-
[show abstract]
[hide abstract]
ABSTRACT: Chlamydia pneumoniae is a common human respiratory pathogen, and sera from infected individuals recognize several proteins of C. pneumoniae. We produced C. pneumoniae-specific proteins in a Bacillus subtilis expression system. We then used these recombinant C. pneumoniae proteins and purified C. pneumoniae elementary bodies as antigens in enzyme immunoassays to assess the kinetics and protein specificity of the systemic and mucosal antibody responses induced by C. pneumoniae intranasal infection in BALB/c mice. The systemic antibodies in mice recognized strong 'key' immunogens of Chlamydia, Omp2 and Hsp60, but weakly targeted the MOMP protein, the major immunogen in chlamydial species other than C. pneumoniae. The IgA antibodies in bronchial secretions specifically recognized the putative surface protein of C. pneumoniae, Omp4. Our preliminary observations point to the necessity of further characterization of the mucosal antibody response during C. pneumoniae infection.
Comparative medicine 09/2006; 56(4):272-8. · 1.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Due to intracellular growth requirements, large-scale cultures of chlamydiae and purification of its proteins are difficult and laborious. To overcome these problems we produced chlamydial proteins in a heterologous host, Bacillus subtilis, a gram-positive nonpathogenic bacterium. The genes of Chlamydia pneumoniae major outer membrane protein (MOMP), the cysteine-rich outer membrane protein (Omp2), and the heat shock protein (Hsp60) were amplified by PCR, and the PCR products were cloned into expression vectors containing a promoter, a ribosome binding site, and a truncated signal sequence of the alpha-amylase gene from Bacillus amyloliquefaciens. C. pneumoniae genes were readily expressed in B. subtilis under the control of the alpha-amylase promoter. The recombinant proteins MOMP and Hsp60 were purified from the bacterial lysate with the aid of the carboxy-terminal histidine hexamer tag by affinity chromatography. The Omp2 was separated as an insoluble fraction after 8 M urea treatment. The purified proteins were successfully used as immunogens and as antigens in serological assays and in a lymphoproliferation test. The Omp2 and Hsp60 antigens were readily recognized by the antibodies appearing after pulmonary infection following intranasal inoculation of C. pneumoniae in mice. Also, splenocytes collected from mice immunized with MOMP or Hsp60 proteins proliferated in response to in vitro stimulation with the corresponding proteins.
Clinical and Diagnostic Laboratory Immunology 06/2003; 10(3):367-75. · 2.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Pulse-chase labelling was used to study the role of the cell wall microenvironment in the functioning of Bacillus subtilis PrsA, an extracellular lipoprotein and member of the parvulin family of peptidylprolyl cis/trans-isomerases. It was found that in protoplasts, and thus in the absence of a cell wall matrix, the post-translocational folding, stability and secretion of the AmyQ alpha-amylase were independent of PrsA, in contrast to the strict dependency found in rods. The results indicate that PrsA is dedicated to assisting the folding and stability of exported proteins in the particular microenvironment of the cytoplasmic membrane-cell wall interface, possibly as a chaperone preventing unproductive interactions with the wall. The data also provide evidence for a crucial role of the wall in protein secretion. The presence of the wall directly or indirectly facilitates the release of AmyQ from the cell membrane and affects the rate of the signal peptide processing.
Microbiology 04/2003; 149(Pt 3):569-77. · 3.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Heat shock protein 60 (Hsp60) and Chlamydia pneumoniae infection have both been associated with cardiovascular diseases. Our aim was to study the role of Hsp60 antibodies as coronary risk predictors and their association with C pneumoniae infection and inflammation. This was a prospective, nested, case-control study. The cases consisted of 239 middle-aged Finnish men who developed myocardial infarction or coronary death during the follow-up. Baseline levels of IgA and IgG antibodies to human-specific and C pneumoniae-specific Hsp60 were measured by enzyme immunoassay. Human Hsp60 IgA, but not IgG or C pneumoniae Hsp60, antibodies were a significant risk factor for coronary events (odds ratio 2.0, 95% CI 1.1 to 3.6, when the fourth and first quartiles are compared). When an elevated human Hsp60 IgA antibody level (above the second quartile) was present simultaneously with a high C pneumoniae IgA antibody level (the third quartile) and an elevated C-reactive protein level (the second quartile), compared with all factors at low levels, the risk was 7.0 (95% CI 2.6 to 19.1) without adjustment and 5.0 (95% CI 1.8 to 14.2) when adjustment was made for age and smoking. In conclusion, an elevated human Hsp60 IgA antibody level was a risk factor for coronary events, especially when it was present together with C pneumoniae infection and inflammation.
Arteriosclerosis Thrombosis and Vascular Biology 04/2002; 22(3):431-7. · 6.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Identification and characterization of a suppressor mutation, sup-15, which partially restored secretion in the protein secretion-deficient Bacillus subtilis ecsA26 mutant, led us to discover a novel function of Clp protease. Inactivation of ClpP improved the processing of the precursor of AmyQ alpha-amylase exposed on the outer surface of the cytoplasmic membrane. A similar improvement of AmyQ secretion was conferred by inactivation of the ClpX substrate-binding component of the ClpXP complex. In the absence of ClpXP, the transcription of the sipS, sipT, sipV, and lsp signal peptidase genes was elevated two- to fivefold, a likely cause of the improvement of the processing and secretion of AmyQ and complementation of ecs mutations. Specific overproduction of SipT enhanced the secretion. These findings extend the regulatory roles of ClpXP to protein secretion. ClpXP also influenced the processing of the lipoprotein PrsA. A concerted regulation of signal peptidase genes by a ClpXP-dependent activator is suggested. In contrast, Ecs did not affect transcription of the sip genes, pointing to a different mechanism of secretion regulation.
Journal of Bacteriology 03/2002; 184(4):1010-8. · 3.83 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A substantial increase in the prevalence of asthma in the Western world during the last few decades has led to a continuous search for novel factors that might be involved in the development of the disease. We carried out a study to clarify whether there is a relationship between severity of asthma and Chlamydia pneumoniae-specific titres at the group level and whether antibodies to the 60 kDa chlamydial heat shock protein (chsp60) are associated with asthma. A total of 116 (31 men, 85 women) consecutive asthma patients from a chest clinic were recruited and divided into 3 groups according to the severity of the disease: there were 13 asthmatics with severe, 54 with moderate and 49 with mild asthma. In addition, 50 (31 men, 19 women) consecutive blood donors were enrolled to serve as a control group. Sera for the measurements of specific IgG, IgA and IgM antibodies using a microimmunofluorescence test and of chsp60 using an enzyme immunoassay were obtained upon enrolment and also 3-4 months later from the asthma patients. Severe and moderate asthma were found to be strongly associated with elevated IgA antibody levels to C. pneumoniae [odds ratio (OR) 5.58, 95% confidence interval (CI) 1.31-23.72 for severe and OR 5.65, 95% CI 2.05-15.53 for moderate asthma] in a logistic regression model. Furthermore, in women, the occurrence of elevated IgA antibody levels and the age-adjusted geometric mean titres of IgA antibodies were significantly higher among the asthmatics than the controls (p = 0.003 and 0.04, respectively). Antibodies to chsp60 occurred more frequently and in higher concentrations among the asthmatics than the controls, although the differences did not reach significance. In conclusion, severe and moderate asthma were significantly associated with elevated IgA antibody levels to C. pneumoniae suggestive of chronic infection. Antibodies to chsp60 did not prove to be a useful marker of such an infection among the asthmatics studied here.
Scandinavian Journal of Infectious Diseases 02/2002; 34(1):22-7. · 1.72 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: ecs is a three-cistron operon of Bacillus subtilis, encoding proteins with similarity to the ATPase (EcsA) and hydrophobic components (EcsB) of ABC transporters. The ecsA26 point mutation was shown to cause a strong processing defect of a secreted α-amylase precursor (preAmyQ) and of three other exoproteins. Northern analysis of the level of amyQ mRNA showed that ecsA26 also decreases amyQ transcription. This effect too was pleiotropic, as judged by a drastic decrease in the expression from an exoprotease promoter of a reporter protein. A knockout mutation of the ecsB cistron caused a processing defect similar to ecsA26 but, unlike ecsA26, did not affect amyQ transcription. There was also no defect in transcription in the ecsA ecsB double mutant. Thus, an intact ecsB product was required for the downregulation of amyQ by the mutant ecsA. These results suggest a dual regulatory function for Ecs, in which Ecs, possibly as part of a signal transduction mechanism, regulates some component(s) of the protein secretion apparatus as well as secretory protein transcription in a co-ordinated fashion.
Molecular Microbiology 12/1998; 31(2):533 - 543. · 5.01 Impact Factor
