BACKGROUND
legionella pneumophila is a gram negative, gamma-proteobacteria organism whose natural hosts are amoebae and protozoa.
this bacterium can infect humans by inhalation of aerosols  <cit>  entering alveolar macrophages causing the well-known, and often lethal, legionnaires' disease  or legionellosis.
despite the great number of isolates of l. pneumophila, the ones belonging to serogroup  <dig> are responsible of about  <dig> to 90% of cases of legionellosis  <cit> .
the first critical event during infection by l. pneumophila involves the macrophages by the action of the type iv secretion system, which prevents the fusion of the phagosome with the lysosome and its acidification  <cit> .
it has been demonstrated that these events start very early after the infection  <cit> .
several mechanisms play an important role in the formation of infection vacuoles.
legionella enters the macrophages by vacuoles that are morphologically similar to macropinosomes by an unusual mechanisms called "coiling phagocytosis"  <cit> .
the vacuole is immediately surrounded by vesicles and mitochondria and moves toward the endoplasmic reticulum escaping or delaying fusion with the lysosome  <cit> .
at this stage, the vacuoles offer a perfect niche for bacteria to multiply safely away from the lysosome.
legionella is also able to mediate the delayed entrance of the cell in apoptosis by modulating the activity of caspase- <dig> and other effectors  <cit> .
in all these stages the dot/icm  system, involved in the formation of type iv secretor machinery, is the main player acting on the transfer of a series of effectors in the host cell  <cit> .
one of the first events in the pathogenic cascade is pore formation, which seems to be caused by a toxin belonging to the rtx family   <cit> .
it was demonstrated that the rtxa gene in l. pneumophila is strictly related to pathogenicity, and its main role involves adherence to the host membranes, thus facilitating all the molecular trafficking of the bacteria during infection processes  <cit> .
in other bacteria like bordetella pertussis, escherichia coli or actinobacillus actinomycetemcomitans, proteins belonging to the rtx family are also described as effectors of immune cell lyses and its action is often mediated by specific host membrane receptors  <cit> .
rtxa in l. pneumophila is a large protein  with several repeated structures belonging to, at least, three protein family domains.
in l. pneumophila strains lens  and paris  a correlation could exists between the number of repeats and greater invasion and virulence properties  <cit> .
in this work, we analyse the mosaic structure of an rtxa toxin from the highly virulent l. pneumophila strain 2300/ <dig>  it was isolated in alcoy  and was retrieved in several outbreaks in  <dig> and  <dig>  during which more than  <dig> patients were infected, a dozen of whom died.
in all cases, transmission was due to aerosol inhalation from out-door installations  <cit> .
the comparative analysis of the rtx locus of closely related l. pneumophila serogroup  <dig> strains showed the existence of a long tandem repeated domain of variable copy number and sequence.
furthermore, we have studied similarity and gene-order conservation of genes flanking the rtx region, finding remarkably high levels of rearrangements and diversity of the rtxa gene as compared to those from flanking regions.
this pattern is completely opposite to that found in several strains of vibrio cholerae, a phylogenetically close extracellular pathogen.
RESULTS
rtx structure
due to the difficulties given by the assembling of such a large and repeated region, the number of repetitions, and consequently, the protein length are approximate and according to what have been published on each released genome.
figure  <dig> shows the structure of rtx region among the different legionella strains and table  <dig> is a summary of the main structural characteristics.
as it can be observed, rtx genes vary in length.
the corresponding orfs are of approximately  <dig> aa,  <dig> aa,  <dig> aa and  <dig> aa for strains lens, paris corby and alcoy , respectively.
in the case of the aa <dig> sequenced contig, two orfs were identified as previously described by cirillo et al.
<cit>  .
in the case of the corby strain, in spite of the high conservation of sequence structure and position of flanking genes, it has been annotated as a "hypothetical protein" and its arrangement is complementary and reversed with respect to paris, lens and philadelphia strains.
in l. pneumophila philadelphia, the rtx gene is broken into two orfs  with an unannotated gap of about  <dig>  nt in 3' with respect to the lpg <dig>  the initial region of rtx gene is highly conserved in the five strains .
however, in the strains analyzed in the present work, the region is followed by a variable number of tandem repeats.
the repeats contain domains involved in host-membrane interaction, with a wide variability either in copy number and, surprisingly, in nucleotide composition .
these repeats ranged from  <dig> nt in the paris strain to  <dig> nt in the lens strain.
in the paris strain,  <dig> type a repeats were described, while in the lens strain two kinds of repeats, namely b <dig> and b <dig>  were observed .
their sequences differ completely, with  <dig> and  <dig> repetitions respectively and with no possible alignment between them .
in the corby strain, we distinguish two different types of repeats, which can be aligned: four named c <dig> and twenty-one named c <dig>  in the alcoy strain, the first repeat spanning from position   <dig> to  <dig> is identical to c <dig> type, whereas the other  <dig> repeats are almost identical to the c <dig> corby type.
finally,  <dig> repeats of type d were found in the philadelphia strain.
the philadelphia repeats of the rtx gene were identified along and ahead of the orf lpg <dig>  finally, in the case of the aa <dig> strain, the analysed sequence was not covering the c-terminal region containing the repeats, probably because the studied fosmid insert did not contain the region.
---
<dig> vwa
<dig> vwa
<dig> vwa
<dig> vwa
spacer
lpg0645
<dig>
681
*length of proteins and number of repeats estimated according to the sequences published in relative genome project.
after searching in the pfam database, several kinds of adhesion related domains were identified as part of repeats type a , b <dig> , c <dig> and c <dig> .
no domains related to adhesion were identified in rtx gene of the philadelphia strain, while only the c <dig> and c <dig> repeats were phylogenetically related .
therefore, the way we approached the modular structure of the repeats was by looking at the function of the domains involved in adhesion, with only one exception: the aa <dig> sequence which spans the region located after the repeats, so it was not possible to include it in our description.
in the four completed genomes  as well as in the contigs sequenced belonging to the alcoy and aa <dig> strains, a von willebrand factor type a domain  was identified, subsequent to the regions with tandem repeats.
in addition, several blocks of tandem repeats identified as hemolysincabind domains were also found.
these blocks were formed by a number of different repeats: 3+ <dig> repeats in paris, 1+3+ <dig> in lens, 3+3+ <dig> in corby, 3+3+ <dig> in alcoy, 3+3+ <dig> in philadelphia and 1+3+ <dig> in aa <dig>  the latter domain was previously described and considered responsible for the virulent activity of the rtxa protein  <cit> .
a summary of all these structural features of the rtx present in different legionella strains is shown in table  <dig>
comparative genomics and phylogenetic analyses
the multi locus sequence typing  analysis carried out following the scheme suggested for l. pneumophila serogroup  <dig>  <cit> , using the information available for the five genomes shows that corby and alcoy strains are close related, while, due to the low bootstrap values, the positioning of an ancestor to these two strains is not univocal .
the whole genome alignments , indicate that the genome back-bone is generally maintained with a high level of synteny.
however, the synteny surrounding the rtx region found in legionella  has not been found in other pathogenic strains.
thus, we performed the same kind of alignment on vibrio cholerae by choosing five rtx genes from two complete genomes plus three partial shotgun contigs .
as it can be observed, there are slight differences in the organization of rtx flanking genes .
moreover, contrary to what observed in legionella, there is a high level of conservation in the rtx region.
strains corby and alcoy, on the other hand, are quite similar in their rtx gene at both protein domains and nucleotide level .
similarly to the other legionella genomes, flanking genes are highly conserved both in order and orientation.
n-terminal repeats are phylogenetically closely related .
the alignment of repeated domains of the genomes considered here is practically impossible at nucleotide level, and even at amino acid level it is very complicated.
additional file  <dig> shows the amino acid alignment for each kind of repeat using clustlalw and corrected by eye.
the domains identified by pfam are highlighted.
it is not possible to identify any phylogenetic relationships either among the repeats or looking at the sole adhesion domains.
discussion
here we report a comparative analysis of the rtx toxin, as well as a fine analysis of repeats, identified in this protein in strains of l. pneumophila serogroup  <dig> from four completed genomes , one shotgun ongoing sequencing project  and one contig coming from a cosmid of the legionella strain aa <dig>  all these strains are known to be virulent  <cit> .
the rtx genes analyzed in the six genomes studied, present modularity.
the toxin appears to be clearly divided in two regions, the n-terminal, involved in adhesion, and the c-terminal region, involved in adhesion and pore formation in the host membranes.
the repeats in the n-terminal region analysed by pfam database searches, have different kinds of adhesion domains.
in all the n-terminal type a repeats of the paris strain, similarity was found with the "thrombospondin type  <dig> repeat"  of the human endothelial cell.
in humans, this domain has been shown to bind fibrinogen, fibronectin, laminin and type v collagen  <cit> .
in the lens strain, repetitions of type b <dig> show domains similar to the "chlamydia polymorphic membrane protein" .
this obligate intracellular human pathogen causes infection of the upper and lower respiratory tract but the role of this membrane protein is still unknown  <cit> .
no similarity with specific domains was found for b <dig> type repeat of the lens strain.
the last kind of repeat-associated domains, identified in type c <dig> and c <dig> of alcoy and corby strains, were similar to the "haemagglutinin"  domain that was found in invasins and hemagglutinins, and is associated with the hep_hag repeats  <cit> .
two other types of domains were commonly identified in all the rtx genes: the vwa domain involved in adhesion processes  <cit> , and another tandem repeated domain related to cytotoxic activity, the haemolysin calcium-binding  site.
the latter domain commonly brings a nonapeptide that is related to the adhesion with other host cell surfaces or vacuole membranes and pores formation  <cit> .
except for alcoy and corby, which are very similar, the n-terminal repeats and their modular structure are highly variable among strains.
despite these differences, the flanking genes at 5' and 3' are highly conserved in sequence and order, which suggests that rtx undergoes a particular intra-genic evolution.
various examples of large non-interspaced repeats within cds  regions were described in bacteria such as e. coli and bacillus subtilis, where recombination events were used to explain the distribution of large repeats among genomes  <cit> .
the case of rtx gene in l. pneumophila is particularly interesting due to the high number of observed intra-genic repeats.
the origin of these repeats is yet unknown, as no similar sequences  have been identified in published data.
as described in gene conversion models, dna can enter to become part of a given gene  <cit>  and afterwards, concerted evolution could be responsible for promoting the different adhesion domains.
in fact, although the repeats type a, b, c, and d do not show any evolutionary relationships, the presence of different adhesion domains points towards a functional evolutionary advantage.
in v. cholerae the configuration of the rtx gene and its flanking regions is extremely different from that observed in l. pneumophila.
comparative analyses of rtx gene among five strains show a high level of conservation and the genes located at 5' of rtx, are strictly conserved, both in sequence and order, whereas the synteny, and sometimes nucleotide similarity, of those located at 3' do not follow the same pattern .
CONCLUSIONS
in legionella spp.
it was previously thought that only strains containing an active rtxa gene were able to produce infection in humans, and that mutants with a frame-shift inactivating rtxa protein were reduced for entry into host cells and pore formation in host membranes  <cit> .
rtxa seems to play a relevant role in the pathogenic activity of legionella, although it also depends on the particular type of host.
the variety of repeats and its homogeneous nature at rtxa n-terminal region of virulent strains of l. pneumophila seems to be acquired by the two mechanisms involved in concerted evolution: intra-genic gene conversion and/or unequal crossing over.
as previously described for similar models in other organisms  <cit> , these mechanisms can be an important source of creating antigenic variability in legionella, affording the ability to increase the host range and escape from the host's immune defences.

 legionella pneumophila is a gram negative, gamma-proteobacteria organism whose natural hosts are amoebae and protozoa. this bacterium can infect humans by inhalation of aerosols entering alveolar macrophages causing the well-known, and often lethal, legionnaires' disease or legionellosis. despite the great number of isolates of l. pneumophila, the ones belonging to serogroup are responsible of about to 90% of cases of legionellosis. the first critical event during infection by l. pneumophila involves the macrophages by the action of the type iv secretion system, which prevents the fusion of the phagosome with the lysosome and its acidification. it has been demonstrated that these events start very early after the infection. several mechanisms play an important role in the formation of infection vacuoles. legionella enters the macrophages by vacuoles that are morphologically similar to macropinosomes by an unusual mechanisms called "coiling phagocytosis". the vacuole is immediately surrounded by vesicles and mitochondria and moves toward the endoplasmic reticulum escaping or delaying fusion with the lysosome. at this stage, the vacuoles offer a perfect niche for bacteria to multiply safely away from the lysosome. legionella is also able to mediate the delayed entrance of the cell in apoptosis by modulating the activity of caspase- and other effectors. in all these stages the dot/icm system, involved in the formation of type iv secretor machinery, is the main player acting on the transfer of a series of effectors in the host cell. one of the first events in the pathogenic cascade is pore formation, which seems to be caused by a toxin belonging to the rtx family. it was demonstrated that the rtxa gene in l. pneumophila is strictly related to pathogenicity, and its main role involves adherence to the host membranes, thus facilitating all the molecular trafficking of the bacteria during infection processes. in other bacteria like bordetella pertussis, escherichia coli or actinobacillus actinomycetemcomitans, proteins belonging to the rtx family are also described as effectors of immune cell lyses and its action is often mediated by specific host membrane receptors. rtxa in l. pneumophila is a large protein with several repeated structures belonging to, at least, three protein family domains. in l. pneumophila strains lens and paris a correlation could exists between the number of repeats and greater invasion and virulence properties. in this work, we analyse the mosaic structure of an rtxa toxin from the highly virulent l. pneumophila strain 2300/ it was isolated in alcoy and was retrieved in several outbreaks in and during which more than patients were infected, a dozen of whom died. in all cases, transmission was due to aerosol inhalation from out-door installations. the comparative analysis of the rtx locus of closely related l. pneumophila serogroup strains showed the existence of a long tandem repeated domain of variable copy number and sequence. furthermore, we have studied similarity and gene-order conservation of genes flanking the rtx region, finding remarkably high levels of rearrangements and diversity of the rtxa gene as compared to those from flanking regions. this pattern is completely opposite to that found in several strains of vibrio cholerae, a phylogenetically close extracellular pathogen.  rtx structure due to the difficulties given by the assembling of such a large and repeated region, the number of repetitions, and consequently, the protein length are approximate and according to what have been published on each released genome. figure shows the structure of rtx region among the different legionella strains and table is a summary of the main structural characteristics. as it can be observed, rtx genes vary in length. the corresponding orfs are of approximately aa, aa, aa and aa for strains lens, paris corby and alcoy, respectively. in the case of the aa sequenced contig, two orfs were identified as previously described by cirillo et al.. in the case of the corby strain, in spite of the high conservation of sequence structure and position of flanking genes, it has been annotated as a "hypothetical protein" and its arrangement is complementary and reversed with respect to paris, lens and philadelphia strains. in l. pneumophila philadelphia, the rtx gene is broken into two orfs with an unannotated gap of about nt in 3' with respect to the lpg the initial region of rtx gene is highly conserved in the five strains. however, in the strains analyzed in the present work, the region is followed by a variable number of tandem repeats. the repeats contain domains involved in host-membrane interaction, with a wide variability either in copy number and, surprisingly, in nucleotide composition. these repeats ranged from nt in the paris strain to nt in the lens strain. in the paris strain, type a repeats were described, while in the lens strain two kinds of repeats, namely b and b were observed. their sequences differ completely, with and repetitions respectively and with no possible alignment between them. in the corby strain, we distinguish two different types of repeats, which can be aligned: four named c and twenty-one named c in the alcoy strain, the first repeat spanning from position to is identical to c type, whereas the other repeats are almost identical to the c corby type. finally, repeats of type d were found in the philadelphia strain. the philadelphia repeats of the rtx gene were identified along and ahead of the orf lpg finally, in the case of the aa strain, the analysed sequence was not covering the c-terminal region containing the repeats, probably because the studied fosmid insert did not contain the region. --- vwa vwa vwa vwa spacer lpg0645 681 *length of proteins and number of repeats estimated according to the sequences published in relative genome project. after searching in the pfam database, several kinds of adhesion related domains were identified as part of repeats type a, b, c and c. no domains related to adhesion were identified in rtx gene of the philadelphia strain, while only the c and c repeats were phylogenetically related. therefore, the way we approached the modular structure of the repeats was by looking at the function of the domains involved in adhesion, with only one exception: the aa sequence which spans the region located after the repeats, so it was not possible to include it in our description. in the four completed genomes as well as in the contigs sequenced belonging to the alcoy and aa strains, a von willebrand factor type a domain was identified, subsequent to the regions with tandem repeats. in addition, several blocks of tandem repeats identified as hemolysincabind domains were also found. these blocks were formed by a number of different repeats: 3+ repeats in paris, 1+3+ in lens, 3+3+ in corby, 3+3+ in alcoy, 3+3+ in philadelphia and 1+3+ in aa the latter domain was previously described and considered responsible for the virulent activity of the rtxa protein. a summary of all these structural features of the rtx present in different legionella strains is shown in table comparative genomics and phylogenetic analyses the multi locus sequence typing analysis carried out following the scheme suggested for l. pneumophila serogroup, using the information available for the five genomes shows that corby and alcoy strains are close related, while, due to the low bootstrap values, the positioning of an ancestor to these two strains is not univocal. the whole genome alignments, indicate that the genome back-bone is generally maintained with a high level of synteny. however, the synteny surrounding the rtx region found in legionella has not been found in other pathogenic strains. thus, we performed the same kind of alignment on vibrio cholerae by choosing five rtx genes from two complete genomes plus three partial shotgun contigs. as it can be observed, there are slight differences in the organization of rtx flanking genes. moreover, contrary to what observed in legionella, there is a high level of conservation in the rtx region. strains corby and alcoy, on the other hand, are quite similar in their rtx gene at both protein domains and nucleotide level. similarly to the other legionella genomes, flanking genes are highly conserved both in order and orientation. n-terminal repeats are phylogenetically closely related. the alignment of repeated domains of the genomes considered here is practically impossible at nucleotide level, and even at amino acid level it is very complicated. additional file shows the amino acid alignment for each kind of repeat using clustlalw and corrected by eye. the domains identified by pfam are highlighted. it is not possible to identify any phylogenetic relationships either among the repeats or looking at the sole adhesion domains. discussion here we report a comparative analysis of the rtx toxin, as well as a fine analysis of repeats, identified in this protein in strains of l. pneumophila serogroup from four completed genomes, one shotgun ongoing sequencing project and one contig coming from a cosmid of the legionella strain aa all these strains are known to be virulent. the rtx genes analyzed in the six genomes studied, present modularity. the toxin appears to be clearly divided in two regions, the n-terminal, involved in adhesion, and the c-terminal region, involved in adhesion and pore formation in the host membranes. the repeats in the n-terminal region analysed by pfam database searches, have different kinds of adhesion domains. in all the n-terminal type a repeats of the paris strain, similarity was found with the "thrombospondin type repeat" of the human endothelial cell. in humans, this domain has been shown to bind fibrinogen, fibronectin, laminin and type v collagen. in the lens strain, repetitions of type b show domains similar to the "chlamydia polymorphic membrane protein". this obligate intracellular human pathogen causes infection of the upper and lower respiratory tract but the role of this membrane protein is still unknown. no similarity with specific domains was found for b type repeat of the lens strain. the last kind of repeat-associated domains, identified in type c and c of alcoy and corby strains, were similar to the "haemagglutinin" domain that was found in invasins and hemagglutinins, and is associated with the hep_hag repeats. two other types of domains were commonly identified in all the rtx genes: the vwa domain involved in adhesion processes, and another tandem repeated domain related to cytotoxic activity, the haemolysin calcium-binding site. the latter domain commonly brings a nonapeptide that is related to the adhesion with other host cell surfaces or vacuole membranes and pores formation. except for alcoy and corby, which are very similar, the n-terminal repeats and their modular structure are highly variable among strains. despite these differences, the flanking genes at 5' and 3' are highly conserved in sequence and order, which suggests that rtx undergoes a particular intra-genic evolution. various examples of large non-interspaced repeats within cds regions were described in bacteria such as e. coli and bacillus subtilis, where recombination events were used to explain the distribution of large repeats among genomes. the case of rtx gene in l. pneumophila is particularly interesting due to the high number of observed intra-genic repeats. the origin of these repeats is yet unknown, as no similar sequences have been identified in published data. as described in gene conversion models, dna can enter to become part of a given gene and afterwards, concerted evolution could be responsible for promoting the different adhesion domains. in fact, although the repeats type a, b, c, and d do not show any evolutionary relationships, the presence of different adhesion domains points towards a functional evolutionary advantage. in v. cholerae the configuration of the rtx gene and its flanking regions is extremely different from that observed in l. pneumophila. comparative analyses of rtx gene among five strains show a high level of conservation and the genes located at 5' of rtx, are strictly conserved, both in sequence and order, whereas the synteny, and sometimes nucleotide similarity, of those located at 3' do not follow the same pattern.  in legionella spp. it was previously thought that only strains containing an active rtxa gene were able to produce infection in humans, and that mutants with a frame-shift inactivating rtxa protein were reduced for entry into host cells and pore formation in host membranes. rtxa seems to play a relevant role in the pathogenic activity of legionella, although it also depends on the particular type of host. the variety of repeats and its homogeneous nature at rtxa n-terminal region of virulent strains of l. pneumophila seems to be acquired by the two mechanisms involved in concerted evolution: intra-genic gene conversion and/or unequal crossing over. as previously described for similar models in other organisms, these mechanisms can be an important source of creating antigenic variability in legionella, affording the ability to increase the host range and escape from the host's immune defences. 
the comparative analysis of rtx gene among strains of l. pneumophila showed modularity in their structures. among compared genomes, the n-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. on the contrary, the c-terminal region is maintained with a fashionable modular configuration, which gives support to its proposed role in adhesion and pore formation. in contrast to the extracellular bacteria vibrio cholerae, in which the rtx gene is highly conserved and flanking genes have lost synteny and similarity, the gene region coding for the rtx toxin in the intracellular pathogen l. pneumophila shows a rapid evolution. the interplay of the rtx toxin with host membranes might lead to the evolution of new variants that are able to escape host cell defences. its role in escaping the host immune system and cytotoxic activity is well known. the repeats in toxin are an important pathogenicity factor involved in host cells invasion of legionella pneumophila and other pathogenic bacteria.
 the repeats in toxin are an important pathogenicity factor involved in host cells invasion of legionella pneumophila and other pathogenic bacteria. its role in escaping the host immune system and cytotoxic activity is well known. its repeated motives and modularity make rtx a multifunctional factor in pathogenicity.  the comparative analysis of rtx gene among strains of l. pneumophila showed modularity in their structures. among compared genomes, the n-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. on the contrary, the c-terminal region is maintained with a fashionable modular configuration, which gives support to its proposed role in adhesion and pore formation. despite the variability of rtx among the considered strains, the flanking genes are maintained in synteny and similarity.  in contrast to the extracellular bacteria vibrio cholerae, in which the rtx gene is highly conserved and flanking genes have lost synteny and similarity, the gene region coding for the rtx toxin in the intracellular pathogen l. pneumophila shows a rapid evolution. changes in the rtx could play a role in pathogenicity. the interplay of the rtx toxin with host membranes might lead to the evolution of new variants that are able to escape host cell defences. 