Lipopolysaccharide of Coxiella burnetii.

A lipopolysaccharide (LPS) is considered to be one of the major determinants of virulence expression and infection of virulent Coxiella burnetii. The LPSs from virulent phase I (LPS I) and from avirulent phase II (LPS II) bacteria were investigated for their chemical composition, structure and biological properties. LPS II is of rough (R) type in contrast to LPS I, which is phenotypically smooth (S) and contains a noticeable amount of two sugars virenose (Vir) and dihydrohydroxystreptose (Strep), which have not been found in other LPSs and can be considered as unique biomarkers of the bacterium. Both sugars were suggested to be located mostly in terminal positions of the O-specific chain of LPS I (O-PS I) and to be involved in the immunobiology of Q fever. There is a need to establish a more detailed chemical structure of LPS I in connection with prospective, deeper studies on mechanisms of pathogenesis and immunity of Q fever, its early and reliable diagnosis, and effective prophylaxis against the disease. This will also help to better understanding of host-pathogen interactions and contribute to improved modulation of pathological reactions which in turn are prerequisite for research and development of vaccines of new type. A fundamental understanding of C. burnetii LPS biosynthesis is still lacking. The intracellular nature of the bacterium, lack of genetic tools and its status as a selected agent have made elucidating basic physiological mechanisms challenging. The GDP-ß-D-Vir biosynthetic pathway proposed most recently is an important initial step in this endeavour. The current advanced technologies providing the genetic tools necessary to screen C. burnetii mutants and propagate isogenic mutants might speed the discovery process.

Proteome of Coxiella burnetii.

Recent proteomic studies of C. burnetii, the etiological agent of Q fever, have brought a deeper insight into the pathogen's physiology and offered new possibilities in investigations of inter- or intra-species relatedness. The data generated from these studies in conjunction with the current genomic sequence databases may reveal additional identities for conserved and unique C. burnetii biomarkers and aid in creating algorithms and/or databases that could develop into diagnostic and detection tools for the pathogen. Moreover, wide scale screening and further characterization of potential C. burnetii protein antigens along with a comprehensive evaluation of the humoral immune response will be of fundamental importance towards research and development of a safe and efficacious vaccine as well as improved serodiagnostic tests for rapid and sensitive detection of the Q fever pathogen. Given these advances, proteomics may make marked contributions to the improvement of human health protection against C. burnetii in the coming years.

An O-Specific Polysaccharide/Tetanus Toxoid Conjugate Vaccine Induces Protection in Guinea Pigs against Virulent Challenge with Coxiella burnetii.

Q fever is caused by the bacterium Coxiella burnetii and is spread to humans from infected animals especially goats, sheep and cattle, predominantly when giving birth. There is an effective human vaccine (Q-VAX) against Q fever, and although Q fever is a worldwide problem, the vaccine is only used in Australia due to difficulties associated with its use and the risk of adverse reactions. The desire to protect humans, particularly farmers and abattoir workers, from Q fever prompted the development of a new safe and effective human vaccine without all the difficulties associated with the current vaccine. Candidate vaccines were prepared using purified O-specific polysaccharide (OSP) extracted from the lipopolysaccharide of virulent (phase 1) C. burnetii, strain Nine Mile, which was then conjugated to a tetanus toxoid (TT) carrier protein. Two vaccines were prepared using OSP from C. burnetii grown in embryonated eggs (vaccine A) and axenic media (vaccine B). Vaccines with or without alum adjuvant were used to vaccinate guinea pigs, which were later challenged by intranasal inoculation with virulent C. burnetii. Both vaccines protected guinea pigs from fever and loss of weight post challenge. Post-mortem samples of the spleen, liver and kidney of vaccinated guinea pigs contained substantially less C. burnetii DNA as measured by PCR than those of the unvaccinated control animals. This study demonstrated that a C. burnetii OSP-TT conjugate vaccine is capable of inducing protection against virulent C. burnetii in guinea pigs. Additionally, OSP derived from C. burnetii grown in axenic media compared to OSP from embryonated eggs is equivalent in terms of providing a protective immune response.

Single-nucleotide-polymorphism genotyping of Coxiella burnetii during a Q fever outbreak in The Netherlands.

Coxiella burnetii is the etiological agent of Q fever. Currently, the Netherlands is facing the largest Q fever epidemic ever, with almost 4,000 notified human cases. Although the presence of a hypervirulent strain is hypothesized, epidemiological evidence, such as the animal reservoir(s) and genotype of the C. burnetii strain(s) involved, is still lacking. We developed a single-nucleotide-polymorphism (SNP) genotyping assay directly applicable to clinical samples. Ten discriminatory SNPs were carefully selected and detected by real-time PCR. SNP genotyping appeared to be highly suitable for discrimination of C. burnetii strains and easy to perform with clinical samples. With this new method, we show that the Dutch outbreak is caused by at least 5 different C. burnetii genotypes. SNP typing of 14 human samples from the outbreak revealed the presence of 3 dissimilar genotypes. Two genotypes were also present in livestock at 9 farms in the outbreak area. SNP analyses of bulk milk from 5 other farms, commercial cow milk, and cow colostrum revealed 2 additional genotypes that were not detected in humans. SNP genotyping data from clinical samples clearly demonstrate that at least 5 different C. burnetii genotypes are involved in the Dutch outbreak.

Low potency of Chlamydophila LPS to activate human mononuclear cells due to its reduced affinities for CD14 and LPS-binding protein.

Chlamydiaceae are small obligate intracellular parasites and classified as Gram-negative bacteria. Among Chlamydiaceae-derived components, LPS is known as an immunomodulator and possesses a unique lipid A structure with longer but fewer acyl chains. In this study, to elucidate the Chlamydiaceae-induced immune responses, we evaluated the actions of Chlamydophila psittaci LPS as a Chlamydiaceae LPS on human PBMCs and compared with those of Escherichia coli LPS. Similar to E. coli LPS, C. psittaci LPS bound to monocytes and induced the pro-inflammatory cytokine production in an LPS-binding protein (LBP)-dependent manner. However, C. psittaci LPS was much less potent than E. coli LPS in both the LPS binding and cytokine production. Interestingly, although the binding of C. psittaci LPS was mediated by CD14, Toll-like receptor 4 (TLR4) and CD11b, CD14 and TLR4 but not CD11b were involved in the cytokine production. Of note, ELISA-based binding assays revealed that C. psittaci LPS directly bound to LBP and CD14; however, the affinities were much less than those of E. coli LPS. Together, these observations possibly suggest that Chlamydiaceae LPS has low binding affinities for LPS recognition molecules such as CD14 and LBP and exhibit weak biological activities against host immune cells including monocytes, thereby contributing to the chronic (persistent) inflammatory reactions during infection.

Detection and identification of Coxiella burnetii based on the mass spectrometric analyses of the extracted proteins.

Rapid and reliable detection, identification, and typing of bacterial species are necessary in response to natural or terrorist-caused outbreaks of infectious diseases and play crucial roles in diagnosis and efficient treatment. We report here two proteomic approaches with a high potential in the detection and identification of Coxiella burnetii, the causative agent of Q fever. The first of them starts with the acetonitrile (ACN) and trichloroacetic acid extractions of inactivated C. burnetii cells followed by the detection of extracted molecules and ions derived from the inactivated cells by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the second approach, identification of the proteins extracted by ACN is accomplished after enzymatic digestion by electrospray tandem mass spectrometry coupled to a nanoscale ultraperformance liquid chromatography (LC-MS/MS). In order to observe morphological differences on the surface structures upon extraction, the inactivated and treated cells of the bacterium were examined by electron microscopy. The LC-MS/MS approach has allowed identification of 20 proteins in the ACN extracts of C. burnetii strain RSA 493 that were observed in more than 3 out of 10 experiments.

Coxiella burnetii isolates originating from infected cattle induce a more pronounced proinflammatory cytokine response compared to isolates from infected goats and sheep.

Coxiella burnetii is the causative agent of Q fever. Although the prevalence of C. burnetii in cattle is much higher than in goats and sheep, infected cattle are rarely associated with human outbreaks. We investigated whether the immune response of humans differs after contact with C. burnetii isolates from different host origins or with different multilocus variable number of tandem repeat analysis (MLVA) genotypes. Cytokine responses were measured in human peripheral blood mononuclear cells (PBMCs) stimulated with 16 C. burnetii isolates with known MLVA genotype from goats, sheep, cattle, acute and chronic Q fever patients. Coxiella burnetii isolates originating from cattle induce significantly more IL-1ß, TNF-α and IL-22 than the isolates from goats, sheep or chronic Q fever patients. Comparing the cytokine induction of the isolates based on their MVLA genotype did not reveal differences in response between the MLVA genotypes. The proinflammatory cytokine response induced in human PBMCs by C. burnetii isolates from cattle may explain the low incidence of human Q fever outbreaks caused by cattle. The cytokine profile of PBMCs stimulated with C. burnetii isolates from chronic Q fever patients resembles isolates from goats. Furthermore, cytokine responses seem to be depending on host origin than on MLVA genotype.

Establishment of a genotyping scheme for Coxiella burnetii.

Coxiella burnetii is the causative agent of Q fever. The bacterium is highly infectious and is classified as a category B biological weapon. The tools of molecular biology are of utmost importance in a rapid and unambiguous identification of C. burnetii in naturally occurring Q fever outbreaks, or in cases of a deliberate release of the infectious agent. In this work, development of a multiple locus variable number tandem repeats (VNTR) analysis (MLVA) for the characterization of C. burnetii is described. Sixteen C. burnetii isolates and five passage history/laboratory variants were characterized. The VNTR markers revealed many polymorphisms resulting in nine unique MLVA types that cluster into five different clusters. This proves that the MLVA system is highly discriminatory. The selected VNTR markers were stable. The MLVA method developed in this report is a promising tool for the characterization of C. burnetii isolates and their epidemiological study.

Structural and functional characterization of the glycan antigens involved in immunobiology of Q fever.

Coxiella burnetii is the causative agent of Q fever. The bacterium is extremely infectious and is classified as a category biological weapon. A lipopolysaccharide I (LPS I) belongs to the main components of the C. burnetii outer membrane and its structure-function relationship studies are of potential interest. Size-exclusion chromatography revealed noticeable differences in distribution and chemical composition of the O-polysaccharide chains in LPS I. It is likely that C. burnetii is capable of synthesizing chemically distinct subclasses of O-specific polysaccharide molecules differing in their antigenic reactivities. Methylation-linkage analysis indicated the presence of terminal virenose (Vir), dihydrohydroxystreptose (Strep), and mannose (Man), 4-substituted Vir, and 4-substituted Man in the O-specific chain. Serological data indicate that Vir and Strep might be involved in the immunobiology of Q fever.

Structural features of lipopolysaccharide from Rickettsia typhi: the causative agent of endemic typhus.

Rickettsia typhi causes endemic typhus, a relatively mild, acute febrile illness characterized by headache and macular rash. It is maintained in rodents and transmitted to humans by flea Xenopsylla cheopis. R. typhi contains a lipopolysaccharide thought to display a noticeable antigenic activity. We examined its structural features and it appears that the O-specific chain of the R. typhi LPS is composed mainly of the alternating Glc and QuiNAc residues linked by 1-->4 bonds.

Coxiella burnetii whole cell lysate protein identification by mass spectrometry and tandem mass spectrometry.

The whole cell lysate of Coxiella burnetii strain RSA 493 was separated by two-dimensional electrophoresis and more than 500 protein spots were found on silver-stained reference map. Spots from the gels were subjected to identification based on peptide mass fingerprinting (PMF). In order to identify additional proteins, tandem mass spectrometry (MS/MS) using electrospray and matrix-assisted laser desorption/ionization techniques was applied. The three independent approaches resulted in the identification of 197 open reading frames (ORFs). Fifty-two proteins were identified by PMF and at least with one of the MS/MS methods, 37 proteins with both MS/MS instruments, and 19 proteins with all three techniques applied. All predicted C. burnetii ORFs were compared with the Clusters of Orthologous Groups database. The data related to identified proteins were stored and indexed in a file that can be read and searched using Microsoft Access.

Physicochemical characterization of the endotoxins from Coxiella burnetii strain Priscilla in relation to their bioactivities.

BACKGROUND: Coxiella burnetii is the etiological agent of Q fever found worldwide. The microorganism has like other Gram-negative bacteria a lipopolysaccharide (LPS, endotoxin) in its outer membrane, which is important for the pathogenicity of the bacteria. In order to understand the biological activity of LPS, a detailed physico-chemical analysis of LPS is of utmost importance. RESULTS: The lipid A moiety of LPS is tetraacylated and has longer (C-16) acyl chains than most other lipid A from enterobacterial strains. The two ester-linked 3-OH fatty acids found in the latter are lacking. The acyl chains of the C. burnetii endotoxins exhibit a broad melting range between 5 and 25 degrees C for LPS and 10 and 40 degrees C for lipid A. The lipid A moiety has a cubic inverted aggregate structure, and the inclination angle of the D-glucosamine disaccharide backbone plane of the lipid A part with respect to the membrane normal is around 40 degrees. Furthermore, the endotoxins readily intercalate into phospholipid liposomes mediated by the lipopolysaccharide-binding protein (LBP). The endotoxin-induced tumor necrosis factor alpha (TNFalpha) production in human mononuclear cells is one order of magnitude lower than that found for endotoxins from enterobacterial strains, whereas the same activity as in the latter compounds is found in the clotting reaction of the Limulus amebocyte lysate assay. CONCLUSIONS: Despite a considerably different chemical primary structure of the C. burnetii lipid A in comparison with enterobacterial lipid A, the data can be well understood by applying the previously presented conformational concept of endotoxicity, a conical shape of the lipid A moiety of LPS and a sufficiently high inclination of the sugar backbone plane with respect to the membrane plane. Importantly, the role of the acyl chain fluidity in modulating endotoxicity now becomes more evident.

Coxiella burnetii lipopolysaccharide blocks p38α-MAPK activation through the disruption of TLR-2 and TLR-4 association.

To survive in macrophages, Coxiella burnetii hijacks the activation pathway of macrophages. Recently, we have demonstrated that C. burnetii, via its lipopolysaccharide (LPS), avoids the activation of p38α-MAPK through an antagonistic engagement of Toll-like receptor (TLR)-4. We investigated the fine-tuned mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement. In macrophages challenged with LPS from the avirulent variants of C. burnetii, TLR-4 and TLR-2 co-immunoprecipitated. This association was absent in cells challenged by the LPS of pathogenic C. burnetii. The disruption makes TLRs unable to signal during the recognition of the LPS of pathogenic C. burnetii. The disruption of TLR-2 and TLR-4 was induced by the re-organization of the macrophage cytoskeleton by C. burnetii LPS. Interestingly, blocking the actin cytoskeleton re-organization relieved the disruption of the association TLR-2/TLR-4 by pathogenic C. burnetii and rescued the p38α-MAPK activation by C. burnetii. We elucidated an unexpected mechanism allowing pathogenic C. burnetii to avoid macrophage activation by the disruption of the TLR-2 and TLR-4 association.

Chemical structure of the carbohydrate backbone of the lipopolysaccharide from Piscirickettsia salmonis.

Elucidation of the carbohydrate backbone structure of the lipopolysaccharide (LPS) from Piscirickettsia salmonis, the etiological agent of the salmonid rickettsial septicemia, is described. Structural information was established by a combination of monosaccharide and methylation analyses of LPS, and by NMR and mass spectrometries of oligosaccharides obtained through the use of various chemical degradations of the native polymer. The following structure of the backbone sugars was determined on the basis of the combined data from these experiments: [formula see text] The presence of two consecutive residues of diacetylated pseudaminic acid (Pse5,7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid) in the LPS appears to be unique among polysaccharides containing this acidic sugar. Similarly, the presence of 4-aminoarabinose (Ara4N, 4-amino-4-deoxy-l-arabinopyranose) on O-4 of the α-GlcN1P of the lipid A moiety is a unique feature of this LPS.

Impaired stimulation of p38α-MAPK/Vps41-HOPS by LPS from pathogenic Coxiella burnetii prevents trafficking to microbicidal phagolysosomes.

Variations in lipopolysaccharide (LPS), a bacterial outer membrane component, determine virulence of the obligate intracellular bacterium Coxiella burnetii, but the underlying mechanisms are unknown. We find that while avirulent C. burnetii LPS (avLPS) stimulates host p38α-MAPK signaling required for proper trafficking of bacteria containing compartments to lysosomes for destruction, pathogenic C. burnetii LPS (vLPS) does not. The defect in vLPS and pathogenic C. burnetii targeting to degradative compartments involves an antagonistic engagement of TLR4 by vLPS, lack of p38α-MAPK-driven phosphorylation, and block in recruitment of the homotypic fusion and protein-sorting complex component Vps41 to vLPS-containing vesicles. An upstream activator of p38α-MAPK or phosphomimetic mutant Vps41-S796E expression overrides the inhibition, allowing vLPS and pathogenic C. burnetii targeting to phagolysosomes. Thus, p38α-MAPK and its crosstalk with Vps41 play a central role in trafficking bacteria to phagolysosomes. Pathogenic C. burnetii has evolved LPS variations to evade this host response and thrive intracellularly.

Proteomic comparison of virulent phase I and avirulent phase II of Coxiella burnetii, the causative agent of Q fever.

Coxiella burnetii, a category B biological warfare agent, causes multiple outbreaks of the zoonotic disease Q fever world-wide, each year. The virulent phase I and avirulent phase II variants of the Nine Mile RSA 493 and 439 strains of C. burnetii were propagated in embryonated hen eggs and then purified by centrifugation through Renografin gradients. Total protein fractions were isolated from each phase and subjected to analysis by one-dimensional electrophoresis plus tandem mass spectrometry. A total of 235 and 215 non-redundant proteins were unambiguously identified from the phase I and II cells, respectively. Many of these proteins had not been previously reported in proteomic studies of C. burnetii. The newly identified proteins should provide additional insight into the pathogenesis of Q fever. Several of the identified proteins are involved in the biosynthesis and metabolism of components of the extracellular matrix. Forty-four of the proteins have been annotated as having distinct roles in the pathogenesis or survival of C. burnetii within the harsh phagolysosomal environment. We propose that nine enzymes specifically involved with lipopolysaccharide biosynthesis and metabolism, and that are distinctively present in phase I cells, are virulence-associated proteins.

Coxiella burnetii glycomics and proteomics--tools for linking structure to function.

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular bacterium and a highly infectious pathogen. The disease is a widespread zoonosis and is endemic throughout the world. An easy aerosol dissemination, environmental persistence, and high infectivity make the bacterium a serious threat for humans and animals. Lipopolysaccharide is considered one of the major factors of virulence expression and infection of the bacterium. Detailed glycomic studies enabled to better understand structural and functional peculiarities of this biopolymer and its role in pathogenesis and immunity of Q fever. Recent proteomic studies of C. burnetii have brought new approaches in accurate detection of the infectious agent and offered new insights into the inter- or intra-species relatedness. Thus, structure/function relationship studies are currently of utmost importance in the field. This paper will focus on glycomic and proteomic approaches providing information on unique glycan and protein species of the microorganism as the candidate molecules for the use in detection/diagnosis, therapy, and prophylaxis.