Jörgen Johansson
RNA-mediated virulence gene regulation: Identification of novel antibacterial compounds
Aim:
1. RNA-mediated virulence regulation:
2. Investigate the role of a blue-light receptor for Listeria monocytogenes physiology and virulence
3. Identification of RNA-dependent and independent antibacterial drugs
In order to establish and maintain a flourishing colonization, bacteria have to constantly survey their surroundings and adjust their gene-expression. Small non-coding RNAs (ncRNAs) can act as an antisense RNA by pairing to complementary regions on target mRNAs, thereby modifying translation or inducing target mRNA degradation. Regulatory RNA regions might also be present at the 5´-end of the transcript itself. One such example are riboswitches, which are able to directly bind specific metabolites and where binding of the metabolite render the riboswitch a conformation where a termination structure is formed and the downstream mRNA is not further synthesized. In the absence of the metabolite, an anti-termination structure is formed and the downstream mRNA is synthesized.
Pathogenic bacteria resistant to one or several types of antibacterial agents are an increasing problem in the society. One possibility to avoid resistance development is to use drugs that instead of killing the bacterium are able to disarm its virulence capabilities, thereby attenuating the pathogen. RNA has already been shown to be a useful antibacterial drug target, with many drugs targeting the ribosomal RNA (rRNA) structures. The interaction between the riboswitch and the metabolite is therefore very rigid, comparable to protein:ligand interactions in structural complexity and selectivity. Part of my suggested research is associated with the identification of novel antibacterial drugs targeting riboswitches.
My research group uses the Gram-positive human pathogen Listeria monocytogenes as a bacterial model system. Analysis of a L. monocytogenes infection has provided considerable knowledge into how bacteria can invade cells, escape the phagosome, move intracellularly and disseminate into deeper tissues. The large amount of information that has been gathered through in vivo studies and complete RNomics investigation makes L. monocytogenes one of the best understood bacterial pathogens.
1. RNA-mediated virulence regulation:
1a) Molecularly dissect the function of short non-coding RNAs in Listeria monocytogenes
I. Characterisation of L. monocytogenes riboswitches acting in trans
Recently, my research group recently demonstrated that a S-adenosylmethionine (SAM) binding riboswitch can function at a distance and control expression of the virulence regulator PrfA in Listeria monocytogenes. How can SreA can down-regulate PrfA-expression? We will examine and determine the exact mechanism using different RNase-mutants. We will also test how SAM affect the SreA:prfA interaction by performing gel-shift and in vitro transcription/translation analysis.
II. Characterisation of a L. monocytogenes ncRNA induced during growth in blood
One ncRNA present only in pathogenic Listeria species was identified by an affymetrix tiling-array method. A strain having the ncRNA knocked out showed a 10-fold decreased capability to colonize the liver and spleen. The decreased survival of the ncRNA strain might be due to an impaired ability to survive in the blood. By a combination of tiling arrays and 2D-gel techniques, we have identified RNA targets possibly interacting with Rli38. To verify putative direct target interactions, both in vivo techniques as well as in vitro techniques will be used.
1b) Examine how RNA-helicases control Listeria monocytogenes motility and cold survival
To identify protein components that could promote a proper interaction between ncRNAs and their targets as well as being important for the function of various 5´-UTRs, we have initiated a project to analyze the biological function of all DEAD-box RNA helicases in L. monocytogenes. To understand the in vitro function of the RNA-helicases, they are being purified and their regulatory role will be investigated using gel-shift, footprint and RNA-structuring experiments. Putative RNA targets will be identified by deep sequencing.
2. Investigate the stress-induction in Listeria monocytogenes
We have identified a gene encoding a blue-light receptor to be important for various biological processes. A transposon-mutant library identified several proteins to be acting in the same pathway as the blue-light receptor. Further characterization of the transposon mutants as well as biochemical analysis of their putative interactions will identify their molecular role.
3. Identification of RNA-dependent and independent antibacterial drugs
3a) Characterize novel antibacterial drugs targeting putative riboswitches.
Two of the riboswitches classes in L. monocytogenes are suggested to use purine and FMN as their natural ligands. Roseoflavin (an analog to riboflavin) has been shown to directly bind FMN riboswitches in Listeria, thereby blocking expression of the essential riboflavin transporter and hence inhibiting bacterial growth. We are trying to further evaluate different analogs to purine and riboflavin as putative antibacterial compounds by genetic and biochemical approaches. Riboswitch analogs will also be used in other bacterial pathogens, such as Staphylococcus and Mycobacterium species, to test if they could be good candidates for novel antibacterial drugs.
3b) Analyze the mechanism by which a small pyridone molecule blocks of Listeria virulence
L. monocytogenes invasion of cultured human epithelial cells was completely inhibited by addition of a small molecule without affecting the survival of L. monocytogenes or the cultured cells. The small molecule block infection by repressing expression of virulence genes. We will use genetic and biochemical tools to identify the mechanism by which this small molecule functions.
