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Background: Members of the phylum Chlamydiae are obligate intracellular pathogens of humans and animals and have a serious impact on host health. They comprise several zoonotic species with varying disease outcomes and prevalence. To investigate differences in virulence, we focused on Chlamydia psittaci, C. abortus and Waddlia chondrophila. Most threatening is C. psittaci, which frequently infects humans and causes psittacosis associated with severe pneumonia. The closest relative of C. psittaci is C. abortus, which shares the vast majority of genes but less frequently infects humans, and causes stillbirth and sepsis. W. chondrophila is more distantly related, and occasional human infections are associated with respiratory diseases or miscarriage. One possible explanation for differences in virulence originate from species-specific genes as well as differentially expressed homologous virulence factors., Results: RNA-sequencing (RNA-Seq) was applied to purified infectious elementary bodies (EBs) and non-infectious reticulate bodies (RBs) in order to elucidate the transcriptome of the infectious and replicative chlamydial states. The results showed that approximately half of all genes were differentially expressed. For a descriptive comparison, genes were categorised according to their function in the RAST database. This list was extended by the inclusion of inclusion membrane proteins, outer membrane proteins, polymorphic membrane proteins and type III secretion system effectors. In addition, the expression of fifty-six known and a variety of predicted virulence and immunogenic factors with homologs in C. psittaci, C. abortus and W. chondrophila was analysed. To confirm the RNA-Seq results, the expression of nine factors was validated using real-time quantitative polymerase chain reaction (RT-qPCR). Comparison of RNA-Seq and RT-qPCR results showed a high mean Pearson correlation coefficient of 0.95., Conclusions: It was shown that both the replicative and infectious chlamydial state contained distinctive transcriptomes and the cellular processes emphasised in EBs and RBs differed substantially based on the chlamydial species. In addition, the very first interspecies transcriptome comparison is presented here, and the considerable differences in expression of homologous virulence factors might contribute to the differing infection rates and disease outcomes of the pathogens. The RNA-Seq results were confirmed by RT-qPCR and demonstrate the feasibility of interspecies transcriptome comparisons in chlamydia.

Background: Members of the order Chlamydiales are known for their potential as human and veterinary bacterial pathogens. Despite this recognition, epidemiological factors such as routes of transmission are yet to be fully defined. Ticks are well known vectors for many other infections with several reports recently describing the presence of bacteria in the order Chlamydiales in these arthropods. Australian wildlife are hosts to an extensive range of tick species. Evidence is also growing that the marsupial hosts these ticks parasitise can also be infected by a number of bacteria in the order Chlamydiales, with at least one species, Chlamydia pecorum, posing a significant conservation threat. In the current study, we investigated the presence and identity of Chlamydiales in 438 ixodid ticks parasitizing wildlife in Australia by screening with a pan-Chlamydiales specific targeting the 16S rRNA gene., Results: Pan-Chlamydiales specific PCR assays confirmed the common presence of Chlamydiales in Australian ticks parasitising a range of native wildlife. Interestingly, we did not detect any Chlamydiaceae, including C. pecorum, the ubiquitous pathogen of the koala. Instead, the Chlamydiales diversity that could be resolved indicated that Australian ticks carry at least six novel Chlamydiales genotypes. Phylogenetic analysis of the 16S rRNA sequences (663 bp) of these novel Chlamydiales suggests that three of these genotypes are associated with the Simkaniaceae and putatively belong to three distinct novel strains of Fritschea spp. and three genotypes are related to the "Ca. Rhabdochlamydiaceae" and putatively belong to a novel genus, Rhabdochlamydia species and strain, respectively., Conclusions: Sequence results suggest Australian wildlife ticks harbour a range of unique Chlamydiales bacteria that belong to families previously identified in a range of arthropod species. The results of this work also suggest that it is unlikely that arthropods act as vectors of pathogenic members of the family Chlamydiaceae, including C. pecorum, in Australian wildlife. The biology of novel Chlamydiales identified in arthropods remain unknown. The pathogenic role of the novel Chlamydiales identified in this study and the role that ticks may play in their transmission needs to be explored further.

Background: To demonstrate the bioinformatics capabilities of a low-cost computer, the Raspberry Pi, we present a comparison of the protein-coding gene content of two species in phylum Chlamydiae: Chlamydia trachomatis, a common sexually transmitted infection of humans, and Candidatus Protochlamydia amoebophila, a recently discovered amoebal endosymbiont. Identifying species-specific proteins and differences in protein families could provide insights into the unique phenotypes of the two species., Results: Using a Raspberry Pi computer, sequence similarity-based protein families were predicted across the two species, C. trachomatis and P. amoebophila, and their members counted. Examples include nine multi-protein families unique to C. trachomatis, 132 multi-protein families unique to P. amoebophila and one family with multiple copies in both. Most families unique to C. trachomatis were polymorphic outer-membrane proteins. Additionally, multiple protein families lacking functional annotation were found. Predicted functional interactions suggest one of these families is involved with the exodeoxyribonuclease V complex., Conclusion: The Raspberry Pi computer is adequate for a comparative genomics project of this scope. The protein families unique to P. amoebophila may provide a basis for investigating the host-endosymbiont interaction. However, additional species should be included; and further laboratory research is required to identify the functions of unknown or putative proteins. Multiple outer membrane proteins were found in C. trachomatis, suggesting importance for host evasion. The tyrosine transport protein family is shared between both species, with four proteins in C. trachomatis and two in P. amoebophila. Shared protein families could provide a starting point for discovery of wide-spectrum drugs against Chlamydiae.

Background: Amoebae are phagocytic protists where genetic exchanges might take place between amoeba-resistant bacteria. These amoebal pathogens are able to escape the phagocytic behaviour of their host. They belong to different bacterial phyla and often show a larger genome size than human-infecting pathogens. This characteristic is proposed to be the result of frequent gene exchanges with other bacteria that share a sympatric lifestyle and contrasts with the genome reduction observed among strict human pathogens., Results: We sequenced the genome of a new amoebal pathogen, Legionella drancourtii, and compared its gene content to that of a Chlamydia-related bacterium, Parachlamydia acanthamoebae. Phylogenetic reconstructions identified seven potential horizontal gene transfers (HGTs) between the two amoeba-resistant bacteria, including a complete operon of four genes that encodes an ABC-type transporter. These comparisons pinpointed potential cases of gene exchange between P. acanthamoebae and Legionella pneumophila, as well as gene exchanges between other members of the Legionellales and Chlamydiales orders. Moreover, nine cases represent possible HGTs between representatives from the Legionellales or Chlamydiales and members of the Rickettsiales order., Conclusions: This study identifies numerous gene exchanges between intracellular Legionellales and Chlamydiales bacteria, which could preferentially occur within common inclusions in their amoebal hosts. Therefore it contributes to improve our knowledge on the intra-amoebal gene properties associated to their specific lifestyle.

Background: Along the chromosome of the obligate intracellular bacteria Protochlamydia amoebophila UWE25, we recently described a genomic island Pam100G. It contains a tra unit likely involved in conjugative DNA transfer and lgrE, a 5.6-kb gene similar to five others of P. amoebophila: lgrA to lgrD, lgrF. We describe here the structure, regulation and evolution of these proteins termed LGRs since encoded by "Large G+C-Rich" genes., Results: No homologs to the whole protein sequence of LGRs were found in other organisms. Phylogenetic analyses suggest that serial duplications producing the six LGRs occurred relatively recently and nucleotide usage analyses show that lgrB, lgrE and lgrF were relocated on the chromosome. The C-terminal part of LGRs is homologous to Leucine-Rich Repeats domains (LRRs). Defined by a cumulative alignment score, the 5 to 18 concatenated octacosapeptidic (28-meric) LRRs of LGRs present all a predicted alpha-helix conformation. Their closest homologs are the 28-residue RI-like LRRs of mammalian NODs and the 24-meres of some Ralstonia and Legionella proteins. Interestingly, lgrE, which is present on Pam100G like the tra operon, exhibits Pfam domains related to DNA metabolism., Conclusion: Comparison of the LRRs, enable us to propose a parsimonious evolutionary scenario of these domains driven by adjacent concatenations of LRRs. Our model established on bacterial LRRs can be challenged in eucaryotic proteins carrying less conserved LRRs, such as NOD proteins and Toll-like receptors.

Background: Chlamydiae species are of much importance from a clinical viewpoint. Their diversity both in terms of their numbers as well as clinical involvement are presently believed to be significantly underestimated. The obligate intracellular nature of chlamydiae has also limited their genetic and biochemical studies. Thus, it is of importance to develop additional means for their identification and characterization., Results: We have carried out analyses of available chlamydiae genomes to identify sets of unique proteins that are either specific for all Chlamydiales genomes, or different Chlamydiaceae family members, or members of the Chlamydia and Chlamydophila genera, or those unique to Protochlamydia amoebophila, but which are not found in any other bacteria. In total, 59 Chlamydiales-specific proteins, 79 Chlamydiaceae-specific proteins, 20 proteins each that are specific for both Chlamydia and Chlamydophila and 445 ORFs that are Protochlamydia-specific were identified. Additionally, 33 cases of possible gene loss or lateral gene transfer were also detected., Conclusion: The identified chlamydiae-lineage specific proteins, many of which are highly conserved, provide novel biomarkers that should prove of much value in the diagnosis of these bacteria and in exploration of their prevalence and diversity. These conserved protein sequences (CPSs) also provide novel therapeutic targets for drugs that are specific for these bacteria. Lastly, functional studies on these chlamydiae or chlamydiae subgroup-specific proteins should lead to important insights into lineage-specific adaptations with regards to development, infectivity and pathogenicity.

Background: The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs)., Results: On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit., Conclusions: A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.