Monosaccharide and Chitin Content of Cell Walls

Transcription

1 JOURNAL OF BACTERIOLOGY, Sept. 1971, p Copyright 1971 Americn Society for Microbiology Vol. 107, No. 3 Printed in U.S.A. Monoscchride nd Chitin Content of Cell Wlls of Histoplsm cpsultum nd Blstomyces dermtitidis JUDITH E. DOMER Deprtment of Microbiology nd Immunology, Tulne University School of Medicine, New Orlens, Louisin Received for publiction 9 June 1971 Cell wlls of Histoplsm cpsultum nd Blstomyces dermtitidis, obtined by mechnicl brekge of yest- nd mycelil-phse cultures, were lipid-extrcted nd then frctionted with ethylenedimine. Unextrcted cell wlls, lipid-extrcted cell wlls, nd the three frctions resulting from ethylenedimine tretment were exmined for monoscchride nd chitin content. The yest-phse cell wlls of five strins of fell into two ctegories, designted chemotypes I nd II, one of which, chemotype II, ws similr to yest-phse cell wlls derived from three strins of. chemotype I cell wlls were chrcterized by lower content of mteril soluble in ethylenedimine, higher chitin content, nd lower monoscchride content thn chemotype II or B. dermtitidis cell wlls. Approximtely 80% of the monoscchrides of chemotype I cell wlls ws combined in forms susceptible to ttck by mild cid hydrolysis, compred with bout 50% of the monoscchrides of chemotype II nd. nd yest-phse cell wlls could be distinguished, however, by their susceptibility to ttck by crude enzyme system derived from Streptomyces sp. incubted with chitin s the only crbon source. Both glucose nd cetylglucosmine were relesed from cell wlls, regrdless of chemotype, during enzymtic hydrolysis, wheres only cetylglucosmine ws relesed from yest-phse cell wlls. Mycelil-phse cell wlls of nd were chrcterized by lower content of mteril soluble in ethylenedimine, higher proportions of mnnose, nd lower chitin content thn their respective yest phses. Glucose nd cetylglucosmine were both relesed from ll mycelil-phse cell wlls, whether or, by the crude enzyme system. In contrst to bcteril cell wlls, dt on the of single strin of (3). However, Pine nd Boone (14) lter reported evi- rchitecture, chemicl composition, nd synthesis of fungl cell wlls hs been somewht meger. dence for two chemicl types of yest-phse cell Histoplsm cpsultum nd Blstomyces dermtitidis re two fungl species which exhibit essry to survey severl strins of wlls of. Therefore, we felt it nec- therml dimorphism. They grow s round, budding yest forms t 37 C but will convert to cell wlls would be more meningful. Although so tht comprtive studies with mycelil (filmentous) forms t 26 C, nd vice chemiclly distinct cell wll types of hve not been reported, we included severl vers; the physiologicl process by which the conversion tkes plce hs not been determined. strins to serch for ny significnt intrspecies In ddition to their dimorphic chrcter, nd re lso pthogenic the monoscchride nd chitin content of the vrition. This report dels with comprison of for mn, cusing host responses tht re similr yest- nd mycelil-phse cell wlls-of nd. nd frequently difficult to differentite if the etiologic gent cnnot be isolted. In ttempting to understnd dimorphism nd pthogenesis better, MATERIALS AND METHODS we previously exmined the composition of the Culturl methods nd cell wll preprtion. The cell wlls of both the yest nd mycelil phses source nd culturl dt for the nd B. 870

2 VOL. 107, 1971 WALLS OF H. CAPSULATUM AND B. DERMATITIDIS 871 dermtitidis strins used for cell wll isoltion re presented in Tble 1. Stock cultures of ll strins were mintined in the yest phse by weekly trnsfer on Brin Hert Infusion gr t 37 C. Three of the H. cpsultum strins, Bon, SwA, nd SwB, were chosen becuse, t the time of isoltion, they ll hd "B" type of mycelil colony s described by Berliner (2), i.e., primrily brown colony with few eril hyphe nd msses of tuberculte chlmydospores. However, SwA nd SwB hve converted over the yers to predominntly "A" type of mycelil growth with profuse white eril mycelium nd smooth, insted of rough, mcroconidi. The G184A nd G184B cultures were used becuse they were representtives of pure "A" nd "B" colonil types. None of the strins ws identified s to serotype. The only criterion used in the selection of the strins ws tht of recent isoltion from humn source. The methods employed in obtining cell wlls were previously described in detil for strin SwB (3). All yest nd mycelil phse cultures of nd were incubted in glucose-yest extrct broth (GYE, reference 7), Soy dilyste broth (Soy, reference 15), respectively, or both (Tble 1). It ws felt necessry to use the two different medi to mke the homologous yest nd mycelil phse comprisons vlid becuse yest cells would not convert completely to myceli in Soy, wheres the yest phse ws not stble in GYE t 37 C nd would begin germinting into mycelil elements fter the second trnsfer. All yest-phse cu!- tures were incubted t 37 C for 3 dys with constnt ertion. Yest phse cells were inoculted into either GYE () or Soy () for conversion to the mycelil phse, nd incubtion ws t 26 C with intermittent gyrtory shking (bout 4 hr per dy). When mechnicl shking ws stopped, the "tide line" ws wshed from the sides of the flsk by mechnicl swirling. Complete conversion to the mycelil phse occurred in pproximtely 4 weeks with weekly trnsfers to fresh medium. The mnner of incubtion resulted in culture which ws devoid of pellets of hyphl growth nd conidi. Once complete conversion hd occurred, the myceli were trnsferred to fresh medium nd incubted, s bove, for 6 dys. The longer incubtion period ws necessry to llow for the ccumultion of enough myceli for cell wll preprtions. To rule out the possiblity tht differences between corresponding yest nd mycelil phses were the result of differences in incubtion time, one strin ech of the yest phse of nd, SwA nd Found, respectively, ws incubted for 6 dys in GYE nd Soy, respectively, nd cell wll preprtions were mde from smples tken t 2-, 4-, nd 6-dy intervls. Cell wlls were prepred in most instnces from t lest two seprtely grown btches of culture nd crried through ll the procedures to scertin the reproducibility of results. The quntity of cell wlls obtined from the myceli of Found nd McCrty ws very low, nd two cell wll preprtions of ech hd to be combined for the extrction procedures described below. Extrction nd frctiontion of cell wlls. Whole unextrcted cell wlls were treted with neutrl nd cidified lipid solvents (12). Three frctions were then obtined from lipid-extrcted cell wlls by frctiontion with ethylenedimine (13); frction C ws resistnt to ethylenedimine; frctions A nd B were both soluble in ethylenedimine nd insoluble in methnol, but were seprted by the fct tht A ws soluble TABLE 1. Source nd culture dt for the Histoplsm cpsultum nd Blstomyces dermtitidis strins used for cell wll isoltion Growth medium for Strin Donorb Yer Phse Conversion cell wll received preprtion received medium YP MP, Bon ( ) FT 1964 MPc BHId GYE, Soye SwA HCS 1964 MP BHI GYE, Soy GYE SwB HCS 1964 MP BHI GYE, Soy GYE G 184A MDB 1969 YP Sb Soy G 184B MDB 1969 YP Sb Soy 3489 BH 1965 YP Sb GYE, Soy Found J DS 1969 YP Sb Soy Soy McCrty J DS 1969 YP Sb Soy Soy All strins were originlly isolted from humns. b Donor initils: FT, Fred Tosh, Ntionl Communicble Disese Center, Knss City Field Sttion, Mo.; HCS, H. C. Sweny, Missouri Stte Sntorium, Mt. Vernon; MDB, Mrth D. Berliner, Hrvrd School of Public Helth, Boston, Mss.; BH nd JDS, Betty Hodges nd John D. Schneidu, Tulne University School of Medicine, New Orlens, L. c MP, Mycelil phse; YP, yest phse. d BHI, Brin Hert Infusion gr; Sb, Sbourud's dextrose gr. egye, glucose-yest extrct medium; Soy, soy dilyste medium. Incubtion ws in both GYE nd Soy, respectively, where both medi re listed for single strin nd phse.

3 872 DOM ER J. BACTERIOL. in wter nd B ws not. Monoscchride deteriintions. Smples (5 mg) of cell wlls nd frctions thereof were subjected to mild hydrolysis with 2 ml of I N HCI in methnol for 40 hr t 80 C. The hydrolyste ws seprted from the residue, nd the hydrolytic products were determined qulittively nd quntittively by gs-liquid chromtogrphy (GLC) of the trimethylsilyl ether derivtives (6, 19) with dulcitol s n internl stndrd. A Brber- Colmn gs chromtogrph equipped with flme ioniztion detector ws employed with the following conditions: pcking, 3% OV-1 on 80/100 Chromosorb W HP (Supelco, Inc., Bellefonte, P.); column temperture, 175 C; injector temperture, 255 C; detector tem. perture, 235 C. The unhydrolyzed residue ws then treted with 2 N queous HCI t 100 C for 4 hr, nd the relesed hexose ws determined by nthrone rection (17). Simultneously, we lso performed nthrone nlyses on 2 N queous HCI hydrolystes of duplicte smples which hd not been subjected to the prior mild cid tretment. Additionl smples were treted with 2 N H2SO4, neutrlized with B(OH)2, nd pssed through n ion-exchnge resin, nd, long with portions of some of the 2 N queous HCI hydrolystes which hd been evported in vcuo over NOH, were subjected to the phenol-sulfuric cid test (4). The vlues cquired in this mnner were ll somewht higher thn those cquired by the nthrone method. However, we found the results obtined by the nthrone method to be more reproducible when processing lrge numbers of smples, probbly due to fewer smple mnipultions in this procedure. Chitin determintions. Smples (5 mg) were treted with crude enzyme system obtined by the method of Reynolds (16) from the culture filtrtes of Streptomyces sp. ATCC which hd been cultured in bsl slts medium (16) contining colloidl crustcen chitin (I ) s the crbon source. The crude enzyme system thus derived contined two known enzymes, chitinse nd d-1,3 glucnse, s scertined by its ction on the substrtes of colloidl chitin nd "insoluble" lminrin (IL23),,B-1,3 glucn obtined from the Institute of Seweed Reserch, Midlothin, Scotlnd. Enzyme ssys were performed ccording to the method of Skujins et l. (18) except tht the ssy mixture ws not pssed through n ion-exchnge resin fter incubtion. It ws centrifuged to remove unhydrolyzed mteril nd lyophilized directly. Preliminry experiments, with pure cetylglucosmine s well s representtive hydrolystes of cell wll smples, showed tht the ion-exchnge procedure resulted in loss of cetylglucosmine nd tht it ws not necessity prior to derivtive formtion. The cetylglucosmine relesed by the enzymtic hydrolysis ws determined by GLC of the trimethylsilyl ethers s described bove for monoscchrides, but with one vrition. The detector response to cetylglucosmine ws less thn its response to n equivlent quntity of the internl stndrd (dulcitol) nd multipliction fctor of 1.6 ws decided upon by experimenttion with known quntities of the two compounds. In selected instnces, the cetylglucosmine in given enzyme hydrolyste ws determined by processing equl portions of ech smple through the GLC procedure nd lso through boric cid modifiction of the Morgn- Elson rection (8). The results were comprble, but GLC quntittion ws the method of choice becuse glucose dt could simultneously be obtined. The residue remining fter the bove enzymtic tretment ws then hydrolyzed with 6 N queous HCI t 100 C for 6 hr in seled tubes nd the glucosmine hydrochloride thus obtined ws determined by modified Elson-Morgn procedure (5). Since the moleculr weight of glucosmine-hci is not significntly different from tht of cetylglucosmine, the results obtined from the clssicl chemicl determintion were simply dded to those obtined by enzymtic hydrolysis to determine the totl quntity of chitin. To scertin tht virtully ll of the glucosmine in the wll ws cetylted nd in the form of chitin, selected smples were treted twice with the enzyme system, nd then the residul glucosmine ws determined s bove. Insignificnt mounts of glucosmine remined in the residue fter the consecutive enzymtic hydrolyses. Additionlly, smples not previously subjected to enzyme tretment were hydrolyzed with 6 N HCI t 100 C for 6 hr, nd the glucosmine in the hydrolystes ws determined by the clssicl Elson-Morgn rection, s well s by modifiction of tht procedure with borte buffer (8). RESULTS The yest-phse cell wll preprtions fell into two chemiclly distinct types. The dt for strins of ech type were so similr tht it ws decided to combine them nd present them on type, rther thn n individul strin, bsis. Therefore, strins Bon, SwA nd SwB will henceforth be referred to s chemotype I; G184A nd G184B will be clled chemotype II. Yestphse cultures of ll three isoltes were similr nd were lso grouped. The mycelil-phse preprtions were more vrible, nd it ws deemed inpproprite to combine them so tht the dt from such preprtions re presented seprtely. Ethylenedimine solubility. Less thn 20% of chemotype I cell wlls, s well s the cell wlls of three of the mycelil-phse preprtions, ws extrctble with ethylenedimine (Tble 2), nd, of this 20% or less, most ws wter soluble. A much higher percentge of H. cpsultum chemotype II nd yest-phse cell wlls ws extrctble under similr conditions, nd most of this extrctble mteril ws wter-insoluble. One of the mycelilphse preprtions, Found, ws unlike the other mycelil phses in tht the wter-insoluble frction extrcted with ethylenedimine represented pproximtely one-qurter of the lipid-extrcted cell wll. Monoscchride determintions. Qulittively, glucose nd mnnose were the only monoscchrides detected by the thin lyer or gs-liquid chromtogrphic procedures employed. The quntittive dt ccumulted for these two monoscchrides re summrized in Tbles 3

4 VOL. 107, 1971 WALLS OF H. CAPSULATUM AND B. DERMATITIDIS 873 nd 4. In those instnces where frction of the tures. The quntity of frction A of Found mycelil-phse wlls ws lso very wll represented less thn 2% of the wll, it ws not nlyzed, e.g., the B frctions of chemotype smll so tht only two procedures could be crried out, the two-step hydrolysis nd the single I yest phse nd three of the mycelil-phse cul- TABLE 2. Results of ethylenedimine (EDA ) frctiontion of lipid-extrcted cell wlls of both yest nd mycelil phses of Histoplsm cpsultum nd Blstomyces dermtitidis Per cent EDA solublemethnol insoluble Per cent EDA Per cent Strin ~~~~~~~~~~~~~resistnt Rercery Strin Per cent wter Per cent wter (frction C)Recovery soluble insoluble (frction A) (frction B) Yest phse chemotype l chemotype c Mycelil phse SwA SwB Found McCrty Includes strins Bon, SwA, nd SwB. h Includes strins G184A nd G184B. Includes strins 3489, Found, nd McCrty. TABLE 3. Monoscchrides of whole, unextrcted (WC W) nd lipid-extrcted (L-E) cell wlls nd Jrctions thereof of the yest phses of Histoplsm cpsultum nd Blstomyces dermtitidis Per cent from GLC Anthrone5 Totl mono- Anthrone' Strin residul scchride totl hexose Mnnose Glucose hexose Unfrctionted Chemotype Id (WCW) 2.2e (L-E) Chemotype 11 (WCW) (L-E) (WCW) (L-E) Frction A Chemotype I Chemotype Frction B Chemotype I Frction C Chemotype I Chemotype I Gs liquid chromtogrphic nlysis of the products of I N methnolic HCI hydrolysis. banthrone nlysis of 2 N queous HCI hydrolysis of the residue remining fter the I N methnolic HCI hydrolysis. c Anthrone nlysis of 2 N queous HCI hydrolysis of smples not subjected to prior hydrolysis. d Chemotype 1, strins Bon, SwA, SwB; chemotype 11, strins G184A nd G184B. eat lest two determintions were mde on cell wlls nd frctions thereof derived from ech strin, nd ech vlue in this tble represents n verge of ll determintions for ll strins included in given ctegory. In instnces where two medi were employed for incubtion, the results from both cell wll preprtions were included.

5 874 DOMER J. BACTERIOL. TABLE 4. Monoscchrides of whole, unextrcted (WC W) nd lipid-extrcted (L-E) cell wlls nd frctions thereof of the mycelil phses of Histoplsm cpsultum nd Blstomyces dermtitidis Per cent from GLC Anthroneb Totl mono- Anthrone' Strin residul scchride totl hexose Mnnose Glucose hexose Unfrctionted SwA (WCW) 7.4d (L-E) SwB (WCW) (L-E) Found (WCW) (L-E) McCrty (WCW) (L-E) Frction A SwA SwB Found McCrty Frction B Found Frction C SwA SwB Found McCrty Gs liquid chromtogrphic nlysis of the products of I N methnolic HCI hydrolysis. b Anthrone nlysis of 2 N queous HCI hydrolysis of the residue remining fter the I N methnolic HCI hydrolysis. c Anthrone nlysis of 2 N queous HCI hydrolysis of smples not subjected to prior hydrolysis. d Most vlues re verges of minimum of two determintions. Lck of sufficient cell wll mteril occsionlly prevented us from doing nthrone totl hexose determintions. 2 N queous HCI hydrolysis. Of the yest phses (Tble 3), 80% or more of the monoscchrides of chemotype I cell wlls, either unextrcted or lipid-extrcted, ws present in form susceptible to mild cid hydrolysis, wheres only bout 50% of the chemotype II or the wll monoscchride ws in susceptible form. Compring the dt of the vrious frctions obtined from the yest-phse cell wlls, while tking into ccount the percentge of the lipidfree cell wll ech frction represented, it cn be seen tht pproximtely one-hlf of the mnnose in the wll ppered in frction A long with lrge quntities of glucose. Frction B contined no mnnose, but lrge quntities of glucose, onehlf or more of which ws present in form resistnt to mild cid hydrolysis, s in the corresponding unextrcted or lipid-extrcted cell wlls. Proportiontely smller mounts of the monoscchrides were retined in frction C s would be expected. However, more polyscchride ws extrcted with ethylenedimine from the yest-phse wlls thn from either type of yest-phse cell wll. The results derived from the vrious mycelilphse preprtions were not s well delineted s those of the yest phse, mking generliztions difficult. However, mnnose ws present in lrger, nd glucose in smller, quntities in the mycelil phse in comprison to the yest phse. In contrst to their corresponding yest-phse cell wlls, more of the glucose of the mycelilphse wlls ws in form resistnt to mild cid hydrolysis. Frction A isolted from mycelil-phse wlls contined proportiontely lrge quntities of mnnose s did

6 VOL. 107, 1971 WALLS OF H. CAPSULATUM AND B. DERMATITIDIS the yest phse, but, when considering the mnnose-glucose-residul glucose rtio, there ws no dermtitidis. Glucose vlues were lwys higher lesed from the cell wlls of the yest phse of B. consistent pttern within the mycelil-phse in the unextrcted cell wlls thn in the lipid-extrcted cell wlls, wheres there ws little differ- preprtions. The one B frction exmined ws very similr to its yest-phse counterprt. The ence between the chitin vlues of the two preprtions. chemotype I contined dt on the C frctions of the mycelil phse reflected the fct tht less of the wll ws soluble pproximtely 40% chitin, chemotype II less thn 15%, nd in ethylenedimine, leving more of the monoscchride ssocited with the C frction. slightly less thn chemotype I. Chitin determintions. The results of the sequentil enzymtic nd cid hydrolyses of the fct tht chitin is resistnt to the solvent ction Incresed vlues in the C frctions reflect the vrious cell wll preprtions of both yest nd of ethylenedimine. The mycelil-phse cell wlls mycelil phses of nd pper in Tble 5. The crude enzyme phse counterprts but were very similr to ech tended to contin less chitin thn their yest- system used in these experiments relesed both other. glucose nd cetylglucosmine from ll yest-phse cell wlls, regrdless of nd chitin determintions of the cell wlls of the Effect of ge of culture. The monoscchride chemotype, nd from ll mycelil-phse cell 2-, 4-, nd 6-dy-old yest-phse cultures of H. wlls, regrdless of species. Glucose ws not re- cpsultum SwA nd Found re- TABLE 5. Glucose nd chitin content of vrious cell wll preprtions.of both yest nd mycelil phses of Histoplsm cpsultum nd Blstomyces dermtitidis Per cent from GLC Residul" Totl Strin Prepn N-cetyl glucosmine glucosmine Glucose $lucosmine (%() Yest phse Chemotype Ic WCWd 11.8e L-E C Chemotype II WCW L-E C WCW L-E C Mycelil phse SwA WCW L-E C SwB WCW L-E C Found WCW L-E C McCrty WCW L-E C Gs liquid chromtogrphic nlysis of the products of n enzymtic hydrolysis. b Elson-Morgn nlysis of 6 N queous HCI hydrolysis of the residue remining fter the enzymtic hydrolysis. c Chemotype 1, strins Bon, SwA, SwB; chemotype II, G 184A, G 184B. d WCW, whole, unextrcted cell wlls; L-E, lipid-extrcted cell wlls; C, cell wll frction insoluble in ethylenedimine. e At lest two determintions were mde on ech preprtion derived from ech strin, nd ech vlue in the tble represents n verge of ll determintions for ll strins included in tht ctegory. In instnces where two medi were employed for incubtion, the results from both cell wll preprtions were included. 875

7 876 DOM ER veled tht there ws no chnge in those two constituents of the cell wll with ge. The cell wll rectivity to mild nd more concentrted cid ws essentilly the sme s tht seen in the respective 3-dy-old cultures, nd there ws no relese of glucose from the yestphse cell wlls with the enzymtic hydrolysis. DISCUSSION The evidence presented in this pper supports the contention tht there re chemiclly distinct cell wll types of the yest phse of, s suggested by Pine nd Boone (14) on the bsis of combined serologicl nd chemicl dt. Erlier, Kufmn nd Blumer (11) hd reported finding severl serotypes of which would suggest differences in chemicl composition between strins lso, but the cellulr source of the serotyping ntigens ws not estblished so they my, or my not, hve been detecting differences in cell wll composition. Our two chemotypes re similr to those described by Pine nd Boone in tht one hs more chitin nd less glucose in its cell wlls thn the other. Additionlly, our dt suggest possible chemicl bsis for the high degree of serologicl cross-rectivity between certin strins of nd B. dermtitidis nd the low cross-rectivity exhibited by others (11); i.e., yestphse cell wlls of chemotype II nd yest-phse cell wlls hd similr solubility chrcteristics in ethylenedimine nd ech lso contined lrge quntities of glucose in form resistnt to mild cid hydrolysis. However, much dditionl work, including serologicl studies nd studies of the proteinceous cell wll components, must be done before firm conclusions cn be drwn. We hve not found evidence for chemotypes of yest-phse cell wlls, but this my simply be due to the fct tht we hve not s yet exmined enough strins. It should be noted, lso, tht we hve not found ny chemicl differences between the yest phses representing the "AA" nd "B" colonil types of single strin of. The differences in rectivity of the vrious cell wll preprtions to mild nd stronger cid hydrolysis, in combintion with the results obtined by enzymtic hydrolysis, re suggestive evidence tht different glycosidic linkges exist in the vrious cell wll preprtions. The results obtined with the crude Streptomyces enzyme strongly suggest the bsence of f,1-3 glycosidic linkges in the cell wll of the yest phse of since both chitinse nd #, 1-3 gluconse re present in our preprtion. Presence of #,1-6 gluconse ctivity would pper to be limited (18). The differences in rectivity could, of J. BACTERIOL. course, be due to msking effect whereby certin linkges would not be exposed nd, therefore, resistnt to ttck. Nevertheless, these results would indicte differences in rrngement of cell wll components, if, in fct, no differences in linkge types cn eventully be demonstrted. Other workers (9), working with nother dimorphic fungus, Prcoccidioides brsiliensis, did indeed find differences in the glycosidic linkges present in the corresponding yest nd mycelil forms. Obviously, more specific nlyticl procedures re necessry to resolve this question in the cse of nd. The dt presented here on the quntities of glucose nd chitin in cell wlls re bsiclly in greement with those in the literture (10). There re some differences between these dt nd those published previously for one strin of (3), prticulrly with respect to the chitin content nd extrctbility of the wlls with ethylenedimine, which re prtly due to improved methodology but my lso be prtly due to chnge in cell wll composition over the yers, especilly with the mycelil phse. Although the ctul vlues reported in the erlier work were somewht different thn those reported here, the sme bsic conclusions concerning the yest nd mycelil phses re still vlid. Additionlly, the dt derived from the 2-, 4-, nd 6-dy-old yest-phse cultures of SwA nd Found confirmed the hypothesis tht observed differences between corresponding yest- nd mycelil-phse wlls were not function of the time of incubtion. ACKNOWLEDGMENTS This investigtion ws supported by Public Helth Service reserch grnt Al from the Ntionl Institute of Allergy nd Infectious Diseses. The uthor wishes to express pprecition to T. Grf nd S. Weiner for technicl ssistnce. LITERATURE CITED 1. Berger, L. R., nd D. M. Reynolds The chitinse system of strin of Streptomyces griseus. Biochem. Biophys. Act 29: Berliner, M. D Primry subcultures of Histoplsm cpsultum, 1. Mcro nd micro-morphology of the mycelil phse. Sbourudi 6: Domer, J. E., J. G. Hmilton, nd J. C. Hrkin Comprtive study of the cell wlls of the yest-like nd mycelil phses of Histoplsm cpsultum. J. Bcteriol. 94: Dubois, M., K. Gilles, J. K. Hmilton, P. A. Rebers, nd F. Smith A colorimetric method for the determintion of sugrs. Nture (London) 168: Elson, L. A., nd W. T. J. Morgn CCXLVIII. A colorimetric method for the determintion of glucosmine nd chondrosmine. Biochem. J. 27: Flint, D. R., T. C. Lee, nd C. G. Huggins A simple nd rpid method for the determintion of myo-inositol

8 on September 16, 2018 by guest VOL. 107, 1971 WALLS OF H. CAPSULATUM AND B. DERMATITIDIS 877 by gs-liquid chromtogrphy. J. Amer. Oil Chem. Soc. 42: Friedmn, L., nd D. Pppginis The inhibitory effect of peptone on the sporultion of three strins of Coccidioides immilis. Amer. Rev. Tuberc. 74: Good, T. A., nd S. P. Bessmn Determintion of glucosmine nd glctosmine using borte buffers for modifiction of the Elson-Morgn nd Morgn-Elson rections. Anl. Biochem. 9: Knetsun, F., nd L. M. Crbonell Cell wll glucns of the yest nd mycelil forms of Prcoccidioides brsiliensis. J. Bcteriol. 101: Knetsun, F., L. M. Crbonell, R. E. Moreno, nd J. Rodriguez Cell wll composition of the yest nd mycelil forms of Prcoccidioides brsiliensis. J. Bcteriol. 97: I 1. Kufmn, L., nd S. Blumer Occurrence of serotypes mong Histoplsm cpsultum strins. J. Bcteriol. 91: Kessler, G., nd W. J. Nickerson Glucomnnnprotein complexes from cell wlls of yests. J. Biol. Chem. 234: Korn, E. D., nd D. H. Northcote Physicl nd chemicl properties of polyscchrides nd glycoproteins. of the yest-cell wll. Biochem. J. 75: Pine, L., nd C. J. Boone Cell wll composition nd serologicl rectivity of Histoplsm cpsultum serotypes nd relted species. J. Bcteriol. 96: Restrepo-Moreno, A., nd J. D. Schneidu, Jr Nture of the skin-rective principle in culture filtrtes prepred from Prcoccidioides brsiliensis. J. Bcteriol. 93: Reynolds, D. M Exocellulr chitinse from Streptomycessp. J. Gen. Microbiol. 11: Seibert, F. B., nd L. F. Affronti Anthrone method for determining polyscchride 11, p , section 11. In Antigen Study Group of the Americn Thorcic Society (ed.), Methodology mnul for investigtion of mycobcteril nd fungl ntigens. Americn Thorcic Society, New York. 18. Skujins, J. J., H. J. Potgieter, nd M. Alexnder Dissolution of fungl cell wlls by streptomycete chitinse nd 6(1-3) glucnse. Arch. Biochem. Biophys. 111: Sweeley, C. C., R. Bentley, M. Mkit, nd W. W. Wells Gs-liquid chromtogrphy of trimethylsilyl derivtives of sugrs nd relted substnces. J. Amer. Oil Chem. Soc. 85: Downloded from