Publications

Trachoma, the leading infectious cause of blindness, is caused by Chlamydia trachomatis (Ct), a bacterium of the phylum Chlamydiae. Recent investigations revealed the existence of additional families within the phylum Chlamydiae, also termed Chlamydia-like organisms (CLOs). In this study, the frequency of Ct and CLOs was examined in the eyes of healthy Sudanese (control) participants and those with trachoma (case). We tested 96 children (54 cases and 42 controls) and 93 adults (51 cases and 42 controls) using broad-range Chlamydiae and Ct-specific (omcB) real-time PCR. Samples positive by broad-range Chlamydiae testing were subjected to DNA sequencing. Overall Chlamydiae prevalence was 36%. Sequences corresponded to unclassified and classified Chlamydiae. Ct infection rate was significantly higher in children (31.5%) compared to adults (0%) with trachoma (p < 0.0001). In general, 21.5% of adults and 4.2% of children tested positive for CLOs (p = 0.0003). Our findings are consistent with previous investigations describing the central role of Ct in trachoma among children. This is the first study examining human eyes for the presence of CLOs. We found an age-dependent distribution of CLO DNA in human eyes with significantly higher positivity in adults. Further studies are needed to understand the impact of CLOs in trachoma pathogenicity and/or protection.

Chlamydiae are obligate intracellular bacteria comprising important human pathogens and symbionts of protists. Molecular evidence indicates a tremendous diversity of chlamydiae particularly in marine environments, yet our current knowledge is based mainly on terrestrial representatives. Here we provide first insights into the biology of marine chlamydiae representing three divergent clades. Our analysis of single-cell amplified genomes revealed hallmarks of the chlamydial lifestyle, supporting the ancient origin of their characteristic developmental cycle and major virulence mechanisms. Surprisingly, these chlamydial genomes encode a complete flagellar apparatus, a previously unreported feature. We show that flagella are an ancient trait that was subject to differential gene loss among extant chlamydiae. Together with a chemotaxis system, these marine chlamydiae are likely motile, with flagella potentially playing a role during host cell infection. This study broadens our view on chlamydial biology and indicates a largely underestimated potential to adapt to different hosts and environments.

'Candidatus Hepatoplasma crinochetorum Ps' is an extracellular symbiont residing in the hepatopancreas of the terrestrial isopod Porcellio scaber. Its genome is highly similar to that of the close relative 'Cand. Hepatoplasma crinochetorum Av' from Armadillidium vulgare. However, instead of a CRISPR/Cas system it encodes a type I restriction modification system.

Chlamydiae are obligate intracellular bacteria found as symbionts and pathogens in a wide range of eukaryotes, including protists, invertebrates, and vertebrates. It was recently proposed that an ancient chlamydial symbiont facilitated the establishment of primary plastids in a tripartite symbiosis with cyanobacteria and early eukaryotes. In this review, we summarize recent advances in understanding of the lifestyle and the evolutionary history of extant Chlamydiae. We reconstruct and describe key features of the ancient chlamydial symbiont. We propose that it was already adapted to an intracellular lifestyle before the emergence of Archaeplastida, and that several observations are compatible with an essential contribution of Chlamydiae to the evolution of algae and plants.

Gene loss, gain, and transfer play an important role in shaping the genomes of all organisms; however, the interplay of these processes in isolated populations, such as in obligate intracellular bacteria, is less understood. Despite a general trend towards genome reduction in these microbes, our phylogenomic analysis of the phylum Chlamydiae revealed that within the family Parachlamydiaceae, gene family expansions have had pronounced effects on gene content. We discovered that the largest gene families within the phylum are the result of rapid gene birth-and-death evolution. These large gene families are comprised of members harboring eukaryotic-like ubiquitination-related domains, such as F-box and BTB-box domains, marking the largest reservoir of these proteins found among bacteria. A heterologous type III secretion system assay suggests that these proteins function as effectors manipulating the host cell. The large disparity in copy number of members in these families between closely related organisms suggests that nonadaptive processes might contribute to the evolution of these gene families. Gene birth-and-death evolution in concert with genomic drift might represent a previously undescribed mechanism by which isolated bacterial populations diversify.

Adelgids (Insecta: Hemiptera: Adelgidae) are known as severe pests of various conifers in North America, Canada, Europe and Asia. Here we present the first molecular identification of bacteriocyte-associated symbionts in these plant sap-sucking insects. Three geographically distant populations of members of the Adelges nordmannianae/piceae complex, identified based on coI and ef1alpha gene sequences, were investigated. Electron and light microscopy revealed two morphologically different endosymbionts, coccoid or polymorphic, that are located in distinct bacteriocytes. Phylogenetic analyses of their 16S and 23S rRNA gene sequences assigned both symbionts to novel lineages within the Gammaproteobacteria sharing less than 92% 16S rRNA sequence similarity with each other and showing no close relationship with known symbionts of insects. Their identity and intracellular location were confirmed by fluorescence in situ hybridisation, and the names ‘Candidatus Steffania adelgidicola’ and ‘Candidatus Ecksteinia adelgidicola’ are proposed for tentative classification. Both symbionts were present in all individuals of all investigated populations and in different adelgid life stages including eggs, suggesting vertical transmission from mother to offspring. An 85 kb genome fragment of ‘Candidatus Steffania adelgidicola’ was reconstructed based on a metagenomic library created from purified symbionts. Genomic features including the frequency of pseudogenes, the average length of intergenic regions and the presence of several genes, which are absent in other long-term obligate symbionts, suggested that ‘Candidatus Steffania adelgidicola’ is an evolutionary young bacteriocyte-associated symbiont, which has been acquired after the diversification of adelgids from their aphid sister group.

Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99 and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees, and together with differences in the composition of the cell wall, reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.

Chlamydia-like bacteria, obligate intracellular relatives of Chlamydia trachomatis and Chlamydophila pneumoniae, are widely distributed in nature. Using a two-step nested and semi-nested PCR approach targeting the 16S rRNA gene, we found DNA of chlamydia-like bacteria in respiratory samples from patients with community-acquired pneumonia. Four out of 387 cases (1.03%) tested positive if only sequences showing less than 99.9% 16S rRNA sequence similarity to known chlamydiae were considered. These included for the first time Protochlamydia amoebophila, Waddlia chondrophila, and ‘Candidatus Rhabdochlamydia porcellionis’-related sequences. This study extends previous findings suggesting an association of chlamydia-like bacteria with respiratory disease, but a causal link between these microorganisms and respiratory tract infections has yet to be established.

Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycle's major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics the reconstruction of genomic data directly from the environment to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organism's special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.

The obligately intracellular coccoid bacterium UWE25, a symbiont of Acanthamoeba spp., was previously identified as being related to chlamydiae based upon the presence of a chlamydia-like developmental cycle and its 16S rRNA gene sequence. Analysis of its complete genome sequence demonstrated that UWE25 shows many characteristic features of chlamydiae, including dependency on host-derived metabolites, composition of the cell envelope and the ability to thrive as an energy parasite within the cells of its eukaryotic host. Phylogenetic analysis of 44 ribosomal proteins further confirmed the affiliation of UWE25 to the 'Chlamydiae'. Within this phylum, UWE25 could be assigned to the family Parachlamydiaceae based on comparative analyses of the 16S rRNA, 23S rRNA and endoribonuclease P RNA genes. The distinct dissimilarities from its closest relative, Parachlamydia acanthamoebae Bn(9)(T) (7.1, 9.7 and 28.8%, respectively), observed in this analysis justify its classification in a new genus. Therefore, the name 'Candidatus Protochlamydia amoebophila' is proposed for the designation of the Acanthamoeba sp. symbiont UWE25 (=ATCC PRA-7).

Chlamydiae are a unique group of obligate intracellular bacteria comprising important pathogens of vertebrates as well as symbionts of free-living amoebae. Although there is ample molecular evidence for a huge diversity and wide distribution of chlamydiae in nature, environmental chlamydiae are currently represented by only few isolates. This paper reports the recovery of a novel environmental chlamydia strain from activated sludge by co-cultivation with Acanthamoeba sp. The recovered environmental chlamydia strain UV-7 showed the characteristic morphology of chlamydial developmental stages as revealed by electron microscopy and was identified as a new member of the family Parachlamydiaceae (98.7 % 16S rRNA sequence similarity to Parachlamydia acanthamoebae). Infection studies suggested that Parachlamydia sp. UV-7 is not confined to amoeba hosts but is also able to invade mammalian cells. These findings outline a new straightforward approach to retrieving environmental chlamydiae from nature without prior, tedious isolation and cultivation of their natural host cells, and lend further support to suggested implications of environmental chlamydiae for public health.

Bacteria living within eukaryotic cells can be essential for the survival or reproduction of the host but in other cases are among the most successful pathogens. Environmental Chlamydiae, including strain UWE25, thrive as obligate intracellular symbionts within protozoa; are recently discovered relatives of major bacterial pathogens of humans; and also infect human cells. Genome analysis of UWE25 predicted that this symbiont is unable to synthesize the universal electron carrier nicotinamide adenine dinucleotide (NAD+). Compensation of limited biosynthetic capacity in intracellular bacteria is usually achieved by import of primary metabolites. Here, we report the identification of a candidate transporter protein from UWE25 that is highly specific for import of NAD+ when synthesized heterologously in Escherichia coli. The discovery of this candidate NAD+/ADP exchanger demonstrates that intact NAD+ molecules can be transported through cytoplasmic membranes. This protein acts together with a newly discovered nucleotide transporter and an ATP/ADP translocase, and allows UWE25 to exploit its host cell by means of a sophisticated metabolic parasitism.

Chlamydiae are the major cause of preventable blindness and sexually transmitted disease. Genome analysis of a chlamydia-related symbiont of free-living amoebae revealed that it is twice as large as any of the pathogenic chlamydiae and had few signs of recent lateral gene acquisition. We showed that about 700 million years ago the last common ancestor of pathogenic and symbiotic chlamydiae was already adapted to intracellular survival in early eukaryotes and contained many virulence factors found in modern pathogenic chlamydiae, including a type III secretion system. Ancient chlamydiae appear to be the originators of mechanisms for the exploitation of eukaryotic cells.

ATP/ADP translocases catalyze the highly specific transport of ATP across a membrane in an exchange mode with ADP. Such unique transport proteins are employed by plant plastids and have among the prokaryotes so far only been identified in few obligate intracellular bacteria belonging to the Chlamydiales and the Rickettsiales. In this study, 12 phylogenetically diverse bacterial endosymbionts of free-living amoebae and paramecia were screened for the presence of genes encoding ATP/ADP transport proteins. The occurrence of ATP/ADP translocase genes was found to be restricted to endosymbionts related to rickettsiae and chlamydiae. We showed that the ATP/ADP transport protein of the Parachlamydia-related endosymbiont of Acanthamoeba sp. strain UWE25, a recently identified relative of the important human pathogens Chlamydia trachomatis and Chlamydophila pneumoniae, is functional when expressed in the heterologous host Escherichia coli and demonstrated the presence of transcripts during the chlamydial developmental cycle. These findings indicate that the interaction between Parachlamydia-related endosymbionts and their amoeba hosts concerns energy parasitism similar to the interaction between pathogenic chlamydiae and their human host cells. Phylogenetic analysis of all known ATP/ADP translocases indicated that the genes encoding ATP/ADP translocases originated from a chlamydial ancestor and were, after an ancient gene duplication, transferred horizontally to rickettsiae and plants.