Manuela Donati

Genomic Approach for Analysis of Surface Proteins in Chlamydia pneumoniae

ABSTRACT Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.

Chlamydial infections in feral pigeons in Europe: Review of data and focus on public health implications.

Feral pigeons (Columba livia domestica), which thrive in most European towns and cities, are commonly infected with the zoonotic bacterium Chlamydophila psittaci, the agent of psittacosis (also known as ornithosis) in humans. A number of surveys carried out over the last thirty years across Europe have detected high seropositivity values and high percentages of infection in feral pigeon populations. Overall, when considering data from 11 European countries, seropositivity values to C. psittaci in the sampled populations ranged from 19.4% to 95.6%. In most surveys, the complement fixation test was used, and antibodies were detected in 19.4-66.3% of the samples, with a median of 46.1%. Indirect immunofluorescence and ELISA tests were employed less frequently, but led to the detection of higher percentages of seropositivity (23.7-67.7% and 35.9-95.6%, respectively). Attempts to grow C. psittaci in cell culture or embryonated chicken eggs were successful in 2-42.3% and 0-57.1% of samples, respectively, antigen detection methods were positive in 2.3-40% of samples, while conventional PCR and real-time PCR using different genomic targets detected the organism in 3.4-50% of samples. Twenty-five C. psittaci isolates from pigeons were typed as ompA genotype B (n=14), E (n=10) and E/B (n=1). The huge increase of feral pigeon populations in Europe is a major cause of concern for the detrimental effect of pigeon droppings on environmental hygiene, in addition to the extensive damage due to the fouling of buildings and monuments. The most important pathogenic organism transmissible from feral pigeons to humans is C. psittaci, with 101 cases of disease reported in the literature. Exposure to C. psittaci-contaminated dust, direct contact with pigeons through handling and, to a lesser extent, through pigeon feeding have been identified as hazardous exposures in more than half of the human cases, while loose or transient contacts with feral pigeons have been mentioned in about 40% of the cases. Education initiatives as to the communication of a health risk resulting from contact with pigeons and pigeon excreta should primarily be targeted at individuals who may be exposed to C. psittaci-contaminated dust, such as demolition/construction workers. Recommendations to this category of workers include wearing protective clothes with hoods, boots, gloves and air filter face masks when removing pigeon faeces from roofs, garrets and buildings, especially if working indoors. Monitoring for C. psittaci infections in these workers over time should also be considered. Children should be warned not to handle sick or dead pigeons, and immunocompromised individuals should be advised to carefully limit their contact to feral pigeons. Culling of pigeons by shooting or poisoning is both unethical and ineffective as the place of the killed birds in the population is quickly filled by new juveniles or immigrating birds from neighbouring areas. Pigeon-deterring systems, such as nets and plastic or metal spikes applied to buildings and monuments will prevent their fouling, and the administration of contraceptive drugs may allow size regulation of the pigeon populations. Nevertheless, the measure that will ultimately lead to permanent reduction and will establish healthy sustainable populations is the restriction of indiscriminate feeding by pigeon lovers. The erection of dovecotes and artificial breeding facilities should be considered for providing shelter and a balanced diet to the birds, as well as a chance of interaction for pigeon lovers in a hygienically controlled environment.

DNA immunization with pgp3 gene of Chlamydia trachomatis inhibits the spread of chlamydial infection from the lower to the upper genital tract in C3H/HeN mice

Chlamydia trachomatis pgp3 DNA immunized (no. 300) and non-immunized (no. 300) C3H/HeN mice were infected by vaginal inoculation with infectious C. trachomatis serotype D elementary bodies (EBs) and the spread of infection to the salpinges was assessed by cell culture isolation from tissue homogenates 7, 14, 21, 28, 35 and 42 days post-infection (p.i.). Overall, the pgp3-DNA immunization prevented salpinx infection in 94 (56%) mice, if compared with the 168 positive animals found among the non-immunized animals (P < 0.001). A group of negative control animals (i.e. mice immunized with plasmid DNA containing an irrelevant insert) was not protected, whereas all the mice of a positive immune control group (mice that had resolved a primary genital C. trachomatis infection) were resistant to re-infection. Pgp3 DNA immunization induced both humoral and mucosal anti-pgp3 antibodies.

Serum specific IgA antibody to Chlamydia trachomatis in patients with chlamydial infections detected by ELISA and an immunofluorescence test.

Sera obtained from 34 men with Chlamydia trachomatis positive non-gonococcal urethritis, 34 men with C trachomatis negative non-gonococcal urethritis, 42 women with acute salpingitis, 38 healthy women, and 34 healthy men were studied for the presence of specific serum C trachomatis IgA and IgG antibodies. Serological results were correlated with C trachomatis isolation in cell culture. An enzyme linked immunosorbent assay (ELISA) for C trachomatis specific serum IgA was employed using highly purified elementary bodies of C trachomatis serotype L2 grown in LLC-MK2 cells. Results obtained for C trachomatis IgA antibody by the ELISA test were compared with results obtained for the same sera by a single antigen immunofluorescence technique. A good correlation (r = 0.91) was found between two methods. Serum IgG antibody was also determined in the same sera by the immunofluorescence technique. Patients with C trachomatis positive non-gonococcal urethritis had a significantly (p less than 0.0005) higher prevalence (94.1%) of serum IgA antibody by ELISA compared with patients with C trachomatis negative non-gonococcal urethritis (20.5%) or healthy men (5.9%). Similarly, women with acute salpingitis had a significantly (p less than 0.005) higher prevalence of serum IgA antibody (45.2%) compared with healthy controls (5.2%). Comparable results were obtained for C trachomatis serum IgA antibody using the immunofluorescence technique. The prevalence of C trachomatis IgG antibody was significantly higher in patients with C trachomatis positive non-gonococcal urethritis (97.0%) compared with those with C trachomatis negative non-gonococcal urethritis (33.3%) and healthy controls (23.5%). The importance of using specific C trachomatis serum IgA in the identification of chlamydial infection is discussed.

Tetracycline-resistant Chlamydia suis isolates in Italy.

ANTIMICROBIAL agents are often used to prevent or treat chlamydial infections. The widespread use of tetracycline antibiotics in this way has encouraged selection for resistant organisms. The aim of this study was to evaluate the sensitivity to tetracycline of some strains of Chlamydia suis isolated

Recombinant outer membrane vesicles carrying Chlamydia muridarum HtrA induce antibodies that neutralize chlamydial infection in vitro

Background Outer membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent vaccines development. Methods Chlamydia muridarum DO serine protease HtrA, an antigen which stimulates strong humoral and cellular responses in mice and humans, was expressed in Escherichia coli fused to the OmpA leader sequence to deliver it to the OMV compartment. Purified OMVs carrying HtrA (CM rHtrA-OMV) were analyzed for their capacity to induce antibodies capable of neutralizing Chlamydia infection of LLC-MK2 cells in vitro. Results CM rHtrA-OMV immunization in mice induced antibodies that neutralize Chlamydial invasion as judged by an in vitro infectivity assay. This was remarkably different from what observed with an enzymatically functional recombinant HtrA expressed in, and purified from the E. coli cytoplasm (CM rHtrA). The difference in functionality between anti-CM rHtrA and anti-CM rHtrA-OMV antibodies was associated to a different pattern of protein epitopes recognition. The epitope recognition profile of anti-CM HtrA-OMV antibodies was similar to that induced in mice during Chlamydial infection. Conclusions When expressed in OMVs HtrA appears to assume a conformation similar to the native one and this results in the elicitation of functional immune responses. These data further support the potentiality of OMVs as vaccine platform.

CT043, a protective antigen that induces a CD4+ Th1 response during Chlamydia trachomatis infection in mice and humans.

ABSTRACT Despite several decades of intensive studies, no vaccines against Chlamydia trachomatis, an intracellular pathogen causing serious ocular and urogenital diseases, are available yet. Infection-induced immunity in both animal models and humans strongly supports the notion that for a vaccine to be effective a strong CD4+ Th1 immune response should be induced. In the course of our vaccine screening program based on the selection of chlamydial proteins eliciting cell-mediated immunity, we have found that CT043, a protein annotated as hypothetical, induces CD4+ Th1 cells both in chlamydia-infected mice and in human patients with diagnosed C. trachomatis genital infection. DNA priming/protein boost immunization with CT043 results in a 2.6-log inclusion-forming unit reduction in the murine lung infection model. Sequence analysis of CT043 from C. trachomatis human isolates belonging to the most representative genital serovars revealed a high degree of conservation, suggesting that this antigen could provide cross-serotype protection. Therefore, CT043 is a promising vaccine candidate against C. trachomatis infection.

Activity of Cathelicidin Peptides against Chlamydia spp

ABSTRACT The in vitro activity of six cathelicidin peptides against 25 strains of Chlamydia was investigated. SMAP-29 proved to be the most active peptide, reducing the inclusion numbers of all 10 strains of Chlamydia trachomatis tested by ≥50% at 10 μg/ml. This peptide was also active against C. pneumoniae and C. felis.

Immunological Evaluation and Cellular Location Analysis of the TprI Antigen of Treponema pallidum subsp. pallidum

ABSTRACT The TprI antigen of Treponema pallidum subsp. pallidum is a putative virulence factor predicted to be located in the outer membrane of the syphilis spirochete. In this study, we analyzed the immune response against TprI and its subunits in sera collected both from rabbits experimentally infected with the Nichols strain and from patients with syphilis, showing a different pattern of reactivity toward the antigen in these two groups of samples. The protective ability of recombinant TprI and its hypothetical outer membrane location were also investigated. Although no rabbit was protected after challenge, immunoelectron microscopy results, to be further investigated, were compatible with the outer membrane location of the antigen.

Helicobacter pylori infection and gastric function in patients with fundic atrophic gastritis.

In the present study we evaluated the relation among histology, H. pylori, IgG to H. pylori, gastric emptying, and acid secretion in 43 patients with fundic atrophic gastritis. On the basis of gastric acid secretion, patients were divided into three subgroups: patients with preserved acid secretion (Group 1), patients with hypochlorhydria (Group 2), and patients with achlorhydria (Group 3). Fundic glandular atrophy was more severe in hypoachlorhydric patients than in those with preserved acid secretion (P < 0.05 vs Group 2, P < 0.005 vs Group 3). H. pylori colonization was found in 94% of patients in Group 1, in 61% of patients in Group 2, and in only 8% of patients in Group 3 (P < 0.001 vs Group 1, P < 0.05 vs Group 2). Conversely, serological positivity to H. pylori was high in all three subgroups of patients (100% in Group 1, 77% in Group 2, 92% in Group 3). Gastric emptying was delayed in atrophic patients, particularly in those with hypoachlorhydria. Our data suggest that fundic atrophic gastritis represents a possible end stage of H. pylori infection, characterized by a progressive disappearance of the bacterium and a progressive deterioration of gastric functions.