Genome‐wide profiling of humoral immune response to Coxiella burnetii infection by protein microarray

Comprehensive evaluation of the humoral immune response to Coxiella burnetii may identify highly needed diagnostic antigens and potential subunit vaccine candidates. Here we report the construction of a protein microarray containing 1901 C. burnetii ORFs (84% of the entire proteome). This array was probed with Q‐fever patient sera and naïve controls in order to discover C. burnetii‐specific seroreactive antigens. Among the 21 seroreactive antigens identified, 13 were significantly more reactive in Q‐fever cases than naïve controls. The remaining eight antigens were cross‐reactive in both C. burnetii infected and naïve patient sera. An additional 64 antigens displayed variable seroreactivity in Q‐fever patients, and underscore the diversity of the humoral immune response to C. burnetii. Nine of the differentially reactive antigens were validated on an alternative immunostrip platform, demonstrating proof‐of‐concept development of a consistent, safe, and inexpensive diagnostic assay alternative. Furthermore, we report here the identification of several new diagnostic antigens and potential subunit vaccine candidates for the highly infectious category B alphaproteobacteria, C. burnetii.     

Protein array of Coxiella burnetii probed with Q fever sera

Coxiella burnetii is the etiological agent of Q fever.To identify its major seroreactive proteins,a subgenomic protein array was developed.A total of 101 assumed virulence-associated recombinant proteins of C.burnetii were probed with sera from mice experimentally infected with C.burnetii and sera from Q fever patients.Sixteen proteins were recognized as major seroreactive antigens by the mouse sera.Seven of these 16 proteins reacted positively with at least 45% of Q fever patient sera.Notably,HspB had the highest fluorescence intensity value and positive frequency of all the proteins on the array when probed with both Q fever patient sera and mouse sera.These results suggest that these seven major seroreactive proteins,particularly HspB,are potential serodiagnostic and subunit vaccine antigens of Q fever.     

Isolation of Coxiella burnetii from Children with Influenza-Like Symptoms in Japan

The prevalence of Coxiella burnetii antibodies was investigated by indirect immunofluorescence (IF) test in 55 paired sera (acute and convalescent phases) of school children who had influenza-like symptoms. Of the convalescent serum samples examined, 18 (32.7%) sera reacted positively to phase II antigen of C. burnetii. Coxiella-like organism was isolated from the sera of 13 children after injection of the 18 acute phase sera into mice. The organism was identified as C. burnetii by Giemsa staining and the IF antigen test of mouse spleen smears, the polymerase chain reaction (PCR) method, electron microscopic observations of the mouse spleen cells, and the IF antibody test of mouse sera. This is the first report of isolation of C. burnetii from serum specimens of children having influenza-like symptoms. The evidence that C. burnetii was isolated from people indigenous to Japan at a considerably high incidence suggested that C. burnetii may be widespread as a cause of influenza-like symptoms in Japan.     

Virulent Coxiella burnetii pathotypes productively infect primary human alveolar macrophages

The intracellular bacterial pathogen Coxiella burnetii is a category B select agent that causes human Q fever. In vivo, C. burnetii targets alveolar macrophages wherein the pathogen replicates in a lysosome‐like parasitophorous vacuole (PV). In vitro, C. burnetii infects a variety of cultured cell lines that have collectively been used to model the pathogen's infectious cycle. However, differences in the cellular response to infection have been observed, and virulent C. burnetii isolate infection of host cells has not been well defined. Because alveolar macrophages are routinely implicated in disease, we established primary human alveolar macrophages (hAMs) as an in vitro model of C. burnetii–host cell interactions. C. burnetii pathotypes, including acute disease and endocarditis isolates, replicated in hAMs, albeit with unique PV properties. Each isolate replicated in large, typical PV and small, non‐fused vacuoles, and lipid droplets were present in avirulent C. burnetii PV. Interestingly, a subset of small vacuoles harboured single organisms undergoing degradation. Prototypical PV formation and bacterial growth in hAMs required a functional type IV secretion system, indicating C. burnetii secretes effector proteins that control macrophage functions. Avirulent C. burnetii promoted sustained activation of Akt and Erk1/2 pro‐survival kinases and short‐termphosphorylation of stress‐related p38. Avirulent organisms also triggered a robust, early pro‐inflammatory response characterized by increased secretion of TNF‐α and IL‐6, while virulent isolates elicited substantially reduced secretion of these cytokines. A corresponding increase in pro‐ and mature IL‐1β occurred in hAMs infected with avirulent C. burnetii, while little accumulation was observed following infection with virulent isolates. Finally, treatment of hAMs with IFN‐γ controlled intracellular replication, supporting a role for this antibacterial insult in the host response to C. burnetii. Collectively, the current results demonstrate the hAM model is a human disease‐relevant platform for defining novel innate immune responses to C. burnetii.     

Electron-microscopic study of the effect of various extractants on the morphology ofCoxiella burnetii

This paper presents the results of the effect of trichloroacetic acid (TCA) at two different temperatures and the effect of a mixture of detergents (D) onCoxiella burnetii. Both TCA and D caused a large destruction ofC. burnetii cells. In both cases complexes of high-molar-mass components from the outer membrane were extracted. In the D case not only proteins but a mixture with other high-molar-mass structures were released from the destroyed cells. By extraction of TCA, the antigenic complex composed of lipopolysaccharides (LPS), proteins and phospholipids was released. The effect of the D mixture onC. burnetii causes their complete destruction. The TCA causes a surface destruction of cells but it does not cause complete disintegration. The small-cell variants (SCV) in both cases were shown to be more stable compared to the large-cell variants (LCV).     

Evaluation of a Recombinant 27-kDa Outer Membrane Protein of Coxiella burnetii as an Immunodiagnostic Reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.     

Seroepidemiological Survey of Coxiella burnetii in Domestic Cats in Japan

Cats are assumed to be one of the most important reservoirs of causative agent of human Q fever especially in urban areas. There is no evidence of Coxiella burnetii infection in cats in Japan prior to this. Sera from 100 cats, collected in various parts of Japan, were examined for antibody against C. burnetii. Sixteen out of the 100 samples contained antibodies against C. burnetii. The prevalence of the antibody decreased from the northeastern to the southwestern part of Japan. A high prevalence of the antibodies was observed in sera from cats of more than four years of age. It is difficult to deny that cats would be one of the important sources of human Q fever in Japan.     

LAMP proteins account for the maturation delay during the establishment of the Coxiella burnetii‐containing vacuole

The obligate intracellular pathogen Coxiella burnetii replicates in a large phagolysosomal‐like vacuole. Currently, both host and bacterial factors required for creating this replicative parasitophorous C. burnetii‐containing vacuole (PV) are poorly defined. Here, we assessed the contributions of the most abundant proteins of the lysosomal membrane, LAMP‐1 and LAMP‐2, to the establishment and maintenance of the PV. Whereas these proteins were not critical for uptake of C. burnetii, they influenced the intracellular replication of C. burnetii. In LAMP‐1/2 double‐deficient fibroblasts as well as in LAMP‐1/2 knock‐down cells, C. burnetii establishes a significantly smaller, yet faster maturing vacuole, which harboured more bacteria. The accelerated maturation of PVs in LAMP double‐deficient fibroblasts, which was partially or fully reversed by ectopic expression of LAMP‐1 or LAMP‐2, respectively, was characterized by an increased fusion rate with endosomes, lysosomes and bead‐containing phagosomes, but not by different fusion kinetics with autophagy vesicles. These findings establish that LAMP proteins are critical for the maturation delay of PVs. Unexpectedly, neither the creation of the spacious vacuole nor the delay in maturation was found to be prerequisites for the intracellular replication of C. burnetii.     

Serological cross-reactivity between sporothrix schenckii and various unrelated fungi

The serological cross-reactivity ofSporothrix schenckii with various unrelated fungi was investigated by use of immunodiffusion tests. A rabbit antiS. schenckii serum was obtained, which reacted withCladosporium werneckii, C. carrionii, C. bantianum, Coccidioides immitis, Phialophora jeanselmei, P. gougerotii, P. dermatitidis, Fonsecaea pedrosoi, Aspergillus fumigatus, Histoplasma capsulatum andTrichophyton mentagrophytes, but not withSaccharomyces cerevisiae antigens. The serological determinants responsible for the cross-reactions were suggested to be D-galactosyl residues.     

Molecular evolution of IgG subclass among nonhuman primates: implication of differences in antigenic determinants among Apes

The cross-reactivity of five different rabbit polyclonal antibodies to human IgG and IgG subclass (IgG1, IgG2, IgG3, and IgG4) was determined by competitive ELISA with nine nonhuman primate species including five apes, three Old World monkeys, and one New World monkey. As similar to those previously reported, the reactivity of anti-human IgG antibody with plasma from different primate species was closely related with phylogenic distance from human. Every anti-human IgG subclass antibody showed low cross-reactivity with plasma from Old World and New World monkeys. The plasma from all apes except for gibbons (Hylobates spp.) showed 60 to 100% of cross-reactivity with anti-human IgG2 and IgG3 antibodies. On the other hand, chimpanzee (Pan troglodytes andPan paniscus) and orangutan (Pongo pygmaeus) plasma showed 100% cross-reactivity with anti-human IgG1 antibody, but gorilla (Gorilla gorilla) and gibbon plasma showed no cross-reactivity. The chimpanzee and gorilla plasma cross-reacted with anti-human IgG4 antibody at different reactivity, 100% in chimpanzee and 50% in gorilla, but no cross-reactivity was observed in orangutan and gibbon plasma. These results suggest the possibilities that the divergence of “human-type” IgG subclasses might occur at the time of divergence ofHomo sapience fromHylobatidae, and that the molecular evolution of IgG1 as well as IgG4 is different from that of IgG2 and IgG3 in great apes, this is probably caused by different in development of immune function in apes during the course of evolution.     

Immunodiffusion test for diagnosing basidiobolomycosis

An immunodiffusion test was developed for the diagnosis of basidiobolomycosis. When culture filtrate antigen (CFA) from Basidiobolus ranarum was reacted against two human patient and two rabbit antisera, 2 precipitin bands, inner (N) and outer (Y), were revealed for both patient and rabbit antisera. A line of identity was also observed between precipitin bands obtained with patient and rabbit sera. When CFA from B. ranarum (B CFA) was reacted against rabbit sera which contained antibody to Conidiobolus coronatus and Pythium insidiosum, 1 precipitin band corresponding to inner band (N) was observed. This finding showed that B. ranarum, C. coronatus and P. insidiosum shared at least one common antigen. After B CFA was absorbed with Pythium rabbit antiserum, the inner precipitin line that occurred between B CFA and rabbit antisera of Pythium and Conidiobolus disappeared. However, with Basidiobolus rabbit antiserum, the result did not change. The antigens which could be demonstrated by inner (N) and outer (Y) precipitin bands were heat stable at 56 ° C for 30 min. The titer of the antibodies specific to these antigens decreased as the lesions subsided. When B. ranarum CFA was reacted against sera from 20 apparently normal persons, 20 diabetes mellitus patients, 5 aspergillosis patients, 2 candidosis patients and 3 pythiosis patients, no precipitin band was found. B. ranarum CFA was also treated with each rabbit antiserum specific to Candida albicans, Malassezia furfur and Aspergillus fumigatus. No precipitin bands occurred with any of these antisera. Thus, this test was found to be practical, sensitive and specific, and can be used to monitor patients infected with Basidiobolus ranarum.     

Analysis of the rnc locus of Coxiella burnetii

A 3.2 kb EcoRI genomic DNA fragment of Coxiella burnetii was isolated by virtue of Its ability to suppress mucoidy in Eschertchia coli. Nucleotide sequence analysis revealed the presence of the genes homologous to rnc, era and recO of E. coli. Suppression of capsule synthesis, measured by β-galactosidase expression in Ion cps-lac fusion strains of E. coli, is caused by gene-dosage effects of the plasmid-borne rnc genes of either C. burnetii or E. coli. The rnc gene of C. burnetii complemented rn– E. coli hosts for lambda plaque morphology and stimulation of lambda N gene expression. We also demonstrated heterologous complementation of an E coli strain defective for the expression of Era, an essential protein in E. coli, using the plasmid-borne C. burnetii era. Under the control of the bacteriophage lambda P_L promoter, this 3.2 kb EcoRI DNA fragment directed the synthesis in E. coli of three proteins with approximate molecular masses of 35,27 and 25 kDa. Antibodies against purified E. coli Era protein cross-reacted with the 35 kDa protein of C. burnetii on Western blots.     

From neglected to dissected: How technological advances are leading the way to the study of Coxiella burnetii pathogenesis

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for severe worldwide outbreaks of the zoonosis Q fever. The remarkable resistance to environmental stress, extremely low infectious dose and ease of dissemination, contributed to the classification of C. burnetii as a class B biothreat. Unique among intracellular pathogens, C. burnetii escapes immune surveillance and replicates within large autophagolysosome‐like compartments called Coxiella‐containing vacuoles (CCVs). The biogenesis of these compartments depends on the subversion of several host signalling pathways. For years, the obligate intracellular nature of C. burnetii imposed significant experimental obstacles to the study of its pathogenic traits. With the development of an axenic culture medium in 2009, C. burnetii became genetically tractable, thus allowing the implementation of mutagenesis tools and screening approaches to identify its virulence determinants and investigate its complex interaction with host cells. Here, we review the key advances that have contributed to our knowledge of C. burnetii pathogenesis, leading to the rise of this once‐neglected pathogen to an exceptional organism to study the intravacuolar lifestyle.     

Rat kappa-chain allotypes

The distribution of rat kappa-chain allotype specificities (RI-1a and 1b) was studied amongRattus rattus and a variety of other Asian rodents. No sera other than those ofRattus norvegicus showed the presence of RI-1a (DA type), whereas many cross-reacted with RI-1b (LEW-type). While manyR. rattus showedtotal cross-reactivity with RI-1b, various sera from the generaRattus, Bandicota, andTokudaia showed different levels ofpartial cross-reactivity. These results indicate that (1) anti-RI-1b reagents can detectmultiple specificities on LEW-type kappa chains, and (2) these RI-1b specificities, butnot RI-1a, are widely distributed among murid rodents, in seeming contradiction to amino acid sequence data suggesting that RI-1b is more closely related to ancestral rat kappa chains.     

Flagellar proteins of motile spores ofActinomycetes

Flagella of some of the actinoplanete genera were purified and the molecular sizes of their flagellin subunits compared by SDS-PAGE analysis to flagellins of cells of other bacteria. Several species ofActinoplanes have a major flagellar protein of subunit sizes of 42–43 kDa and a lesser amount of a second protein, possibly a minor flagellin subunit, of 60 kDa. The flagellar protein sizes of other actinoplanetes ranged from 32–43 kDa (major) and 48–58 kDa (minor). Antibodies formed against the 42-kDa protein ofA. rectilineatus showed cross-reactivity in Western blots against flagellar proteins of spores of otherActinoplanes species, two species ofDactylosporangium and anAmpullariella species. Cross-reactivity was also observed with motile cells of two other actinomycetes,Arthrobacter atrocyaneus and aGeodermatophilus species, and withBacillus subtilis. No cross-reactivity was observed withEscherichia coli orPlanomonospora parontospora flagellar proteins. The amino acid composition and partial N-terminal sequence of the 42-kDa flagellar protein ofA. rectilineatus was compared to literature data for other bacterial flagellins and found to be most similar toB. subtilis 168.     

Seroepidemiology of Feline Chlamydiosis by Microimmunofluorescence Assay with Multiple Strains as Antigens

The prevalence of anti-chlamydia antibodies was examined in 232 cat sera collected in 1985 and from 1993 to 1995 from laboratories and veterinary hospitals located in 11 prefectures of Japan. The antibodies were determined by an indirect microimmunofluorescence test using six strains of feline Chlamydia: one strain each of avian- and guinea pig-derived C. psittaci and one strain each of C. pecorum, C. pneumoniae and C. trachomatis. Positive rates of IgG antibodies to chlamydiae were 34.4% in 1985 and 16.5–21.4% from 1993 to 1995. Positive rates of IgM antibodies to chlamydiae were 8.2% in 1985 and 6.6–14.3% from 1993 to 1995. Variations in antibody reactivity to the different feline strains were observed. The results suggest the wide prevalence of chlamydial infection in cats in Japan, and antigenic diversity in the feline strains of C. psittaci.     

Antibodies Are Generated during Infection to Coxiella burnetii Macrophage Infectivity Potentiator Protein (Cb-Mip)

Antisera from rabbits immunized with formalin inactivated Coxiella burnetii isolates associated with either acute (Nine Mile, phase I or phase II) or chronic (Priscilla) Q fever showed reactivity to a C. burnetii macrophage infectivity potentiator protein (Cb-Mip) cloned in Escherichia coli. Further, antisera generated in BALB/c mice after infection with live Nine Mile phase I or Priscilla isolates also showed reactivity to Cb-Mip by immunoblot analysis. In addition, human serum from an individual with previous serological and clinical evidence of Q fever showed reactivity to Cb-Mip. This study indicates that Cb-Mip is immunogenic in both experimental and natural infections, and is the first report on the presence of antibodies to Mip/Mip-like proteins of intracellular bacteria in human sera. Cb-Mip may serve as a potential target antigen for developing recombinant vaccines or diagnostic assays for Q fever.     

The Coxiella burnetii type IV secretion system substrate CaeB inhibits intrinsic apoptosis at the mitochondrial level

Manipulation of host cell apoptosis is a virulence property shared by many intracellular pathogens to ensure productive replication. For the obligate intracellular pathogen Coxiella burnetii anti‐apoptotic activity, which depends on a functional type IV secretion system (T4SS), has been demonstrated. Accordingly, the C. burnetii T4SS effector protein AnkG was identified to inhibit pathogen‐induced apoptosis, possibly by binding to the host cell mitochondrial protein p32 (gC1qR). However, it was unknown whether AnkG alone is sufficient for apoptosis inhibition or if additional effector proteins are required. Here, we identified two T4SS effector proteins CaeA and CaeB (C . burnetii a nti‐apoptotic e ffector) that inhibit the intrinsic apoptotic pathway. CaeB blocks apoptosis very efficiently, while the anti‐apoptotic activity of CaeA is weaker. Our data suggest that CaeB inhibits apoptosis at the mitochondrial level, but does not bind to p32. Taken together, our results demonstrate that C. burnetii harbours several anti‐apoptotic effector proteins and suggest that these effector proteins use different mechanism(s) to inhibit apoptosis.     

The Effect of C. burnetii Infection on the Cytokine Response of PBMCs from Pregnant Goats

In humans, infection with Coxiella burnetii, the causative agent of Q fever, leads to acute or chronic infection, both associated with specific clinical symptoms. In contrast, no symptoms are observed in goats during C. burnetii infection, although infection of the placenta eventually leads to premature delivery, stillbirth and abortion. It is unknown whether these differences in clinical outcome are due to the early immune responses of the goats. Therefore, peripheral blood mononuclear cells (PBMCs) were isolated from pregnant goats. In total, 17 goats were included in the study. Six goats remained naive, while eleven goats were infected with C. burnetii. Toll-like receptor (TLR) and cytokine mRNA expression were measured after in vitro stimulation with heat-killed C. burnetii at different time points (prior infection, day 7, 35 and 56 after infection). In naive goats an increased expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-10 and interferon (IFN)-γ mRNA upon C. burnetii stimulation was detected. In addition, TLR2 expression was strongly up-regulated. In goats infected with C. burnetii, PBMCs re-stimulated in vitro with C. burnetii, expressed significantly more TNF-α mRNA and IFN-γ mRNA compared to naive goats. In contrast, IL-10 mRNA production capacity was down-regulated during C. burnetii infection. Interestingly, at day 7 after inoculation a decreased IFN-γ protein level was observed in stimulated leukocytes in whole blood from infected goats, whereas at other time-points increased production of IFN-γ protein was seen. Our study shows that goats initiate a robust pro-inflammatory immune response against C. burnetii in vitro. Furthermore, PBMCs from C. burnetii infected goats show augmented pro-inflammatory cytokine responses compared to PBMCs from non-infected goats. However, despite this pro-inflammatory response, goats are not capable of clearing the C. burnetii infection.     

<Emphasis Type="Italic">Coxiella burnetii</Emphasis> Nine Mile II proteins modulate gene expression of monocytic host cells during infection

Background  

Coxiella burnetii is an intracellular bacterial pathogen that causes acute and chronic disease in humans. Bacterial replication occurs within enlarged parasitophorous vacuoles (PV) of eukaryotic cells, the biogenesis and maintenance of which is dependent on C. burnetii protein synthesis. These observations suggest that C. burnetii actively subverts host cell processes, however little is known about the cellular biology mechanisms manipulated by the pathogen during infection. Here, we examined host cell gene expression changes specifically induced by C. burnetii proteins during infection.