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Introduction to Current Research
The overarching research themes in the Gerdes group are Bacterial Stress Responses and Antibiotic Multidrug Tolerance (Persistence). Research within these fields facilitated the establishment of a research centre funded by the Danish National Research Foundation and the Novo Nordisk Foundation called Centre for Bacterial Stress Responses and Persistence (BASP). This long-term funding enables us to attack difficult but important basic research questions.
Most bacteria live in constantly changing environments and, accordingly, have evolved highly sophisticated regulatory mechanisms that allow them to withstand stressful conditions. In particular, almost all bacteria depend on the ubiquitous regulatory molecules tetra and penta-guanosine phosphate, collectively called (p)ppGpp or Magic Spot, for the survival in the environment and during infections. Thus, the (p)ppGpp-mediated response is required for almost all pathogenic bacteria to be virulent, and thus our basic research will lead to an increased general understanding of bacterial survival and virulence mechanisms.
Magic Spot was discovered in experiments with the model organism E. coli undergoing amino acid starvation elicits the “stringent response”. In that response, (p)ppGpp reprograms cellular metabolism from rapid to slow growth or dormancy. Here, (p)ppGpp increases dramatically in concentration and profoundly influences gene expression such that the cells manage to adapt to and survive the limited nutrient supply. Importantly, rRNA synthesis is severely curtailed while transcription of amino acid biosynthetic operons is stimulated. Thus, a primary role of (p)ppGpp is to adjust cell growth to the available nutrient resources. However, (p)ppGpp affects many other cellular processes, such as replication, transcription and protein turnover, either directly or indirectly. Stunningly, even though (p)ppGpp has been known for almost 50 years, it is not yet understood how its synthesis is controlled. Magic Spots is synthesized and hydrolysed by the bifunctional Rel enzymes (RelA and SpoT) that are regulated by a number of factors including ribosome-bound tRNA and essential GTPases. However, at the molecular level surprisingly little is known about how Rel enzymes are regulated. As described further below, a long-term goal of our research is to understanding how the enzymatic activities of RelA and SpoT are regulated and how (p)ppGpp contributes to bacterial virulence and persistence (multidrug tolerance).
Current Research Projects
Magic Spot and Bacterial Persistence
(Etienne Maisonneuve & Elsa Germain).
Persisters are bacterial cells that are multidrug tolerant and in a slow-growing or dormant state. In contrast antibiotic resistance, persistence is a transient, non-inherited state. For example, it is well known that penicillin, which inhibits cell wall synthesis, efficiently kills growing bacteria whereas slow or non-growing bacteria are much less sensitive to the drug, a difference anticipated to be due to a much lower rate of cell wall turn-over of the latter cells. It has been known for many years that almost all bacteria form slow-growing variants that exhibit multidrug tolerance and that such “persistent” bacteria pose a health threat because they cannot be eradicated by antibiotics. TA modules encode inhibitors that reversibly halt cell growth. The model organism E. coli has at least eleven TA loci, all of which encode inhibitors of translation.
The “toxins” encoded by TA modules are activated when the antitoxins are degraded. Interestingly all eleven antitoxins of E. coli are degraded by Lon protease and, consistently, Lon is required for persistence of E. coli. We were able to show that, in E. coli, (p)ppGpp switch to a high level in single cells and thereby induce TA-encoded toxins and persistence (Maisonneuve et al., Cell, 2013) (Figure 1B). We also uncovered a linear, hierarchical signalling pathway connecting (p)ppGpp to TA genes: a high level of (p)ppGpp inhibits the enzyme exopolyphosphatase (PPX) that degrades inorganic polyphosphate (PolyP). Thus a high level of (p)ppGpp leads to a high level of PolyP. In turn, PolyP combines with and activates Lon to degrade TA-encoded antitoxins and thereby activate the toxins. Even though it’s largely accepted that slow growth per se is a common mechanism that leads to drug tolerance and persistence, we have shown recently that this is not the case. We found that cells characterized by a high level of (p)ppGpp and therefore grew slowly surprisingly were not persisters if they lacked the known type II Toxin – Antitoxin (TA) loci.
Toxin – Antitoxins
(Mohammad Roghanian and Kathryn Turnbull)
Usually, TA modules code for two components, a toxin that reversibly inhibits cell growth and an antitoxin that counteracts toxin activity; three types of TA loci have been identified. Type I and type III TA loci encode small RNAs that counter the toxins at the translational and posttranslational levels, respectively. Toxins encoded by type II TA loci are inhibited by protein antitoxins via direct protein – protein interaction. Owing to sequence conservation of the toxins, type II TA loci have been divided into families that are broadly conserved in bacteria, or in some cases even in both bacteria and archaea. Thus, members of the relBE, vapBC, and hicAB families are abundant in these two domains of
Both of these gene modules function in persistence but we do not exclude that the genes have other functions as well.
Toxin – Antitoxins of Mycobacterium tuberculosis
(Kristoffer Skovbo Winther)
Some organisms have mystifying high numbers of TA genes. For example, the highly pathogenic and extremely persistent deadly bacterium M. tuberculosis has approximately 80 TA loci. Remarkably, at least 45 of these modules are vapBC genes that encode PIN domain RNA endonucleases. We are now identifying the targets of these difficult-to-analyse toxins, using an array of different approaches. Using a method developed in David Tollervey’s lab, we have now identified the singular molecular targets of a substantial number of these exciting RNases.
(p)ppGpp and Toxin – Antitoxins of Photorhabdus luminescence
(Ragnhild Jørgensen Bager in collaboration with David Clarke, Cork, Ireland)
P. luminescens is a Gram-negative belonging to the family Enterobacteriaceae. Being both a pathogen of a wide range of insects, as well as mutualistic associated with nematodes from the family Heterorhabditis, the bacterium is an excellent model system in which to study the genetics of both mutualism and pathogenicity. Similar to M. tuberculosis, the P. luminescence has a cohort of TA genes. We are now validating some of the TA modules experimentally. We are also investigating the effects of deleting different regulatory genes (relA, spoT, lon, ppk, ppx and TAs) on various phenotypes of this insect pathogen, primarily the effects on its persistence, symbiosis and virulence.
(p)ppGpp and Toxin – Antitoxins of Burkholderia cenocepacia
(Mustafa Fazli in collaboration with Tim Tolker-Nielsen, Costerton Biofilm Center, UCHP)
B. cenocepacia is a member of a group of closely related Gram-negative bacteria referred to as the Burkholderia cepacia complex. It is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. B. cenocepacia has more than 20 know type II TA genes, most of which have not been validated experimentally. In addition to the TA genes, we are also investigating the effects of deleting different regulatory genes on various phenotypes of this pathogen, primarily the effects on its persistence and virulence.
The highly conserved RtcB RNA ligase
RtcB is a phylogenetically conserved RNA ligase with homologues in all three domains of life. In E. coli, RtcB is expressed from the rtcBA operon that also encodes a 3’-terminal RNA cyclase, RtcA. The rtcBA operon is transcribed by σ54-associated RNA polymerase and requires the product of the neighbouring gene rtcR, which encodes a transcriptional activator that binds to an upstream activating sequence (UAS). However, the signal that activates RtcR has not been identified. Moreover, the function of bacterial RtcB ligase is unknown, but a range of RNA ligation activities have been demonstrated. Recombinant RtcB from E. coli complemented the normally lethal deletion of Trl1 RNA ligase in Yeast and was found to seal both tRNA and mRNA halves after intron removal. We are trying to identify the natural substrate(s) of bacterial RtcB and the signal that activates RtcR.
(p)ppGpp controls cell wall synthesis
(Patricia Dominguez Cuevas)
Observations in the literature indicate that the stringent response controls cell wall synthesis. We are investigating the underlying molecular mechanism.