| BackgroundCryptococcus neoformans (CN) is important human pathogenic fungi with capsular. It mainly caused cryptococcal meningitis (CM). Patients with cryptococcal meningitis have vital symptoms and highly mortality. Immunocompromised patients are susceptible population. The main invasion pathway of CN is that spore in the natural environment such as pigeon manure and soil is inhaled through respiratory tract. The fungus can also be found in the normal microbial population and its infection happens when the persons is in immunocompromised state, such as patients with AIDS, diabetes, leukemia and other diseases. In recent years, Cryptococcus neoformans infection rate increased greatly due to the AIDS epidemic, transplantation immune inhibitor and antineoplastic chemotherapy drug usage. CN infection has become one of the most common complications of the AIDS in Europe and the United States. Each year there is one million new cases, leading 700 thousands deaths around the world. Limiting anti-fungal drugs, side effects and drug resistance make great difficulties to get effective treatment.The cryptococcal meningitis is the most serious among CN infections. In Europe and the United States,80% of the cryptococcal meningitis happens in HIV patients. But in China, in recent years, the number of non-HIV patients and immunocompromised people suffering from cryptococcal meningitis is increased gradually. It is unknown that whether the cryptococcal meningitis mainly occurred in HIV patients or non-HIV patients. It is also unknown that whether it happened in persons with underlying diseases that result in human immunodeficiency. So the epidemiological characteristics of Chinese cryptococcal meningitis should be analysis.Back in 1894, people have isolated Cryptococcus neoformans from the human body and peach juice, then found it widely distributed in the soil, pigeon droppings, eucalyptus and other natural environment. Cryptococcus spore in pigeon droppings, eucalyptus and other spread in the air, inhaled through the respiratory tract, caused pneumonia in a majority of patients, eliminated by alveolar phagocytic. If not destroyed, cryptococcus neoformans cross through the pulmonary blood barrier into the blood and caused bacteremia. Due to the neurotropic, cryptococcus neoformans cross through blood brain barrier (BBB) and lead to serious cryptococcal meningitis or encephalitis. Cryptococcus neoformans crossing through the body’s first line of barrier-the alveolar capillary barrier is key step for the fungus hematogenous spread to brain. But the mechanism of bacteria crossing through pulmonary-blood barrier consisted of human alveolar epithelial cells, basement membrane and pulmonary microvascular endothelial cell is not yet clear, relevant research and is also less. If make clear the key receptor in human alveolar epithelial cells and the related signal pathways of cryptococcus neoformans intrusive,we will further reveal the pathogenic mechanism of Cryptococcus neoformans infection, and provide important pharmacologic targets for the treatment of the disease.The main virulence factor of Cryptococcus neoformans is capsular polysaccharide (such as GXM and GalXM) and soluble extracellular components which are related with anti-phagocytic function. Ambrose. Jong found hyaluronic acid capsule (HA) is an important virulence factor of Cryptococcus neoformans invasion to human brain microvascular endothelial cells. Cryptococcus neoformans CPS1 gene were identified by Ambrose Jong, and this gene encoding hyaluronic acid synthase, responsible for synthetic hyaluronic acid. His further study found that:HA in cps1 knockout strainscould not be detected in the membrane, and the adhesion and invasion rate of this strain to the brain microvascular endothelial cell decreased significantly. Cryptococcus neoformans HA in capsule binds with receptor CD44 on the brain microvascular endothelial cell lipid rafts, induce cytoskeletal rearrangement by a series of signal molecules such as PKC-a and dyrk3, then cryptococcus neoformans invade into the cell through caveolae/lipid raft mediated endocytic pathway.CD44 is widely expressed in many endothelial cells, epithelial cells, mesenchymal cells and mediates the adhesion between cells, or extracellular matrix and cell. It is receptor of hyaluronic acid (HA). Its molecular weight is about 80-95KD and. CD44 bind with HA to activate the downstream signaling molecules such as Src kinase, regulating the cytoskeletal rearrangement process. In tumor cells, CD44 extracellular domain binds with HA in host cells or in the stroma, intracellular domain in a single locus binds Src kinase with high affinity. The interaction of HA and CD44 to stimulate Src kinase activity, then cytoskeletal protein-cortactin are phosphorylated on tyrosine, cross-linking ability of cortactin and filamentous actin are reduced, leading to cytoskeletal rearrangement and tumor cell migration and invasion. Studies have shown that Src kinase signaling involved in the process of Escherichia coli invade into human brain microvascular endothelial cells. Src kinase also relates to Staphylococcus aureus invasion embryo kidney cell, Shigella invasion of epithelial cells and urinary tract pathogenic Escherichia coli invade bladder epithelial cells.In order to understand the clinical features and epidemiological characteristics of Chinese cryptococcal meningitis, our project reviews and analysis the case reports in China from 1981 to 2013. At the same time, Cryptococcus neoformans wild strains B4500FO2, cps1 knockdown strains TYCC645 and cps1 covering plant PCIP-39 were used to infect the human alveolar epithelial cell line A549 monolayer, then adhesion and invasion rate was detected, CD44 receptor expression and cytoskeletal changes were also detected. Futhermore, we observed the inhibition effect of CD44 receptor inhibitor and the Src kinase inhibitor on Cryptococcus neoformans invasion into A549 cells. The aim is to explore the mechanism that CD44/lipid raft-Src kinase signal pathway mediated Cryptococcus neoformans invasion into the human alveolar epithelial cells.ObjectiveTo analyze the epidemical characteristics of Chinese cryptococcal meningitis and to investigate mechanism of CD44/lipid raft-Src kinase signal pathway mediating cryptococcus neoformans invasion into human alveolar epithelial cells. The aim is to seek the drug targets that it can block Cryptococcus neoformans invasion across pulmonary-blood barrier and provide a theoretical basis for the prevention and treatment of the cryptococcal meningitis.Methods1. Epidemiological analysis of the cryptococcal meningitisA comprehensive analysis of the reported cases of cryptococcal meningitits in China was conducted, covering publications from 1981 to 2013 from CBMdisk database (China Biology and Medicine data disc, Institute of Medical Information, Chinese Academy of Medical Sciences). Case definition:Positive cerebrospinal fluid (CSF) India ink examination or positive CSF cryptococcal antigen test or positive CSF cryptococcal culture. Some cases were confirmed through the pathology examination of brain tissue. Cryptococcal was found in the brain tissue taken by biopsy or operation. Reading the documents selected carefully in the literature according to the inclusion and exclusion criteria, recording gender, age, occupation, contact history, basic disease, time of onset, symptoms of onset, laboratory examination, imaging findings, diagnosis, treatment and prognosis of each reported patient. Statistical data were analyzed. 2. Detection of AS49 cytoskeleton actin by fluorescence immunoassay104A549 cell were cultured in eight well plate coated with collagen for 30 min and grew into monolayer. Then 105 Cryptococcus neoformans B4500FO2 and TYCC645#32 were added to the cell monolayer, incubated for 3 hours in 37 ℃, washed 3 times with cold PBS, fixed with 4% paraformaldehyde, blocked with 5%BSA. Then phalloidin labled with Rhodamine (Sigma,1:300) was added to the corresponding wells. After lhour at 4 ℃, the wells were washed with cold PBS. Finally, a drop of DAPI (Sigma) was added in each well. The results were observed by fluorescence microscopy.3. Detection of the adhesion and invasion rate of Cryptococcus neoformans to A549 cellsThe 24 wells plate was coated with collagen for 30min, and then 105 A549 cells were vaccinated into each well and grew into monolayer. Cryptococcus neoformans were cultured in YPD complete medium at 30 ℃ 220rpm for 12 hours and suspended in the PBS dilution after washing 2 times with PBS. Cryptococcus neoformans wild strain B4500FO2, mutant TYCC645#32 and revertant strain PCIP-39(1:10 inoculation) were added on A549 monolayer for 1,3,5h at 37 ℃ and 5%CO2 enviroment. A549 monolayer was washed 3 times with PBS and lyses with 0.5% tritonX-100. Cellular dissolved substances were cultured in YPD plate for 24 hours; the number of yeast colony was counted. The adhesion rate was calculated by colony counting method. CD44 inhibitor Bikunin 40mg/L was added to A549 cell monolayer for 1 hour, above experiment was repeated. HA enzyme was incubated with wild strain B4500 for 1 hour, above experiment was repeated again.4. Inhibition role of Src kinase inhibitor PP2 in cryptococcus neoformans invasion into A549 cell105 A549 cells were inoculated in each well after the 24 wells plate was coated with collagen for 30min and grew into monolayer.106 Cryptococcus neoformans B4500FO2, TYCC645#32 and PCIP-39 were used to infect the cells. And 0,10,20, 30 mmol/L PP2 were added to cells at same time. Cells were incubated for 3h at 37 ℃ and 5% CO2 enviroment, then washed 3 times with PBS, lysed with 0.5% tritonX-100. Cellular dissolved substances were cultured in YPD plate for 24 hours, the number of yeast colony was counted. The adhesion rate was calculated by colony counting method.5. Detection of CD44 on A549 cell by immunoblotting 106 A549 cells were inoculated in 100mm petri dish and grew into monolayer cell after the dish was coated with collagen for 30min. Then 107 Cryptococcus neoformans B4500FO2 and TYCC645#32 were added to the cell monolayer and incubated for 3 hours,37 ℃ , wash 3 times with PBS, then lyses cell RIPA, cell total protein extraction. SDS-PAGE electrophoresis, transfer film, sealing, washed 3 times with PBST,5% skimmed milk shaker closed 60min, plus anti CD44 (1:500), beta-actin (1:300), sealed 4 degree C for the night. Washed 3 times with PBST, plus HRP Goat anti rabbit two anti shake, room temperature 60min. Wash the membrane 3 times, ECL chemical luminescence.6. Detection of CD44 in the lung of mice by fluorescence-immunohistochemistry6 weeks Balb/c mice and cyclophosphamide (CTX) were used to establish the immunosuppressive mice model, and then Cryptococcus neoformans suspension atomized steam fog particles were inhaled by mice through spontaneous breathing path. After 6h infection, mice were sacrificed and the lung tissue was extracted. The expression of CD44 in the lung of mice was detected by the fluorescence-immunohistochemistry.Result1. Epidemical characteristics of the cryptococcal meningitis in ChinaA comprehensive analysis of the reported cases of the cryptococcal meningitits in China was conducted, covering publications from 1981 to 2013 from CBMdisk database (China Biology and Medicine data disc, Institute of Medical Information, Chinese Academy of Medical Sciences), by two separate authors. All cases were collected from the above database with key words cryptococcus and meningitits. Among the 306 reports and 3961 cases about cryptococcal meningitits, there were 21% patients with HIV infection,39% with other underlying diseases and 40% patients without underlying diseases. Main underlying diseases in non-HIV patients were tuberculosis (10.46%), liver disease (9.83%), SLE (7.00%), DM(Diabetes 5.26%), Kidney disease (3.16%), lung disease (2.97%), cancer (2.01%). Clinical manifestations of Cryptococcal meningitis at onset were headache and fever in many cases (40.22%). About 60.8% of the cases were reported in males. In the total 3961 cases,1605 cases clearly described their treatment protocols and outcome. Patients who received treatment of amphotericin B (AmB) with 5-flucytosine, AmB with Fluconazole, and the combination of AmB+5-FU+FCZ were 495,411,266 persons and got a total efficiency of 70.1%,70.8%, and 74.8% respectively.2 A549 cell cytoskeleton rearrangemen was induced by the infection of Cryptococcus neoformansAfter infected by cryptococcus neoformans, A549 cells were stained with phalloidin labeled by rhodamine and observed by the fluorescence microscopy. It was found that the actin lines of A549 cell were unclear and concentrated on the membrane in wild strains-infection group. The similar situation was appeared in Acpsl strain-infection group, but the change degree is significantly weaker than the wild strain-infection group. A549 actin distribution has no any change in non-infection group.3. Capsular HA on Cryptococcus neoformans contributes to its invasion into A549 cellsA549 cell was infected by the wild strains, mutant strains and revertant strains of Cryptococcus neoformans for 1,3,5 hours respectively. The adhesion and invasion rate is 0.062±0.0025%,0.08 ± 0.0020%,0.05 ± 0.01040% respectively in the wild strains-infection group. The adhesion and invasion rate is 0.012 ±.0017%,0.029± 0.0037%,0.036 ± 0.0028% in the mutant strains-infection group. The adhesion and invasion rate is 0.032±0.0030%,0.039±0.0040%,0.026±0.0041% in the revertant strains-infection group. The adhesion and invasion rate was higher in the wild strains-infection group than it is in the mutant strains-infection group. The difference was statistically significant (p<0.05).4. Effect of the hyaluronidase on the association of C. neoformans with A549 cell.Hyaluronidase can specifically decompose the capsular HA on Cryptococcus neoformans. A549 cell monolayer was treated with the hyaluronidase of 0,5,10,50U for 1 hours, and then infected by the wild strain B4500F02 of Cryptococcus neoformans for 1,3,5 hours respectively. We found that the adhesion rate were 0.062 ± 0.0025%,0.08 ± 0.0020%,0.05 ± 0.01040% in 0U group. The adhesion rate were 0.050±0.0030%,0.051±0.0020%,0.040±0.0020% in 5U group. The adhesion rate were 0.040±0.0030%,0.048±0.0030%,0.037±0.0028% in 10U group. The adhesion rate were 0.030±0.00205,0.035±0.0040%,0.024±0.0061% in 50U group. The results show that the hyaluronidase reduced the adherence ability of wild strain B4500F02 to A549 cells in dose-dependent way. The difference was statistically significant (p<0.05).5. Expression of CD44 on A549 cells infected by Cryptococcus neoformansA549 cells were infected with the wild strain or cps1 mutant strain of Cryptococcus neoformans for 3 hours. Total protein was extracted and CD44 was detected by western blot using CD44 monoclonal antibody. It was found that the specific bands appeared in the position of molecular weight 90KD. CD44 expression was not different in wild strain-infection group and mutant strain-infection group.6. Expression of CD44 in mouse lung tissuesFluorescence-immunohistochemical method was used to detect the CD44 expression in the lung tissue of immunosuppressed mice with Cryptococcus neoformans infection. Results showed that CD44 fluorescence concentrated in the alveolar margin in the wild strains-infection group, while CD44 fluorescence is not obvious expression in the cpsl mutant-infection group and the normal group.7. Effects of CD44 inhibitor Bikunin on the association of Cryptococcus neoformans with A549 cellsA549 cell monolayer was pretreated with 40mg/L Bikunin for 1 hour, and then infected with the wild strain and cps1 mutant of Cryptococcus neoformans. The adhesion rate of the fungus to A549 cells was detected. Presuming the relative adhesion rate is 100% in Bikunin untreated and wild strain infected group, it is 55.7% in Bikunin treated and wild strain infected group. They were different significantly (P<0.05). A549 cells relative adhesion rate in Bikunin untreated and treated group that they were infected with cps1 mutations were 8.3% and 6.7% respectively, they had no significant difference (P> 0.05).8. The block effect of Src kinase inhibitor PP2 to Cryptococcus neoformans invasion into A549 cellsA549 cells were infected with the wild strain, cps1 mutant strain and the revertant strains of Cryptococcus neoformans for 3 hours respectively. And 0,10,20,30 mmol/L PP2 were added to cells at same time.The results show that the adhesion rate of Cryptococcus neoformans was reduced in PP2 treated A549 cells in dose-dependent manner. The difference is significant statistically (P< 0.05).Statistical analysisData are expressed as mean ± standard deviation. Comparation of the adhesion and invasion rate between two groups is treated with Mann-Whitney Test. P< 0.05 was considered statistically significant differences. The level of test is alpha=0.05. Data were analyzed using SPSS 13.0 software.Conclusion Among the 306 reports and 3961 cases about cryptococcal meningitits, there were 21% patients with HIV infection,39% with other underlying diseases and 40% patients without underlying diseases. Main underlying diseases in non-HIV patients were tuberculosis (10.46%), liver disease (9.83%), SLE (7.00%), DM(Diabetes 5.26%), Kidney disease (3.16%), lung disease (2.97%), cancer (2.01%). Clinical manifestations of Cryptococcal meningitis at onset were headache and fever in many cases (40.22%). About 60.8% of the cases were reported in males. In the total 3961 cases,1605 cases clearly described their treatment protocols and outcome. Patients who received treatment of amphotericin B (AmB) with 5-flucytosine, AmB with Fluconazole, and the combination of AmB+5-FU+FCZ were 495,411,266 persons and got a total efficiency of 70.1%,70.8%, and 74.8% respectively.Human alveolar epithelial cell line A549 cytoskeletal rearrangement was found after the cells were infected by the wild type strain of Cryptococcus neoformans. The adhesion and invasion rate was significantly higher in the wild type strains-infected group than it is in △ cps1 strain-infected group. The hyaluronidase reduced the adherence ability of wild strain B4500F02 to A549 cells in dose-dependent way. The results above show that capsular HA on Cryptococcus neoformans contributes to its invasion into A549 cells. CD44 expression on A549 cells was found in wild strain-infection group and △ cpsl strain-infected group. And CD44 fluorescence concentrated in the alveolar margin in the wild strains-infection mice lung tissue. HA enzyme treatment, wild strains adhesion invasion rate decreased significantly, showed capsular HA play a role in cryptococcus neoformans adhesion and invasion to A549 cell. CD44 inhibitor Bikunin and Src kinase inhibitor PP2 have block effect on Cryptococcus neoformans invasion into A549 cells. The results above shows that CD44/lipid raft -Src kinase signaling pathway is involved in the process of Cryptococcus neoformans invasion into the alveolar epithelial cells. |
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