Whole genome de novo sequencing and comparative genomic analyses suggests that Chlamydia psittaci strain 84/2334 should be reclassified as Chlamydia abortus species.

BACKGROUND: Chlamydia abortus and Chlamydia psittaci are important pathogens of livestock and avian species, respectively. While C. abortus is recognized as descended from C. psittaci species, there is emerging evidence of strains that are intermediary between the two species, suggesting they are recent evolutionary ancestors of C. abortus. Such strains include C. psittaci strain 84/2334 that was isolated from a parrot. Our aim was to classify this strain by sequencing its genome and explore its evolutionary relationship to both C. abortus and C. psittaci. RESULTS: In this study, methods based on multi-locus sequence typing (MLST) of seven housekeeping genes and on typing of five species discriminant proteins showed that strain 84/2334 clustered with C. abortus species. Furthermore, whole genome de novo sequencing of the strain revealed greater similarity to C. abortus in terms of GC content, while 16S rRNA and whole genome phylogenetic analysis, as well as network and recombination analysis showed that the strain clusters more closely with C. abortus strains. The analysis also suggested a closer evolutionary relationship between this strain and the major C. abortus clade, than to two other intermediary avian C. abortus strains or C. psittaci strains. Molecular analyses of genes (polymorphic membrane protein and transmembrane head protein genes) and loci (plasticity zone), found in key virulence-associated regions that exhibit greatest diversity within and between chlamydial species, reveal greater diversity than present in sequenced C. abortus genomes as well as similar features to both C. abortus and C. psittaci species. The strain also possesses an extrachromosomal plasmid, as found in most C. psittaci species but absent from all sequenced classical C. abortus strains. CONCLUSION: Overall, the results show that C. psittaci strain 84/2334 clusters very closely with C. abortus strains, and are consistent with the strain being a recent C. abortus ancestral species. This suggests that the strain should be reclassified as C. abortus. Furthermore, the identification of a C. abortus strain bearing an extra-chromosomal plasmid has implications for plasmid-based transformation studies to investigate gene function as well as providing a potential route for the development of a next generation vaccine to protect livestock from C. abortus infection.

Differential cytokine expression in Chlamydophila psittaci genotype A-, B- or D-infected chicken macrophages after exposure to Escherichia coli O2:K1 LPS.

Chlamydophila (Cp.) psittaci and avian pathogenic Escherichia (E.) coli infections contribute to the respiratory disease complex observed in turkeys. Secondary infection with E. coli exacerbates Cp. psittaci pathogenicity and augments E. coli excretion. The innate immune response initiated by both pathogens in their avian host is unknown. We therefore determined the cytokine responses following Cp. psittaci infection and E. coli superinfection of avian monocytes/macrophages by examining gene transcripts of IL-1beta, IL-6, CXCLi2 (IL-8), CXCLi1 (K60), IL-10, IL-12alpha/beta, IL-18, TGF-beta4 and CCLi2 at 4h post-inoculation with different Cp. psittaci strains or 4h post-treatment with avian E. coli LPS of Cp. psittaci pre-infected HD11 cells. Cp. psittaci strains used were 84/55 and 92/1293 (highly virulent), CP3 (low virulent) and 84/2334 (phylogenetically intermediate between Cp. psittaci and Chlamydophila abortus). At 4h post chlamydial infection, an increased expression of IL-1beta and IL-6 as well as CXCLi2, CXCLi1 and CCLi2 was observed compared to levels in uninfected HD11 controls. This effect was less pronounced for the milder CP3 strain. The pro-inflammatory response of Cp. psittaci infected cells to E. coli LPS was significantly lowered compared to uninfected controls, especially when the cells were pre-infected with highly virulent Cp. psittaci strains. In both experiments, exceptionally high IL-10 and no TGF-beta4 responses were observed, and we propose that this could induce macrophage deactivation and NF-kappaB suppression. Consequently, pro-inflammatory and Th1-promoting responses to both the primary Cp. psittaci infection and E. coli would be inhibited, thus explaining the observed aggravated in vivo pathology.

Vaccination of turkeys against Chlamydophila psittaci through optimised DNA formulation and administration.

We have demonstrated that vaccination of turkeys with an unformulated DNA vaccine induces significant protection against Chlamydophila (Cp.) psittaci infections. Nevertheless, the immunogenicity of the DNA vaccine can still be improved by increasing translation and transfection efficiency. Therefore, the ompA codon was adapted to the codon usage in birds, resulting in pcDNA1/MOMP(opt). To increase gene transfer, polyplexes of pcDNA1/MOMP(opt)-EGFP with different cationic polymers, such as linear and branched polyethyleneimine (lPEI and brPEI) and starburst PAMAM dendrimers, and lipoplexes with cationic DOTAP/DOPE liposomes were created. Transfection of lPEI and brPEI polyplexes with an N/P ratio of 8 resulted in the highest transfection efficiencies, but lPEI polyplexes were completely destroyed following nebulisation. Secondly, we examined the capacity of nebulised or intramuscularly (IM) administered brPEI-pcDNA1/MOMP(opt) to induce a significant protective immune response in SPF turkeys experimentally infected with 10(8) TCID(50) of a virulent Cp. psittaci strain. Results were compared to IM administration of naked plasmid DNA and to results of non-vaccinated animals. Intramuscular administration of brPEI-pcDNA1/MOMP(opt) increased the immunogenicity of the Cp. psittaci DNA vaccine as compared to IM administration of pcDNA1/MOMP(opt) or aerosol delivery of brPEI-pcDNA1/MOMP(opt). Improved immunogenicity was correlated with increased protection. Vaccinated groups were significantly protected against Cp. psittaci challenge.

Biology and intracellular pathogenesis of high or low virulent Chlamydophila psittaci strains in chicken macrophages.

Within a few days post infection of SPF turkeys, highly pathogenic Chlamydophila (Cp.) psittaci genotype A and D strains can be found in blood monocytes/macrophages, while this effect is less pronounced for infection with a milder genotype B strain. To elucidate on the observed difference, we studied the developmental cycle of avian Cp. psittaci strains of varying virulence in a matched avian monocyte/macrophage cell line (HD11) by electron microscopy and immunofluorescence and determined the gene transcription of 26 Type III secretion related genes and six control genes upon infection of HD11 cells. The genotype A (84/55) and D (92/1293) strains (1) clearly induced actin recruitment to the site of entry, (2) initiated host cell degeneration at earlier time points, and (3) survived and proliferated better when compared to the milder CP3 strain. Strain 84/2334, genetically intermediate between Cp. psittaci and Cp. abortus, did not induce actin recruitment. Limited mRNA transcripts for the cell division genes ftsW and ftsK were in agreement with the observed low replication of Cp. psittaci in these host cells. The results also indicated that genes coding for the structural components of the Type III secretion system were transcribed earlier compared to an infection in epithelial cells. Based on the presented results, we postulate that upon infection of blood monocytes/macrophages, Cp. psittaci deliberately limits its replication and immediately arms itself to infect other cells elsewhere in the host, whilst using the monocytes/macrophages as a quick transport vehicle.

Bacterial secretion systems with an emphasis on the chlamydial Type III secretion system.

Numerous bacterial proteins exert their function outside the prokaryotic cell. To this end, both Gram-negative and Gram-positive bacteria have evolved specialized mechanisms to transport their proteins to the bacterial supernatant or host cell cytoplasm, so called secretion systems. These different strategies will be briefly discussed, followed by an in depth description of the Type III secretion system, an efficient molecular syringe assisting Gram-negative bacteria in entrance, growth and survival in eukaryotic host cells. Topics addressed include classification and role of multiple Type III secretion systems, the mechanism of protein translocation into the host cell as well as substrate recognition and chaperoning. Chlamydiales have also been found to encode a Type III secretion system and associated effector proteins. In contrast to the genetic organization in other bacteria, the encoding genes are scattered throughout the genome. To date, no structural information is available on the chlamydial Type III secretion system. We therefore propose a model of the chlamydial Type III secretion system and summarize current knowledge on the role of Type III secretion in the different stages of the chlamydial developmental cycle.

Identification and characterization of a type III secretion system in Chlamydophila psittaci.

Chlamydiaceae are obligate intracellular Gram-negative bacteria replicating in vacuoles inside eukaryotic cells. It has been proven that most of them possess a type III secretion system (T3SS) allowing them to transfer effector molecules in the host cell. We examined the existence of a T3SS in Chlamydophila psittaci by studying the expression of three essential structural proteins SctW, SctC, and SctN, and one putative effector protein IncA. Immunofluorescence assays showed SctW and IncA to be associated with the bacteria and the inclusion membrane, while SctC and SctN were only localized to the bacteria itself. Immuno electron microscopy could confirm these results for SctW, IncA, and SctC. Unfortunately, SctN was not investigated with this technique. Additionally, we sequenced 14 full-length T3S genes (scc1, sctW, sctJ, sctL, sctR, sctS, scc2, copD1, sctN, sctQ, sctC, incA, ca037, and cadd) and examined the transcription of 26 Cp. psittaci T3S genes namely cluster 1 (scc1, sctW, sctV, sctU), cluster 2 (sctJ, sctL, sctR, sctS, sctT, scc2, copB1, copD1), cluster 3 (sctD, sctN, ca037, sctQ, pkn5, sctC) and non-clustered genes (incA, incC, scc3, copD2, cap1, tarp, ca530, cadd). The gene expression study indicated the T3S structural protein encoding genes to be transcribed from mid-cycle (12-18 h post infection (p.i.)) on. Genes encoding effector proteins and putative T3S related proteins were expressed early (1.5 h-8 h p.i.) or late (>24 h p.i.) during the developmental cycle. We hereby provided evidence for the existence of a T3SS and possible effectors in avian Cp. psittaci.

Effect of ovotransferrin and lactoferrins on Chlamydophila psittaci adhesion and invasion in HD11 chicken macrophages.

The effect of ovotransferrin (ovoTF), human lactoferrin (hLF) and bovine lactoferrin (bLF) on the obligate intracellular pathogen Chlamydophila (Cp.) psittaci was evaluated using a model of Buffalo Green Monkey kidney (BGM) cells and HD11 chicken macrophages as artificial hosts. Firstly, the effect of transferrins on the infectivity of the bacteria was evaluated. Pre-incubation of Cp. psittaci with 0.5 to 5 mg/mL ovoTF prior to infecting BGM cells significantly lowered the infection rate (P < 0.05). For both lactoferrins, the infection rate could only be reduced with 5 mg/mL, albeit not significantly as compared to the infection rate created by the untreated bacteria. Secondly, transferrins were tested for their ability to influence bacterial adhesion and entry in HD11 cells. Maximal non-cytotoxic and non-bactericidal concentrations of 0.05 mg/mL ovoTF and 0.5 mg/mL hLF and bLF were used. Overall, ovoTF was more effective than human and bovine LF in inhibiting bacterial irreversible attachment and cell entry and the latter was accompanied by a dose-dependent reduction of actin recruitment at the bacterial entry site. However, once bacteria had entered HD11 cells, transferrins had apparently no effect on intracellular replication. The present findings suggest a possible role for transferrins and especially ovoTF, in preventing avian Cp. psittaci infections.