High-yield culture and purification of Chlamydiaceae bacteria

Li, Dan; Vaglenov, A.; Kim, Teayoun; Wang, Chengming; Gao, Dongya; Kaltenboeck, Bernhard

doi:10.1016/j.mimet.2004.10.020

Cited by 40 publications

(23 citation statements)

References 28 publications

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“…We further analyzed the data set using the R/QTL package 19 and found the chromosome 17 QTL to be significant when analyzing male and University and A/J and B6 control mice (Jackson Labs., Bar Harbor, ME, USA) were infected intranasally following the procedure described previously 12 with 5 Â 10 7 Cp organisms at 10-16 weeks of age. Chlamydia pneumoniae strain CDC/CWL-029 (ATCC VR-1310) was prepared as described, 30 using Buffalo Green Monkey Kidney cells (Diagnostic Hybrids Inc., Athens, OH, USA) as host cells and purifying elementary bodies by sedimentation and centrifugation. 30 The level of bacteria in the lung was measured by quantitative PCR using Chlamydia-specific 23S rRNA as described 16 at 15 days post infection in all mice.…”

Section: Resultsmentioning

confidence: 99%

“…Chlamydia pneumoniae strain CDC/CWL-029 (ATCC VR-1310) was prepared as described, 30 using Buffalo Green Monkey Kidney cells (Diagnostic Hybrids Inc., Athens, OH, USA) as host cells and purifying elementary bodies by sedimentation and centrifugation. 30 The level of bacteria in the lung was measured by quantitative PCR using Chlamydia-specific 23S rRNA as described 16 at 15 days post infection in all mice. The distribution of measurable Cp organisms in F2 mice is shown for males (a) and females (b) separately, in comparison to parental strains from the same infection.…”

Section: Resultsmentioning

confidence: 99%

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Genetic control of susceptibility to pulmonary infection with Chlamydia pneumoniae in the mouse

et al. 2007

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A mouse model was used to study the genetic control of differential host response to pulmonary infection with Chlamydia pneumoniae. The A/J and C57BL/6 strains show differential response to intranasal infection with respect to their ability to clear pulmonary bacterial load and the extent of lung pathology developed by 2 weeks post infection. The genetic basis of this interstrain difference was studied by whole-genome scan in an informative [A/J Â C57BL/6J] F2 cross using the pulmonary microbial load as a phenotypic readout of host response. We detected a highly significant linkage (LOD score ¼ 11.5) on chromosome 17 that overlaps with the major histocompatibility (MHC) locus. This quantitative trait locus (QTL) accounts for B30% of the phenotypic variance with B6 alleles conferring susceptibility and inherited in a recessive fashion. Significant linkage was also detected to chromosome 5 in female mice, while chromosome 6 showed suggestive linkage in male mice, pointing to additional complexity in the genetic control of the difference in susceptibility observed in A/J and C57BL/6J.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Genetic control of susceptibility to pulmonary infection with Chlamydia pneumoniae in the mouse

et al. 2007

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“…The EBs were purified from 48-h cultures as described previously (27), and the chlamydial outer membrane complex (COMC) was extracted from the purified EB organisms as described elsewhere (6,31,41) with minor modifications. Approximately 2 to 3 mg of purified EBs was resuspended in 5 ml of phosphate-buffered saline (PBS; pH 7.4) containing 2% (wt/vol) N-lauroylsarcosine sodium salt (Sarkosyl [catalog no.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Pgp3, aChlamydia trachomatisPlasmid-Encoded Immunodominant Antigen

Chen¹,

Lei²,

Lu³

et al. 2010

J Bacteriol

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Human antibody recognition of Chlamydia trachomatis plasmid-encoded Pgp3 protein is dependent on the native conformation of Pgp3. The structural basis for the conformation dependence and the function of Pgp3 remain unknown. Here, we report that Pgp3 trimerization is required for the recognition of Pgp3 by human antibodies. In a native polyacrylamide gel, Pgp3 purified from a bacterial expression system migrated as stable trimers that were dissociated into monomers only by treatment with urea or sodium dodecyl sulfate (SDS) but not nonionic detergents. Human antibodies recognized trimeric but not monomeric Pgp3, suggesting that Pgp3 is presented to the human immune system as trimers during C. trachomatis infection. The endogenous Pgp3 secreted into the chlamydial outer membrane complex or host cell cytosol is always trimerized. Intact Pgp3 trimers were eluted from the outer membrane complex by a combination of nonionic detergents with reducing agents but not by the presence of either alone. These observations have provided important information for further understanding the role of Pgp3 in chlamydial pathogenesis and potentially optimizing Pgp3 as a subunit vaccine candidate antigen.

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“…The mouse pneumonitis biovar, C. muridarum, was originally obtained from Xi Yang (University of Manitoba, Winnipeg, Manitoba, Canada) and propagated in McCoy cells (ATCC, Manassas, VA) according to procedures described previously (63,32). Briefly, McCoy cells were infected for 48 to 72 h and harvested with sterile glass beads.…”

Section: Methodsmentioning

confidence: 99%

“…The supernatants were collected and spun at 30,000 ϫ g for 30 min to pellet the organism. The elementary bodies (EBs) were then further purified by discontinuous density gradient centrifugation using 30% Isovue-370 (Bracco Diagnostics, Princeton, NJ) and 50% sucrose (Sigma, Oakville, Ontario, Canada) as described previously (32). Purified EB preparations were stored in sucrose-phosphate-glutamic acid (SPG) buffer in small aliquots and frozen at Ϫ80°C until needed.…”

Section: Methodsmentioning

confidence: 99%

Critical Role of the Interleukin-17/Interleukin-17 Receptor Axis in Regulating Host Susceptibility to Respiratory Infection withChlamydiaSpecies

Zhou¹,

Chen²,

Moore³

et al. 2009

Infect Immun

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The specific contribution of interleukin-17/interleukin-17 receptor (IL-17/IL-17R)-mediated responses in regulating host susceptibility against obligatory intracellular Chlamydia infection was investigated in C57BL/6 and C3H/HeN mice during Chlamydia muridarum respiratory infection. We demonstrated that Chlamydia stimulated IL-17/IL-17R-associated responses in both Chlamydia-resistant C57BL/6 and Chlamydia-susceptible C3H/HeN mice. However, C3H/HeN mice developed a significantly greater IL-17/IL-17R-associated response than C57BL/6 mice did. This was reflected by an increase in IL-17 mRNA expression, a higher recall IL-17 production from splenocytes upon antigen restimulation, and higher production of Chlamydia trachomatis is an obligate intracellular gram-negative bacterium that primarily infects epithelial cells lining the ocular, respiratory, and urogenital tract surfaces and causes many human diseases including trachoma, pneumonia, and pelvic inflammatory disease (2). Although effective antibiotics are available, the incidence of C. trachomatis infections continues to increase worldwide (41). In the United States alone, it is estimated that there are approximately 2.8 million new cases of urogenital C. trachomatis infection each year (58). An effective and safe Chlamydia vaccine is needed to address the global C. trachomatis epidemic, and a comprehensive understanding of the means of protective immunity and immunopathology of C. trachomatis infection is essential for vaccine development.C. trachomatis infection results in a wide variety of clinical manifestations, ranging from asymptomatic to mild or severe symptoms, acute or chronic inflammatory responses, and a wide range of chronic complications (5,47,59). Host genetic factors appear to be important in determining the outcome of Chlamydia infections. It has been reported that the increased incidence of Chlamydia-induced chronic diseases, such as tubal infertility and scarring trachoma, is correlated with certain human leukocyte antigen (HLA) haplotypes and polymorphism of genes encoding interleukin-10 (IL-10), CD14, and tumor necrosis factor alpha (6,7,16,25,44,54). However, how these specific genes are involved in shaping the specific immune responses during Chlamydia infection in humans remains unclear. As in humans, inbred mouse strains, such as, and DBA/2J (H-2 d ) mice respond to respiratory (1,39,40,63), genital (9-12, 52), and intraperitoneal (i.p.) (36) Chlamydia infections differently. C57BL/6 mice are regarded as a resistant strain, whereas BALB/c, DBA/2, and C3H/HeN mice are reported as susceptible strains with higher mortality, more prolonged bacterial burden, more severe tissue inflammatory responses (such as neutrophil infiltration), and higher rates of infertility following Chlamydia infection. Thus, these inbred mouse strains have been used extensively for identification of specific host factors that regulate immune responses and immune mechanisms underlying the pathogenesis of Chlamydia infection.Based on animal models (5,8,38,50) and hum...

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High-yield culture and purification of Chlamydiaceae bacteria

Cited by 40 publications

References 28 publications

Genetic control of susceptibility to pulmonary infection with Chlamydia pneumoniae in the mouse

Genetic control of susceptibility to pulmonary infection with Chlamydia pneumoniae in the mouse

Characterization of Pgp3, aChlamydia trachomatisPlasmid-Encoded Immunodominant Antigen

Critical Role of the Interleukin-17/Interleukin-17 Receptor Axis in Regulating Host Susceptibility to Respiratory Infection withChlamydiaSpecies

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