Extracellular Enzymes and Mycotoxins as Virulence Factors...
Transcript of Extracellular Enzymes and Mycotoxins as Virulence Factors...
BIOSCIENCES BIOTECHNOLOGY RESEARCH ASIA, August 2014. Vol. 11(2), 479-490
* To whom all correspondence should be addressed.Mob.: +91-9486120820; Fax: 0091422 2591865;E-mail: [email protected]
Extracellular Enzymes and Mycotoxins as Virulence Factors in Fusarium and Aspergillus Keratitis
Kanesan Panneer Selvam1, Y. Randhir Babu Singh2, Coimbatore Subramanian Shobana2*, Nair Kalesh Karunakaran2,
Csaba Vágvölgyi3, László Kredics3, Raid Saleem Al-Baradie4 and Palanisamy Manikandan4-5
1Department of Microbiology, MR Government Arts College, Mannargudi - 614 001, India2Department of Microbiology, Dr. GR Damodaran College of Science, Coimbatore – 641 014, India.
3Faculty of Science and Informatics, Department of Microbiology, University of Szeged, Szeged, Hungary.4Department of Medical Laboratory, Applied Medical Sciences College, Majmaah University,
Kingdom of Saudi Arabia.5Department of Microbiology, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology,
Coimbatore - 641 014, Tamilnadu, India
doi: http://dx.doi.org/10.13005/bbra/1298
(Received: 09 May 2014; accepted: 06 June 2014)
The incidence of Fusarium and Aspergillus keratitis is on alarming rise with its burden increasing by the fact that they can produce an array of extra cellular enzymes and mycotoxins which act as virulence factors. In this context, 154 isolates of Fusarium spp. and Aspergillus spp. were screened for extracellular enzymes production. Further, representative isolates from the two genera were analyzed for mycotoxin production. Both genera were able to produce an array of extracellular enzymes. It was noted that protease and lipase are actively involved in Fusarium and Aspergillus keratitis. Aflatoxins were detected from Aspergillus spp., however no mycotoxins were detected from Fusarium spp. The study concludes that the extra cellular protease and lipase are cause of concern since it play important roles in pathogenesis of Fusarium and Aspergillus keratitis.
Key words: Fusarium, Aspergillus, Keratitis, Extracellular enzymes, Mycotoxins.
Eye diseases affecting the cornea are a major cause of blindness worldwide 1 next to cataract. In developing countries such as India, it has been recognized as a “silent epidemic” 2. Over the decades, there has been an increase in the percentage of infectious keratitis caused by filamentous fungi 3-6. Further, its burden is
worsened by the fact that fungi secrete several extracellular hydrolytic enzymes 7 with potential role as virulence factors 8-10, however, research on extracellular enzymes production as a virulence factor of Fusarium and Aspergillus isolated from ocular infection is very less. Gopinathan et al. 11, Zhu et al. 12, Burda & Fisher 13 and Dudley et al. 14 have reported that extracellular protease excreted from Fusarium and Aspergillus spp. isolated from corneal ulcer have been implicated in corneal matrix degradation. Mycotoxins are a group of secondary metabolites produced by various filamentous fungi and are involved in the infectious process of the fungi and may serve as virulence
480 SELVAM et al., Biosci., Biotech. Res. Asia, Vol. 11(2), 479-490 (2014)
factors 15-18. The present study was designed to evaluate the correlation between extracellular enzymes and mycotoxin production by Fusarium and Aspergillus with clinical characteristics of the mycotic keratitis patients so as to establish that extracellular enzymes and mycotoxin can act as virulence factors in mycotic keratitis.
MATERIALS AND METHODS
Patients All the patients studied in the present series were presented at Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore, India, between July 2010 and June 2012. Further, the clinical details of selected patients were collected and analyzed accordingly.Collection and processing of specimens Corneal scraping was done by an ophthalmologist with the aid of Kimura’s spatula (sterilized by flaming or wiping with 70% ethyl alcohol and drying), applying multiple, moderately firm, unidirectional strokes, under slit lamp illumination and directly inoculated on blood agar, chocolate agar and potato dextrose agar (PDA). The culture plates were incubated at 37ºC for 5 days except for PDA, which was incubated at 25ºC for 7 days. The identification of fungi was based on colony morphology and microscopic observation using lactophenol cotton blue wet mount. In vitro screening of extracellular enzymes among Fusarium and Aspergillus spp. In the present study seven different enzymes viz., protease 19 cellulase 20, keratinase 21, elastase 22, lipase 23, DNase 24 and a- amylase 25 was screened based on the methodologies described in the literature. Briefly, the conidial suspension of the test isolates was prepared to achieve a concentration of 105 conidia / mL and 50 µl of the spore suspension was inoculated on respective assay medium. The enzyme activity index was calculated for the test isolate by dividing the enzyme lysis diameter by the colony diameter as described by Blanco et al. 26.In vitro screening of aflatoxigenic Aspergillus spp. isolated from keratitis patients Screening of aflatoxigenic A. flavus was performed based on the method described by Saito and Machida 27. Briefly, the test isolate of Aspergillus spp. was inoculated at the center of the
PDA and incubated at 25°C for 3-4 days and the colonies were subsequently exposed to ammonia vapour. A change in the color of the colony reverse and formation of pink orange coloration upon exposing it to ammonium vapour was taken as a positive activity of aflatoxin.Determination of aflatoxins and T2 toxin from Fusarium and Aspergillus spp. Mycotoxin extract was prepared as described by Shih and Marth 28 and Dai et al. 29. The mold was grown on PDA slants for 7 days at 28°C. Spores were harvested by adding sterile distilled water to the slants. Precisely, 2ml of the spore suspension (with an optical density value of 0.5 at 550 nm) was inoculated into sterile glucose–salt medium (for aflatoxin) and T2-Toxin extraction medium (for T2-Toxin) and incubated at 25°C for 15 days. Later, the culture was filtered using Whatman No.1 filter paper and the filtrate was extracted with chloroform (2 volume of chloroform / volume of the filtrate). The extract was evaporated in a water bath at a temperature of 80°C and the residue thus obtained was dissolved in 0.2 ml of chloroform and mixed thoroughly. The extract thus obtained was subjected to Gas Chromatography–Mass Spectrometry (GC–MS) analysis for T2-Toxin. Similarly, presence of aflatoxins was determined using High Performance Liquid Chromatography (HPLC).
RESULTS
A total of 68 Fusarium spp. and 86 Aspergillus spp. (A. flavus; 63, A. fumigatus; 14, A. niger; 5, A. terreus; 3, and A. tamarii; 1) were evaluated for their extracellular enzyme activity and the representative results of each enzyme assay are shown in figure 1 (A-G). Among the isolates, Aspergillus spp. showed more protease (82.5% of 86), and a- amylase (88.3% of 86) activities than that of Fusarium spp. (protease; 70.5% of 68, a- amylase; 57.3% of 68). However, Fusarium spp. were comparatively active with cellulase and lipase than Aspergillus spp. (cellulase; 72% of 86 and lipase; 68.6% of 86). Interestingly, the rest of the enzymes viz., DNase, elastase and keratinase showed to have considerably lower activities in both Fusarium and Aspergillus spp. Among Aspergillus spp. the predominant species of the study, A. flavus were noted to have considerable
481SELVAM et al., Biosci., Biotech. Res. Asia, Vol. 11(2), 479-490 (2014)
Table 1. Percentage (%) of Fusarium and Aspergillus species/isolates with extracellular enzyme activities
Isolates Extracellular enzymes
Protease α- amylase Cellulase Lipase DNase Elastase Keratinase
A. flavus 53 57 45 44 21 18 21 (n = 63) (84.1%) (90.4%) (71.4%) (69.8%) (33.4%) (28.5%) (33.4%)A. fumigatus 10 10 11 8 3 3 6 (n = 14) (71.4%) (71.4%) (78.5%) (57.1%) (21.4%) (21.4%) (42.8%)A. niger 4 5 4 4 1 Nil Nil(n = 5) (80%) (100%) (80%) (80%) (20%) A. terreus 3 3 1 2 1 Nil Nil (n = 3) (100%) (100%) (33.4%) (66.7%) (33.4%) A. tamari 1 1 1 1 1 Nil Nil(n = 1) (100%) (100%) (100%) (100%) (100%) Fusarium 48 39 61 59 26 13 8 (n = 68) (70.5%) (57.3%) (89.7%) (86.7%) (38.2%) (19.1%) (11.7%)
Nil- No activity
activities of protease (84.1%, 53 of 63) and lipase (69.8%, 44 of 63) enzymes (table 1). Except two of the 154 isolates involved in this study, all the isolates had a minimum of two different enzyme activities. A total of 51 different sets/groups of enzyme combinations were identified to be secreted combining both the fungi and are given in table 2. A maximum activity was noted with respect to protease, α - amylase, cellulase, and lipase (14.2%; 22 of 154) combinations, followed by protease, α - amylase, cellulase, lipase, and DNase (9% of 154). Interestingly, though many of the Aspergillus species had extracellular protease activities compared to Fusarium spp., the latter was found to have a higher EAI (1.1) than Aspergillus (1.09), particularly from A. terreus. A. terreus also had a higher cellulase EAI of1.19. However, there were no comparatively significant differences existing in enzyme activity indices of a- amylase, DNase, and elastase amongst Fusarium and Aspergillus (table 3). Clinical data of a total of 63 representative patients suffering from Fusarium and Aspergillus keratitis were retrieved to ascertain the correlation of extracellular enzyme activity with clinical conditions of the patient. It was noted that elastase, keratinase and DNase activity have little influence on clinical character of fungal keratitis patient. On the other hand, protease and lipase activities were found to have strong associations with severity, healing etc., amongst infectious Fusarium and
Aspergillus keratitis cases. While protease activity was found to be associated with 71% (10 of 14 cases) of Fusarium keratitis cases who were earlier characterized to have a ‘severe’ infection, lipase activity was noted in all the cases. The observations were noted to be more similar among Aspergillus keratitis also. The detailed analyses of extracellular enzyme activities and the recorded clinical features are provided in tables 4 and 5 for Fusarium and Aspergillus keratitis, respectively. In total, 124 Aspergillus spp. (82 A. flavus, 11 A. niger, 24 A. fumigatus, 5 A. terreus and 2 A. tamari) were screened for aflatoxin producing ability, of which 41 (33%) isolates were detected with aflatoxin activity and were categorized to be aflatoxigenic (figure 1 H&I). Majority of these isolates were identified to be A. flavus (39, 95.1% of 41) and 1 isolate each of A. fumigatus and A. terreus. Further, 4 isolates of A. flavus, (2 each positive and negative aflatoxigenic A. flavus) were specifically identified for B1, B2, G1 and G2 toxins using HPLC and the overall findings are presented in table 6. Of the two non-aflatoxigenic strains, one (Cu34/11) was confirmed to produce aflatoxin B1, B2, G1 and G2 using HPLC. Remarkably, the A. flavus strain Cu670/10 was detected with higher concentrations (1.096 µg/ml) of G1 mycotoxin and the HPLC peaks indicating different types of aflatoxin identified are shown in figure 2. Similarly, among fusaria, four representative isolates of F. solani were screened for T2 mycotoxin production using GC-MS analysis. However, no isolate was
482 SELVAM et al., Biosci., Biotech. Res. Asia, Vol. 11(2), 479-490 (2014)
Table 2. Percentage distribution of hydrolytic enzyme activities among Fusarium and Aspergillus spp
Enzyme/s Aspergillus Fusarium Total (n=86) (n=68) (%)
α - Amylase 1 - 1 (0.65%)Lipase - 1 1 (0.65%)Keratinase 1 1 2 (1.3%)Protease, α - amylase 3 - 3 (2%)Protease, cellulase - 1 1 (0.65%)Protease, Keratinase - 1 1 (0.65%)Protease, α - amylase, cellulase 5 1 6 (3.9%)Protease, α - amylase, lipase 3 2 5 (3.2%)Protease, lipase, elastase, keratinase 1 - 1(0.65%)Protease, α - amylase, cellulase, lipase 12 10 22 (14.2%)Protease, α - amylase, lipase, DNase 3 - 3(2%)Protease, α - amylase, cellulase, elastase 1 - 1(0.65%)Protease, α - amylase, cellulase, DNase 3 - 3(2%)Protease, α - amylase, cellulase, lipase, DNase 7 7 14 (9%)Protease, α - amylase, cellulase, lipase, elastase 1 3 4 (2.6%)Protease, α - amylase, cellulase, DNase,keratinase 1 - 1(0.65%)Protease, α - amylase, cellulase, lipase, keratinase 4 1 5(3.2%)Protease, α - amylase, cellulase, lipase, DNase, elastase 2 3 5(3.2%)Protease, α - amylase, cellulase, lipase, DNase, keratinase 3 - 3(2%)Protease, α - amylase, cellulase, lipase, elastase, keratinase 2 1 3(2%)Protease, α - amylase, cellulase, lipase, DNase, elastase, keratinase - 1 1(0.65%)Protease, α - amylase, cellulase, keratinase 1 - 1(0.65%)Protease, α - amylase, cellulase, elastase 1 - 1(0.65%)Protease, α - amylase, cellulase, elastase, keratinase 1 - 1(0.65%)Protease, α - amylase, cellulase, DNase, elastase 1 - 1(0.65%)Protease, α - amylase, lipase, elastase 1 - 1(0.65%)Protease, α - amylase, lipase, keratinase 3 - 3(2%)Protease, α - amylase, lipase, elastase, keratinase 2 - 2(1.3%)Protease, α - amylase, keratinase 3 - 3(2%)Protease, cellulase, lipase 2 9 11 (7.1%)Protease, cellulase, lipase, DNase - 3 3(2%)Protease, cellulase, lipase, DNase, elastase - 1 1(0.65%)Protease, cellulase, lipase, DNase, keratinase 1 1 2(1.3%)Protease, cellulase, keratinase 2 - 2(1.3%)Protease, cellulase, DNase, 1 2 3(2%)Protease, lipase, DNase 1 - 1(0.65%)α - Amylase, lipase, DNase, elastase 1 - 1(0.65%)α - Amylase, cellulase, lipase 4 4 8 (5.2%)α - Amylase, lipase - 1 1(0.65%)α - Amylase, cellulose, lipase, DNase 1 2 3(2%)α - Amylase, cellulose, lipase, elastase - 1 1(0.65%)α - Amylase, cellulase, lipase, keratinase 1 1 2(1.3%)α - Amylase, cellulase, elastase 1 - 1(0.65%)α - Amylase, cellulase - 1 1(0.65%)α - Amylase, cellulase, DNase 1 - 1(0.65%)α - Amylase, cellulase, lipase, DNase, elastase - 1 1(0.65%)α - Amylase, cellulase, lipase, DNase, keratinase 1 - 1(0.65%)α - Amylase, cellulase, lipase, elastase, keratinase 1 - 1(0.65%)α - Amylase, elastase 1 - 1(0.65%)Cellulase, lipase 1 4 5(3.2%)Cellulase, lipase, DNase, elastase - 1 1(0.65%)Cellulase, lipase, elastase - 1 1(0.65%)Cellulase, lipase, keratinase - 1 1(0.65%)DNase, lipase - 1 1(0.65%)Total 86 68 154 (100%)
483SELVAM et al., Biosci., Biotech. Res. Asia, Vol. 11(2), 479-490 (2014)
Table 3. Mean extracellular enzymes activity indices (EAI) of Fusarium and Aspergillus spp.
Fungi Mean enzyme activity index
Protease α-amylase Cellulase Lipase DNase Elastase
A. flavus 1.04 1.1 1.09 1.19 1.1 1.2A. fumigatus 1.06 1.1 1.1 1.17 1.1 1.3A. niger 1.07 1.1 1.07 1.1 NA NAA. terreus 1.09 1.1 1.19 1.19 NA NAA. tamarii NA NA NA NA NA NAFusarium 1.1 1.1 1.13 1.2 1.08 1.2
NA – Not applicable
Fig. 1. Extracellular enzyme activity of Fusarium and Aspergillus and aflatoxin screening of Aspergillus spp.α-amylase (a), cellulase (b), elastase (c), DNase (d), lipase (e) and protease (f) indicated with formation of clear hallow zone around the fungal colonies. Keratinase activity (g) was indicated by diffusion of keratine azure in basal media. Aflatoxigenic positive (h) and negative (i) A. flavus based on ammonium vapour method. The arrows in horizontal and vertical directions indicates zone of enzyme hydrolysis and zone of fungal colonies respectively
484 SELVAM et al., Biosci., Biotech. Res. Asia, Vol. 11(2), 479-490 (2014)
Tab
le 4
. Per
cent
age
dist
ribu
tion
of
posi
tive
cor
rela
tion
bet
wee
n re
cord
ed c
lini
cal f
eatu
res
and
the
extr
acel
lula
r en
zym
e ac
tivi
ty a
mon
g F
usar
ium
ker
atit
is p
atie
nts
Cli
nica
l fea
ture
s E
xtra
cell
ular
enz
ymes
act
ivit
ies
(%)
P
rote
ase
Lip
ase
Ela
stas
e K
erat
inas
e C
ellu
lase
D
Nas
e α
-Am
ylas
e
Sev
erit
y
M
ild
(n =
6)
3 (5
0.0)
4
(66.
7)
1 (1
6.7)
4
(66.
7)
3 (5
0.0)
-
3 (5
0.0)
Mod
erat
e (n
= 1
1)
7 (6
3.6)
9
(81.
8)
2 (1
8.1)
2
(18.
1)
11 (
100)
3
(27.
2)
9 (8
1.8)
Seve
rity
(n
= 1
4)
10 (
71.4
) 14
(10
0)
4 (2
8.5)
-
10 (
71.4
) 6
(42.
8)
6 (4
2.8)
Fin
al o
utco
me
Hea
led
(n =
26)
18 (
69.2
) 22
(84
.6)
5 (1
9.2)
5
(19.
2)
19 (
73.0
) 7
(26.
9)
15 (
57.6
)N
ot h
eale
d (n
= 3
) 2
(66.
7)
3 (1
00)
1 (3
3.4)
1
(33.
4)
3 (1
00)
1 (3
3.4)
1
(33.
4)N
o fo
llow
up(
n =
2)
- 2
(100
) 1
(50.
0)
- 2
(100
) -
1 (5
0.0)
Surg
ical
inte
rven
tion
Req
uire
d (n
= 6
) 3
(50.
0)
6 (1
00)
1 (1
6.7)
-
3 (5
0.0)
2
(33.
4)
3 (5
0.0)
Not
req
uire
d (n
= 2
5)
17 (
68.0
) 21
(84
.0)
6 (2
4.0)
6
(24.
0)
21 (
84.0
) 7
(28.
0)
15 (
60.0
)D
ata
not a
vail
able
-
- -
- -
- -
Sym
ptom
sP
ain
and
redn
ess
#(n
= 1
4)
6 (4
2.8)
10
(71
.4)
2 (1
4.2)
4
(28.
5)
10 (
71.4
) 4
(28.
5)
8 (5
7.1)
Oth
ers
* (n
= 1
7)
14 (
82.3
) 17
(10
0)
5 (2
9.4)
2
(11.
7)
14 (
82.3
) 5
(29.
4)
10 (
71.4
)D
ata
not a
vail
able
-
- -
- -
- -
Per
iod
requ
ired
for
reco
very
<1
mon
th (
n =
11)
5
(45.
4)
9 (8
1.8)
2
(18.
1)
5 (4
5.4)
8
(72.
7)
2 (1
8.1)
6
(54.
5)>
1mon
th –
4 m
onth
s(n
= 1
1)
10 (
90.9
) 9
(81.
8)
1 (9
.0)
- 9
(81.
8)
5 (4
5.4)
7
(63.
6)>
5 m
onth
s (n
= 6
) 4
(66.
7)
6 (1
00)
2 (3
3.4)
1
(16.
7)
4 (6
6.7)
-
4 (6
6.7)
Dat
a no
t ava
ilab
le(n
= 3
) 1
(33.
4)
3 (1
00)
2 (6
6.7)
-
3 (1
00)
2 (6
6.7)
1
(33.
4)
# -
Sin
ce p
ain
and
redn
ess
was
the
mos
t com
mon
sym
ptom
, it w
as p
rior
itiz
ed a
gain
st th
e re
st.
* -
Sym
ptom
s in
clud
ed w
ater
ing,
pho
toph
obia
, irr
itat
ion,
itch
ing,
dis
char
ge, s
wel
ling
, def
ecti
ve v
isio
n et
c.
485SELVAM et al., Biosci., Biotech. Res. Asia, Vol. 11(2), 479-490 (2014)
Tab
le 5
. Per
cent
age
dist
ribu
tion
of
posi
tive
cor
rela
tion
bet
wee
n re
cord
ed c
lini
cal f
eatu
res
and
the
extr
acel
lula
r en
zym
e ac
tivi
ty a
mon
g A
sper
gill
us k
erat
itis
pat
ient
s
Cli
nica
l fea
ture
s E
xtra
cell
ular
enz
ymes
act
ivit
ies
(%)
P
rote
ase
Lip
ase
Ela
stas
e K
erat
inas
e C
ellu
lase
D
Nas
e α
-Am
ylas
e
Sev
erit
y
M
ild
(n =
1)
1 -
- -
1 -
1M
oder
ate
(n =
14)
12
(85
.7)
6 (4
2.8)
2
(14.
2)
4 (2
8.5)
8
(57.
1)
3 (2
1.4)
13
(92
.8)
Seve
rity
(n
= 1
7)
16 (
94.1
) 12
(70
.5)
3 (1
7.6)
2
(11.
7)
15 (
88.2
) 4
(23.
5)
16 (
94.1
)F
inal
out
com
eH
eale
d (n
=23
) 23
(10
0)
11 (
47.8
) 2
(8.6
) 4
(17.
3)
18 (
78.2
) 6
(26.
0)
22 (
95.6
)N
ot h
eale
d -
- -
- -
- -
No
foll
ow u
p(n
= 9
) 6
(66.
7)
7 (7
7.8)
3
(33.
4)
2 (2
2.3)
6
(66.
7)
1 (1
1.2)
8
(88.
9)Su
rgic
al in
terv
enti
onR
equi
red
(n =
12)
12
(10
0)
7 (5
8.4)
2
(16.
7)
2 (1
6.7)
9
(75.
0)
3 (2
5.0)
12
(10
0)N
ot r
equi
red
(n =
20)
17
(85
.0)
11 (
55.0
) 3
(15.
0)
4 (2
0.0)
15
(75
.0)
4 (2
0.0)
18
(90
.0)
Dat
a no
t ava
ilab
le
- -
- -
- -
-Sy
mpt
oms
Pai
n an
d re
dnes
s #(
n =
13)
12
(92
.3)
7 (5
3.8)
1
(7.6
) 1(
7.6)
11
(84
.6)
1(7.
6)
13 (
100)
Oth
ers
* (n
= 1
9)
17 (
89.4
) 11
(57
.8)
4 (2
1.0)
5
(26.
3)
13 (
68.4
) 6
(31.
5)
17 (
89.4
)D
ata
not a
vail
able
-
- -
- -
- -
Per
iod
requ
ired
for
reco
very
<1
mon
th (
n =
5)
5 (1
00)
2 (4
0.0)
-
1 (2
0.0)
4
(80.
0)
1 (2
0.0)
4
(80.
0)>
1mon
th –
4 m
onth
s(n
= 1
0)
10 (
100)
4
(40.
0)
2 (2
0.0)
1
(10.
0)
7 (7
0.0)
3
(30.
0)
10 (
100)
> 5
mon
ths
(n =
9)
9 (1
00)
6 (6
6.7)
1
(11.
2)
2 (2
2.3)
7
(77.
8)
2 (2
2.3)
9
(100
)D
ata
not a
vail
able
(n =
8)
5 (6
2.5)
6
(75.
0)
2 (2
5.0)
2
(25.
0)
6 (7
5.0)
1
(12.
5)
7 (8
7.5)
486 SELVAM et al., Biosci., Biotech. Res. Asia, Vol. 11(2), 479-490 (2014)
detected with mycotoxin activities, interestingly the compounds such as vitamins (Cu482/11), sterols
(Cu598/11) and a carotenoid such as rhodopin were detected from them.
Table 6. Screening of aflatoxins based on preliminary (ammonium vapor method)
and HPLC methods among Aspergillus spp. isolated from corneal ulcer
S. Isolates(Strain) Ammonium vapour HPLC
No. technique Aflatoxin Result Concentration(µg/ml)
1. A. flavus(Cu34/11) Negative B1 Positive 0.047 B2 Positive 0.012 G1 Positive 0.002 G2 Positive 1.4702. A. flavus(Cu1149/10) Negative B1 Negative NA B2 Negative NA G1 Negative NA G2 Negative NA3. A. flavus(Cu670/10) Positive B1 Negative NA B2 Positive 0.802 G1 Positive 1.096 G2 Positive 0.5444. A. flavus(Cu832/10) Positive B1 Negative NA B2 Negative NA G1 Negative NA G2 Negative NA
NA – Not applicable
DISCUSSION
In pursuit to find the possible role of the extracellular enzymes of Fusarium and Aspergillus as a virulence factor in causing keratitis, it was observed that both the species of Fusarium and Aspergillus were able to produce an array of
extracellular enzymes and the production spectrum differed between them. It was also noted that enzymes (cellulase and α- amylase) related to plant pathogenesis were produced by both Fusarium (89.7% in α- amylase and 57.3% in cellulase) and Aspergillus spp. (88.3% in α- amylase and 72% in cellulase). The observation implied that Fusarium
Fig. 2. Production of aflatoxins (B1, B2, G1 and G2) by A. flavus (Cu34/11) as shown by HPLC chromatogram
487SELVAM et al., Biosci., Biotech. Res. Asia, Vol. 11(2), 479-490 (2014)
and Aspergillus isolated from corneal ulcer could have chiefly originated from plant materials. F. solani isolated from mycotic keratitis patients and plants had demonstrated the same pathogenicity in young bean, corn, and tomato plants and also invoked an eye reaction (erythema) in rabbit eye tested under laboratory conditions30. Ajayi et al. 31 reported amylase production by Fusarium spp. and Mishra and Dadhich 32 determined higher activity of amylase and xylanase in A. niger than Fusarium spp. The rate of cellulase activity as recorded in the present study was found to be in agreement with a previous study 33, although Kwon et al. 34 observed no cellulase activity in Fusarium spp. Additionally, the present study found Fusarium and Aspergillus had lesser activities of DNase, elastase and keratinase compared to protease and lipase but presumably with no active role in causing and keratinase. The detection of elastase and keratinase activity has been documented from clinical fungi isolated from invasive aspergillosis 22, 26, 35. Although the study detected no keratinase activity from A. niger, A. terreus and A. tamarii, previous studies 36-37 have reported higher activity of these enzymes in these species compared to Fusarium spp. More importantly, Rhodes et al. 38 stated that all clinical isolates involved in invasive aspergillosis produced elastase, but not all isolates having elastase activity were associated with invasive disease. Alp and Arikan 35 reported the extracellular elastase, acid proteinase and phospholipase activities of clinical isolates of A. fumigatus, A. flavus and A. niger and suggested that the existence and degree of these enzyme activities in clinical Aspergillus strains tend to show species and strain based variations and potentially significant correlation between the existence of high phospholipase activity and development of invasive disease. The fact that Fusarium (89.7% of 68) had higher activity of lipase than that of Aspergillus (68.8% of 86) in the present study was more or less congruent with the available literature39-42. In the present study, protease activity was profoundly seen in Aspergillus spp. (83% of 86), especially in A. flavus (84% of 63) compared to Fusarium spp. (71% of 68). The finding is a cause of concern among ophthalmologists since protease was proven to be associated with cornea that showed progressive ulcer along with the
presence of dense inflammatory cells 11. Further, the pathogenic role of protease in microbial keratitis has been documented by various authors 12, 43. The strong reports on the virulent nature of protease suggested that protease activity was strongly associated with severe Fusarium and Aspergillus keratitis. It was further found that all the patients of Aspergillus caused keratitis and 50% of the Fusarium keratitis cases with a positive protease activity had to undertake surgical intervention. The Fusarium isolates from the all patients with severe infection, who further required surgical intervention and longer period of healing showed a positive lipase activity. Fliesler 44 commented that lipids and lipid soluble compounds are essential constituents of the cells and tissues that comprise the eye and defect in their synthesis, intracellular and extracellular transport and turnover underlie a variety of significant, common and often severely debilitating eye diseases. These probably answer the critical role of lipase produced by the pathogenic isolates of fusaria and aspergilli of the present study towards the overall increase in the severity of keratitis. The toxic effects of aflatoxins in the cornea involve haziness of the cornea and separation of corneal lamellae, in addition to infiltration by polymorphonuclear leucocytes 45. Leema et al. 46 reported aflatoxin production from A. flavus of which 80% was found to be aflatoxin B1. However, the present study observed aflatoxin B2, G1 and G2 being more common compared to aflatoxin B1. It was emphasized that if an A. flavus strain isolated from a human lesion is found to possess aflatoxin-producing ability, it may be necessary to treat the lesion with not only standard antifungals, but also with molecules that suppress the production, or neutralize the deleterious effects of aflatoxins 46. Although, the present study could not detect any mycotoxin from the species of Fusarium a study47 reported as much as 76% of Fusarium spp. including F. solani, F. oxysporum, F. incarnatum, F. dimerum, F. verticilloides, and F. chlamydosoprum producing fusaric acid, moniliformin or fumonisin B1. Similarly, other study 48 reported detection of T-2 Toxin, nivalenol, diacetoxyacirpenol and deoxynevalenol from Fusarium keratitis using GC-MS. Interestingly, the authors had also concluded that in vitro toxin production does not influence severity of the
488 SELVAM et al., Biosci., Biotech. Res. Asia, Vol. 11(2), 479-490 (2014)
infection. Conversely, stated Fusarium infections of the eye after traumatic inoculation were more severe when trichothecenes produced 47. The high frequency of aflatoxin production by the clinical A. flavus compared to environmental strain possibly represented a response to pressures (antifungal chemotherapy, toxic factors released from corneal epithelial cells, or infiltrating inflammatory cells) that are not conducive to an ideal existence 46. However, it was observed that mycotoxin profiles of clinical and environmental A. flavus and A. fumigatus isolates were homogenous, except for gliotoxin production, which was detected only in the group of clinical isolates of A. fumigatus 49. The reports of mycotoxin being detected from various species of Fusarium keratitis isolates 47-48 and the contradiction with the present study could be due to non-expression of mycotoxin producing genes. However, the study points out the fact that fusaria were able to implicate with keratitis in high frequency, thus confirming the aggressive nature of Fusarium spp. causing mycotic keratitis. Though the screening of aflatoxigenic Aspergillus using ammonium vapour method as in the present study was not highly reliable and confirmative (as only 50% of the isolates was re-confirmed by HPLC, this approach could still provide provisional information on the virulent nature of the isolates. Screening of aflatoxigenic Aspergillus based on emission of bright blue or blue-green fluorescent area around the colonies has been reported for testing mycotoxin production 50. Similarly, different other approaches have also been reported for screening afltoxigenic Aspergillus 27,
51-53. The GC-MS based detection of compounds such as vitamin, sterol from Fusarium spp. in this study had been already reported 48, 54. Nevertheless, in vitro sterol production was not related to the severity of the infection 48.
CONCLUSION
The burden of Fusarium and Aspergillus tends to be increasing with the fact that they are able to produce various extra cellular enzymes which play important roles in the pathogenesis of the disease. Protease and particularly lipase are actively involved in severe mycotic keratitis which warrants further investigation.
ACKNOWLEDGEMENTS
The authors of this study were supported by the Indian National Science Academy and the Hungarian Academy of Sciences within the frames of the Indo – Hungarian bilateral exchange program no. IA/INSA-HAS Project 2013-2015/189. The research of Cs.V. was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP 4.2.4.A/2-11-1-2012-0001 'National Excellence Program'.
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