Subtle Changes in the Combining Site of the Chlamydiaceae-Specific mAb S25-23 Increase the Antibody-Carbohydrate Binding Affinity by an Order of Magnitude.

Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2→8)αKdo(2→4)αKdo (where Kdo is 3-deoxy-α-d- manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26. We determined the high-resolution crystal structures of S25-23 and S25-5 antigen binding fragments in complex with a pentasaccharide derived from the LPS of Chlamydia and measured the affinity of S25-5 for chlamydial LPS antigens using isothermal titration microcalorimetry. The 1.75 Å resolution structure of S25-23 shows how subtle conservative mutations Arg(L)-27E to lysine and Ser(H)-56 to threonine lead to an order of magnitude increase in affinity. Importantly, comparison between previous S25-26 structures and the 1.99 and 2.05 Å resolution liganded and unliganded structures of S25-5, respectively, shows how a Ser(L)-27E mutation results in an intermediate affinity due to the reduced enthalpic penalty associated with complex formation that would otherwise be required for arginine in this position. This strategy allows for subtle adjustments in the combining site via affinity maturation that have dramatic consequences for the affinity of an antibody for its antigen.

Chlamydiae interaction with the endoplasmic reticulum: contact, function and consequences.

Chlamydiae and chlamydiae-related organisms are obligate intracellular bacterial pathogens. They reside in a membrane-bound compartment termed the inclusion and have evolved sophisticated mechanisms to interact with cellular organelles. This review focuses on the nature, the function(s) and the consequences of chlamydiae-inclusion interaction with the endoplasmic reticulum (ER). The inclusion membrane establishes very close contact with the ER at specific sites termed ER-inclusion membrane contact sites (MCSs). These MCSs are constituted of a specific set of factors, including the C. trachomatis effector protein IncD and the host cell proteins CERT and VAPA/B. Because CERT and VAPA/B have a demonstrated role in the non-vesicular trafficking of lipids between the ER and the Golgi, it was proposed that Chlamydia establish MCSs with the ER to acquire host lipids. However, the recruitment of additional factors to ER-inclusion MCSs, such as the ER calcium sensor STIM1, may suggest additional functions unrelated to lipid acquisition. Finally, chlamydiae interaction with the ER appears to induce the ER stress response, but this response is quickly dampened by chlamydiae to promote host cell survival.

Helicobacter pylori induced gastric immunopathology is associated with distinct microbiota changes in the large intestines of long-term infected Mongolian gerbils.

BACKGROUND: Gastrointestinal (GI) inflammation in mice and men are frequently accompanied by distinct changes of the GI microbiota composition at sites of inflammation. Helicobacter (H.) pylori infection results in gastric immunopathology accompanied by colonization of stomachs with bacterial species, which are usually restricted to the lower intestine. Potential microbiota shifts distal to the inflammatory process following long-term H. pylori infection, however, have not been studied so far. METHODOLOGY/PRINCIPAL FINDINGS: For the first time, we investigated microbiota changes along the entire GI tract of Mongolian gerbils after 14 months of infection with H. pylori B8 wildtype (WT) or its isogenic ΔcagY mutant (MUT) strain which is defective in the type IV secretion system and thus unable to modulate specific host pathways. Comprehensive cultural analyses revealed that severe gastric diseases such as atrophic pangastritis and precancerous transformations were accompanied by elevated luminal loads of E. coli and enterococci in the caecum and together with Bacteroides/Prevotella spp. in the colon of H. pylori WT, but not MUT infected gerbils as compared to naïve animals. Strikingly, molecular analyses revealed that Akkermansia, an uncultivable species involved in mucus degradation, was exclusively abundant in large intestines of H. pylori WT, but not MUT infected nor naïve gerbils. CONCLUSION/SIGNIFICANCE: Taken together, long-term infection of Mongolian gerbils with a H. pylori WT strain displaying an intact type IV secretion system leads to distinct shifts of the microbiota composition in the distal uninflamed, but not proximal inflamed GI tract. Hence, H. pylori induced immunopathogenesis of the stomach, including hypochlorhydria and hypergastrinemia, might trigger large intestinal microbiota changes whereas the exact underlying mechanisms need to be further unraveled.

Clinicoimmunopathologic findings in Atlantic bottlenose dolphins Tursiops truncatus with positive Chlamydiaceae antibody titers.

Sera from free-ranging Atlantic bottlenose dolphins Tursiops truncatus inhabiting the Indian River Lagoon (IRL), Florida, and coastal waters of Charleston (CHS), South Carolina, USA, were tested for antibodies to Chlamydiaceae as part of a multidisciplinary study of individual and population health. A suite of clinicoimmunopathologic variables was evaluated in Chlamydiaceae-seropositive dolphins (n = 43) and seronegative healthy dolphins (n = 83). Fibrinogen, lactate dehydrogenase, amylase, and absolute numbers of neutrophils, lymphocytes, and basophils were significantly higher, and serum bicarbonate, total alpha globulin, and alpha-2 globulin were significantly lower in dolphins with positive Chlamydiaceae titers compared with seronegative healthy dolphins. Several differences in markers of innate and adaptive immunity were also found. Concanavalin A-induced T lymphocyte proliferation, lipopolysaccharide-induced B lymphocyte proliferation, and granulocytic phagocytosis were significantly lower, and absolute numbers of mature CD 21 B lymphocytes, natural killer cell activity and lysozyme concentration were significantly higher in dolphins with positive Chlamydiaceae antibody titers compared to seronegative healthy dolphins. Additionally, dolphins with positive Chlamydiaceae antibody titers had significant increases in ELISA antibody titers to Erysipelothrix rhusiopathiae. These data suggest that Chlamydiaceae infection may produce subclinical clinicoimmunopathologic perturbations that impact health. Any potential subclinical health impacts are important for the IRL and CHS dolphin populations, as past studies have indicated that both dolphin populations are affected by other complex infectious and neoplastic diseases, often associated with immunologic perturbations and anthropogenic contaminants.

First report of Chlamydiaceae seroprevalence in Tibetan pigs in Tibet, China.

The seroprevalence of Chlamydiaceae infection in Tibetan pigs in Tibet, China, was examined by indirect hemagglutination assay (IHA), between April, 2010, and December, 2010. A total of 71 of 427 serum samples (16.63%, 95% confidence interval [CI] 15.31-17.95] were positive for Chlamydiaceae antibodies. Forty Chlamydiaceae seropositives from 232 samples were recorded in sera from Nyingchi (17.24%, 95% CI 15.40-19.08) and 31 positives were recorded in 195 serum samples from Mainling (15.90%, 95% CI 14.02-17.78). The investigation showed that the prevalence in female animals was 17.61% (95% CI 15.22-20.00), and in male animals it was 12.72% (95% CI 11.07-14.37). The prevalence ranged from 0% to 20.61% (95% CI 17.81-23.48) among different age groups, with a higher prevalence in growing pigs (p<0.01). The results indicated that Chlamydiaceae infection was widespread in Tibetan pigs in Tibet, China, which is of public health concern in this region of the world. To our knowledge, this is the first report of Chlamydiaceae seroprevalence in Tibetan pigs in Tibet, China.

The alternative translational profile that underlies the immune-evasive state of persistence in Chlamydiaceae exploits differential tryptophan contents of the protein repertoire.

One form of immune evasion is a developmental state called "persistence" whereby chlamydial pathogens respond to the host-mediated withdrawal of L-tryptophan (Trp). A sophisticated survival mode of reversible quiescence is implemented. A mechanism has evolved which suppresses gene products necessary for rapid pathogen proliferation but allows expression of gene products that underlie the morphological and developmental characteristics of persistence. This switch from one translational profile to an alternative translational profile of newly synthesized proteins is proposed to be accomplished by maximizing the Trp content of some proteins needed for rapid proliferation (e.g., ADP/ATP translocase, hexose-phosphate transporter, phosphoenolpyruvate [PEP] carboxykinase, the Trp transporter, the Pmp protein superfamily for cell adhesion and antigenic variation, and components of the cell division pathway) while minimizing the Trp content of other proteins supporting the state of persistence. The Trp starvation mechanism is best understood in the human-Chlamydia trachomatis relationship, but the similarity of up-Trp and down-Trp proteomic profiles in all of the pathogenic Chlamydiaceae suggests that Trp availability is an underlying cue relied upon by this family of pathogens to trigger developmental transitions. The biochemically expensive pathogen strategy of selectively increased Trp usage to guide the translational profile can be leveraged significantly with minimal overall Trp usage by (i) regional concentration of Trp residue placements, (ii) amplified Trp content of a single protein that is required for expression or maturation of multiple proteins with low Trp content, and (iii) Achilles'-heel vulnerabilities of complex pathways to high Trp content of one or a few enzymes.

Analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from Chlamydophila psittaci.

Chlamydiae contain a rough-type lipopolysaccharide (LPS) of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid residues (Kdo). Two Kdo trisaccharides, 2.8/2.4- and 2.4/2.4-linked, and a branched 2.4[2.8]2.4-linked Kdo tetrasaccharide occur in Chlamydiaceae. While the 2.8/2.4-linked trisaccharide contains a family-specific epitope, the branched Kdo oligosaccharide occurs only in Chlamydophila psittaci and antibodies against it will be useful in human and veterinarian diagnostics. To overcome the generation of cross-reactive antibodies that bind with high affinity to a dominant epitope formed by 2.4/2.4-linked Kdo, we immunized mice with a synthetic 2.4[2.8]-linked branched Kdo trisaccharide and used phage display of scFv to isolate recombinant antibody fragments (NH2240-31 and SAG506-01) that recognize the branched Kdo oligosaccharide with a K(D) of less than 10 nM. Importantly, although these antibodies used germline genes coding for an inherited Kdo recognition site, they were able clearly to distinguish between 2.4[2.8]2.4- and 2.4/2.4-linked Kdo. Sequence determination, binding data, and X-ray structural analysis revealed the basis for the improved discrimination between similar Kdo ligands and indicated that the alteration of a stacking interaction from a phenylalanine residue in the center of the combining site to a tyrosine residue facing away from the center favors recognition of branched 2.4[2.8]2.4-linked Kdo residues. Immunofluorescence tests of infected cell monolayers using this antibody show specific staining of C. psittaci elementary bodies that allow it to be distinguished from other pathogenic chlamydiae.

Antibodies raised against chlamydial lipopolysaccharide antigens reveal convergence in germline gene usage and differential epitope recognition.

The structures of antigen-binding fragments from two related monoclonal antibodies have been determined to high resolution in the presence of several carbohydrate antigens raised against chlamydial lipopolysaccharide. With the exception of CDR H3, antibodies S54-10 and S73-2 are both derived from the same set of germline gene segments as the previously reported structures S25-2 and S45-18. Despite this similarity, the antibodies differ in specificity and the mechanism by which they recognize their cognate antigen. S54-10 uses an unrelated CDR H3 to recognize its antigen in a fashion analogous to S45-18; however, S73-2 recognizes the same antigen as S45-18 and S54-10 in a wholly unrelated manner. Together, these antibody-antigen structures provide snapshots into how the immune system uses the same set of inherited germline gene segments to generate multiple possible specificities that allow for differential recognition of epitopes and how unrelated CDR H3 sequences can result in convergent binding of clinically relevant bacterial antigens.

High prevalence of antibodies against Chlamydiaceae and Chlamydophila abortus in wild ungulates using two "in house" blocking-ELISA tests.

Few data are available on the prevalence and relevance of chlamydiae in wild mammals, and even fewer studies have been conducted to determine the prevalence of Chlamydophila abortus in wildlife hosts, most probably due to the absence of suitable species-specific serological assays for testing sera from wild animals. In light of this, we have developed two in-house blocking-ELISA tests for detection of antibodies against Chlamydiaceae and C. abortus in wild ungulates, and analyzed the relationship between geographical and biological factors and the prevalence of antibodies against Chlamydiaceae and C. abortus in 434 wild ungulates from Spain, including sera from European wild boar, Red deer, Fallow deer, Roe deer, Mouflon, Barbary sheep, Southern chamois, and Iberian ibex. Serology revealed that 41.7+/-4% of the sera were positive for the b-ELISA-LPS (Chlamydiaceae-specific) and 18.9+/-3% for the b-ELISA-rPOMP (C. abortus-specific). Antibodies against Chlamydiaceae lipopolysaccharide (LPS) were detected in sera from all eight ungulate species, the prevalence ranging from 23 to 60%. Iberian ibex was the only wild ungulate not showing seropositivity to the C. abortus specific polymorphic outer membrane protein (POMP). The prevalence of anti-POMP antibodies in the other seven wild ungulate species ranged from 7 to 40%. While significant seroprevalence differences were detected among species and among sampling regions, no effect of age and sex was observed. The high prevalence levels found should be considered with regards to livestock and human health, and warrant further research.

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.

Lack of microbial DNA in tissue specimens of patients with abdominal aortic aneurysms and positive Chlamydiales serology.

In a case-control study that included a total of 98 patients and 83 controls, the possible link between various pathogens and abdominal aortic aneurysms was investigated. For 68 patients with abdominal aortic aneurysm and age-matched controls, no differences were detected in the levels of immunoglobulin (Ig)A and IgG Chlamydiaceae and Chlamydophila pneumoniae antibodies. Patients with IgA titers positive for Chlamydophila pneumoniae showed progressive disease (defined as an annual increase of the aneurysm diameter of > or = 0.5 cm) more frequently than patients with negative IgA titers (p = 0.046). Polymerase chain reactions performed to detect DNA for Chlamydophila pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, human cytomegalovirus, Borrelia burgdorferi and Helicobacter pylori in tissue specimens of 30 patients and 15 controls were negative. In summary, Chlamydophila pneumoniae may contribute to aortic aneurysm disease progression, but DNA of this and other pathogens was not found in patients' specimens.

Pathogenic potential of novel Chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria.

Novel chlamydiae are newly recognized members of the phylum Chlamydiales that are only distantly related to the classic Chlamydiaceae, i.e., Chlamydia and Chlamydophila species. They also exhibit an obligate biphasic intracellular life cycle within eukaryote host cells. Some of these new chlamydiae are currently considered potential emerging human and/or animal pathogens. Parachlamydia acanthamoebae and Simkania negevensis are both emerging respiratory human pathogens, Waddlia chondrophila could be a novel abortigenic bovine agent, and Piscichlamydia salmonis has recently been identified as an agent of the gill epitheliocystis in the Atlantic salmon. Fritschea spp. and Rhabdochlamydia spp. seem to be confined to arthropods, but some evidence for human exposure exists. In this review, we first summarize the data supporting a pathogenic potential of the novel chlamydiae for humans and other vertebrates and the interactions that most of these chlamydiae have with free-living amoebae. We then review the diagnostic approaches to infections potentially due to the novel chlamydiae, especially focusing on the currently available PCR-based protocols, mammalian cell culture, the amoebal coculture system, and serology.

Immuno-histochemical demonstration of the role of chlamydiaceae in renal, uterine and Salpingeal disease of the koala, and demonstration of chlamydiaceae in novel sites.

Numerous bacteria, including Chlamydophila pecorum and Chlamydophila pneumoniae, are known to occur in diseased sites in koalas. In the present study the significance of such organisms was investigated by demonstrating their distribution in situ, in tissues collected opportunistically from wild koalas. Chlamydiaceae were demonstrated in epithelial cells and macrophages in association with pyogranulomatous pyelonephritis (8/11 kidneys), focal interstitial nephritis (3/21), and active inflammation and fibrosis of the entire upper female reproductive tract (10/10). In one case of pyelonephritis, Gram-positive cocci were also demonstrated in association with Chlamydiaceae and, in another, haematogenous filamentous bacteria appeared to be the sole aetiological agent. Three cases of chlamydial metritis were also superficially co-infected by a mixture of other bacteria. Chlamydiaceae were also demonstrated in pulmonary alveolar macrophages and epithelial cells in association with pneumonitis, and in hepatic and splenic macrophages in one koala. The study illustrated the prominent role of Chlamydiaceae in renal disease and disease of the uterus, uterine tube and ovarian bursa, with implications for pathogenesis and therapy. In addition, macrophages appeared to be a potential site of latent persistence from which systemic spread of infection might occur.

Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera.

Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.

DNA vaccination against Chlamydiaceae: current status and perspectives.

DNA vaccination (also called genetic vaccination) recently celebrated its ten years of existence. This new method of immunization presents several advantages, including the induction of both humoral and cellular immune responses. This vaccination strategy has been very successful and has served as a basis for numerous experiments that had the aim of resolving parasitic, viral, and bacterial infections. In particular, DNA vaccination has been evaluated against Chlamydiaceae, small obligate intracellular bacteria, that induce many pathologies in humans and animals. Despite promising protective effects obtained in murine and turkey models with genes encoding outer membrane proteins and heat shock proteins, DNA vaccination against Chlamydiaceae must be optimized by further investigations and could benefit from the genomic sequencing in terms of the identification of new antigens.

'Candidatus Xenohaliotis californiensis', a newly described pathogen of abalone, Haliotis spp., along the west coast of North America.

Withering syndrome is a fatal disease of wild and cultured abalone, Haliotis spp., that inhabit the west coast of North America. The aetiological agent of withering syndrome has recently been identified as a member of the family Rickettsiaceae in the order Rickettsiales. Using a combination of morphological, serological, life history and genomic (16S rDNA) characterization, we have identified this bacterium as a unique taxon and propose the provisional status of 'Candidatus Xenohaliotis californiensis'. The Gram-negative, obligate intracellular pleomorphic bacterium is found within membrane-bound vacuoles in the cytoplasm of abalone gastrointestinal epithelial cells. The bacterium is not cultivable on synthetic media or in fish cell lines (e.g. CHSE-214) and may be controlled by tetracyclines (oxytetracycline) but not by chloramphenicol, clarithromycin or sarafloxicin. Phylogenetic analysis based on the 16S rDNA of 'Candidatus Xenohaliotis californiensis' places it in the alpha-subclass of the class Proteobacteria but not to the four recognized subtaxa of the alpha-Proteobacteria (alpha-1, alpha-2, alpha-3 and alpha-4). The bacterium can be detected in tissue squashes stained with propidium iodide, microscopic examination of stained tissue sections, PCR or in situ hybridization. 'Candidatus Xenohaliotis californiensis' can be differentiated from other closely related alpha-Proteobacteria by its unique 16S rDNA sequence.

Simkania negevensis strain ZT: growth, antigenic and genome characteristics.

Simkania negevensis is the type species of Simkaniaceae, a recently proposed family in the order Chlamydiales. In the current study, growth, antigenic and genomic characteristics of this intracellular bacterium were investigated and compared to those of members of the family Chlamydiaceae. Growth of the organism, as assessed by infectivity assays, reached a plateau in 2-3 d although by light microscopy the cytopathic effect on the host cells increased for 12 or more days after infection. S. negevensis growth was unaffected by sulfadiazine. Cells infected by S. negevensis strain ZT were not recognized by either of two monoclonal antibodies specific for Chlamydiaceae LPS and several specific Chlamydiaceae ompA primers were unable to PCR amplify a S. negevensis gene. The S. negevensis genome contained one copy of the ribosomal operon. The genome size of S. negevensis strain ZT was determined by PFGE to be 1.7 Mbp, and the G + C content was 42.5 mol%. These data, taken together with other published data, are consistent with the proposal that S. negevensis belongs to a distinct family in the order Chlamydiales.

Pneumonia with serological evidence of acute infection with the Chlamydia-like microorganism "Z".

"Z" is a recently discovered microorganism that may belong to a new genus in the family Chlamydiaceae. Using an ELISA test we developed, we measured levels of serum antibody against "Z" for 308 paired sera obtained from adult patients hospitalized with community-acquired pneumonia (CAP). In 114 patients (37%), serological evidence of past infection with "Z" was found. In eight patients (2.6%) there was serological evidence of acute infection with this pathogen. In four of these eight patients, no other pathogen for CAP was identified despite an intensive serological investigation encompassing 13 etiological agents. The four patients were about 30 yr old, and three of them had no history of chronic illness. Their illness was characterized by high fever, a nonproductive cough, gastrointestinal symptoms, a shift to the left in the white blood cell count, and a prompt, dramatic response to erythromycin therapy. We conclude that the microorganism "Z", or a close variant, is infectious for humans, in some cases causing CAP. In these cases the disease is mild and responds quickly to treatment with erythromycin.