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Comparative analysis of pathogenicity of Cryptococcus neoformans serotypes A, D and AD in murine cryptococcosis

Comparative analysis of pathogenicity of Cryptococcus neoformans serotypes A, D and AD in murine cryptococcosis
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  Comparative analysis of pathogenicity of  Cryptococcus neoformans  serotypes A, D and AD inmurine cryptococcosis F.Barchiesi a, *,M.Cogliati b ,M.C.Esposto b ,E.Spreghini a ,A.M.Schimizzi a ,B.L. Wickes c , G. Scalise a , M.A. Viviani b a Istituto di Malattie Infettive e Medicina Pubblica, Universita` Politecnica delle Marche, AziendaOspedaliera Umberto I 8 , Via Conca 60020 Torrette di Ancona, Ancona, Italy  b Istituto di Igiene e Medicina Preventiva, Universita` degli Studi di Milano, IRCCS Ospedale Maggiore,Milano, Italy  c Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA Accepted 21 July 2004Available online 16 September 2004 KEYWORDS Cryptococcusneoformans ;Serotype;Diploid;Haploid;Hybrid Abstract  Objectives . To characterize the pathogenicity of 15 strains of  Crypto-coccus neoformans  belonging to several serotype/mating type allele patterns (D a ,D a , A a , A a , A a /D a  and D a /A a ) in experimental models of murine cryptococcosis. Methods . CD1-infected mice were examined for survival and fungal loads in eitherbrain or lung during the course of infection. Results . All strains, with the exception of one D a  strain, produced melanin in vitro.Similarly, all strains were encapsulated and produced phospholipase. When CD1 micewere challenged intravenously (i.v.) with 5 ! 10 5 CFU/mouse and observed for 60days post-infection, a significant variation of mortality rate was observed amongmice infected with different strains. A a  and A a /D a  strains all produced 100%mortality within the study period with mean survivals significantly shorter than thoseof mice infected with strains belonging to any other allele type ( P  ! 0.0001). A widerange of pathogenicity was shown by haploid and diploid strains presenting D a allele.This finding was confirmed by an intranasal model of challenge. To investigate theprogression of infection, the mice were challenged i.v. with 5 ! 10 4 CFU/mouse andtissue burden experiments (brain and lung) were performed on days 6 and 12 post-infection. Only the mice infected with A a  and A a /D a  strains showed a  O 1 log 10 increase of CFU/g in both tissues throughout the study period. Conclusions . Our results suggest that the presence of the A a  mating type allele ineither haploid or diploid strains is correlated with virulence, while the presence ofthe A a  or D a  allele in haploid strains is associated with moderate or no virulence. Journal of Infection (2005)  51 , 10–$30.00 Q 2004 The British Infection Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.jinf.2004.07.013* Corresponding author. Tel.: C 39-071-5963467; fax: C 39-071-5963468. E-mail address: (F. Barchiesi).  Finally, either haploid or diploid strains presenting D a  allele vary in virulence. Q 2004 The British Infection Society. Published by Elsevier Ltd. All rights reserved. Introduction The yeast  Cryptococcus neoformans  is an encap-sulated fungus that can cause infection in bothimmunocompetent and immunocompromisedindividuals. Its incidence has dramaticallyincreased during the last two decades becauseof the AIDS epidemic, although impaired cell-mediated immunity in general represents themain predisposing factor for development ofcryptococcosis. Meningoencephalitis is the mostfrequent clinical presentation, although anyorgan can be affected. Cryptococcus neoformans  has two mating types, MAT  a  and  MATa , and two varieties, var.  neofor-mans  and var.  gatti , which can be further subdivided into four serotypes (A, B, C, D). 1 Anadditional class, serotype, AD, has been shown tobe diploid and results from serotype A and Dhybridization. 2,3 A relationship between matingtype and virulence, with  MAT  a  found to be morevirulent than  MATa  strains, was reported by KwonChung et al. 4 Recent data also document a differentdegree of virulence among serotypes and matingtypes. 5–7 In an attempt to further elucidate the relation-ship between mating type, serotype, and virulence,representative haploid and hybrid strains of each  C.neoformans  serotype were compared in terms ofvirulence factors and pathogenicity in an exper-imental murine model of cryptococcosis. Materials and methods Sources of isolates and characterization A total of 15 strains of  C. neoformans  wereselected in order to have representative strainsof each mating type allelic pattern (Table 1).Eleven strains were isolated from humans, twofrom the environment and two others werelaboratory strains. All the isolates were pre-viously serotyped by slide agglutination with theCrypto Check kit (Iatron Labs., Tokyo, Japan)and genotyped by PCR fingerprinting. 8 Matingtype was determined by PCR of  MFa ,  MF  a  and STE20   alleles. 3 DNA content was measured byflow cytometry. 9 Analysis of virulence factors Strains were grown on YPD agar (1% yeast extract,2% peptone, 2% dextrose, 2% agar) for 24 h at 30  8 Cbefore each assay. Phospholipase activity wastested by inoculating 1 ! 10 8 yeast/ml on egg yolkagar. 10 Cultures were observed for formation of aprecipitate around the colony for up to 14 days at30  8 C. Melanin production was tested by dropping1  m l of yeast suspension (1 ! 10 8 ml K 1 ) onto L-DOPAagar using a Steer inoculator. 11 The development ofa dark brown colony within 24–48 h at 30  8 Cindicated a positive reaction. Capsule formationwas tested on low iron medium. 11 Plates wereinoculated by dropping 1  m l of yeast suspension (1 ! 10 8 ml K 1 ) onto plates using a Steer inoculator andincubating for 7 days at 25  8 C. The capsule was Table 1  List of the  Cryptococcus neoformans  strainsstudiedStrain a Mating typeallelic pattern b Source c H99 d A a  CSF (HIV K )UA1993 A a  CSF (HIV K )IUM96-2828 e A a  SoilIUM99-3617 f A a  CSF (HIV K )JEC20 g D a  LaboratoryIUM99-5509 D a  CSF (HIV C )JEC21 g D a  LaboratoryUA477 D a  Blood (HIV C )UA491 D a  Blood (HIV C )UA4223 D a  CSF (HIV K )CBS132 h A a /D a  Peach juiceUA486 A a /D a  CSF (HIV C )UA1094 A a /D a  CSF (HIV C )UA2341 A a /D a  CSF (HIV C )UA2715 A a /D a  CSF (HIV C ) a IUM, Istituto di Igiene e Medicina Preventiva, Universita`degli Studi, Milano, Italy; NIH, National Institutes of Health,Bethesda, MD, USA; CBS, Centraalbureau voor Schimmelcul-tures, Baarn, The Netherlands; UA, Universita` degli Studi,Ancona, Italy. b Mating type was determined by PCR of  MFa ,  MF  a ,  STE20a ,and  STE20  a  genes specific for serotype A and D. c CSF, cerebrospinal fluid. d Strain kindly supplied by J. Perfect, Duke University,Durham, NC, USA. e Viviani et al., Med Mycol 2001; 39, 383–6. f Viviani et al. Emerg Infect Dis 2003; 9, 1179–80. g Kwon Chung et al. Infect Immun 1992; 60, 602–5. h Sanfelice  Cryptococcus neoformans  type strain. Pathogenicity of  Cryptococcus neoformans  ser. A, D and AD 11  visualized under the microscope in a suspension ofIndia ink. Animal studies A murine model of systemic cryptococcosis wasestablished in CD1 male mice (weight, 25 g; CharlesRiver Laboratories, Calco, Italy) by intravenousinjection of viable yeast cells grown overnight inbrain-heart infusion broth. In survival studies, themice were challenged with approximately 5 ! 10 5 CFU/mouse, using 10 animals per each group.The mice were observed throughout day 60, anddeaths were recorded daily. In tissue burdenstudies, the mice were challenged with approxi-mately 5 ! 10 4 CFU/mouse. After 6 and 12 dayspost-infection, five mice in each group wereeuthanized by CO 2 -induced asphyxia, and thenumber of viable CFU per gram of brain and lungsof each animal was determined by quantitativeplating of organ homogenates on Sabouraud dex-trose agar (SDA) plates. A selected group of isolateswere also tested in mice by using a model ofpulmonary cryptococcosis. Briefly, mice wereanesthetized with ketamine. A 6  m l droplet contain-ing approximately 1 ! 10 7 CFU/mouse was placedon the nares while the diaphragm was compressed.The diaphragm was released and the mouse inhaledthe droplet. Either in the intravenous or pulmonarymodel of infection, the CFU/mouse was confirmedby plating the final inoculum onto SDA plates.Animal experiments were conducted with theapproval of the University of Ancona ethicscommittee. Statistical analysis Survival data from the mouse experiments wereanalyzed by using a Kruskal–Wallis test. Student’s  t test was used in evaluating the yeast colony countsfrom mice. A  P  ! 0.05 was considered significant. Results All strains, with the exception of JEC20 (serotype D MATa ), produced melanin (Fig. 1A). Similarly, allstrains were encapsulated (data not shown) andproduced phospholipase (Fig. 1B). Results of survi-val studies are presented in Table 2. The A a  strains,UA1993 and H99, showed 100% mortality betweenday 26 and 29 post-infection, respectively ( P  Z 0.5).There was a marked difference in the survival ofmice infected with the 4 serotype D  MAT  a  isolates.Only UA4223 yielded 100% mortality within thestudy period. Mortality among the other 3 isolatesranged from 10 to 70%. A clear rank order ofpathogenicity within this group of strains was foundwith UA4223 O JEC21 O UA477 y UA491. None of themice infected with JEC20 (serotype D  MATa )succumbed within the study period. Although 40%of mice infected with the other serotype D  MATa (IUM99-5509) died, the difference in percentage ofmortality caused by the two strains did not reach astatistical significance ( P  Z 0.055). A mortality of 40and 60% resulted from infection with the twoserotype A  MATa  isolates, IUM96-2828 and IUM99-3617, respectively ( P  Z 0.2).For the hybrid strains, the three A a /D a  typescaused 100% mortality within 60 days (range 25–45days). The survival of mice infected with UA 486,but not with CBS 132, was significantly longer thanthat observed in mice infected with UA 1094 ( P  Z 0.03).Strains presenting the A a /D a  mating type pat-tern caused 30% (UA2715) and 90% (UA2341)mortality within the study period ( P  Z 0.003).Fig. 2 shows cumulative survivals of miceinfected with all strains grouped by mating type.Only A a  and A a /D a  types yielded 100% of mortalitywithin the study period. Mortality of the other typesranged from 20% (D a  types) and 60% (A a /D a ). Nodifference was found in the survival rate betweenthe groups of mice infected with A a  and A a /D a types ( P  Z 0.1), while both types were found to besignificantly more pathogenic than the other fourmating types ( P  ! 0.0001). Similarly, no differenceswere found in the survival rates among the groupsof mice infected with A a /D a , D a  and A a  types ( P  ranging from 0.1 to 0.5). While both A a /D a  and D a types were found to be significantly more patho-genic than D a  types ( P  Z 0.01 and 0.03, respect-ively), the survival rate of mice infected with A a types did not differ with that of mice infected withD a  types ( P  Z 0.165).Since we observed a wide virulence variationamong D a types, these isolates were also tested in a Figure 1  Analysis of virulence factors. (A) Melaninproduction on L-DOPA agar. (B) Phospholipase productionon egg yolk agar. Negative controls,  Candida lusitaniae and  Candida krusei , are the last two samples in the rightbottom of the plates both in figures A and B. F. Barchiesi et al.12  pulmonary model of murine cryptococcosis. Theresults are shown on Fig. 3. Again,  C. neoformans 4223 was the most pathogenic strain showing 60% ofmortality on day 60, while the percent of mortalityfor the other three strains ranged from 10 to 50%( P  ! 0.05, 4223 vs. 491 and JEC 21).To investigate the progression of fungal infec-tions caused by strains belonging to differentmating types, six strains were selected for tissueburden experiments (Table 3). The mice infectedwith H99 (A a ) showed the highest increase of CFU/gin the brain tissue between day 6 and 12 post-infection ( P   ranging from ! 0.0001 to 0.038 vs. allstrains) followed by the mice infected with UA1094(A a /D a ). Similar to that observed in the brain, themice infected with H99 and UA1094 showed thehighest increase of CFU/g of lung between day 6and 12 post-infection ( P   ranging from ! 0.0001 to0.021 vs. IUM 96-2828, IUM 99-5509 and UA 1094). Discussion To our knowledge this is the first study in which therepresentative haploid and hybrid strains of each  C.neoformans  var.  neoformans  serotype were Table 2  Survival of mice infected intravenously with equal number of yeast cellsStrains Mating type allelicpatternMortality on day 60 post-infection (%) Mean survival(days G SD)UA1993 A a  100 19 G 4H99 A a  100 21 G 5IUM96-2828 A a  40 53 G 11IUM99-3617 A a  60 45 G 17UA491 D a  10 58 G 11UA477 D a  30 56 G 11JEC21 D a  70 43 G 16 a UA4223 D a  100 30 G 8 a,b JEC20 D a  0  O 60IUM99-5509 D a  40 50 G 17UA486 A a /D a  100 26 G 10CBS132 A a /D a  100 26 G 12UA1094 A a /D a  100 18 G 5 c UA2715 A a /D a  30 51 G 17UA2341 A a /D a  90 34 G 15 d a vs. UA 491 and UA 477. b vs. JEC21. c vs. UA 486. d vs. UA 2715. Figure 2  Cumulative survivals of mice infected intravenously with all the strains grouped by mating type. Pathogenicity of  Cryptococcus neoformans  ser. A, D and AD 13  compared in terms of virulence factors and patho-genicity in an experimental murine model ofsystemic cryptococcosis. The majority of thesestrains were clinical isolates. Our results showedthat haploid and hybrid strains presenting the A a mating type locus are highly virulent in the murinetail vein injection model. These results are consist-ent with data reported by Chaturvedi et al. whofound that eight A a /D a  hybrid strains tested in anintravenous animal infection model were all asvirulent as the highly pathogenic strain H99. 6 Similarly, although in a different murine model ofinfection (inhalation), Lengeler et al. tested fourhybrid strains (two strains each of A a /D a  and A a /D a ) andfound that one ofthetwo A a /D a  strains wasvirulent, but less than H99. 3 The remaining threestrains were moderately virulent. The second A a /D a  strain was CBS132, which in our model gave 100%mortality as did H99. The different route ofinoculation may be the cause of the divergence inthe results for this strain. When yeasts areinoculated by inhalation, several factors play animportant role in the host infection: the yeast size(quite variable in  C. neoformans  and crucial toreach the alveoli), the capacity of adhesion to thepulmonary epithelial cells and the ability to invadethehost. Inthe tail vein injectionmodelthere likelyare other factors distinct from a pulmonary infec-tion route that lead to a different response toinfection.Though our sample size is admittedly small, theresults also showed that haploid A a strains are morevirulent than haploid A a  and D a  strains. None-theless, these results are consistent with data fromtwo other studies which showed that haploid A a strains, 125.91 and IUM96-2828, were markedly lessvirulent than H99. 12,13 In a recent report, Nielsenet al., showed the absence of relationship betweenvirulence and mating type in serotype A isolates. 7 However, the strains used in this study were heavilytransformed in order to improve their fertility andto obtain congenic strains. In addition, such con-genic strains have only the H99 background, themost virulent of the two parent strains.With regard to the D a  haploid strains, theydisplayed a wide and heterogeneous level ofpathogenicity as did the A a /D a  hybrid strains. Thisfinding was confirmed by the additional model of   Figure 3  Survivals of mice infected intranasally with the four strains belonging to D a  types. Table 3  Yeast cells count increase between day 6and 12 post-infection in tissues of mice infected withsix strains of  Cryptococcus neoformans Strain Matingtype allelicpatternMean CFUs increase(log 10 G SD) in the follow-ing tissuesBrain LungH99 A a  1.7 G 0.3 a 1.1 G 0.2  b,c,d IUM96-2828A a  0.9 G 0.4 0.1 G 0.0UA4223 PDF D a  0.7 G 0.2 0.8 G 0.3 d IUM99-5509D a  0.7 G 0.8 0.7 G 0.0 d UA1094 A a /D a  1.3 G 0.2 b,e 1.3 G 0.4 b,c,d UA2715 A a /D a  0.7 G 0.1 0.7 G 0.2 d a vs. Any strain. b vs. UA2715. c vs. IUM99-5509. d vs. IUM96-2828. e vs. UA4223. F. Barchiesi et al.14
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