Electrophoretic and immunologic comparative analysis of Mycoplasma pneumoniae and Mycoplasma genitalium proteins

The Mycoplasma pneumoniae FH strain routinely used in our laboratory for over 25 years as antigen in serological tests, 2 reference M. pneumoniae strains from ATCC (29342 and M129) and 3 isolates of M. pneumoniae obtained in 1995 from pneumonia patients were compared by SDS-PAGE, complement fixation test (CFT) and by Western-immunoblotting against human and rabbit serum samples with high level of mycoplasmal antibodies. On SDS-PAGE all M. pneumoniae strains showed the same number of 23 polypeptides on the gel with identical molecular weights. The same strains on immunoblotting against human and rabbit serum samples showed six bands: 170, 89, 75, 55, 38 and 33 kDa with the strongest antibody staining in 170-(P1 protein) and 89-kDa bands. Because of its known antigenic relationships Mycoplasma genitalium was used for comparison. The pattern of M. genitalium proteins on SDS-PAGE was similar to pattern of M. pneumoniae but distinguishable. On immunoblotting six proteins of M. genitalium (135, 127, 110, 95, 75 and 45 kDa) reacted with human and rabbits immunoglobulins for M. pneumoniae antigens. Furthermore in complement fixation test both antigens, prepared from M. pneumoniae and M. genitalium, reacted as well with human and rabbit immunoglobulins for M. pneumoniae and with rabbit immunoglobulins for M. genitalium. These cross-reactions observed in serological techniques could give false positive results in routine diagnosis of M. pneumoniae infections. In such situations showing on immunoblott of presence in tested serum sample of antibodies to 170- and 89 kDa proteins could confirm M. pneumoniae infection.     

Specificity of the microimmunofluorescence assay for the serodiagnosis of Chlamydia pneumoniae infections.

Chlamydia pneumoniae infections are mostly confirmed using an indirect microimmunofluorescence test for which potential cross-reactions between antigens from different chlamydial species are not well documented. Using this assay, 928 sera (507 subjects) submitted for Chlamydia pneumoniae serology were tested for specific IgM and IgG to this bacteria using the TW-183 antigen. IgM and IgG reactivities to Chlamydia trachomatis serotypes C, D, E, and L2 and Chlamydia psittaci strain 6BC antigens were also tested. A sample was interpreted as positive only when evenly fluorescent elementary bodies were observed. Twenty-five subjects (4.9%) showed serological evidence of recent Chlamydia pneumoniae infection (IgM positive and (or) IgG seroconversion); 11 of them also showed serological evidence of recent infection with at least one other chlamydial species. Specificity was 50 and 63% for IgM and IgG detection, respectively. These results suggest that mixed or temporally related infections might occur, or, more likely, that some Chlamydia pneumoniae IgM or IgG reactivities might be due to heterotypic antibodies.     

Identification of the outer membrane proteins of Campylobacter pyloridis and antigenic cross-reactivity between C. pyloridis and C. jejuni

The outer membrane and surface exposed proteins of four strains of the gastric Campylobacter-like organism Campylobacter pyloridis were identified by SDS-PAGE of Sarkosyl-insoluble membranous material and 125I-surface-labelled whole bacteria. Although constant outer membrane proteins (molecular mass 61, 54 and 31 kDa) were observed in these strains, several variable 125I-labelled surface proteins were detected. C. pyloridis does not appear to express a single surface-exposed major outer membrane protein like that of C. jejuni and C. coli. Putative flagella proteins were identified from isolated flagella and acid-extractable surface material and by immunoblotting with anti-flagella antibodies. Several major protein antigens were observed by immunoblotting with anti-C. pyloridis antisera. At least two of these antigens cross-reacted with anti-C. jejuni antiserum. This cross-reaction appears to be caused primarily by flagellar antigens. However, one major protein antigen (61 kDa) was not cross-reactive with C. jejuni and may, therefore, be useful in serological tests for the specific diagnosis of C. pyloridis infections.     

Immunoreactive proteins of Campylobacter concisus, an emergent intestinal pathogen

Campylobacter concisus is an emerging pathogen of the human gastrointestinal tract. Recently, a significantly higher prevalence of C.?concisus DNA and higher levels of antibodies specific to C.?concisus was detected in children with Crohn's disease when compared with controls. The aim of this study was to identify C.?concisus immunoreactive antigens. Proteins from C.?concisus were separated using two-dimensional gel electrophoresis, and sera from 10 C.?concisus-positive children with Crohn's disease were employed for immunoprobing. The patients' sera reacted with 69 spots, which corresponded to 31 proteins identified by mass spectrometry. The proteins were functionally classified as involved in chemotaxis, signal transduction, flagellar motility, surface binding and membrane protein assembly. Although the individual patients' sera reacted to different sets of proteins, common antigens that were recognized by all patients were flagellin B, ATP synthase F1 alpha subunit, and outer membrane protein 18. Cross-reactivity between proteins of the Campylobacter genus was tested using patients' sera absorbed with Campylobacter showae, Campylobacter jejuni and Campylobacter ureolyticus. Most of the C.?concisus immunoreactive proteins identified in this study showed cross-reactivity with other species except for three antigens. In conclusion, this study has identified C.?concisus proteins that are immunoreactive within patients with Crohn's disease.     

Detection of antibodies to Campylobacter jejuni in pediatrics patients with Gullain-Barré syndrome using different antigen preparations

Diagnosis of previous C. jejuni infections in GBS patients are mostly based on serological findings. However, there are not consensus what kind of antigen should be used in the serological assays. In our study we used ELISA with four different antigen preparations for investigation of specific antibodies to C. jejuni in serum samples obtained from 6 children with GBS. In all patients the high level of IgA and IgG antibodies to LPS were diagnosed. The antibodies in particular classes of immunoglobulins to recombinant proteins (Mikrogen), termostabile antigen and whole-cell antigen (Virion/Serion) of C. jejuni were diagnosed only in some of the children with GBS. However, in comparison to the control groups, the ELISA with recombinant proteins was most specific. Moreover, in two of the patients a characteristic decline of the level of antibodies to recombinant proteins in sera obtained in acute and chronic phase of disease have been observed. The results of this study showed that serodiagnosis, specially based on recombinant antigens, may be a reliable marker of recent or previous infection caused by C. jejuni in patients with GBS.     

Serological profiling with Chlamycheck, a commercial multiplex recombinant antigen Western blot assay of chlamydial infections

A new chlamydial test system, the Chlamycheck assay, which uses 4 purified recombinant antigens of Chlamydia trachomatis and Chlamydophila pneumoniae and one antigen of Chlamydophila psittaci, has been developed and commercialized. We investigated the reactivities of the recombinant antigens with sera from a group of 30 patients with acute Chlamydia trachomatis infection, 88 patients consulting for sexually transmitted infections, and 46 patients with serological evidence of Chlamydophila pneumoniae infection. The results obtained from human and infected mouse sera suggest that Chlamycheck serology against multiple proteins may provide additional useful information that is not available by conventional whole elementary body microimmunofluorescence or single-antigen enzyme-linked immunosorbent assay serology. Specific serological profiles were associated with acute versus past Chlamydia trachomatis infection or with Chlamydia trachomatis primo-infection versus infection in a Chlamydophila pneumoniae history context.     

Serological characterizations of tandem repeat proteins for detection of African trypanosome infection in cattle

Serological diagnosis is a useful method to detect African trypanosome infection in livestock animals. Currently available serological tests utilize whole parasites or crude antigens, and recombinant antigens may improve reproducibility/standardization and reduce production costs. With a goal of identifying such recombinant proteins, we computationally identified proteins with tandem repeat (TR) domain from the parasite proteomes and evaluated their potential for serological diagnosis of African trypanosome infections in cattle. Among those tested, Tbg4 demonstrated the best performance with 92% sensitivity, followed by TbbGM6 (85%), TcoGM6 (85%), Tbg2 (65%) and Tbg5 (65%). Although further evaluations such as investigating cross-reactivity to other infections are needed, our data indicate the potential of these antigens for detection of African trypanosome infection in cattle.     

Serological study on Chlamydophila pneumoniae in patients with community-acquired pneumonia

This study aimed to evaluate the incidence of Chlamydophila pneumoniae antibodies in patients with community acquired pneumonia (CAP) by a new ELISA test (EIA CP-IgG, IgA, IgM--Eurospital, Trieste, Italy). From January 1999 to July 2001 141 patients with clinical signs of CAP were enrolled in sixteen Italian Hospitals. Specific IgM and IgG antibodies anti-C. pneumoniae in serum and IgA in both serum and sputum were detected. At a primary inspection (time T-0) serum and sputum samples were taken from 115/141 patients, whereas serum was collected from only 100/141 patients after 30 days (time T-30). At T-0 24/115 (20.8%) patients showed serological markers thus suggesting an acute C. pneumoniae infection. In 23/24 patients the overall serological pattern found at T-0 was confirmed at T-30. In 32/115 patients (27.8%) serological markers of C. pneumoniae past infection were found positive and were confirmed 30 days later. These data support the role of C. pneumoniae as an important aetiological agent of CAP throughout different geographic areas of Italy. The test was suitable for the laboratory diagnosis of C. pneumoniae infection. In particular, the presence of specific IgA anti- C. pneumoniae in both serum and sputum proved useful to define different stages and evolution of infection.     

Immunoproteomic identification and serological responses to novel Chlamydia pneumoniae antigens that are associated with persistent C. pneumoniae infections

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.     

Serodiagnosis of clinical infections caused by Mycoplasma pneumoniae in Poland in 1970-2010

The aim of the study was evaluation of the reliability of serodiagnosis of mycoplasmosis in Poland after replaced of classical assays, as complement fixation test (CFT) and immunoelectroprecipitation test (IEPT), by the ELISA method. The data were obtained from National Public Health Institute in Warsaw (NPHI), which receives quarterly reports of serologically confirmed infection from Sanitary and Epidemiological Stations through the country. Previously, from the 1970 to 1999 the serodiagnosis in Poland was performed only by uniform CFT using the same M. pneumoniae FH antigen prepared at the Mycoplasma Laboratory of NPHI. The first data of M. pneumoniae serological investigation performed by commercial ELISA, mainly Virotech, which gradually replaced the CFT, were obtained in 2000. In the studied period 1970-2010 a total of over 300 thousand patients with respiratory tract infections (85% were children below 18 years old) were tested. During these studies five epidemics of mycoplasmosis were noted in Poland before 2000. However, after introduction of ELISA for serodiagnosis this characteristic distinct difference in epidemic and endemic occurrence of M pneumoniae infections have not been seen. In our opinion it may be caused by changes in the epidemiological pattern of M. pneumoniae infections in Poland as well as, paradoxical, by the decrease of sensitivity and specificity of serological investigation performed by ELISA since 2000. First of all, for the economical reasons 98% of the patients were tested by ELISA only once, secondly, the mycoplasmosis in many laboratories was confirmed only on the basis of the presence of IgG antibodies. The results of our analysis showed also usefulness in the serodiagnosis of mycoplasmosis, mainly during the first 2 weeks of the disease, of IEPT, which detect only specific IgM antibodies to M pneumoniae antigens.     

Dengue and Zika virus multi-epitope antigen expression in insect cells

Dengue virus and Zika virus are arthropod-borne flaviviruses that cause millions of infections worldwide. The co-circulation of both viruses makes serological diagnosis difficult as they share high amino acid similarities in viral proteins. Antigens are one of the key reagents in the differential diagnosis of these viruses through the detection of IgG antibodies in serological assays during the convalescent-phase of infections. Here, we report the expression of Dengue virus (DENV) and Zika virus (ZIKV) antigens containing non-conserved and immunodominant amino acid sequences using the baculovirus expression vector system in insect cells. We designed DENV and ZIKV antigens based on the domain III of the E protein (EDIII) after analyzing previously reported epitopes and by multiple alignment of the most important flaviviruses. The ZIKV and DENV multi-epitope genes were designed as tandem repeats or impaired repeats separated by tetra- or hexa-glycine linkers. The biochemical analyses revealed adequate expression of the antigens. Then, the obtained multi-epitope antigens were semi-purified in a sucrose gradient and tested using patients’ sera collected during the convalescent-phase that were previously diagnosed positive for anti-DENV and -ZIKV IgG antibodies. The optimal serum dilution was 1:200, and the mean absorbance values in the preliminary tests show that multi-epitope antigens have been recognized by human sera. The production of both antigens using the multi-epitope strategy in the eukaryotic system and based on the EDIII regions provide a proof of concept for the use of antigens in the differentiation between DENV and ZIKV.

     

Enzyme-linked immunosorbent assay in the diagnosis of campylobacteriosis

Campylobacter jejuni and Campylobacter coli are the bacterial cause of human gastroenteritis commonly reported worldwide. The serodiagnosis of Campylobacter infections is not routinely done in Poland so the aim of this study was to evaluation of ELISA in the diagnosis ofcampylobacteriosis. Serum samples obtained from 145 patients with gastroenteritis were tested by ELISA with 7 different heat-stable antigens of C. jejuni and one of C. coli and by the commercial Virion/Serion ELISA with purified 45 kDa outer membrane protein of C. jejuni. Antibodies for heat-stable antigens of C. jejuni were detected statistically more often than antibodies for heat-stable antigens of C. coli and for purifled protein of C. jejuni. We found significant differences in the frequency of detection of antibodies to different heat-stable antigens, ranged from 18.6% to 68.9% of positive results, what indicate for serological heterogenicity of C. jejuni strains isolated in Poland. The results of our study showed usefulness of ELISA in serological diagnosis of campylobacteriosis. However it is necessary to serotype the C. jejuni strains isolated in Poland to find the appropriate C. jejuni serotype for using in ELISA.     

The use of serologic tests for the diagnosis of chlamydial infections

Serology is commonly used for the diagnosis of acute Chlamydia pneumoniae infections and also for the diagnosis of complicated Chlamydia trachomatis infections. Furthermore, recent sero-epidemiological studies have linked C. pneumoniae infection with several diseases traditionally considered non-infectious. The objectives of this mini-review are to critically review and discuss some selected analytical and methodological aspects, controversies and current problems in chlamydial serodiagnosis. To illustrate our views we present some original data of the comparison of current technologies. The review of the literature revealed high variability in methodologies applied to different studies. This observation was supported by our own data, which explains occasional conflicting clinical interpretation. Although the microimmunofluorescence (MIF) technique is generally considered as the gold standard for serodiagnosis of chlamydial infections, assay conditions are highly variable and hence pose a major problem in the interpretation of the results. For instance, many recent studies linking C. pneumoniae and atherosclerosis have utilized MIF techniques with variable threshold criteria for the positivity, in combination with selection bias of cases and controls possibly leading to conflicting results. Variability of assay conditions is also a common problem with Western blots, and interpretation is problematic when both anti-C. pneumoniae and anti-C. trachomatis antibodies are present. Furthermore, there is a lot of disagreement in serological criteria applied to recently emerged enzyme immunoassay (EIA) techniques when these assays are used for acute and non-acute clinical conditions and their association with Chlamydiae. In conclusion, standardization of serological techniques and the development of uniform criteria for interpretation of serologic findings is necessary to increase our knowledge of the biology of Chlamydiae, pathogenesis of any chlamydial infection and chronic infections in particular.     

检测梅毒螺旋体抗体的纤维膜条方法的建立及其在诊断中的应用

目的：建立以纤维膜为载体的检测梅毒螺旋体抗体的方法,检查病人血清中对梅毒螺旋体多种抗原的抗体,用于梅毒感染的诊断。方法：将基因工程表达及纯化的梅毒螺旋体蛋白tp15、tp17、tp42和tp47分别结合在纤维膜上,用载抗原的纤维膜条检查血清中的抗体,抗体阳性者在相应抗原位置显示出特异条带。结果：梅毒螺旋体感染者血清中存在特异性抗体,在检查的460份临床诊断的患者血清中,对tp15、tp17、tp42和tp47抗原的抗体检出率分别为41.3%、100%、98.7%和51.7%;134份献血员血清抗体阴性。结论：建立的检测梅毒螺旋体感染的方法可同时检查对多种抗原的抗体,以纤维膜条作为诊断条检测血清抗体方法简便,用于临床诊断更特异、更敏感。     

肺炎克雷伯氏菌特异性抗原的筛选与鉴定

目的寻找肺炎克雷伯氏菌（Klebsiella pneumoniae）血清学检测用特异性抗原。方法双向电泳分离K.pneumoniae总蛋白,通过免疫印迹（Western blotting）与常见病原菌的多抗反应筛选特异性抗原蛋白,原核表达该蛋白并用ELISA法验证。结果获得K.pneumoniae的双向电泳图谱。寻找Western blotting中与K.pneumoniae自身多抗反应而不与与其它病原菌多抗反应的蛋白,质谱鉴定为酸性磷酸酶（Acid phosphatase,GI：238894261）。经表达纯化并以酶联免疫吸附试验（ELISA）验证,证明该蛋白作为包被抗原的灵敏度高,特异性强。结论 K.pneumoniae酸性磷酸酶是适用于该菌感染检测特异性抗原蛋白。     

Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis

Klebsiella pneumoniae is an opportunistic pathogen which causes pneumoniae, urinary tract infections and septicemia in immunocompromised patients. Hospital outbreaks of multidrug-resistant K. pneumoniae, especially those in neonatal wards, are often caused by strains producing the extended-spectrum-beta-lactamases (ESBLs). An immunoproteome based approach was developed to identify candidate antigens of K. pneumoniae for vaccine development. Sera from patients with acute K. pneumoniae infections (n = 55) and a control group of sera from healthy individuals (n = 15) were analyzed for reactivity by Western blot against ESBL K. pneumoniae outer membrane proteins separated by 2-DE. Twenty highly immunogenic protein spots were identified by immunoproteomic analysis. The immunogenic proteins that are most frequently recognized by positive K. pneumoniae sera were OmpA, OmpK36, FepA, OmpK17, OmpW, Colicin I receptor protein and three novel proteins. Two of the vaccine candidate genes, OmpA (Struve et al. Microbiology 2003, 149, 167-176) and FepA (Lai, Y. C. et al.. Infect Immun 2001, 69, 7140-7145), have recently been shown to be essential in colonization and infection in an in vivo mouse model. Hence, these two immunogenic proteins could serve as potential vaccine candidates.     

实验大鼠泰泽菌检测方法的比较研究

目的　比较实验大鼠泰泽菌检测方法─nested PCR、IFA、免疫抑制诱发试验 触片染色镜检和组织病理学诊断。方法　根据泰泽菌 16SrDNA合成引物 ,对 16个菌株作nested PCR扩增并进行特异性、敏感性试验、验证。将此PCR应用于 2 0只免疫抑制Wistar大鼠和 5只非免疫抑制SD大鼠泰泽菌检测 ,并作IFA、常规细菌学检测和组织病理学诊断。结果　nested PCR中仅有泰泽菌出现 196bp特异性扩增条带 ;而 15株非泰泽菌均未出现此扩增条带。该PCR能检出 10pg泰泽菌DNA。将此PCR应用于大鼠泰泽菌检测 ,结果未检出阳性样品。nested PCR与常规细菌学检测、组织病理学诊断结果相一致。采用IFA方法 ,以购得的大鼠泰泽菌抗原片对上述 2 5份大鼠血清进行检测 ,结果有 6份血清产生非特异性反应。结论　采用IFA对动物群进行筛查出现阳性结果 ,须采用免疫抑制诱发试验、PCR和组织病理学诊断技术组合进一步验证。本研究建立的nested PCR方法 ,特异、敏感、快速 ,结合组织病理学诊断技术对实验动物泰泽菌感染可做出精确诊断。     

Developing a new clinical tool for diagnosing chronic Q fever: the Coxiella ELISPOT

Definitively establishing a clinical diagnosis of chronic Q fever remains challenging, as the diagnostic performance of both conventional serological tests and PCR is limited. Given the importance of an early diagnosis of chronic Q fever, there is a need for a reliable diagnostic test. We developed an enzyme-linked immunospot assay to measure Coxiella burnetii (C.?burnetii)-specific T-cell responses (Coxiella ELISPOT) to both phase I and phase II antigens and tested convalescent Q fever patients (without chronic disease, n?=?9) and patients with an established diagnosis of chronic Q fever (n?=?3). The Coxiella ELISPOT adequately identified convalescent Q fever patients from healthy controls by demonstrating C.?burnetii-specific T-cell interferon-γ production to both phase I and phase II antigens. Compared to convalescent Q fever patients, chronic Q fever patients showed a distinct Coxiella ELISPOT profile characterized by a much higher spot count for both phase I and phase II (18-fold for phase II, 8-fold higher for phase I) and a consistent shift towards more phase I reactivity. The diagnostic potential of the Coxiella ELISPOT is promising and warrants further investigation.     

Immunological Analysis of Mycoplasma Membranes

The antigens responsible for the production of antibodies to Mycoplasma laidlawii and M. gallisepticum causing growth and metabolic inhibition of these organisms were localized in the cell membrane. Various membrane fractions were tested for serological activity. Membrane lipids were completely or almost completely inactive, whereas several preparations of defatted membrane proteins retained some serological activity, shown by their ability to stimulate metabolic inhibition antibody in rabbits and to adsorb metabolic inhibition antibody and form precipitation lines with an antiserum to the membrane. When the membranes were heated to 65 C for 1 hr, they virtually lost their ability to adsorb metabolic inhibition antibody, which suggests that the antigenic determinants are proteins. Serological activity was retained in reaggregated membranes obtained by dialysis against Mg²⁺ of membranes solubilized in sodium dodecyl sulfate. The amount of solubilized membrane protein and lipid incorporated into the reaggregated membranes could be regulated by varying the Mg²⁺ concentration. As the serological tests indicated that the various membrane antigens were selectively incorporated into the different reaggregated membranes, the use of controlled reaggregation of solubilized membranes is suggested as a new tool for the fractionation and antigenic analysis of membrane proteins.     

Another turn of the screw in shaving Gram-positive bacteria: Optimization of proteomics surface protein identification in Streptococcus pneumoniae

Bacterial surface proteins are of outmost importance as they play critical roles in the interaction between cells and their environment. In addition, they can be targets of either vaccines or antibodies. Proteomic analysis through "shaving" live cells with proteases has become a successful approach for a fast and reliable identification of surface proteins. However, this protocol has not been able to reach the goal of excluding cytoplasmic contamination, as cell lysis is an inherent process during culture and experimental manipulation. In this work, we carried out the optimization of the "shaving" strategy for the Gram-positive human pathogen Streptococcus pneumoniae, a bacterium highly susceptible to autolysis, and set up the conditions for maximizing the identification of surface proteins containing sorting or exporting signals, and for minimizing cytoplasmic contamination. We also demonstrate that cell lysis is an inherent process during culture and experimental manipulation, and that a low level of lysis is enough to contaminate a "surfome" preparation with peptides derived from cytoplasmic proteins. When the optimized conditions were applied to several clinical isolates, we found the majority of the proteins described to induce protection against pneumococcal infection. In addition, we found other proteins whose protection capacity has not been yet tested. In addition, we show the utility of this approach for providing antigens that can be used in serological tests for the diagnosis of pneumococcal disease.