Chlamydia Spreads to the Large Intestine Lumen via Multiple Pathways.

Chlamydia in the genital tract is known to spread via the blood circulation system to the large intestine lumen to achieve long-lasting colonization. However, the precise pathways by which genital Chlamydia accesses the large intestine lumen remain unclear. The spleen was recently reported to be critical for chlamydial spreading. In the current study, it was found that following intravaginal inoculation with Chlamydia, mice with and without splenectomy both yielded infectious Chlamydia on rectal swabs, indicating that the spleen is not essential for genital Chlamydia to spread to the gastrointestinal tract. This conclusion was validated by the observation that intravenously inoculated Chlamydia was also detected on the rectal swabs of mice regardless of splenectomy. Careful comparison of the tissue distribution of live chlamydial organisms following intravenous inoculation revealed redundant pathways by which Chlamydia can reach the large intestine lumen. The intravenously inoculated Chlamydia was predominantly recruited to the spleen within 12 h and then detected in the stomach lumen by 24 h, in the intestinal lumen by 48 h, and on rectal swabs by 72 h. These observations suggest a potential spleen-to-stomach pathway for hematogenous Chlamydia to reach the large intestine lumen. This conclusion was supported by the observation made in mice under coprophagy-free condition. However, in the absence of spleen, hematogenous Chlamydia was predominantly recruited to the liver and then simultaneously detected in the intestinal tissue and lumen, suggesting a potential liver-to-intestine pathway for Chlamydia to reach the large intestine lumen. Thus, genital/hematogenous Chlamydia may reach the large intestine lumen via multiple redundant pathways.

Gastrointestinal Chlamydia-Induced CD8+ T Cells Promote Chlamydial Pathogenicity in the Female Upper Genital Tract.

Chlamydia is known to both ascend to the upper genital tract and spread to the gastrointestinal tract following intravaginal inoculation. Gastrointestinal Chlamydia was recently reported to promote chlamydial pathogenicity in the genital tract since mice intravaginally inoculated with an attenuated Chlamydia strain, which alone failed to develop pathology in the genital tract, were restored to develop hydrosalpinx by intragastric coinoculation with wild-type Chlamydia. Gastrointestinal Chlamydia promoted hydrosalpinx via an indirect mechanism since Chlamydia in the gut did not directly spread to the genital tract lumen. In the current study, we further investigated the role of CD8+ T cells in the promotion of hydrosalpinx by gastrointestinal Chlamydia. First, we confirmed that intragastric coinoculation with wild-type Chlamydia promoted hydrosalpinx in mice that were inoculated with an attenuated Chlamydia strain in the genital tract 1 week earlier. Second, the promotion of hydrosalpinx by intragastrically coinoculated Chlamydia was blocked by depleting CD8+ T cells. Third, adoptive transfer of gastrointestinal Chlamydia-induced CD8+ T cells was sufficient for promoting hydrosalpinx in mice that were intravaginally inoculated with an attenuated Chlamydia strain. These observations have demonstrated that CD8+ T cells induced by gastrointestinal Chlamydia are both necessary and sufficient for promoting hydrosalpinx in the genital tract. The study has laid a foundation for further revealing the mechanisms by which Chlamydia-induced T lymphocyte responses (as a 2nd hit) promote hydrosalpinx in mice with genital Chlamydia-triggered tubal injury (as a 1st hit), a continuing effort in testing the two-hit hypothesis as a chlamydial pathogenic mechanism.

Chlamydia exploits filopodial capture and a macropinocytosis-like pathway for host cell entry.

Pathogens hijack host endocytic pathways to force their own entry into eukaryotic target cells. Many bacteria either exploit receptor-mediated zippering or inject virulence proteins directly to trigger membrane reorganisation and cytoskeletal rearrangements. By contrast, extracellular C. trachomatis elementary bodies (EBs) apparently employ facets of both the zipper and trigger mechanisms and are only ~400 nm in diameter. Our cryo-electron tomography of C. trachomatis entry revealed an unexpectedly diverse array of host structures in association with invading EBs, suggesting internalisation may progress by multiple, potentially redundant routes or several sequential events within a single pathway. Here we performed quantitative analysis of actin organisation at chlamydial entry foci, highlighting filopodial capture and phagocytic cups as dominant and conserved morphological structures early during internalisation. We applied inhibitor-based screening and employed reporters to systematically assay and visualise the spatio-temporal contribution of diverse endocytic signalling mediators to C. trachomatis entry. In addition to the recognised roles of the Rac1 GTPase and its associated nucleation-promoting factor (NPF) WAVE, our data revealed an additional unrecognised pathway sharing key hallmarks of macropinocytosis: i) amiloride sensitivity, ii) fluid-phase uptake, iii) recruitment and activity of the NPF N-WASP, and iv) the localised generation of phosphoinositide-3-phosphate (PI3P) species. Given their central role in macropinocytosis and affinity for PI3P, we assessed the role of SNX-PX-BAR family proteins. Strikingly, SNX9 was specifically and transiently enriched at C. trachomatis entry foci. SNX9-/- cells exhibited a 20% defect in EB entry, which was enhanced to 60% when the cells were infected without sedimentation-induced EB adhesion, consistent with a defect in initial EB-host interaction. Correspondingly, filopodial capture of C. trachomatis EBs was specifically attenuated in SNX9-/- cells, implicating SNX9 as a central host mediator of filopodial capture early during chlamydial entry. Our findings identify an unanticipated complexity of signalling underpinning cell entry by this major human pathogen, and suggest intriguing parallels with viral entry mechanisms.

A nanovaccine formulation of Chlamydia recombinant MOMP encapsulated in PLGA 85:15 nanoparticles augments CD4+ effector (CD44high CD62Llow) and memory (CD44high CD62Lhigh) T-cells in immunized mice.

Vaccine developmental strategies are utilizing antigens encapsulated in biodegradable polymeric nanoparticles. Here, we developed a Chlamydia nanovaccine (PLGA-rMOMP) by encapsulating its recombinant major outer membrane protein (rMOMP) in the extended-releasing and self-adjuvanting PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. PLGA-rMOMP was small (nanometer size), round and smooth, thermally stable, and exhibited a sustained release of rMOMP. Stimulation of mouse primary dendritic cells (DCs) with PLGA-rMOMP augmented endosome processing, induced Th1 cytokines (IL-6 and IL-12p40), and expression of MHC-II and co-stimulatory (CD40, CD80, and CD86) molecules. BALB/c mice immunized with PLGA-rMOMP produced enhanced CD4+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) phenotypes and functional antigen-specific serum IgG antibodies. In vivo biodistribution of PLGA-rMOMP revealed its localization within lymph nodes, suggesting migration from the injection site via DCs. Our data provide evidence that the PLGA (85:15) nanovaccine activates DCs and augments Chlamydia-specific rMOMP adaptive immune responses that are worthy of efficacy testing.

Advances and Obstacles in the Genetic Dissection of Chlamydial Virulence.

Obligate intracellular pathogens in the family Chlamydiaceae infect taxonomically diverse eukaryotes ranging from amoebae to mammals. However, many fundamental aspects of chlamydial cell biology and pathogenesis remain poorly understood. Genetic dissection of chlamydial biology has historically been hampered by a lack of genetic tools. Exploitation of the ability of chlamydia to recombine genomic material by lateral gene transfer (LGT) ushered in a new era in chlamydia research. With methods to map mutations in place, genetic screens were able to assign functions and phenotypes to specific chlamydial genes. Development of an approach for stable transformation of chlamydia also provided a mechanism for gene delivery and platforms for disrupting chromosomal genes. Here, we explore how these and other tools have been used to test hypotheses concerning the functions of known chlamydial virulence factors and discover the functions of completely uncharacterized genes. Refinement and extension of the existing genetic tools to additional Chlamydia spp. will substantially advance understanding of the biology and pathogenesis of this important group of pathogens.

Manipulation of the Host Cell Cytoskeleton by Chlamydia.

Chlamydiae are obligate intracellular pathogens. They undergo a biphasic developmental cycle differentiating between the infectious but metabolically quiescent elementary body and the vegetative, but non-infectious reticulate body. Chlamydia spends a significant portion of its development in the non-infectious stage, demanding an effective strategy of manipulating the host cells to ensure its intracellular survival and replication. A common target of all Chlamydia species studied so far is the host cell cytoskeleton, with past and recent findings revealing crucial roles in invasion, inclusion maintenance, nutrient acquisition, and egress. The molecular details of how Chlamydia co-opts the cytoskeleton is becoming clearer, with bacterial factors and their corresponding host cell targets identified.

Subversion of Cell-Autonomous Host Defense by Chlamydia Infection.

Obligate intracellular bacteria entirely depend on the metabolites of their host cell for survival and generation of progeny. Due to their lifestyle inside a eukaryotic cell and the lack of any extracellular niche, they have to perfectly adapt to compartmentalized intracellular environment of the host cell and counteract the numerous defense strategies intrinsically present in all eukaryotic cells. This so-called cell-autonomous defense is present in all cell types encountering Chlamydia infection and is in addition closely linked to the cellular innate immune defense of the mammalian host. Cell type and chlamydial species-restricted mechanisms point a long-term evolutionary adaptation that builds the basis of the currently observed host and cell-type tropism among different Chlamydia species. This review will summarize the current knowledge on the strategies pathogenic Chlamydia species have developed to subvert and overcome the multiple mechanisms by which eukaryotic cells defend themselves against intracellular pathogens.

Deconstructing the Chlamydial Cell Wall.

The evolutionary separated Gram-negative Chlamydiales show a biphasic life cycle and replicate exclusively within eukaryotic host cells. Members of the genus Chlamydia are responsible for many acute and chronic diseases in humans, and Chlamydia-related bacteria are emerging pathogens. We revisit past efforts to detect cell wall material in Chlamydia and Chlamydia-related bacteria in the context of recent breakthroughs in elucidating the underlying cellular and molecular mechanisms of the chlamydial cell wall biosynthesis. In this review, we also discuss the role of cell wall biosynthesis in chlamydial FtsZ-independent cell division and immune modulation. In the past, penicillin susceptibility of an invisible wall was referred to as the "chlamydial anomaly." In light of new mechanistic insights, chlamydiae may now emerge as model systems to understand how a minimal and modified cell wall biosynthetic machine supports bacterial cell division and how cell wall-targeting beta-lactam antibiotics can also act bacteriostatically rather than bactericidal. On the heels of these discussions, we also delve into the effects of other cell wall antibiotics in individual chlamydial lineages.

Molecular Pathogenesis of Chlamydia Disease Complications: Epithelial-Mesenchymal Transition and Fibrosis.

The reproductive system complications of genital chlamydial infection include fallopian tube fibrosis and tubal factor infertility. However, the molecular pathogenesis of these complications remains poorly understood. The induction of pathogenic epithelial-mesenchymal transition (EMT) through microRNA (miRNA) dysregulation was recently proposed as the pathogenic basis of chlamydial complications. Focusing on fibrogenesis, we investigated the hypothesis that chlamydia-induced fibrosis is caused by EMT-driven generation of myofibroblasts, the effector cells of fibrosis that produce excessive extracellular matrix (ECM) proteins. The results revealed that the targets of a major category of altered miRNAs during chlamydial infection are key components of the pathophysiological process of fibrogenesis; these target molecules include collagen types I, III, and IV, transforming growth factor ß (TGF-ß), TGF-ß receptor 1 (TGF-ßR1), connective tissue growth factor (CTGF), E-cadherin, SRY-box 7 (SOX7), and NFAT (nuclear factor of activated T cells) kinase dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1a (Dyrk1a). Chlamydial induction of EMT resulted in the generation of α-smooth muscle actin (α-SMA)-positive myofibroblasts that produced ECM proteins, including collagen types I and III and fibronectin. Furthermore, the inhibition of EMT prevented the generation of myofibroblasts and production of ECM proteins during chlamydial infection. These findings may provide useful avenues for targeting EMT or specific components of the EMT pathways as a therapeutic intervention strategy to prevent chlamydia-related complications.

The Roles of Unfolded Protein Response Pathways in Chlamydia Pathogenesis.

Chlamydia is an obligate intracellular bacterium that relies on host cells for essential nutrients and adenosine triphosphate (ATP) for a productive infection. Although the unfolded protein response (UPR) plays a major role in certain microbial infectivity, its role in chlamydial pathogenesis is unknown. We hypothesized that Chlamydia induces UPR and exploits it to upregulate host cell uptake and metabolism of glucose, production of ATP, phospholipids, and other molecules required for its replicative development and host survival. Using a combination of biochemical and pathway inhibition assays, we showed that the 3 UPR pathway transducers-protein kinase RNA-activated (PKR)-like ER kinase (PERK), inositol-requiring enzyme-1α (IRE1α), and activating transcription factor-6α (ATF6α)-were activated during Chlamydia infection. The kinase activity of PERK and ribonuclease (RNase) of IRE1α mediated the upregulation of hexokinase II and production of ATP via substrate-level phosphorylation. In addition, the activation of PERK and IRE1α promoted autophagy formation and apoptosis resistance for host survival. Moreover, the activation of IRE1α resulted in the generation of spliced X-box binding protein 1 (sXBP1) and upregulation of lipid production. The vital role of UPR pathways in Chlamydia development and pathogenesis could lead to the identification of potential molecular targets for therapeutics against Chlamydia.

From genomes to genotypes: molecular epidemiological analysis of Chlamydia gallinacea reveals a high level of genetic diversity for this newly emerging chlamydial pathogen.

BACKGROUND: Chlamydia (C.) gallinacea is a recently identified bacterium that mainly infects domestic chickens. Demonstration of C. gallinacea in human atypical pneumonia suggests its zoonotic potential. Its prevalence in chickens exceeds that of C. psittaci, but genetic and genomic research on C. gallinacea is still at the beginning. In this study, we conducted whole-genome sequencing of C. gallinacea strain JX-1 isolated from an asymptomatic chicken, and comparative genomic analysis between C. gallinacea strains and related chlamydial species. RESULTS: The genome of C. gallinacea JX-1 was sequenced by single-molecule, real-time technology and is comprised of a 1,059,522-bp circular chromosome with an overall G + C content of 37.93% and sequence similarity of 99.4% to type strain 08-1274/3. In addition, a plasmid designated pJX-1, almost identical to p1274 of the type strain, except for two point mutations, was only found in field strains from chicken, but not in other hosts. In contrast to chlamydial species with notably variable polymorphic membrane protein (pmp) genes and plasticity zone (PZ), these regions were conserved in both C. gallinacea strains. There were 15 predicted pmp genes, but only B, A, E1, H, G1 and G2 were apparently intact in both strains. In comparison to chlamydial species where the PZ may be up to 50 kbp, C. gallinacea strains displayed gene content reduction in the PZ (14 kbp), with strain JX-1 having a premature STOP codon in the cytotoxin (tox) gene, while tox gene is intact in the type strain. In multilocus sequence typing (MLST), 15 C. gallinacea STs were identified among 25 strains based on cognate MLST allelic profiles of the concatenated sequences. The type strain and all Chinese strains belong to two distinct phylogenetic clades. Clade of the Chinese strains separated into 14 genetically distinct lineages, thus revealing considerable genetic diversity of C. gallinacea strains in China. CONCLUSIONS: In this first detailed comparative genomic analysis of C. gallinacea, we have provided evidence for substantial genetic diversity among C. gallinacea strains. How these genetic polymorphisms affect C. gallinacea biology and pathogenicity should be addressed in future studies that focus on phylogenetics and host adaption of this enigmatic bacterial agent.

Culture-independent genomics of a novel chlamydial pathogen of fish provides new insight into host-specific adaptations utilized by these intracellular bacteria.

Several Chlamydiales families are associated with epitheliocystis, a common condition of the fish gill epithelium. These families share common ancestors with the Chlamydiaceae and environmental Chlamydiae. Due to the lack of culture systems, little is known about the biology of these chlamydial fish pathogens. We investigated epitheliocystis in cultured Orange-spotted grouper (Epinephelus coioides) from North Queensland, Australia. Basophilic inclusions were present in the gills of 22/31 fish and the presence of the chlamydial pathogen in the cysts was confirmed by in situ hybridization. Giant grouper (Epinephelus lanceolatus) cultured in the same systems were epitheliocystis free. 16S rRNA gene sequencing revealed a novel member of the Candidatus Parilichlamydiaceae: Ca. Similichlamydia epinephelii. Using metagenomic approaches, we obtained an estimated 68% of the chlamydial genome, revealing that this novel chlamydial pathogen shares a number of key pathogenic hallmarks with the Chlamydiaceae, including an intact Type III Secretion system and several chlamydial virulence factors. This provides additional evidence that these pathogenic mechanisms were acquired early in the evolution of this unique bacterial phylum. The identification and genomic characterization of Ca. S. epinephelii provides new opportunities to study the biology of distantly-related chlamydial pathogens while shining a new light on the evolution of pathogenicity of the Chlamydiaceae.

Chlamydia spp. development is differentially altered by treatment with the LpxC inhibitor LPC-011.

BACKGROUND: Chlamydia species are obligate intracellular bacteria that infect a broad range of mammalian hosts. Members of related genera are pathogens of a variety of vertebrate and invertebrate species. Despite the diversity of Chlamydia, all species contain an outer membrane lipooligosaccharide (LOS) that is comprised of a genus-conserved, and genus-defining, trisaccharide 3-deoxy-D-manno-oct-2-ulosonic acid Kdo region. Recent studies with lipopolysaccharide inhibitors demonstrate that LOS is important for the C. trachomatis developmental cycle during RB- > EB differentiation. Here, we explore the effects of one of these inhibitors, LPC-011, on the developmental cycle of five chlamydial species. RESULTS: Sensitivity to the drug varied in some of the species and was conserved between others. We observed that inhibition of LOS biosynthesis in some chlamydial species induced formation of aberrant reticulate bodies, while in other species, no change was observed to the reticulate body. However, loss of LOS production prevented completion of the chlamydial reproductive cycle in all species tested. In previous studies we found that C. trachomatis and C. caviae infection enhances MHC class I antigen presentation of a model self-peptide. We find that treatment with LPC-011 prevents enhanced host-peptide presentation induced by infection with all chlamydial-species tested. CONCLUSIONS: The data demonstrate that LOS synthesis is necessary for production of infectious progeny and inhibition of LOS synthesis induces aberrancy in certain chlamydial species, which has important implications for the use of LOS synthesis inhibitors as potential antibiotics.

Clinical characteristics of genital chlamydia infection in pelvic inflammatory disease.

BACKGROUND: Chlamydia infection in acute pelvic inflammatory disease (PID) is associated with serious complications including ectopic pregnancy, tubal infertility, Fitz-Hugh-Curtis syndrome and tubo-ovarian abscess (TOA). This study compared clinical and laboratory data between PID with and without chlamydia infection. METHODS: The medical records of 497 women who were admitted with PID between 2002 and 2011 were reviewed. The patients were divided into two groups (PID with and without chlamydia infection), which were compared in terms of the patients' characteristics, clinical presentation, and laboratory findings, including inflammatory markers. RESULTS: The chlamydia and non-chlamydia groups comprised 175 and 322 women, respectively. The patients in the chlamydia group were younger and had a higher rate of TOA, a longer mean hospital stay, and had undergone more surgeries than the patients in the non- chlamydia group. The erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and CA-125 level were higher in the chlamydia group than in the non-chlamydia group, but there was no significant difference in the white blood cell count between the two groups. The CA-125 level was the strongest predictor of chlamydia infection, followed by the ESR and CRP level. The area under the receiving operating curve for CA-125, ESR, and CRP was 0.804, 0.755, and 0.663, respectively. CONCLUSIONS: Chlamydia infection in acute PID is associated with increased level of inflammatory markers, such as CA-125, ESR and CRP, incidence of TOA, operation risk, and longer hospitalization.

Hijacking of Membrane Contact Sites by Intracellular Bacterial Pathogens.

Intracellular bacterial pathogens have evolved sophisticated mechanisms to hijack host cellular processes to promote their survival and replication inside host cells. Over the past two decades, much attention has been given to the strategies employed by these pathogens to manipulate various vesicular trafficking pathways. But in the past 5 years, studies have brought to light that intracellular bacteria also target non-vesicular trafficking pathways. Here we review how three vacuolar pathogens, namely, Legionella, Chlamydia, and Coxiella hijack components of cellular MCS with or without the formation of stable MCS. A common theme in the manipulation of MCS by intracellular bacteria is the dependence on the secretion of bacterial effector proteins. During the early stages of the Legionella life cycle, the bacteria connects otherwise unrelated cellular pathways (i.e., components of ER-PM MCS, PI4KIIIα, and Sac1 and the early secretory pathway) to remodel its nascent vacuole into an ER-like compartment. Chlamydia and Coxiella vacuoles establish direct MCS with the ER and target lipid transfer proteins that contain a FFAT motif, CERT, and ORP1L, respectively, suggesting a common mechanism of VAP-dependent lipid acquisition. Chlamydia also recruits STIM1, an ER calcium sensor involved in store-operated calcium entry (SOCE) at ER-PM MCS, and elucidating the role of STIM1 at ER-Chlamydia inclusion MCS may uncover additional role for these contacts. Altogether, the manipulation of MCS by intracellular bacterial pathogens has open a new and exciting area of research to investigate the molecular mechanisms supporting pathogenesis.

Chlamydia felis: Lack of association between clinical signs and the presence of the cryptic plasmid.

Chlamydia felis is an obligate intracellular bacterial pathogen that infects cats, causing severe conjunctivitis associated with upper respiratory tract disease (URTD). In the present study, 186 cats from three non-commercial catteries in São Paulo, SP, Brazil were evaluated. The detection of Chlamydia felis was performed by PCR. The clinical severity was scored from 1 to 4, with a score of 4 as the most severe manifestation. The total occurrence of C. felis was of 18.82% (35/186) of cats overall, but notably, 58.06% (18/31) of infected cats originated from a single cattery. All animals harboring C. felis had URTD clinical signs and higher scores (3 and 4). In addition, C. felis occurrence was associated with the presence of cryptic plasmid. However, the virulence and clinical severity were not correlated.

[A FITZ-HUGH-CURTIS SYNDROME AS A PREMISE OF THE INFLAMMATORY DISEASES OF A HEPATOPANCREATOBILIARY ZONE ORGANS].

A content of a biliary ways microflora and its correlation with clinical form and severity of inflammatory-destructive processes in hepatopancreatobiliary zone were studied up. The investigation objective was the optimization of a treatment--diagnostic tactics for the complications and remote recurrences rate reduction in a complicated biliary calculous disease. There was established, that a standard bacteriological investigation do not give a possibility to estimate some causes of a biliary ducts affection, a chlamidial infection in particular. So on, for the individual antibacterial therapy choosing a more detailed and modern examination of patients is needed.

Genetic diversity in the plasticity zone and the presence of the chlamydial plasmid differentiates Chlamydia pecorum strains from pigs, sheep, cattle, and koalas.

BACKGROUND: Chlamydia pecorum is a globally recognised pathogen of livestock and koalas. To date, comparative genomics of C. pecorum strains from sheep, cattle and koalas has revealed that only single nucleotide polymorphisms (SNPs) and a limited number of pseudogenes appear to contribute to the genetic diversity of this pathogen. No chlamydial plasmid has been detected in these strains despite its ubiquitous presence in almost all other chlamydial species. Genomic analyses have not previously included C. pecorum from porcine hosts. We sequenced the genome of three C. pecorum isolates from pigs with differing pathologies in order to re-evaluate the genetic differences and to update the phylogenetic relationships between C. pecorum from each of the hosts. METHODS: Whole genome sequences for the three porcine C. pecorum isolates (L1, L17 and L71) were acquired using C. pecorum-specific sequence capture probes with culture-independent methods, and assembled in CLC Genomics Workbench. The pairwise comparative genomic analyses of 16 pig, sheep, cattle and koala C. pecorum genomes were performed using several bioinformatics platforms, while the phylogenetic analyses of the core C. pecorum genomes were performed with predicted recombination regions removed. Following the detection of a C. pecorum plasmid, a newly developed C. pecorum-specific plasmid PCR screening assay was used to evaluate the plasmid distribution in 227 C. pecorum samples from pig, sheep, cattle and koala hosts. RESULTS: Three porcine C. pecorum genomes were sequenced using C. pecorum-specific sequence capture probes with culture-independent methods. Comparative genomics of the newly sequenced porcine C. pecorum genomes revealed an increased average number of SNP differences (~11 500) between porcine and sheep, cattle, and koala C. pecorum strains, compared to previous C. pecorum genome analyses. We also identified a third copy of the chlamydial cytotoxin gene, found only in porcine C. pecorum isolates. Phylogenetic analyses clustered porcine isolates into a distinct clade, highlighting the polyphyletic origin of C. pecorum in livestock. Most surprising, we also discovered a plasmid in the porcine C. pecorum genome. Using this novel C. pecorum plasmid (pCpec) sequence, a) we developed a pCpec screening assay to evaluate the plasmid distribution in C. pecorum from different hosts; and b) to characterise the pCpec sequences from available previously sequenced C. pecorum genome data. pCpec screening showed that the pCpec is common in all hosts of C. pecorum, however not all C. pecorum strains carry pCpec. CONCLUSIONS: This study provides further insight into the complexity of C. pecorum epidemiology and novel genomic regions that may be linked to host specificity. C. pecorum plasmid characterisation may aid in improving our understanding of C. pecorum pathogenesis across the variety of host species this animal pathogen infects.

Identification, characterisation and expression analysis of natural killer receptor genes in Chlamydia pecorum infected koalas (Phascolarctos cinereus).

BACKGROUND: Koalas (Phascolarctos cinereus), an iconic Australian marsupial, are being heavily impacted by the spread of Chlamydia pecorum, an obligate intracellular bacterial pathogen. Koalas vary in their response to this pathogen, with some showing no symptoms, while others suffer severe symptoms leading to infertility, blindness or death. Little is known about the pathology of this disease and the immune response against it in this host. Studies have demonstrated that natural killer (NK) cells, key components of the innate immune system, are involved in the immune response to chlamydial infections in humans. These cells can directly lyse cells infected by intracellular pathogens and their ability to recognise these infected cells is mediated through NK receptors on their surface. These are encoded in two regions of the genome, the leukocyte receptor complex (LRC) and the natural killer complex (NKC). These two families evolve rapidly and different repertoires of genes, which have evolved by gene duplication, are seen in different species. METHODS: In this study we aimed to characterise genes belonging to the NK receptor clusters in the koala by searching available koala transcriptomes using a combination of search methods. We developed a qPCR assay to quantify relative expression of four genes, two encoded within the NK receptor cluster (CLEC1B, CLEC4E) and two known to play a role in NK response to Chalmydia in humans (NCR3, PRF1). RESULTS: We found that the NK receptor repertoire of the koala closely resembles that of the Tasmanian devil, with minimal genes in the NKC, but with lineage specific expansions in the LRC. Additional genes important for NK cell activity, NCR3 and PRF1, were also identified and characterised. In a preliminary study to investigate whether these genes are involved in the koala immune response to infection by its chlamydial pathogen, C. pecorum, we investigated the expression of four genes in koalas with active chlamydia infection, those with past infection and those without infection using qPCR. This analysis revealed that one of these four, CLEC4E, may be upregulated in response to chlamydia infection. CONCLUSION: We have characterised genes of the NKC and LRC in koalas and have discovered evidence that one of these genes may be upregulated in koalas with chlamydia, suggesting that these receptors may play a role in the immune response of koalas to chlamydia infection.