Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin

Introduction

The autolysis taking place in submerged carbon-de-pleted cultures of Penicillium chrysogenum is influ-

enced by numerous intrinsic (vacuolation, age-relatedhydrolase production, aging) and extrinsic (powerinput, shearing forces, nutrient and O2 limitations,penicillin side-chain precursors) factors (Harvey et al.,1998; McIntyre et al., 1999, 2000; Nielsen andKrabben, 1995; Paul et al., 1994; Paul and Thomas,1996; Pócsi et al., 2000; Pusztahelyi et al., 1997a, b;White et al., 1999). In some aspects, the autolysis of P.chrysogenum is reminiscent of the apoptosis of highereucaryotes under these conditions, e.g., it is an en-

Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production

and antifungal effect of allosamidin†

László Sámi,1 Tünde Pusztahelyi,1,2 Tamás Emri,1 Zoltán Varecza,1 Andrea Fekete,3

Ágnes Grallert,3 Zsolt Karányi,4 László Kiss,2 and István Pócsi1,*

1 Department of Microbiology and Biotechnology, Faculty of Sciences, University of Debrecen, P. O. Box 63, H-4010 Debrecen, Hungary

2 Department of Biochemistry, Faculty of Sciences, University of Debrecen, P. O. Box 55, H-4010 Debrecen, Hungary

3 Department of Genetics, Faculty of Sciences, University of Debrecen, P. O. Box 56, H-4010 Debrecen, Hungary

4 First Department of Medicine, Faculty of Medicine, University of Debrecen, P. O. Box 19, H-4012 Debrecen, Hungary

(Received February 6, 2001; Accepted July 9, 2001)

In carbon-depleted cultures of Penicillium chrysogenum, age-related chitinases were shown toplay a crucial role in both autolysis and fragmentation as indicated by in vivo enzyme inhibitionexperiments using allosamidin. This pseudotrisaccharide even hindered significantly the out-growth of new hyphal tips from the surviving yeastlike fragments after glucose supplementation.The antifungal effect of allosamidin on autolyzing P. chrysogenum mycelia was fungistatic ratherthan fungicidal. In growing hyphae, membrane-bound microsomal chitinase zymogen(s) weredetected, which may be indicative of some compartmentalization of these hydrolases. Later, dur-ing autolysis, no zymogenic chitinase was detected in any enzyme fraction studied, includingmicrosomes. These observations may explain the different sensitivity of growing and autolyzingmycelia to allosamidin. Chitinases taking part in the age-related fragmentation of hyphae and theoutgrowth of surviving hyphal fragments seem to be potent targets for future antifungal drug re-search.

Key Words——allosamidin; autolysis; chitinase; cryptic growth; glutathione; hyphal fragmentation; Peni-cillium chrysogenum; zymogen activation

J. Gen. Appl. Microbiol., 47, 201–211 (2001)

† The first two parts of this series of papers were published byPusztahelyi et al. (1997a, b).

* Address reprint requests to: Dr. István Pócsi, Department ofMicrobiology and Biotechnology, Faculty of Sciences, Universityof Debrecen, P. O. Box 63, H-4010 Debrecen, Hungary.

E-mail: [email protected]

Full Paper

ergy-consuming process in contrast to necrotic celldying (McIntyre et al., 1999). After the autolytic phaseof growth, a new equilibrium between cell lysis andgrowth can be observed, which was termed as a cryp-tic growth phase (Pusztahelyi et al., 1997a, b). Round-ended yeastlike hyphal fragments typically consistingof two cells are the dominant surviving morphologicalforms in these cultures that are subject to aging duringa prolonged incubation (Pócsi et al., 2000; Pusztahelyiet al., 1997a, b).

Autolysis and fragmentation of hyphae have beenshown to be associated with increased intracellular hy-drolase accumulation, including protease (Harvey etal., 1998; McIntyre et al., 2000; Pusztahelyi et al.,1997b), b-1,3-glucanase (Harvey et al., 1998), and N-acetyl-b-D-hexosaminidase (N-acetyl-b-D-glucosamini-dase, GlcNAc-ase) (Pusztahelyi et al., 1997b). Thesehydrolytic enzymes may play a crucial role in the mobi-lization of intracellular reserves and, later, in the weak-ening of the cell walls that results in the disintegrationof ordered mycelial structures (Harvey et al., 1998;McIntyre et al., 2000; Pusztahelyi et al., 1997b).

Allosamidin-sensitive chitinases have been demon-strated to take a sizable part in the age-related frag-mentation processes of the b-lactam producer filamen-tous fungi P. chrysogenum and Acremonium chryso-genum (Pócsi et al., 2000; Sándor et al., 1998). Thepseudotrisaccharide allosamidin, which is a well-known potent inhibitor of numerous fungal chitinases(Cabib et al., 1992; Dickinson et al., 1989; Escott etal., 1998; Gooday et al., 1992; Hodge et al., 1996;Sándor et al., 1998), also inhibited the outgrowth ofsurviving hyphal fragments in aging P. chrysogenumcultures relieved of carbon starvation by the addition ofan extra dose of glucose (Pócsi et al., 2000). To thebest of our knowledge, this is the first indication of apossible antifungal effect of allosamidin on a filamen-tous growth form. Earlier in vivo enzyme inhibitionstudies have demonstrated that allosamidin inhibitedeffectively the cell separation of the yeasts Saccha-romyces cerevisiae (Cabib et al., 1992; Izumida et al.,1995) and Candida albicans (Gooday et al., 1992), aswell as the age-related fragmentation of Acremoniumchrysogenum hyphae (Sándor et al., 1998). Thus farthe nature of growth inhibition by allosamidin (fungicid-al or fungistatic) has remained to be elucidated in theaging mycelia of P. chrysogenum (Pócsi et al., 2000).More recently, the formation of Penicillium marneffeicells with yeastlike morphology was also reported dur-

ing the infection of human tissues (Cooper and Hay-cocks, 2000), which may give this study additional im-portance.

Materials and Methods

Organism, growth conditions, monitoring hydrolaseproduction, and other physiological parameters. P.chrysogenum NCAIM (National Collection of Agricul-tural and Industrial Microorganisms) 00237 was main-tained and grown as described before (Pócsi et al.,2000; Pusztahelyi et al., 1997a, b). Culture flasks(500 ml) containing 100 ml of a complex culturemedium (Pusztahelyi et al., 1997a) were inoculatedwith 108 spores and incubated at 250 rev./min at 25°C.To investigate the effect of allosamidin and glucose onthe carbon-depleted cultures, 9.6 mM allosamidin (EliLilly and Company, Indianapolis, IN, USA) and 51 mM

extra glucose were added to selected flasks at 35 (de-celeration phase) and 115 h (autolytic phase) incuba-tion times, respectively (Pócsi et al., 2000; Pusztahelyiet al., 1997a, b; Sándor et al., 1998). Samples weretaken at 21, 24, 28, 35, 44, 66, 88, 115, 120, 133, and164 h of incubation and were centrifuged or filtered,depending on age, as described before (Pusztahelyi etal., 1997a, b). Filtrates and supernatants (5–10 ml)were used directly in GlcNAc-ase activity measure-ments and in pH and glucose concentration determina-tions (Pócsi et al., 1993, 1999; Pusztahelyi et al.,1997b). Alternatively, they were dialyzed twice against250-fold volumes of 0.1 M citric acid-sodium citrate (pH5.5) buffer and were used further for the estimation ofchitinase activity (Pócsi et al., 1999; Sándor et al.,1998).

In chitinase assays, purified colloidal crab shellchitin (0.15%, w/v) was dissolved in 0.1 M citric acid-sodium citrate buffer (pH 5.5), and the reaction mix-tures (2.0 ml) were also supplied with 0.6 nkat P.chrysogenum GlcNAc-ase as an auxiliary enzyme(Pócsi et al., 1999; Sándor et al., 1998). After 1 h of in-cubation with shaking at 37°C, the reactions were ter-minated by boiling, and the liberated N-acetyl-D-glu-cosamine (GlcNAc) was determined spectrophotomet-rically (Pócsi et al., 1999; Sándor et al., 1998). In Glc-NAc-ase activity measurements, p-nitrophenyl N-acetyl-b-D-glucosaminide was used as a substrate asdescribed earlier [1.0 mM substrate concentration in0.1 M citric acid-sodium citrate buffer (pH 4.5); 37°C](Pócsi et al., 1993, 1999; Pusztahelyi et al., 1997b).

202 SÁMI et al. Vol. 47

For glutathione (GSH) and glutathione disulfide(GSSG) determinations, mycelia were separated from5–10 ml aliquots of the cultures by centrifugation(10,000�g, 10 min, 4°C), were resuspended in ice-cold 5% (w/v) 5-sulfosalicylic acid by vigorous mixing,and were left at 0°C for 30 min (Emri et al., 1999).After centrifugation at 10,000�g for 10 min, the super-natants were neutralized with triethanolamine at 0°C,and the intracellular GSH and GSSG concentrationswere determined according to Anderson (1985).

Extracellular pH values, glucose concentrations, drycell weights (DCWs), and intracellular protein concen-trations were determined as described in previouspublications (Emri et al., 1997, 1999; Pusztahelyi etal., 1997a, b).

For in vitro enzyme inhibition studies with al-losamidin, centrifuged 115 h culture fluid (nontreatedby allosamidin during cultivation) was used as an ex-tracellular chitinase sample. Allosamidin stock solutionwas diluted with 0.1 M citric acid-sodium citrate buffer(pH 5.5) and was added to the chitinase reaction mix-ture covering the concentration range of 0–20 mM. IC50

value was calculated by using the GraFit, Version 2.10data analysis and graphics program (Erithacus Soft-ware Ltd., Horley, UK).

A chemical analysis of the cell wall was performedduring cryptic growth both in the allosamidin-treatedand the nontreated cultures. P. chrysogenum cells har-vested at 164 h incubation time were boiled in 0.5 M

NaOH for 1 h. The alkali insoluble residue waslyophilized, and the chitin content was calculated fromelemental analysis data as before (Pócsi et al., 1999;Sándor et al., 1998).

Quantification of fragmentation and testing cell sur-vival in the presence and absence of allosamidin.The number of single cells, round-ended hyphal frag-ments consisting of two cells and other hyphal ele-ments with more than two cells (regarded as “hy-phae”), was determined and compared in allosamidin-treated and nontreated cultures at 116, 134, and 165 hincubation times. In four independent experiments,900–1,800 hyphal elements were analyzed in total ateach incubation time tested.

The cell numbers were also estimated by viablecounts (Nógrády et al., 1998) at 116, 134, and 165 h ofincubation. Tenfold serial dilutions of cell suspensionswere prepared in sterile distilled water. Five drops(100 ml in total) of appropriate dilutions were thenpipetted onto 2.0% nutrient agar containing the same

components as the complex culture medium used inshake flask experiments. The plates were incubated at25°C for one week.

Cell survival and vitality in the presence of al-losamidin was also estimated and compared with con-trols at 88, 115, 120, and 133 h incubation times.Mycelia from 30 ml culture aliquots were washed andtransferred into allosamidin-free complex culturemedium, and changes in DCW were registered for35–50 h (Emri et al., 1997, 1999). The outgrowth fre-quencies of the hyphal fragments (no outgrowth, out-growth at one end, outgrowths at two ends, branchinghyphae) were evaluated after 15 h incubation withshaking. The outgrowth of 100–130 hyphal fragmentswas registered at each incubation time selected (88,115, and 133 h).

Microsomal, cytosolic, and extracellular chitinases inthe deceleration and autolytic phases of growth.These experiments were performed in Dr. David J.Adams’ Laboratory in the Department of Microbiology,University of Leeds, United Kingdom. Deceleration(38 h) and autolytic (76 h) phase cultures were har-vested and disrupted by using a Dyno-Mill Type KDLdisintegrator (Willy A. Bachofen, Basel, Switzerland)as described before (Dickinson et al., 1989, 1991; Es-cott and Adams, 1995). Cell lysates were centrifugedat 9,000�g for 20 min at 4°C to remove unbroken cellsand mycelial debris. Following that, supernatants werecentrifuged further at 145,000�g for 1 h at 4°C to sep-arate cytosolic and microsomal enzyme fractions(Dickinson et al., 1989, 1991; Escott and Adams,1995).

In these experiments, chitinase activities were mea-sured according to Dickinson et al. (1989) by using[3H]chitin. Reaction mixtures contained 60 ml aliquotsof [3H]chitin suspensions [2.6 mg ml�1; 1.9 mCi (mgchitin)�1; 70 kBq (mg chitin)�1], 80–100 ml assay buffer(50 mM Bis-Tris-HCl, pH 6.5), and 20–40 ml enzymepreparation. After 1 h incubation at 37°C with vigorousshaking, the reactions were terminated by the additionof 180 ml 10% (w/v) trichloroacetic acid, and the re-maining chitin was separated by filtration through glasswool. Samples (180 ml) of the filtrates were mixed with4 ml LKB Optiphase Safe scintillant (Amersham Inter-national, Amersham, United Kingdom), and radioactiv-ity was measured in an LKB Rachbeta 1217 liquidscintillation counter (Dickinson et al., 1989). GlcNAcequivalents of solubilized chitin were calculated.

Chitinase zymogens were chased in both decelera-

2001 Chitinase production of P. chrysogenum 203

tion and autolytic phases (Dickinson et al., 1991). Mi-crosomal enzyme preparations (200 ml) were incubatedwith 200 ml aliquots of 50 mM Bis-Tris-HCl (pH 6.5)buffer containing bovine pancreatic trypsin (final con-centrations were of 50–500 mg ml�1) for 10 min at25°C. The activation was stopped by the addition ofsoybean trypsin inhibitor (50 ml; final concentrationswere twice trypsin concentrations), and the reactiontubes were cooled. Reaction mixtures were assayedfor chitinase activity by using tritiated chitin as de-scribed before (Dickinson et al., 1991).

Microscopy. The cell morphology was examinedunder an OLYMPUS BH-2 microscope equipped with aSPlan 20NH phase contrast objective (Pócsi et al.,2000).

Statistics. The variations between experimentswere estimated by standard deviations (SD) as before(Emri et al., 1997, 1999; Sándor et al., 1998). The sta-tistical significance of changes in physiological param-eters, including DCW, hydrolase production, and viablecell count, were estimated by the Student’s t-test. Onlythe probability levels of p�5% were regarded as in-dicative of the statistical significance. For a statisticalanalysis of fragmentation of the SAS for Windows sys-tem, Version 6.12 was used. The significance of thedifferences between cell counts changing in time andas a function of the presence of allosamidin was ex-amined by the two-way analysis of variance (Sándor etal., 1998).

Chemicals. Unless otherwise indicated, all chemi-cals were purchased from the Sigma-Aldrich Ltd., Bu-dapest, Hungary, and Sigma Chemicals, Poole, UnitedKingdom.

Results

Growth, hydrolase production, and intracellular GSHlevels

Physiological changes as a result of the addition of9.6 mM allosamidin at 35 h incubation time and/or51 mM glucose at 115 h of incubation to P. chryso-genum cultures are summarized in Figs. 1–4. In accor-dance with previous observations by Pócsi et al.(2000), allosamidin significantly hindered the loss ofbiomass in the autolytic phase of growth after 88 h ofincubation (Fig. 1). In contrast to control cultures, theaddition of an extra dose of glucose initiated no in-crease in DCW in the presence of the chitinase in-hibitor (Fig. 1) (Pócsi et al., 2000).

As shown in Fig. 2, both the extracellular chitinaseand GlcNAc-ase activities started to increase in thestationary phase of growth and reached a plateau of 330–420 pkat/ml and 250 nkat/ml, respectively, at115 h of incubation. After the addition of extra glucose,both tested enzyme activities dropped (Fig. 2A and C).It is worth noting that allosamidin did not significantlyinfluence the extracellular hydrolase production (Fig.2B and D). Nevertheless, when allosamidin-treatedcultures were supplemented with glucose, the extra-cellular chitinase and GlcNAc-ase activities decreasedsignificantly, but more moderately, than in the absenceof the chitinase inhibitor (Fig. 2).

Age-related changes in the glutathione metabolismof P. chrysogenum (Fig. 3) are discussed elsewhere(Pócsi, I., pers. comm.). Here we put emphasis only onhow the GSH metabolism of the fungus was affectedby allosamidin. As shown in Fig. 3B and D, the addi-


Fig. 1. Changes in DCWs because of the addition of 9.6 mM

allosamidin and/or 51 mM glucose at 35 and 115 h incubationtimes, respectively (A and B).

The symbols stand for �, control; �, addition of allosamidin at35 h; �, glucose supplementation at 115 h; �, allosamidin andglucose supplementation at 35 and 115 h of incubation. In con-trol (�) and glucose supplementation (�) experiments, symbolsand bars represent Mean�SD values, calculated from 6 inde-pendent experiments. To demonstrate the physiological effectsof allosamidin (�, �) one typical set of curves is presented.


Fig. 2. The effects of allosamidin (9.6 mM; 35 h of incubation) and/or glucose supplementation (51 mM; 115 h) on the extracellularchitinase (A and B) and GlcNAc-ase (C and D) activities.

Symbols and the statistical evaluation of data are the same as in Fig. 1.

Fig. 3. Specific intracellular GSH (A and B) and GSSG (C and D) concentrations as well as GSH/GSSG redox balances (E andF) in control and allosamidin-treated and glucose-supplemented cultures.

Symbols and the statistical evaluation of data are the same as in Fig. 1.

tion of the chitinase inhibitor decreased both the spe-cific GSH and GSSG concentrations during autolysisand cryptic growth. It is important to note that thesechanges were clearly attributable to allosamidin-de-pendent increases in DCWs (Fig. 1B) and were sta-tistically insignificant at any incubation time tested. The addition of allosamidin did not influence theGSH/GSSG redox balance either, with the exception of164 h incubation time when this parameter was signifi-cantly lower in allosamidin-treated culture (Fig. 3F).After carbon supplementation at 115 h of incubation,the specific GSH concentrations did not decrease inthe presence of the chitinase inhibitor (Fig. 3F), but avast decrease was recorded in glucose-supplementedcontrol cultures (Fig. 3E). Because the intracellularGSSG levels greatly decreased (Fig. 3D), significantincreases in the GSH/GSSG redox ratios were ob-served in the presence of allosamidin (Fig. 3F). Mean-while, a drop in the GSH/GSSG redox ratio was foundin controls (Fig. 3E).

Experimental data presented in Fig. 4 clearly indi-cate that both allosamidin-treated and nontreated cul-tures effectively metabolized the 51 mM extra glucoseadded to carbon-depleted cultures at 115 h incuba-tion time. Nevertheless, the decreases in the glucoseconcentration (Fig. 4C) and the extracellular pH (Fig.4A and B) observed from 115 to 117 h of incubationwere profoundly less in the presence than in the ab-sence of the chitinase inhibitor (Dcglucose, allosamidin�

15.5 mM, Dcglucose, control�29.0 mM; DpHallosamidin�0.87,DpHcontrol�1.59).

Inhibition of extracellular chitinase activity by al-losamidin

In physiological experiments, chitinase activitieswere always measured in culture fluid samples dia-lyzed exhaustively against 0.1 M citric acid-sodium cit-rate (pH 5.5) buffer. In samples with no treatment orsubjected to buffer exchange on PD-10 columns(Pharmacia Biotech, Uppsala, Sweden) instead of dial-ysis, the apparent chitinase activity was negligibleeven at 165 h incubation time because of the inhibitoryeffect of allosamidin. In in vitro enzyme inhibition ex-periments, allosamidin was proved to be a potent in-hibitor of extracellular chitinase activity with an IC50

value of 1.4 mM. At 9.6 mM allosamidin concentration,which was selected in physiological experiments, theinhibition was complete.

Chitin content of the cell wallAn elemental analysis of alkali insoluble cell wall

residues indicated that the chitin contents of 165 h al-losamidin-treated and nontreated dried mycelia wereabout 7.0 and 2.6%, respectively. In cryptic growthphase cultures (after 148 h of incubation; Pusztahelyiet al., 1997a, b), the greatly increased chitin content ofthe cell wall resulted in no morphological changes ob-servable by phase contrast microscopy. The breakageof allosamidin-treated mycelia eventually gave rise toround-ended hyphal fragments very similar to those incontrol cultures (Pócsi et al., 2000).

Fragmentation, cell lysis, cell survival, and vitalityThe statistical evaluation of morphological data by a

two-way analysis of variance demonstrated that thetotal number of hyphal elements and the number of hy-phal fragments consisting of two cells decreased sig-nificantly from 116 to 165 h incubation times. It is im-portant to note that allosamidin significantly hinderedthese changes (Fig. 5). Meanwhile, the number of hy-phal elements consisting of the minimum three cells(termed as “hyphae”) decreased, and the number ofone-cell fragments increased slightly as a function oftime, but these changes were statistically not signifi-cant. The number of these morphological forms wasnot influenced by allosamidin either.

The cell numbers measured by viable counts weresignificantly lower than the numbers of hyphal ele-


Fig. 4. Decreased acidification (A and B) and glucose uti-lization (C) rates in the presence of allosamidin.

The symbols are the same as in Fig. 1. Symbols and barsrepresent Mean�SD values calculated from 3 independent ex-periments in Fig. 4A and B, and one typical set of curves is pre-sented in Fig. 1C.

ments based on microscopic cell counting at each in-cubation time tested, with the exception of 116 h al-losamidin-treated samples (Table 1). It is interestingthat no significant differences were found between theviable cell numbers of allosamidin-treated and controlcultures (Table 1). In accordance with this, allosamidin-treated and nontreated mycelia transferred into al-losamidin-free culture medium at 88, 115, 120, and

133 h of incubation showed very similar growth pat-terns and outgrowth frequencies (Fig. 6).

Cellular distribution of chitinasesThe distribution of chitinase activities between mi-

crosomal, cytosolic, and extracellular fractions were0.06, 15.04, and 84.90%, respectively, in the decelera-tion phase (38 h) and 0.94, 25.01, and 74.05%, re-spectively, in the autolytic phase of growth (76 h). Thetotal chitinase activity found at 76 h of cultivation [6.0mkat (kg protein)�1] was 4 times higher than that foundat 36 h [1.5 mkat (kg protein)�1]. Chitinases associatedwith the cell surface and the cell wall (Hearn et al.,1996) were not analyzed in this study.

Zymogen activation by trypsinThe effects of trypsin on the deceleration phase and

the autolytic phase microsomal chitinase activities arecompared in Fig. 7. Although the specific activity of de-celeration phase enzyme preparation increased siz-ably in a dose-dependent manner in the presence oftrypsin, no significant change, neither activation nor in-activation, was observed with the autolytic phase en-zyme.

Discussion

A comparative statistical analysis of the average


Fig. 5. Changes in the average number of hyphal frag-ments (A) and in the distribution of hyphal elements betweenone-cell and two-cell fragments and hyphae (B) as a function ofincubation time and the addition of allosamidin at 35 h of incu-bation.

Letters C and A stand for control and allosamidin-treated cul-tures, respectively. By definition, fragments consisting of threeor more cells were regarded as “hyphae.” The mean values,calculated from four independent experiments, are shown inFig. 5A and B; the bars represent SD values in Fig. 5A.

Table 1. Comparison of P. chrysogenum cell counts determined by microscopic counting (A)

and viable counting (B).

Assay methodsCell numbersa

and samplesControl Allosamidin-treated

A. Microscopic count[10�6�hyphal elements (ml)�1]116 h culture 11.7�1.1 9.8�0.3134 h culture 10.0�0.6 9.6�0.7165 h culture 6.4�1.0 5.9�0.4

B. Viable count[10�6�colony-forming units (ml)�1]116 h culture 9.0�0.1 7.7�2.7134 h culture 6.9�1.1 7.1�0.7165 h culture 2.1�0.1 2.4�0.3

a Cell numbers are expressed as Mean�SD, calculated fromfour independent experiments.

numbers of hyphal fragments and the distribution ofhyphal elements between different morphologicalforms in the presence and in the absence of al-losamidin has provided us with prima facie evidenceconcerning the involvement of chitinase(s) in the frag-mentation process and autolysis of P. chrysogenum(Fig. 5) (Pócsi et al., 2000). Chitinases are thought toparticipate in the morphogenesis of chitin-containingfungi in several ways, including the swelling and ger-mination of spores, branching and apical extension ofgrowing hyphae, cell separation of yeasts and age-re-lated fragmentation, and autolysis of hyphae (Gooday,

1997; Gooday et al., 1992). In fact, most of the evi-dence supporting these roles has come from in vivoenzyme inhibition experiments by allosamidin (Goodayet al., 1992; Pócsi et al., 2000; Sándor et al., 1998),besides the disruption of genes coding for fungal chitin-ases (Gooday, 1995; Kuranda and Robbins, 1991;Takaya et al., 1998).

Relying on the unitary model for hyphal growth pro-posed by Bartnicki-García (1973 and 1999), which pre-sumes an enzymatic loosening of the existing wall as amajor component of the growth of the hyphal apex, al-losamidin and its semisynthetic derivatives have beenregarded as potential antifungal agents since the endof the ’80s (Dickinson et al., 1989; Gooday, 1989; Rastet al., 2000). Unfortunately, all attempts to inhibit theexponential growth of either yeasts or filamentousfungi (including P. chrysogenum) by allosamidin havefailed (Dickinson et al., 1989; Escott et al., 1998; Goo-day et al., 1992; Pócsi et al., 2000; Sándor et al.,1998). Although chitinases are always found in grow-ing fungal mycelia together with chitin synthase (Goo-day et al., 1986; Hearn et al., 1997; Rast et al., 1991) itis possible that they are protected from the inhibitoryeffect of allosamidin (Gooday, 1995; Gooday et al.,1997). Alternatively and according to the steady-statemodel for hyphal growth proposed by Wessels (1984,1986, 1999), these hydrolases may play no role in theapical wall extension. Nevertheless, growth-relatedfungal chitinases seem to be unsuitable targets for an-tifungal drug design, and allosamidin is therefore un-likely to be a suitable tool to get clear-cut evidence onthe involvement of chitinases in the apical growth.

In our experiments, allosamidin was added in thedeceleration phase of growth (at 35 h incubation time),


Fig. 6. The growths (A) and outgrowth frequencies (B) ofallosamidin-treated and nontreated P. chrysogenum myceliaafter transferring them into allosamidin-free complex culturemedium.

In Fig. 6A, symbols stand for �, control; �, the addition of al-losamidin at 35 h of incubation, and growths observed aftertransfer at 133 h are shown. Very similar growth patterns wereobserved following transfers at 88, 115, and 120 h of incuba-tions. In Part B, letters A and C stand for allosamidin-treatedand control cultures, respectively, and a typical set of distribu-tion data is presented; these data were recorded after 15 h incu-bation of mycelia in allosamidin-free culture medium.

Fig. 7. In vitro zymogen activation with trypsin in decelera-tion (�) and autolytic (�) phase chitinase preparations.

Reaction conditions were essentially the same as before withCandida albicans microsomal chitinase (Dickinson et al., 1991).Values shown are Mean�SD calculated from 3 parallel experi-ments and are expressed as a percentage of the specific en-zyme activity in a parallel reaction mixture that did not containtrypsin.

which gave us the opportunity to map the physiologicaleffects of this pseudotrisaccharide on aging P. chryso-genum mycelia via the inhibition of age-related chitin-ases. Besides the hindrance of fragmentation and au-tolysis, a sizable increase in the chitin content of thefungus (7.0 versus 2.6% of DCW in comparison withcontrol cultures) was also observed. The thickening ofthe chitin layer within the wall might be a consequenceof two independent events. Namely, the equilibriumbetween cell wall biosynthesis and lysis (Bartnicki-García, 1973, 1999; Peter and Schweikart, 1990)might be thrown out of balance because cryptic growthcontinued during carbon starvation (Pusztahelyi et al.,1997a, b; White et al., 1999), and/or the autolyticdegradation of cell wall matrix polymers (Harvey et al.,1998; Reyes et al., 1988, 1989) was hindered by thechitinase inhibitor. The increased chitin content of thecells together with the substantial inhibition of chitin-ases (IC50�1.4 mM for extracellular enzymes) precededthe blockage of growth observed later, after glucosesupplementation at 115 h of incubation (Fig. 1B) (Pócsiet al., 2000).

In contrast to apical extension in exponentially grow-ing cultures where the involvement of lytic enzymes isdebated, chitinases are widely accepted to play an im-portant role in branch formation and germination,where the local softening of the rigid cell wall is of pri-mary importance (Gooday et al., 1997; Wessels,1984). Our data fully support this view. After the addi-tion of an extra dose of glucose to allosamidin-treatedcultures, new hyphal tips emerged only very rarelyfrom the surviving fragments as small protrusions withno significant apical extension even after a prolongedincubation period (Pócsi et al., 2000).

The physiological effects of allosamidin were disad-vantageous for the microorganism. For example, afterglucose supplementation at 115 h, the decreases inthe extracellular chitinase and GlcNAc-ase activities(Fig. 2B and D), pH (Fig. 4B), and glucose concentra-tion (Fig. 4C) were moderated in comparison with con-trol cultures (Figs. 2A, 2C, 4A, and 4C). Moreover, theintracellular GSH levels did not change in allosamidin-treated cultures after carbon supplementation (Fig.3B), though GSH was extensively used in controls asan easily available endogenous N and S source afterthe reinitiation of growth (Fig. 3A).

Nevertheless, GSH/GSSG redox ratios even in-creased greatly in allosamidin-treated and carbon-sup-plemented cultures (Fig. 3F), though they decreased in

controls (Fig. 3E) when the autolytic loss of biomasswas reversed by the addition of an extra dose of glu-cose (Fig. 1A). This observation and because al-losamidin did not significantly affect the intracellularGSH and GSSG levels during carbon starvation (Fig.3B and D) made any severe cell injuries as a result ofallosamidin unlikely. Therefore allosamidin-treated andnontreated mycelia showed very similar growth pat-terns and outgrowth frequencies following transfer intoan allosamidin-free culture medium (Fig. 6); neitherwas there any difference between the viable cell num-bers (Table 1). Taking into consideration all these find-ings, we concluded that the overall effect of al-losamidin on autolyzing P. chrysogenum mycelia wasfungistatic and not fungicidal.

Similar to previous observations (Dickinson et al.,1991; Humphreys and Gooday, 1984; Manocha andBalasubramanian, 1988), P. chrysogenum microsomalchitinase(s) have been demonstrated to be mem-brane-bound zymogens at least during exponentialgrowth (Fig. 7). The association of chitinase(s) withmembranes may lead to some form of cellular com-partmentalization of the hydrolases, which may conferprotection against the effects of allosamidin in vivo(Dickinson et al., 1991). Later, during autolysis, no zy-mogen activation was observed (Fig. 7), i.e., all thechitinases had been activated and might have beenexposed to allosamidin. This may explain the fungista-tic effect of this chitinase inhibitor in the autolyzing cul-tures of P. chrysogenum.

The results presented here further strengthen theview that chitinases play an important role in the age-related breakage of hyphae and the reinitiation of thegrowth of hyphal fragments, which may contribute tothe invasion of host organisms by filamentous fungalpathogens. These hydrolases together with chitinolyticenzymes participating in the separation and spread ofyeast cells should be regarded as promising targets forantifungal drug research. The 1.4 mM IC50 value for P.chrysogenum extracellular chitinase activity is at mid-position within the range of IC50 values (0.010–67 mM)observed for fungal chitinases with allosamidin (Cabibet al., 1992; Dickinson et al., 1989; Hodge et al., 1996;McNab and Glover, 1991; Rast et al., 2000), and thepreparation of new, even more effective semisyntheticallosamidin derivatives is in progress (Rast et al.,2000; Sakuda and Sakurada, 1998). This means thatthe antifungal effect of compounds targeting fungalchitinolytic enzymes can soon be increased further.


Acknowledgments

The authors are indebted to Dr. David J. Adams (University ofLeeds) for his valuable help in the analysis of the cellular distri-bution and zymogen activation of chitinases and in the prepara-tion of the manuscript. One of us (T.P.) spent 5 months at theUniversity of Leeds with the financial support of the HungarianScientific Research Fund (OTKA) and also received an OTKApostdoctoral fellowship (grant reference number D34568). TheHungarian Ministry of Education granted Széchenyi Scholar-ships for Professors to L.K. and I.P., which are greatly acknowl-edged. Allosamidin was generously supplied by the Lilly Re-search Laboratories (Indianapolis, IN, USA), and we especiallythank the valuable contributions of Dr. H. A. Kirst and Mrs. M.H. Niedenthal. This project was supported financially by OTKA(grant reference numbers F017620, T025174, T032106, andT034315) and the Hungarian Creative Art Foundation (13-914-97/P1).

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Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin

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