In vitro Growth Characteristics of Two Cryptococcus neoformans Isolates

Transcription

1 Journal of the Arkansas Academy of Scence Volume 52 Artcle In vtro Growth Characterstcs of Two Cryptococcus neoformans Isolates Krsty L. Jones John Brown Unversty Juneann W. Murphy Unversty of Oklahoma Follow ths and addtonal works at: Part of the Pathogenc Mcrobology Commons Recommended Ctaton Jones, Krsty L. and Murphy, Juneann W. (1998) "In vtro Growth Characterstcs of Two Cryptococcus neoformans Isolates," Journal of the Arkansas Academy of Scence: Vol. 52, Artcle 14. Avalable at: Ths artcle s avalable for use under the Creatve Commons lcense: Attrbuton-NoDervatves 4.0 Internatonal (CC BY-ND 4.0). Users are able to read, download, copy, prnt, dstrbute, search, lnk to the full texts of these artcles, or use them for any other lawful purpose, wthout askng pror permsson from the publsher or the author. Ths Artcle s brought to you for free and open access by ScholarWorks@UARK. It has been accepted for ncluson n Journal of the Arkansas Academy of Scence by an authorzed edtor of ScholarWorks@UARK. For more nformaton, please contact scholar@uark.edu.

2 80 Journal of the Arkansas Academy of Scence, Vol. 52 [1998], Art. 14 In Vtro Growth Characterstcs of Two Cryptococcus neoformans Isolates Krsty L.Jones* and Juneann W. Murphy *Department of Bology John Brown Unversty P.O. Box 2365 Sloam Sprngs, AR Department of Mcrobology and Immunology Unversty of Oklahoma Health Scences Center P.O. Box Oklahoma Cty, OK * Correspondng Author Abstract Cryptococcus neoformans s an opportunstc pathogen whch attacks ndvduals wth weakened mmune systems. Two C. neoformans solates, 184A and NU-2, were examned to determne characterstcs that contrbute to ther dfference n vrulence. Both solates were grown under tssue culture condtons, and dfferent characterstcs were tested at tmed ntervals. Isolate 184A was found to grow at a faster rate than solate NU-2, wth doublng tmes of 4 and 12 hours respectvely. The polysaccharde capsule ofsolate NU-2 doubled n sze n four hours, whle that of solate 184A stayed approxmately the same. The NU-2 yeast cell dameter wthout the polysaccharde capsule was ntally bgger than 184A at 3.62 pm and contnued to ncrease although the dameter of solate 184A stayed approxmately the same at 2.41 pm. Isolate NU-2 shed excessve capsular materal nto the meda, but solate 184A shed lttle. These fndngs hghlght some major dfferences n the characterstcs of the two solates whch may contrbute to the dfferng degrees of vrulence. Introducton Cryptococcus neoformans s a ubqutous yeast-lke organsm whch causes cryptococcoss. The occurrence of ths dsease has ncreased n recent years due to the ncrease n mmunocompromsed ndvduals such as AIDS patents. Humans are frequently exposed to C. neoformans, yet the occurrence ofcryptococcoss n mmunocompetent ndvduals s relatvely low. In contrast, ndvduals wth depressed cell-medated mmunty (CMI) functon due to chemotherapy, underlyng malgnancy, or nfectous dsease such as AIDS are extremely susceptble to a C. neoformans nfecton (Murphy, 1989). The organsm s acqured through nhalaton of the desccated yeast cells n contamnated dust (Kwon-Chung and Bennett, 1992) and may cause an asymptomatc to mld pulmonary nfecton before t spreads to extrapulmonary stes. C. neoformans has a predlecton for the central nervous system, and the majorty ofcryptococcal dsease s dagnosed as menngts or menngoencephalts (Murphy, 1989). There are four serotypes of C. neoformans (A, B. C, D), but vrtually 100 percent of the AIDSpatents n the Unted States wth cryptococcoss are nfected wth serotype A cryptococc (Chernak and Sundstrom, 1994). Among the many solates of serotype A are cultures 184A and NU-2. These two solates are known to be qute dfferent from one another wth respect to host clearance (Murphy unpub. observ.). In an experment desgned to dsplay ths dfference, each solate was njected ntratracheally nto nave mce. Sxty percent of the 184A nfected mce survved to 95 days post-nfecton whch was the end pont of the experment, but 100% of the NU-2 nfected mce ded by the 67th day of nfecton (Murphy unpublshed observ.). In ths stuaton, solate NU-2 was clearly more vrulent than solate 184A. Ths research focuses on determnng the morphologcal and growth dfferences between these two solates of C. neoformans. Knowng the combnaton of host and pathogen factors whch are assocated wth ether mld or severe dsease may allow for the development of procedures whch nterfere wth the vrulence factors of the pathogen therebye reducng the ncdence or extent of the dsease n mmunocompromsed patents. The frst characterstc of the solates examned was rate Journal of the Arkansas Academy of Scence, Vol. 52, 1998 Publshed by Arkansas Academy of Scence,

3 81 Journal of the Arkansas Academy of Scence, Vol. 52 [1998], Art. 14 In VtroGrowth Characterstcs of Two Cryptococcus neoformans Isolates ofgrowth. One possble reason for the ncreased level of vrulence observed n mce of solate NU-2 over 184A may be due to faster rates of growth and reproducton. To test ths possblty, the growth rate of each solate was determned under tssue culture condtons. A second characterstc of the solates that was examned was the dameter of the yeast cell wthout the polysaccharde capsule. The cell dameter wth and wthout the polysaccharde capsule was measured, and the data from the two parameters was used to characterze the yeast cells. A thrd characterstc that was examned was the knetcs of polysaccharde capsule formaton. The presence of a polysaccharde capsule s known to contrbute to the vrulence of C. neoformans (Bulmer et al.,1967; Fromtlng et al., 1982; Kozel and Cazn, 1971; Kwon-Chung and Rhodes, 1986). Both solates have a polysaccharde capsule, but as tme progresses n tssue culture medum, solate NU-2 s able to ncrease capsule synthess (Chernak and Sundstrom, 1994). Usng dfferental dsplay PCR, dfferentally expressed genes for capsule producton or other possble vrulence factors can be examned. For ths procedure, however, mrna must be solated, and the excessve capsule producton of NU-2 can nterfere wth RNA solaton. By examnng the knetcs of capsule producton, the optmal tme for RNA solaton when the transcrpts nvolved n capsule producton are abundant, but lttle capsular materal s produced can be found. Ths wllpermt the study of the capsular genes whch wllbe helpful ndentfyng vrulence mechansms. Ths n turn may allow for the augmentaton of host resstance or therapeutc nhbton of the vrulence factors. The polysaccharde capsule producton was also tested wth a latex agglutnaton test. Ths test measures the amount of capsular materal the solate has shed nto the culture medum. Ths wll help determne when the solates begn capsular producton and the peak of that producton. Ths along wth the other data yelds the optmal tme for mrna retreval. Allof these factors may combne to make solate NU-2 more vrulent. The nformaton obtaned from these experments may provde further nsght nto the causes of ths vrulence whch could lead to more effectve control of cryptococcoss. Materals and Methods Cryptococcus neoformans solate 184 A was solated from he sputum of a patent at Charty Hosptal nnew Orleans, Lousana n C. neoformans solate NU-2 was solated at he Unversty of Nebraska School of Medcne, Department of Mcrobology. Both solates are serotype A wth smlar glucuronoxylomannan (GXM) structure and are alpha matng type. Isolates were ntally grown on Modfed Sabourauds Agar (MSA) slants. For each experment and solate, threeday cultures were transferred to fresh slants and two days later used for the experments. Isolates were washed off the MSA slants wth sterle physologcal salne soluton (SPSS). Organsms were washed 3 tmes n SPSS and resuspended n RPMI, ph 6.8. They were then counted on a hemacytometer and resuspended at a concentraton of 10 4 organsms/ml. One mlof the organsms nrpmi soluton ph 6.8 was then added to each of three 14 ml tubes per solate per tme pont. The noculaton of the tubes was done under the hood, and the tubes were ncubated at 37 C, n a 7% atmosphere of CO 2. The growth curve was determned by hemacytometer counts at 0, 4, 8, 12, 24, and 48 hours after noculaton. The number of C. neoformans cells/ml from the three tubes was averaged to obtan the mean for each tme pont. The tme ponts for the measurement of the dameter of the yeast cells wthout the polysaccharde capsules and the dameter of the polysaccharde capsules themselves were 0, 4, 8, 12, 24, and 48 hours. At each tme pont the yeast cells n the noculated tubes used for the hemacytometer count were measured. After the yeast cells were removed for the hemacytometer count, the rest of the soluton was transferred to three 1.6 ml centrfuge tubes per solate. The three tubes were centrfuged for 10 mnutes at 600 g. Two-hundred ul of the supernatant flud was removed from each of the tubes and pooled nto a 1.6 ml tube and frozen at -20 C. Ten ul of remanng soluton was put on a mcroscope slde wth 37 ul of Inda Ink. A coverslp was then added. The dameters of the yeast cells both wth and wthout the capsules were measured wth an ocular mcrometer. The dameters of the capsules were obtaned by subtractng the dameters of the yeast cells wthout the capsules from the dameters of the yeast cells wth the capsules. Approxmately 100 cells/solate were measured for each tme pont. These numbers were averaged to get a mean value for both measurements. The Latex-Crypto Antgen Detecton System (Immuno- Mycologcs, Inc., Norman, OK) was used for the latex agglutnaton test or the soluble polysaccharde determnaton. Frozen supernatant was thawed for approxmately 15 mnutes; then 25 ul was added to the rng slde provded by the kt along wth 25 jul of the Ant-Cryptococcus Globuln Reagent (ACGR). They were mxed and shaken at 100 rpm for 10 mnutes. The reacton was then read accordng to the manufacturer's nstructons. If the reacton was postve, 50 ulof the supernatant was added to 50 ul of a 1:10 dluton of the dluent wth deonzed water creatng a 1:2 tter.twentyfve ulof the 1:2 tterand 25 lof the ACGR were added to a rng slde, mxed, and shaken for 10 mnutes at 100 rpm. The reacton was then read. The dluton was ncreased log- Journal of the Arkansas Academy of Scence, Vol. 52,

4 82 Journal of the Arkansas Academy of Scence, Vol. 52 [1998], Art. 14 Krsty L.Jones andjuneann W. Murphy arthmcally untl a negatve reacton occurred. Means, standard errors of the means (SEM) and Student's t test were used for statstcal analyss of the data. Results were consdered sgnfcant fthe P value was < Results The frst objectve of the experment was to construct a growth curve for each solate of C. neoformans. Separate cultures of each solate were ntated wth equvalent numbers of cells/ml. The hemacytometer count values were used to construct the growth curves. The mean values for cells/ml at each tme pont are shown n Fg. 1. The data from ths experment show that the doublng tme for solate 184A was approxmately 4 hours whle the doublng tme of solate NU-2 was approxmately 12 hours. The data shown are representatve of three experments. These data demonstrates that solate 184A reproduces more rapdly n culture than solate NU-2. Fg. 2. Yeast cell dameter wthout the polysaccharde capsule for C. neoformans solates 184A and NU-2. Error bars represent mean ± standard error of the mean of 100 determnants. Tme (hours) Fg. 1. Growth rate of C. neoformans solates 184A or NU-2 n RPMI at 37 C n the presence of 7% CO 2. Error bars represent mean ± standard error of the mean of three determnatons. The dameter of the yeast cells wthout the polysaccharde capsule was another aspect of the two solates that was measured. Ths measurement helped make the polysaccharde capsule measurements more sgnfcant because t showed a trend n the sze of the nner part of the cells wthout the capsule. The mean values of the dameters of the nner part of the cells of the solates at the successve tme Tme (hours) capsule dameter of C. neoformans so- Fg. 3. Polysaccharde lates 184A or NU-2. Error bars represent error of the mean of 100 determnatons. mean ± standard ponts are shown n Fg. 2. The cells of solate NU-2 possessed dameters that were larger at tme zero than solate 184A and contnued to ncrease n sze whereas the dameters of the yeast cells of solate 184A stayed relatvely constant throughout the course of the experment. These data represent two experments. Journal of the Arkansas Academy of Scence, Vol. 52, 1998 Publshed by Arkansas Academy of Scence,

5 83 Journal of the Arkansas Academy of Scence, Vol. 52 [1998], Art. 14 In Vtro Growth Characterstcs of Two Cryptococcus neoformans Isolates Table 1. Latex agglutnaton results for C. neoformans solates 184A or NU-2 at varous tme ponts. Hours Isolate 184A Isolate NU-2 0 = + UNDILUTED 4 I +(1:4) 8 +(1:32) + J2 UNDILUTED +(1:128) 24 +(l:64) a +(1:512) 48 + (1:256) + (1:2048) a Values are the tter from latex agglutnaton kt. Next, we observed the knetcs of the polysaccharde capsular producton. The mean values of the dameter of the polysaccharde capsule over 48 hours are shown n Fg. 3. By eght hours the average dameter of the yeast cells' capsules of solate NU-2 had doubled whle the mean dameter of capsules for solate 184A had decreased slghtly. Yeast cells ofsolate NU-2 possessed capsule dameters that stayed roughly the same from 8 to 48 hours. The dameter of yeast cell capsules of solate 184A dd not change sgnfcantly durng culture. These data represent two experments. Both solates are known to have a polysaccharde capsule, but t s not clear at what pont the capsule producton peaks. A specfc latex agglutnaton test was used to determne the amount of polysaccharde capsular materal shed nto the medum. Isolate 184 A dd not show a postve reacton untl 12 hours (Table 1), but ts tters dd begn to ncrease at subsequent hours. Isolate NU-2 showed a postve reacton at zero hours whch means that tprobably was not washed well enough, but t contnued to ncrease ts tters by at least 2 ncrements at each subsequent tme pont. Ths demonstrates that solate NU-2 s producng consderably more capsule than solate 184 A and s releasng t nto the medum at a greater rate than solate 184A. Dscusson These data gve growth rate estmates for two solates of C. neoformans. The 184A solate exhbts a much faster growth rate than the NU-2 solate. Ths s an mportant dference n the twosolates. Ithas been postulated that solate MU-2 s more vrulent n mce than solate 184A because t grows faster and smply overwhelms the host wth cell numbers. The data present suggests otherwse. The growth rates also gve the approxmate doublng tme of both solates. Isolate 184 A cell numbers doubled approxmately every 4 hours, whereas solate NU-2 cell numbers doubled approxmately every 12 hours. These data suggest an optmal tme of retrevng mrna from the cells whch would be after 4 hours for solate 184A and after 8-12 hours for solate NU-2. The doublng tme s mportant for mrna retreval because the cells must be adequately growng and producng RNA. Ths doublng tme wllbe consdered wth the tme frame of the capsular producton to fnd the optmal tme for mrna solaton. The knetcs of the yeast cell sze wthout the polysaccharde capsule s also an mportant dfference between the twosolates because ttoo may contrbute to the vrulence of solate NU-2 The greater cell sze may cause the organsm to occlude arways nto the lung. The data from ths experment ndcated that the yeast cell dameter wthout the polysaccharde capsule of yeast cells from solate 184A stayed approxmately the same throughout the 48 hour perod. In contrast, the yeast cell dameter of solate NU-2 ncreased over the frst 24 hours and then leveled off. The queston surroundng ths proven growth n sze of NU-2 cells s whether the cell s actually producng more proten materal or just swellng wth water ntake. Ths s sgnfcant because f solate NU-2 s producng more proten materal, ths materal may contrbute to ts vrulence. Ths mechansm s also sgnfcant because tcould allow treatment for reducng the sze ofnu-2 to reduce pulmonary and dessmnated systemc nfectons. Further studes on the bomass wllhave to be completed to understand the mechansm by whch cell sze ncreases. We also explored the producton of the polysaccharde capsule. Ths s an mportant dfferng characterstc of the two C. neoformans solates because the capsule s a factor n the vrulence of solate NU-2. A larger capsule may more effectvely protect the yeast from host effecter cells such as alveolar macrophages n the lung (Bulmer and Sans, 1968; Swenson and Kozel, 1978). Ths experment examned the knetcs of the capsular producton. There was lttle capsule producton by solate 184A whle solate NU-2 produced excessve amounts of capsule, especally wthn the frst eght hours. Usng ths nformaton, the optmal tme to extract capsule gene mrna from solate NU-2 wthout the nterference of excessve capsular materal would be n the Journal of the Arkansas Academy of Scence, Vol. 52,

6 84 Journal of the Arkansas Academy of Scence, Vol. 52 [1998], Art. 14 Krsty L.Jones and Juneann W. Murphy frst eght hours. More knetc studes wth smaller tme ntervals would be needed to fnd the specfc optmal tme pont n ths eght hour span. Alatex agglutnaton test was also performed to observe the knetcs of capsular materal that was shed nto the culture medum. Its well documented that ncreasng cryptococcal antgen tters are characterstc of a poor prognoss whle decreasng antgen tters sgnfy a good prognoss (Damond and Bennett, 1974). Excess capsular polysaccharde lkely affects host reactvty. The experment results show that solate NU-2 was producng and sheddng capsular materal throughout the entre experment. The tters were constantly ncreasng whch confrms the results from the measurement of the actual polysaccharde capsule. These data help to explan why solate NU-2 s more often fatal than 184A n expermental anmals. Isolate 184A dd not begn producng capsular materal for 12 hours but the tters ncreased thereafter. Ths fact seems to ndcate that an optmal tme for RNA retreval from solate 184A would be after 12 hours, and the RNA from solate NU-2 should be retreved as soon as possble. More precse experments wth smaller tme ntervals wllhave to be done to get the precse tme when the solates are both actvely growng and reproducng and not producng excessve capsular materal. These data narrow down the optmal tme for retreval, but t s stllqute broad. Cryptococcoss. In Medcal Mycology. Lea & Febger, Phladelpha, 397 pp. Murphy, J.W Immunoregulaton n Cryptococcoss. In Immunology of Fungal Dsease (E. Kurstak, ed.) Marcel Dekker, Inc., New York, pp Swenson, J.F. and T.R. Kozel Phagocytoss of Cryptococcus neoformans by normal and thoglycolateactvated macrophages. Infect. Immun. 21: »?. t < Lterature Cted Bulmer, G.S., M.D. Sans and CM. Gunn Cryptococcus neoformans. I.Nonencapsulated mutants. J. Bacterol. 94: Bulmer, G.S. and M.D.Sans Cryptococcus neoformans. III.Inhbton of phagocytoss. J. Bacterol. 95:5-8. Chernak, R. and J.B. Sundstrom Polysaccharde antgens of the capsule of Cryptococcus neoformans. Infect. Immun. 62: Damond, R.D. and J.E. Bennett Prognostc factors n cryptococcal menngts. A study n 111 cases. Ann. Intern. Med. 80: Frontlng, R.A., HJ. Shadomy and E.S. Jacobson Decreased vrulence n stable, acapsular mutants of Cryptococcus neoformans. Mycopathologa. 79: Kozel, T.R. andj. Cazn, Jr Non-encapsulated varant of Cryptococcus neoformans. I.Vrulence studes and characterzaton of soluble polysaccharde. Infect. Immun. 3: Kwon-Chung, K.J. and J.C. Rhodes Encapsulaton and melann formaton as ndcators of vrulence n Cryptococcus neoformans. Infect. Immun. 51: Kwon-Chung, K.J. and J.E. Bennett I < < «Publshed by Arkansas Academy of Scence, 1998 Journal of the Arkansas Academy of Scence, Vol. 52, «*