Our lab is focused on understanding the interactions between the human bacterial pathogen Listeria monocytogenes and mammalian cells, specifically, macrophage cells. We explore different strategies of L. monocytogenes that allow it to infect and replicate efficiently within mammalian cells, evade recognition by the innate immune system and manipulate host cellular pathways. We combine computational, biochemical and genetic approaches to decipher these mechanisms.
The current research projects conducted in the lab are:
Studying pathogen-phage-host interactions: Active Lysogeny
Most bacterial pathogens are lysogens, namely carry DNA of infective and cryptic phage elements within their genome (in many cases more than one), yet the impact of this phenomenon on their behaviour during mammalian infection is not well understood. Several years ago, we uncovered a novel and highly dynamic example of a pathogen-prophage interaction, in which a prophage promotes the virulence of its host, the intracellular bacterial pathogen Listeria monocytogenes (Lm), via an adaptive behaviour. We identified an infective prophage, ϕ10403S, that stably inhabits the Lm 10403S chromosome, serving as an intervening DNA element that regulates bacterial gene expression (the com genes), some of which are important for virulence (Cell, Rabinovich et al., 2012).
It has long been known that certain Listeria strains, especially those associated with foodborne illness outbreaks, carry a ~40-kb-long infective prophage of the Siphoviridae family of double-stranded DNA viruses, integrated within the comK gene. These Listeria-specific phages are known to reproduce through both lysogenic and lytic cycles. In the lysogenic cycle, the phage’s genome is integrated at a specific attachment site located within the comK gene, resulting in its inactivation. Production of infective virions is induced upon DNA damage (SOS conditions), and is accompanied by bacterial lysis, driven by the phage-encoded holin and endolysin. Because of the prophage insertion, the listerial comK gene was considered to be non-functional. In Bacillus subtilis, comK encodes the master transcriptional activator of the competence system (the com genes), a system that is known to facilitate DNA uptake. During transcriptome studies of Lm bacteria grown intracellularly in macrophage cells, we noticed that the com genes are highly transcribed. Further investigation showed that two components of the Com system, ComEC and ComG, are required for Lm efficient phagosomal escape, while the others are dispensable. Notably, the expression of com genes during Lm intracellular growth in macrophage cells was found to require the formation of a functional comK gene via precise excision of the prophage. Prophage excision was highly induced when bacteria were located within the macrophage phagosomes, yet, unlike in classic phage induction, this did not lead to the production of progeny virions and bacterial lysis. These observations indicated an intriguing adaptive behaviour of the prophage to the intracellular lifestyle of its host, serving as a molecular switch that controls bacterial gene expression to promote virulence. We termed this type of phage behaviour active lysogeny, representing cases where prophages control bacterial gene expression via genomic excision, without triggering the lytic cycle (Nature Reviews Microbiology, Feiner et al., 2015, Current Opinion in Microbiology, Argov et al., 2017, Cell reports, Pasechnek et al., 2020).
In the frame of this project we study:
1) The regulation of active lysogeny in L. monocytogenes.
2) The crosstalk between L. monocytogenes and its prophages during mammalian infection.
3) The function of the Com system in L. monocytogenes phagosomal escape.
4) The interaction of ϕ10403S-phage with other phage elements that inhabit the Lm chromosome.
Cross-regulation of metabolism and virulence in L. monocytogenes
Intracellular bacterial pathogens are metabolically adapted to grow within mammalian cells. While these adaptations are fundamental to the ability to cause a disease, we know little about the relationship between the pathogen’s metabolism and virulence. Several years ago, we took a combined approach using the integrative Metabolic Analysis Tool (iMAT), which combines transcriptome data with genome scale metabolic models, to define the metabolic requirements of L. monocytogenes during growth in mammalian cells. Twelve metabolic pathways were identified as highly activated during L. monocytogenes intracellular growth, among them de novo synthesis of histidine, arginine, purine and branch chain amino acids (BCAAs). The importance of each metabolic pathway during Lm infection was confirmed. Next, we investigated the association of these metabolic requirements with the regulation of L.monocytogenes virulence gene expression. We found that limiting BCAA concentrations, primarily of isoleucine, results in robust induction of the master virulence activator gene, prfA, and its down-stream regulated genes. This response was specific and required the nutrient responsive regulator CodY, which is known to bind isoleucine. Further analysis demonstrated that CodY is directly involved in prfA regulation, playing a role in its activation under limiting BCAAs conditions (such as in mammalian cells). This study revealed a novel regulatory mechanism, placing CodY at the crossroads between metabolism and virulence (PLoS Genetics Lobel et al., 2012, Molecular Microbiology Lobel et al., 2015, PLoS Genetics, Lobel and Herskovits, 2016).
In the frame of this project we study:
- The role of CodY in L. monocytogenes virulence.
- The regulation of BCAAs biosynthesis in L. monocytogenes.
- Additional metabolic signals and pathways that affect L. monocytogenes virulence.