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Outer Membrane Protein OmpB Methylation May Mediate Bacterial Virulence
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Review
Outer Membrane Protein OmpB Methylation May Mediate Bacterial Virulence David C.H. Yang,1,* Amila H. Abeykoon,2 Bok-Eum Choi,1 Wei-Mei Ching,3 and P. Boon Chock4 Methylation of outer membrane proteins (OMPs) has been implicated in bacterial virulence. Lysine methylation in rickettsial OmpB is correlated with rickettsial virulence, and N- and O-methylations are also observed in virulencerelevant OMPs from several pathogenic bacteria that cause typhus, leptospirosis, tuberculosis, and anaplasmosis. We summarize recent findings on the structure of methylated OmpB, biochemical characterization, and crystal structures of OmpB methyltransferases. Native rickettsial OmpB purified from highly virulent strains contains multiple clusters of trimethyllysine, in contrast with mostly monomethyllysine, and no trimethyllysine is found in an avirulent strain. Crystal structure of the methyltransferases reveals mechanistic insights for catalysis, and a working model is discussed for this unusual post-translational modification.
Trends Protein methylation plays vital roles in cellular regulation and signaling. Bacterial chemotaxis is regulated by methylation of specific membrane proteins to mediate excitation and adaptation in response to chemotaxis. O-[23_TD$IF]Methylation of Msp4, a surface protein, is required for Anaplasma phagocytophilum[23_TD$IF]–mediated infection, and [129_TD$IF]multisite N-methylation of OmpB correlates with typhus rickettsial virulence. Structural and mechanistic analysis of the newly characterized rickettsial methyltransferases led us to propose a mechanism in which the enzyme catalyzes multimethylation of OmpB.
Methylation of OMPs Covalent modification of proteins, such as methylation, alters their enzymatic activity and/or affinity to interact with other proteins, membranes, or nucleic acid [1,2]. Thus, protein methylation, generally catalyzed by methyltransferases, serves as a regulatory mechanism in epigenetics and cellular signaling [3]. Studies of protein methylation have primarily focused on histones [4,5]. More recently, such studies have extended to nonhistone proteins [6,7]. In bacteria, the methyl group is primarily known to attach to Lys, Asp, Glu, and Gln [8]. In the case of methylation at the lysyl residues, additional repertories of one, two, or three methyl groups would further expand their potential varying functionalities. The OMPs provide the first line of communication with their extracellular environment. They often function as adhesin, [234_TD$IF]invasin, or transporter, as well as playing a dominant role in host immune response. Methylation of OMPs has been implicated in the virulence of infectious diseases mediated by Gram-negative bacteria. Contribution of OMPs to bacterial virulence has been widely reported [9–13]. Examples include OmpB from Rickettsia prowazekii in typhus, LipL32 from Leptospira interrogans in leptospirosis, major surface protein [235_TD$IF]4 (Msp4) from Anaplasma phagocytophilum in human granulocytic anaplasmosis, Ail of Yersinia pestis in plague, BBK32, GAG from Borrelia burgdorferi in Lyme disease, and AlpB from Helicobacter pylori in gastric ulcers. Unlike integral membrane proteins from most other sources, the autotransporter domains of many OMPs do not consist of transmembrane a-helices, but instead fold into antiparallel b-barrels [14,15]. Recent investigation on methylation of bacterial OMPs has uncovered novel mechanisms with respect to protein structure and catalytic function in OMP methylation.
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We believe that knowing the catalytic and regulatory mechanism of OMP methylation, and how methylated OMP exerts its virulent effect, would provide new insights to facilitate the development of novel therapeutic and diagnostic strategies.
1
Department of Chemistry, Georgetown University, Washington[231_TD$IF], DC 20057, USA 2 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892, USA 3 Viral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, MD 20910, USA 4 Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA
http://dx.doi.org/10.1016/j.tibs.2017.09.005 © 2017 Elsevier Ltd. All rights reserved.
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In this review, we focus on methylation of OMPs from pathogenic bacteria and in-depth methylation studies of rickettsial OmpB, in view of the fact that the mechanism and the potential physiological functions of OmpB methylation have been extensively investigated recently. While the function of OMP methylation could be multifaceted, evidence suggests pivotal roles in bacterial virulence. The virulence of A. phagoctyophilum and infection of L. interrogans have recently been demonstrated to require methylation of their OMPs. Note that the biological function of protein reversible methylation in regulating bacterial chemotaxis, has been reviewed [16,17] and will not be covered here.
N-[23_TD$IF]Methylation of OmpB in R. prowazekii and Rickettsia [236_TD$IF]typhi Correlation of Rickettsial Virulence with OmpB Methylation Rickettsiae are Gram-negative, obligatory intracellular bacteria, and etiologic agents of typhus and spotted fever [18–20]. R. prowazekii and R. [237_TD$IF]typhi belong to the typhus group, while R. rickettsii and Rickettsia conorii belong to the spotted fever group. Virulent rickettsial strains induce symptoms such as fever, nausea, or diarrhea, and eventually may lead to multiorgan failure, while avirulent strains do not [19]. At the cellular level, virulent rickettsial strains grow fast in host cells, and induce cytokines or chemokines [21]. Rickettsial OMPs are localized at the outer leaflet of the outer membrane [22–25] and they play prominent roles in mediating host cell adhesion and invasion [26–29], as well as facilitating intracellular movement [30,31]. Amino acid analysis revealed that OmpBs from virulent strains of R. prowazekii contain more trimethylated lysyl residues than those of avirulent bacteria [32–34]. Consistent with a notion that lysine methylation may play an important role in rickettsial virulence, Zhang et al[238_TD$IF] showed that the gene Rp028/027, which encodes the lysine methyltransferase, was mutated by frameshift in the avirulent R. prowazekii Madrid E strain but not in the virulent revertant Evir and the virulent strain Breinl [35]. Bechah et al[238_TD$IF] investigated the correlation between lysine methylation in OmpB with the degree of virulence by carrying out a comparative investigation of four phenotypes of R. prowazekii that differed in virulence, using genomic, transcriptomic, and proteomic analysis [36]. They found that several genes that mediate the differences in virulence are mainly associated with surface proteins and post-translational modification. Together, results from genetic and biochemical studies suggest that lysine methylation of OMPs regulates bacterial virulence. Thus, current investigations are focusing on gaining new structural insights into OmpB methylation as well as the catalytic mechanisms governing the methylation of the OMPs. Methylation in Native OmpB from R. prowazekii and R. typhi To identify both methylated amino acids and the nature of methylation in native OmpB, OmpBs were isolated and purified from different species and strains of R. prowazekii and R. typhi for structural analysis using LC-tandem mass spectrometry (MS/MS) methods. LC-MS/MS analysis provides data on the methylation status of OmpB. The observed methylation profiles reveal that in the entire passenger domain of OmpB, multiple lysyl residues are either mono-, di-, and/ or trimethylated, and the methylation profile varies with the degree of virulence [37]. Figure 1 depicts the methylation profiles of OmpB from four different strains and species with varying virulence. The patterns of methylation in these profiles show extensive and widespread methylation throughout OmpB. No apparent consensus sequences at the methylated sites are discernable, but the differences in these profiles are apparent. OmpB from the avirulent Madrid E contains mono- and dimethyllysine residues but is devoid of trimethyllysine. In contrast, the highly virulent strains, Breinl and RP22, contain appreciable levels of trimethyllysine at relatively selective lysine residues, with several trimethyllysines occurring in clusters. OmpB from the R. typhi species, which is mildly virulent relative to Breinl and RP22 of R. prowazekii, showed intermediate levels of trimethylation. Thus the levels of trimethyllysine in OmpB from the four species and strains apparently correlate with their degree of virulence. In addition to the observations of pervasive mono- and trimethylation and the accompanied microheterogeneity of OmpB, the results raised two new aspects of post-translational
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*Correspondence: [email protected] (David C.H. Yang).
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Breinl
Methyl
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1.0 0.8 0.6 0.4 0.2 0.0
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120 130 156 204 231 272 278 296 309 352 382 387 414 417 425 487 546 574 594 623 634 666 710 722 762 775 787 791 828 837 891 925 926 954 984 986 1026 1059 1185 1228 1289 1319
Typhi
Methyl
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1.0 0.8 0.6 0.4 0.2 0.0 118 131 149 157 205 222 226 232 273 279 297 310 353 379 383 388 415 426 458 488 547 624 635 667 676 711 723 763 776 788 792 829 838 891 926 954 984 986 1026 1039 1056 1185 1228 1289 1319
Normalized fracons
RP22 1.0 0.8 0.6 0.4 0.2 0.0
Madrid E
Methyl
Dimethyl
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120 130 156 204 231 272 278 296 309 352 382 387 414 417 425 487 546 574 594 623 634 666 710 722 762 775 787 791 828 837 891 925 926 954 984 986 1026 1059 1185 1228 1289 1319
1.0 0.8 0.6 0.4 0.2 0.0
Lysine residue numbers Figure 1. Methylation Profiles of Native OmpB Purified from Rickettsia. The location, methylation state, and normalized fraction of methylated Lys residues in native OmpBs purified from highly virulent Rickettsia prowazekii, Breinl strain (top panel) and RP22 strain (second), mildly virulent Rickettsia typhi (third) and avirulent R. prowazekii, Madrid E strain (fourth) are shown [37]. Monomethyllysine residues are shown in green, dimethyllysine in blue and trimethyllysine in red. Lysine residue numbers and the normalized fractions are shown on the x- and y-axes, respectively. The amino acid sequences of OmpBs from R. prowazekii and R. typhi differ, thus different numbering of lysyl residues. The observed number of peptide sequence matches was normalized using the total number of peptide sequence matches at all methylation states at each site.
modifications. First, this class of methyltransferases must be able to recognize multiple lysyl residues with different amino acid sequence contexts. Second, a complex enzyme mechanism is needed to achieve the observed wide range of methylation profiles throughout OmpB. To elucidate this catalytic mechanism, one needs to identify the enzymes involved. However, genetic manipulation of rickettsial genes is hampered by rickettsial obligate intracellular growth. Thus, bioinformatic analysis of genomic DNA of Rickettsia was carried out to identify the putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli [38] to characterize the methylation systems. OmpB Methyltransferases and Methylation of OmpB [239_TD$IF]in vitro Methylation of rOmpB (recombinant OmpB) fragments using the putative methyltransferases revealed the existence of lysine methyltransferases that target rOmpB but not histones or
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proteins from E. coli [38]. LC-MS/MS analysis of the OmpB modifications catalyzed by the methyltransferases demonstrated the existence of two types of protein lysine methyltransferases (PKMT). Figure 2 shows the methylation profiles of rOmpB catalyzed by PKMT1 and PKMT2. PKMT1 primarily catalyzes monomethylation, and PKMT2 catalyzes essentially trimethylation [37]. The two types of enzymes exhibit 45% sequence identity. PKMT2 methylates at relatively specific consensus sequences, KX(G/A/V/I) and KT(I/L/F), while monomethylation by PKMT1 occurs pervasively throughout OmpB. Similar to the known mono- and trimethyltransferases, the catalytic efficiency of PKMT1 is appreciably higher than that of PKMT2. However, these protein lysine methyltransferases are unusual in that they consist of >500 amino acid residues relative to the majority of known methyltransferases (220–350 residues). Comparing the locations of trimethyllysyl residues observed with the purified native OmpB from R. typhi (depicted in the third panel of Figure 1) with those found in rOmpB catalyzed by PKMT2 (Figure 2, lower panel) reveals that their methylation profiles are correlated. Similarly, the locations of monomethyllysyl residues found in the native OmpB from R. typhi (Figure 1, third panel) also correlated well with the methylation observed in rOmpB catalyzed by PKMT1 (Figure 2, top panel). Together, the close correlations of the methylation profiles found in native OmpB with those catalyzed by PKMT1 and PKMT2 using OmpB(AN) and (K) fragments suggests that these enzymes are likely the ones that catalyze the methylation of OmpB in vivo. The 3D structures of these enzymes are necessary to provide additional structural insight about substrate recognition and catalysis. Crystal Structures of Methyltransferases PKMT1 and PKMT2 The crystal structural data of PKMT1 and PKMT2 provide the first look at the 3D structures of protein lysine methyltransferases from Gram-negative bacteria. PKMT1 from R. prowazekii and
Methylaon catalyzed by RtPKMT1 Dimethyl
Trimethyl
118 131 149 157 205 222 226 232 273 279 297 310 353 379 383 388 415 426 458 488 547 624 635 667 676 711 723 763 776 788 792 829 838 891 926 954 984 986 1026 1039 1056 1185 1228 1289 1319
Methylaon catalyzed by RtPKMT2 Methyl
Dimethyl
Trimethyl
0.4 0.3 0.2 0.2 0.1 0.0 118 131 149 157 205 222 226 232 273 279 297 310 353 379 383 388 415 426 458 488 547 624 635 667 676 711 723 763 776 788 792 829 838 891 926 954 984 986 1026 1039 1056 1185 1228 1289 1319
Normalized fracons
Methyl 1.0 0.8 0.6 0.4 0.2 0.0
Lysine residue numbers Figure 2. Methylation Profiles of OmpB Catalyzed in vitro Using PKMT1 and PKMT2. Rickettsia typhi PKMT1 (RT776) (upper panel) and R. typhi PKMT2 (RT0101) (lower panel) catalyzed reactions using R. typhi rOmpB(AN) (residues 33–744) and (K) (residues 745–1353) as the substrates [36]. Color code is the same as that shown in Figure 1. Abbreviations: PKMT, protein lysine methyltransferase.
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of PKMT2 from R. typhi were examined, both the apo form (PDB ID 5DO[240_TD$IF]0 and 5DOO for PKMT1 and PKMT2, respectively) and complexes with its cofactor, AdoMet (5DPD for PKMT1) and AdoHcy (5DNK and 5DPL for PKMT1 and PKMT2, respectively) [39]. The overall 3D structures of PKMT1 and PKMT2 are superimposable with an RMSD of 1.51 Ǻ. PKMT1 and PKMT2 share four common structural domains consisting of an AdoMet binding domain, a dimerization domain, a middle domain, and a C-terminal domain. The four domains in PKMT1 and PKMT2 are spatially arranged as an open palm where the AdoMet binding domain occupies the center of the palm, the dimerization domain orients as the thumb, and the middle domain together with the C-terminal domain position as the fingers. The seven-strand b-sheet with a core Rossmann fold in the AdoMet binding domain conforms to type 1 methyltransferases but differs from the SET domain methyltransferases identified in almost all histone lysine methyltransferases [40,41]. In addition, AdoMet in the cofactor–PKMT complex opens to the centrally located cleft, in contrast to the SET domain of typical histone lysine methyltransferase with shielded AdoMet. The centrally located cleft along the base of the AdoMet binding domain may serve as the protein substrate-binding site. Consistent to this notion, alanine mutants such as W123A, Y219A, and F291A drastically reduce their catalytic activity for OmpB methylation [39]. The feature of a centrally open cleft resembles the structures of a few processive enzymes such as HIV reverse transcriptase (PDB ID: 1RTD), hyaluronate lyase, cellulase Cel6a (1QK2), and exonuclease I (1FXX) [42]. One plausible mechanism by which the enzyme could methylate multiple lysyl residues located at diverse sequences in a protein is that, initially, the enzyme may bind to a limited number of high affinity sites on the OmpB. The enzyme can then slide on the surface of OmpB to progressively methylate additional lysyl residues that would have an advantage of a reduced dimensionality effect [43,44]. In accordance with this notion, the proposed working model (Figure 3) shows that AdoMet binds closely to the bound OmpB in an extended conformation threaded along its putative binding site. Kinetic Mechanisms of [241_TD$IF]Multisite Methylation In-depth analysis of the kinetic mechanisms of PKMT1 and PKMT2 is complex because each OmpB molecule would serve as substrate of the enzyme multiple times, and Km and kcat for each lysyl residue at various locations may not be the same and may also be affected by methylation of any lysine residues [45]. Therefore, kinetic constants reported in the literature for
OmpB (model)
Putave OmpB binding site
90°
OmpB (model) AdoMet binding site AdoMet binding site
Figure 3. A Working Model of Substrate Binding by PKMT2. The working model of PKMT2–OmpB peptide complex shows the binding of an extended peptide fragment in green to PKMT2. The AdoMet binding site and the putative OmpB binding site are indicated. The dashed circle indicates the location of the elongated loop in PKMT2 [39]. Abbreviations: PKMT2, protein lysine methyltransferase 2.
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rickettsial methyltransferases can only be considered as apparent kinetic constants. Their apparent catalytic and Michaelis–Menten constants reveal that the catalytic efficiency, kcat/Km, of monomethyltransferase, PKMT1, is much greater than that of trimethyltransferase, PKMT2. The slow rate of trimethylation may involve the slow release of S-adenosylhomocysteine and subsequent binding of the structurally similar S-adenosylmethionine in consecutive methylation steps in processive trimethylation [46]. The methylation mechanism of OmpB in vivo is not known. Neither PKMT1 nor PKMT2 contains a signal peptide implying that they are localized in the cytoplasm. Thus, methylation of OmpB likely occurs in the bacterial cytoplasm before or during OmpB secretion to the outer leaflet of the outer membrane. Yet, despite the fact that PKMT2 shows disparate kinetic disadvantage relative to PKMT1, the majority of trimethyllysine in native OmpBs from virulent strains exhibits high level of trimethylation (Figure 1). Thus, methylation of OmpB in vivo likely requires additional factors that could enhance the process in favor of trimethylation relative to monomethylation. This could be achieved by cellular proteins and/or allosteric effectors that enhance binding of the trimethyltransferase relative to that of monomethyltransferase to the OmpB protein, and leading to trimethylation reaction.
O-Methylation of Msp4 Is Required for Infection by A. phagocytophilum Recent studies elegantly revealed that O-methylation of OMP Msp4 in A. phagocytophilum, the causative agents of human granulocytic anaplasmosis ([47,48], Adela S.O. Chávez, PhD thesis, University of Minnesota, 2014), plays important roles in infection of tick cells. Transmission of infectious diseases to humans frequently involves specific vectors as additional hosts. Ticks are the vectors that transmit anaplasmosis. Interestingly, an O-methyltransferase (OMT) is required for the bacterial infection of tick cells but not for the infection or survival in human cells. The OMT in the wild-type bacteria is upregulated 34-fold within 4 h after bacteria binding to tick cells, and the OMT upregulation is correlated with bacteria binding to and entry into tick cells. Through random transposon mutagenesis of A. phagocytophilum, an OMT deletion mutant, DOMT, was selected in HL60 cells. These mutants failed to grow in tick cells (ISE6). Furthermore, the mutant DOMT showed reduced binding to tick cells and the internalized bacteria were unable to replicate within tick cells [47]. The primary target of OMT was identified to be Msp4 by proteomic analysis using iTRAQ [49]. Figure 4 shows that two glutamic acid residues in Msp4 are specifically methylated, as revealed by LC-MS/MS analysis. Thus, the OMT, which specifically methylates Msp4, is required for its infection and the survival of A. phagocytophilum in tick cells. Complementary secondary mutation is yet to be carried out. Recombinant OMT catalyzes the methylation of Msp4 in vitro specifically and efficiently. The crystal structures of the 226-residue dimeric O-methyltransferase have been determined in its apo form (PDB ID:4OA8) and in complex with AdoMet (4OA5) or AdoHcy (4PCA). The presence of the sevenstrand b-sheet in the Ado-Met binding domain indicates that the O-methyltransferase belongs to type 1 methyltransferases.
N-[23_TD$IF]Methylation of Heparin-Binding Hemagglutinin (HBHA) in Mycobacterium tuberculosis (Mtb) Tuberculosis (TB), caused by Mtb-mediated infection, remains as one of the leading causes of morbidity and mortality worldwide. Mtb is a slow-growing and waxy bacterium. It generates HBHA, an important Mtb virulence factor, which functions as an adhesin involved in the attachment of mycobacteria to epithelial cells and leads to dissemination of Mtb from the site of primary infection. HBHA is located at the outer leaflet of the outer membrane of Mtb, and could bind to the sulfated glycoconjugates through its C-terminal Lys-rich repeats [50–52]. Importantly, HBHA serves as the major diagnostic target that differentiates active TB from asymptomatic latent TB [53,54], and it is a major vaccine candidate against TB [50]. LC-MS/MS
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Msp4
1
282
E E 126 124
HBHA 199
1 K K K K K K K K KKK K K 194 193 189 188 184 182 179 178 173 172 167 166 161
LipL32
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272 KK
199
246 245
178 172
K
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29
K K K K 152
K
OmpL32
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1 E E
E
EEE
E
54 45 43
115 106
172
205 200 190
235
E 261
EE E
Figure 4. The Sites of N- and O-Methylation in OMPs of Selected Pathogenic Bacteria. The residue numbers of methylated Glu in Msp4 of Anaplasma phagocytophilum [47], of methylated Lys in HBHA of Mycobacterium tuberculosis [109_TD$IF][55], of methylated Lys in LipL32 of Leptospira interrogans [18_TD$IF][62], and of methylated Glu in OmpL32 of L[29_TD$IF]. interrogans [64] are shown. Abbreviations: HBHA, heparin-binding hemagglutinin; LipL32, outer membrane protein from L[230_TD$IF]. interrogans; Msp4, major surface protein 4; OmpL32, outer membrane protein L32.
analysis revealed that the native HBHA contains 13 mono- and dimethylated lysyl residues at its C-terminal Lys-rich repeats (Figure 4) [55]. In vitro methylation of rHBHA catalyzed by enzyme[24_TD$IF](s) in mycobacterial cell extract yielded a similar methylation pattern as native HBHA [56]. These results indicate that the mycobacterial cell lysate contains all the methyltransferases for proper methylation [55]. The methylated HBHA exhibits a high degree of protection against Mtb challenge in mice, whereas unmethylated HBHA does not. Methylation of HBHA is also important for the induction of both T cell antigenicity and protective immunity to Mtb infection [52]. This notion is consistent with the observation that rHBHA is not immunogenic and methylation of rHBHA is required to exhibit full immunological properties of the protein. Thus, methylation of HBHA correlates with the immune response modulation, and may indirectly affect the virulence. The HBHA methyltransferase has yet to be identified.
N-[23_TD$IF]Methylation of LipL32 in L. interrogans Leptospirosis is re-emerging as the most widespread zoonotic disease worldwide. Leptospira are Gram-negative, long thin spirochetes that cause leptospirosis [57,58]. Infected rats are asymptomatic and serve as carriers of the pathogenic bacteria and shed the bacteria in their urine to transmit the bacteria through breaches in the skin. The most abundant OMP of Leptospira, LipL32, is specific to pathogenic Leptospira species and is implicated in the pathogenesis of leptospirosis [59–61]. LC-MS/MS analyses of native LipL32 purified from L. interrogans showed that LipL32 is trimethylated at several Lys residues of the protein (Figure 4) purified from Leptospira in the urine of infected rats, but not in the protein purified from in vitro cultivated bacteria [62]. The demonstration of trimethylation of Lys residues in LipL32 from in vivo isolated Leptospira exclusively implies the infection-generated modification may play a role during the course of infection. The crystal structures of LipL32 and its calcium-
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bound form indicate the trimethylated Lys residues are located on the surface of LipL32, and one trimethyllysine residue is located at the C-terminal sequence as the dominant epitope and the binding site of collagen [63]. The LipL32 methyltransferase has yet to be identified.
O-[23_TD$IF]Methylation of OmpL32 in L. interrogans Comparative proteome analysis of L. interrogans exposed to different growth conditions by varying the presence or absence of iron, fetal calf serum, or both, reveals differential expression of OmpL32 (gene locus, LIC11848) and the presence of multiple forms of OmpL32 [64]. OmpL32 is a surface exposed protein. LC-MS/MS analysis revealed a widespread methylation with differential patterns of O-methylation at 11 glutamic acid residues of OmpL32 under different growth conditions (Figure 4). The O-methylation of OmpL32 has yet to be correlated with virulence and their methyltransferases have not been identified. Nonetheless, the results demonstrated the presence of regulatory mechanisms in vivo for O-methylation of OMP in L. interrogans.
Concluding Remarks and Future Perspective In this review we focus [243_TD$IF]on the methylation of OMPs and bacterial virulence. Recent in-depth investigations, including biochemical characterization, and crystallographic and mechanistic analysis of methyltransferases that catalyze multimethylation of rickettsial OmpB have provided significant advances. Reports on methylation of OMPs of pathogenic Gram-negative bacteria have revealed not only insight into processes in infection, but also the weaknesses in host defense against these Gram-negative bacteria. Nevertheless, our knowledge on the roles of OMP methylation in bacterial virulence, including the effect of protein methylation on OMP structure, the catalytic and regulatory mechanism of OMP methylation remains largely unexplored. Figure 3 shows a working model for OmpB methylation catalyzed by rickettsial methyltransferase. This model was derived from the crystal structures of AdoMet-bound methyltransferases and observations that rickettsial methyltransferases catalyze OmpB methylation at multiple sites with limited specificity. In this model, a fully extended OmpB fragment is threading along its putative binding site to illustrate how OmpB may bind into the cleft above the cofactor-binding site. Note that examples of peptides in extended conformation binding to proteins have been reported in the literature [65–69]. It is conceivable that PKMT binding to an extended conformation of OmpB could facilitate enzymatic methylation at multiple lysyl residues. At the molecular level, multiple issues need to be addressed. To name a few, they include elucidating the mechanism by which methyltransferases carry out multisite methylation of protein substrates consisting of diverse amino acids sequences; revealing the effect of lysine mono- [24_TD$IF]7and trimethylation on the structures and export of OMP; investigating potential roles of processivity on the enzyme–protein substrates interaction to facilitate multisite methylation; analyzing the crystal structure of methyltransferase complexed with peptide-mimetic substrate to verify the working model shown in Figure 3; and developing an algorithm for analyzing the kinetics of multisite methylation (see Outstanding Questions). In addition, the biology that links methylation of OMPs and virulence is largely elusive. While the correlation between methyltransferases and certain rickettsial virulence has been demonstrated, the roles of methylation of OMPs and methyltransferases have yet to be clearly addressed. Furthermore, advances to overcome the technical hurdles of genetic manipulation of the obligatory intracellular pathogenic bacteria will undoubtedly aid moving this field forward. It is interesting to note that the methylated lysyl residues generated by PKMT1- and PKMT2catalyzed reactions are mainly monomethylated and trimethylated lysines, respectively, with
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Outstanding Questions What is the mechanism by which methyltransferases catalyze multisite methylation of OMPs with diverse amino acid sequences? Are OmpB methyltransferases required for typhus rickettsial virulence? How do various forms of OMP methylation exert their virulent effect? In vitro experiments reveal that the rate of OMP monomethylation is significantly faster than that of trimethylation. However, the levels of trimethylated lysyl residues in virulent OmpB are high relative to the monomethylated lysyl residues. How does virulent rickettsia overcome this kinetic deficiency? What biochemical factors regulate various types of OMP methylation?
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limited quantity of dimethyl lysine formed. These observations indicate that the PKMT2catalyzed trimethylation reaction likely proceeds via a processive mechanism, where the partially methylated intermediates are enzyme bound, in contrast to a distributive mechanism. Consistent to this notion, the OmpB purified from the avirulent strain Madrid E, which is highly monomethylated, does not serve as a good substrate for PKMT2 [36], indicating that trimethyltransferase uses mainly the unmethylated OmpB as its substrate. Thus, inactivating trimethyltransferase by mutation or specific inhibitors, and/or activating monomethyltransferase may lead to a reduction in virulent activity of rickettsial bacteria. We believe that future advances in our understanding of the nature of outer membrane methyltransferases, their catalytic and regulatory mechanisms, the effect of protein methylation on OMP structure, and the resulting biochemical effect on bacterial virulence would provide new insights to facilitate the development of novel diagnostic and therapeutic strategies. Acknowledgements The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the US Government. This work was supported/funded by work unit number 6000.RAD1.J.A0310 (WMC) and a contract (N32938-15-P-0254) to DCHY. WMC is employee of the U.S. Government. This work was prepared as part of their official duties. Title 17 U.S.C. x105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. x101 defines a U.S. Government work as a work prepared by military service member or employee of the U.S. Government as part of that person’s official duties.
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Review
Outer Membrane Protein OmpB Methylation May Mediate Bacterial Virulence David C.H. Yang,1,* Amila H. Abeykoon,2 Bok-Eum Choi,1 Wei-Mei Ching,3 and P. Boon Chock4 Methylation of outer membrane proteins (OMPs) has been implicated in bacterial virulence. Lysine methylation in rickettsial OmpB is correlated with rickettsial virulence, and N- and O-methylations are also observed in virulencerelevant OMPs from several pathogenic bacteria that cause typhus, leptospirosis, tuberculosis, and anaplasmosis. We summarize recent findings on the structure of methylated OmpB, biochemical characterization, and crystal structures of OmpB methyltransferases. Native rickettsial OmpB purified from highly virulent strains contains multiple clusters of trimethyllysine, in contrast with mostly monomethyllysine, and no trimethyllysine is found in an avirulent strain. Crystal structure of the methyltransferases reveals mechanistic insights for catalysis, and a working model is discussed for this unusual post-translational modification.
Trends Protein methylation plays vital roles in cellular regulation and signaling. Bacterial chemotaxis is regulated by methylation of specific membrane proteins to mediate excitation and adaptation in response to chemotaxis. O-[23_TD$IF]Methylation of Msp4, a surface protein, is required for Anaplasma phagocytophilum[23_TD$IF]–mediated infection, and [129_TD$IF]multisite N-methylation of OmpB correlates with typhus rickettsial virulence. Structural and mechanistic analysis of the newly characterized rickettsial methyltransferases led us to propose a mechanism in which the enzyme catalyzes multimethylation of OmpB.
Methylation of OMPs Covalent modification of proteins, such as methylation, alters their enzymatic activity and/or affinity to interact with other proteins, membranes, or nucleic acid [1,2]. Thus, protein methylation, generally catalyzed by methyltransferases, serves as a regulatory mechanism in epigenetics and cellular signaling [3]. Studies of protein methylation have primarily focused on histones [4,5]. More recently, such studies have extended to nonhistone proteins [6,7]. In bacteria, the methyl group is primarily known to attach to Lys, Asp, Glu, and Gln [8]. In the case of methylation at the lysyl residues, additional repertories of one, two, or three methyl groups would further expand their potential varying functionalities. The OMPs provide the first line of communication with their extracellular environment. They often function as adhesin, [234_TD$IF]invasin, or transporter, as well as playing a dominant role in host immune response. Methylation of OMPs has been implicated in the virulence of infectious diseases mediated by Gram-negative bacteria. Contribution of OMPs to bacterial virulence has been widely reported [9–13]. Examples include OmpB from Rickettsia prowazekii in typhus, LipL32 from Leptospira interrogans in leptospirosis, major surface protein [235_TD$IF]4 (Msp4) from Anaplasma phagocytophilum in human granulocytic anaplasmosis, Ail of Yersinia pestis in plague, BBK32, GAG from Borrelia burgdorferi in Lyme disease, and AlpB from Helicobacter pylori in gastric ulcers. Unlike integral membrane proteins from most other sources, the autotransporter domains of many OMPs do not consist of transmembrane a-helices, but instead fold into antiparallel b-barrels [14,15]. Recent investigation on methylation of bacterial OMPs has uncovered novel mechanisms with respect to protein structure and catalytic function in OMP methylation.
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We believe that knowing the catalytic and regulatory mechanism of OMP methylation, and how methylated OMP exerts its virulent effect, would provide new insights to facilitate the development of novel therapeutic and diagnostic strategies.
1
Department of Chemistry, Georgetown University, Washington[231_TD$IF], DC 20057, USA 2 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892, USA 3 Viral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, MD 20910, USA 4 Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA
http://dx.doi.org/10.1016/j.tibs.2017.09.005 © 2017 Elsevier Ltd. All rights reserved.
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In this review, we focus on methylation of OMPs from pathogenic bacteria and in-depth methylation studies of rickettsial OmpB, in view of the fact that the mechanism and the potential physiological functions of OmpB methylation have been extensively investigated recently. While the function of OMP methylation could be multifaceted, evidence suggests pivotal roles in bacterial virulence. The virulence of A. phagoctyophilum and infection of L. interrogans have recently been demonstrated to require methylation of their OMPs. Note that the biological function of protein reversible methylation in regulating bacterial chemotaxis, has been reviewed [16,17] and will not be covered here.
N-[23_TD$IF]Methylation of OmpB in R. prowazekii and Rickettsia [236_TD$IF]typhi Correlation of Rickettsial Virulence with OmpB Methylation Rickettsiae are Gram-negative, obligatory intracellular bacteria, and etiologic agents of typhus and spotted fever [18–20]. R. prowazekii and R. [237_TD$IF]typhi belong to the typhus group, while R. rickettsii and Rickettsia conorii belong to the spotted fever group. Virulent rickettsial strains induce symptoms such as fever, nausea, or diarrhea, and eventually may lead to multiorgan failure, while avirulent strains do not [19]. At the cellular level, virulent rickettsial strains grow fast in host cells, and induce cytokines or chemokines [21]. Rickettsial OMPs are localized at the outer leaflet of the outer membrane [22–25] and they play prominent roles in mediating host cell adhesion and invasion [26–29], as well as facilitating intracellular movement [30,31]. Amino acid analysis revealed that OmpBs from virulent strains of R. prowazekii contain more trimethylated lysyl residues than those of avirulent bacteria [32–34]. Consistent with a notion that lysine methylation may play an important role in rickettsial virulence, Zhang et al[238_TD$IF] showed that the gene Rp028/027, which encodes the lysine methyltransferase, was mutated by frameshift in the avirulent R. prowazekii Madrid E strain but not in the virulent revertant Evir and the virulent strain Breinl [35]. Bechah et al[238_TD$IF] investigated the correlation between lysine methylation in OmpB with the degree of virulence by carrying out a comparative investigation of four phenotypes of R. prowazekii that differed in virulence, using genomic, transcriptomic, and proteomic analysis [36]. They found that several genes that mediate the differences in virulence are mainly associated with surface proteins and post-translational modification. Together, results from genetic and biochemical studies suggest that lysine methylation of OMPs regulates bacterial virulence. Thus, current investigations are focusing on gaining new structural insights into OmpB methylation as well as the catalytic mechanisms governing the methylation of the OMPs. Methylation in Native OmpB from R. prowazekii and R. typhi To identify both methylated amino acids and the nature of methylation in native OmpB, OmpBs were isolated and purified from different species and strains of R. prowazekii and R. typhi for structural analysis using LC-tandem mass spectrometry (MS/MS) methods. LC-MS/MS analysis provides data on the methylation status of OmpB. The observed methylation profiles reveal that in the entire passenger domain of OmpB, multiple lysyl residues are either mono-, di-, and/ or trimethylated, and the methylation profile varies with the degree of virulence [37]. Figure 1 depicts the methylation profiles of OmpB from four different strains and species with varying virulence. The patterns of methylation in these profiles show extensive and widespread methylation throughout OmpB. No apparent consensus sequences at the methylated sites are discernable, but the differences in these profiles are apparent. OmpB from the avirulent Madrid E contains mono- and dimethyllysine residues but is devoid of trimethyllysine. In contrast, the highly virulent strains, Breinl and RP22, contain appreciable levels of trimethyllysine at relatively selective lysine residues, with several trimethyllysines occurring in clusters. OmpB from the R. typhi species, which is mildly virulent relative to Breinl and RP22 of R. prowazekii, showed intermediate levels of trimethylation. Thus the levels of trimethyllysine in OmpB from the four species and strains apparently correlate with their degree of virulence. In addition to the observations of pervasive mono- and trimethylation and the accompanied microheterogeneity of OmpB, the results raised two new aspects of post-translational
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*Correspondence: [email protected] (David C.H. Yang).
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Breinl
Methyl
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Trimethyl
120 130 156 204 231 272 278 296 309 352 382 387 414 417 425 487 546 574 594 623 634 666 710 722 762 775 787 791 828 837 891 925 926 954 984 986 1026 1059 1185 1228 1289 1319
1.0 0.8 0.6 0.4 0.2 0.0
Methyl
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120 130 156 204 231 272 278 296 309 352 382 387 414 417 425 487 546 574 594 623 634 666 710 722 762 775 787 791 828 837 891 925 926 954 984 986 1026 1059 1185 1228 1289 1319
Typhi
Methyl
Dimethyl
Trimethyl
1.0 0.8 0.6 0.4 0.2 0.0 118 131 149 157 205 222 226 232 273 279 297 310 353 379 383 388 415 426 458 488 547 624 635 667 676 711 723 763 776 788 792 829 838 891 926 954 984 986 1026 1039 1056 1185 1228 1289 1319
Normalized fracons
RP22 1.0 0.8 0.6 0.4 0.2 0.0
Madrid E
Methyl
Dimethyl
Trimethyl
120 130 156 204 231 272 278 296 309 352 382 387 414 417 425 487 546 574 594 623 634 666 710 722 762 775 787 791 828 837 891 925 926 954 984 986 1026 1059 1185 1228 1289 1319
1.0 0.8 0.6 0.4 0.2 0.0
Lysine residue numbers Figure 1. Methylation Profiles of Native OmpB Purified from Rickettsia. The location, methylation state, and normalized fraction of methylated Lys residues in native OmpBs purified from highly virulent Rickettsia prowazekii, Breinl strain (top panel) and RP22 strain (second), mildly virulent Rickettsia typhi (third) and avirulent R. prowazekii, Madrid E strain (fourth) are shown [37]. Monomethyllysine residues are shown in green, dimethyllysine in blue and trimethyllysine in red. Lysine residue numbers and the normalized fractions are shown on the x- and y-axes, respectively. The amino acid sequences of OmpBs from R. prowazekii and R. typhi differ, thus different numbering of lysyl residues. The observed number of peptide sequence matches was normalized using the total number of peptide sequence matches at all methylation states at each site.
modifications. First, this class of methyltransferases must be able to recognize multiple lysyl residues with different amino acid sequence contexts. Second, a complex enzyme mechanism is needed to achieve the observed wide range of methylation profiles throughout OmpB. To elucidate this catalytic mechanism, one needs to identify the enzymes involved. However, genetic manipulation of rickettsial genes is hampered by rickettsial obligate intracellular growth. Thus, bioinformatic analysis of genomic DNA of Rickettsia was carried out to identify the putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli [38] to characterize the methylation systems. OmpB Methyltransferases and Methylation of OmpB [239_TD$IF]in vitro Methylation of rOmpB (recombinant OmpB) fragments using the putative methyltransferases revealed the existence of lysine methyltransferases that target rOmpB but not histones or
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proteins from E. coli [38]. LC-MS/MS analysis of the OmpB modifications catalyzed by the methyltransferases demonstrated the existence of two types of protein lysine methyltransferases (PKMT). Figure 2 shows the methylation profiles of rOmpB catalyzed by PKMT1 and PKMT2. PKMT1 primarily catalyzes monomethylation, and PKMT2 catalyzes essentially trimethylation [37]. The two types of enzymes exhibit 45% sequence identity. PKMT2 methylates at relatively specific consensus sequences, KX(G/A/V/I) and KT(I/L/F), while monomethylation by PKMT1 occurs pervasively throughout OmpB. Similar to the known mono- and trimethyltransferases, the catalytic efficiency of PKMT1 is appreciably higher than that of PKMT2. However, these protein lysine methyltransferases are unusual in that they consist of >500 amino acid residues relative to the majority of known methyltransferases (220–350 residues). Comparing the locations of trimethyllysyl residues observed with the purified native OmpB from R. typhi (depicted in the third panel of Figure 1) with those found in rOmpB catalyzed by PKMT2 (Figure 2, lower panel) reveals that their methylation profiles are correlated. Similarly, the locations of monomethyllysyl residues found in the native OmpB from R. typhi (Figure 1, third panel) also correlated well with the methylation observed in rOmpB catalyzed by PKMT1 (Figure 2, top panel). Together, the close correlations of the methylation profiles found in native OmpB with those catalyzed by PKMT1 and PKMT2 using OmpB(AN) and (K) fragments suggests that these enzymes are likely the ones that catalyze the methylation of OmpB in vivo. The 3D structures of these enzymes are necessary to provide additional structural insight about substrate recognition and catalysis. Crystal Structures of Methyltransferases PKMT1 and PKMT2 The crystal structural data of PKMT1 and PKMT2 provide the first look at the 3D structures of protein lysine methyltransferases from Gram-negative bacteria. PKMT1 from R. prowazekii and
Methylaon catalyzed by RtPKMT1 Dimethyl
Trimethyl
118 131 149 157 205 222 226 232 273 279 297 310 353 379 383 388 415 426 458 488 547 624 635 667 676 711 723 763 776 788 792 829 838 891 926 954 984 986 1026 1039 1056 1185 1228 1289 1319
Methylaon catalyzed by RtPKMT2 Methyl
Dimethyl
Trimethyl
0.4 0.3 0.2 0.2 0.1 0.0 118 131 149 157 205 222 226 232 273 279 297 310 353 379 383 388 415 426 458 488 547 624 635 667 676 711 723 763 776 788 792 829 838 891 926 954 984 986 1026 1039 1056 1185 1228 1289 1319
Normalized fracons
Methyl 1.0 0.8 0.6 0.4 0.2 0.0
Lysine residue numbers Figure 2. Methylation Profiles of OmpB Catalyzed in vitro Using PKMT1 and PKMT2. Rickettsia typhi PKMT1 (RT776) (upper panel) and R. typhi PKMT2 (RT0101) (lower panel) catalyzed reactions using R. typhi rOmpB(AN) (residues 33–744) and (K) (residues 745–1353) as the substrates [36]. Color code is the same as that shown in Figure 1. Abbreviations: PKMT, protein lysine methyltransferase.
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of PKMT2 from R. typhi were examined, both the apo form (PDB ID 5DO[240_TD$IF]0 and 5DOO for PKMT1 and PKMT2, respectively) and complexes with its cofactor, AdoMet (5DPD for PKMT1) and AdoHcy (5DNK and 5DPL for PKMT1 and PKMT2, respectively) [39]. The overall 3D structures of PKMT1 and PKMT2 are superimposable with an RMSD of 1.51 Ǻ. PKMT1 and PKMT2 share four common structural domains consisting of an AdoMet binding domain, a dimerization domain, a middle domain, and a C-terminal domain. The four domains in PKMT1 and PKMT2 are spatially arranged as an open palm where the AdoMet binding domain occupies the center of the palm, the dimerization domain orients as the thumb, and the middle domain together with the C-terminal domain position as the fingers. The seven-strand b-sheet with a core Rossmann fold in the AdoMet binding domain conforms to type 1 methyltransferases but differs from the SET domain methyltransferases identified in almost all histone lysine methyltransferases [40,41]. In addition, AdoMet in the cofactor–PKMT complex opens to the centrally located cleft, in contrast to the SET domain of typical histone lysine methyltransferase with shielded AdoMet. The centrally located cleft along the base of the AdoMet binding domain may serve as the protein substrate-binding site. Consistent to this notion, alanine mutants such as W123A, Y219A, and F291A drastically reduce their catalytic activity for OmpB methylation [39]. The feature of a centrally open cleft resembles the structures of a few processive enzymes such as HIV reverse transcriptase (PDB ID: 1RTD), hyaluronate lyase, cellulase Cel6a (1QK2), and exonuclease I (1FXX) [42]. One plausible mechanism by which the enzyme could methylate multiple lysyl residues located at diverse sequences in a protein is that, initially, the enzyme may bind to a limited number of high affinity sites on the OmpB. The enzyme can then slide on the surface of OmpB to progressively methylate additional lysyl residues that would have an advantage of a reduced dimensionality effect [43,44]. In accordance with this notion, the proposed working model (Figure 3) shows that AdoMet binds closely to the bound OmpB in an extended conformation threaded along its putative binding site. Kinetic Mechanisms of [241_TD$IF]Multisite Methylation In-depth analysis of the kinetic mechanisms of PKMT1 and PKMT2 is complex because each OmpB molecule would serve as substrate of the enzyme multiple times, and Km and kcat for each lysyl residue at various locations may not be the same and may also be affected by methylation of any lysine residues [45]. Therefore, kinetic constants reported in the literature for
OmpB (model)
Putave OmpB binding site
90°
OmpB (model) AdoMet binding site AdoMet binding site
Figure 3. A Working Model of Substrate Binding by PKMT2. The working model of PKMT2–OmpB peptide complex shows the binding of an extended peptide fragment in green to PKMT2. The AdoMet binding site and the putative OmpB binding site are indicated. The dashed circle indicates the location of the elongated loop in PKMT2 [39]. Abbreviations: PKMT2, protein lysine methyltransferase 2.
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rickettsial methyltransferases can only be considered as apparent kinetic constants. Their apparent catalytic and Michaelis–Menten constants reveal that the catalytic efficiency, kcat/Km, of monomethyltransferase, PKMT1, is much greater than that of trimethyltransferase, PKMT2. The slow rate of trimethylation may involve the slow release of S-adenosylhomocysteine and subsequent binding of the structurally similar S-adenosylmethionine in consecutive methylation steps in processive trimethylation [46]. The methylation mechanism of OmpB in vivo is not known. Neither PKMT1 nor PKMT2 contains a signal peptide implying that they are localized in the cytoplasm. Thus, methylation of OmpB likely occurs in the bacterial cytoplasm before or during OmpB secretion to the outer leaflet of the outer membrane. Yet, despite the fact that PKMT2 shows disparate kinetic disadvantage relative to PKMT1, the majority of trimethyllysine in native OmpBs from virulent strains exhibits high level of trimethylation (Figure 1). Thus, methylation of OmpB in vivo likely requires additional factors that could enhance the process in favor of trimethylation relative to monomethylation. This could be achieved by cellular proteins and/or allosteric effectors that enhance binding of the trimethyltransferase relative to that of monomethyltransferase to the OmpB protein, and leading to trimethylation reaction.
O-Methylation of Msp4 Is Required for Infection by A. phagocytophilum Recent studies elegantly revealed that O-methylation of OMP Msp4 in A. phagocytophilum, the causative agents of human granulocytic anaplasmosis ([47,48], Adela S.O. Chávez, PhD thesis, University of Minnesota, 2014), plays important roles in infection of tick cells. Transmission of infectious diseases to humans frequently involves specific vectors as additional hosts. Ticks are the vectors that transmit anaplasmosis. Interestingly, an O-methyltransferase (OMT) is required for the bacterial infection of tick cells but not for the infection or survival in human cells. The OMT in the wild-type bacteria is upregulated 34-fold within 4 h after bacteria binding to tick cells, and the OMT upregulation is correlated with bacteria binding to and entry into tick cells. Through random transposon mutagenesis of A. phagocytophilum, an OMT deletion mutant, DOMT, was selected in HL60 cells. These mutants failed to grow in tick cells (ISE6). Furthermore, the mutant DOMT showed reduced binding to tick cells and the internalized bacteria were unable to replicate within tick cells [47]. The primary target of OMT was identified to be Msp4 by proteomic analysis using iTRAQ [49]. Figure 4 shows that two glutamic acid residues in Msp4 are specifically methylated, as revealed by LC-MS/MS analysis. Thus, the OMT, which specifically methylates Msp4, is required for its infection and the survival of A. phagocytophilum in tick cells. Complementary secondary mutation is yet to be carried out. Recombinant OMT catalyzes the methylation of Msp4 in vitro specifically and efficiently. The crystal structures of the 226-residue dimeric O-methyltransferase have been determined in its apo form (PDB ID:4OA8) and in complex with AdoMet (4OA5) or AdoHcy (4PCA). The presence of the sevenstrand b-sheet in the Ado-Met binding domain indicates that the O-methyltransferase belongs to type 1 methyltransferases.
N-[23_TD$IF]Methylation of Heparin-Binding Hemagglutinin (HBHA) in Mycobacterium tuberculosis (Mtb) Tuberculosis (TB), caused by Mtb-mediated infection, remains as one of the leading causes of morbidity and mortality worldwide. Mtb is a slow-growing and waxy bacterium. It generates HBHA, an important Mtb virulence factor, which functions as an adhesin involved in the attachment of mycobacteria to epithelial cells and leads to dissemination of Mtb from the site of primary infection. HBHA is located at the outer leaflet of the outer membrane of Mtb, and could bind to the sulfated glycoconjugates through its C-terminal Lys-rich repeats [50–52]. Importantly, HBHA serves as the major diagnostic target that differentiates active TB from asymptomatic latent TB [53,54], and it is a major vaccine candidate against TB [50]. LC-MS/MS
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Msp4
1
282
E E 126 124
HBHA 199
1 K K K K K K K K KKK K K 194 193 189 188 184 182 179 178 173 172 167 166 161
LipL32
20
272 KK
199
246 245
178 172
K
166
29
K K K K 152
K
OmpL32
276
1 E E
E
EEE
E
54 45 43
115 106
172
205 200 190
235
E 261
EE E
Figure 4. The Sites of N- and O-Methylation in OMPs of Selected Pathogenic Bacteria. The residue numbers of methylated Glu in Msp4 of Anaplasma phagocytophilum [47], of methylated Lys in HBHA of Mycobacterium tuberculosis [109_TD$IF][55], of methylated Lys in LipL32 of Leptospira interrogans [18_TD$IF][62], and of methylated Glu in OmpL32 of L[29_TD$IF]. interrogans [64] are shown. Abbreviations: HBHA, heparin-binding hemagglutinin; LipL32, outer membrane protein from L[230_TD$IF]. interrogans; Msp4, major surface protein 4; OmpL32, outer membrane protein L32.
analysis revealed that the native HBHA contains 13 mono- and dimethylated lysyl residues at its C-terminal Lys-rich repeats (Figure 4) [55]. In vitro methylation of rHBHA catalyzed by enzyme[24_TD$IF](s) in mycobacterial cell extract yielded a similar methylation pattern as native HBHA [56]. These results indicate that the mycobacterial cell lysate contains all the methyltransferases for proper methylation [55]. The methylated HBHA exhibits a high degree of protection against Mtb challenge in mice, whereas unmethylated HBHA does not. Methylation of HBHA is also important for the induction of both T cell antigenicity and protective immunity to Mtb infection [52]. This notion is consistent with the observation that rHBHA is not immunogenic and methylation of rHBHA is required to exhibit full immunological properties of the protein. Thus, methylation of HBHA correlates with the immune response modulation, and may indirectly affect the virulence. The HBHA methyltransferase has yet to be identified.
N-[23_TD$IF]Methylation of LipL32 in L. interrogans Leptospirosis is re-emerging as the most widespread zoonotic disease worldwide. Leptospira are Gram-negative, long thin spirochetes that cause leptospirosis [57,58]. Infected rats are asymptomatic and serve as carriers of the pathogenic bacteria and shed the bacteria in their urine to transmit the bacteria through breaches in the skin. The most abundant OMP of Leptospira, LipL32, is specific to pathogenic Leptospira species and is implicated in the pathogenesis of leptospirosis [59–61]. LC-MS/MS analyses of native LipL32 purified from L. interrogans showed that LipL32 is trimethylated at several Lys residues of the protein (Figure 4) purified from Leptospira in the urine of infected rats, but not in the protein purified from in vitro cultivated bacteria [62]. The demonstration of trimethylation of Lys residues in LipL32 from in vivo isolated Leptospira exclusively implies the infection-generated modification may play a role during the course of infection. The crystal structures of LipL32 and its calcium-
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bound form indicate the trimethylated Lys residues are located on the surface of LipL32, and one trimethyllysine residue is located at the C-terminal sequence as the dominant epitope and the binding site of collagen [63]. The LipL32 methyltransferase has yet to be identified.
O-[23_TD$IF]Methylation of OmpL32 in L. interrogans Comparative proteome analysis of L. interrogans exposed to different growth conditions by varying the presence or absence of iron, fetal calf serum, or both, reveals differential expression of OmpL32 (gene locus, LIC11848) and the presence of multiple forms of OmpL32 [64]. OmpL32 is a surface exposed protein. LC-MS/MS analysis revealed a widespread methylation with differential patterns of O-methylation at 11 glutamic acid residues of OmpL32 under different growth conditions (Figure 4). The O-methylation of OmpL32 has yet to be correlated with virulence and their methyltransferases have not been identified. Nonetheless, the results demonstrated the presence of regulatory mechanisms in vivo for O-methylation of OMP in L. interrogans.
Concluding Remarks and Future Perspective In this review we focus [243_TD$IF]on the methylation of OMPs and bacterial virulence. Recent in-depth investigations, including biochemical characterization, and crystallographic and mechanistic analysis of methyltransferases that catalyze multimethylation of rickettsial OmpB have provided significant advances. Reports on methylation of OMPs of pathogenic Gram-negative bacteria have revealed not only insight into processes in infection, but also the weaknesses in host defense against these Gram-negative bacteria. Nevertheless, our knowledge on the roles of OMP methylation in bacterial virulence, including the effect of protein methylation on OMP structure, the catalytic and regulatory mechanism of OMP methylation remains largely unexplored. Figure 3 shows a working model for OmpB methylation catalyzed by rickettsial methyltransferase. This model was derived from the crystal structures of AdoMet-bound methyltransferases and observations that rickettsial methyltransferases catalyze OmpB methylation at multiple sites with limited specificity. In this model, a fully extended OmpB fragment is threading along its putative binding site to illustrate how OmpB may bind into the cleft above the cofactor-binding site. Note that examples of peptides in extended conformation binding to proteins have been reported in the literature [65–69]. It is conceivable that PKMT binding to an extended conformation of OmpB could facilitate enzymatic methylation at multiple lysyl residues. At the molecular level, multiple issues need to be addressed. To name a few, they include elucidating the mechanism by which methyltransferases carry out multisite methylation of protein substrates consisting of diverse amino acids sequences; revealing the effect of lysine mono- [24_TD$IF]7and trimethylation on the structures and export of OMP; investigating potential roles of processivity on the enzyme–protein substrates interaction to facilitate multisite methylation; analyzing the crystal structure of methyltransferase complexed with peptide-mimetic substrate to verify the working model shown in Figure 3; and developing an algorithm for analyzing the kinetics of multisite methylation (see Outstanding Questions). In addition, the biology that links methylation of OMPs and virulence is largely elusive. While the correlation between methyltransferases and certain rickettsial virulence has been demonstrated, the roles of methylation of OMPs and methyltransferases have yet to be clearly addressed. Furthermore, advances to overcome the technical hurdles of genetic manipulation of the obligatory intracellular pathogenic bacteria will undoubtedly aid moving this field forward. It is interesting to note that the methylated lysyl residues generated by PKMT1- and PKMT2catalyzed reactions are mainly monomethylated and trimethylated lysines, respectively, with
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Outstanding Questions What is the mechanism by which methyltransferases catalyze multisite methylation of OMPs with diverse amino acid sequences? Are OmpB methyltransferases required for typhus rickettsial virulence? How do various forms of OMP methylation exert their virulent effect? In vitro experiments reveal that the rate of OMP monomethylation is significantly faster than that of trimethylation. However, the levels of trimethylated lysyl residues in virulent OmpB are high relative to the monomethylated lysyl residues. How does virulent rickettsia overcome this kinetic deficiency? What biochemical factors regulate various types of OMP methylation?
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limited quantity of dimethyl lysine formed. These observations indicate that the PKMT2catalyzed trimethylation reaction likely proceeds via a processive mechanism, where the partially methylated intermediates are enzyme bound, in contrast to a distributive mechanism. Consistent to this notion, the OmpB purified from the avirulent strain Madrid E, which is highly monomethylated, does not serve as a good substrate for PKMT2 [36], indicating that trimethyltransferase uses mainly the unmethylated OmpB as its substrate. Thus, inactivating trimethyltransferase by mutation or specific inhibitors, and/or activating monomethyltransferase may lead to a reduction in virulent activity of rickettsial bacteria. We believe that future advances in our understanding of the nature of outer membrane methyltransferases, their catalytic and regulatory mechanisms, the effect of protein methylation on OMP structure, and the resulting biochemical effect on bacterial virulence would provide new insights to facilitate the development of novel diagnostic and therapeutic strategies. Acknowledgements The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the US Government. This work was supported/funded by work unit number 6000.RAD1.J.A0310 (WMC) and a contract (N32938-15-P-0254) to DCHY. WMC is employee of the U.S. Government. This work was prepared as part of their official duties. Title 17 U.S.C. x105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. x101 defines a U.S. Government work as a work prepared by military service member or employee of the U.S. Government as part of that person’s official duties.
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