j med vet mycol 1991 kiryu 395 406, kedokteran hewa

5/19/2018 J Med Vet Mycol 1991 Kiryu 395 406, kedokteran hewa

1/1

Journal of Medical and Veterinary Mycology

1991),

29,

3 9 5 4 0 6

I n v a s i o n a n d s u r v iv a l o f

usar ium solan i

n t h e

d e x a m e t h a s o n e t r e a t e d c o rn e a o f ra b b it s

H . K I R Y U 1, S . Y O S H I D A 2 , Y . S U E N A G A ~ A N D M . A S A H P

~Department of Dermatology and 2Department o f Microbiology, School of Medicine,

University of Occupational and Environmental Health, 1-1 Iseigaoka Yahata-nishi-ku,

Kitakyushu 807, Japan

Accepted 2 September 1991

Electron microscop ic observations, as we ll as

in vitro

experiments, on experimental

Fusarium

solani

keratitis o f rabbits were performed to study the m ode o f fungal invasion into the corneal

stroma, the interactions between

E solani

and inflamm atory cells und er the influence of topical

dexamethasone DXM ) treatment, and the survival mechanism of the fungi in the DXM -treated

cornea. Electron microscop y showed that, wh ile the fungus invaded into the corneal stroma, diges-

tion of collagen fibrils occurred aroun d the hyphae, where amorphous material w as often noted.

In DXM-nontreated cornea, the fungal hyphae were entrapped by pseudopodia of the neutrophils

and destruction of the hyphae was noted on da y 3 of infection, most hyphae having disappeared

by day 7. In the DX M-treated cornea, however, neutrophils could no t ingest and destroy the

hyphae. In qualitative nitroblue tetrazolium NB T)-reduction tests using rabbit peripheral blood

neutrophils , DXM significantly suppressed the rate o f NBT-reduction and the rate of adherence to

the fungal microconidia. In the D XM -treated corneal lesions, a considerable increase in both num-

ber and s ize of fungal peroxisomes was noted. Furthermore, the hyphae, surrounded by neu-

trophils , showed double or tr iple c ell wa ll formation or som etimes a hypha-in-hypha structure.

Similar hypha-in-hypha structures were also observed when the organisms were treated

in vitro

with a fungistatic concentration of H20> W e suggest that t his special structure is a protective

device produced for the survival of E solaniwhen subject to neutrophil attack in the DXM -treated

cornea.

Fusarium s p e c i e s a re w i d e l y d i s t r i b u t e d i n n a t u r e , b e i n g d e t e c t e d m a i n l y i n s o il s a n d o n

p la n ts . T h e y a r e i m p o r t a n t p l a n t p a t h o g e n s [2 3] a n d a r e n o w r e c o g n i z e d a s o p p o r -

t u n i st i c p a t h o g e n s c a u s i n g k e r a t i ti s [ 11 , 2 1 , 2 7 ], i n f e c t i o n s o f b u r n s [1 , 2 8 ] a n d s y s t e m i c

i n f e c ti o n in i m m u n o l o g i c a l l y c o m p r o m i s e d p a t i e n ts [1 8, 3 0] . A m o n g t h e

Fusarium

s p e c i e s , Fusarium solani is t h e m o s t c o m m o n l y f o u n d e t io l o g ic a g e n t o f m y c o t i c k e ra t i -

t is i n J a p a n w h e r e i ts i n c i d e n c e h a s i n c r e a s e d [ 15 ]. C o r n e a l t r a u m a i s p r o b a b l y a m a j o r

p r e d i s p o s i n g f a c t o r f o r E solani i n f e c t i o n [ 11 , 2 1 , 2 7 ] . I t is a l s o w e l l k n o w n t h a t c l i n ic a l

u s e o f c o r t i c o s t e r o i d e y e d r o p s e x a c e r b a t e s t h e i n f e c t i o n [ 9, 1 9] .

I n o c u l a t i o n o f c u l t u r e d

E solani

i n t o r a b b i t c o r n e a c a u s e d k e r a t it i s w h e r e t h e f u n g i

g r e w i n t h e c o r n e a l s t r o m a [ 8, 1 4 ]. I n t h e s e l e s io n s , t h e k i l li n g p r o c e s s b y t h e i n f i l t r a ti n g

n e u t r o p h i l s w a s n o t e d 3 d a y s a f te r i n o c u l a t io n a n d t h e l e si o ns h e a l e d s p o n t a n e o u s l y

t h e r e a ft e r . W h e n c o r t i c o s t e r o id e y e s o l u t io n w a s a p p l i e d t o pi ca l l y , t h e f u n g i g r e w m u c h

m o r e r a p i d l y a n d s u r v i v e d m u c h l o n g e r in t h e c o r n e a [ 14 ]. H o w e v e r , t h e m e c h a n i s m s

o f in v a s i o n o f th e f u n g i , k il li n g o f t h e f u n g i b y n e u t r o p h i l s , a n d e x a c e r b a t i o n o f t h e

e x p e r i m e n t a l k e r a t i ti s b y t o p i c a l c o r t i c o s t e r o i d s , a r e s t il l n o t c l e a r. T o i n v e s t ig a t e t h e s e

Correspondence address: H. Kiryu, Departm ent o f Dermatology, School of Medicine, University of

Occu pational and Environm ental Health, 1-1 Iseigaoka Yahata-nishi-ku, Kitakyushu 807 , Japan.

395

atPNUMedicalLibonSeptember10,2014

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2/1

96 K RYU

ET AL

p o i n t s f u r th e r , w e p e r f o r m e d e l e c t r o n m i c r o s c o p i c a n d c y t o c h e m i c a l s t u d i e s o n r a b b i t

c o r n e a e x p e r i m e n t a l l y i n f e c t e d w i t h

E s o l a n i

I n add i t i on ,

in v i t ro

s t u d i e s w e r e p e r -

f o r m e d t o i n v e st ig a te t h e e f fe c t o f d e x a m e t h a s o n e ( D X M ) o n t h e s u p e r o x id e g e n e r a t -

i ng a c t iv i ty o f r a b b i t n e u t r o p h i ls . T h e e f fe c t s o f h y d r o g e n p e r o x i d e ( H 2 0 2 ) t r e a t m e n t o n

f u n g a l s t ru c t u r e w e r e a l so s t u d i e d

in v i t ro

M E T H O D S

Fungus

A s t r a i n o f

E S o l a n i

i s o l at e d f r o m a c a s e o f m y c o t i c k e r a ti ti s w i t h c o r n e a l u l c e r a t i o n

a n d h a z e w a s u s e d i n t h i s s t u d y . T h e f u n g u s w a s c u l t u r e d o n S a b o u r a u d g l u c o s e a g a r

( S A B ) f o r 7 d a y s a t 2 7 C b e f o r e u s e .

A n i m a l s

S m a l l t y p e w h i t e ra b b i t s d e r i v e d b y m a t in g P o l i s h a n d D u t c h r a b b i t s w e r e u s e d . T h e y

w e r e b r e d f o r 1 3 g e n e r a t i o n s o f b r o t h e r - t o - s is t e r m a t i n g a t T o k y o M e d i c a l a n d D e n t a l

Un i ve r s i t y [ 14 ]. Tw e l ve r abb i t s o f bo t h s e xes we i gh i ng 1 - 5 -2 . 0 kg w e r e u s ed .

D e x a m e t h a so n e D X M ) t re a tm e n t

T w o d r o p s o f 0 . 1 ( w : v i n H 2 0 ) D X M s o d i u m so l u t i o n ( C o m m e r c i a l p r o d u c t f o r c lin -

i ca l u s e , S c h e r in g A G , G e r m a n y ) w e r e d r o p p e d i n t o b o t h e y e s o f s ix ra b b i t s t h r e e t i m e s

d a i ly a t 6 h i n te r v al s. T h e t r e a t m e n t s t a r t e d 2 d a y s b e f o r e f u n g a l i n o c u l a t i o n a n d w a s

c o n t i n u e d f o r 9 da y s. N o D X M t r e a t m e n t w a s a p p l ie d i n t h e c o n t r o l g r o u p .

In fec t ion exper im ent

I n t r a c o r n e a l i n o c u l a t i o n w i t h E s o l a n i w a s p e r f o r m e d b y th e m e t h o d d e s c r i b e d b y

I s h i ba s h i

et a l

[ 13 ] wi t h s l igh t m od i f i c a ti ons . Sp or e s a nd m y ce l i um o f

E s o l a n i

w e r e h a r -

v e s t e d a f t e r 1 w e e k o f c u l t u r e a n d t h e s p o r e fr a c ti o n , 9 5 - 9 8 m a c r o c o n i d i a a n d 2 - 5

m i c r o c o n i d i a , w a s s e p a r a t e d b y f il tr a ti o n t h r o u g h c o t t o n w o o l a n d t h e n s u s p e n d e d i n

D u l b e c c o ' s p h o s p h a t e b u f f e r e d s a li n e ( P B S ) . J u s t p r i o r t o in o c u l a t io n , a n i n t r a c o r n e a l

p o c k e t w a s m a d e w i t h a 2 7 - g a u g e i n j e c t i o n n e e d l e b y i n s e r t i n g i t i n t o t h e c o r n e a a n d

m o v i ng i t s i deways . The con i d i a s us pe ns i on was ad j us t ed t o 106 ce l ls m 1-1 and 10 p l o f

t h e s u s p e n s i o n w a s i n j e c t e d i n t o t h e c o r n e a l p o c k e t u s i n g a M i c r o S y r i n g e ( K u s a n o

K a g a k u K i k a i C o ., T o k y o ) . O n d a y s 3 a n d 7 p o s t - in o c u l a t i o n , t h e c o r n e a l l e s i o n s w e r e

b i o p s i e d b y tr e p h i n e ( G r i e s h a b e r , S w i t z e r la n d ) a n d a p o r t i o n e x a m i n e d b y l ig h t a n d

e l e c t r o n m i c ro s c o p y . T h e r e m a i n d e r o f t h e b i o p s i e d s p e c i m e n s w e r e c u l t u r e d o n S A B

p l a te s t o o b s e r v e f u n g a l g r o w t h .

Light microscopy

T h e s p e c i m e n s w e r e f ix e d w i t h 6 g l u t a r a l d e h y d e i n 0 .1 M c a c o d y l a t e b u f f e r ( p H 7 . 4)

f o r 6 h a t 4 C a n d p r o c e s s e d r o u t in e l y . B o t h h e m a t o x y l i n - e o s i n a n d p e r i o d i c a c i d -S c h i ff

( P A S ) s t a in i n g w e r e p e r f o r m e d .



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97

E l e c t r o n m i c r o s c o p y E M )

After washing twice in 0.1 M cacodylate buffer, all samples were post-fixed with 1

osmic acid in 0.1 M cacodylate buffer for 12 h at room temperature , dehydrated in

ethanol and propylene oxide, and then embedded in Epon 812 (Taab, England).

Ultrathin sections were cut on a LKB ultra tome (LKB, Sweden) with a diamond knife

(Diatome, Switzerland), post-stained with 1 uranyl acetate and examined under a

JEM-100CX (JEOL, Japan) electron microscope.

C y t o c h e m i c a l e le c t r o n m i c r o sc o p y

The tissues, fixed as above, were used to demonstrate the peroxidase activity of the neu-

trophils and the catalase activity of the fungi after washing with cacodylate buffer for

12 h at 4C. The specimens were incubated in 0-1 M cacodylate buffer (pH 7.4) with

0-5 mg m1-1 3,3'-diaminobenzidine (DAB, Sigma) and 0.01 H202 for 4 h at 37C to

demonstrate peroxidase activity of the neutrophils [4]. For examination of catalase

activity, the samples were incubated in 2 mg ml -~ DAB and 0.03 H202 in 0.05 M

propandiol buffer (pH 9.5) for 4 h at 37C. To demonstrate acid phosphatase activity in

the neutrophi ls [7], corneal tissues were fixed in 5 formaldehyde and 4 glutaral-

dehyde in 0.1 M cacodylate buffer, pH 7.4 with 7 sucrose for 6 h. They were washed

twice in cacodylate buffer and incubated for 4 h at 37C in a modified Gomori medium

containing 0.1 M acetate buffer, pH 5.0, 1 mM sodium glycerophosphate and 0.1 lead

nitrate. These cytochemically stained specimens were post-fixed with osmic acid and

trea ted as described above for EM.

n vitro H z O z t r ea t m e n t o f E solani

The spores of

F s o l a n i

were preincubated in Sabouraud glucose broth for 6 h at 30C.

After centrifugation, they were washed in PBS and suspended at a concentration of

1 107 m1-1 in Sabouraud glucose broth, which included 2mM

H 2 2

and 0.1 M phos-

phate buffer, pH 7.4. After incubation for 2 h at 30C, the samples were fixed with 6

glutaraldehyde for 6 h at 4C, and examined under an elec tron microscope.

Qu al i ta ti ve n i t rob lue t e t razo l ium NB T) -red uc t ion t e st s

Qualitative NBT-reduction was performed as described by Ando

e t a l

[2] with some

modifications. The NBT reagent (grade III, Sigma) was dissolved in PBS (3 mg ml 1),

centrifuged at 3 000 g for 10 min to remove any insoluble material, and then stored in

the dark. Rabbit polymorphonuclear leukocytes (PMN) were prepared by centrifuga-

tion on double Ficoll-Hypaque density gradients. The cell pellets were washed three

timed in PBS and resuspended at a concentration of 5 106 cells ml 1 in RPMI 1640.

DXM was dissolved in ethanol (70 mg m1-1) and diluted with water as appropriate.

Spores of E s o l a n i were suspended in PBS, pH 7.4, at a concentration of 5 106 spores

ml 1 and 0.2 ml of the suspension was preincubated with 0.1 ml of rabbit serum at 37C

for 10 min in plastic tubes. Then 0-2 ml of the PMN suspension, 0-2 ml of the NBT solu-

tion and 10/A of DXM solution (final concentrations were 0, 0.001 and 0.01 , respec-

tively) were added. The incubation was continued for a further 30 min at 37C. As a

control, zymosan was used instead of

F s o l a n i

spores. Smears of each sample were fixed

in methanol and s tained with Wright-Giemsa reagents. The smears were examined by



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98 KIRYUET AL

l ig h t m i c r o s c o p y t o c o u n t u p t o 1 0 0 P M N i n e a c h s m e a r , a n d t h e n u m b e r o f P M N

a d h e r i n g t o t h e s p o r e s w a s c o u n t e d a s p o s i t i v e a d h e r e n c e . F o r c a l cu l a ti n g N B T -

r e d u c t i o n , t h e n u m b e r s o f P M N w h i c h c o n t a i n e d a l a rg e b lu i s h p r e c i p i t a te o f f o r m a z a n

o n t h e a d h e r i n g s p o r e s w e r e c o u n t e d a s p o s i ti v e a m o n g 1 0 0 P M N c o u n t e d . E a c h e x p e r -

i m e n t w as done i n t r ip l ic a t e . D a t a w e r e an a l ysed s t a t is t ic a l l y by t he S t u den t ' s t t e s t .

R E S U L T S

l i n i c a l c o u r s e a n d h i s t o l o g i c a l f e a t u r e s o f solani k e r a t i t i s

I n t h e ra b b i t s th a t d i d n o t u n d e r g o D X M t r e a t m e n t , c o r n e a l u l c e rs a n d h a z e a p p e a r e d

2 o r 3 d a y s a f t e r i n o c u l a t i o n . T h e l e s i o n s b e g a n t o r e s o l v e a f t e r 7 - 1 0 d a y s a n d h a d

h e a l e d b y 1 4 d a y s . T h e P A S - s t a i n e d s p e c i m e n s s h o w e d t h a t t h e h y p h a e o f

E s o l a n i

s p r e a d m a i n l y i n p a r a l l e l a n d s o m e t i m e s p e r p e n d i c u l a r t o t h e c o l l a g e n f i b r e s i n t h e

s t ro m a , a n d r e a c h e d t h e d e e p l a y e r o f t h e c o r n e a a t d a y 3, w h e n i n f l a m m a t o r y c e l ls

w e r e r a r e ly o b s e r v e d . B y d a y 7 , n u m e r o u s i n f la m m a t o r y c e l ls h a d i n f i lt r a te d i n t o t h e

supe r f ic i a l and m i dd l e l aye r s o f the co r nea . I n t he s e s eve r e l y i n f i l tr a t ed a r ea s , no f un -

g a l e l e m e n t s r e m a i n e d , b u t s o m e h y p h a e w e r e s t il l n o t e d i n th e d e e p l a y er . T h e i n fi lt ra t-

i ng c e ll s c o n s i s te d m a i n ly o f n e u t r o p h i l s t h r o u g h o u t t h e c o u r s e o f h ea l in g . A t d a y 1 4,

w h e n t h e h y p h a e h a d m o s t l y d i s a p p e a r e d , s o m e m a c r o p h a g e i n f i l t r a t i o n w a s n o t e d .

D X M - t r e a t m e n t e x a c e r b a t e d t h e c o rn e a l le s io n s a n d n o i m p r o v e m e n t w a s n o t e d

d u r i n g t h e e x p e r i m e n t a l p e r i o d o f 1 4 d a y s . H i s t o l o g i c a l e x a m i n a t i o n s h o w e d t h a t

i n fi lt ra t io n o f i n f l a m m a t o r y c e l ls b y d a y 7 w a s s c a r c e a n d t h a t t h e h y p h a e h a d g r o w n

m o r e v i g o ro u s ly in th e D X M - t r e a t e d c o r n e a . W h e n t h e D X M - t r e a t e d c o r n e a l sp e c i-

m e n s w e r e c u l t u r e d o n S A B , f u n g a l g r o w t h w a s a l w a y s p o s i t i v e a t d a y 3 , 7 a n d 1 4 o f

infec t ion .

M o d e o f i n v a si o n o f solani n t o t h e c o r n e a l s t r o m a

E M s t u d i e s w e r e p e r f o r m e d t o i n v e s t ig a t e t h e m o d e o f i n v a s io n o f t h e f u n g u s t h r o u g h

t h e c o r n e a l s t r o m a . A l t h o u g h m o s t c o l l a g e n f i b r i l s e x h i b i t e d l a m i n a t e d a n d p e r i o d i c

a r r a n g e m e n t s , t h o s e a r o u n d t h e h y p h a e s h o w e d m a r k e d i r r e g u la r it ie s i n t h e i r a r ra n g e -

m e n t . M a n y c o l l a g e n f i b r i l s w e r e t o r n t o s m a l l p i e c e s a n d t h e i r e l e c t r o n d e n s i t y w a s

u n e v e n (F ig . 1 A , B , D ) . T h e s e c h a n g e s i n co l la g e n f ib r il s w e r e o b s e r v e d o n l y a r o u n d t h e

h y p h a e , a n d w e r e e s p e c ia l ly c o n s p i c u o u s a t t h e a p i c a l e n d , s u g g e s ti n g t h e d e s t r u c t i v e

i n fl u en c e o f th e h y p h a e . M o r e o v e r , e l e c tr o n d e n s e a m o r p h o u s m a t e ri a l, w h i c h l o o k e d

as if i t w a s en t r app i n g and d i ge s t i ng co l l agen f ib rils , w a s o f t en o bse r ve d i n f r on t o f t he

a p i c al e n d o f th e h y p h a e (F ig . 1 A ) . T h e a m o r p h o u s m a t e r i a l a l s o h a d c o n t a c t w i t h t h e

su r f ace o f t he f unga l c e l l w a l l ( Fig . 1B, C) . F ea t u r e s o f d i s so l v ing co l l agen f ib r il s w e r e

a l s o d e t e c t e d a r o u n d t h e o t h e r p a r t s o f t h e h y p h a e ( F i g . 1 D ) . S o m e v a c a n t a r e a s w e r e

t h e n c r e a t e d b e t w e e n t h e h y p h a e a n d t h e s u r r o u n d i n g s t r o m a (F ig . 2 ). S u c h s p a c e s

w e r e f r e q u e n t l y o b s e r v e d , e s p e c ia l ly in f r o n t o f t h e a p i c a l e n d o f t h e h y p h a e .

I n t e r a c t i o n b e t w e e n solani a n d n e u t r o p h i l s in t h e c o r n e a

U l t r a s t r u c t u r a l s t u d i e s w e r e p e r f o r m e d t o e x a m i n e t h e i n t e r a c t i o n b e t w e e n t h e f u n g a l

h y p h a e a n d t h e n e u t r o p h i l s i n t h e c o r n e a l l e si o ns . T h e f e a t u r e s o f th e h y p h a e , w h i c h

w er e f r ee o f i n f l am m at o r y cell s, w e r e s i m i l ar t o t hose c u l t u r ed

in v i t ro

O n d a y 3 , n e u -

t ro p h i ls e n c i r c l ed t h e h y p h a e w i t h t h e ir p s e u d o p o d i a a n d s o m e s i g ns o f d e g e n e r a t i o n ,

s u c h a s l ar g e v e s ic l es in t h e c y t o p l a s m a n d p l a sm o l y s is , w e r e o b s e r v e d w i t h in t h e e n -



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o f

E solani

g r o w i n g in t h e s tr o m a o f D X M - t r e a t e d c o r n e a . ( A ) E l e c t r o n

dense

m a t e r i a l ( a r r o w s ) i s t r a p p i n g a n d d i g e s t i n g t h e c o l l a g e n f i b r il s ( d a y 3 o f i n f e c t io n , x 4 0 00 0 ).

( B ) E l e c t r o n d e n s e m a t e r i a l ( a r r o w s ) i s e x t e n d i n g f r o m t h e c e l l w a l l ( d a y 3 o f i n f e c t i o n ,

x 3 6 50 0 ). ( C ) D i g e s t i o n o f t h e c o l l a g e n f ib r i ls i n t h e a m o r p h o u s m a t e r i a l ( d a y 7 o f i n f e c t i o n , x

2 2 0 0 0 ) . ( D ) T r a p p i n g a n d d i g e s t i o n o f t h e c o l l a g e n f ib ri ls . A s m a l l a m o u n t o f a m o r p h o u s m a t e r i a l i s

a l s o n o t e d ( a r r o w ) ( d a y 3 o f i n f e c t i o n , x 3 6 0 0 0 ) . F H = Fusarium h y p h a , C F = c o l l a g e n fi br il s, C W

= cell wall.

c i r cl e d h y p h a e ( F ig . 3 A ) . I n s e c ti o n s s t a i n e d f o r a c i d p h o s p h a t a s e a c ti vi ty , n o r e a c t i v e

p r o d u c t s w e r e f o u n d a r o u n d t h e h y p h a e , i n d i c a t i n g t h a t p h a g o s o m e - l y s o s o m e f u s i o n

w a s n o t t a k in g p l a ce ( d a ta n o t s h o w n ) . D a m a g e t o th e c y t o p l a sm p r e c e d e d , i n g e n e r a l,

t h e c h a n g e s i n t h e f u n g a l c e ll w a l l . O n d a y 7 , i n t h e D X M - t r e a t e d c o r n e a , m i g r a t in g

n e u t r o p h il s h a d h a rd l y t o u c h e d a n d h a d n o t e n c i r c le d t h e h y p h a e , a n d d e g e n e r a t i o n o f

t h e h y p h a e w a s r a r e ly o b s e r v e d ( F ig . 3 B ) .



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F I G . 2. I n t e r s p a c e f o r m e d b e t w e e n t h e a p i c al e n d o f th e h y p h a a n d t h e c o l la g e n s t r o m a i n t h e D X M - t r e a t e d

corn ea day 7 of in fec t ion , x 8000) .

. v . - ; . . -

F I G . 3 . I n t e r a c t i o n b e t w e e n E solani a n d n e u t r o p h i l s in t h e c o r n e a l le s io n . A ) D a y 3 o f in f e c t i o n w i t h o u t

D X M - t r e a t m e n t . T h e h y p h a i s t r a p p e d b y p s e u d o p o d i a o f t h e n e u t r o p h il s . A l a r g e c e n t r a l v a c u o l e

a n d d e g e n e r a t i o n o f th e c y t o p l a s m i c o r g a n e U e s c a n b e s e e n i n t h e h y p h a . T h e r e a c t i o n p r o d u c t s o f

p e r o x i d a s e a r e n o t e d in s o m e g r an u l e s o f n e u t r o p h i l s a r r o w s ). S t a i n e d fo r m y e l o p e r o x i d a s e a c-

t iv it y, x 1 0 0 0 0. ) B ) D a y 3 o f i n f e c t io n w i th D X M - t r e a t m e n t . T h e s u r r o u n d i n g n e u t r o p h i l s s h o w n o

p h a g o c y t o s is o f t h e h y p h a . N o d e g e n e r a t i v e c h a n g e is n o t e d i n t h e h y p h a . S t a i n e d f o r m y e l o p e r o x -

idase act ivi ty, x 9500.) FH = Fusarium h y p h a , N P = n e u t r o p h i l .



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I N V A S IO N A N D S U R V I V A L OF

F SO L A N I

4 0 1

E f f e c t o f D X M o n t h e q u a l it a ti v e N B T - r e d u c t io n

Q ual i ta t ive NBT-reduct ion t e s t s were done i n v i t r o on rabbi t PM N af ter contac t wi th

s p o r e s o f E s o l a n i i n t h e p r e s e n c e o r a b s e n c e o f D X M . A d h e r e n c e o f th e P M N t o th e

s p o r e s o f

E s o l a n i

was s ign i f icant ly inh ib i ted by D X M at a concentrat ion o f 0-01

(P < 0 .05, Fig . 4) . At the same t ime, a s igni f icant decrease in the NBT-reduct ion pos i -

t i ve PM N was obser ved in the presen ce o f DX M at a concen trat ion o f 0.001 and 0 -01

(P < 0 .01 , F ig . 4 ) . W hen ph agocytos i s and NB T-redu ct ion were as sayed wi th zym ozan

a s a c o n t r o l , 8 0 - 9 0 o f P M N p h a g o c y t o s e d z y m o s a n a n d N B T - r e d u c ti o n w a s 7 0 - 8 0

p o s i t iv e in t h e a b s e n c e o f D X M ( d a ta n o t s h o w n ) .

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ET AL

. w - J ~

F I G . 5 . P e r o x i s o m e s a r r o w s ) in h y p h a s u r r o u n d e d b y n e u t r o p h i l s i n t h e D X M - t r e a t e d c o r n e a o n d a y 7 o f

i n f e c t i o n . A ) c a t a l a s e s t a i n i n g , x 1 8 0 0 0. B ) a c i d - p h o s p h a t a s e s ta i n i n g , x 1 2 5 0 0. N o r e a c t i o n p r o d -

u c t s f o r a c id p h o s p h a t a s e c a n b e s e e n b e t w e e n t h e h y p h a a n d th e n e u t r o p h i l s B ) . S o m e m e s o s o m e -

l i k e s t r u c t u r e s c a n b e o b s e r v e d a t t h e i n n e r s u r f a c e o f t h e c e l l w a l l A ) .

F I G . 6 . F o r m a t i o n o f d u p l i c a te c e l l w a l l o r h y p h a - i n - h y p h a s tr u c t u r e s o f

E solani

h y p h a s u r r o u n d e d b y n e u -

t r o p h i l s i n t h e D X M - t r e a t e d c o r n e a o n d a y 7 o f i n f e ct i o n . A ) T r ip l e c e ll w a l l f o r m a t i o n x 1 2 5 0 0 ).

B ) H y p h a - i n - h y p h a s t ru c t u r e. M a n y i n t a c t o r g a n e l l e s c a n b e r e c o g n i z e d in t h e i n n e r h y p h a i n co n -

t r a s t t o s ev e r e d e g e n e r a t i o n o f t h e c y t o p l a s m i n t h e o u t e r h y p h a x 2 2 0 0 0 ).

D I S C U S S I O N

T h e m o d e o f in v a s io n o f solani n t o t h e c o r n e a o f r a b b it s w a s i n v e s t i g a t e d i n t h e p r e -

s e n t s t ud y . A r o u n d t h e h y p h a e i n t h e c o r n e a , d e s t r u c t io n o f t h e c o l l a g e n fib rils w a s

n o t e d a n d a t t h e a p ic a l en d o f th e g r o w i n g h y p h a e , a m o r p h o u s m a t e r ia l w a s s o m e t i m e s

o b s e r v e d F i g . 1 A , B , C ) . T h i s a m o r p h o u s m a t e r ia l , a s w e l l a s t h e c e ll w a l l o f t h e h y p h a e

F i g . 1 D ) , s e e m e d t o e n g u l f a n d l y s e t h e su r r o u n d i n g c o l l a g e n f ib rils . W e s u g g e s t t h a t



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INVASION AND SURVIVAL OF

E SOLANI 403

FIG. 7. Hypha-in-hypha structure of F solani induced in vitro after processing with a low concentrat ion of

H202 12 000).

t h is m a t e r i a l i s a p r o du c t o f t he fungus , con t a i n i ng ab un dan t co l l ageno l y t i c enzym es ,

a l t h o u g h w e c a n n o t d e n y t h e p o s s i b i li ty t h a t t h e a m o r p h o u s m a t e r i a l i s d e g r a d e d c o l-

l agen o r s l oughed - o f f c e l l w a l l m a t e r i a l . I t c an be s een t ha t a f t e r d i ge s t ing t he co l l agen

f i b r i l s , vacan t spaces w i t hou t co l l agen f i b r i l s w e r e f o r m ed a r ound t he f unga l hyphae

F i g . 1D , 2 ) . I t i s no t ye t c l ea r w he t he r solani p r o d u c e s c o l l a g e n o l y t i c e n z y m e s

a l t houg h so m e au t ho r s have su gges t ed t he pa r t i c i pa t i on o f such an enzy m e [ 6, 12 ]. Th i s

s p a c e f o r m a t i o n m i g h t b e f a v o u r a b l e f o r f u n g a l g r o w t h a n d a t t h e s a m e t i m e t h e

d i ges t ed p r o t e i n s m i gh t s e r ve a s nou r i sh i ng subs t ances f o r t he f ung i . Such space f o r -

m a t i o n c a n n o t b e f i x a ti o n a r te f a c t s in c e n o o b v i o u s s h r in k a g e a r t e f a c t h a s b e e n e x p e r i -

e n c e d s o f a r fo r t h e g l u t a r a l d e h y d e - o s m i c a c id f ix a t io n p r o c e d u r e : i n fa c t, n o t a l l b u t

s o m e h y p h a e s h o w e d s p a c e f o r m a t i o n i n o u r st ud y . A t t h e a p i c a l e n d o f t h e h y p h a e ,

w h e r e s p a c e f o r m a t i o n w a s m o s t r e m a r k a b l e , n o m o r p h o l o g i c a l f e a t u r e s o f sh r i n k a g e

s u c h a s d e f o r m a t i o n o f t h e c e l l w a l l w e r e o b s e r v e d .

W h e n solani w a s i n o c u l a t e d i n t o t h e c o r n e a o f r a b b it s , th e i n f l t r a t in g n e u -

t r oph i l s enc i r c l ed t he f unga l hyp hae w i t h t he i r p s eu do po d i a F ig . 3A ) . solani is

t houg h t t o be ve r y s ens i t ive t o t he f ung i c i da l a c ti v i ty o f i n f il tr a ti ng ne u t r oph i l s b eca use

t he f unga l hyphae i n t he neu t r oph i l - i n f i l t r a t ed a r ea d i s appea r ed du r i ng i n f i l t r a t i on .

N e u t r oph i l s a r e t hus t hou gh t t o be t he f ir s t l ine o f hos t de f ense ag a i ns t

solani

inva-

s i o n i n t h e c o r n e a . B y E M , m a n y v a c u o l e s w e r e s e e n t o h a v e a p p e a r e d i n si d e th e . f u n g i

bu t the f unga l w a l l r em a i ne d i n t ac t on day 3 o f i n f ec t i on F ig . 3A ) . O n day 7, t he w ho l e

c y t o p l a s m h a d s e v e r e l y d e g e n e r a t e d a n d t h e c e l l w a l l s h o w e d c o n s i d e r a b l e d e s t r u c t i o n

d a t a n o t s h o w n ) . T h e f u n g ic i d al a c ti v it y o f t h e n e u t r o p h i l s d e p e n d s m a i n l y o n l y s o s o -

m a l en zym es [ 17 ] and r ea c t i ve ox ygen i n t e r m ed i a t e s [5 , 16 ]. I n t he p r e sen t s t udy , a c i d

p h o s p h a t a s e , w h i c h is a m a r k e r e n z y m e o f p h a g o s o m e - l y s o s o m e f u s io n , w a s n o t

d e m o n s t r a t e d a r o u n d t h e h y p h a e F ig . 5 B ) . T h e s u p e r o x i d e g e n e r a t i o n s y s t e m , t h e r e -

f o r e , w ou l d p l ay an i m p or t an t r o l e i n f ung i c ida l a c t i v i ty in t he co r n ea l l e s i on .



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4 4 KIRYU E T A L

T o p i c al a p p l i c a ti o n o f c o r t ic o s t e r o i d e y e d r o p s h a d a c o n s i d e r a b l e e f f e c t o n t h e

i n te r a ct io n b e t w e e n E solani and i n f i l t r a t i ng neu t r oph i l s i n t he expe r i m en t a l co r nea l

l e s i ons . I n f i l t r a t i ng neu t r oph i l s d i d no t enc i r c l e t he f unga l hyphae and t he hyphae

r em a i ne d i n t ac t i n t he D X M - t r e a t e d l e s i on (F ig . 3B) . Suppr e s s i ve e f f ec t s o f g l ucoc o r t i -

c o s t e r o i d s o n n e u t r o p h i l f u n c t i o n h a v e b e e n r e p o r t e d , i n cl u d in g e f f e c ts o n m i g r a t io n

[ 20 , 29 ], phag ocy t os i s and a t t a c hm en t [ 24 ], l y sosom a l enzy m e r e l ea se a nd s up e r ox i de

p r o d u c t i o n [1 0]. O u r m o r p h o l o g i c a l o b s e r v a t i o n s p r e s e n t f u r t h e r m o r p h o l o g i c a l e v i -

d e n c e o f t h e s u p p r e s s i v e e f fe c t s o f c o r ti c o s t e r o id s o n n e u t r o p h i l f u n c t i o n i n t h e c o r n e a l

l e s i on . We a l so s t ud i ed t he in vitro i n te r a c ti o n b e t w e e n r a b b i t P M N a n d E solani

c o n i d ia . A d h e r e n c e o f t h e n e u t r o p h i l s t o t h e c o n i d ia a n d s u p e r o x i d e g e n e r a t i o n b y t h e

n e u t r o p h i l s w e r e s t u d i e d b y t h e q u a l it a t iv e N B T - r e d u c t i o n te s t. W h e n a d h e r e n c e a n d

N B T - r e d u c t i o n w e r e a s s a y e d w i t h f u n g a l c o n i d ia , a n d t h e r e su l t s c o m p a r e d w i t h t h o s e

o b t a i n e d w it h z y m o s a n , o v e r 4 0 o f t h e d e c r e a s e s w e r e o b s e r v e d i n D X M - n o n t r e a t e d

c u lt u re s . W e t h i n k t h e d e c r e a s e s w e r e d u e t o t h e i n t ri n si c d i f f e re n c e s b e t w e e n t h e t ar -

g e t s , t h a t i s b e t w e e n z y m o s a n a n d

Fusarium

con i d i a . I n sp i t e o f the i n i ti a l dec r ea se s ,

D X M had a de f i n i t e i nh i b i t o r y e f f ec t on t h e se f unc t i ons (F ig . 4 ). Th ese

in vitro

s t u d i e s s u p p o r t o u r in vivo o b s e r v a t i o n o f t h e i n h i b i t o r y e f f e c t o f t o p i c a l l y a p p l i e d

D X M .

I n t he s t e r o i d - t r ea t ed l e si ons , E solani c o n t i n u e d t o i n v a d e i n to t h e c o r n e a a n d t h e

h y p h a e e l o n g a t e d i n s p i te o f s o m e n e u t r o p h i l i n fi lt ra ti o n . E M o b s e r v a t i o n o f su c h s u r -

v i vi n g h y p h a e s h o w e d s e v e r a l m o r p h o l o g i c a l d e v i c e s f o r su r v iv a l, s u c h a s a n i n c r e a s e i n

i n tr a c e ll u la r p e r o x i s o m e s a n d t h e f o r m a t i o n o f d o u b l e o r tr ip l e c e ll w a l l s a n d / o r h y p h a -

i n - h y p h a s t r u c t u r e s . A n i n c r e a s e i n p e r o x i s o m e s i n E solani h y p h a e w a s n o t e d a f t e r

d a y 7 o f i n f e ct io n i n t h e D X M - t r e a t e d c o r n e a (F ig . 5) . A p e r o x i s o m e is a n o r g a n e l l e

s u r r o u n d e d b y a s i n g l e -l a y e r e d u n it m e m b r a n e a n d c o n t a i n s c a t a la s e [2 2, 2 5 ]. A l t h o u g h

p e r o x i s o m e s w e r e r a r e l y f o u n d i n t h e c u l t u r e d h y p h a e o f E solani an i nc r ea se i n pe r -

o x i s o m e s w a s o b s e r v e d a f t e r E solani w a s t r e a t e d in vitro w i t h a l o w c o n c e n t r a t i o n o f

H 2 0 2 ( u n p u b l i s h e d o b s e r v a t i o n ) . A n i n c r e a s e i n p e r o x i s o m e s w a s a ls o r e p o r t e d i n b o t h

Candida albicans

[3] and

Hansenula polyrnorpha

[2 6], w h e n t h e f u n g i w e r e t r e a t e d w i t h

f u n g i st a ti c a g e n ts , o r m e t h a n o l , r e s p ec t iv e l y . F r o m t h e s e o b s e r v a t i o n s , t h e p e r o x i s o m e

i s cons i de r ed t o be an o r gane l l e w h i ch r eac t s t o and p r o t ec t s aga i ns t s t r e s s .

F o r m a t i o n o f a d o u b l e o r t r ip l e c e ll w a ll s u g g es ts t h e s u r v i v a l m e c h a n i s m s o f

E solani i n t i s sues . The ce l l w a l l i s na t u r a l l y a ve r y r e l i ab l e de f ense s t r uc t u r e f o r t he

f ung i aga i ns t ha r m f u l env i r onm en t a l i n f l uences [ 13 ] . The ou t s i de ce l l w a l l w ou l d p r o -

t e c t t h e c e ll s f r o m h a r m f u l f a c t o r s o f h o s t d e f e n s e m e c h a n i s m s . I n t h e D X M - t r e a t e d

c o r n e a , a sp e c i al s t ru c t u r e, c o m p o s e d o f a n i n n e r h y p h a w r a p p e d i n a n o u t e r h y p h a , w a s

a l so d e t e c t e d o n d a y 7 o f i n fe c t io n . W e d e s i g n a t e d i t a s a ' h y p h a - i n - h y p h a ' s t ru c t u r e .

T h i s s t r u c t u r e w a s o b s e r v e d o n l y i n D X M - t r e a t e d l es io n s , s u g g e s ti n g t h a t i t w a s f o r m e d

b y s ti m u l a t io n f r o m s t e r o i d - t r e a t e d n e u t r o p h il s . A n a l m o s t s i m i la r h y p h a - i n - h y p h a

s t r u c tu r e w a s f o r m e d in vitro b y p r o c e s s in g t h e c u l t u r e d h y p h a e w i t h a fu n g i s ta t ic c o n -

cen t r a t i on ( 2 m M) o f H 202 ( F ig . 7) . I t i s un l i ke l y t ha t f o r m a t i on o f t he se s t r uc t u r e s is

d u e t o s o m e d i r ec t a c ti o n o f D X M o n t h e f u n g u s b e c a u s e s u ch s t r u c tu r e s w e r e n o t

d e t e c t e d i n c u lt u r e s in vitro w i th 0 .0 01 a n d 0 . 0 1 D X M ( d a t a n o t s h o w n ) . T h e D X M -

t r e a t e d n e u t r o p h i l s a c c o r d i n g ly g e n e r a t e d a l o w c o n c e n t r a t io n o f re a c t i v e o x y g e n i n te r -

m e d i a t e s w h i c h w o u l d c o in c i d e w i t h t h e a p p r o p r i a t e c o n d i t i o n f o r fo r m a t i o n o f

h y p h a - i n - h y p h a s t r u c t u r e s . W e s u g g e s t t h a t i n t h e D X M - t r e a t e d c o r n e a l l e s i o n , t h e E

solani

h y p h a e t h u s d e v e l o p e d r e s i s ta n c e a g a i n st t h e d e s t r u c t i v e a t t a c k o f n e u t r o p h i ls .

The supp r e s s i ve e f f ec t o f top i ca l l y app l i ed c o r t i cos t e r o i d o n i n f l am m at o r y ce l l i n f il tr a -



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E SOLANI

4 5

t i o n t o g e t h e r w i t h th e i n c r e a s e i n f u n g a l r e s is t a n c e w o u l d e x a c e r b a t e t h e c o r n e a l

l e s i on .

I n s u m m a r y s o l a n i w a s k i l l e d b y n e u t r o p h i l s i n t h e r a b b i t c o r n e a l l e s i o n b u t t h i s

f u n g i c id a l e f f e c t w a s l o s t o r l o w e r e d b y c o r t i c o s t e r o id - t r e a t m e n t . I n t h e c o r t i c o s t e r o i d -

t r e a t ed c o r n e a t h e f u n g i s e e m e d t o su r v i v e b y fo r m i n g a h y p h a - i n - h y p h a s t ru c t u re .

T h i s s t ru c t u r e p r o b a b l y p l a y s a n i m p o r t a n t r o l e in p r o t e c t i o n a g a i n s t h o s t d e f e n s e s . W e

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s o l a n i

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A C K N O W L E D G E M E N T S

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