GATC-3'-sites in the genomes of several Gram-negative bacterial species like Campylobacter coli. (Wright et al., 2010) and Campylobacter jejuni (Gu et al., ...
Epigenetic Modifications in Gram-Negative Bacteria and the Induction of Patho-Epigenetic Modifications in Host Tissue Andreas Erich Zautner Abteilung für Medizinische Mikrobiologie Universitätsmedizin Göttingen, Germany
Hagen Frickmann Fachbereich Tropenmedizin am Bernhard-Nocht-Institut Bundeswehrkrankenhaus Hamburg, Germany
1
Introduction
Epigenetic modification plays a dual role in bacterial infection. On the one hand, the expression of certain genes in the bacterial organism is regulated by epigenetic mechanisms. For example, the expression of components of the type IV secretion system in Gram-negative bacteria like enteroaggregative Escherichia coli (EAggEC), which is responsible for the secretion of proteins involved in host cell invasion and bacterial killing, is regulated by DNA methylation (Brunet et al., 2011). Differences in the digestion-pattern of endonucleases recognizing methylated adenine at 5’GATC-3’-sites in the genomes of several Gram-negative bacterial species like Campylobacter coli (Wright et al., 2010) and Campylobacter jejuni (Gu et al., 2009) suggest that further genes are regulated by epigenetic modifications as well. Bioinformatic investigations demonstrated epigenetic regulation of type III secretion system (TTSS) associated toxicity in the non-fermentative Gram-negative facultative pathogen Pseudomonas aeruginosa (Filopon et al., 2006). A generalized logical analysis indicated epigenetic control of Pseudomonas aeruginosa’s mucoidy (Guespin-Michel & Kaufman, 2001). There are even hints for epigenetic regulation of multistationary of bacterial growth (Guespin-Michel & Kaufman, 2001). On the other hand, bacterial infections induce epigenetic changes in the cells of the infected organism. It is a well described phenomenon that viral proteins affect the activity of cellular promoters via epigenetic mechanisms. In the last years similar effects were demonstrated for bacterial infections. Promotor silencing by hypermethylation at CpG dinucleotides was described for Campylobacter rectus in placenta tissue (Bobetsis et al., 2007) and for Helicobacter pylori in gastric mucosa (Chan et al., 2003). This epigenetic reprogramming might play a critical role in fetal growth and ontogenetic programming but also in carcinogenesis. In contrast, methylation of bacterial 16S rRNA is a resistance mechanism providing high-level resistance to aminoglycosides in actinomycetes, Enterobacteriaceae and Gram-negative, non-fementing, rod-shaped bacteria like Pseudomonas aeruginosa (Yokoyama et al., 2003; Doi et al., 2004; Périchon et al., 2008). However, this phenomenon is no epigenetic mechanism as gene expression is not affected. Infection with Gram-negativ bacteria induces epigenetic modifications not only in the invading bacterial cells, but also in the host tissue. Epigenetic consequences of bacterial infection are important for the understanding of the pathomechanisms and may have principal therapeutic implications.
2
Basic Mechanisms of DNA Methylation in Bacteria
Methylation of chromosomal DNA widely occurs in bacteria. Restriction-modification by both N6methyladenine and 5-methycytosine is well described for a broad variety of bacterial species (Roberts, 1985; Kessler & Holtke, 1986). One central role of such methylation systems is the differentiation between own and extraneous DNA. Unmethylated or inappropriately methylated DNA can be selectively destroyed. Methylated adenine has been detected predominantly in many Gram-negative bacteria (Barbeyron et al., 1984; Hughes and Johnson, 1990; Roberts, 1985; Kessler & Holtke, 1986), but also less frequently in Gram-positive bacterial species (Lacks et al., 1986; MacNeil, 1988; Cerritelli et al., 1989; Dingman, 1990). The mechanisms of DNA methylation have been extensively studied in Escherichia coli. The vast majority of E. coli isolates contains three site-specific DNA methylases. The dam
gene of E. coli encodes a DNA adenine methylase that catalyzes the transfer of methyl groups from Sadenosyl-L-methionine to the N6-position of the adenine residues in GATC-motifs of double-stranded DNA during DNA replication (Marinus & Morris, 1973; Geier & Modrich, 1979; Barbeyron et al., 1984; Bakker & Smith, 1989). The Dcm methylase (DNA cytosine methylase, formerly referred to as the Mec methylase) encoded by the dcm gene, methylates the C5 position of cytosine residues within the sequences 5’-CCAGG-3’ and 5’-CCTGG-3’ (Marinus & Morris, 1973; May & Hattman, 1975). The third but less important enzyme that has to be mentioned here is the EcoKI methylase (HsdM subunit), which modifies adenine residues in the motifs 5’-AAC(N6)GTGC-3’ and 5’-GCAC(N6)GTT-3’. EcoKI methylation sites (~1 site per 8 kb) are much less common than Dam methylation sites (~1 site per 256 bp) or Dcm methylation sites (~1 site per 512 bp) in random DNA sequences with equal GC and AT content (Marinud, 1987; Cooper et al., 1994; Powel et al., 1998a & b; O’Neill et al. 2001, Roberts et al., 2003). 2.1
Methylation of Adenine in 5’-GATC-3’ Sequences
It was demonstrated that transient hemimethylation of adenine in 5’-GATC-3’ sequences is involved in DNA strand discrimination during mutHLS-dependent mismatch repair (Bakker & Smith, 1989; Barras & Marinus, 1989; Lahue et al. 1989, Modrich, 1989). Additionally it was suggested that this mechanism has a pivotal role in the initiation of DNA replication of E. coli, because the 245 bp oriC sequence contains a high abundance of GATC motifs (eleven at all) and fully methylated it is twice as active in comparison to the unmethylated state (Barras & Marinus, 1988; Boye, 1991). Furthermore N6-methyladenine is involved in the transposition of IS10, the component insertion sequence of transposon Tn10 and the compound transposon Tn5 (Roberts et al., 1985; Yin et al., 1988). Adenine methylation by the EcoRI methylase plays a role in the K restriction-modification system of deoxyribonucleic acid self-recognition (Mamelak & Boyer, 1970). Dam methylation has been demonstrated in many bacterial species of several genera for example: Bacillus, Clostridium, Enterobacter, Escherichia, Erwinia, Haemophilus, Proteus, Salmonalla, Serratia, and Streptococcus (Barbeyron et al., 1984; Dingman, 1990; Dreiseikelmann & Wackernagel, 1981; Gomez-Eichelmann, 1979; Hattman et al., 1978, Urieli-Shoval et al., 1983). 2.1.1
5’-GmATC-3’ Specific Restriction Enzyme Isoschizomer Digestion Assay
A very simple approach to detect methylated adenine in GATC sequences is the digestion of genomic/chromosomal bacterial DNA with restriction enzymes specific for this motif (Edmonds et al., 1992). A restriction endonuclease cutting specifically at this site is DpnI. The dpnI gene was originally derived from Streptococcus (Diplococcus) pneumoniae G41 (Lacks, 1980). Very useful crosschecks for this assay should be performed using the restriction endonucleases MboI and Sau3AI (Edmonds et al., 1992). The mboI gene originates from Moraxella bovis ATCC 10900 (Ueno et al., 1993). MboI is responsive to dam methylation. That means, GATC sequences that are adenosyl-methylated by the dam methylation system of E. coli become refractory to cleavage by MboI (Dreiseikelmann et al., 1979). Thus, the degree of dam methylation of various bacterial DNAs can be estimated by MboI digestion (Dreiseikelmann et al., 1979). In contrast to MboI the restriction endonuclease Sau3AI, which originates from Staphylococcus aureus strain 3A (Sussenbach et al., 1976) is insensitive to adenosyl-methylation by the dam system and cleaves at all 5’-GATC-3’ sequences whether they are adenosyl-methylated or not (Edmonds et al., 1992). Studies on the presence of methylated adenines in 5’-GATC-3’ sequences in
Campylobacter and Helicobacter species demonstrated that these are only present in certain strains (Edmonds et al., 1992).
Figure 1: Overview of the specific cutting sites of the methyl-adenine sensitive endonucleases DpnI and MboI, as well as the methylation-insensitive endonuclease Sau3AI
2.2
Methylation of Cytosine in 5’-CC(A/T)GG-3’ Sequences
Although the presence of methylated cytosine in bacterial DNA was reported over 35 years ago, the biological role of 5-methylcytosine still remains unclear (Palmer & Marinus, 1994, Militello et al., 2012). E. coli dcm-knockout mutants expressed no obvious associated phenotypic abnormality. (Marinus, 1987; Marinus & Morris, 1973). Deamination of 5-methylcytosine leads to thymine-guanine mismatches that can cause mutations by C-to-T transitions (Coulondre et al., 1978). Thus the second cytosines in two CCAGG motifs in the lacI gene were identified as hot spots for mutations, which were not observed in dcm mutants (Coulondre et al., 1978). These mutagenic effects of accidental deamination of 5methylcytosine are corrected by the very short patch repair sytem (vsr) that was discovered in E. coli K12 (Sohail et al., 1990). Genetic recombination is stimulated by hemimethylated 5’-CCWGG-3’ motifs in dcm deficient bacterial cells with the arl mutation (Korba & Hays, 1982). Dcm methylation was observed in E. coli K-12, E. coli C, Escherichia blattae, Escherichia fergusoni, Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella enterica serovar Poona, serovar Thyphimurium and serovar Thyphi, Shigella sonnei, Shigella flexneri but not in Aeromonas caviae, Aeromonas hydrophilia, Agrobacterium tumefaciens, Bacillus sp., Enterobacter cloacae, E. coli B, Clostridium difficile, Lactobacillus casei, Morganella morgani, Proteus mirabilis, Pseudomonas aeruginosa, Rhizobium phaseoli, Serratia marcescens, Staphylococcus aureus, Streptomyces phaeochromogenes and Yersinia enterocolitica (Dreiseikelmann & Wackernagel, 1981, Marinus, 1987; GomezEichelmann et al., 1991). 2.2.1
5’-Cmcwgg-3’ Specific Restriction Enzyme Isoschizomer Digestion Assay
The presence of Dcm methylated cytosines in bacteria can be easily detected performing a 5’-CmCWGG3’ specific restriction enzyme isoschizomer digestion assay using the endonucleases EcoRII and BstNI (Gomez-Eichelmann et al., 1991). EcoRII cleaves specific at unmethylated 5'-CCWGG-3’ sequence motifs (Gomez-Eichelmann et al., 1991, Golovenko et al., 2009). It possesses a self-contained DNA methyltransferase activity (M.EcoRII) (Liu et al., 2001; Subach et al. 2004). The EcoRII dimer consists of three structural subunits. There is one central catalytic core, which is composed of two copies of the C-terminal domain (EcoRII-C) and two N-terminal effector DNA binding domains (EcoRII-N) (Golovenko et al., 2009). The enzyme bound to one 5'-CCWGG-3’ alone has no activity. EcoRII cleavage activation re-
quires binding of one or two additional target sites as allosteric effectors (Tamulaitis et al. 2006, Shlyakhtenko et al. 2007, Takahashi et al. 2008, Golovenko et al., 2009). EcoRII’s enzymatic activity is precisely regulated by autoinhibition via its N-terminal domain, which sterically occludes the catalytic site (Szczepek et al., 2009). In contrast to EcoRII, BstNI cleaves methylated and unmethylated 5'-C[m]CWGG-3’ sequences (Gomez-Eichelmann et al., 1991). BstNI is an isoschizomer of EcoRII but it cuts DNA at a distinct location. While EcoRII cuts before the two C residues BstNI cuts between the the two C residues and the W (see Figure 2). Thus, BstNI digestion leads to DNA fragments with a single-base 5´ extension (Repin et al., 1995). Parallel digestion with EcoRII and BstNI is suitable to detect methylated 5'-CC[m]WGG-3’ sequences by additional EcoRII digestion (Gomez-Eichelmann et al. 1979)
Figure 2: Overview of the specific cutting sites of the methyl-cytosine sensitive endonucleases EcoRII and BstNI
2.2.2
Dcm Methylation Protects the Bacterial Genome
In some bacteria like S. enterica and H. pylori methyltransferases are associated with an inactive restriction enzyme gene (Ibanez et al., 1997; Kong et al., 2000; Lin et al., 2001), while others, for example the Dcm gene of E. coli, are not associated with a restriction enzyme homologue (Palmer et al., 1994). Thus, initially the reason for the existence of the dcm-vsr system was not fully clear, as its function as protection system from foreign viral or plasmid DNA seemed to be no satisfactory explanation (Kobayashi, 2001). As mentioned above, the EcoRII complex consists of a restriction endonuclease and a cognate methyltransferase that methylates the same recognition motifs to protect them from digestion, and these recognition motifs are identical as described above for Dcm (Roberts & Marcelis, 2001). It is not surprising that the methyltransferase subunit M.EcoRII and Dcm share a high degree of evolutionary relatedness (Sohail et al., 1990). The loss of an intact type II restriction-modification system (RM) like EcoRII RM results in the inability to modify a sufficient number of restriction recognition sites on the bacterial chromosome, which consecutively leads to cell death due to a restriction attack on the genome (Naito et al., 1995, Handa & Kobayashi, 1999; Handa et al., 2000). Overexpression of dcm was able to prevent this way of cell killing (Takahashi et al., 2002). Thus, it seems that dcm can assure the methylation of potential R.EcoRII restriction sites in cells with a disturbed EcoRII RM gene complex and thereby it protects from R.EcoRII initiated cell killing (Takahashi et al., 2002). 2.3
Differences in the DNA Methylation Capability
While adenine (dam) and cytosine (dcm) methylation occur in parallel in prokaryotic organisms, DNA methylation occurs exclusively at cytosine residues in higher eukaryotes (Razin & Riggs, 1980). Alto-
gether more or less 2% of adenines are transformed to N6-methyladenine and about 1% cytosines are transformed to 5-methlycytosine in E. coli K-12 (Marinus, 1987). Whereas (hemi-)methylation of adenine in GATC sequences plays an important role in the enhancement of gene expression (Barras & Marinus, 1988; Boye, 1991) methylation of cytosine in 5'-CCWGG-3’ sequences protects cellular DNA from endonuclease EcoRII digestion (Schlagman et al. 1976). To date there are no detailed studies on the intra species variability of the presence and methylation state of the different methylation sites. One study about C. jejuni suggests that there should be specific subclades, in which specific methylation sites are missing or just unmethylated (Gu et al., 2009). A second pioneer study examining the ability of MboI to digest genomic DNA from C. coli originating from turkeys and swine indicates the presence of a hostassociated DNA modification system for GATC sites in C. coli of porcine origin, because genomic DNA of C. coli isolates belonging to seven swine-associated multilocus sequence types was resitants to MboI digestion (Wright et al., 2010).
3
Epigenetic Regulation of Bacterial Protein Expression
3.1
DNA Methylation Regulates the Expression Secretion Components
Gram-negative bacteria developed seven different types of secretion systems T(I-VII)SS under evolution that were - among other mechanisms – used to export virulence-associated factors (Tseng et al., 2009). Particularly, Type VI secretion systems (T6SS) are relatively common among Gram-negative bacteria (Beingle et al., 2008; Cascales, 2008; Filloux et al., 2008). T6SS consist of about 13 subunits bridging the bacterial cell membranes and the cell wall (Cascales, 2008). The whole secretory apparatus is anchored in the peptidoglycan layer (Aschtgen et al., 2010a+b). A family of T6SS proteins, the VgrGs (valine–glycine repeat protein G), includes some members that contain C-terminal extensions, which act as effector-domains triggering the translocation by the T6SS (Pukatzki et al. 2009). T6SS are important virulence factors playing a crucial role in the invasion and lysis of eukaryotic cells (Pukatzki et al. 2006). Furthermore, anti-bacterial toxins involved in inter-bacterial competition and secreted via the T6SS have been demonstrated for Pseudomonas aeruginosa, Burkholderia spp., and Vibrio cholera (Hood et al., 2010; Schwarz et al., 2010a+b; MacIntyre et al., 2010). On the other hand, T6SS function is not restricted to rivalry with competing organisms. It has also been demonstrated that T6SSs are involved in biofilm formation, resisting amoeba predation, and stress sensing (Aschtgen et al., 2008; Jani & Cotter, 2010; Weber et al.; 2009) Several regulation mechanisms for the modulation of the expression of the T6SS components have been reported like quorum sensing, transcriptional factors, two-component systems, histone-like proteins, alternative sigma factors (Bernhard et al., 2010; Bernhard et al., 2011; Leung et al., 2011) and epigenetic switch mechanisms (Brunet et al., 2011). The players in the epigenetic switch mechanism are the sci1 T6SS gene cluster of enteroaggregative E. coli (EAEC) and the ferric uptake regulator – Fur (Brunet et al., 2011). Fur has been shown to play a role in iron homeostasis and in the acid stress response (Escolar et al., 1999). It forms dimers acting as the main repressor of the sci1 gene cluster. In the presence of iron, it functions as a transcriptional repressor of iron-dependent promoters (Escolar et al., 1999). Iron-complexed Fur specifically binds to the so-called Fur box, a concensus binding motif consisting of the 19 nucleotides 5’GATAATGATAATCATTATC-3’, which is part of these iron-dependent promoters (Escolar et al.,
1999). At sufficient concentrations of iron Fur is bound to the respective promotors and represses the expression of the genes controlled. In the absence of iron Fur is relieved from the Fur boxes giving access for the RNA polymerase (Escolar et al., 1999). The sciH gene is the first gene of the sci1 gene cluster and the non-coding sequence starting 578 basepairs upstream of sciH forms the sci1 promotor (Brunet et al., 2011). Two Fur boxes are present in the promotor region of the sci1 gene cluster, designated as fur1 and fur2 box (Brunet et al., 2011). Additional sequence analysis of the sci1 promotor region revealed three GATC motifs (I-III), the potential sites for Dam methylation (Brunet et al., 2011). The fur2 box is located upstream of GATC-III and of the putative -10 element of the σ70 promotor, whereas the fur1 box overlaps both with the -35 element of the σ70 promotor and the GATC-I motif. Thus, especially the position and sequence of fur1 indicate a direct interference between the Fur-dimer and the RNA polymerase (Brunet et al., 2011). In vivo assays demonstrated a higher affinity of iron-complexed Fur to the fur1 box in comparison to the fur2 box (Brunet et al., 2011). Isoschizomer digestion assays (see above) showed that Dam could methylate alle three GATC motifs. Addition of Fur prevented the digestion by methylation-sensitive restriction endonucleases only at the GATC-I site, indicating that Fur binding prevents methylation within the fur1 box by the Dammethylase (Brunet et al., 2011). On the other hand, electromobility shift assays demonstrated a lowering of the Fur affinity to the fur1 box as a result of Dam-dependent methylation (Brunet et al., 2011). In consequence, sci1 promotor expression is regulated by the competing processes of its own Dam-dependent methylation and Fur binding (Brunet et al., 2011). These regulatory mechanisms led to the postulation of an epigenetic switch mechanism that switches between an ON (expressing) and an OFF (nonexpressing) expression state of the sci1 gene cluster. During the OFF phase, that means the repression of the sci1 transcription under iron rich conditions, Fur prevents the binding of the RNA polymerase as well as the GATC-I methylation by Dam. This OFF phase (the nonmethylated state) should be principally understood as the ground level state. Low iron ion concentrations result in a lowered forming rate of Fur complexes that were able to bind the fur1 box. Thus, the fur1 box is accessable for the RNA polymerase (ON phase) as well as for Dam. Dam-dependent methylation of the GATC-I site stabilizes the ON phase by preventing de novo Fur binding (Brunet et al., 2011) The model of the sci1 epigenetic switch suggests that the transition from the OFF to the ON state is fundamentally dependent on the availability of iron. Methylation of the GATC-I site is an essential prerequisite for the maintenance of a stable ON state. Fur binding to a hemimethylated fur1 box is suggested to be feasible. Consequently, an ON to OFF switch during DNA replication under iron repletion could still be possible. The ON to OFF switch, a change from a methylated GATC-I site to a nonmethylated, can only occur during DNA replication. Thus, it was hypothesized that the absence of DNA replication should maintain the expression of the sci1 cluster in the ON state irrespective of environmental iron concentrations. In this model, Dam methylation prevents de novo Fur binding in the absence of DNA replication and de novo Fur binding upon DNA replication (Brunet et al., 2011). 3.2
Epigenetic Regulation of Uropathogenic Escherichia coli Adhesin Expression
Urinary tract infections are a very common disease especially in women with about 150 million cases per year worldwide (Foxman, 2010). The so-called uropathogenic E. coli – UPEC are the most prevalent bacterium causing urinary tract infections (Song et al., 2008, Weichhart et al., 2008). These E. coli strains
express specific pathogenicity factors like type I pili, adhesin P (pap), adhesin S (sfa), afimbrial adhesin I (afa), siderophore aerobactin (aer), alpha-hemolysin (hly), cytotoxic necrotizing factor type 1 (cnf1), and the outer membrane protein TraT, which is associated with serum resistance (Wiles et al., 2008, Oliveira et al. 2011). Additionally, biofilm formation is an important virulence factor, especially for the colonization of urinary catheters by UPEC (Ponnusamy et al., 2012). The most crucial UPEC virulence factor is adhesin P that is encoded by the pyelonephritisassociated pili operon (pap) consisting of 11 genes (Hale et al. 1998). E. coli strains, which do not express this Pap pili are not able to bind to the urothel and are simply rinsed out of the urinary tract (Hagberg et al., 1983, Roberts et al., 1989). The Pap pili expression is regulated by a phase variation mechanism (van der Woude et al., 1992), which itself is regulated by DNA methylation (Braaten et al., 1994). This results in a variable expression of the Pap pili within a particular UPEC population. Bacteria in the ON phase express the Pap pili, whereas bacteria in the OFF phase do not express Pap pili (Blyn et al. 1989, Low et al., 1987, van der Woude et al., 1996). It was supposed, that this ON/OFF phase switch is necessary to make retrograde movement up the urinary tract feasible through alternating phases of free swimming and attachment (Hale et al., 1998). Thus, it is an essential mechanism in the pathogenesis of urinary tract infections. The regulatory region of the pap operon contains six binding sites for the leucine-responsive regulatory protein (Lrp). Sequence analysis revealed two GATC sites in this region: one within the Lrp binding site 5 (GATC-I) and the second within the Lrp binding site 2 (GATC-II) (Kaltenbach et al., 1995). These GATC sites are also called GATCdist (= GATC-I) and GATCprox (= GATC-II) (van der Woude & Bäumler, 2004). Occupation of the respective binding sites No. 1-3 by Lrp blocks Dam-dependent methylation of GATC-II in the OFF phase, whereas occupation of the binding sites 4-6 hinders methylation of GATC-I in the ON phase (van der Woude et al., 1996). On the other hand, methylation of the GATC-I site in the OFF phase reduces the affinity of Lrp to the binding sites 4-6 (Nou et al., 1993, Hale et al., 1998). The regulatory protein PapI controls the translocation of Lrp from the binding sites 1-3 to the sites 4-6. This translocation is required for the ON phase (Kaltenbach et al., 1995, Nou et al., 1995). PapI interacts directly with Lrp bound to pap DNA, and increases therewith the binding strength of Lrp for the binding sites 4 and 5 (Nou et al., 1995). Thus, it was suggested that phase switching takes place immediately after DNA replication; in a time frame while the GATC-I is hemimethylated and binding of Lrp-PapI to the binding sites 4-6 is possible (Braaten et al., 1994, Blyn et al., 1990). 3.3
Phase and Antigenic Variation Controlled by DNA Methylation in Salmonella
Phase variation is a common reversible mechanism of gene regulation in bacteria (Torres-Cruz & van der Woude, 2003). It is a natural “tool” to yield phenotypic heterogenecity in a clonal population that contributes to the success of a pathogen to evade the adaptive immune response and can be understood as additional virulence strategy (van der Woude, 2011). Phase variation can be reached by slipped strand mispairing, site-specific recombination and, of course, by epigenetic regulation (Torres-Cruz & van der Woude, 2003, van der Woude, 2011). Probably the best-known example of phase variation is the modification of the O-antigen of Salmonella spp. that affects the serotype (Broadbent et al., 2010). Responsible for the modification of the Oantigen are the proteins encoded by the glycosyltransferase operon (gtr). The gtr gene cluster consists of the three genes gtrA, gtrB, and gtrC encoding for a bactophenol-linked glucosyl translocase or flippase
(gtrA), a bactophenol glucosyl transferase (gtrB), and a glucosy ltransferase (gtrC) mediating the attachment of glucose to the O-antigen (Allison &Verma, 2000). Epigenetic control was especially demonstrated for the phage P22 Salmonella gtr cluster (gtrP22) associated with the O1 serotype (Broadbent et al., 2010). The key factors in the regulation of the gtrP22 promotor are the Dam methyltransferase and the oxidative stress regulator OxyR (Broadbent et al., 2010). Within the region 115 bp upstream of the gtrP22 transcription start site (that is 25 bp upstream of the gtrA start codon) four GATC motifs (GATC1-4) are present (Broadbent et al., 2010). These four GATC motifs are arranged in two pairs. OxyR usually interacts as dimer of dimers with its DNA binding motifs. There are three (A-C) potential interaction sites for OxyR dimers within the 115 bp upstream region, which are referred as OxyR binding half sites. The reduced form of the OxyR dimer of dimers binds to two of these binding half sites in parallel. Thus there are two complete binding sites OxyR(AB) and OxyR(BC) (Toledano et al., 1994). Half site OxyR(A) overlappes with GATC1/GATC2 and half site OxyR(C) overlaps with GATC3/GATC4 (Broadbent et al., 2010). Thus, ligands binding to one of these overlapping binding sites compete with each other. The start site of transcription is located 33 bp downstream of GATC4 (Broadbent et al., 2010). Thus, GATC4 and OxyR(BC) are sited in the promotor region. The epigenetic mechanism involving Dam and OxyR controlling gtrP22 phase variation was postulated as follows: During the ON phase GATC1+2 are unmethylated whereas GATC3+4 are Dam methylated. In consequence OxyR is bound to OxyR(AB), while OxyR binding to OxyR(C) is prevented by methylation. On the other hand OxyR binding prevents Dam methylation of GATC1+2. Thus, methylation protection of GATC1+2 by OxyR stabilizes the ON phase and results in full activation of the gtrP22 promotor (Broadbent et al., 2010). We find the inverse situation during the OFF phase: GATC1+2 are methylated and GATC3+4 are unmethylated and OxyR is bound to OxyR(BC), while OxyR binding to OxyR(A) is prevented by methylation. While both, the OxyR(C) half site and GATC3+4 overlap the RNA polymerase binding site transcription of the gtrP22 gene cluster is downregulated in the OFF phase (Broadbent et al., 2010). A role of the hemimethylated state during the DNA replication as demonstrated for the epigenetically controlled pap and agn43 phase variation systems was supposed (Correnti et al., 2002, Hernday et al., 2004, Kaminska & van der Woude, 2010). Oxidative stress was assumed as external factor triggering phase switching (Broadbent et al., 2010). 3.4
Phase Variation of the Outer Membrane Protein Family Ag43 in E. coli
A further protein that’s expression is controlled by an epigenetic mechanism is the outer membrane protein Ag43 of E. coli (Henderson & Owen, 1999, Haagmans & van der Woude, 2000). Ag43 encoded by the agn43 gene was shown to play a role in autoagglutination and biofilm formation (Danese et al., 2000, Henderson et al., 1997). Ag43 and fimbrial expression are coordinately regulated. If fimbrial proteins are expressed agn43 transciption is repressed and vice versa (Schembri & Klemm, 2001). The key factors in the regulation of the agn43 gene are also the Dam methyltransferase and the oxidative stress regulator OxyR (Wallecha et al., 2002). Three GATC sites have been identified in the upstream region of agn43 (Wallecha et al., 2002). The ON phase is maintained by the methylation of these GATC sites by Dam preventing the binding of OxyR to the regulatory region (Henderson et al., 1999). OxyR binding to the unmethylated upstream region of agn43 inhibits RNA polymerase binding and therewith Ag43 expression, resulting in an OFF
phase (Henderson et al., 1999). OxyR binding to the regulatory upstream region is only feasible if all three GATC motifs are unmethylated (Haagmans & van der Woude, 2000). The repression of agn43 transcription by OxyR is independent of its oxidation state (Wallecha et al., 2003). Comparable to the other switch mechanisms described above OxyR binding prevents Dam methylation (Correnti et al., 2002). Explicitly for agn43 it was demonstrated that the hemimethylated state, that means when only one strand of the DNA double helix is methylated during DNA replication, is a key event for the switch from ON to OFF (Kaminska & van der Woude, 2010). Thus, a model was postulated in which OxyR at the replication fork initiates the ON-to-OFF switch (Kaminska & van der Woude, 2010). 3.5
The Impact of DNA Methylation on Bacterial Virulence and the Pathogenesis of Infectious Diseases
The presence or absence of Dam shows direct effects on bacterial pathogenesis by a regulation of gene expression and virulence (Low et al., 2001). Dam provides both clock-like controls that permit transient gene transcription during specific states of DNA replication and switch-like controls in which transcription is regulated by DNA methylation patterns (Low et al., 2008), thus leading to versatile and widespread regulation of bacterial virulence functions (Heusipp et al., 2007). Dam mediated effects on bacterial virulence have been shown for Aeromonas hydrophila, Pseudomonas multocida, Salmonella enterica, Yersinia pestis, Yersinia pseudotuberculosis, and Vibrio cholerae in vivo (Heusipp et al., 2007). E.g., Dam negative mutants of S. Typhimurium are incapable of colonizing deep tissue sites, thus leading to a complete loss of virulence. A depletion of Dam activity affects the regulation of 20 genes that are usually activated in the course of a Salmonella infection, including genes coding for betagalactosidase and PhoP-activated genes. Accordingly, selective Dam inhibitors might be useful as antimicrobial agents (Heithoff et al., 1999). Further, both Salmonella strains without functional Dam and with Dam overproduction are suitable to induce protective immune responses, an observation with relevance for vaccine development (Julio et al., 2001). Moreover, this phenomenon is also common in other Enterobacteriaceae including Y. pseudotuberculosis and V. cholerae, in which Dam overproduction leads to attenuation (Julio et al., 2001) in spite of increased tissue culture invasion (Fälker et al., 2005). Loss of the Dam gene function leads to a loss of viability in these species. In Y. pseudotuberculosis, a Dam overregulation-associated ectopic secretion of virulence proteins even leads to protective immune response (Julio et al., 2001). The Dam regulated processes in the bacterial cell with critical relevance of bacterial pathogenesis include mismatch repair, chromosome replication, expression of bacterial virulence genes, transposition, and transcription (Fälker et al., 2005, Heusipp et al., 2007). In detail, Dam affects phase variations in various fimbriae and pili as shown for E. coli and S. enterica (see above), regulation of various in vivo expressed genes, effector translocation as shown for A. hydrophila, membrane (in-)stability by the release of membrane vesicles as shown for S. enterica, cell motility as shown for A. hydrophila, E. coli, and Y. enterocolitica, as well as host cell invasion, e.g. in cell lines, and adhesion to cellular surfaces (Heusipp et al., 2007). Based on Dam depleted or modified strains, oral live vaccine candidates were introduced for H. influenzae, S. enterica, Y. pestis and Y. pseudotuberculosis (Heusipp et al., 2007), once more demonstrating the relevance of Dam function for pathogenicity or non-pathogenicity of strains of these species.
4
DNA Methylation Induced by Bacterial Infection
4.1
Epigenetic Mechanisms in Eukaryotic Cells
Bacteria do not only methylate their own DNA at certain sites dependent on specific environmental circumstances. They also induce hypermethylation of key cellular promoters of mammalian cells during the process of infection (Minarovits, 2009). Thus, they may induce pathological processes by epigenetic reprogramming. In contrast to bacterial cells, eukaryotic cell DNA is predominantly methylated at cytosine residues (Razin & Riggs, 1980). In mammals the DNA meythyltransferase I (DNMTI), which arose phylogenetically by fusion of the prokaryotic modification methyltransferase gene, encoding the C-terminal catalytic domain and a second, not further identified gene, encoding the N-terminal regulatory domain, is the key enzyme for DNA methylation. Cytosine methylation by DNMTI occurs specifically at 5’-CpG-3’ (short writing: CpG) sites (Bestor et al., 1988; Yen et al., 1992; Deaton & Bird, 2011). The unmethylated CpG islands are typically located upstream of the exons of constitutively expressed housekeeping genes (Bird, 1986 & 1987). To ensure the maintainance of methylation patterns, DNMT1 preferes hemimethylated DNA as substrate, which is produced during DNA replication if the original strands are methylated (Van Emburgh & Robertson, 2008). De novo methylation (of unmethylated DNA) is predominatly performed by the mammalian methyltransferases DNMT3A and B (Van Emburgh & Robertson, 2008). The discrimination between methylated and unmethylated occurs via differing N-terminal domains (Xie et al., 1999). Methyl-CpG binding proteins like the transcriptional repressor MeCP2 cofractionate with histone deacetylases and histone methylases in cluster analysis. Deacetylation of histone tails and histone methylation lead to a remodelling of the chromatin structure. In consequence, DNA methylation dominantly silences transcription, whereas active promoters are usually unmethylated at the CpG sites (Jones et al., 1998; Jenuwein & Allis, 2001; Fuks et al., 2003). Additionally, the polycomb-trithorax protein group regulates gene expression independently of DNA methylation (Bird, 2002; Cernilogar & Orlando, 2005; Laue et al. 2008). DNA methylation regulates a wide spectrum of cellular processes like neutralization of potentially harmful genomic elements (Hendrich & Tweedie, 2003), ontogenetic processes, genomic imprinting – the epigenetic modification leading to differential expression of the two parental alleles (Robertson, 2005), fetal growth and placental development (Isles & Holland, 2005). 4.2
DNA Hypermethylation Triggered by Bacterial Infection
Bacterial infection, especially with Gram-negative bacteria, induces DNA hypermethylation in eukaryotic host cells as demonstrated in several models. Statistical analysis pointed out that maternal bacterial infections and the resulting inflammatory immune response at some stages in pegnancy could lead to preterm delivery and growth restriction (Andrews et al., 2000; Goldenberg et al., 2000). Especially periodontal infections with Prevotella intermedia, Fusobacterium nucleatum, Peptostreptococcus micros, Campylobacter rectus, Eikenella corrodens, Selenomonas noxia and Streptococcus intermedius have been associated with an increased risk of pre-term delivery (Offenbacher et al., 2001, Madianos et al., 2001, Buduneli et al., 2005). In particular, C. rectus was identified as key pathogen in this process (Offenbacher et al., 2005; Buduneli et al., 2005).
4.2.1
Silencing of the Igf2 Promotor as a result of Campylobacter rectus Infection
Using an infection/pregnancy mouse model (Offenbacher et al., 2005), it was possible to demonstrate that C. rectus leads to a significant decrease in Insulin-like growth factor 2 (IGF2) mRNA levels after translocation to the placenta (Bobetsis et al., 2007). IGF2 has been previously demonstrated to affect the growth of the fetus as well as placental growth (Constancia et al., 2002). The IGF2 gene is located in a cluster of imprinted genes. This cluster contains an intronic CpG island (designated as region 2), in the second intron. This region 2 was identified as the imprinting element of this gene cluster. It inherits a methylation imprint from the maternal gamete (DeChiara et al., 1991; Wutz & Barlow, 1998). This cluster is located on chromosome 7 in the mouse and on chromosome 11p15.5 in the human (Paulsen et al., 1998). Knockout of the igf2 promotor region P0 in mice was shown to cease placental igf2 expression and results in reduced placental and consecutive fetal growth (Constancia et al., 2002). Infection with C. rectus leads to similar effects. A significant increase (10.3%) in hypermethylation of 4 out of the 16 CpG loci of the igf2 promotor region P0 was detected by bisulfite sequencing in placental DNA of infected intra-uterine growth-restricted placentas (Bobetsis et al., 2007). Thus, infection with C. rectus suppresses IGF2 gene expression and therewith placental and fetal somatic growth (Bobetsis et al., 2007). 4.2.2
Promotor Silencing in Consequence of Helicobacter pylori Infections
H. pylori infections, which are known to be one key risk factor for the development of gastric cancer (McNamara & El-Omar, 2008), have been demonstrated to trigger aberrant DNA methylation in gastral mucosa cells (Maekita et al., 2006; Nakajima et al., 2006). A well-described specific methylation-site is the promotor region of the E-cadherin gene, an adhesion molecule that plays a crucial role in tumor invasion, dissemination and metastatic disease (Chan et al., 2003). It could be demonstrated by methylation specific polymerase chain reaction that the CpG sites of the E-cadherin promotor region in gastral mucosa cells of H. pylori-positive patients are significantly more often methylated and thereby silenced in comparison to healthy individuals (Chan et al., 2003). It is suggested that silencing of the E-cadherin could be an early event in gastric carcinogenesis (Chan et al., 2003). Similar effects on the methylation rate of the CpG sites in the E-cadherin promotor could be observed after interleukin-1β treatment of human gastric cell lines (Qian et al., 2008). The supposed tumor suppressor gene RUNX3 is a second factor whose expression is downregulated by H. pylori induced promotor hypermethylation (Kitajima et al., 2002; Li et al., 2002). The question arises how to distinguish between cancer-associated (Kaneda et al., 2002) and H. pylori-induced hypermethylation (Yoo et al., 2008). It was shown that genes occupied by so-called polycomb proteins in embryonic stem cells are more vulnerable to aberrant DNA hypermethylation in malignomas. Because most of the H. pylori-associated hypermethylated genes contain at least one polycomb repressive mark, it was suggested that these marks determine the H. pylori-induced DNA hypermethylation sites (Yoo et al., 2008). Some of these alterations in the methylation pattern are only temporary and reversible after H. pylori-eradication (Miyazaki et al., 2007), whereas others are permanent in stem or differentiated cells (Ushijima, 2007).
4.2.3
Epigenetic Alterations by Uropathogenic Bacteria (UPEC)
UPEC are able to invade urothelial cells, replicate intracellularly and thereby establish a reservoir for chronic recurrent infections (Dhakal et al. 2008). It was observered that UPEC residing inside urothelial carcinoma cells lead to an increased DNA methyl-transferase I (DNMT1) RNA synthesis and protein expression as well as enzymatic activity. In consequence, DNA hypermethylation was detected in the CpG-island of the tumor suppressor gene cdkn2A (cyclin-dependent kinase inhibitor 2A), which was consecutively downregulated (Tolg et al., 2011; Schulz, 2011). Additionally, a second tumor suppressor gene – the DNA repair enzyme MGMT (O(6)-methyl-guanine- DNA-methyl-transferase) – was also downregulated, but no change in the methylation state of this gene could be detected (Tolg et al., 2011). In contrast, the H. pylori triggered downregulation of MGMT was found to be associated with an increased CpG methylation (Sepulveda et al. 2010). The downregulation of these two tumor suppresser genes was suggested to be an initial step in the tumor transformation process, because it is well known that CDKN2A gene deletion in consequence of the impact of chemical carcinogens is the major reason for CDKN2A inactivation in urothelial cancer (Florl et al., 2000). In contrast to these supposed initial mechanisms of tumor transformation, epidemiological data failed to confirm an association between bacterial infection of the urinary tract and the incidence of urothelial cancer (Wu et al., 2008; MurtaNascimento et al., 2007). It should be mentioned here that Schistosoma haematobium, causing inflammatory cystitis, is associated with squamous cell carcinoma of the urine bladder (Abol-Enein, 2008). One study demonstrated an increased DNA methylation in these carcinomas in comparison to urothelial carcinomas (Guiérrez et al., 2004). 4.2.4
Epigenetic Control of Toll-like Receptor 4 by Commensal Bacteria
A further interesting observation was described by Takahashi and coworkers. It is well known that intestinal epithelial cells are constantly exposed to bacteria of the intestinal flora but they are usually insensitive to these bacteria. The maintenance of this commensal flora, that exists in a symbiotic relationship with its host, is the basic meachnism to avoid an intestinal inflammatory reaction (Takahashi et al., 2011). Intestinal epithelial cells receive stimulation from this normal flora by Toll-like receptors (TLR; Rakoff-Nahoum et al., 2004). The decreased expression of particular TLRs in intestinal epithelial cells is one key mechnism of this intestinal tolerance (Abreu et al., 2001 & 2003; Melmed et al., 2003). The TLR4 gene is methylated at its 5’ CpG motifs to a higher degree in human intestinal cell lines as well as in murine primary intestinal cells compared to other cells. Thus, lower receptor amounts are expressed. In contrast, the TLR4 gene methylation level is significantly lower in intestinal epithelial cells of germ free mice than in mice with a normal intestinal flora. This finding suggests that bacteria of the normal intestinal flora contribute via epigenetic mechanisms to the maintenance of the intestinal symbiosis (Takahashi et al., 2011).
5
Outlook
As described in this chapter, epigenetic mechanisms play a significant role in the pathogenesis of bacterial infections, both on the side of the bacteria as well as on the side of the infected host organism.
However, many open questions remain. So far we cannot assign any appropriate regulatory mechanisms to the most of the potential methylation sites in the bacterial and in the host’s genome. Triggering signals and the mechanisms for the switches between ON-to-OFF and OFF-to-ON are only poorly understood. And ultimately, the mechanisms that ensures the inheritance of the expression state in the next generation are largely unclear. Future scientific approaches in this field should respond to these questions.
Acknowledgments This publication was funded by the Open Access support program of the Deutsche Forschungsgemeinschaft and the publication fund of the Georg August Universität of Göttingen.
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