Chinese Journal of Mycology

The morphological diversity and virulence factorssecretory activity of Candida parapsilosis

LI Xiao-ling, JIN Fang, CHEN Yu-ru, MU Li-li, LIU Tao-hua, LV Qian, KANG Ying-qian

2015, 10(2): 65-69.

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Objective Compared the colonies and cells morphology and the secreted aspartic proteinase (SAP) enzyme activity of different clinical isolates of Candida parapsilosis,and elucidated the morphological diversity and the differences of virulence of the pathogenic fungi.Method Seven clinical isolates of Candida parapsilosis were cultured respectively on different solid media,and the morphology characteristics of colonies and cells were observed and recorded.Strains were also cultured on solid medium of bovine serum albumin (BSA) at 30 ℃ for determining the secretory activity of the virulence factors (SAP) by observing the proteolytic halos.Result Diverse colonies and phenotypes of the above strains were checked under several sorts of conditions—smooth,crepe and crater in various shades of stained color,and the ability of filamentation were also different from each other among the tested strains.In addition,the sizes of proteolytic halos were various from 2.55 cm to 1.55 cm.Conclusion There are diverse phenotypes which can be shown for the clinical isolates of Candida parapsilosis,and the SAP enzyme activities of them are distinct,which may imply the significant virulent differences among the strains.

A study of function in Candida albicans mutant ORF19.2500

NIU Li-da, CHEN Xiao-qing, ZHAO Jing, WU Jian-hua, ZHENG Qing-hu

2015, 10(2): 70-74.

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Objective For searching some genes which impacted the formation of Candida albicans biofilm.Then we screened of 50 Candida albicans mutants.So as to identify potential pathogen mechanism of Candida albicans.Methods Screening 50 Candida albicans mutants in the method of cultivating biofilm in vivo.Biofilm was quantified by XTT.Then the mutants of growth rate and hyphal formation were analyzed.Results It was defected for Candida albicans mutant orf19.2500 to form biofilm which was quantified by XTT.The mutant grows slowly,which was quantified by growth curve and spot assay.Then we find the mutant can not form hyphal in liquid spider media,while it can normally form hyphal in liquid YPD plus 10% fetal bovine serum media.Conclusion The Candida albicans mutant orf19.2500 can not utilize liquid media which impacted morphogenetic switching and the biofilm formation.

Analysis of relationship between transcription factor RPN4 and drug susceptibility of Candida albicans

XU Hong-tao, TANG Ruo-yu, QU Ying-ge, WANG Xiao-juan, JIANG Yuan-ying, CAO Yong-bing, YAN Tian-hua

2015, 10(2): 75-79.

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Objective To study the influence on the drug susceptibility of Candida albicans after transcription factor RPN4 was knocked out and the mechanism how this transcription factor regulates the drug susceptibility in Candida albicans.Methods The broth microdilution method was used to determine the minimal inhibitory concentrations (MICs) of 30 strains transcription factor knocked out Candida albicans .The drug susceptibility was reassured by spot assay and growth curve.The total RNA of parental strain SN250 and susceptible strains were extracted and the expression of multidrug resistance genes CDR1 and MDR1 was determined by real-time PCR,and the pump ability was examined by the ability of pump out rhodamine 6G.Results Compaired to the parental strain SN250,transcription factor RPN4 knocked out strain was more susceptible to fluconazole.The expression of CDR1 and MDR1 was lower than SN250 and the pump ability was also weaker than SN250.Conclusion The knockout of transcription factor PRN4 results susceptible to fluconazole in Candida albicans,and this transcription factor may be involved in the regulation of drug susceptibility in Candida albicans.

Identification and knocking-out of transcription co-activation factor gene MBF1 in Cryptococcus neoformans

ZHOU Zhao-jing, MENG Yun-fang, ZHAO Jing-yu, SANG Jun-jun, ZHANG Chao, FA Zhen-zong, FANG Wei, LIAO Wan-qing

2015, 10(2): 80-83.

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Objective To construct the mutant strain of transcriptional co-activated factor Mbf1 in Cryptococcus neoformans.Methods Amplified the homologous recombination cassette containing report gene NEO and flanking DNA fragments of MBF1 via overlap PCR,which was then transformed into Cryptococcus neoformans through gene gun.The positive transformants was screened by diagnostic PCR and then confirmed by DNA sequencing.Results The target gene MBF1 was successfully knocked out in Cryptococcus neoformans.Conclusion Construction of the mutant strain would lay the foundation for further pathogenetic studies research of transcriptional co-activated factor Mbf1 in Cryptococcus neoformans.

Gene expression profiling analysis of mouse brain microvascular endothelial cells incubated with glycuronoxylomannan of Cryptococcus neoformans

NIE Shu, LI Ping, ZHU Hong-mei, WEN Hai

2015, 10(2): 84-87.

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Objective To explore whether cryptococcal capsular polysaccharide GXM can influence the gene expression of cerebrovascular endothelial cells and providing clues for further study of the molecular mechanism of cryptococcus addicted to central.Methods The Mouse 12×135K Gene Expression Array manufactured by Roche NimbleGen was used to identify the differentially expressed genes of bEnd.3 cells after co-culturing with GXM in different levels.TopGO method is used to analyze the differentially expressed genes.We analyze differences in gene function with the GO classification function and find information about the literature data.The changes of some important genes were found and then validated by RT-PCR.Results After comparison,we got 698 differentially expressed genes,including 402 up-regulated and 296 down-regulated in the GXM (90 μg/mL) group and 985 differentially genes,including 421 up-regulated and 564 down-regulated in the GXM (180 μg/mL) group.Cryptococcal capsular polysaccharide GXM can regulate vascular endothelial cell gene expression.Genes relating to tight junction,cell transduction,cytoskeleton,metabolism of cell membrane componentsetc have been impacted.Considering literature and GO analysis results,we selected gene PIK3C2G and gene ADAMDEC1 and validated their expression levels by RT-PCR.The results were in line with the microarray data.Conclusions Cryptococcal capsular polysaccharide GXM can influence the gene expression of cerebrovascular endothelial cells Increase in genetic PIK3C2G,ADAMDEC1 may relate to the ability of cryptococcus invading blood brain barrier.

To establish and optimize the genetic transformation technology of Penicillium marneffei by Agrobacterium tumefaciens-mediated transformation

LI Xin-lei, LI Xin, CAO Cun-wei

2015, 10(2): 88-91.

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Objective To construct and optimize the condition of Agrobacteriumtumefaciens-mediated transformattechology in Penicillium marneffei.Method A.tumefaciens transformed by Calcium chloride with pDHt/SK::pyrG plasmids insert pyrG gene into SPM4 (pyrG^-,niaD^-),medium without uracil screening positive transformat,Using PCR verification restructuring.It aimed at finding out the optimal protocol by changing some important effecting factors including A.tumefaciens strains,concentration,transformat medium,co-cultivation temperature,period,AS.The positive transformant growed well in the media without uracil,and PCR analysis showed that T-DNA was inserted into the transformants.Result The transformation efficiency was about 300 transformants/10⁶ spores by optimizing.The optimal condition for AGL-1,concentration of A.tumefaciens OD₆₀₀=0.8,the AS concentration of 200 μmol/L,without nitrocellulose filters,co-culture 48 h in 25℃.Conclusion A.tumefaciens-mediated transformattechology in P.marneffei.was successfully established,simplified and optimized.The method can be used for PM gene function research.

Construction and application of blood-brain-barrier model in vitro in cryptococcal infection

MENG Yun-fang, FA Zhen-zong, FANG Wei, ZHOU Zhao-jing, YI Jiu, GU Ju-lin, LIAO Wan-qing

2015, 10(2): 92-95.

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Objective To establish an in vitro Blood-Brain-Barrier (BBB) model,which was then applied to evaluate the capacities of different cryptococcal strains in crossing Blood-Brain-Barrier.Methods Mouse brain endothelium cell bEND.3 was used to construct the Blood-Brain-Barrier model in vitro.In order to test its feasibility,we next examined the efficacy of several cryptococcal strains with different serotype or gene mutation in central nervous system (CNS) invasion.Results The transendothelial electrical resistance (TEER) measurement suggested that BBB model was constructed successfully.Compared with C.neoformans serotype A strain H99,JEC21 (C.neoformans serotype D) displayed a reduced efficacy in BBB invasion while the negative control strain (non pathogenic Saccharomyces cerevisiae strain Y187) with the lowest efficacy.Furthermore,the ure1 mutant (urease deletion) and the cap59 mutant (capsule deletion) showed a 50% and 80% decrease of their efficacy in crossing BBB compared to their parent strain H99,respectively (P<0.001).Conclusion The in vitro model of cryptococcal CNS infection was established successfully.Serotype,urease,and capsule was essential for C.neoformans to cross the Blood-Brain-Barrier.

In vitro susceptibility test of clinical isolates of Pseudallescheria boydii complex to four anti-fungus agents

WANG Hong, WAN Zhe, YU Jin

2015, 10(2): 96-99.

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Objective To determine the in vitro susceptibility of Pseudallescheria boydii complex to voriconazole(VRC),posaconazole(POS),itraconazole(ITC)and amphotericin B(AMB).Methods The MICs of VRC,POS,ITC and AMB were determined according to M38-A2 methods in nine isolates of P.boydii,six isolates of P.ellipsoidea,one isolate of P.angusta,one isolate of P.fusoidea and six isolates of S.apiospermum.Results The MIC range of VRC and POS were 0.25-1 μg/mL and 2-4 μg/mL.ITC had higher MIC range which was 2->16 μg/mL against P.ellipsoidea and S.apiospermum than P.boydii,P.angusta,and P.fusoidea(2-4 μg/mL).The MIC range of AMB against all the species was 4->16 μg/mL.Conclusion VRC,POS,ITC and AMB have the similar susceptibility patterns against various types of Pseudallescheria boydii complex.Since ITC was more sensitive to P.boydii,P.angusta and P.fusoidea than P.ellipsoidea and S.apiospermum in our study,VRC is the most sensitive drug against all the types and than POS and AMB is the worst one among the four drugs.

Detection of fungal infection from 458 balanoposthitis patients and antibiotic susceptibility test

CAO Wei-sheng, CHEN Shao-nan, WEI Fang-quan, CHEN Wei-hua, ZHANG Feng-xia

2015, 10(2): 100-103.

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Objective To investigate the infection and antibiotic susceptibility of fungi in the patients with balanoposthitis in Zhaoqing area.Methods Microscopy,CHROM agar candida,ATB ID32C of France Bio-Merieux Company and Rosco Disk diffusion method were applied to examine and culture and identify candida strains and for yeast susceptibility testing.Results 153 cases were positive fungal microscopy examination in 458 patients.There were 157 strains candida by culture and identify and the positive detection rate was 33.4%.116 of 157 isolates (73.9%) were albicans,41 of 157 isolates (26.1%) were non-C.albicans.17 cases were cultured positive for candida in healthy control group and the positive detection rate was 14.2%.6 of 17 isolates (35.3%) were albicans,11 of 17 isolates (64.7%) were non-C.albicans.The detection rate of candida culture and the constituent rate of albicans in patient group were higher than those in healthy control group,the difference has statistical significance (χ²=16.95,χ²=10.72,P<0.05).The susceptibility rate of isolated pathogenic strain in nystatin and ketoconazole were all more than 90%,were 97.5% and 93.0%.The rate in fluconazole was 79.6%,while the lowest susceptibility rate was terbinafine (33.8%).Conclusion Candida infection is the most important pathogenetic factor for balanoposthitis and the main species of pathogenetic yeasts is albicans in Zhaoqing area.Pathogenic candida have a higher drug susceptibility rate to polyene drugs and have different degree of cross resistance to azole drugs.There are differences among the antibiotic sensitivity spectrums of various candida.

The antifungal effect of 5-aminolevulinic acid photodynamic therapyat different concentrations on C.albicans

XU Hui, CHEN Hong-xia, ZOU Xian-biao

2015, 10(2): 104-107.

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Objective To contribute theoretically to the selection of the optimal concentration of the photosensitizer through the determination of the level at which C.albicans generate protoporphyrin IX (PpIX) under different concentrations of the photosensitizer and of the bacteriostatic rate of 5-amino ketones pentanoic acid photodynamic therapy (5-ALA PDT).Methods C.albicans suspension was prepared and incubated with ALA in the dark.The generation of PpIX was observed under a laser confocal microscope.The inhibitory rate of 5-ALA PDT on C.albicans was determined by MTT method.Results After dark incubation,C.albicans and ALA produced fluorescent PpIX.ALA concentration was positively correlated with the intensity of PpIX.When drug concentration reached 300mmol/L,the intensity of PpIX strength ceased to increase.There was no fluorescent material in control group.Conclusions The inhibitory effect of ALA PDT on C.albicans was closely related to the concentration of the photosensitizer,which provides data for treatment of C.albicans infection.

CT features of pulmonary fungal infection mimicking tumor

ZHOU Ying, FAN Li, YANG Zhen-yue, LI Hua, LIU Shi-yuan

2015, 10(2): 108-112.

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Objective To evaluate the CT features of patients with pulmonary fungal infection mimicking thoracic tumor and to increase awareness for both clinicans and radiologists to avoid misdiagnosis.Methods The clinical and CT findingsfrom 53 patients with pulmonary fungal infection mimicking tumor were reviewed retrospectively.Results Of 53 patients with pulmonary fungal infection,32 (60.4%) were cryptococcosis,21 (39.6%) were aspergillosis,29 (54.7%) were single lesion and 24 (45.3%) were multiple lesions.CT findings included nodules 30 (56.6%),mass 10 (18.9%),or both 13 (24.5%).The median lesion size was 2.5 cm (range 1.0-7.0 cm).The lesions were in 53 patients,32 (60.4%) were adjacent to the pleura,21 (39.6%) were in other places.There was no statistically significant difference in characteristics,shape,lobulation,spiculation,halo sign and other additional lesions such aspatchinvasiveinfection,miliary nodules,lyphadenopathy,pleural thickening and pleural effusion (P>0.05).There was statistically significant difference in location (χ²=10.633),margin (χ²=4.828) and cavity (χ²=18.702)(P<0.05).Conclusion Cryptococcosis and aspergillosis were more common in pulmonary fungal infection mimicking tumor.The CT findings were characteristic in two different fungal infection.The differential diagnosis from pulmonary malignancy should combine CT findings with clinical.

The efficacy and safety of sertaconazole cream on infant dermatitis candidiasis

CAI Qing, DAI He-jun, GAO Zhiqing, YANG Hong, REN Xiang-yu, ZHANG Min, YANG Lian-juan

2015, 10(2): 115-117.

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Objective To investigate the clinical efficacy and safety of sertaconazole cream on infant suffering from dermatitis candidiasis.Method Twenty-six patients with clinical and mycological diagnosis of dermatitis candidiasis were enrolled.Patients were treated with twice daily applications of sertaconazole cream for 14 days and were followed-up for 2 further weeks.Results Twenty-two Candida albicans and four non-Candida albicans were isolated in twenty-six infants.The treatment evaluation after 7 days treatment showed that more than 75% of average signs decreased from 6 to 1.35.There was a total clinical cure in 57.69%,improvement in 92.3% and mycological cure in 88.46% after 7 days treatment.At the time of 14 days follow-up,there was a total clinical and mycological cure in 92.3%.91.67% cure infants had not recurred at the end of the follow-up period.There was side effect in only one patient.Conclusions Based on its safety and effectiveness,sertaconazole cream may be considered a new alternative for infant dermatitis candidiasis treatment.

New advance in the study of the molecular biological identification and typing of Prototheca

LI Wen-jing, LIU Jin-yan, SHI Ce, WANG Ying, SUN Jing-yong, XIANG Ming-jie

2015, 10(2): 126-128.

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The molecular biology method based on gene sequencing which is objective,highly reproducible and so on,is considered the "gold standard" of Prototheca's molecular identification.To summarize the new molecular biology methods and techniques applied to Prototheca's identification and typing,such as real-time quantitative PCR (RT-PCR) technology,single strand conformation polymorphism (SSCP) analysis,PCR-restriction fragment length polymorphism (RFLP)analysis,high resolution melting (HRM) analysis,mass spectrometry(MS) analysis.

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