The Role Of α-mannosidase Activity In Chlamydia Trachomatis-infecting Cells And Its Diagnostic Value

The a-mannosidase is a kind of glycoside hydrolases, which plays an important role in the synthesis and processing of N-linked glycoprotein. Currently, studies on a-mannosidase mainly focus on eukaryotes, but there was so few literatures aiming at prokaryotes especially pathogenic prokaryotes. With rapid development of specific enzyme-chromogenic technology, it is possible to detect a-mannosidase activity by using this technology.There are various well-known methods for detecting Chlamydia trachomatis, including cell culture, immunology, molecular biology, and biochemistry-based methods, but all of these methods harbor some problems. The sensitivities of both cell culture method and colloidal gold immunoassay method are too low, and it is difficult for culture method to meet the requirements of the actual application in clinical screening. Because the specificity of biochemical assay is not high, so clinic is relatively less. Molecular biology method has not only operation complexity, but also exists timeliness problem. These problems have affected clinical detection involved in C.trachomatis.At present, with the development of chromogenic medium and enzyme-chromogenic technology, rapid screening for microganisms using special enzymes has been one novel direction of clinical microbiological analysis. It will determine whether α-mannosidase activity has potential diagnostic value for the clinical detection of C.trachomatis, which common pathogenic microorganisms in urogenital tract and most popular strains of C.trachomatis have α-mannosidase activity or not. These problems are worthy of further studies such as methods of separation and purification for a-mannosidase of C.trachomatis, α-mannosidase affecting C.trachomatis-infecting cells or not, the specificity of this effect, the performance of enzymatic method, and this technology transformed into a product or not, if a-mannosidase activity can be used as a diagnostic marker.In order to study these issues in detail, this paper is divided into the following three parts to clarify the role of a-mannosidase activity during C.trachomatis-infecting cells and its diagnostic value.PartⅠ Studies on a-mannosidase of genitourinary tract common microorganismsObjectiveTo study a-mannosidase activity of genitourinary tract common microorganisms by rapid detection, which will determine whether a-mannosidase activity for C.trachomatis infection has the rapid diagnostic value.Methods1.Twenty-eight standard strains, forty clinical isolates related to genitourinary tract common microorganisms as well as two serotypes of C.trachomatis were cultured.2.Collecting variety cultures by either washing-centrifugation or no treatment, respectively.3.Collecting cultures of C.trachomatis and Streptococcus pyogenes by either ultrasonic crushing or no treatment, respectively. Collecting cultures of Candida albicans by either freeze grinding or no treatment, respectively.4. Measuring the obsorbance density (OD) value at570nm and512nm after different time (10,30and60min) of incubation at37°C using5-Br-4-Cl-3-indoly-a-D-mannopyranoside and6-Cl-3-indoly-a-D-mannopyranoside as chromogenic agent, respectively.Results 1. The rates of HeLa229cells infected with C.trachomatis serotype serotype D strain (18.75%,24.06%and17.65%) were significantly lower than those of HeLa229cells infected with serotype E (59.69%,62.96%and53.85%). The difference about infection rates of probability distribution were statistically significant (χ2=16.273, P<0.05) by chi-square analysis.2. A more high level of a-mannosidase activity was showed for C.trachomatis serotype D strain and E strain than for other microorganisms, no matter what kinds of chromogenic reagents were used, and the differences were statistically significant (P<0.05). a-mannosidase activity for C.trachomatis infection has the rapid diagnostic value. Trichomonas vaginalis and some species of candida showed weak a-mannosidase activity, whose OD values were from0.150to0.250.3. Repeated measures analysis of variance was done to OD57o and OD512of various microbial culture collections under different conditions. It was showed that such factors as different chromogenic reagent (P=103.251, P<0.05), washing or not (F=88.253, P<0.05), and chromagenic time (P=213.235, P<0.05), affected the results in varying degrees.4. Color slowly and light using5-Br-4-Cl-3-indoly-a-D-mannopyranoside as chromogenic substrate (OD570of C.trachomatis serotype D strain unbroken were0.159,0.221and0.389at10,30and60min, respectively. Meanwhile, OD570of C.trachomatis serotype E strain unbroken were0.147,0.198and0.358at10,30and60min, respectively. The difference of OD570between serotype D strain and serotype E strain was statistically significant), however, color fast and deep using6-chloro-3-indole-a-D-mannopyranoside as chromogenic substrate (OD512of C.trachomatis serotype D strain unbroken were0.519,0.607and1.826at10,30and60min, respectively. Additionally, OD512of C.trachomatis serotype E strain unbroken were0.588,0.957and2.025at10,30and60min, respectively. The difference of OD512between serotype D strain and serotype E strain was statistically significant).PartⅡ Purification for a-mannosidase of C.trachomatis and effect of α-mannosidase activity on C.trachomatis-infecting cellsObjectiveTo study purification method for a-mannosidase of C.trachomatis and effect of α-mannosidase activity on C.trachomatis-infecting cells.Methods1. α-Mannosidase was isolated and purified from broken cell culture infected by C.trachomatis serotype D strain after centrifugalization, ultrasonic crushing, fraction precipitation by ammonium sulfate and SephadexG-100column chromatography. The purity and molecular weight of purified α-mannosidase were identified by SDS-PAGE.2. Preparing anti-α-mannosidase serum by immunizing rabbits with purified α-mannosidase, purifying antiserum successively treated with ammonium sulfate precipitation and Sepharose CL-4B affinity chromatography, and detecting antibody titer using agarose gel double diffusion method.3. HeLa229monolayers treated with DEAE-dextran solution were added with purified a-mannosidase, anti-α-mannosidase polyclonal antibody and a-mannosidase from jack bean at three concentrations, respectively, and infected with C.trachomatis serotype D strain. Observing differences in infection rates (infected cells/total cells) of C.trachomatis after stained with direct fluorescent antibody kit.Results1. Purified α-mannosidase of Chlamydia trachomatis serotype D strain exhibited one band by SDS-PAGE, and the molecular weight is about50kDa.2. Antibody titer of anti--mannosidase serum was1/64, and antibody titer of purified anti-a-mannosidase polyclonal antibody was also1/64.3. The difference about infection rates of probability distribution was statistically significant (χ2=137.062, P<0.05) when adding anti-α-mannosidase polyclonal antibody as experimental group, furthermore, infection rates under such conditions existed a down trendency with the increase of antibody addition (χ2=65.328, P<0.05). The difference about infection rates of probability distribution was statistically significant (χ2=117.539, P<0.05) when adding purified a-mannosidase as experimental group, furthermore, infection rates under such conditions existed a rising trending with the increase of purified a-mannosidase antibody addition (χ2=98.227, P<0.05). However, The difference about infection rates of probability distribution was not statistically significant (χ2=0.096, P>0.05) when adding a-mannosidase from jack bean.Part Ⅲ Establishment and clinical evaluation of a novel assay based on a-mannosidase activity to detect C.trachomatisObjectiveTo establish a novel assay based on a-mannosidase activity to detect C.trachomatis using6-chloro-3-indole-a-D-mannopyranoside as substrate, which should meet the requirements of the actual application in clinical screening and might be commercialized.Methods1. The enzymatic formula based on basic detecting system, was established after single-factor design with multiple levels, orthogonal test and extreme difference analysis, and orthogonal verification, taken account of adjusting for reaction time. It was the jumping-off place that "M" values need to be greater in the following formulas. M=(AD+AE-B-C)/2"M" represented the differences between positive OD512and negative OD512."AD" represented OD512of the test result of C.trachomatis serotype D strain."AE" represented OD512of the test result of C.trachomatis serotype E strain."B" represented the average of OD512of the test results of two strains of C.albicans."C" represented the average of OD512of the test results of two strains of S.pyogenes.2. Confirming the limits of detection (LOD) of the enzymatic assay containing the LOD for C.trachomatis and the LOD for a-mannosidase. Evaluting initially positive rates of such kinds of specimens as urethral discharge specimens (198samples), vaginal secretion specimens (232samples), cervical secretion specimens (157samples), prostate massage liquid specimens (33samples), and semen specimens (45samples), to determine appropriate types of specimens for the enzymatic assay.3. Cell culture and ligase chain reaction method (LCR) were used to evaluate the clinical performance of the enzymatic method. Adopting an "expanded gold standard" described as follows:any positive by either culture or LCR was classified as a true positive, whatever the result of the enzymatic method.4. Confirming period of validity of the enzymatic method established and the control reference.Results1. The concentration of fast violet B salt had greatest impact on results, while the pH value of0.1mol/L citric acid/sodium citrate buffer solution had the least influence on it. However, because the formula of experiment6in the orthogonal design table was superior to the optimal combination of factors level by verifying, and when clinical value of time and influence of Triton X-100concentration on this basis were taken account of, the best formula selected in being incubated for15min at37℃was as follows:chromogenic solution contained2mg/ml6-chloro-3-indolyl-a-D-mannoside,0.1mol/L citric acid/sodium citrate buffer solution (pH4.0), and0.75%Triton X-100; concentration of fast violet B salt was1.5mg/ml whose solvent was normal saline; sample solution was chromogenic solution without addition of substrate.2. The LOD of the enzymatic assay for C.trachomatis was617IFU/ml, and the LOD of the enzymatic assay for a-mannosidase was10-3u/ml. Positive rates of urethral discharge, cervical secretion, vaginal secretion, prostate massage liquid, and semen were11.11%,12.74%,9.91%,27.27%and93.33%, respectively, as suggested that semen should make interference with the enzymatic assay, and prostate massage liquid might make interference with the enzymatic assay.3. Prostate massage liquid had interference on the enzymatic assay. In those specimens other than prostate fluid samples, the sensitivity and specificity of the enzymatic method were91.8%(95%CI,86.0%to97.6%) and98.3%(95%CI,97.1%to99.5%). There were no significant differences in performance between the enzymatic method and the expanded gold standard (P>0.05).4. The period of validity of the enzymatic method established and the control reference was23months (stored at2℃～8℃) and5.5months (stored at-20℃), respectively.Conclusions1. a-Mannosidase activity is one diagnostic marker of C.trachomatis, which has very high potential value.2. The molecular weight of purified a-mannosidase of C. trachomatis serotype D strain is about50kDa.3. The level of a-mannosidase activity may significantly affect ability of C. trachomatis serotype D strain to infect sensitive cells.4. The method for rapid detection a-mannosidase activity established in this paper can be used to diagnose clinical infection of C.trachomatis, and the enzymatic method is worthy to be extended in clinics.