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Title: Listeria monocytogenes Dampens the DNA Damage Response}
Article:
Abstract:
The DNA damage response( DDR) is an essential signaling pathway that detects DNA lesions, which constantly occur upon either endogenous or exogenous assaults, and maintains genetic integrity. An infection by an invading pathogen is one such assault, but how bacteria impact the cellular DDR is poorly documented. Here, we report that infection with Listeria monocytogenes induces host DNA breaks. Strikingly, the signature response to these breaks is only moderately activated. We uncover the role of the listerial toxin listeriolysin O( LLO) in blocking the signaling response to DNA breaks through degradation of the sensor Mre11. Knocking out or inactivating proteins involved in the DDR promotes bacterial replication showing the importance of this mechanism for the control of infection. Together, our data highlight that bacterial dampening of the DDR is critical for a successful listerial infection.
Introduction:
In human cells, the genomic integrity is continuously challenged by environmental and endogenous factors, which induce DNA damage. Cells have evolved an impressive array of repair and signaling pathways to restore the structure of DNA which are collectively termed the DNA damage response( DDR). Briefly, eukaryotic double strand DNA breaks are detected by PARP1 and PARP2 proteins that assemble poly( ADP-ribose) chains on histone H1 and H2B to mediate recruitment of the MRE11-RAD50-NBS1( MRN) complex, which is assembled at the damaged site and is crucial for engaging the downstream response[1],[2]. The MRN complex recruits the Ataxia-telangiectasia mutated( ATM) protein that contributes to the phosphorylation of H2AX on serine 139( γH2AX) and of the Mediator of DNA-Damage Checkpoint 1( MDC1). MDC1 bound to γH2AX acts as an interaction platform for other DDR components including the ubiquitin E3 ligase Ring finger protein 8( RNF8), which ubiquitinates H2A-type histones and associates with the ubiquitin E2 enzyme UBC13[3]. H2A ubiquitination is essential for recruitment of another E3 ubiquitin ligase RNF168, itself important for p53 binding protein( 53BP1) and breast cancer 1 early onset( BRCA1) foci formation[4],[5]. When the DNA lesion is not repaired, the DDR slows down the cell cycle, leading to either cell cycle arrest, senescence or death[1],[6]. Thus, maintenance of genome integrity is critical for cell homeostasis and defects in the DDR lead to severe genetic diseases or premature ageing[7]. Due to its important role in genomic stability, the DDR is a common target of many viruses[8]. Indeed, the DDR benefits the replication of some viruses, in particular a number of DNA viruses, but for other, such as Herpes virus, the DDR is an obstacle to overcome during infection. Some bacterial pathogens have also been shown to induce genomic instability upon infection, but the impact of the DNA-damage response on infection efficiency is largely unknown[9],[10],[11],[12],[13],[14]. It has, however, been hypothesized that the cell cycle arrest provoked by bacteria-induced DNA damage could prolong bacterial colonization in vivo by slowing down the epithelium renewal or exfoliation[14],[15]. More studies are thus needed to understand the action of the DDR components on either bacterial clearance or persistence. L. monocytogenes is the etiological agent of listeriosis, a food borne disease acquired by ingestion of contaminated food. Listeriosis manifests itself as febrile gastroenteritis, or in severe cases as meningoencephalitis, abortion and septicaemia leading to death in 30% of cases[16]. At the cellular level, L. monocytogenes is a facultative intracellular bacterium that is well equipped to survive in the cytoplasm of the infected cell. Indeed, L. monocytogenes promotes its internalization into host cells via two bacterial proteins InlA and InlB that bind the host receptors E-cadherin and c-Met respectively. Activation of these receptors leads to a series of events involving the recruitment of endocytic effectors, and actin cytoskeletal rearrangements, that induce entry of Listeria into the host cell[17]. Once inside the cell, L. monocytogenes escapes from the endocytic vacuole using the pore-forming toxin listeriolysin O( LLO), and multiplies in the cytoplasm. There, Listeria overexpresses ActA, which allows escape from autophagy and also polymerizes host actin, in order to move and spread to the neighboring cells[18]. Importantly, in addition to mediating vacuole escape, LLO also targets several host functions such as, host gene transcription, mitochondrial dynamics, host protein synthesis or SUMOylation[19],[20]. Thus, L. monocytogenes has evolved many mechanisms to manipulate the host to its advantage. In this study we investigate the effects of L. monocytogenes infection on the host DNA integrity. We show that infection with L. monocytogenes induces DNA breaks and activates the DDR in host cells in vitro and in vivo. Strikingly, L. monocytogenes is able to dampen activation of the DNA-damage signaling pathway through the bacterial factor LLO. We show that LLO induces the degradation of Mre11, one of the major sensors of DNA damage through a mechanism independent of proteasome degradation. In addition, our results reveal that dampening of the DDR is important for infection as inhibition of proteins implicated in this pathway increases bacterial infection. Thus, our study uncovers a subversion of the DDR during infection by L. monocytogenes and a role for the DDR in blocking bacterial proliferation.
Discussion:
In this study, we report that the bacterium L. monocytogenes provokes DNA breaks in its host while at the same time controlling the ensuing host DDR. Indeed, we found that although wild type bacteria modestly induce the DDR, a Δhly mutant lacking LLO did so to a significantly higher level. We further reveal that LLO dampens the DDR, through degradation of the host protein Mre11( figure 7). Interestingly and consistent with these observations, we find that deletion or inhibition of proteins implicated in the DDR lead to higher levels of infection demonstrating for the first time that the DDR can be a negative regulator of a bacterial infection. Several bacteria have been reported to induce host DNA damage, a phenotype which had not yet been studied during a Listeria infection. Extracellular pathogenic bacteria such as Pseudomonas aeruginosa, Neisseria gonorrhoeae, E. coli, or Helicobacter pylori and intracellular bacteria such as S. flexneri and Chlamydia trichomatis are able to induce host DNA double-strand breaks( DBS)[9],[10],[12]–[14]. Very recently a report has shown that Listeria monocytogenes delays the host cell cycle by increasing the levels of host DNA strand breaks[32]. However, in most cases, the bacterial factor( s) responsible for inducing these DNA breaks has/have not been identified. To our knowledge, only two bacterial virulence factors have been shown to directly induce DNA breaks: Cytolethal distending toxin( CTD) produced by several Gram negative pathogens such as E. coli, and Typhoid toxin( TT) that is expressed by the intracellular S. typhi[15],[33],[11]. Both types of toxin are related and share a common subunit, CdtB, responsible for DNA breaks. However, no homologues of these virulence factors are present in L. monocytogenes. It has recently been reported that C. trachomatis could induce DSB through generation of reactive oxygen species( ROS)[10]. We explored this possibility during infection with L. monocytogenes using the DCFDA reporter, but failed to observe ROS production under our conditions during infection. Thus, ROS production does not appear to contribute to DNA breaks induced by Listeria. A very recent report using multiple bacterial, fungal and plant pathogen species which induce DSBs in plant DNA illustrates the complexity of identifying the cause of pathogen-induced DNA damage[34] and that other factors besides ROS are involved. In our case, we still observe the same increase in γH2AX levels during infection with L. monocytogenes deleted for inlB and impaired in cell invasion, and therefore this suggests that a secreted virulence factor or a surface component is necessary to induce the observed DNA breaks. Along with the genotoxic stress induced by S. flexneri and C. trachomatis, both bacteria were also shown to impair the DDR( figure 4). Shigella flexneri infection leads to DNA breaks, γH2AX accumulation along with a degradation of the p53 protein, which blocks the p53-dependent DNA repair response[9]. Similarly, C. trachomatis was very recently shown to induce extensive double strand breaks during infection with no associated cell cycle arrest[10]. Indeed, C. trachomatis blocked the formation of 53BP1 foci and phosphorylation of Mre11 through an unknown mechanism. Remarkably, in our report we find that L. monocytogenes, via LLO, also prevents excessive DNA damage signaling, similarly to S. flexneri and C. trachomatis, but through a different mechanism. Interestingly, we show in this report that the DDR is a negative regulator of a listerial infection. Although the inhibitory effect of the DDR on infection had been suggested for S. flexneri and C. trachomatis, this had not been shown experimentally. Our results therefore indicate that dampening of the DDR might be a common and beneficial feature of multiple intracellular pathogen infections. It is clear that viruses, being obligate intracellular pathogens have an advantage to block the host DDR in order to inhibit cell-cycle checkpoints and p53-mediated apoptosis and keep the cell alive. However, inhibition of the DDR by a bacterial pathogen such as L. monocytogenes, which can survive very well without the host cell, is less intuitive. Nevertheless, It is well known that the DDR induces activation of NF-κB[35],[36]. Furthermore, a very recent report shows that DNA damage induces an immune response[37]. In addition, it has been suggested that the Mre11 protein could be a sensor of exogenous DNA leading to activation of type I interferon[38]. Therefore, our data along with a growing body of evidence suggests that the DDR could have an important role in inducing inflammation during infection. To our knowledge, this is the first report to show that a bacterium induces Mre11 degradation. Mre11 degradation has only been shown to occur during infection with a virus. Indeed, infection with adenovirus has been shown to result in both reorganization and degradation of members of the Mre11–Rad50–NBS1 complex[39]. Furthermore, and similarly to our findings, inhibition of the DDR is essential for adenovirus infection as in the absence of this block, viral replication is inhibited[39]. Therefore, the DDR is a negative regulator of adenovirus infection. Likewise, in the case of Listeria, we show that downregulation of Mre11 or ATM or H2AX favors infection. This is the first report showing that the DDR has an impact on bacterial infection. Finally we show here that LLO induces a decrease in the levels of Mre11 in a pore dependent manner. Indeed, point mutants of LLO which are reduced in their hemolytic activity lead to a lesser effect on Mre11 and γH2AX compared to native LLO. LLO is a pore forming toxin which was originally shown to be essential for Listeria escape from the internalization vacuole[40]. Since then, LLO has been shown to have many different extracellular activities on the host cell[19]. Remarkably, the pore formation of LLO has been shown to lead to degradation of at least 2 other proteins during infection, the catalytic subunit of the human telomerase complex( hTERT), and the human E2 SUMO enzyme Ubc9[20],[41]. Although the mechanism by which hTERT levels were decreased remains to be determined, the degradation of Ubc9 was shown to be partially dependent on an aspartyl-protease. Similarly, we find here that Mre11 is degraded, in a manner that appears to be dependent on an aspartyl-protease. Therefore, the downstream events of LLO pore formation leading to cellular protein degradation are beginning to emerge, however, the specificity of the targeted protein and the molecular mechanism by which degradation occurs remain elusive. Surprisingly, we find that Mre11 levels remain low 24 h after removal of LLO, even though pores have been shown to be repaired very rapidly[42]. The mechanism by which the long lasting effect on Mre11 is achieved will be of particular interest in future studies, and have a wide ranging impact on understanding the long term effects of bacterially induced DNA damage.
In eukaryotic cells both normal metabolic activities and environmental factors such as UV radiation can cause DNA lesions or mutations . The ability of a cell to restore integrity to its genome is vital , and depends on a signaling cascade called the DNA damage response ( DDR ) that both senses and responds to the assaults . Bacterial infection is one such assault , but its effect on the DDR of the invaded cell remains elusive . Here we used the bacterial pathogen Listeria monocytogenes to study its effect on host DNA damage and its impact on the DDR . Our results show that although Listeria is able to induce DNA damage , the ensuing response is surprisingly low , demonstrating that this bacterium is able to dampen the DDR . We have also shown that the listerial toxin listeriolysin O ( LLO ) is responsible for the observed block in the DDR . In fact , we find that LLO induces protein degradation of the main DNA damage sensor , Mre11 , thereby blocking downstream signaling . Furthermore , we have studied the impact of mutating the DDR on the infectious process and find that it negatively regulates infection with Listeria . In conclusion , our findings reveal that dampening of the DDR is crucial for a productive infection .