Techn. Universiẗat, Diss., 2005--München.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

There are two main human pathogens in the family of Chlamydiaceae. Different serovars of Chlamydia trachomatis cause sexually-transmitted disease and eye infections whereas C. pneumoniae (TWAR) is a common cause of community-acquired respiratory infection. Chlamydia species are obligate, intracellular bacteria sharing a unique developmental cycle that occurs within a protected vacuole termed an inclusion. These microorganisms can be distinguished by two different forms: the infectious, metabolically inert elementary body (EB) and the reproducing non-infectious form, termed the reticulate body (RB). The cycle is terminated when re-differentiation of RBs back to infectious EBs occurs. Chlamydia possesses a type III secretion system (T3SS) essential for delivery of effector proteins into the host for host-cell interactions. This virulence system has been systematically characterized in several mammalian pathogens. Due to lack of a tractable genetic system for Chlamydia species, we have employed chemical genetics as a strategy to investigate molecular aspects of the T3SS. We have identified that the T3S-inhibitors INP0010 and INP0400 block the developmental cycle and interfere with secretion of T3S effector proteins in C. pneumoniae and C. trachomatis, without any cytotoxic effect. We have further shown that INP0010 decreases initiation of transcription in C. pneumoniae during the early mid-developmental cycle as demonstrated by a novel calculation, useful for measurement of transcription initiation in any intracellular pathogen. The mechanism regulating the signal(s) for primary as well as terminal differentiation of RBs has not been defined in Chlamydia. We show using T3S-inhibitors that INP0010 targets the T3SS and thereby arrests RB proliferation as well as RB to EB re-differentiation of C. pneumoniae as where INP0400 targets the T3SS and provokes a bacterial dissociation from the inclusion membrane presumed to mimic the natural occurrence of terminal differentiation. The effect of INP0010 on iron-responsive genes indicates a role for T3S in iron acquisition. Accordingly, our results suggest the possibility that C. pneumoniae acquires iron via the intracellular trafficking pathway of endocytosed transferrin. Moreover, we have for the first time presented data showing generalized bone loss from C. pneumoniae infection in mice. The infection was associated with increased levels of the bone resorptive cytokines IL-6 and IL-1beta. In addition, an increased sub-population of T-cells expressed RANKL during infection. Additionally, C. pneumoniae established an infection in a human osteoblast cell line in vitro with a similar cytokine profile as seen in vivo, supporting a causal linkage. Collectively, these data may indicate a previously unknown pathological role of C. pneumoniae in generalized bone loss.

A large number of studies have reported on the association between the obligate intracellular bacterium, Chlamydia pneumoniae and atherosclerosis. These studies suggest that C. pneumoniae may potentially play a role in the atherosclerotic process, as not all the current atherosclerotic risk factors account for the resulting complications, such as angina, myocardial infarction, heart failure and stroke. The research presented in this thesis analysed whether there are any reliable markers of chronic C. pneumoniae vascular infection, including chlamydial sero-prevalence as defined by two commercial serological tests, detection of C. pneumoniae DNA in the peripheral circulation, the presence or absence of risk factors and symptomatic status. The presence of the bacterium in atherosclerotic carotid specimens was diagnosed directly using a C. pneumoniae-specific polymerase chain reaction (PCR) and a genus-specific immunofluorescent (IF) assay. Eighteen of the 54 (33%) carotid artery diseased (CAD) specimens were positive for the presence of C. pneumoniae DNA by PCR detection, whereas the IF assay detected only six positive samples. PCR analysis found that only two of 43 (5%) patients had C. pneumoniae DNA present within their peripheral blood mononuclear cell (PBMC) fraction. Chlamydial antibodies were detected by Focus microimmunofluorescence and/or Medac recombinant enzyme-linked immunosorbert assay (rELISA) in 56% (24/43) of CAD patients tested. Traditional risk factors, symptomatic status, antigen detection and PCR-based detection of C. pneumoniae in PBMCs, all failed to correlate with the presence of a chlamydial vascular infection. In conclusion, the existing non-invasive diagnostic tests (serology and peripheral blood-based PCR detection) are inefficient for diagnosing a vascular Chlamydia infection, suggesting that a different chlamydial antigen should be tested targeted to identify a chronic C. pneumoniae infection in CAD patients. Given the observation that numerous previously published studies have detected C. pneumoniae in atherosclerotic arterial tissue, yet at widely different detection rates (0% to 100%), it was clear that the location and quantity of clinical specimen could directly affect the detection rate. Previous reports have not used a standard and validated procedure for sampling arterial specimens for C. pneumoniae DNA. The inconsistent detection rates of chlamydial DNA in atherosclerotic plaque are a result of low concentration and irregular distribution of the bacterium, as reported in this study. Our research concluded that a minimum of 15 (30ìm-thick) sections should be analysed by PCR to minimize these sampling variables and obtain a 95% chance of detecting all true C. pneumoniae-positive samples. All previous studies may have under estimated the prevalence of C. pneumoniae, as stringent sampling and repeat testing of the bacterium is required to minimise false-negative results. An interesting finding was that C. pneumoniae DNA was present in all 10 atherosclerotic arteries, although extensive sampling of the carotid was crucial for detection. The third area of research examined the question of possible strain differences between C. pneumoniae isolates infecting human atherosclerotic carotid arteries. Whole genome sequencing as well as specific gene typing suggests that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. We analysed the genotypes of C. pneumoniae present in human atherosclerotic carotid plaque and found several polymorphisms in the variable domain-4 (VD4) region of the outer membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD-urk) genes. Our research identified four different genotypes of C. pneumoniae in human atherosclerotic carotid arteries, including an isolate that appears genetically identical to a strain previously detected in koalas. Two genotypes of C. pneumoniae were present in both human carotid specimens and koala PBMC fractions, suggesting that these genotypes of C. pneumoniae may be capable of crossing the host barrier. The study showed that diversity exists in both the ompAVD4 gene and the ygeD-urk intergenic region enabling fine-detailed differentiation between five different genotypes found in respiratory and/or vascular C. pneumoniae isolates. The importance of the diversity of C. pneumoniae isolates in its role in atherogenesis needs to be further studied.

Universiẗat, Diss., 2005--Jena.

Abstract Chlamydia pneumoniae is an obligate intracellular gram-negative bacterium, which causes respiratory infections in humans and may participate in the development of chronic diseases like atherosclerosis, chronic obstructive lung disease, adult-onset asthma and late-onset Alzheimer’s disease. It can infect various cell types, e.g. vascular endothelial cells, smooth muscle cells and monocyte-derived macrophages in vitro. It has been speculated that circulating macrophages disseminate the infection in the body, and that genetically susceptible individuals become chronically infected. Quantification of C. pneumoniae growth inside cultured cells is needed when studying e.g. the effect of drugs or host cell factors on infectivity and replication. Conventionally this has been done by immunofluorescence staining and microscopic counting of chlamydial inclusions. However, this method is usable only if the cell numbers do not fluctuate in cell culture vials and the inclusions are uniform. In macrophages, inclusions are often aberrant, their sizes vary and multiple inclusions are also seen. Therefore we developed a new method based on the real-time PCR quantification of chlamydial genomes adjusted to the number of human genomes and used it to quantify the exact amounts of C. pneumoniae in infected cells. The susceptibility of monocyte-macrophages from healthy individuals to C. pneumoniae infection in vitro was studied first. Intracellular growth of C. pneumoniae was used as an indicator of susceptibility to infection, and it was compared to serum levels of CRP, soluble CD14, human HSP-IgG, human HSP-IgA, C. pneumoniae IgG and IgA antibodies. The growth of C. pneumoniae in infected macrophages was highly variable, ranging from 0 to 638 chlamydial genomes per human genome. C. pneumoniae growth associated positively with serum C. pneumoniae IgA (titer ≥10) and hHSP-IgG and negatively with soluble CD14 concentration. The association between chlamydial IgA antibodies, hHSP-IgG and C. pneumoniae growth was statistically significant only among men. Age did not correlate with the growth. Therefore we hypothesize that persons whose macrophages cannot restrict the growth of C. pneumoniae are more prone to chronic infection by this agent. In the next study, we evaluated the effects of innate immunity genes CD14 -260 C>T, TLR2 Arg753Gln, TLR4 Asp299Gly, LBP Phe436Leu and IL-6 -174 G>C polymorphisms on C. pneumoniae growth in human macrophages in vitro. The growth of C. pneumoniae was highest in CD14 -260 C>T TT genotype cells and the difference to CC or CT genotype was statistically significant. The G-allele of the IL6 -174 G>C polymorphism had a positive influence on chlamydial growth; the difference was statistically significant only between CC and GC genotypes. TLR2 Arg753Gln, TLR4 Asp299Gly, LBP Phe436Leu polymorphisms showed no effect on chlamydial growth.

Abstract Chlamydia pneumoniae is a common, widespread pathogen that causes acute and chronic infections. Serological diagnosis of C. pneumoniae infection is primarily based on the microimmunofluorescence (MIF) method, but only a fourfold IgG antibody increase between paired sera and the presence of IgM antibodies have generally been accepted as markers of acute infection. At the present, no commonly accepted, reliable serological or other methods for the diagnosis of chronic C. pneumoniae infection exist. We evaluated C. pneumoniae specific serological tests in different populations, followed the kinetics of C. pneumoniae antibodies in multiple sera obtained from the same individuals, compared anti-human IgA FITC conjugates in MIF test and evaluated C. pneumoniae specific antibody tests before and after coronary events in case-control pairs matched for the time point of serum sampling, place of residence, and treatment. We showed that reinfection or reactivation is needed for the persistence of elevated IgG and IgA antibody levels. In chronic infections and upon reactivation, chronic processes may be better diagnosable based on IgA persistence than IgG levels because of the rapid disappearance of IgA levels after seroconversions. The cycle of reinfection and reactivation seems to be faster than previously thought in crowded conditions, such as in military service, since we recorded several antibody changes between the arrival and departure sera of military recruits during 6-month service. The presence of antibodies does not provide protection from reinfection. Commercial anti-human IgA conjugates act differently in MIF tests, and there is marked variation in their ability to detect IgA antibodies. The EIA test used here overestimated the prevalence and persistence of IgA antibodies when compared to MIF. The best compability between MIF and EIA antibody levels was seen in the participants with high titers. Only high IgA MIF titers to C. pneumoniae at the baseline predicted future coronary events. In the present study, seroconversions both in the participants who developed a coronary event and in the controls were detected by MIF and EIA, but mostly in different persons. Seroconversion suggesting reinfection or reactivation of persistent infection may have a role in accelerating chronic processes, because the participants with MIF seroconversion between consecutive sera had a slightly higher risk for coronary events than the controls. EIA seroconversions were more common in the controls than in the cases before the coronary event. The difference in the kinetics of EIA and MIF antibodies warrants future research and supports the use of the MIF method as a golden standard in the measurement of C. pneumoniae IgG and IgA antibody levels and seroconversions. In their diagnostic practice, laboratories should use, compare, and validate more C. pneumoniae IgA antibody tests in addition to IgG tests. Unspecific findings in C. pneumoniae EIA tests require re-estimation and a new way to interpret the results. Chlamydia experts should speak for MIF and rethink the meaning of IgA antibodies and recommendations in the diagnosis of C. pneumoniae infections.

<p><em>Chlamydia pneumoniae </em>is a Gram-negative, obligate intracellular bacterium which shares its unique biphasic developmental cycle, genus-specific lipopolysaccharide, and complement fixation antigen with the other <em>Chlamydia</em> species. Intracellular bacteria, like <em>Chlamydia</em>, require strategies to invade host cells, evade host detection, commandeer host processes, and absorb nutrients in order to support their developmental cycle and survive. The type III secretion (T3S) system meets these needs by transporting bacterial effector proteins across the bacterial membrane and through the host cell membrane. The T3S system in <em>C. pneumoniae </em>is composed of approximately twenty different proteins, whose encoding genes are dispersed throughout ten operons in the <em>Chlamydia</em> genome. CdsD (<em>Cpn0712</em>), a basal body protein component of the T3S apparatus, is suggested to localize to the inner membrane and anchor other T3S structural components of the inner membrane ring. However, the cytoplasmic N-terminal domain contains two putative forkhead-associated (FHA) domains which may play an additional functional role in cellular signalling. This large hypothetical inner-membrane protein is poorly characterized in <em>C. pneumoniae </em>and the role of the predicted phospho-threonine binding, N-terminal FHA domains has yet to be elucidated. Herein, we provide evidence that CdsD has a high affinity for five cytoplasmic (CdsQ, CdsL, CdsN, PknD and SycH) and one periplasmic (CdsF) T3S-associated proteins. We also provide the first evidence that the phosphorylation of CdsD may permit the phosphorylation-dependent oligomerization or interaction with other phosphorylated components of the T3S apparatus. Future research will clarify the role of phosphate signalling in the T3S virulence mechanism. Ultimately, this may lead to a greater understanding of signalling mechanisms that regulate the secretion of bacterial effectors into host eukaryotic cells.</p> / Master of Science (MSc)

Genomic analysis is a new approach for the characterization and investigation of novel genes, gene clusters, the function of uncharacterized proteins, and genetic diversity in microorganisms. These approaches are important for the study of chlamydiae, a system in which several genomes have been sequenced but in which techniques for genetic manipulation are not available. The objective of this thesis is to combine computer-based analysis of chiamydial inclusion membrane proteins (Incs) with cellular and molecular biological analysis of the bacteria. Three different experimental lines of investigation were examined, focusing on Incs of C. trachomatis and C. pneumoniae. Chlamydiae are obligate intracellular bacteria that develop within a nonacidified membrane bound vacuole termed an inclusion. Putative Inc proteins of C. trachomatis and C. pneumoniae were identified from genomic analysis and a unique structural motif. Selected putative Inc proteins are shown to localize to the inclusion membrane. Chiamydia trachomatis variants with unusual multiple-lobed, nonfusogenic, inclusion were identified from a large scale serotyping study. Fluorescence microscopy showed that IncA, a chiamydial protein localized to the inclusion membrane, was undetectable on non-fusogenic inclusions of these variants. Sequence analysis of incA from non-fusogenic variant isolates revealed a defective incA in most of the variants. Some variants lack not only IncA on the inclusion membrane but also CT223p, an additional Inc protein. However, no correlation between the absence of CT223p and distinctive inclusion phenotype was identified. Nucleotide sequence analysis revealed sequence variations of C. trachomatis incA and CT223 in some variant and wild type isolates. Comparative analyses of the three recently published C. pneumoniae genomes have led to the identification of a novel gene cluster named the CPn1O54 gene family. Each member of this family encodes a polypeptide with a hydrophobic domain characteristic of proteins localized to the inclusion membrane. These studies provided evidence that gene variation might occur within this single collection of paralogous genes. Collectively, the variability within this gene family may modulate either phase or antigenic variation, and subsequent physiologic diversity, within a C. pneumoniae population. These studies demonstrate the genetic diversity of Inc proteins and candidate Inc proteins, within and among the different chiamydial species. This work sets the stage for further investigations of the structure and function of this set of proteins that are likely critical to chlamydial intracellular growth. / Graduation date: 2003

Abstract Chronic Chlamydia pneumoniae infections have been associated with cardiovascular diseases (CVD), but the treatment is difficult. Some drugs used for CVD have been found to have an inhibitory effect on the C. trachomatis infection, which is not considered to be associated with CVD. The purpose of this study was to investigate the effects of heparan sulfate-like glycosaminoglycans, COX inhibitors and rapamycin on the C. pneumoniae infection with cell culture methods. Almost any conceivable factors may affect the results of cell cultures. This study showed the complex interaction between temperature, time and medium during the pre-treatment before inoculation. The influences of these factors on the results overlapped and interlaced. The simple washing procedure could enhance the infectivity of C. pneumoniae although it is generally considered to cause the loss of chlamydial EBs and sequentially decrease the chlamydial infectivity. Although the detailed mechanisms were not studied, the results of this study showed that selective COX inhibitors and rapamycin can inhibit the infectivity of C. pneumoniae by inhibiting the growth and maturation, whereas heparan sulfate-like glycosaminoglycans perhaps inhibit the attachment of C. pneumoniae EBs onto the host cells. Recovery and repassage results showed that the growth can be only delayed by selective COX inhibitors, and it can recover to normal level once the drugs were removed. However, rapamycin inhibited the maturation of chlamydial EBs and therefore the infectivity fell down further even when the rapamycin was removed. This study also presented the variations of pathogenicity between different C. pneumoniae strains in vitro. This study is based on in vitro experiments with an acute infection model. Thus, any definite conclusions on the possible antichlamydial effects of the drugs tested in the treatment of cardiovascular diseases which are associated with chronic C. pneumoniae infections cannot be drawn on the basis of this study.