Hidden from plain sight: Novel Simkaniaceae and Rhabdochlamydiaceae diversity emerging from screening genomic and metagenomic data.

Chlamydiota are an ancient and hyperdiverse phylum of obligate intracellular bacteria. The best characterized representatives are pathogens or parasites of mammals, but it is thought that their most common hosts are microeukaryotes like Amoebozoa. The diversity in taxonomy, evolution, and function of non-pathogenic Chlamydiota are slowly being described. Here we use data mining techniques and genomic analysis to extend our current knowledge of Chlamydiota diversity and its hosts, in particular the Order Parachlamydiales. We extract one Rhabdochlamydiaceae and three Simkaniaceae Metagenome-Assembled Genomes (MAGs) from NCBI Short Read Archive deposits of ciliate and algal genome sequencing projects. We then use these to identify a further 14 and 8 MAGs respectively amongst existing, unidentified environmental assemblies. From these data we identify two novel clades with host associated data, for which we propose the names "Sacchlamyda saccharinae" (Family Rhabdochlamydiaceae) and "Amphrikana amoebophyrae" (Family Simkaniaceae), as well as a third new clade of environmental MAGs "Acheromyda pituitae" (Family Rhabdochlamydiaceae). The extent of uncharacterized diversity within the Rhabdochlamydiaceae and Simkaniaceae is indicated by 16 of the 22 MAGs being evolutionarily distant from currently characterised genera. Within our limited data, there was great predicted diversity in Parachlamydiales metabolism and evolution, including the potential for metabolic and defensive symbioses as well as pathogenicity. These data provide an imperative to link genomic diversity in metagenomics data to their associated eukaryotic host, and to develop onward understanding of the functional significance of symbiosis with this hyperdiverse clade.

The Fish Pathogen "Candidatus Clavichlamydia salmonicola"-A Missing Link in the Evolution of Chlamydial Pathogens of Humans.

Chlamydiae like Chlamydia trachomatis and Chlamydia psittaci are well-known human and animal pathogens. Yet, the chlamydiae are a much larger group of evolutionary ancient obligate intracellular bacteria that includes predominantly symbionts of protists and diverse animals. This makes them ideal model organisms to study evolutionary transitions from symbionts in microbial eukaryotes to pathogens of humans. To this end, comparative genome analysis has served as an important tool. Genome sequence data for many chlamydial lineages are, however, still lacking, hampering our understanding of their evolutionary history. Here, we determined the first high-quality draft genome sequence of the fish pathogen "Candidatus Clavichlamydia salmonicola", representing a separate genus within the human and animal pathogenic Chlamydiaceae. The "Ca. Clavichlamydia salmonicola" genome harbors genes that so far have been exclusively found in Chlamydia species suggesting that basic mechanisms important for the interaction with chordate hosts have evolved stepwise in the history of chlamydiae. Thus, the genome sequence of "Ca. Clavichlamydia salmonicola" allows to constrain candidate genes to further understand the evolution of chlamydial virulence mechanisms required to infect mammals.

Diversity of Chlamydiales detected in pet birds privately kept in individual homes in Japan.

Chlamydia-related bacteria of the Chlamydiales order have recently been described as emerging pathogens that cause pneumonia and abortion in animals and humans. We investigated the presence of Chlamydiales using real-time polymerase chain reaction (PCR) by targeting the 16S rRNA gene of a broad range of Chlamydiales in 827 fecal samples from pet birds kept in individual homes in Japan. Of the 827 samples, 493 (59.6%) tested positive for the Chlamydiales 16S rRNA gene in the real-time PCR assay. We determined the nucleic acid sequences of PCR products from 17 Chlamydiales strains. A homology search and phylogenetic analysis using these sequences confirmed that the detected Chlamydiales included C. pecorum and a broad range of Chlamydia-related bacteria. To the best of our knowledge, this is the first study to detect a wide range of Chlamydia-related bacteria in birds.

Phylogenetic analyses of Candidatus Branchiomonas cysticola refine the taxonomic classification of Betaproteobacteria associated with epitheliocystis in fish.

Candidatus Branchiomonas cysticola is recognized as the most prevalent bacterial agent causing epitheliocystis in Atlantic salmon (Salmo salar). Based on its partial 16S rRNA sequence, the bacterium has previously been found to be a member of Burkholderiales in the class Betaproteobacteria. Multilocus Sequence Analysis (MLSA) of the bacterium and 60 type strains of Betaproteobacteria using newly identified housekeeping genes (dnaK, rpoC, and fusA) and ribosomal subunit sequences (16S and 23S), instead supported the bacterium's affiliation to Nitrosomodales. Taxonomic rank normalization by Relative Evolutionary Divergence (RED) showed the phylogenetic distinction between Cand. B. cysticola and its closest related type strain to be at the family level. A novel bacterial family named Branchiomonaceae has thus been proposed to include a monophyletic clade of Betaproteobacteria exclusively associated with epitheliocystis in fish.

The role of Chlamydiales in adverse pregnancy outcome: a community-based nested case-control study.

OBJECTIVES: Over the last decades, the Chlamydiales order has expanded and a new group of Chlamydia-related bacteria has emerged, covering species such as Waddlia chondrophila associated with bovine abortion. However, it is unknown whether they compromise human reproduction such as Chlamydia trachomatis. We therefore aimed to investigate the association between vaginal colonization of selected species of the Chlamydiales order with spontaneous abortion, preterm birth, and animal exposure. METHODS: Pregnant women were enrolled at the nuchal translucency scan or when admitted for suspected miscarriage at Aarhus University Hospital, Denmark. Cases were defined as spontaneous abortion <22 weeks and preterm birth <37 weeks. Controls were defined as term birth ≥37 weeks. Vaginal samples from 1203 women were assessed using C. trachomatis, W. chondrophila, and pan-Chlamydiales-specific real-time PCRs targeting the 16S rRNA gene. RESULTS: A total of 1120 women of primarily Caucasian ancestry were enrolled, including 193 spontaneous abortions, 88 preterm births, and 839 term births. After sequencing for verification, the prevalence of Chlamydiales was 3 of 193 (1.6%; 95% CI, 0.5-4.8) in women experiencing spontaneous abortion, 2 of 88 (2.3%; 95% CI, 0.6-8.9) in women with preterm birth, and 20 of 839 (2.4%; 95% CI, 1.6-3.7) in women giving birth at term. Thus, Chlamydiales infection was neither significantly associated with spontaneous abortion (OR, 0.68; 95% CI, 0.15-2.01) nor preterm birth (OR, 1.02; 95% CI, 0.15-3.60) compared with women giving birth at term. Amplicons from the pan-Chlamydiales assay revealed close sequence homology and were primarily identified as uncultured Chlamydiales bacteria. DISCUSSION: Among Danish pregnant women, the prevalence of Chlamydiales was low and not associated with adverse pregnancy outcomes.

First evidence for the existence of Simkania negevensis in the genitalia of human females.

INTRODUCTION: Simkania negevensis has been linked to some respiratory and non-respiratory diseases. However, there is still a serious lack of clinical investigations that attempt to determine possible body sites that could be inhabited by this microorganism and evaluate its true pathogenic capacity. The goal of this study was to examine the potential presence of Simkania and its prevalence in the genital tract of human adult females. METHODS: Lower vaginal swabs from 169 Jordanian adult females who attended Obstetrics and Gynecology clinic were collected and tested for Simkania DNA by PCR method. RESULTS: The presence of bacterial nucleic acids was confirmed in the genital system of adult females with an overall prevalence of 24.26% (41/169). Interestingly, the positivity of Simkania DNA was significantly higher in women of reproductive age than females of non-reproductive age (28.03% versus 10.81%; p≤0.05). Moreover, the presence of S. negevensis was evident in approximately 43% of females suffering from vaginal itching and/or abnormal discharge, exhibiting about two-fold increase in the positivity rate compared to detection rates assessed for women who attended the clinic for routine checkup or menstruation problems. However, the current work failed to find any link between the bacterial agent and spontaneous abortion (miscarriage). CONCLUSIONS: This study showed for the first time the presence of S. negevensis in the genitalia of human females. These novel data could provide a basis to clarify the exact role of S. negevensis in the female genitalia and its potential involvement in genital system disorders.

Identification of Parachlamydiaceae DNA in nasal and rectal passages of healthy dairy cattle.

AIMS: The order Chlamydiales comprises a broad range of bacterial pathogens and endosymbionts, which infect a wide variety of host species. Within this order, members of the family Parachlamydiaceae, which includes Parachlamydia and Neochlamydia species, have been particularly associated with infections in both humans and cattle, including having a potential pathogenic role in cases of bovine abortion. While the route of transmission has yet to be defined, it has been hypothesised that asymptomatic carriage and contamination of the immediate environment may be a route of inter-animal transmission. We investigated the asymptomatic carriage of Chlamydia-related organisms in healthy cattle. METHODS & RESULTS: DNA was isolated from nasal and rectal swabs obtained from 38 healthy dairy heifers. A Chlamydiales sp. 16S rRNA qPCR was performed on each sample. A total of 18/38 nasal samples and all 38/38 rectal samples were identified as positive for Chlamydiales sp. Each positive sample was sequenced confirming the presence of DNA belonging to the Parachlamydiaceae. CONCLUSIONS: The presence of Parachlamydiaceae DNA in nasal and rectal swab samples of healthy cattle provides evidence for the asymptomatic carriage of parachlamydial organisms within cattle. SIGNIFICANCE & IMPACT OF THE STUDY: The study provides evidence of potential routes of environmental contamination that could provide a route for inter-animal and animal transmission of Parachlamydiaceae.

Conservation of the glycogen metabolism pathway underlines a pivotal function of storage polysaccharides in Chlamydiae.

The order Chlamydiales includes obligate intracellular pathogens capable of infecting mammals, fishes and amoeba. Unlike other intracellular bacteria for which intracellular adaptation led to the loss of glycogen metabolism pathway, all chlamydial families maintained the nucleotide-sugar dependent glycogen metabolism pathway i.e. the GlgC-pathway with the notable exception of both Criblamydiaceae and Waddliaceae families. Through detailed genome analysis and biochemical investigations, we have shown that genome rearrangement events have resulted in a defective GlgC-pathway and more importantly we have evidenced a distinct trehalose-dependent GlgE-pathway in both Criblamydiaceae and Waddliaceae families. Altogether, this study strongly indicates that the glycogen metabolism is retained in all Chlamydiales without exception, highlighting the pivotal function of storage polysaccharides, which has been underestimated to date. We propose that glycogen degradation is a mandatory process for fueling essential metabolic pathways that ensure the survival and virulence of extracellular forms i.e. elementary bodies of Chlamydiales.

Development of a new TaqMan-based PCR assay for the specific detection and quantification of Simkania negevensis.

Simkania negevensis is an emerging Chlamydia-like bacterium related to human respiratory diseases. An early and accurate detection of this pathogen could be useful to monitor the potential infectious risks and to set suitable outbreak control measures. In Tunisia, distribution and abundance of S. negevensis remain until now largely unknown. In the present work, a qPCR assay, targeting the 16S rRNA gene, for fast detection and quantification of S. negevensis was developed and validated. A high specificity for S. negevensis detection displaying no cross-reaction with the closely related Chlamydia spp. or the other tested microorganisms was noticed. qPCR assay performance was considered very satisfying with detection limits of 5 DNA copies per reaction. qPCR assay validation was performed by screening 37 clinical specimens and 35 water samples. S. negevensis wasn't detected in respiratory samples, but it was found in four cases of water samples. We suggest that the qPCR assay developed in this study could be considered sufficiently characterized to initiate the quantification of S. negevensis in environmental samples.

Marine Sediments Illuminate Chlamydiae Diversity and Evolution.

The bacterial phylum Chlamydiae is so far composed of obligate symbionts of eukaryotic hosts. Well known for Chlamydiaceae, pathogens of humans and other animals, Chlamydiae also include so-called environmental lineages that primarily infect microbial eukaryotes. Environmental surveys indicate that Chlamydiae are found in a wider range of environments than anticipated previously. However, the vast majority of this chlamydial diversity has been underexplored, biasing our current understanding of their biology, ecological importance, and evolution. Here, we report that previously undetected and active chlamydial lineages dominate microbial communities in deep anoxic marine sediments taken from the Arctic Mid-Ocean Ridge. Reaching relative abundances of up to 43% of the bacterial community, and a maximum diversity of 163 different species-level taxonomic units, these Chlamydiae represent important community members. Using genome-resolved metagenomics, we reconstructed 24 draft chlamydial genomes, expanding by over a third the known genomic diversity in this phylum. Phylogenomic analyses revealed several novel clades across the phylum, including a previously unknown sister lineage of the Chlamydiaceae, providing new insights into the origin of pathogenicity in this family. We were unable to identify putative eukaryotic hosts for these marine sediment chlamydiae, despite identifying genomic features that may be indicative of host-association. The high abundance and genomic diversity of Chlamydiae in these anoxic marine sediments indicate that some members could play an important, and thus far overlooked, ecological role in such environments and may indicate alternate lifestyle strategies.

A predation assay using amoebae to screen for virulence factors unearthed the first W. chondrophila inclusion membrane protein.

Waddlia chondrophila is an intracellular bacterium phylogenetically related to the well-studied human and animal pathogens of the Chlamydiaceae family. In the last decade, W. chondrophila was convincingly demonstrated to be associated with adverse pregnancy outcomes in humans and abortions in animals. All members of the phylum Chlamydiae possess a Type Three Secretion System that they use for delivering virulence proteins into the host cell cytosol to modulate their environment and create optimal conditions to complete their life cycle. To identify W. chondrophila virulence proteins, we used an original screening approach that combines a cosmid library with an assay monitoring resistance to predation by phagocytic amoebae. This technique combined with bioinformatic data allowed the identification of 28 candidate virulence proteins, including Wimp1, the first identified inclusion membrane protein of W. chondrophila.

Multilocus Sequence Typing (MLST) of Chlamydiales.

Developed two decades ago as a molecular method to provide definite characterization of a bacterial isolate, Multilocus Sequence Typing (MLST) is today globally adopted as a universal fine-detailed molecular typing tool and has been applied to numerous pathogenic and nonpathogenic bacterial as well eukaryotic organisms. MLST utilizes DNA sequence of several conserved housekeeping (HK) genes which are assigned an allelic number, which then collectively constitute an allelic profile or sequence type (ST), a "molecular barcode" of the interrogated bacterial strain or a eukaryotic organism. Here, we describe the principles and molecular approaches for generating MLST data for an analysis of a bacteria in the order Chlamydiales, using a Chlamydia pecorum-specific MLST scheme as an example.

Sequencing the Obligate Intracellular Rhabdochlamydia helvetica within Its Tick Host Ixodes ricinus to Investigate Their Symbiotic Relationship.

The Rhabdochlamydiaceae family is one of the most widely distributed within the phylum Chlamydiae, but most of its members remain uncultivable. Rhabdochlamydia 16S rRNA was recently reported in more than 2% of 8,534 pools of ticks from Switzerland. Shotgun metagenomics was performed on a pool of five female Ixodes ricinus ticks presenting a high concentration of chlamydial DNA, allowing the assembly of a high-quality draft genome. About 60% of sequence reads originated from a single bacterial population that was named "Candidatus Rhabdochlamydia helvetica" whereas only few thousand reads mapped to the genome of "Candidatus Midichloria mitochondrii," a symbiont normally observed in all I. ricinus females. The 1.8 Mbp genome of R. helvetica is smaller than other Chlamydia-related bacteria. Comparative analyses with other chlamydial genomes identified transposases of the PD-(D/E)XK nuclease family that are unique to this new genome. These transposases show evidence of interphylum horizontal gene transfers between multiple arthropod endosymbionts, including Cardinium spp. (Bacteroidetes) and diverse proteobacteria such as Wolbachia, Rickettsia spp. (Rickettsiales), and Caedimonas varicaedens (Holosporales). Bacterial symbionts were previously suggested to provide B-vitamins to hematophagous hosts. However, incomplete metabolic capacities including for B-vitamin biosynthesis, high bacterial density and limited prevalence suggest that R. helvetica is parasitic rather than symbiotic to its host. The identification of novel Rhabdochlamydia strains in different hosts and their sequencing will help understanding if members of this genus have become highly specialized parasites with reduced genomes, like the Chlamydiaceae, or if they could be pathogenic to humans using ticks as a transmission vector.

The first detection of the sequence of bacteria from the Simkaniaceae family in surface waters in Poland.

One of the most common reasons for horse lameness is subchondral bone cysts (SBCs), which are especially evident in young horse athletes. It is believed that SBC development is strongly associated with an individual's bone growth and/or bone microstructure impairment. Current methods of SBC treatment include pharmacological treatment or surgical procedures which may allow the bone within the cyst to rebuild and be restored to properly developed bone tissue. Thus, we propose filling the SBCs with a 3D complex of alginate hydrogel and autologous adipose derived mesenchymal stem cells (ASCs). We have observed at the in vitro level, that this hydrogel complex induces osteogenic and chondrogenic differentiation potential through the upregulation of bone morphogenetic protein, osteopontin, collagen type I and aggrecan mRNA levels. Moreover, we detected the creation of a 3D extracellular matrix (EM). To investigate the complex in vivo, we chose 8 horses of varying age suffering from SBC, which resulted in lameness, to undergo experimental surgery. We documented the horses' clinical appearance, lameness and radiographic appearance, to determine that there was clinical improvement in 87.75% of the patients (n=7, out of 8 horses) 6 months postoperatively and 100% (n=8, out of 8 horses) a year after surgery. These results are promising for the potential of this procedure to become the standard in SBC treatment.

Identification of new DNA-associated proteins from Waddlia chondrophila.

Transcriptional regulation in Chlamydiae is still poorly understood. The absence until recently of genetic tools is the main cause of this gap. We discovered three new potential DNA-associated proteins of Waddlia chondrophila, a Chlamydia-related bacterium, using heparin chromatography coupled to mass spectrometry (Wcw_0377, Wcw_1456, and Wcw_1460). By ChIP-seq analysis, we determined the regulatory landscape of these three proteins and we showed that Wcw_0377 binds all along the genome whereas Wcw_1456 and _1460 possess a wide regulon with a large number of co-regulated genes. Wcw_1456 and Wcw_1460 interact with RpoD (σ66), emerging as potential RpoD regulators. On the other hand, Wcw_0377 is able to reach the host nucleus, where it might interact with eukaryotic histones through its putative chromatin-remodelling SWIB/MDM2 domain.

Metagenomic Analysis of Fish-Associated Ca. Parilichlamydiaceae Reveals Striking Metabolic Similarities to the Terrestrial Chlamydiaceae.

Chlamydiae are an example of obligate intracellular bacteria that possess highly reduced, compact genomes (1.0-3.5 Mbp), reflective of their abilities to sequester many essential nutrients from the host that they no longer need to synthesize themselves. The Chlamydiae is a phylum with a very wide host range spanning mammals, birds, fish, invertebrates, and unicellular protists. This ecological and phylogenetic diversity offers ongoing opportunities to study intracellular survival and metabolic pathways and adaptations. Of particular evolutionary significance are Chlamydiae from the recently proposed Ca. Parilichlamydiaceae, the earliest diverging clade in this phylum, species of which are found only in aquatic vertebrates. Gill extracts from three Chlamydiales-positive Australian aquaculture species (Yellowtail kingfish, Striped trumpeter, and Barramundi) were subject to DNA preparation to deplete host DNA and enrich microbial DNA, prior to metagenome sequencing. We assembled chlamydial genomes corresponding to three Ca. Parilichlamydiaceae species from gill metagenomes, and conducted functional genomics comparisons with diverse members of the phylum. This revealed highly reduced genomes more similar in size to the terrestrial Chlamydiaceae, standing in contrast to members of the Chlamydiae with a demonstrated cosmopolitan host range. We describe a reduction in genes encoding synthesis of nucleotides and amino acids, among other nutrients, and an enrichment of predicted transport proteins. Ca. Parilichlamydiaceae share 342 orthologs with other chlamydial families. We hypothesize that the genome reduction exhibited by Ca. Parilichlamydiaceae and Chlamydiaceae is an example of within-phylum convergent evolution. The factors driving these events remain to be elucidated.

Virulence-related comparative transcriptomics of infectious and non-infectious chlamydial particles.

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.

Genotyping of Candidatus Syngnamydia salmonis (chlamydiales; Simkaniaceae) co-cultured in Paramoeba perurans (amoebozoa; Paramoebidae).

Candidatus Syngnamydia salmonis (Chlamydiales, Simkaniaceae) was described as an epitheliocystis-causing bacterium from the gills of Atlantic salmon (Salmo salar) in Norway. A bacterium showing 99.2% 16S rRNA identity to Cand. S. salmonis is able to multiply in Paramoeba perurans and based on the classification criteria this bacterium could represent the same species as Cand. S. salmonis. Sequencing the genome of the cultured bacterium has made it possible to fulfill the minimal standards for genetic characterization of species within the order Chlamydiales. The complete rRNA genes, the amino acid sequences of SucA, PepF, Adk, HemL, DnaA, FtsK and FabI, are presented in addition to the morphology of the Chlamydia-like morphs in the cytoplasm of P. perurans.