A mutation inactivating the methyltransferase gene in avirulent Madrid E strain of Rickettsia prowazekii reverted to wild type in the virulent revertant strain Evir

Zhang, Jian Zhi; Hao, Jun; Walker, David H.; Yu, Xue Jie

doi:10.1016/j.vaccine.2005.11.044

Cited by 37 publications

(26 citation statements)

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“…Two fl ying squirrel isolates from the United States were differentiated by a single nucleotide substitution at position 480 in rp028. E strain and its revertant Evir strain differed by a single nucleotide insertion in E strain at position 732 in rp028, which we reported previously (9). Breinl and Cairo strains were closely related but were differentiated by several deletion/insertion mutations in rp181 and the spacer between rp272 and rp273.…”

Section: Discussionsupporting

confidence: 60%

“…We also sequenced rp028, the methyltransferase gene, because we wanted to know if this gene was inactivated in any virulent strain of R. prowazekii. Pseudogene rp028 was inactivated in a virulent E strain but not in its virulent revertant Evir strain (9). Coincident with inactivation of the methyltransferase gene, E strain is defi cient in methylation of surface proteins (10,11).…”

Section: Discussionmentioning

confidence: 99%

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GenotypingRickettsia prowazekiiIsolates

Zhu

Medina-Sanchez

Bouyer

et al. 2008

Emerg. Infect. Dis.

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Section: Discussionsupporting

confidence: 60%

Section: Discussionmentioning

confidence: 99%

GenotypingRickettsia prowazekiiIsolates

Zhu

Medina-Sanchez

Bouyer

et al. 2008

Emerg. Infect. Dis.

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“…This protein encoded by open reading frames RP028 and RP027 was expressed in the virulent Breinl strain but not in the avirulent Madrid E strain (8). The finding was supported by a frameshift mutation of the gene only in the avirulent Madrid E strain (46). BLASTP analysis showed that a hypothetical protein of R. parkeri (spot 29) shared 86 and 87% amino acid sequence identity to R. prowazekii proteins encoded by RP027 and RP028, respectively, suggesting the expression of methyltransferase in R. parkeri.…”

Section: Discussionmentioning

confidence: 85%

Proteomic Analysis of Rickettsia parkeri Strain Portsmouth

et al. 2009

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Rickettsia parkeri, a recently recognized pathogen of human, is one of several Rickettsia spp. in the United States that causes a spotted fever rickettsiosis. To gain insights into its biology and pathogenesis, we applied the proteomics approach to establish a two-dimensional gel proteome reference map and combined this technique with cell surface biotinylation to identify surface-exposed proteins of a low-passage isolate of R. parkeri obtained from a patient. We identified 91 proteins by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. Of these, 28 were characterized as surface proteins, including virulence-related proteins (e.g., outer membrane protein A [OmpA], OmpB, ␤-peptide, and RickA). Two-dimensional immunoblotting with serum from the R. parkeri-infected index patient was utilized to identify the immunoreactive proteins as potential targets for diagnosis and vaccine development. In addition to the known rickettsial antigens, OmpA and OmpB, we identified translation initiation factor 2, cell division protein FtsZ, and cysteinyl-tRNA synthetase as immunoreactive proteins. The proteome map with corresponding cell surface protein analysis and antigen detection will facilitate a better understanding of the mechanisms of rickettsial pathogenesis.Rickettsia parkeri, a member of the spotted fever group Rickettsia (SFGR), was first isolated from the Gulf Coast tick, Amblyomma maculatum, in 1937 (29). In 2004, the first confirmed human infection with R. parkeri was reported in a 40-year-old man from the Tidewater area of coastal Virginia. The agent was isolated in cell culture from an eschar biopsy specimen and designated the Portsmouth strain (28). Recently, the first recognized case of tick bite-associated human infection was described (43); however, the epidemiology of R. parkeri is not well defined. In the United States, R. parkeri has been detected in A. maculatum and A. americanum; the geographical overlap between R. parkeri and these ticks with that of the vectors of R. rickettsii (the etiological agent of Rocky Mountain spotted fever [RMSF]) suggests that many cases of R. parkeri infection have been misidentified as RMSF (27,35). For example, Western blot analysis of serum specimens from 15 U.S. patients previously diagnosed with RMSF identified four serum specimens reactive with a 120-kDa protein of R. parkeri, suggesting infection with R. parkeri rather than R. rickettsii (30). However, a serologic test specific for this pathogen is not available (43), and little is known about its biology.Due to their obligate intracellular nature, genetic manipulation of Rickettsia has proven difficult. Alternatively, protein expression profiles (proteomes) are utilized to identify the mechanisms of pathogenesis and differentiate rickettsial species recognizing host immune response specificity to cell surface molecules, referred to as outer membrane proteins (Omps). The presence or absence of some Omps allows for differentiation between the typhus group and the SFGR, and the response...

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“…Genomic sequence and transcription analyses showed that the mutation of RP027/RP028 in the Madrid E strain reverts back to the wild type (39). The gene encoding one of the putative methyltransferases RP027/RP028 was found to have a frameshift mutation in the avirulent strain, resulting in two split genes, the RP027 and RP028 genes, which are detectable only in avirulent strains (4).…”

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Two Protein Lysine Methyltransferases Methylate Outer Membrane Protein B from Rickettsia

Abeykoon

Chao

Wang³

et al. 2012

J Bacteriol

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c Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickelnitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10-to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatographytandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates. Rickettsial OmpBs belong to the outer membrane protein autotransporter family and contain large passenger domains. Rickettsial OmpBs have been shown to participate in adhesion to mammalian cells in vitro (8,36). Ectopically expressed rickettsial OmpB in Escherichia coli has been demonstrated to be sufficient in mediating bacterial attachment and invasion of cultured mammalian cells (8,24). Unlike the ␣-helical transmembrane proteins in plasma membranes, the autotransporter domains of these outer membrane proteins have structural characteristics of ␤-barrel integral membrane proteins (3, 33). As the immunodominant antigenic surface protein, native OmpB induces strong humoral and cellular immune responses in animal models and in patients (6,12,15,16,23). Methylation of OmpA of Rickettsia canadensis and ompB of Rickettsia typhi is known, but whether this is important for any other pathogenic rickettsia is unknown.One important but not well understood structural feature of rickettsial OmpB is the methylation at ε-amino groups of lysine residues (28). Postt...

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A mutation inactivating the methyltransferase gene in avirulent Madrid E strain of Rickettsia prowazekii reverted to wild type in the virulent revertant strain Evir

Cited by 37 publications

References 20 publications

GenotypingRickettsia prowazekiiIsolates

GenotypingRickettsia prowazekiiIsolates

Proteomic Analysis of Rickettsia parkeri Strain Portsmouth

Two Protein Lysine Methyltransferases Methylate Outer Membrane Protein B from Rickettsia

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