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Title: Histone Methylation by NUE, a Novel Nuclear Effector of the Intracellular Pathogen Chlamydia trachomatis}
Article:
Abstract:
Sequence analysis of the genome of the strict intracellular pathogen Chlamydia trachomatis revealed the presence of a SET domain containing protein, proteins that primarily function as histone methyltransferases. In these studies, we demonstrated secretion of this protein via a type III secretion mechanism. During infection, the protein is translocated to the host cell nucleus and associates with chromatin. We therefore named the protein nuclear effector( NUE). Expression of NUE in mammalian cells by transfection reconstituted nuclear targeting and chromatin association. In vitro methylation assays confirmed NUE is a histone methyltransferase that targets histones H2B, H3 and H4 and itself( automethylation). Mutants deficient in automethylation demonstrated diminished activity towards histones suggesting automethylation functions to enhance enzymatic activity. Thus, NUE is secreted by Chlamydia, translocates to the host cell nucleus and has enzymatic activity towards eukaryotic substrates. This work is the first description of a bacterial effector that directly targets mammalian histones.
Introduction:
Chlamydiae spp. are responsible for a variety of significant diseases in both animals and humans. Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen, infecting an estimated 92 million people a year, and leads to severe pathologies including infertility, ectopic pregnancy and pelvic inflammatory disease. Additionally, C. trachomatis infection of the ocular epithelium is the leading cause of blindness by an infectious agent and C. pneumoniae is a prevalent respiratory pathogen that has been implicated in coronary artery diseases[1]. Chlamydiae are obligate intracellular pathogens that target epithelial cells and have a specific biphasic developmental cycle. The infectious form of the bacteria, called elementary bodies( EBs), are characterized by a rigid cell wall, densely packed DNA and metabolic inactivity. Upon entry of a host cell, EBs rapidly convert to reticulate bodies( RBs), the metabolically active but noninfectious form of the bacteria. RBs replicate within a membrane-bound vacuole in the host cell called an inclusion. The bacteria remain within inclusions until they eventually convert back to infectious EBs and exit the host cell as a result of cell lysis or via fusion of the inclusion with the cell membrane[2]. Like other pathogenic gram negative bacteria, C. trachomatis encodes a type three secretion( TTS) system that enables the translocation of proteins across a eukaryotic host membrane. In the case of chlamydiae, TTS occurs both across the plasma membrane during entry and across the inclusion membrane during the intracellular developmental cycle. There are no common signal sequences found in proteins secreted by TTS although it is generally accepted that the signal is located at the N-terminus[3]. It is therefore infeasible to identify effector proteins by sequence alone. As chlamydiae are intractable for genetic manipulation, it is also not possible to identify secreted proteins using bacterial mutants. Despite these experimental obstacles, several groups have identified chlamydial proteins secreted by TTS[4],[5],[6],[7] Although the specific function of most of these proteins remains unknown, they presumably target various cellular processes and allow the bacteria to subvert host defense mechanisms. To date, no such effectors have been found to target the host cell nucleus. The SET domain is a 130-residue domain originally defined in proteins capable of changing the expression of heterochromatin-embedded gene sequences[8]. Subsequent studies identified these proteins as histone methyltransferases( HMTs) whose enzymatic function is covalent attachment of methyl groups to lysine residues of histones. A SET domain containing protein has been identified in every eukaryote studied[9], yet these proteins are notably underrepresented in prokaryotes presumably because they lack the target substrate, histones. The majority of non-eukaryotic SET domain proteins are found in species that interact with eukaryotes, such as pathogenic bacteria or viruses. Although these enzymes are generally presented as specific for one particular histone modification, several reports have found multiple histone and/or non-histone cellular substrates for a single SET domain protein[10],[11],[12]. In some cases, this includes methylation of more than one histone or methylation of multiple residues within a particular histone[13],[14],[15]. Regardless of the substrate, all SET domain proteins require the presence of S-adenosyl-L-methionine( SAM) as the methyl donor. Eukaryotic DNA is tightly packaged in the nucleus as nucleosomes, a repeating unit of 146 bp of DNA encircling an octamer of core histones( two copies each of H2A, H2B, H3 and H4). Higher order chromatin structure is regulated in part by N-terminal histone tails of the core histones that protrude from the nucleosome and are available for inter-nucleosomal interactions. These histone tails are the target of various post-translational modifications including acetylation, phosphorylation, ubiquitination and methylation. Each of these modifications contributes to reduced or enhanced accessibility of the transcriptional machinery at a particular locus and ultimately dictates whether a gene is expressed or repressed. The biological consequence of histone methylation varies greatly depending on the targeted histone, the specific lysine modified and the number of methyl groups added to this lysine( mono-, di- or tri-methylation). The effects of these modifications range from gene suppression to gene activation, either directly due to a change in chromatin structure or indirectly through the recruitment of additional modifying proteins[9],[16]. Among histone modifications, methylation is thought to be a relatively stable modification resulting in longterm changes in cellular function. Once thought to be a permanent modification, the growing number of histone demethylases being identified suggests that histone methylation is a dynamic process[17],[18]. Unlike eukaryotes, prokaryotes do not have histones nor highly ordered chromatin. However, several reports have identified histone-like proteins in bacteria[19]. The specific function of these proteins remains largely undefined. Chlamydia trachomatis encodes two histone-like proteins called histone H1-like chlamydial protein 1 and 2, or Hc1 and Hc2. Both are thought to play a critical role in compacting bacterial DNA during the transition from RBs to EBs[20],[21],[22]. The metabolic inactivity of EBs due to extreme condensation of DNA is thought to be regulated by tight interactions with Hc1 and Hc2. In support of this hypothesis, Hc1 expression is decreased during the EB to RB transition which corresponds temporally to the onset of transcriptional activity in the bacteria[23]. Pathogenic bacteria employ a wide range of strategies to avoid elimination by their host. Targeting histone modifications could allow a pathogen to inhibit transcriptional activation of host defense genes. In this study, we set out to characterize the chlamydial SET domain protein CT737. We found the protein to be secreted from bacteria and localized to the mammalian nucleus. Additionally, we report its enzymatic activity as a histone methyltransferase. Altogether, we propose to designate CT737 and its homologs in other chlamydiae genomes as NUE, as it is the first nuclear effector identified in chlamydiae.
Discussion:
Bacterial effector proteins play crucial roles in pathogenicity. In this work, we have identified a new chlamydial effector protein, NUE, which contains a SET domain predictive of histone methyltransferase activity. We identified NUE as a novel TTS substrate of chlamydiae based on( i) the presence of NUE in the host cell nucleus during C. trachomatis infection, indicative that the protein is secreted outside the inclusion and( ii) the presence of a TTS signal in the N-terminal part of NUE from three different chlamydiae species. In vitro enzymatic assays confirmed the activity of NUE as a histone methyltransferase that modified mammalian histones H2B, H3 and H4. This is the first identified bacterial SET domain protein to directly enter the nucleus of the host cell and target mammalian histones. Our observation that NUE methylates multiple histones is unusual but not unique. There are a growing number of mammalian examples in which a single histone methyltransferase has multiple histone and/or non-histone substrates. G9a is a well-characterized human SET domain protein that methylates both H3K9 and H3K27[13]. A separate study identified G9a methylation of several non-histone proteins including chromodomain Y-like protein, widely interspaced zinc finger motifs protein, Cockayne syndrome group B protein and G9a itself( automethylation)[14]. Although not a SET domain protein, protein arginine N-methyltransferase 8( PRMT8) is a histone methyltransferase that methylates both histones H2A and H4 and automethylates[29]. Interestingly, other pathogen effector proteins have been reported to have multiple mammalian targets. For example, Listeria monocytogenes induced dephosphorylation of histone H3 as well as deacetylation of both histones H3 and H4[30]. In a separate study, the Shigella flexneri effector OspF was identified as a phosphatase that dephosphorylates two mitogen-activated protein kinases, ERK1 and p38, which ultimately leads to inhibition of histone H3 phosphorylation[31]. It is therefore entirely feasible that bacteria use their limited genome to target multiple substrates with a single effector. Studies are ongoing to determine the in vivo lysines targeted by NUE in order to analyze its specific effect on host cell chromatin. Chlamydiae histone-like proteins Hc1 and Hc2 play a role in compacting DNA during the RB to EB transition[21],[22]. It was previously reported that Hc1 acts as a substrate of the C. pneumoniae NUE homolog in addition to histone H3( other histones were not tested)[32]. To the contrary, we did not find methylation of Hc1 in our in vitro studies. Slight variations in protocol may contribute to the observed differences. Murata et al. used C. pneumoniae NUE with truncated Hc1 fragments as substrates. We used C. trachomatis with full-length Hc1. Additionally, we found NUE to be secreted by TTS, and substrates of TTS are often retained unfolded before secretion via association with a chaperone protein. If this is true for NUE, enzymatic activity within the bacteria would be unlikely. In addition to its activity as a histone methyltransferase, NUE is an automethyltransferase. To date, there are two SET domain proteins reported to automethylate. G9a was shown to methylate its K239 residue[33]. Mutation analysis revealed the importance of this methylation event for G9a interaction with heterochromatin protein 1( HP1) which therefore increased the function of the protein to act as a transcriptional repressor. In contrast, automethylation was shown to have little effect on the methyltransferase activity of Metnase but inhibited its decatenation activity[34]. Additionally, two non-SET domain HMTs, PRMT6 and PRMT8, automethylate. Automethylation of PRMT8 was shown to inhibit its function as a methyltransferase and the automethylation function of the fourth example, PRMT6, remains unexplored[29],[35]. Here we observed that mutation of the putative automethylation sites of NUE resulted in a decreased activity towards histone substrates. This suggests automethylation of NUE may enhance its methyltransferase activity perhaps by increasing its affinity for the target substrates. One important question left opened by our study is the identity of the genes regulated by NUE translocation in the nucleus during Chlamydia infection. In the absence of a genetic system to manipulate Chlamydia it is difficult to answer. We have observed that NUE is expressed in low abundance during infection, a condition which is not reproduced by transfection approaches. Transcriptional studies have shown that, not surprisingly, numerous host genes are upregulated or downregulated during Chlamydia infection[36],[37]. One study that addresses global changes on host gene expression on a large time scale indicates that about 150 genes are up-regulated 36 hrs after infection of HeLa cells by C. trachomatis L2, against only about 75 genes 24 hrs post infection[38]. These data are in favor of the existence of mechanisms of control of gene expression by the bacteria that might be turned on only late in the infectious cycle, when NUE is expressed. While the cell is responding to dramatic changes triggered by infection, only some of its transcriptional response may result directly from NUE activity. Identification of NUE specific targets might come from the identification of its nuclear binding partner( s), which we are currently undertaking. We are also developing anti-NUE antibodies that would be suitable for chromatin immunoprecipitation approaches with infected cells. SET domain proteins are present in all eukaryotes, and the few examples of such domains in bacterial proteins were initially interpreted to be the result of horizontal gene transfer. A more recent study favors the hypothesis that prokaryotic SET domain proteins evolved independently from a bacterial ancestor[39]. While our findings do not solve this intriguing question, we do show that the chlamydial SET domain protein evolved into a secreted protein targeting eukaryotic histones. Additionally, the ability of chlamydiae to sustain chronic infections presents an opportunity and motive for the bacteria to exert long-term epigenetic control of host cells. Any link between cleared chlamydial infection and dysregulation of genetic information due to epigenetic reprogramming, such as in cancer, will be challenging to establish as infections are often not diagnosed. Regardless of the long term implications, taking control of the host cell at the level of gene expression is certainly advantageous for a bacterium. Manipulating the histone code is one way to achieve this, and we can speculate that other pathogens that possess a SET domain protein may employ this strategy to sustain their own development.
Chlamydia trachomatis is a particularly prevalent human pathogen responsible for loss of eyesight through trachoma and is the most common sexually transmitted disease of bacterial origin . Unlike most other bacterial pathogens , chlamydiae survive only within another cell and thus must develop sophisticated mechanisms to subvert immune clearance by their host . To this end , the bacteria secrete proteins into the cells they occupy which disrupt normal cellular function . In this work , we identify one such protein , NUE , which is injected into the nucleus of human cells during infection . Sequence analysis of NUE revealed the presence of a SET domain which suggests its involvement in chromatin remodeling . Indeed , we found the protein associated with chromatin both during infection and when transfected into human cells . Importantly , we demonstrate NUE is an active histone methyltransferase that targets host cell histones but does not modify bacterial histone-like proteins . This is the first bacterial protein identified which is able to penetrate the nucleus and directly modify mammalian histones .