Cryptococcus neoformans Csn1201 Is Associated With Pulmonary Immune Responses and Disseminated Infection.

Cryptococcus neoformans is a major etiological agent of fungal meningoencephalitis. The outcome of cryptococcosis depends on the complex interactions between the pathogenic fungus and host immunity. The understanding of how C. neoformans manipulates the host immune response through its pathogenic factors remains incomplete. In this study, we defined the roles of a previously uncharacterized protein, Csn1201, in cryptococcal fitness and host immunity. Use of both inhalational and intravenous mouse models demonstrated that the CSN1201 deletion significantly blocked the pulmonary infection and extrapulmonary dissemination of C. neoformans. The in vivo hypovirulent phenotype of the csn1201Δ mutant was attributed to a combination of multiple factors, including preferential dendritic cell accumulation, enhanced Th1 and Th17 immune responses, decreased intracellular survival inside macrophages, and attenuated blood-brain barrier transcytosis rather than exclusively to pathogenic fitness. The csn1201Δ mutant exhibited decreased tolerance to various stressors in vitro, along with reduced capsule production and enhanced cell wall thickness under host-relevant conditions, indicating that the CSN1201 deletion might promote the exposure of cell wall components and thus induce a protective immune response. Taken together, our results strongly support the importance of cryptococcal Csn1201 in pulmonary immune responses and disseminated infection.

Ribosomal Protein L40e Fused With a Ubiquitin Moiety Is Essential for the Vegetative Growth, Morphological Homeostasis, Cell Cycle Progression, and Pathogenicity of Cryptococcus neoformans.

Ubiquitin is a highly conserved protein required for various fundamental cellular processes in eukaryotes. Herein, we first report the contribution of the ubiquitin fusion protein Ubi1 (a ubiquitin monomer fused with the ribosome protein L40e, Rpl40e) in the growth and pathogenicity of Cryptococcus neoformans. UBI1 deletion resulted in severe growth restriction of C. neoformans, whose growth rate was positively correlated with UBI1 expression level. The growth defect of the ubi1Δ strain could be closely associated with its morphological abnormalities, such as its reduced ribosome particles. In addition, the ubi1Δ mutant also displayed increased cell ploidy, cell cycle arrest, and decreased intracellular survival inside macrophages. All these phenotypes were reversed by the reconstitution of the full-length UBI1 gene or RPL40a domain. Mouse survival and fungal burden assays further revealed a severely attenuated pathogenicity for the ubi1Δ mutant, which is probably associated with its reduced stress tolerance and the induction of T-helper 1-type immune response. Taken together, Ubi1 is required for maintaining the vegetative growth, morphological homeostasis, cell cycle progression, and pathogenicity in vivo of C. neoformans. The pleiotropic roles of Ubi1 are dependent on the presence of Rpl40e and associated with its regulation of cryptococcal ribosome biogenesis.

microRNA-625 inhibits tumorigenicity by suppressing proliferation, migration and invasion in malignant melanoma.

Dysregulated microRNA (miR)-625 expression has been observed in several kinds of cancer. MicroRNAs are important factors in the development and progression of malignant melanoma, though the clinical significance and function of miR-625 in human malignant melanoma remain unclear. Levels of miR-625 expression were therefore determined in 36 pairs of malignant melanoma and adjacent non-tumor tissue using qPCR. The effects of miR-625 dysregulation on malignant melanoma cell proliferation, wound healing, migration and invasion in vitro and tumorigenicity in vivo were investigated using CCK-8, transwell assays, and a nude mouse subcutaneous tumor model. Bioinformatics analysis and luciferase reporter system were used to predict and confirm the target gene of miR-625. miR-625 levels were frequently decreased in malignant melanoma. Ectopic expression of miR-625 suppressed proliferation, wound healing, migration, and tumorgenicity in malignant melanoma. Moreover, miR-625 acted, at least in part, by suppressing potential target SOX2. These results show that miR-625 is a tumor suppressor that inhibits the development and progression of malignant melanoma, which suggests miR-625 is potentially a new diagnostic marker and therapeutic target of malignant melanoma.

Complex Roles of Annexin A2 in Host Blood-Brain Barrier Invasion by Cryptococcus neoformans.

INTRODUCTION: Fungal transversal across the brain microvascular endothelial cells (BMECs) is the essential step for the development of cryptococcal meningoencephalitis. Annexin A2 (AnxA2) is an important signaling protein involved in several intracellular processes such as membrane trafficking, endocytosis, and exocytosis. AIM: To investigate the roles and mechanism of AnxA2 during cryptococcal transversal of BMECs. RESULTS: Cryptococcus neoformans infection initiated upregulation of AnxA2 in mouse BMECs. Blockade with anti-AnxA2 antibody led to a reduction in fungal transcytosis activity but no change in its adhesion efficiency. Intriguingly, AnxA2 depletion caused a significant increase in fungal association activity but had no effect on their transcytosis. AnxA2 suppression resulted in marked reduction in its partner protein S100A10, and S100A10 suppression in BMECs significantly reduced the cryptococcal transcytosis efficiency. Furthermore, AnxA2 dephosphorylation at Tyr23 and dephosphorylation of downstream cofilin were required for cryptococcal transversal of BMECs, both of which might be primarily involved in the association of C. neoformans with host cells. CONCLUSIONS: Our work indicated that AnxA2 played complex roles in traversal of C. neoformans across host BMECs, which might be dependent on downstream cofilin to inhibit fungal adhesion but rely on its partner S100A10 to promote cryptococcal transcytosis.

Systemic Review of Published Reports on Primary Cutaneous Cryptococcosis in Immunocompetent Patients.

Primary cutaneous cryptococcosis (PCC) has been confirmed as a distinct clinical entity with secondary cutaneous cryptococcosis from systematic infection since 2003. Although it has been confirmed as a distinct clinical entity, little has progressed on PCC in immunocompetent hosts compared to their immunocompromised counterpart. We reviewed the literature on cases of PCC in immunocompetent patients from 2004 to 2014, and 21 cases from 16 reports were identified. Males are more likely to develop PCC infections, with a ratio of 17:4 male to female. These patients were found to be almost all senior population except for patients from Asia. Asymptomatic or moderate itching manifesting in a painful nodule is the most common presentation, although there is no typical clinical manifestation recorded. Upper limbs are the most common site of infection, accounting for 71.4 % of all patients. Of the 12 identified isolates, 6 strains are identified as C. neoformans, 5 as C. gattii, and 1 as C.laurentii. Fluconazole was used in 10 cases; however, only 80 % of the 10 cases could confirm that fluconazole was effective in clearing the infections. Interestingly although not approved as a treatment option, Itraconazole was effective in the seven cases it was used to treat cryptococcosis, with a dosage range of 100-400 mg/d and duration from 3 to 6 months. Even though the prognosis of these patients was generally good, more data are need to determine which antifungal azole is the better treatment option and whether primary skin infections could disseminate to systematic infection.

Cryptococcosis in kidney transplant recipients in a Chinese university hospital and a review of published cases.

BACKGROUND: Cryptococcosis is a severe fungal infection with a high mortality rate among solid-organ transplant recipients. Today, China is among the countries performing the most kidney transplants worldwide, however data on the association of cryptococcosis with kidney transplantation in mainland China remain scarce and fragmented. METHODS: We retrospectively analyzed cases of culture-confirmed cryptococcosis following kidney transplantation that have occurred at our hospital and reviewed the published cases in China over the last 30 years. RESULTS: Cryptococcosis in kidney transplant recipients was mainly caused by Cryptococcus neoformans var. grubii VNI strains and occurred most frequently in patients aged 41-50 years (37.9%, 11/29). The average time to infection after kidney transplantation was 5.16 ± 3.97 years. The clinical manifestations were found to be diverse, with slight to moderate headache and fever, meningeal irritation, and high cerebrospinal fluid pressure being relatively common. Physicians should be alert to these symptoms among kidney transplant recipients. CONCLUSIONS: Cryptococcosis is a serious infection among kidney transplant recipients in mainland China. It has unique characteristics, such as a relatively long time to onset after kidney transplantation, and diverse clinical manifestations. Treatment with intrathecal injection of amphotericin B is considered effective for central nervous system involvement. The findings of this study also highlight the urgent need for multicenter, prospective, and multidisciplinary clinical studies and education on cryptococcosis in kidney transplant recipients in China.

Design, synthesis and antifungal activity of novel triazole derivatives containing substituted 1,2,3-triazole-piperdine side chains.

Due to increasing incidence of invasive fungal infections and severe drug resistance to triazole antifungal agents, a series of novel antifungal triazoles with substituted triazole-piperidine side chains were designed and synthesized. Most of the target compounds showed good inhibitory activity against a variety of clinically important fungal pathogens. In particular, compounds 8t and 8v were highly active against Candida albicans and Cryptococcus neoformans with MIC values in the range of 0.125 µg/mL to 0.0125 µg/mL. They represent promising leads for the development of new generation of triazole antifungal agents. Molecular docking studies revealed that the target compounds interacted with CACYP51 mainly through hydrophobic and Van der Waals interactions.

Proteomic analysis of human umbilical vein endothelial cells incubated with Cryptococcus neoformans var. neoformans.

Cryptococcus neoformans is a medically important fungus and can infect all the organs of the body. As vascular endothelial cell is an important target for C. neoformans to penetrate any organs, the differential protein expression of human umbilical vascular endothelial cell (HUVEC) after incubating with C. neoformans may be the key to penetration. The proteins of HUVECs incubated with C. neoformans and normal HUVECs were collected and purified. After two-dimensional electrophoresis, the differential protein expression was identified by matrix-assisted laser desorption/ionisation mass spectrometry. The mRNA levels of some proteins were measured by real-time PCR. Three proteins were found significantly overexpressed in HUVECs incubated with C. neoformans, and nine other proteins were downregulated. The mRNA levels of S100A10 and peroxiredoxin I fluctuated with the protein levels. These results suggested that the expressions of peroxiredoxin I and S100A10 were regulated during the process of invasion of HUVECs by C. neoformans. We hypothesise that these proteins take part in the modifications of HUVEC cytoskeleton and the tolerance to oxidative stress, which may affect the process of invasion by C. neoformans.

Efficacy of intravenous amphotericin B-polybutylcyanoacrylate nanoparticles against cryptococcal meningitis in mice.

Amphotericin B deoxycholate (AmB), a classic antifungal drug, remains the initial treatment of choice for deep fungal infections, but it is not appropriate for treatment of cryptococcal meningitis due to its inability to pass through the blood-brain barrier (BBB). We examined the efficacy of amphotericin B-polybutylcyanoacrylate nanoparticles (AmB-PBCA-NPs) modified with polysorbate 80 that had a mean particle diameter less than 100 nanometers (69.0 ± 28.6 nm). AmB-PBCA-NPs were detected in the brain 30 minutes after systemic administration into BALB/c mice and had a higher concentration than systemically administered AmB liposome (AmB-L, P < 0.05); AmB was not detected in the brain. Following infection for 24 hours and then 7 days of treatment, the survival rate of mice in the AmB-PBCA-NP group (80%) was significantly higher than that of the AmB (0%) or AmB-L (60%) treatment groups. Fungal load was also lower when assessed by colony-forming unit counts obtained after plating infected brain tissue (P < 0.05). Our study indicates that AmB-PBCA-NPs with polysorbate 80 coating have the capacity to transport AmB across the BBB and is an efficient treatment against cryptococcal meningitis in a mouse model.

S100A10 downregulation inhibits the phagocytosis of Cryptococcus neoformans by murine brain microvascular endothelial cells.

Cryptococcus neoformans invades the central nerve system through endocytosis by the vascular endothelia. S100A10 interacts with Ca(2+) channel proteins in the vascular endothelia and regulates the filamentous actin network. We hypothesized that S100A10 was involved in the pathogenesis of cryptococcosis and sought to investigate here the effect on the phagocytosis and growth of C. neoformans of S100A10 downregulation by small interfering RNA in murine brain microvascular endothelial cell (MBMECs). We found that S100A10 downregulation significantly reduced the intracellular Ca(2+) concentration (P < 0.05). Additionally, suppression of S100A10 expression was associated with significantly reduced rate of phagocytosis and budding of C. neoformans. The capsule of intracellular C. neoformans also became thicker after S100A10 was knocked down. The findings demonstrated that knockdown of S100A10 in MBMEC could attenuate the growth of intracellular C. neoformans. We inferred that the S100A10 may play an important role in transpenetration of C. neoformans across the vascular endothelia.