Study On The Role Of Hyaluronic Acid In Cryptococcus Neoformans Penetrating The Alveolar-capillary Barrier

BackgroundCryptococcus neoformans is an encapsulated pathogenic fungus that primarily afflicts immunocompromised individuals, and rarely cause the disease in the healthy people, Cryptococcosis is a kind of chronical, sub-acute even acute infection and can invade into meninges, brain, lung, bone or skin. In the recent years, rates of infection by C. neoformans have increased considerably for the increasingly expanded use of immunosuppressive drugs and the onset of AIDS, Therefore, the study of Cryptococcus neoformans has been attached great importance to scholars of global fungal researcher.Dehydrated haploid yeast or basidiospore of C. neoformans is the usual form of inhalation.The organisms are likely to cause asymptomatic infection in the lungs and then penetrate alveolar-capillary barrier to extrapulmonary tissues and show a remarkable propensity in spreading to the brain and meninges. The key step of the pathogenic process is that C. neoformans penetrating alveolar blood barrier, which locats between the alveoli and alveolar capillaries, and composed of six layers:the liquid layer of the pulmonary surfactant; alveolar epithelial cell layer; the basal membrane; the gap between the alveoli and capillaries; capillary basement membrane; pulmonary microvascular endothelial cells. Its function is to make sure O2and CO2finish successful exchange, and defense bacteria invasion and prevent the loss of other substances within the capillary. Because the pulmonary microvascular endothelial cells is the most important part of alveolar blood barrier, Cryptococcus neoformans must adhere and invade into the pulmonary microvascular endothelial cells for its invasion. At present, people know very little about the molecular mechanisms of the invasion of alveolar blood barrier for Cryptococcus neoformans, which is the important problem need to be solved in Cryptococcus neoformans disease prevention.Cryptococcus neoformans is encompassed by its capsule polysaccharide, which is a well-known major virulent factor in this yeast. Ambrose Jong have identified a C. neoformans capsule gene, CPS1and demonstrated that CPS1encodes a hyaluronic acid synthase. It synthesizes cell surface hyaluronic acid, which is important for yeast infection in vitro and in vivo. Deletion of CPS1from C. neoformans resulted in alterations of several phenotypes important for the virulence of C. neoformans, including modification of the ultrastructure between the cell wall and the capsule, temperature sensitivity, and a reduced ability to associate with HBMEC in vitro. These results suggest that CPS1plays an important role in the pathobiology of C. neoformans. The association of hyaluronic acid with cell surface receptors was CD44, and this interaction between hyaluronic acid and host CD44may lead to the transduction of signals that participate in cytoskeletal rearrangement during pathogen entry. CD44is a trans-membrane glycoprotein widely expressed on many kinds of cell from epithelial, mesen-chymal, hematopoietic and mesodermal tissues,and can combined with HA, GAG, collagen, Iaminin, fibronectin, select protein combination. And the molecular weight of CD44is80-95KD. Because this interaction between hyaluronic acid and host CD44may exist in cell types,we speculate this response might take place via interaction between hyaluronic acid and the host receptor on HPMEC.Animal experiments is crucial for the systematic study of Cryptococcus neoformans infection mechanism, there are four animal models used commonly:mice, guinea pigs, rats and rabbits, in which mice is the most sensitive animals to Cryptococcus neoformans, According to the need of experiment and the site of infection, the mouse is the most commonly used animal models of C. neoformans research. The mainly infection modeling methods are intraperitoneal, intravenous, nasal cavity, trachea, and intracranial injection. Because we want to observe the ability of C. neoformans across the alveolar blood barrier and simulate the normal infection, the method of infection is through aerosol inhalation device; Ultrasonic atomizing inhalation has been successfully applied to a wide range of animals inhaling bacterial infection.Furthermore cryptococcus neoformans infection occurs mainly in the immunocompromised population,so the study of cryptococcus neoformans pathogenesis should include the relation between immunosuppressive animal model and pathogenesis of cryptococcus neoformans.ObjectiveThe in vitro adhesion and invasion assay will be performed; using immunoblot technique to compare the differences between wild strains and the mutant induced expression of CD44on HPMEC; immunofluorescence techniques will be used to compare the CD44expression on HPMEC and actin rearrangement; we will establish immunosuppressed Balb/c mice by ultrasonic atomized inhalation of Cryptococcus neoformans to find wether HA affect C. neoformans across the alveolar blood barrier.Methods and results1. The in vitro assay of the binding of C.neoformans to HPMEC were performed asfollows:Briefly, HPMEC were grown until confluence in collagen-coated24-well tissue culture plates. An inoculum of106yeast cells suspended in0.5ml experimental medium was added to the HPMEC monolayer (multiplicity of infection,10) for1,3and5hours.at37℃. Yeast cells unattached to the HPMEC monolayer were removed by washing the monolayer three times with experimental medium. HPMEC were lysed with0.5%Triton, diluted, and plated onto a YPD agar plate to determine the number of CFU that were associated with HPMEC.Results are presented as percentages of the level of adhesion of the inoculum,calculated as [(number of Cryptococcus cells recovered)/(number of Cryptococcus cells inoculated)] X100%. The adhesion rates of B4500FO2at1,3, and5h were0.0123±0.0025%、0.055±0.0041%and0.012±0.0041%respectively. However, the adhesion rates of TYCC645#32reduced to0.005±0.0017%、0.025±0.0044%and0.0004±0.0007%.2.Samples for immunofluorescence microscopy were prepared as follows.HPMEC were plated onto glass coverslips (22mm, square), which had been previously coated with type I collagen from rat tail (Upstate,5-10μg/cm2) in an8-well square culture system for30min. HPMEC (-1×104cells) were seeded onto one coverslip24hours prior to the experiment. HPMEC were prewashed three times with PBS, then fixed with4%formaldehyde for30min at room temperature. After an additional three-washes with PBS, the HPMEC were blocked with5%milk/PBS for30min and then incubated with anti-CD44monoclonal antibody (1:100dilution) into some wells at4℃overnight.The coverslips were then washed3times with PBS, anti-rabbit IgG FITC conjugate (1:100dilution) were added into compared wells and phalloidin-rhodamine conjugate (1:100dilution)were also added for1hour at4℃. Another three washes were applied before adding one drop of Vectashield mounting solution with DAPI to seal the coverslips onto slides. Samples were examined under an immunofluorescence microscope after24hours. CD44was stained with a specific antibody conjugated with FITC (green), the CD44signals in B4500FO2-treated HBMEC increased, but not in TYCC645#32-treated cells. Actin molecules were stained by the phalloidin-rhodamine conjugate (red), In the absence of C. neoformans or in the presence of TYCC645#32, the actin staining was in general distributed throughout the HPMEC, whereas, in comparison,in the presence of B4500FO2, the actin molecules were clustered or concentrated on the marginal regions of HPMEC.3. Samples for western blots were prepared as follows, HPMEC was seeded and grown in60mm Petri dish for3days. On the day of experiment,the cells were individually incubated with either medium(control),1×108C neoformans B4500FO2or TYCC645#32individually for3h in the experimental medium with1%human serum. After incubation, the cells were washed with PBS three times, scraped in PBS and spun down at2000r.p.m. at4℃. Cell pellets were lysed in RIPA Buffer on ice and incubated for30min on ice. Samples were mixed with4xSDS. Forty microlitres of each fraction was used for western blots. The antibody dilution was chosen as follows:CD44(Santa Cruz, sc-7964,1:500dilution), actin(Santa Cruz, sc-8432,1:300dilution), and anti-rabbit-HRP conjugate (1:6000dilution). We observed that incubation with strain B4500FO2led to an increased association of CD44. In contrast, incubation with isogenic strain TYCC645#32(ACPS1) resulted in much less CD44detected in the low-density fraction.4. Animal model:The in vitro experiments demonstrated that the CPS1deletion mutant TYCC64532was less adherent and invasive than its parent strain B4500FO2in the HPMEC cell culture model. To further investigate the role of CPS1on the development of Cryptococcosis, the Balb/c mice model of experimental Cryptococcosis was used to determine the in vivo virulence phenotype. the Balb/c mice immunosuppressed with cyclophosphamide (CTX), and then inhaled Cryptococcus neoformans using ultrasonic atomized inhalation.Mice were sacrificed at6h、day3and day7post-inoculation. The number of CFU of Cryptococci was quantized on YPD plates prepared from serial dilutions of peripheral blood and homogenates of whole lungs and brains, respectively, Lung tissues were sectioned and stained for pathologic examination. The magnitude of Cryptococcus neoformans induced by the mutant strain was similar to that of the wild-type strain at6h post-inoculation. However,there were significant differet magnitude of Cryptococcus neoformans between B4500FO2and TYCC645#32at day3and day7post-inoculation. Our in vitro and in vivo studies demonstrated that the CPS1gene was required for Cryptococcus neoformans B4500FO2penetration across the the alveolar blood barrier.Statistical analysisAll values are expressed as mean±standard deviation. Statistical analysis was performed with Mann-Whitney Test for comparison of two groups, In vivo experiments, the data of WBCs was analyzed by Student’s t-test.while the data of CFU were analyzed by kruskal-wallis test.Differences with p<0.05were considered to be statistically significant.ConclusionIn order to determine the role of HA in the pathogenesis of Cryptococcus neoformans penetrating the pulmonary microvascular endothelial cell of alveolar-capillary barrier, we used the CPS1deletion mutants to study its related mechanism by in vitro and in vivo models of the alveolar-capillary barrier. We showed that the mutant TYCC645’32was considerably less adherent and invasive for HPMEC, and significantly less virulent in the entry into the the alveolar-capillary barrier in the Balb/c model model of experimental Cryptococcosis. Our in vitro and in vivo studies demonstrated that the CPS1gene was required for Cryptococcus neoformans B4500FO2penetrating across the the alveolar blood barrier.To test the influence of the expression of CD44on HPMEC induced by CPS1deletion mutant and the wild-type strain, We used immune fluorescence and western blotting technology to detect the expression of CD44on HPMEC, which was more in the B4500FO2group than the normal HPMEC, but less in TYCC645#32-treated cells, the expressions of CD44was increased little, but it was less than that of B4500FO2.Remodelling of the host-cell actin cytoskeleton has been observed during pathogenic invasion. The Cryptococcus neoformans adhesion and invasion of the HPMEC leading to cytoskeletal reorganization and downstrem signal activiation is important for the Cryptococcus neoformans penetrating across the the alveolar blood barrier. To test the cytoskeletal reorganization in the HPMEC induced by CPS1deletion mutant and the wild-type strain, the rhodamine-labelled phalloidin was used to make the rearrangement of actin filaments induced by Cryptococcus neoformans visible under the fluorescence microscope. Our findings showed that the actin filaments rearrangement in the HPMEC induced by TYCC645#32was significantly weaker than that induced by the parent strain B4500FO2.Taken together, our findings suggest that the HA plays an important role in B4500FO2penetrating across the the alveolar blood barrier. After hyaluronic acid of cryptococcus neoformans combining with CD44on HPMEC, what is the specific downstrem signal activiation and mechanism in the penetration across the the alveolar blood barrie for Cryptococcus neoformansr,which is our follow-up studies.