmental pathogens - organisms that survive in the outside environment but maintain the capacity to cause disease in mammals - navigate the challenges of life in habitats that range from water and soil to the cytosol of host cells. The bacterium Listeria monocytogenes has served for decades as a model organism for studies of host-pathogen interactions and for fundamental paradigms of cell biology. This ubiquitous saprophyte has recently become a model for understanding how an environmental bacterium switches to life within human cells. This review describes how L. monocytogenes balances life in disparate environments with the help of a critical virulence regulator known as PrfA. Understanding L. monocytogenes survival strategies is important for gaining insight into how environmental microbes become pathogens.

Figure 1. The varied habitats of Listeria monocytogenes

The bacterium Listeria monocytogenes survives and replicates within diverse environments, ranging from ground water and soil to the cytosol of infected mammalian cells. L. monocytogenes is thought to live as a saprophyte in the outside environment and it has been isolated from soil, decaying plant matter, sewage, silage and water. Animals ingesting L. monocytogenes may become infected and/or may shed the bacterium in feces, facilitating transmission via oral–fecal routes. Food-borne outbreaks of L. monocytogenes have been associated with contaminated fruit and vegetables, and from bacterial contamination of food produced within food-processing plants.

Figure 2. Multiple regulatory check-points control prfA expression and protein activity

PrfA plays an essential role in facilitating Listeria monocytogenes survival within host cells, and the activity of this critical virulence regulator is itself tightly regulated by a number of mechanisms, including transcriptional, post-transcriptional and post-translational modes of control. (A) Transcriptional control of prfA expression is mediated by the presence of three separate promoter elements. P_prfAP1 (P1) and P_prfAP2 (P2) are located immediately upstream of prfA, and both direct monocistronic transcripts of prfA. The P_plcA promoter is located upstream of plcA and directs both a monocistronic plcA transcript and a bicistronic plcA and prfA transcript. P_prfAP1 and P_prfAP2 are responsible for maintaining basal levels of PrfA protein, but both promoters are negatively (-)influenced by high levels of PrfA, whereas P_plcA is positively (+) influenced, resulting in the production of the bicistronic mRNA to generate the high levels of PrfA required for intracellular growth and spread. (B) Post-transcriptional control of prfA expression involves the presence of a thermosensor riboswitch in the 5′ untranslated region of the prfAP1-directed mRNA promoter region that forms a stem-loop structure at temperatures of 30°C or lower. This stem-loop structure effectively masks the prfA mRNA ribosome-binding site to inhibit PrfA protein synthesis. At higher temperatures (37°C), the thermosensor stem-loop is destabilized; however, a trans-acting S-adenosyl methionine-responsive riboswitch (SreA) is then able to bind to a complementary region in the prfA transcript in the prfAP1 promoter region to inhibit translation and reduce PrfA protein synthesis. (C) Post-translational modification of PrfA is required to fully activate PrfA within the host. Binding of a small-molecule cofactor induces structural changes that activate PrfA and that are associated with the high levels of PrfA-dependent virulence gene expression required for survival within the host.

Figure 3. Location of the putative PrfA cofactor-binding pocket and of mutations that influence PrfA activation

(A) Electrostatic modeling of wild-type PrfA protein demonstrating the potential distribution of solvent-accessible surface charges on the protein dimer and indicating binding-pocket mutations. Positive charge is shown in blue and negative charge is shown in red, with electrostatic potentials ranging from −4 kT/e (red) to +4 kT/e (blue). Arrows point to the lysine residues that contribute to the positive charge of the putative cofactor-binding pocket within PrfA. The positive charge of the DNA-binding region is also highlighted at the bottom of the PrfA monomer. (B) Ribbon modeling of PrfA, highlighting the putative cofactor-binding pocket described by Eiting et al. [62], as indicated by the thick black arrow, and identifying amino acid substitutions that influence PrfA activation. The monomers that make up the dimer are colored either light or dark gray, and the DNA-binding helix-turn-helix motifs are shown in blue. PrfA* mutations resulting in high levels of PrfA-dependent virulence gene expression are colored in green, while specific mutations abrogating or reducing PrfA activation are colored in red. (A) Reproduced with permission from [74].

Figure 4. Listeria monocytogenes regulates PrfA activity so as to increase bacterial fitness in multiple environments

Experimental evidence indicates that the expression and activity of PrfA must be carefully regulated in order to optimize Listeria monocytogenes fitness in diverse environments. Outside of host cells, the expression of prfA is low, as is PrfA activity, resulting in low levels of PrfA-dependent virulence gene expression. Under these conditions, the bacterium readily grows on preferred carbon sources such as glucose and cellobiose, with glycolysis being the predominant metabolic pathway. The bacteria exhibit robust flagella-mediated swimming motility, resistance to salt and acid stress and PrfA-enhanced biofilm formation on abiotic surfaces. Following entry of L. monocytogenes into a mammalian host, PrfA becomes highly activated and increases the expression and secretion of multiple gene products that enable bacterial survival within host cells. These gene products include those with direct roles in pathogenesis, as well as those that contribute to bile resistance and the metabolism of alternative carbon sources that are prevalent within the cytosol. BCAA: Branched chain amino acid; PTS: Phosphotransferase system.