פרופ' ענת הרשקוביץ

סגל אקדמי בכיר במקרוביולוגיה מולקולרית
ראש ביה"ס במנהלת הפקולטה למדעי החיים
מקרוביולוגיה מולקולרית סגל אקדמי בכיר
ניווט מהיר:
פרופ' ענת הרשקוביץ
טלפון פנימי: 03-6407502
טלפון נוסף: 03-6407505
פקס: 03-6409407
משרד: גרין ביוטכנו, 107

Research Interests

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:

  1. The role of CodY in L. monocytogenes virulence.
  2. The regulation of BCAAs biosynthesis in L. monocytogenes.
  3. Additional metabolic signals and pathways that affect L. monocytogenes virulence.

 

Recent Publications

 

 

Listeria monocytogenes TcyKLMN Cystine/Cysteine Transporter Facilitates Glutathione Synthesis and Virulence Gene Expression

Moran BrennerSivan FriedmanAdi HaberNurit Livnat-LevanonIlya BorovokNadejda SigalOded LewinsonAnat A Herskovits

mBio 2022 Apr 18;e0044822

A dual-function phage regulator controls the response of cohabiting phage elements via regulation of the bacterial SOS response

Gil AzulayAnna PasechnekOlga StadnyukShai Ran-SapirAna Mejia FleisacherIlya BorovokNadejda SigalAnat A Herskovits

Cell Rep​ 2022 Apr 19;39(3):110723

Generation of Markerless Gene Deletion Mutants in Listeria monocytogenes Using a Mutated PheS for Counterselection

Shai Ran Sapir, Etai Boichis, and Anat A. Herskovits

Bacterial Virulence, 1st ed. 2022, Methods and Protocols, Methods in Molecular Biology Series, Vol. 2427

 

Bone Marrow-Derived Macrophage (BMDM) Infection by Listeria monocytogenes

Etai Boichis, Shai Ran Sapir, and Anat A. Herskovits

Bacterial Virulence, 1st ed. 2022, Methods and Protocols, Methods in Molecular Biology Series, Vol. 2427

A Metzincin and TIMP-Like Protein Pair of a Phage Origin Sensitize Listeria monocytogenes to Phage Lysins and Other Cell Wall Targeting Agents

Etai Boichis, Nadejda Sigal, Ilya Borovok and Anat A. Herskovits

Microorganisms, 2021, 9(6), 1323

Active Lysogeny in Listeria Monocytogenes Is a Bacteria-Phage Adaptive Response in the Mammalian Environment.

Anna Pasechnek, Lev Rabinovich, Olga Stadnyuk, Gil Azulay, Jessica Mioduser, Tal Argov, Ilya Borovok, Nadejda Sigal and Anat A. Herskovits

Cell Reports, 2020

 

Coordination of cohabiting phage elements supports bacteria-phage cooperation.

Tal Argov, Shai Ran Sapir, Anna Pasechnek, Gil Azulay, Olga Stadnyuk, Lev Rabinovich, Nadejda Sigal, Ilya Borovok and Anat A. Herskovits

Nature Communications,2019

Metabolism of the Gram-Positive Bacterial Pathogen Listeria monocytogenes.

John-Demian Sauer, Anat A. Herskovits and Mary X.D. O'riordan.

Microbiology Spectrum,2019

Controlled branched-chain amino acids auxotrophy in Listeria monocytogenes allows isoleucine to serve as a host signal and virulence effector. 
Moran Brenner, Lior Lobel, Ilya Borovok, Nadejda Sigal, Anat A Herskovits (2018)
PLoS Genet 14(3): e1007283.https://doi.org/10.1371/journal.pgen.1007283

 

Temperate bacteriophages as regulators of host behavior.
Tal Argov, Gil Azulay, Anna Pasechnek, Olga Stadnyuk, Shai Ran-Sapir, Ilya Borovok, Nadejda Sigal and Anat A Herskovits.
Current Opinion in Microbiology 2017, 38:81–87

 

Metabolic Genetic Screens Reveal Multidimensional Regulation of Virulence Gene Expression in 

Listeria monocytogenes and an Aminopeptidase That Is Critical for PrfA Protein Activation.

Sivan Friedman, Marika Linsky, Lior Lobel, Lev Rabinovich, Nadejda Sigal and Anat A. Herskovits

Infect Immun. 2017 May 23;85(6). pii: e00027-17. doi: 10.1128/IAI.00027-17. 

 

An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S.

Tal Argov, Lev Rabinovich, Nadejda Sigal, Anat A. Herskovits

Appl Environ Microbiol. 2017 Mar 2;83(6). pii: e02927-16. doi: 10.1128/AEM.02927-16.

 

L-glutamine Induces Expression of Listeria monocytogenes Virulence Genes.

Adi Haber, Sivan Friedman, Lior Lobel , Tamar Burg-Golani , Nadejda Sigal , Jessica Rose , Nurit Livnat-Levanon , Oded Lewinson, Anat A. Herskovits

PLoS Pathog. 2017 Jan 23;13(1):e1006161. doi: 10.1371/journal.ppat.1006161.

 

RNA Purification from Intracellularly Grown Listeria monocytogenesin Macrophage Cells.

Nadejda Sigal, Anna Pasechnek and Anat A. Herskovits

J. Vis. Exp. (112), e54044, doi:10.3791/54044 (2016).

 

Systems Level Analyses Reveal Multiple Regulatory Activities of CodY Controlling Metabolism, Motility and Virulence in Listeria monocytogenes.

Lior Lobel and Anat A. Herskovits 

PLoS Genet. 2016 Feb 19;12(2):e1005870.

 

A new perspective on lysogeny: prophages as active regulatory switches of bacteria
Ron Feiner, Tal Argov, Lev Rabinovich, Nadejda Sigal, Ilya Borovok and Anat A. Herskovits

Nature Reviews Microbiology 13,641–650, (2015)

 

The Human P-Glycoprotein Transporter Enhances the Type I Interferon Response to Listeria monocytogenes Infection.

Nadejda Sigal, Millie Kaplan Zeevi, Shiri Weinstein, Dan Peer and Anat A. Herskovits

Infect Immun. 2015 Mar 30, 83:2358–2368.  

 

The metabolic regulator CodY links Listeria monocytogenes metabolism to virulence by directly activating the virulence regulatory gene prfA.

Lior Lobel, Nadejda Sigal, Ilya Borovok, Boris R. Belitsky, Abraham L. Sonenshein and Anat A. Herskovits

Mol Microbiol. 2015 Feb;95(4):624-44. 2015 Feb;95(4):624-44.

 

Listeria monocytogenes MDR transporters are involved in LTA synthesis and triggering of innate immunity during infection.

Keren Tadmor, Yair Pozniak, Tamar Burg Golani, Lior Lobel, Moran Brenner, Nadejda Sigal, and Anat A. Herskovits

Front Cell Infect Microbiol. 2014; 4: 16.

  

Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Tamar Burg-Golani, Yair Pozniak, Lev Rabinovich, Nadejda Sigal, Ran Nir Paz and Anat A. Herskovits

J. Bacteriol. December 2013 vol. 195 no. 23 5262-5272

 

Listeria monocytogenes MDR transporters and c-di-AMP that contribute to Type I interferons induction, play a role in cell wall stress.

Millie Kaplan Zeevi, Nirit S. Shafir, Shira Shaham, Sivan Friedman, Nadejda Sigal, Ran Nir-Paz, Ivo G. Boneca and Anat A. Herskovits

J. Bacteriol. December 2013 vol. 195 no. 23 5250-5261

 

Prophage Excision Activates Listeria Competence Genes that Promote Phagosomal Escape and Virulence.

Lev Rabinovich, Nadejda Sigal, Ilya Borovok, Ran Nir-Paz, and Anat A. Herskovits
Cell 150, 792–802, August 17, 2012

 

Integrative Genomic Analysis Identifies Isoleucine and CodY as Regulators of Listeria monocytogenes Virulence
Lior Lobel, Nadejda Sigal, Ilya Borovok, Eytan Ruppin, and Anat A. Herskovits
PLoS Genet. 2012 Sep;8(9):e1002887.

 

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