Physcion, a novel anthraquinone derivative against Chlamydia psittaci infection.

Physcion, a natural anthraquinone derivative, has been reported to exert remarkable antibacterial activities against Staphylococcus aureus,Staphylococcus epidermidis and Pseudomonas aeruginosa. However, it is not fully illustrated as anti-Chlamydia substance. In the present study, minimum inhibitory concentration(MIC)values for physcion against Chlamydia psittaci(C.psittaci) 6BC, C.psittaci SBL and C.psittaci HJ were 128 µg/mL,256 µg/mL and 128 µg/mL while minimum bactericidal concentration (MBC) values were 256 µg/mL,512 µg/mL and 256 µg/mL,respectively. Moreover, Chlamydial adhesion to Hela 229 cells was blocked in a dose-dependent manner and RB-to-EB differentiation was inhibited by physcion from 28 to 48 hpi.Post treatment,upregulation of LC3-II was in a dose-dependent manner, indicating physcion activated autophagy and bacterial clearance.To validate clinical efficacy,49 SPF chickens aged 21days were divided into 5 groups and infected intra-laryngeally with 0.2 mL of 1 × 107 IFU/mL C.psittaci 6 BCE.Three days later, birds received orally with serial doses of physcion (4 mg/kg to 9 mg/kg), or 3 mg/kg of doxycycline for 6 days.Chickens with difficulty in breathing were alleviated significantly with increasing concentrations of physicon.Postmortem,lesions of air sacs were reduced significantly in a dose-dependent manner.More importantly,birds with 9 mg/kg of physcion could alleviate lesions of air sacs and lungs, and reduce bacterial loads in spleens, which was comparable to doxycycline treatment. Based on above evidences, physcion is a promising cost-effective natural drug by blocking Chlamydial adhesions to host cells, RB-to-EB differentiation and activating bacterial autophagy and it will be a good alternative to doxycycline combating virulent C.psittaci infection, contributing to eradication of Chlamydial transmission from animals to human beings.

Chlamydia psittaci inclusion membrane protein CPSIT_0842 induces macrophage apoptosis through MAPK/ERK-mediated autophagy.

Chlamydia psittaci is a multi-host zoonotic pathogen, which mainly infects poultry and inflicts an appreciable economic burden on the livestock farming industry. C. psittaci inclusion membrane proteins are uniquely positioned at the host-pathogen interface and are important virulence proteins. We have previously confirmed that Incs regulate host cell survival to help Chlamydia sp. evade host-cell-mediated defense mechanisms. However, the role of the Inc, CPSIT_0842, in the regulation of cell death following the establishment of persistent C. psittaci infection remains unknown. This study explored the effect of CPSIT_0842 on the crosstalk between the autophagic and apoptotic pathways in macrophages. Results showed that CPSIT_0842 initiated autophagy and blocked autophagic flux in human macrophages, as indicated by autophagy-related protein LC3-II, Beclin-1, and p62 upregulation, autophagosome accumulation, and lysosomal protein LAMP1 diminution. We also showed that the disruption of autophagic flux had a regulatory effect on CPSIT_0842-induced apoptosis. Moreover, the suppression of autophagy initiation by 3-methyladenine attenuated CPSIT_0842-induced apoptosis. By contrast, the induction of autophagic flux by rapamycin did not significantly affect CPSIT_0842-induced apoptosis. Taken together, these findings demonstrate that CPSIT_0842 induced macrophage apoptosis by initiating incomplete autophagy through the MAPK/ERK/mTOR signaling pathway, which may be instrumental to the ability of C. psittaci to evade the host innate immune response and establish persistent infection. The improved understanding of the autophagic and cell death pathways triggered upon bacterial inclusion will likely help in the development of novel treatment strategies for chlamydia infection.

Multi-organ failure with necrotic skin lesions due to infection with Chlamydia psittaci.

Presented is a patient with dyspnea and painful ulcers finally resulting in multi-organ failure. A detailed history resulted in positive PCR testing for Chlamydia psittaci. We emphasize the importance of a definitive history in establishing the correct diagnosis. When clinicians observe dyspnea with multi-organ failure, they should be aware of psittacosis.

Complement and Chlamydia psittaci: Early Complement-Dependent Events Are Important for DC Migration and Protection During Mouse Lung Infection.

The zoonotic intracellular bacterium Chlamydia psittaci causes life-threatening pneumonia in humans. During mouse lung infection, complement factor C3 and the anaphylatoxin C3a augment protection against C. psittaci by a so far unknown mechanism. To clarify how complement contributes to the early, innate and the late, specific immune response and resulting protection, this study addresses the amount of C3, the timing when its presence is required as well as the anaphylatoxin receptor(s) mediating its effects and the complement-dependent migration of dendritic cells. Challenge experiments with C. psittaci on various complement KO mice were combined with transient decomplementation by pharmacological treatment, as well as the analysis of in vivo dendritic cells migration. Our findings reveal that a plasma concentration of C3 close to wildtype levels was required to achieve full protection. The diminished levels of C3 of heterozygote C3+/- mice permitted already relative effective protection and improved survival as compared to C3-/- mice, but overall recovery of these animals was delayed. Complement was in particular required during the first days of infection. However, additionally, it seems to support protection at later stages. Migration of CD103+ dendritic cells from the infected lung to the draining lymph node-as prerequisite of antigen presentation-depended on C3 and C3aR and/or C5aR. Our results provide unique mechanistic insight in various aspects of complement-dependent immune responses under almost identical, rather physiological experimental conditions. Our study contributes to an improved understanding of the role of complement, and C3a in particular, in infections by intracellular bacteria.

Characterization and comparison of differentially expressed genes involved in Chlamydia psittaci persistent infection in vitro and in vivo.

Chlamydia psittaci is an obligate intracellular zoonotic pathogen that can enter a persistence state in host cells. While the exact pathogenesis is not well understood, this persistence state may play an important role in chronic Chlamydia disease. Here, we assess the effects of chlamydial persistence state in vitro and in vivo by transmission electron microscopy (TEM) and cDNA microarray assays. First, IFN-γ-induced C. psittaci persistence in HeLa cells resulted in the upregulation of 68 genes. These genes are involved in protein translation, carbohydrate metabolism, nucleotide metabolism, lipid metabolism and general stress. However, 109 genes were downregulated following persistent C. psittaci infection, many of which are involved in the TCA cycle, expression regulation and transcription, protein secretion, proteolysis and transport, membrane protein, presumed virulence factor, cell division and late expression. To further study differential gene expression of C. psittaci persistence in vivo, we established an experimentally tractable mouse model of C. psittaci persistence. The C. psittaci-infected mice were gavaged with either water or amoxicillin (amox), and the results indicated that the 20 mg/kg amox-exposed C. psittaci were viable but not infectious. Differentially expressed genes (DEGs) screened by cDNA microarray were detected, and interestingly, the results showed upregulation of three genes (euo, ahpC, prmC) and downregulation of five genes (pbp3, sucB_1, oppA_4, pmpH, ligA) in 20 mg/kg amox-exposed C. psittaci, which suggests that antibiotic treatment in vivo can induce chlamydial persistence state and lead to differential gene expression. However, the discrepancy on inducers between the two models requires more research to supplement. The results may help researchers better understand survival advantages during persistent infection and mechanisms influencing C. psittaci pathogenesis or evasion of the adaptive immune response.

Polymorphic Membrane Protein 17G of Chlamydia psittaci Mediated the Binding and Invasion of Bacteria to Host Cells by Interacting and Activating EGFR of the Host.

Chlamydia psittaci (C. psittaci) is an obligate intracellular, gram-negative bacterium, and mainly causes systemic disease in psittacine birds, domestic poultry, and wild fowl. The pathogen is threating to human beings due to closely contacted to employees in poultry industry. The polymorphic membrane proteins (Pmps) enriched in C. psittaci includes six subtypes (A, B/C, D, E/F, G/I and H). Compared to that of the 1 pmpG gene in Chlamydia trachomatis (C. trachomatis), the diverse pmpG gene-coding proteins of C. psittaci remain elusive. In the present study, polymorphic membrane protein 17G (Pmp17G) of C. psittaci mediated adhesion to different host cells. More importantly, expression of Pmp17G in C. trachomatis upregulated infections to host cells. Afterwards, crosstalk between Pmp17G and EGFR was screened and identified by MALDI-MS and Co-IP. Subsequently, EGFR overexpression in CHO-K1 cells and EGFR knockout in HeLa 229 cells were assessed to determine whether Pmp17G directly correlated with EGFR during Chlamydial adhesion. Finally, the EGFR phosphorylation was recognized by Grb2, triggering chlamydial invasion. Based on above evidence, Pmp17G possesses adhesive property that serves as an adhesin and activate intracellular bacterial internalization by recognizing EGFR during C. psittaci infection.

Species, sex and geographic variation in chlamydial prevalence in abundant wild Australian parrots.

Chlamydia psittaci (order: Chlamydiales) is a globally distributed zoonotic bacterium that can cause potentially fatal disease in birds and humans. Parrots are a major host, yet prevalence and risk factors for infection in wild parrots are largely unknown. Additionally, recent research suggests there is a diverse range of novel Chlamydiales circulating in wildlife. We therefore sampled seven abundant parrot species in south-eastern Australia, taking cloacal swabs and serum from n = 132 wild adults. We determined C. psittaci and Chlamydiales prevalence and seroprevalence, and tested for host species, sex, geographical and seasonal differences, and temporal changes in individual infection status. Across all species, Chlamydiales prevalence was 39.8% (95% CI 31.6, 48.7), C. psittaci prevalence was 9.8% (95% CI 5.7, 16.3) and C. gallinacea prevalence was 0.8% (95% CI 0.1, 4.5). Other Chlamydiales species were not identified to species level. We identified two C. psittaci strains within the 6BC clade, which is highly virulent in humans. Seroprevalence was 37.0% (95% CI 28.5, 46.4). Host species (including crimson rosellas, galahs, sulphur-crested cockatoos and blue-winged parrots) differed in seroprevalence and Chlamydiales prevalence. Galahs had both highest Chlamydiales prevalence (54.8%) and seroprevalence (74.1%). Seroprevalence differed between sites, with a larger difference in males (range 20-63%) than females (29-44%). We reveal a higher chlamydial prevalence than previously reported in many wild parrots, with implications for potential reservoirs, and transmission risks to humans and other avian hosts.

Metagenomic next-generation sequencing in the diagnosis of severe pneumonias caused by Chlamydia psittaci.

PURPOSE: Chlamydia psittaci infection in humans can lead to serious clinical manifestations, including severe pneumonia, adult respiratory distress syndrome, and, rarely, death. Implementation of metagenomic next-generation sequencing (mNGS) gives a promising new tool for diagnosis. The clinical spectrum of severe psittacosis pneumonia is described to provide physicians with a better understanding and to highlight the rarity and severity of severe psittacosis pneumonia. METHODS: Nine cases of severe psittacosis pneumonia were diagnosed using mNGS. Retrospective analysis of the data on disease progression, new diagnosis tool, treatments, and outcomes, and the findings were summarised. RESULTS: Frequent symptoms included chills and remittent fever (100%), cough and hypodynamia (100%), and headache and myalgia (77.8%). All patients were severe psittacosis pneumonia developed respiratory failure, accompanied by sepsis in 6/9 patients. mNGS takes 48-72 h to provide the results, and help to identify diagnosis of psittacosis. Laboratory data showed normal or slightly increased leucocytes, neutrophils, and procalcitonin but high C-reactive protein levels. Computed tomography revealed air-space consolidation and ground-glass opacity, which began in the upper lobe of one lung, and spread to both lungs, along with miliary, nodular, or consolidated shadows. One patient died because of secondary infection with Klebsiella pneumoniae, while the other eight patients experienced complete recoveries. CONCLUSIONS: The use of mNGS can improve accuracy and reduce the delay in diagnosis of psittacosis. Severe psittacosis pneumonia responds well to the timely use of appropriate antibiotics.

A parrot-type Chlamydia psittaci strain is in association with egg production drop in laying ducks.

Chlamydophila psittaci (C. psittaci) is an avian pathogen associated with systemic wasting disease in birds, as well as a public health risk. Although duck-related cases of psittacosis have been reported, the pathogenicity and shedding status of C. psittaci in ducks are unclear. In this study, we reported that C. psittaci (genotype A) is responsible for a disease outbreak characterized by poor laying performance and severe lesions in multiple organs of ducks. Oral administration of antibiotic, doxycycline, was found to effectively control the C. psittaci infection in laying ducks. Collectively, our new findings provide evidence that C. psittaci was the major pathogen responsible for the outbreak of this disease in ducks. In order to reduce economic losses incurred by this disease, effective control measures must be taken to prevent infection in laying duck farms.

Immunization with Chlamydia psittaci plasmid-encoded protein CPSIT_p7 induces partial protective immunity against chlamydia lung infection in mice.

The present study evaluated the immune-protective efficacy of the Chlamydia psittaci (C. psittaci) plasmid protein CPSIT_p7 and analyzed the potential mechanisms of this protection. The current study used recombinant CPSIT_p7 protein with Freund's complete adjuvant and Freund's incomplete adjuvant to vaccinate BALB/c mice. Adjuvants alone or PBS formulated with the same adjuvants was used as negative controls. Mice were intranasally challenged with 105 inclusion-forming units (IFU) of C. psittaci. We found that CPSIT_p7 vaccination significantly decreased the mouse lung chlamydial load, interferon-γ (IFN-γ) level, and pathological injury. This protection correlated well with specific humoral and cellular immune responses against C. psittaci. In vitro or in vivo neutralization of C. psittaci with sera harvested from immunized mice did not reduce the number of recoverable C. psittaci in the infected lungs, but CD4+ spleen cells collected from CPSIT_p7-immunized mice significantly decreased the chlamydial load via adoptive transfer to native mice. These results reveal that the protection conferred by CPSIT_p7 is dependent on CD4+ T cells.

Disease burden of psittacosis in the Netherlands.

Psittacosis (infection with Chlamydia psittaci) can have diverse presentations in humans, ranging from asymptomatic infection to severe systemic disease. Awareness of psittacosis and its presentations are low among clinicians and the general public. Therefore, underdiagnosis and thereby underestimation of the incidence and public health importance of psittacosis is very likely. We used the methodology developed for the Burden of communicable diseases in Europe toolkit of the European Centre for Disease Prevention and Control, to construct a model to estimate disease burden in disability-adjusted life years (DALYs) attributable to psittacosis. Using this model, we estimated the disease burden caused by psittacosis in the Netherlands to have been 222 DALY per year (95% CI 172-280) over the period 2012-2014. This is comparable with the amount of DALYs estimated to be due to rubella or shigellosis in the same period in the Netherlands. Our results highlight the public health importance of psittacosis and identify evidence gaps pertaining to the clinical presentations and prognosis of this disease.

The JAK/STAT3 signaling pathway mediates inhibition of host cell apoptosis by Chlamydia psittaci infection.

The JAK-STAT3 signaling pathway is a key regulator of cell growth, motility, migration, invasion and apoptosis in mammalian cells. Infection with intracellular pathogens of the genus Chlamydia can inhibit host cell apoptosis, and here we asked whether the JAK-STAT3 pathway participates in chlamydial anti-apoptotic activity. We found that, compared with uninfected cells, levels of JAK1 and STAT3 mRNA as well as total and phosphorylated JAK1 and STAT3 protein, were significantly increased in C. psittaci-infected HeLa cells. Moreover, the apoptosis rate of infected cells was higher after treatment with the tyrosine kinase inhibitor AG-490 (2-cyano-3-(3, 4-dihydroxyphenyl)-N-(phenylmethyl)-2-propenamide). Immunoblotting of apoptosis-related proteins showed that C. psittaci infection reduces Bax, but increases Bcl-2, protein levels, resulting in reduced activation of caspase-3, caspase-7, caspase-9 and PARP; AG490 attenuates these effects. Together, our data suggest that the JAK/STAT3 signaling pathway facilitates the anti-apoptotic effect of C. psittaci infection by reducing the Bax/Bcl-2 apoptotic switch ratio, and by inhibiting the intracellular activation of key pro-apoptotic enzymes.

Persistence of Chlamydia psittaci in Various Temperatures and Times.

Chlamydia psittaci, an obligate intracellular gram-negative bacteria, causes an important zoonotic disease in humans, namely, psittacosis. The objective of this study was to determine the persistent viability of C. psittaci at various temperature conditions. The cloacal swab samples were collected from feral and racing pigeons to find a C. psittaci field strain. The bacterial isolation showed that 1.3% of feral pigeons were PCR positive, while all samples of racing pigeons were PCR negative. Also, bacterial characterization suggested that it belonged to genotype B, which had bacterial titers 3.2 and 3.89 log 50% lethal dose/ml, respectively. A bacterial persistence test was performed, and the results showed that C. psittaci could survive at 56 C for up to 72 hr. In conclusion, C. psittaci could be found in feral pigeons in central Thailand. The bacteria can survive in equatorial temperature areas. This study was the first to report that C. psittaci could survive and has infectivity at 56 C for 72 hr. Therefore, awareness of C. psittaci infection in humans is necessary and should be a public health concern.

First Experimental Evidence for the Transmission of Chlamydia psittaci in Poultry through Eggshell Penetration.

Eggshell penetration by pathogens is considered a potential route for their transmission in poultry flocks. Additionally, in case of zoonotic pathogens, contact with infected eggs or their consumption can result in human infection. Chlamydia psittaci is a zoonotic bacterium that causes a respiratory disease in poultry and humans. In this study, we provide an experimental evidence for eggshell penetration by C. psittaci. Additionally, we show that after eggshell penetration, C. psittaci could eventually infect the growing embryo. Our findings portend the potential of horizontal trans-shell transmission as a possible route for the spread of C. psittaci infection in poultry flocks. Considering that horizontal transmission of pathogens via eggs mainly occurs in hatcheries and hatching cabinets, we suggest the latter as critical control points in the transmission of C. psittaci to hatching chicks and broilers, as well as to the hatchery workers and consumers of table eggs.

Chlamydia-host cell interaction not only from a bird's eye view: some lessons from Chlamydia psittaci.

Chlamydia psittaci causes psittacosis/ornithosis in birds and is an economically important pathogen for poultry farming. It also infects nonavian domestic animals as well as rodents, and is a zoonotic human pathogen responsible for atypical pneumonia. The bacterium efficiently disseminates in host organisms causing pulmonary and systemic disease. Its rapid entry, fast replication cycle, and tight control of intracellular transport routes contribute to the host-to-host transmission and efficient growth observed with C. psittaci. Recent studies have revealed that the pathogen copes better than other chlamydial strains with proinflammatory effectors produced during the early immune reaction of infected hosts. These features likely contribute to successful infections and might explain the potent adaptation and evasion characteristics of the agent. Current findings on cell-autonomous, innate, and adaptive defenses against C. psittaci provide novel insights into the concerted immune mechanisms involved in the clearance of the pathogen. Further in-depth studies on C. psittaci and other related agents in cellular as well as animal models are needed to develop more efficient antichlamydial therapies and vaccination strategies.

Oral Uptake of Chlamydia psittaci by Ducklings Results in Systemic Dissemination.

Enteric infections caused by Chlamydia (C.) psittaci are frequent in ducks, but mostly remain subclinical under field conditions. To emulate natural infection, we investigated the pathogenic potential of a C. psittaci field strain in orally inoculated 4-day-old ducklings. Three different challenge doses were tested and seven contact animals were also mock-inoculated with buffer in each group. Over the course of ten days, the birds were monitored for clinical symptoms and chlamydial dissemination before final examination of tissues using histopathology and immunohistochemistry. While the challenge strain disseminated systemically to all internal organs, mild signs of diarrhea were confined to ducklings inoculated with the highest dose (4.3 x 108 IFU/mL, Group 1). No other clinical symptoms or histopathological lesions were seen. The chlamydial load in internal organs as measured by PCR depended on the challenge dose and was unevenly distributed, i.e. high loads in spleen, liver, and distal small and large intestinal tract (ileum, cecum and rectum) vs. ten times lower values in lungs and proximal small intestinal tract (duodenum and jejunum). Notably, the C. psittaci infection of contact birds became evident on day 10 post-infection, with bacterial loads comparable to those of experimentally-infected animals, thus suggesting rapid bird-to-bird transmission of the challenge strain.

Chlamydia psittaci: update on an underestimated zoonotic agent.

Chlamydia (C.) psittaci is an economically relevant pathogen in poultry and pet birds, where it causes psittacosis/ornithosis, and also a human pathogen causing atypical pneumonia after zoonotic transmission. Despite its well-documented prevalence, the agent has received less attention by researchers than other Chlamydia spp. in the last decades. In the present paper, we review recently published data on C. psittaci infection and attempt to single out characteristic features distinguishing it from related chlamydial agents. It is remarkable that C. psittaci is particularly efficient in disseminating in the host organism causing systemic disease, which occasionally can take a fulminant course. At the cellular level, the pathogen's broad host cell spectrum (from epithelial cells to macrophages), its rapid entry and fast replication, proficient use of intracellular transport routes to mitochondria and the Golgi apparatus, the pronounced physical association of chlamydial inclusions with energy-providing cell compartments, as well as the subversive regulation of host cell survival during productive and persistent states facilitate the characteristic efficient growth and successful host-to-host spread of C. psittaci. At the molecular level, the pathogen was shown to upregulate essential chlamydial genes when facing the host immune response. We hypothesize that this capacity, in concert with expression of specific effectors of the type III secretion system and efficient suppression of selected host defense signals, contributes to successful establishment of the infection in the host. Concerning the immunology of host-pathogen interactions, C. psittaci has been shown to distinguish itself by coping more efficiently than other chlamydiae with pro-inflammatory mediators during early host response, which can, to some extent, explain the effective evasion and adaptation strategies of this bacterium. We conclude that thorough analysis of the large number of whole-genome sequences already available will be essential to identify genetic markers of the species-specific features and trigger more in-depth studies in cellular and animal models to address such vital topics as treatment and vaccination.

Identification of in vivo-induced bacterial protein antigens during calf infection with Chlamydia psittaci.

Chlamydia (C.) psittaci, the causative agent of ornithosis, is an obligate intracellular pathogen with a unique developmental cycle and a high potential for zoonotic transmission. Various mammalian hosts, such as cattle, horse, sheep and man that are in close contact with contaminated birds can get infected (referred to as psittacosis). Since little is known about long-term sequelae of chronic disease and the molecular mechanisms of chlamydial pathogenesis, a key step in understanding the in vivo situation is the identification of C. psittaci infection-associated proteins. For this, we investigated sera of infected calves. Using the immunoscreening approach In Vivo Induced Antigen Technology (IVIAT) including all relevant controls, we focused on C. psittaci proteins, which are induced in vivo during infection. Sera were pooled, extensively adsorbed against in vitro antigens to eliminate false positive results, and used to screen an inducible C. psittaci 02DC15 genomic expression library. Screening and control experiments revealed 19 immunogenic proteins, which are expressed during infection. They are involved in transport and oxidative stress response, heme and folate biosynthesis, DNA replication, recombination and repair, cell envelope, bacterial secretion systems and hypothetical proteins of so far unknown functions. Some of the proteins found may be considered as diagnostic markers or as candidates for the development of vaccines.

Genome-wide DNA methylation profiles according to Chlamydophila psittaci infection and the response to doxycycline treatment in ocular adnexal lymphoma.

PURPOSE: To compare genome-wide DNA methylation profiles according to Chlamydophila psittaci (Cp) infection status and the response to doxycycline treatment in Korean patients with ocular adnexal extranodal marginal zone B-cell lymphoma (EMZL). METHODS: Twelve ocular adnexal EMZL cases were classified into two groups (six Cp-positive cases and six Cp-negative cases). Among the 12 cases, eight were treated with doxycycline as first-line therapy, and they were divided into two groups according to their response to the treatment (four doxy-responders and four doxy-nonresponders). The differences in the DNA methylation states of 27,578 methylation sites in 14,000 genes were evaluated using Illumina bead assay technology. We also validated the top-ranking differentially methylated genes (DMGs) with bisulfite direct sequencing or pyrosequencing. RESULTS: The Infinium methylation chip assay revealed 180 DMGs in the Cp-positive group (74 hypermethylated genes and 106 hypomethylated genes) compared to the Cp-negative group. Among the 180 DMGs, DUSP22, which had two significantly hypomethylated loci, was validated, and the correlation was significant for one CpG site (Spearman coefficient=0.6478, p=0.0262). Regarding the response to doxycycline treatment, a total of 778 DMGs were revealed (389 hypermethylated genes and 336 hypomethylated genes in the doxy-responder group). In a subsequent replication study for representative hypomethylated (IRAK1) and hypermethylated (CXCL6) genes, the correlation between the bead chip analysis and pyrosequencing was significant (Spearman coefficient=0.8961 and 0.7619, respectively, p<0.05). CONCLUSIONS: Ocular adnexal EMZL showed distinct methylation patterns according to Cp infection and the response to doxycycline treatment in this genome-wide methylation study. Among the candidate genes, DUSP22 has a methylation status that was likely attributable to Cp infection. Our data also suggest that the methylation statuses of IRAK1 and CXCL6 may reflect the response to doxycycline treatment.

Chlamydia psittaci inclusion membrane protein IncB associates with host protein Snapin.

Chlamydia (C.) psittaci, the causative agent of psittacosis in birds and humans, is the most important zoonotic pathogen of the family Chlamydiaceae. During a unique developmental cycle of this obligate intracellular pathogen, the infectious elementary body gains access to the susceptible host cell, where it transforms into the replicative reticulate body. C. psittaci uses dynein motor proteins for optimal early development. Chlamydial proteins that mediate this process are unknown. Two-hybrid screening with the C. psittaci inclusion protein IncB as bait against a HeLa Yeast Two-hybrid (YTH) library revealed that the host protein Snapin interacts with IncB. Snapin is a cytoplasmic protein that plays a multivalent role in intracellular trafficking. Confocal fluorescence microscopy using an IncB-specific antibody demonstrated that IncB, Snapin, and dynein were co-localized near the inclusion of C. psittaci-infected HEp-2 cells. This co-localization was lost when Snapin was depleted by RNAi. The interaction of Snapin with both IncB and dynein has been shown in vitro and in vivo. We propose that Snapin connects chlamydial inclusions with the microtubule network by interacting with both IncB and dynein.