Established epithelial cell lines equipped with pattern recognition receptors such as the Toll-like receptor (TLR)-2 are common tools for immune response studies on invading pathogens, e.g. the ...obligate intracellular species of Chlamydia. Moreover, such models are widely used to elucidate fatty acid-mediated immune effects. In several transformed cell lines, however, unusual loss of metabolic functions was described. The cell lines A549 and HeLa are poorly characterized in this respect. Therefore, we comparatively assessed the metabolic capacity of A549 and HeLa prior to proposed application as in vitro model for fatty acid effects on chlamydial infection.
We incubated both cell lines either with substrates (C18:2n-6 or C18:3n-3) or products (C18:3n-6, C18:4n-3) of fatty acid desaturase-2 (FADS2), and analysed the fatty acid profiles after 24 h and 72 h by gas chromatography. Based on these data, we suspected that the complete discontinuation of normal biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) in HeLa was due to loss of FADS2 function. Consequently, prostaglandin E2 (PGE2) formation was less inducible by TLR2 stimulation in HeLa, likely as a result of not only insufficient supply of precursors but also weak cyclooxygenase-2 (COX-2) response. In accordance, Chlamydia infection rates were consistently lower in HeLa than in A549. Sequence analysis revealed no alteration within the FADS2 gene in HeLa. The FADS2 expression level, however, was significantly lower and, in contrast to A549, not regulated by C18:2n-6. A549 exhibited regular fatty acid metabolism and enzyme functionality.
Our data show that HeLa cells considerably differ from A549 at several stages of fatty acid metabolism. The poor metabolic potential of HeLa, mainly concerning FADS2 upstream of COX-2 function, calls into question whether these cells represent a good model to unveil fatty acid or downstream eicosanoid effects in the course of intracellular bacterial infection.
Celotno besedilo
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
A concise overview of current knowledge on Chlamydia psittaci, its pathogenic features and strategies to deal with host defense.
Recurrent airway obstruction (RAO) in horses is a naturally occurring dust-induced disease mainly characterized by bronchiolitis which shows histological and pathophysiological similarities to human ...chronic obstructive pulmonary disease (COPD). In human COPD previous investigations indicated an association with Chlamydophila psittaci infection. The present study was designed (1) to clarify a possible role of this infectious agent in RAO and (2) to investigate the suitability of this equine disorder as a model for human COPD.
Clinico-pathological parameters of a total of 45 horses (25 horses with clinical signs of RAO and 20 clinically healthy controls) were compared to histological findings in lung tissue samples and infection by Chlamydiaceae using light microscopy, immunohistochemistry, and PCR.
Horses with clinical signs of RAO vs. controls revealed more inflammatory changes in histology (p = 0.01), and a higher detection rate of Chlamydia psittaci antigens in all cells (p < 0.001) and bronchiolar epithelial cells alone (p < 0.001) by immunohistochemistry. The abundance of chlamydial inclusions increased with the severity of disease. PCR was positive in 60% of horses with RAO vs. 45% of the controls (p = 0.316). OmpA sequencing identified Chlamydophila psittaci (n = 9) and Chlamydophila abortus (n = 13) in both groups with no significant differences. Within the group of clinically healthy horses subgroups with no changes (n = 15) and slight inflammation of the small airways (n = 5) were identified. Also in the group of animals with RAO subgroups with slight (n = 16) and severe (n = 9) bronchiolitis could be formed. These four subgroups can be separated in parts by the number of cells positive for Chlamydia psittaci antigens.
Chlamydophila psittaci or abortus were present in the lung of both clinically healthy horses and those with RAO. Immunohistochemistry revealed acute chlamydial infections with inflammation in RAO horses, whereas in clinically healthy animals mostly persistent chlamydial infection and no inflammatory reactions were seen. Stable dust as the known fundamental abiotic factor in RAO is comparable to smoking in human disease. These results show that RAO can be used as a model for human COPD.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Chlamydophila (Cp.) psittaci, the causative agent of psittacosis in birds and humans, is the most important zoonotic pathogen of the family Chlamydiaceae. These obligate intracellular bacteria are ...distinguished by a unique biphasic developmental cycle, which includes proliferation in a membrane-bound compartment termed inclusion. All Chlamydiaceae spp. possess a coding capacity for core components of a Type III secretion apparatus, which mediates specific delivery of anti-host effector proteins either into the chlamydial inclusion membrane or into the cytoplasm of target eukaryotic cells. Here we describe the interaction between Type III-secreted protein IncA of Cp. psittaci and host protein G3BP1 in a yeast two-hybrid system. In GST-pull down and co-immunoprecipitation experiments both in vitro and in vivo interaction between full-length IncA and G3BP1 were shown. Using fluorescence microscopy, the localization of G3BP1 near the inclusion membrane of Cp. psittaci-infected Hep-2 cells was demonstrated. Notably, infection of Hep-2 cells with Cp. psittaci and overexpression of IncA in HEK293 cells led to a decrease in c-Myc protein concentration. This effect could be ascribed to the interaction between IncA and G3BP1 since overexpression of an IncA mutant construct disabled to interact with G3BP1 failed to reduce c-Myc concentration. We hypothesize that lowering the host cell c-Myc protein concentration may be part of a strategy employed by Cp. psittaci to avoid apoptosis and scale down host cell proliferation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To study the specific antibody response to infection with Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), the agent of Contagious Bovine Pleuropneumonia (CBPP), we examined three panels of ...sera collected during three experimental infection trials in African cattle. The methods used included an in-house complement fixation test (CFT), a commercially available CFT, a competitive antibody ELISA (cELISA) and the immunoblotting test (IBT). In addition, lung tissue samples were examined by culture.
A total of 89% (51/59) of all experimentally infected animals tested positive on at least one of the serological tests throughout the trial. The specific antibody titres to the MmmSC infection became positive first by CFT (6 to 9 days post infection dpi), followed by IBT (9 to 13 dpi) and cELISA (13 to 16 dpi). Individual animals were found to display remarkably distinct seroconversion patterns, which allowed their classification into i) early high responders, ii) late high responders, and iii) low responders. In accordance with other studies, none of the present serological tests was capable of detecting all CBPP infected animals.
Comparison of the assays' performance in terms of sensitivity and specificity raises serious questions as to their reliability for identification of infected individuals in the field. In view of these limitations, a combination of CFT and cELISA can markedly improve CBPP diagnosis at single-animal level.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PCR has become a widely used tool for detection, identification and differentiation of pathogenic microorganisms in diagnosis of animal and human diseases. However, quite a number of currently used ...protocols can be further optimized to exclude nonspecific reactions. On the one hand, target sequences as defined by primer binding sites should be checked carefully for the absence of significant homologies to other organisms in order to insure high specificity of detection. A major part of PCR assays is still based on target sequences in the ribosomal RNA operon, but, as the differentiating potential of this region is limited, genes encoding cellular proteins, such as toxins, surface antigens or enzymes, have been shown to be a viable alternative in many instances. On the other hand, various approaches are available to improve the performance of the amplification reaction itself. The kinetics of amplification is known to be heavily dependent on primer-to-template ratio, efficiency of primer annealing and enzyme-to-template ratio. In the present paper, recently published PCR detection assays for microorganisms, particularly bacterial pathogens, are reviewed and optimization strategies are explained. The practical implications and epidemiological consequences of routine use of PCR in the diagnostic laboratory are also discussed.
Epidemiological data indicate that infection of cattle with chlamydiae such as
Chlamydophila (
C.)
pecorum,
C. abortus,
C. psittaci and
Chlamydia suis, is ubiquitous with mixed infections occurring ...frequently. The apparent lack of association between infection and clinical disease has resulted in debate as to the pathogenic significance of these organisms, and their tendency to sub-clinical and/or persistent infection presents a challenge to the study of their potential effects. However, recent evidence indicates that chlamydial infections have a substantial and quantifiable impact on livestock productivity with chronic, recurrent infections associated with pulmonary disease in calves and with infertility and sub-clinical mastitis in dairy cows. Data also suggest these infections manifest clinically when they coincide with a number of epidemiological risk factors. Future research should: (1) use relevant animal models to clarify the pathogenesis of bovine chlamydioses; (2) quantify the impact of chlamydial infection at a herd level and identify strategies for its control, including sub-unit vaccine development; and (3) evaluate the zoonotic risk of bovine chlamydial infections which will require the development of species-specific serodiagnostics.
The recently introduced bacterial species Chlamydia gallinacea is known to occur in domestic poultry and other birds. Its potential as an avian pathogen and zoonotic agent is under investigation. The ...whole-genome sequence of its type strain, 08-1274/3, consists of a 1,059,583-bp chromosome with 914 protein-coding sequences (CDSs) and a plasmid (p1274) comprising 7,619 bp with 9 CDSs.
Rapid detection of slow-growing or non-culturable microorganisms, such as Mycoplasma spp. and Chlamydia spp., is still a challenge to diagnosticians in the veterinary field. In addition, as ...epidemiological evidence on the frequency of mixed infections involving two and more bacterial species has been emerging, detection methods allowing simultaneous identification of different pathogens are required. In the present chapter, we describe DNA microarray-based procedures for the detection of 83 Mollicutes species (Mycoplasma assay) and 11 Chlamydia spp. (Chlamydia assay). The assays are suitable for use in a routine diagnostic environment, as well as in microbiological research.
The recent description of Chlamydia (C.) avium and C. gallinacea as new species of the reunited genus Chlamydia can be expected to have implications on the perception of avian chlamydiosis. We ...discuss possible effects on epidemiology, diagnosis and our understanding of aetiopathogenesis resulting from this discovery.
This commentary explains the implications of the recent introduction of two new Chlamydia spp.
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