The research projects conducted in our lab are designed to address different aspects of the following fundamental questions.
Project 1 - European Research Council (ERC) project Co-Patho-Phage:
Studying Host-Pathogen-Phage 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).
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 prophage during mammalian infection.
3) The function of the Com system in L. monocytogenes phagosomal escape.
4) The interaction of ϕ10403S-prophage with other prophage elements that inhabit the Lm chromosome.
Project 2 - 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.
Project 3 - Studying L. monocytogenes interactions with the host innate immune system.
L. monocytogenes activates a robust type I interferon response upon infection. We have previously shown that this response is dependent on the expression of the multi-drug resistance transporter, MdrM (Herskovits and Crimmins et al., 2008, PNAS). It was also shown by others that the induction of type I interferons relies on the secretion of cyclic-di-AMP by the bacteria during intracellular growth. While it was suggested that MdrM mediates c-di-AMP secretion, the physiological role of this function was not clear. In this research project we previously found that it is not MdrM alone, but a cohort of MDR transporters that together induce the type I interferon response during L. monocytogenes infection. In a search for a physiological function of these transporters, we identified a role under cell wall stress. A mutant deleted of four MDR transporters, MTAC mutant, was found to be sensitive to sub-lethal concentration of vancomycin due to its inability to produce and shed peptidoglycan under this stress. Remarkably, c-di-AMP was involved in this phenotype, as over-expression of the c-di-AMP phosphodiesterase (PdeA) resulted in increased susceptibility of the MTAC mutant to vancomycin, whereas over-expression of the c-di-AMP diadenylate cyclase (DacA) reduced its susceptibility to this drug. These observations established a functional association between c-di-AMP and the MDR transporters, supporting the premise that MDR transporters and c-di-AMP regulate peptidoglycan synthesis in response to cell wall stress (Journal of Bacteriology, Kaplan Zeevi et al., 2013). In addition, we further investigated MdrM’s physiological function using a genetic approach. We performed a genetic screen looking for mutants that modulate MdrM’s induction of Type I interferons. We identified three genes of the lipoteichoic acid (LTA) biosynthesis pathway that when deleted lead to even higher induction of Type I interferons. We further found that deletion of the MTAC transporters results in aberrant LTA production. Distinct LTA variants were detected in the MDR mutants in comparison to wild type bacteria. The LTA variants activated different levels of cytokines response, which correlated with the response elicited by their cognate bacterial strains. Overall this study linked for the first time MDR transporters to LTA biosynthesis, providing further insights into the mechanisms by which L. monocytogenes activates innate immune responses in mammalian cells (Frontiers in Cellular and Infection Microbiology, Tadmor et al., 2014).
In the frame of this project we are currently studying how c-di-AMP is involved in the regulation of cell wall synthesis.
We think Listeria monocytogenes is an amazing bacterium; it serves us well in exploring a wide array of biological questions and topics that are related to bacterial pathogenesis and physiology, immunity, gene-regulation and bacterial-phage interactions. We enjoy every day working on this bacterium.