Molecular Evolution of Rickettsia Surface Antigens: Evidence of Positive Selection
http://www.100md.com
分子生物学进展 2005年第10期
* Information Génomique et Structurale, UPR 2589, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France; and Unité des rickettsies, IFR 48 CNRS UMR 6020, Faculté de médecine, Université de la Méditerranée, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
E-mail: guillaume.blanc@igs.cnrs-mrs.fr.
Abstract
The Rickettsia genus is a group of obligate intracellular parasitic -proteobacteria that includes human pathogens responsible for the typhus disease and various types of spotted fevers. rOmpA and rOmpB are two members of the "surface cell antigen" (Sca) autotransporter (AT) protein family that may play key roles in the adhesion of the Rickettsia cells to the host tissue. These molecules are likely determinants for the pathogenicity of the Rickettsia and represent good candidates for vaccine development. We identified the 17 members of this family of outer-membrane proteins in nine fully sequenced Rickettsia genomes. The typical architecture of the Sca proteins is composed of an N-terminal signal peptide and a C-terminal AT domain that promote the export of the central passenger domain to the outside of the bacteria. A characteristic of this family is the frequent degradation of the genes, which results in different subsets of the sca genes being expressed among Rickettsia species. Here, we present a detailed analysis of their phylogenetic relationships and evolution. We provide strong evidence that rOmpA and rOmpB as well as three other members of the Sca protein family—Sca1, Sca2, and Sca4—have evolved under positive selection. The exclusive distribution of the predicted positively selected sites within the passenger domains of these proteins argues that these regions are involved in the interaction with the host and may be locked in "arms race" coevolutionary conflicts.
Key Words: adaptive evolution ? Rickettsia ? autotransporter ? adhesin ? antigen
Introduction
The Rickettsia are obligate intracellular parasites belonging to the -protobacteria. They are associated with arthropods (i.e., flea, tick, mite, and lice) and often pathogenic for humans. Members of the Rickettsia genus include the etiologic agents of epidemic and murine typhus (Rickettsia prowazekii and Rickettsia typhi), as well as the etiologic agents of the Rocky Mountain spotted fever (Rickettsia rickettsii) and the Mediterranean spotted fever (Rickettsia conorii), two tick-borne diseases. Due to their medical importance, this group of bacteria has justified considerable sequencing efforts over the last 7 years. To date, the genome sequences of three Rickettsia species have been published (R. conorii [Ogata et al. 2001], R. prowazekii [Andersson et al. 1998], and R. typhi [McLeod et al. 2004]) and at least six other sequencing projects are under way for Rickettsia belli, Rickettsia africae, Rickettsia sibirica, R. rickettsii, Rickettsia akari, and Rickettsia felis. This abundance of data, coupled to a well-preserved colinearity of the genes, make them a very good model to study bacterial evolution.
Rickettsia cells are surrounded by a crystalline proteic layer (Palmer, Martin, and Mallavia 1974), also referred to as S-layer, which represents 10% to 15% of their total protein mass (Ching et al. 1996) and is composed of immunodominant surface protein antigens (SPA) (Dasch 1981; Ching et al. 1990; Ching, Carl, and Dasch 1992). Two SPAs, i.e., rOmpA (Anacker et al. 1987; Vishwanath, McDonald, and Watkins 1990) and rOmpB (Gilmore, Joste, and McDonald 1989; Gilmore et al. 1991), have been identified in several Rickettsia species and are the major antigenic determinants eliciting an immune response in patients infected by rickettsioses (Teysseire and Raoult 1992). rOmpA and rOmpB are two large proteins (about 2,000 and 1,600 aa, respectively) that share no significant sequence similarity except that they exhibit a highly conserved 300-aa C-termini. This region, the autotransporter (AT) domain, folds into a ?-barrel structure and inserts into the outer membrane. This structure forms a pore through which the central passenger domain of the proprotein is transported across the membrane (Desvaux, Parham, and Henderson 2004). Experimental studies have demonstrated that following export, the rOmpB proprotein is proteolytically cleaved to release the passenger peptide at the cell surface (Ching et al. 1990; Gilmore et al. 1991; Ching, Carl, and Dasch 1992; Hackstadt et al. 1992). In addition, rOmpA and rOmpB possess an N-terminal signal peptide allowing their passage through the inner membrane presumably by means of the Sec secretion system (Henderson et al. 2004).
Members of the AT protein superfamily are found in -, ?-, -, and -proteobacteria as well as in Chlamydia species (Henderson and Nataro 2001). They all share a homologous C-terminal AT domain. In contrast, the passenger domains are not all homologous and encode for a wide variety of virulence factors that can catalyze proteolysis, serve as adhesins, mediate actin-promoted bacterial motility, or act as cytotoxins to animal cells (Henderson and Nataro 2001). Experimental studies have suggested that rOmpA and rOmpB function as adhesin in Rickettsia (Li and Walker 1998; Uchiyama 2003).
Analyses of the genome sequences of R. conorii (Ogata et al. 2001) and R. prowazekii (Andersson et al. 1998) have revealed the existence of three additional genes encoding a highly conserved C-terminal AT domain. These genes are annotated as "surface cell antigen" (sca) genes (i.e., sca1, 2, 3), together with rOmpA (sca0) and rOmpB (sca5). In addition, "GeneD" (Sekeyova, Roux, and Raoult 2001) has been renamed sca4 (Andersson et al. 1998), though the protein product lacks the AT domain. So far, only rOmpA and rOmpB proteins have been detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis or western blotting in R. conorii (Teysseire and Raoult 1992) as well as Sca4 in R. japonica (Uchiyama, Zhao, and Uchida 1996).
Given the seemingly prominent role of rOmpA and rOmpB proteins in the virulence of rickettsiae, we further characterized the Sca protein family by using the data from six publicly available Rickettsia genomes as well as the complete genome sequences of R. belli, R. felis, and R. africae that have been recently determined in our laboratory. We constructed an updated catalog of 17 subfamilies of sca genes found in nine Rickettsia spp. and analyzed their phylogenetic relationships. In addition, we have investigated the selective pressure acting on the Sca proteins. Our results suggest that the passenger domains of rOmpA, rOmpB, Sca1, and Sca2 as well as the Sca4 protein have evolved under positive selection.
Methods
Data
The genome sequences of R. conorii (NC_003103), R. typhi (NC_006142), R. sibirica (NZ_AABW00000000), R. rickettsii (NZ_AADJ00000000), R. felis (CP000053, CP000054) R. akari (NZ_AAFE00000000), and R. prowazekii (NC_000963) were downloaded from GenBank. For these species, the complete set of proteins was constructed using the GenBank annotations. We used the complete genome sequences of R. belli and R. africae that have been recently determined in our laboratory and will be published elsewhere. Every open reading frames (ORFs) of more than 100 nonstop codons were collected from the six possible reading frames and virtually translated into proteins. For these organisms, the ORF nucleotide sequences of the sca genes were released in GenBank. Locus names and GenBank accession numbers of all analyzed sequences are available as Supplementary Material online. For the study of selective constraints acting on sca genes, only the second halves of the gene sequences were available in most organisms for rOmpA and sca1. In consequence, subsequent analyses were restricted to these regions of proteins, which contain part of the passenger domain and the complete AT domain.
Sequence analysis
ORF products with sequence similarity with the Sca proteins previously identified in R. conorii and R. prowazekii (i.e., rOmpA, rOmpB, Sca1, Sca2, Sca3, and Sca4) were searched in the nine complete rickettsial proteomes using the BlastP program (E value < 1 x 10–10; Altschul et al. 1997). The AT domains were searched in the Sca protein sequences and the GenBank protein database with the program HMMSEARCH from the HMMER package (R. S. Eddy, unpublished data, http://hmmer.wustl.edu/), using the Pfam AT domain HMM profile (PF03797.6) as query. Hits were considered significant when they matched the Pfam profile with E values 10–5. Orthologous relationships between sca ORFs were determined by the reciprocal best BlastP match criterion. Except for R. bellii, the Rickettsia genomes exhibit a nearly perfect colinearity and few genomic rearrangements (Ogata et al. 2001). Hence, we confirmed the orthologous relationships by verifying that the genes surrounding each orthologous sca genes were in collinear order. Repeated peptide motifs were identified using the dotter program (Sonnhammer and Durbin 1995). Predictions of N-terminal signal peptides were done using the TMHMM web tool at http://www.cbs.dtu.dk/services/TMHMM/ (Krogh et al. 2001). The significance of sequence similarity between paralogous passenger domains was assessed using the program PRDF from the FASTA2 package (Pearson 1990) with default parameters. The threshold of significance was set to P value (opt delta) < 0.01.
Phylogenetic analysis
Protein and nucleotide alignments were carried out using the programs MUSCLE (Edgar 2004) and ClustalW (Higgins and Sharp 1988), respectively, and corrected manually. We constructed the phylogenetic trees of the sca5 nucleotide sequences and AT domains (fig. 2) using the Neighbor-Joining (NJ) method implemented in the MEGA software (Kumar, Tamura, and Nei 1994). Phylogenetic distances were calculated using the Tamura-Nei method (Tamura and Nei 1993) for nucleotide sequences and a gamma correction (alpha = 3.34) for the AT domains. The shape parameter alpha for the gamma correction was determined by maximum likelihood using the CODEML program from the PAML package (Yang 1997). For all tree constructions, bootstrap supports for branches were assessed using 100 pseudoreplicates.
a single ORF encoding a protein of less than 50% of the longest orthologous protein), or absent (flag "-"). Split genes and gene fragments may be the result of ongoing gene degradation, a common feature in rickettsial genomes that is concomitant with reductive evolution (Andersson et al. 1998; Ogata et al. 2001). Thus, these may be incapable of producing functional proteins and could be pseudogenes. None of the nine Rickettsia genomes studied here harbor the 17 intact sca genes (fig. 1). Rickettsia prowazekii is remarkable as it retained only two intact sca genes (sca3 and rOmpB). On the other extreme, the R. felis genome contains the largest complement of sca genes with 10 complete and 3 split sca genes. rOmpB is the only sca gene found complete in all the nine genomes studied.
Table 2 Likelihood Ratio Tests of Positive Selection
Positively selected amino acid sites were identified under model M8 using the Bayesian method developed by Nielsen and Yang (1998). Consistent with our hypothesis that only the passenger domains are involved in host-parasite interactions, the predicted positively selected sites are exclusively located within this region of Sca1, Sca2, rOmpA, and rOmpB (fig. 3). The distributions of the predicted positively selected sites between passenger and AT domains are highly nonrandom (P < 3.2 x 10–4; binomial distribution), which further confirms that the two domains have evolved under different selection regimes.
//www.mbe.oxfordjournals.org/).
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E-mail: guillaume.blanc@igs.cnrs-mrs.fr.
Abstract
The Rickettsia genus is a group of obligate intracellular parasitic -proteobacteria that includes human pathogens responsible for the typhus disease and various types of spotted fevers. rOmpA and rOmpB are two members of the "surface cell antigen" (Sca) autotransporter (AT) protein family that may play key roles in the adhesion of the Rickettsia cells to the host tissue. These molecules are likely determinants for the pathogenicity of the Rickettsia and represent good candidates for vaccine development. We identified the 17 members of this family of outer-membrane proteins in nine fully sequenced Rickettsia genomes. The typical architecture of the Sca proteins is composed of an N-terminal signal peptide and a C-terminal AT domain that promote the export of the central passenger domain to the outside of the bacteria. A characteristic of this family is the frequent degradation of the genes, which results in different subsets of the sca genes being expressed among Rickettsia species. Here, we present a detailed analysis of their phylogenetic relationships and evolution. We provide strong evidence that rOmpA and rOmpB as well as three other members of the Sca protein family—Sca1, Sca2, and Sca4—have evolved under positive selection. The exclusive distribution of the predicted positively selected sites within the passenger domains of these proteins argues that these regions are involved in the interaction with the host and may be locked in "arms race" coevolutionary conflicts.
Key Words: adaptive evolution ? Rickettsia ? autotransporter ? adhesin ? antigen
Introduction
The Rickettsia are obligate intracellular parasites belonging to the -protobacteria. They are associated with arthropods (i.e., flea, tick, mite, and lice) and often pathogenic for humans. Members of the Rickettsia genus include the etiologic agents of epidemic and murine typhus (Rickettsia prowazekii and Rickettsia typhi), as well as the etiologic agents of the Rocky Mountain spotted fever (Rickettsia rickettsii) and the Mediterranean spotted fever (Rickettsia conorii), two tick-borne diseases. Due to their medical importance, this group of bacteria has justified considerable sequencing efforts over the last 7 years. To date, the genome sequences of three Rickettsia species have been published (R. conorii [Ogata et al. 2001], R. prowazekii [Andersson et al. 1998], and R. typhi [McLeod et al. 2004]) and at least six other sequencing projects are under way for Rickettsia belli, Rickettsia africae, Rickettsia sibirica, R. rickettsii, Rickettsia akari, and Rickettsia felis. This abundance of data, coupled to a well-preserved colinearity of the genes, make them a very good model to study bacterial evolution.
Rickettsia cells are surrounded by a crystalline proteic layer (Palmer, Martin, and Mallavia 1974), also referred to as S-layer, which represents 10% to 15% of their total protein mass (Ching et al. 1996) and is composed of immunodominant surface protein antigens (SPA) (Dasch 1981; Ching et al. 1990; Ching, Carl, and Dasch 1992). Two SPAs, i.e., rOmpA (Anacker et al. 1987; Vishwanath, McDonald, and Watkins 1990) and rOmpB (Gilmore, Joste, and McDonald 1989; Gilmore et al. 1991), have been identified in several Rickettsia species and are the major antigenic determinants eliciting an immune response in patients infected by rickettsioses (Teysseire and Raoult 1992). rOmpA and rOmpB are two large proteins (about 2,000 and 1,600 aa, respectively) that share no significant sequence similarity except that they exhibit a highly conserved 300-aa C-termini. This region, the autotransporter (AT) domain, folds into a ?-barrel structure and inserts into the outer membrane. This structure forms a pore through which the central passenger domain of the proprotein is transported across the membrane (Desvaux, Parham, and Henderson 2004). Experimental studies have demonstrated that following export, the rOmpB proprotein is proteolytically cleaved to release the passenger peptide at the cell surface (Ching et al. 1990; Gilmore et al. 1991; Ching, Carl, and Dasch 1992; Hackstadt et al. 1992). In addition, rOmpA and rOmpB possess an N-terminal signal peptide allowing their passage through the inner membrane presumably by means of the Sec secretion system (Henderson et al. 2004).
Members of the AT protein superfamily are found in -, ?-, -, and -proteobacteria as well as in Chlamydia species (Henderson and Nataro 2001). They all share a homologous C-terminal AT domain. In contrast, the passenger domains are not all homologous and encode for a wide variety of virulence factors that can catalyze proteolysis, serve as adhesins, mediate actin-promoted bacterial motility, or act as cytotoxins to animal cells (Henderson and Nataro 2001). Experimental studies have suggested that rOmpA and rOmpB function as adhesin in Rickettsia (Li and Walker 1998; Uchiyama 2003).
Analyses of the genome sequences of R. conorii (Ogata et al. 2001) and R. prowazekii (Andersson et al. 1998) have revealed the existence of three additional genes encoding a highly conserved C-terminal AT domain. These genes are annotated as "surface cell antigen" (sca) genes (i.e., sca1, 2, 3), together with rOmpA (sca0) and rOmpB (sca5). In addition, "GeneD" (Sekeyova, Roux, and Raoult 2001) has been renamed sca4 (Andersson et al. 1998), though the protein product lacks the AT domain. So far, only rOmpA and rOmpB proteins have been detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis or western blotting in R. conorii (Teysseire and Raoult 1992) as well as Sca4 in R. japonica (Uchiyama, Zhao, and Uchida 1996).
Given the seemingly prominent role of rOmpA and rOmpB proteins in the virulence of rickettsiae, we further characterized the Sca protein family by using the data from six publicly available Rickettsia genomes as well as the complete genome sequences of R. belli, R. felis, and R. africae that have been recently determined in our laboratory. We constructed an updated catalog of 17 subfamilies of sca genes found in nine Rickettsia spp. and analyzed their phylogenetic relationships. In addition, we have investigated the selective pressure acting on the Sca proteins. Our results suggest that the passenger domains of rOmpA, rOmpB, Sca1, and Sca2 as well as the Sca4 protein have evolved under positive selection.
Methods
Data
The genome sequences of R. conorii (NC_003103), R. typhi (NC_006142), R. sibirica (NZ_AABW00000000), R. rickettsii (NZ_AADJ00000000), R. felis (CP000053, CP000054) R. akari (NZ_AAFE00000000), and R. prowazekii (NC_000963) were downloaded from GenBank. For these species, the complete set of proteins was constructed using the GenBank annotations. We used the complete genome sequences of R. belli and R. africae that have been recently determined in our laboratory and will be published elsewhere. Every open reading frames (ORFs) of more than 100 nonstop codons were collected from the six possible reading frames and virtually translated into proteins. For these organisms, the ORF nucleotide sequences of the sca genes were released in GenBank. Locus names and GenBank accession numbers of all analyzed sequences are available as Supplementary Material online. For the study of selective constraints acting on sca genes, only the second halves of the gene sequences were available in most organisms for rOmpA and sca1. In consequence, subsequent analyses were restricted to these regions of proteins, which contain part of the passenger domain and the complete AT domain.
Sequence analysis
ORF products with sequence similarity with the Sca proteins previously identified in R. conorii and R. prowazekii (i.e., rOmpA, rOmpB, Sca1, Sca2, Sca3, and Sca4) were searched in the nine complete rickettsial proteomes using the BlastP program (E value < 1 x 10–10; Altschul et al. 1997). The AT domains were searched in the Sca protein sequences and the GenBank protein database with the program HMMSEARCH from the HMMER package (R. S. Eddy, unpublished data, http://hmmer.wustl.edu/), using the Pfam AT domain HMM profile (PF03797.6) as query. Hits were considered significant when they matched the Pfam profile with E values 10–5. Orthologous relationships between sca ORFs were determined by the reciprocal best BlastP match criterion. Except for R. bellii, the Rickettsia genomes exhibit a nearly perfect colinearity and few genomic rearrangements (Ogata et al. 2001). Hence, we confirmed the orthologous relationships by verifying that the genes surrounding each orthologous sca genes were in collinear order. Repeated peptide motifs were identified using the dotter program (Sonnhammer and Durbin 1995). Predictions of N-terminal signal peptides were done using the TMHMM web tool at http://www.cbs.dtu.dk/services/TMHMM/ (Krogh et al. 2001). The significance of sequence similarity between paralogous passenger domains was assessed using the program PRDF from the FASTA2 package (Pearson 1990) with default parameters. The threshold of significance was set to P value (opt delta) < 0.01.
Phylogenetic analysis
Protein and nucleotide alignments were carried out using the programs MUSCLE (Edgar 2004) and ClustalW (Higgins and Sharp 1988), respectively, and corrected manually. We constructed the phylogenetic trees of the sca5 nucleotide sequences and AT domains (fig. 2) using the Neighbor-Joining (NJ) method implemented in the MEGA software (Kumar, Tamura, and Nei 1994). Phylogenetic distances were calculated using the Tamura-Nei method (Tamura and Nei 1993) for nucleotide sequences and a gamma correction (alpha = 3.34) for the AT domains. The shape parameter alpha for the gamma correction was determined by maximum likelihood using the CODEML program from the PAML package (Yang 1997). For all tree constructions, bootstrap supports for branches were assessed using 100 pseudoreplicates.
a single ORF encoding a protein of less than 50% of the longest orthologous protein), or absent (flag "-"). Split genes and gene fragments may be the result of ongoing gene degradation, a common feature in rickettsial genomes that is concomitant with reductive evolution (Andersson et al. 1998; Ogata et al. 2001). Thus, these may be incapable of producing functional proteins and could be pseudogenes. None of the nine Rickettsia genomes studied here harbor the 17 intact sca genes (fig. 1). Rickettsia prowazekii is remarkable as it retained only two intact sca genes (sca3 and rOmpB). On the other extreme, the R. felis genome contains the largest complement of sca genes with 10 complete and 3 split sca genes. rOmpB is the only sca gene found complete in all the nine genomes studied.
Table 2 Likelihood Ratio Tests of Positive Selection
Positively selected amino acid sites were identified under model M8 using the Bayesian method developed by Nielsen and Yang (1998). Consistent with our hypothesis that only the passenger domains are involved in host-parasite interactions, the predicted positively selected sites are exclusively located within this region of Sca1, Sca2, rOmpA, and rOmpB (fig. 3). The distributions of the predicted positively selected sites between passenger and AT domains are highly nonrandom (P < 3.2 x 10–4; binomial distribution), which further confirms that the two domains have evolved under different selection regimes.
//www.mbe.oxfordjournals.org/).
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