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Variation in gene expression patterns as the insect pathogen Metarhizium anisopliae adapts to different host cuticle or nutrient deprivation in vitro


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Metarhizium anisopliae infects a broad range of insects by direct penetration of the host cuticle. To explore the molecular basis of this process, its gene expression responses to diverse insect cuticles were surveyed, using cDNA microarrays constructed from an expressed sequence tag (EST) clone collection of 837 genes. During growth in culture containing caterpillar cuticle (Manduca sexta), M. anisopliae upregulated 273 genes, representing a broad spectrum of biological functions, including cuticle-degradation (e.g. proteases), amino acid/peptide transport and transcription regulation. There were also many genes of unknown function. The 287 down-regulated genes were also distinctive, and included a large set of ribosomal protein genes. The response to nutrient deprivation partially overlapped with the response to Man. sexta cuticle, but unique expression patterns in response to cuticles from another caterpillar (Lymantria dispar), a cockroach (Blaberus giganteus) and a beetle (Popilla japonica) indicate that the pathogen can respond in a precise and specialized way to specific conditions. The subtilisins provided an example of a large gene family in which differences in regulation could potentially allow virulence determinants to target different hosts and stages of infection. Comparisons between M. anisopliae and published data on Trichoderma reesei and Saccharomyces cerevisiae identified differences in the regulation of glycolysis-related genes and citric acid cycle/oxidative phosphorylation functions. In particular, M. anisopliae has multiple forms of several catabolic enzymes that are differentially regulated in response to sugar levels. These may increase the flexibility of M. anisopliae as it responds to nutritional changes in its environment.
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Variation in gene expression patterns as the
insect pathogen Metarhizium anisopliae adapts
to different host cuticles or nutrient deprivation
in vitro
Florian M. Freimoser,3 Gang Hu and Raymond J. St Leger
Raymond J. St Leger
Department of Entomology, University of Maryland, 4112 Plant Sciences Building, College
Park, MD 20742, USA
Received 16 August 2004
Revised 25 October 2004
Accepted 28 October 2004
Metarhizium anisopliae infects a broad range of insects by direct penetration of the host cuticle.
To explore the molecular basis of this process, its gene expression responses to diverse insect
cuticles were surveyed, using cDNA microarrays constructed from an expressed sequence tag
(EST) clone collection of 837 genes. During growth in culture containing caterpillar cuticle
(Manduca sexta), M. anisopliae upregulated 273 genes, representing a broad spectrum of
biological functions, including cuticle-degradation (e.g. proteases), amino acid/peptide transport
and transcription regulation. There were also many genes of unknown function. The 287
down-regulated genes were also distinctive, and included a large set of ribosomal protein genes.
The response to nutrient deprivation partially overlapped with the response to Man. sexta
cuticle, but unique expression patterns in response to cuticles from another caterpillar
(Lymantria dispar), a cockroach (Blaberus giganteus) and a beetle (Popilla japonica) indicate
that the pathogen can respond in a precise and specialized way to specific conditions. The
subtilisins provided an example of a large gene family in which differences in regulation could
potentially allow virulence determinants to target different hosts and stages of infection.
Comparisons between M. anisopliae and published data on Trichoderma reesei and
Saccharomyces cerevisiae identified differences in the regulation of glycolysis-related genes
and citric acid cycle/oxidative phosphorylation functions. In particular, M. anisopliae has multiple
forms of several catabolic enzymes that are differentially regulated in response to sugar levels.
These may increase the flexibility of M. anisopliae as it responds to nutritional changes in its
Current molecular and genomic methods are being applied
to Metarhizium anisopliae, the causative agent of green
muscardine disease, because of its importance for the bio-
logical control of insect pests. It is a very versatile fungus,
being able to infect a broad range of insects, 200 species
from over 50 insect families (Samuels et al., 1989), and is
also adapted to life in the root rhizosphere (Hu & St Leger,
2002). Consi stent with its promiscuous nature, an array of
expressed sequence tags (ESTs) from M. anisopliae strain
2575 identified large numbers of genes dedicated to host
interaction and countering insect defences, as well as regu-
lators for coordinating their implementation (Freimoser
et al., 2003). Sequence comparisons and conserved motifs
suggest that about 60 % of the ESTs of strain 2575 expressed
during growth on cuticle encode secreted enzymes and
toxins. Acting collectively, the number and diversity of these
effectors may be the key to this pathogen’s ability to infect a
wide variety of insects. In contrast, ESTs from the specialized
locust pathogen M. anisopliae sf. acridum strain 324 revealed
very few toxins (Freimoser et al., 2003). This relates to life-
styles. Strain 2575 kills hosts quickly via toxins, and grows
saprophytically in the cadaver. In contrast, strain 324 causes
a systemic infection of host tissues before the host dies. This
shows that by utilizing ESTs, multiple virulence factors and
pathways can be viewed simultaneously, and the different
lifestyles that exist in insect–fungus interactions can be
understood from a broader perspective.
3Present address: Institute of Plant Sciences, ETH Zurich,
Universita¨tsstr. 2, CH-8092 Zurich, Switzerland.
The expression ratios for Metarhizium anisopliae ESTs in different
cuticle-containing media are shown in Supplementary Table S1 with
the online version of this paper at
Abbreviations: BC, beetle (Popilla japonica) cuticle; CC, cockroach
(Blaberus giganteus) cuticle; ESTs, expressed sequence tags; GC, gypsy
moth (Lymantria dispar) cuticle; HL, haemolymph; MC, Manduca sexta
cuticle; RT-PCR, reverse-transcription PCR.
2005 SGM Printed in Great Britain 361
Microbiology (2005), 151, 361–371 DOI 10.1099/mic.0.27560-0
In this report, we use cDNA microarrays for high-throughput
expression profiling of how M. anisopliae strain 2575
responds over a 24 h period to cuticle from tobacco horn-
worm caterpillars (Manduca sexta). As a control, we also
define the response of M. anisopliae to nutrient deprivation.
In addition we obtained snapshots of gene expression at
24 h to compare and contrast the responses of M. anisopliae
to gypsy moth caterpillar cuticle (Lymantria dispar), and
hard (sclerotized) cuticles from a beetle (Popilla japonica)
and a cockroach (Blaberus giganteus). Each of these insects is
a susceptible host for M. anisopliae.
These studies demonstrated that M . anisopliae can rapidly
adjust its genomic expression patterns to adapt to insect
cuticle, and identified specific responses to different cuticles.
Genes specifically induced by cuticle incl uded a plethora of
cuticle-degrading enzymes, transporters for cuticle degra-
dation products and a subset of transcription factors.
Strains and culture conditions. To measure the variation in the
expression of genes during starvation conditions or during adapta-
tion to growth on different insect cuticles, we transferred cultures to
minimal medium (MM) or cuticle-containing media after a period
of unrestricted growth on a nutrient-rich medium. This is an effec-
tive and reproducible procedure for obtaining proteins that require
release from catabolite repression and/or specific induction by a
cuticular component (St Leger et al., 1994). M. anisopliae sf. aniso-
pliae (strain ARSEF 2575) was routinely grown at 27 uC, either in
liquid (SDB) or on solid (SDA) Sabouraud dextrose medium sup-
plemented with 0? 5 % yeast extract. For RNA extraction, the fungus
was grown for 48 h in 50 ml liquid SDB broth. The cultures were
then washed with sterile distilled water and 2 g wet weight of the
fungal biomass was transferred for up to 24 h to 10 ml MM con-
taining 0?1% KH
,0?05 % MgSO
and 50 % tap water, supple-
mented with 1 % of the following additives: tobacco hornworm
caterpillar (Man. sexta cuticle, MC); cockroach cuticle (CC, B.
giganteus); beetle cuticle (BC, P. japonica); gypsy moth cuticle (GC,
L. dispar ). Cuticles were prepared as described previously (St Leger
et al., 1986b). Alternatively, M. anisopliae sf. anisopliae was trans-
ferred to 10 ml of Man. sexta haemolymph (HL) obtained and trea-
ted as described by Grundschober et al. (1998).
cDNA microarray experiments. All unique ESTs with significant
BLAST matches (Freimoser et al., 2003) were amplified using T3
and T7 primers and standard PCR protocols. It should be noted
that, as with most other bioinformatic studies, gene identities are
based on computer-predicted homologies, and in very few cases (e.g.
serine proteases and hydrophobins) have the protein products of
these genes been demonstrated experimentally. Genes found among
the EST sequences of M. anisopliae sf. acridum (ARSEF 324), such
as chitinases and chitosanase (Freimoser et al., 2003), which were
absent from the M. anisopliae sf. anisopliae (ARSEF 2575) EST col-
lection, were amplified from M. anisopliae sf. anisopliae genomic
DNA with specific primers and included on the array. This
resulted in 837 clones, which were precipitated and resuspended
in 36 SSC (16 SSC=0?15 M sodium chloride, 0?015 M sodium
citrate, pH 7?0) to give a final DNA concentration between 100 and
300 ng ml
Printing, hybridization and scanning of slides were performed with
an Affymetrix 417 Arrayer and 418 Scanner (see http://www. for detailed protocols)
at the University of Maryland Biotechnology Institute’s Microarray
Core Facility located at the Center for Biosystems Research. PCR
products were spotted in triplicate on poly-lysine-coated glass slides,
with a mean spot diameter of 100
mm and a spot spacing of 375 mm.
Following printing and cross-linking, slides were washed with 1 %
SDS to remove background, treated with blocking solution (0?2M
succinic anhydride, 0?05 M sodium borate, prepared in 1-methyl-
2-pyrrolidinone) and washed with 95 uC water and 95 % ethanol. After
drying, slides were kept in the dark at room temperature.
RNA was extracted as previously described for M. anisopliae (Joshi &
St Leger, 1999). For experiments comparing different media, RNA
from a culture transferred to SDB was used as the reference sample.
For time-course experiments, mycelium was collected after 1, 2, 4, 8,
12, 18 and 24 h from MM or from medium containing MC. RNA
from the 0 h time-point was used as a reference. Hybridizations
were done with Cy3- and Cy5-labelled probes derived from 50–80
of total RNA. All hybridizations were repeated at least three times
with RNA from independent experiments and with switched labelling
for the reference and test RNA samples.
Analysis of microarray data. The images of the scanned slides
were analysed with Scanalyse (available from Eisen Lab: http://rana. and the data obtained from each scanned slide were nor-
malized using global normalization, as performed by J-Express
(Dysvik & Jonassen, 2001). All data were log
-transformed, and for
further analysis the mean (Em) and the standard deviation (
the log-transformed expression ratios of the replicates were calcu-
lated for all genes. A gene was defined as differently regulated if the
expression varied by at least a factor of two (1<Em<21). Expres-
sion ratios not fulfilling this requirement (21<Em<1) were defined
as zero, and the same was done for cases where the interval
SD (95 % confidence interval around the mean value for
the three replicate spots) included the value 0. Further analysis of the
processed data was performed using J-Express (Dysvik & Jonassen,
EPCLUST ( and Excel.
Validation of differentially expressed clones through real-
time PCR. A total of 16 clones predicted to be differentially
expressed by microarray analysis were tested by quantitative reverse-
transcription PCR (RT-PCR) by using an Applied Biosystems
GeneAMP 5700 sequence detection system and an Applied
Biosystems TaqMan RT kit. Transcript abundance was calculated
by using the comparative
DCt method relative to the amount of
the tubulin alpha chain transcript AJ273998 or 18S rRNA in the
sample, with primers and conditions as described by Parsley et al.
(2002). Differential expression based on RT-PCR measurements
was defined as a change in transcript abundance accumulation of
twofold or more.
Overall patterns of cuticle-induced gene
The libraries we employed to obtain ESTs were made
from fungi growin g on Man. sexta cuticle (MC) for 24 h
(Freimoser et al., 2003). The complete list of ESTs class-
ified into functional groups is available at http:// These
ESTs were hybridized with labelled RNA probes isolated
from mycelium harvested up to 24 h after the transfer from
a nutrient-rich medium (SDB) to media containing an
insect cuticle or HL. As a control for time-cou rse studies
with MC, mycelium was challenged by transfer to MM,
362 Microbiology 151
F. M. Freimoser, G. Hu and R. J. St Leger
revealing the response to nutrient deprivation after growth
in SDB. The responses to MC and MM were studied in
parallel time-course experiments, each with seven time-
points (1–24 h) that, together with the redundant sequence
representation in the microarrays, ensured the robustness of
the expression profiles. The expression ratios for ESTs in the
different media are shown in Supplementary Table S1 with
the online version of this paper at http://mic.sgmjournals.
org. In addition, the ESTs are characterized in Table S1 with
their accession numbers, E values and a description of the
BLAST hit.
An overview of the microarray results is presented in Fig. 1.
They illustrate the rapid changes in expression of some genes
in response to MC. Overall, these changes increased in
magnitude with time. Thus at 4 and 18 h post-inoculation
in MC medium, 88 and 154 genes were upregulated, res-
pectively. Similarly, 66 genes were down-regulated at 8 h,
and 143 genes were down-regulated at 18 h. During the
first hour, there was no overlap between genes upregulated
in response to MC and those upregulated in resp onse to
starvation conditions (MM). However, by 18 and 24 h, 30 %
of the genes were concomitantly upregulated in MC and
MM, indicating that catabolite repression is involved in
regulating at least some cuticle-induced genes. A cluster of
41 gen es was rapidly activated (<2 h) by MM, but down-
regulated in response to cuticle (Fig. 1). Only eight of
these genes had homologues with known biological activity
in databases, and these included the subtilisin Pr1G and
ribosomal proteins. At least with respect to the regulation of
these 41 genes, nutrient deprivation may be perceived as
having an effect distinct from and even opposite to that of
induction by cuticle.
In contrast, the magnitude of expression of most genes
upregulated by nutrient deprivation, including the majority
of the secreted proteases, was sharply increased by the pre-
sence of cuticle (Supplementary Table S1). In addition, a
large subset of diverse genes was upregulated by MC, and
not by MM, during the first 2 h, suggesting that these genes
are specifically involved in adaptation to growth on cuticle
(Fig. 2). A broad view of the nature of the adaptations
made by M. anisopliae following transfer from nutrient-rich
(SDB) medium to MC was obtain ed by grouping func-
tionally related genes (Fig. 3). Changes involving upregula-
tion, measured on the microarray for each functional
category during the 24 h of growth on MC, were either
gradual following the first hour, as for secreted proteases,
or abrupt during the first hour followed by a slow decline, as
for genes for amino acid/peptide uptake. Down-regulated
genes included many for protein synthesis machinery,
excluding RNA synthesis and processing. Genes encoding
ribosomal proteins and translational machinery were coor-
dinately regulated, showing an initial decrease, followed
by an increase and a decrease, resulting in an 8- to 13-fold
down-regulation. The repression of ribosomal protein
genes has been reported in yeasts during multiple stress
responses, including glucose deprivation (Warner, 1999),
and may therefore be a general feature of fungi transferred
to a low-nutrient medium. Overall, ho usekeeping genes for
cell metabolism, including endocellular proteases, showed
stable expression.
Fig. 1. Gene expression patterns of M. anisopliae in response
to starvation conditions (MM), haemolymph (HL) from Man.
sexta, or cuticles from a beetle (BC), a cockroach (CC) and
caterpillars (Man. sexta, MC, and Lymantria dispar, GC).
Mycelia growing on MC or in starvation conditions were assayed
in time-course experiments. The 837 cDNA clone set was ana-
lysed by hierarchical clustering based on their expression pat-
terns. Genes showing at least twofold regulation, compared
with a reference probe from mycelia grown on SDB, are shown
in red (upregulated) and green (down-regulated). Colour inten-
sity is directly relative to magnitude of differential expression
ratios. Experiments were carried out in triplicate, and represen-
tative clusters are shown. 363
Microarray analysis of Metarhizium anisopliae
Based on the time-course experiments under starvation
conditions or in MM supplemented with MC, we chose the
24 h time-point to obtain a snapshot of gene expression
during growth on other insect cuticles. The large-scale
features of the expression patterns illustrate shared features
between the responses to different cuticles and to starva-
tion conditions, indicative of a stereotyped programme
of gene expression. However, no two expression patterns
were identical in terms of the genes affected and the
magnitude of expression alteration (Fig. 1). Of the 136
genes upregulated on MC at 24 h, 87 (64 %) were similarly
regulated on CC, 96 (71 %) were similarly regulated on
BC and 95 (70 %) were similarly regulated on GC. Among
these commonly regulated genes, 64 were upregulated on
all four cuticles at 24 h. The balance of genes demonstrated
specific responses to different cuticles, including up- or
down-regulation of genes not observed at any time-point
on MC (Fig. 4). This implies that the pathogen can pre-
cisely respond to different conditions. In some cases, genes
coordinately upregulated on a particular cuticle were func-
tionally related. Thus, several sequences upregulated at
24 h on GC, but not MC, CC or BC at this time-point,
have homologues in yeast that are involved in integrating
nutrient and growth signals with morphogenesis. These
include LAS1, a nuclear protein required for cell-surface
growth and bud formation (Doseff & Arndt, 1995); SLA2,
required for morphogenesis and polarization of the
membrane cytoskeleton (Holtzman et al., 1993); Ecm15p,
involved in yeast cell-wall biogenesis (Goffeau et al., 1996);
and PIG-L, essential in the synthes is of glycosylpho-
sphatidylinositol, used as a membrane anchor by cell-
surface proteins (Watanabe et al., 1999).
Fig. 2. The cluster of M. anisopliae genes upregulated within 2 h in medium containing Man. sexta cuticle, and which were
not upregulated in minimal medium (starvation conditions). Samples were reordered from Fig. 1 according to the time-scale
shown across the top, and genes were hierarchically clustered. Gene names and accession numbers are shown to the right of
the figure.
364 Microbiology 151
F. M. Freimoser, G. Hu and R. J. St Leger
Identification of genes regulated by nutrient
deprivation and by insect cuticles
Energy metabolism. While the overall expression of sev-
eral functional categories, including cell metabolism, was
largely unaltered by transfe r to cuticle (Fig. 3), individual
ESTs among categories were altered in regulation, possibly
indicative of pivotal enzymes involved in metabo lic repro-
gramming (Supplementary Table S1). Although pathways
are incomplete in the M. anisopliae array, infere nces can
be made from the differential expression of representative
genes, as this likely reflects alterations in the pathways
in which these genes are involved. We thus compared our
results with experiments performed with Saccharomyces
cerevisiae (DeRisi et al., 1997) and Trichoderma reesei
(Chambergo et al., 2002), using very similar nutrient-rich
and nutrient-poor media. In all three species, the regula-
tion of many genes that participate in key metabolic pro-
cesses is not affected by being in sugar-rich media, such
as SDB (Supplementary Table S1). However, in T. reesei,
expression of genes encoding tricarboxylic acid (TCA)
cycle components and mitochondrial proteins favours
the oxidation of pyruvate via the TCA cycle, rather than
its reduction to ethanol by fermentation. In contrast,
S. cerevisiae preferentially ferments glucose, even in the
presence of oxygen. Only when glucose is exhausted do
yeast cells use the ethanol as a carbon and energy source
for aerobic respiration (the ‘diauxic shift’). M. anisopliae
resembled T. reesei in that the abundance of transcripts
encoding enzymes of the glycolytic pathway and TCA
cycle (e.g. isocitrate dehydrogenase, AJ272972) was mostly
unaffected upon transfer from a sugar-rich (SDB) to a
sugar-deficient medium (MM). In yeast, these genes are
strongly repressed in sugar-rich media. Yeast mitochon-
drial genes are also subject to strong repression by glu-
cose. However, levels of M. anisopliae transcripts encoded
by the mitochondrial genome (e.g. NADH ubiquinone
dehydrogenase, AJ273010) and nuclear genes encoding
mitochondrial proteins (e.g. cytochrome c oxidase chain
V, AJ272726) were the same or higher in sugar-rich media
than in MM. These results indicate that, like T. reesei, but
unlike yeast, M. anisopliae will respire in the presence of
However, M. anisopliae appears to differ from T. reesei in
the extent to which aerobic respiration prevails. As in
yeast and T. reesei,aM. anisopliae pyruvate decarboxy-
lase (AJ274332) is upregulated in the presence of sugar.
Fig. 3. Regulation of functionally related
genes. The curves represent the average
induction or repression ratios for all the
genes in each indicated group. The total
number of genes in each group was as fol-
lows: cell metabolism, 71; cofactors/vitamins,
6; energy metabolism, 27; ribosomal pro-
teins, 25; translation, 15; tRNA synthesis, 4;
secreted protease, 23; intracellular pro-
teases, 12; transport proteins, 14; amino
acid/peptide transporters, 6; cell wall struc-
ture/formation, 26; stress response, 26;
RNA metabolism, 28. 365
Microarray analysis of Metarhizium anisopliae
However, in contrast to these fungi, M. anisopliae has
an additional pyruvate decarboxylase (AJ274298) that is
repressed in nutrient-rich medium but upregulated within
1 h on MC (Fig. 2). In S. cerevisiae, the acetaldehyde
formed from pyruvate decarboxylase is reduced to ethanol
by alcohol dehydrogenase, and is not converted to acetate,
due to repression of aldehyde dehydrogenase by glucose.
Two paralogous genes for aldehyde dehydrogenase hav e
been identified in T. reesei, only one of which is repressed
by nutrient-rich conditions. In contrast , both the aldehyde
dehydrogenases (AJ272833 and AJ273869) in M. anisopliae
are down-regulated in SDB compared to cuticle-containing
media, suggesting that readily utilized nutrients repress
acetate production. It is of interest that AJ272833 is
upregulated at an earlier time-point on MC than in
MM (Fig. 2). We also identified two paralo gues of
acetyl coenzyme A synthetase: the AJ273955 transcript is
upregulated early during growth on cuticle and late
during growth in MM (Fig. 2), while regulation of
AJ274191 is not affected. If both enzymes have compara-
ble specificity, production of acetyl coenzyme A in glucose-
poor media such as cuticle will increase the entry of
acetate, produced via the pyruvate bypass route, into the
TCA cycle. Interestingly, M. anisopliae also has two
paralogous genes for alcohol dehydrogenase. AJ273792 is
regulated in a similar fashion to pyruvate decarboxylase
AJ274332 (upregulated in SDB), whereas AJ273547, like
pyruvate decarboxylase AJ274298, is repressed in SDB.
Thus, M. anisopliae has multiple gene families of cata-
bolic enzymes, some of which include isoforms that are
differentially regulated by sugars. These alternative forms
may give M. anisopliae the flexibility to shunt any avail-
able pyruvate into fermentation or the TCA cycle,
irrespective of sugar levels.
Fig. 4. Subclusters of genes specifically upregulated on only one of the cuticles (BC, beetle; CC, cockroach; MC, Man.
sexta; GC, Lymantria dispar). Samples were reordered from Fig. 1 according to the time-scale shown across the top, and
genes were hierarchically clustered. Gene names and accession numbers are shown to the right of the figure.
366 Microbiology 151
F. M. Freimoser, G. Hu and R. J. St Leger
Amino acid, carbohydrate and lipid metabolism. Genes
with homologues involved in amino acid catabolism and
which were upregulated on cuticle included glutami nase
A (AJ273512) and NADH-specific glutamate dehydrogen-
ase (AJ274362). Glutamate is the preferred amino acid
substrate for M. anisopliae (St Leger et al., 1986a).
Otherwise, diverse genes involved in amino acid synthesis
were commonly down-regulated in MM and on cuticle,
consistent with the reduced availability of raw materials
for biosynthesis. Insect cuticle also contains diverse lipids,
and seven of 13 genes for lipid metabolism were upre gu-
lated on at least one cuticle. Only a cytochrome P450
monooxygenase (AJ274003) wa s also upregulated during
growth in MM. Lipases are the last class of depolymerases
to be secreted in insect cuticle (St Leger et al., 1986 b),
consistent with which, lipase AJ274124 was upregulated
in late cuticle-containing cultures (24 h) only. Enzyme
assays have also detected a secreted DNase activity during
growth on cuticle (St Leger et al., 1986b), and in this
study DNase (AJ273950) was upregulated in cuticle-
containing media.
Protein a side, the major component of insect cuticle is
chitin, and predictably therefore chitinases were upregu-
lated on cuticle. Chitinase AJ274366 was exp ressed
within 1 h on MC, but was not expressed in MM (Fig. 2).
Chitosanase was only produced on GC (Fig. 4). As this
coincides with the GC-specific expression of genes involved
in morphogenesis, it is possible that the chitosanase may
be involved in modifying cell wall components. However,
five additional enzymes involved in metabolizing carbo-
hydrates not known to occur in cuticle were also upregu-
lated in one or more of the cuticle media: formate
dehydrogenase (AJ274347), usually involved in detoxifica-
tion reactions; 1,2-
a-D-mannosidase (AJ273630); b-D-
galactosidase (AJ273808);
L-sorbosone dehydrogenase
(AJ273834); and b-glucosidase (AJ273623). These could
be involved in digesting glycoproteins, but were also
more weakly upregulated in starvation conditions, consis-
tent with catabolite repression in SDB . Only one of
the genes for carbohydrate metabolism (AJ272928)
was upregulated in response to HL, while seven genes
were down-regulated (Supplementary Table S1). Seven
carbohydrate-metabolizing enzymes were down-regulated
on cuticle-containing media, including a transketolase
(AJ274194) and fructose-bisphosphate aldolase (AJ273952).
RNA synthesis. Elements required for mRNA synthesis,
such as RNA polymerase (AJ272996) and RNA polymer-
ase transcription factor (AJ274125) were upregulated in
cuticle-containing media, but not in MM or HL. This
presumably adapts the fungus for the rapid synthesis of
cuticle-degrading enzymes.
Transport proteins. The ESTs included homologues
of two distinct peptide transport systems, one for di-/
tripeptides (PTR transporter AJ273551 and PTR-2 trans-
porter AJ272830) and another for tetra-pentapeptides
(OPT transporter AJ273568), as well as diverse amino
acid transporters (e.g. the INDA1 homologue AJ272773).
These all required induction by cuticle, and most were
upregulated 8- to 12-fold within 1 h on MC (Figs 2 and
3). In contrast, the PTR transporter in T. reesei is upregu-
lated by glucose exhaustion alone (Chambergo et al., 2002),
consistent with the M. anisopliae transporters having
acquired more specialized functions in pathogenicity.
Only the M. anisopliae oligopeptide transporter OPT2
(AJ273118) was not upregulated in cuticle-containing
media. Regulation of peptide/amino acid transporters was
not altered in HL compared to growth on SDB.
Proteolytic enzymes. It had been shown previ ously
that total subtilisin activity is produced in response to
nutrient deprivation, but that production is enhanced by
the addition of cuticle to media (Paterson et al., 1994).
Consistent with this, subtilisins Pr1A and Pr1B were upre-
gulated on MM, and to a greater extent on insect cuticles
(Fig. 5). Increased induction by cuticle compared with
nutrient deprivation alone suggests that subtilisin produc-
tion is controlled by multiple regulatory systems evoked
under different environmental conditions. In contrast,
Pr1C, Pr1D, Pr1E, Pr1F, Pr1I and Pr1J were down-
regulated at most time-p oints in MM. Of these, Pr1C and
Pr1D were rapidly upregulated (Pr1C within 1 h of trans-
fer to MC; Fig. 2), while upregulation of Pr1E and Pr1K
in MC was delayed by 4 and 8 h, respectively. Pr1J was
upregulated on all the cuticles, except BC. Pr1G was shar-
ply down-regulated in CC. Pr1F and Pr1I were upregu-
lated on MC and on GC. Expression of Pr1H was slightly
upregulated by transfer to MC and MM.
The exo-acting carboxypeptidase AJ274343 was upregulated
after 18 h in MM, but showed earlier and much stronger
upregulation in all cuticle-containing media. Most other
categories of exopeptidases (e.g. aminopeptidases AJ273806
and AJ274061) and endopeptidases, including trypsin
(AJ272743), chymotrypsin (AJ273663), metalloprotease
(AJ273481) and aspartyl protease (pepsinogen)
(AJ274168) were only upregulated in the presence of cuticle.
Transcription factors and signal transduction. Of the
17 arrayed ESTs enc oding homologues of proteins known
to be involved in transcription in other organisms, ten
(AJ272823, AJ272967, AJ273078, AJ273134, AJ273171,
AJ273219, AJ273260, AJ273589, AJ273694 and AJ274235)
were upregulated on at least one cuticle. The positive
sulfur transcription regulator homologue (AJ273134)
was down-regulated in MM and BC, suggestive of parti-
cularly low sulfur levels in these media (sulfite reductase,
AJ273620, but not sulfite oxidase, AJ272866, was upregu-
lated on cuticle, within 1 h in MC, but not in MM and
HL). In contrast, the pH signalling transcription factor
PacC (AJ273219) was upregulated on cuticle, but not in
MM or HL . AJ272977 was unique in being upregulated in
HL. In contrast, AJ273694 was very strongly down-regulated
in HL and strongly upregulated on the lepidopteran 367
Microarray analysis of Metarhizium anisopliae
cuticles GC and MC. Among gene products involved
in signalling (category 4f), adenylate cyclase (AJ251971)
(the enzyme that produces cAMP) and protein kinase A
(AF116597) (PKA: the major effector of cAMP responses)
were not upregulated on cuticle-containing media, while a
downstream activity, MAP kinase kinase 2 (AJ273356)
was upregulated in GC- and BC-containing media, and at
two time-points in MC.
Cell wall proteins. Of 30 genes encoding proteins
involved in cell structure and function, 18 were upregu-
lated in at least one cuticle-containing medium. The
hydrophobins are differenti ally regulated. Thus, AJ273847
was upregulated in HL and MM, and down-regulated in
cuticle-containing media, while AJ274156 was upregulated
in MM and on sclerotized cuticles (CC and BC), un-
altered on lepidopteran cuticles (GC and MC) and down-
regulated in HL. The other cell wall proteins upregulated
on cuticle were AJ273845, a homologue to an antigenic
cell wall protein from the human pathogen Aspergillus
fumigatus, and AJ274019, which is very similar to the
antifungal glucan 1,3-
b-glucosidase fro m Trichoderma
atroviride (Donzelli et al., 2001). Clearly, besides cell wall
biosynthesis and structure, these proteins may have addi-
tional functions in pathogenicity or in protecting scarce
resources from competitors.
Stress response. Several arrayed M. anisopliae ESTs
are similar to peptide synthases, reductases and other
enzymes that take part in the synthesis of fungal toxins,
such as destruxins, trichothecene and enniatin (Freimoser
et al., 2003). This is in agreement with the observation
that M. anisopliae strain 2575 rapi dly kills its host after
infection through the action of toxins, and subsequently
colonizes the insect host by saprob ic growth (Samuels
et al., 1989). Genes upregulated in at least one cuticle-
containing medium included those encoding a peptide
synthase (AJ272930, in BC and GC), a protein involved
in sterigmatocystin biosynthesis (AJ273515, in GC), versi-
colorin B synthase (AJ272697, in CC, GC, MC and HL)
and a bacteriolytic enzyme (AJ272917, in CC, BC, GC,
early in MC and after 12 h in MM).
Validation of microarray results. An external quality
control check on the lists of differentially expressed clones
generated through microarray profiling was provided by
analysing a subset of 16 clones by quantitative RT-PCR.
Each clone tested by RT-PCR was measured in triplicate
for each of two independent RNA isolations. All 16 clones
were confirmed by RT-P CR, indicating a very high success
rate for predicting differentially expressed clones. How-
ever, expression ratios were consistently underestimated at
least 10-fold by cDNA microarrays, compared to PCR-
based methods.
The construction of a M. anisopliae cDNA microarray
provides many advantages over previous labour-intensive
techniques to monitor transcriptional responses to host
tissues. For this study, it provided a powerful tool with
which to examine the influence of culture conditions on the
magnitude and spectrum of cuticle-induced gene expres-
sion. The analysis presented here has expanded the number
of identified M. anisopliae genes that respond to cuticle
from about 20 (Joshi & St Leger, 1999; Joshi et al., 1997) to
more than 200. Expression patterns of known pathogeni-
city genes, incl uding Pr1A subtilisin, hydrophobin, trypsins,
chymotrypsin and carboxypeptidase, matched previously
published data (Screen & St Leger, 2000; St Leger et al.,
1986b, 1987, 1992, 1996). This provides a high level of
confidence that the arrays accurately identify differentially
expressed clones. For selec ted genes, including subtilisins
(Pr1A, Pr1H and Pr1K), a trypsin (Try1) and tubulin, the
Fig. 5. Subtilisin gene expression during growth on cuticle from Man. sexta compared with expression during nutrient
deprivation. The curves show average expression ratios for different subtilisins in minimal medium (MM) and on Man. sexta
cuticle (MC): &, Pr1A; %, Pr1B; #, Pr1I;
, Pr1E; +, Pr1J; n, Pr1D; m, Pr1C; x, Pr1K.
368 Microbiology 151
F. M. Freimoser, G. Hu and R. J. St Leger
expression patterns were also verified by quantitative real-
time RT-PCR. Normally (e.g. Yuen et al., 2002), expression
ratios are greatly underestimated by cDNA microarrays,
compared to PCR-based methods. Together with our strict
analysis of the replicates within and between different arrays,
this suggests that our estimates of the magnitude of changes
in expression are conservative.
The demonstration of the differential regulation of genes
encoding cuticle-degrading enzymes, cell w all proteins,
toxins and toxin-producing enzymes on the different
cuticles, and in HL and MM suggests that M. anisopliae
may have the ability to target the production of these
proteins to different hosts. Like other ascomycete patho-
gens, M. anisopliae secretes a great variety of proteases (Hu
and St Leger, 2004), some of which have been associated
with virulence, because they allow rapid physical ingress,
nutrient solubilization and the disabling of antimicrobial
peptides (St Leger et al., 1996). The subtilisin cluster pro-
vides a good example of de novo protein synthesis req uired
for adaptation to growth on cuticle (Figs 1 and 5, Supple-
mentary Table S1), particularly as the differences in regula-
tion of subtilisins imply differences in their function. This
supports homology-modelling studies based on seque nces
that predict differences between the Pr1s in their secondary
specificities, adsorption properties to cuticle and alkaline
stability (Bagga et al., 2004). It is likely that these differences
in regulation and structure–function allow M. anisopliae to
respond flexibly, producing proteases that are appropriate
to the composition of the environment, consistent with its
opportunistic lifestyle. Thus the proteases such as Pr1A
produced as part of a general response to nutrient depriva-
tion could also function outside of pathogenesis by scaveng-
ing for nutrients during saprophytic existence. During early
infection processes, they could also function in concert with
the exopeptidases to provide host degradation products.
These may include specialized signals that allow the fungus
to ‘sample’ the cuticle and then respond with the secretion of
a plethora of cuticle-induced proteins. This will include the
proteases that require cuticle for induction, as they
presumably have specialized roles in breaching host barriers.
The very early induction of peptide/amino acid transport
systems (Figs 2 and 3) would enhance the ability of the
fungus to rapidly and precise ly monitor host degradation
Hydrophobins provide another example besides subtilisins
where members of a family are differentially regulated,
consistent w ith different functions. Thus, AJ273847 was
upregulated in HL, while AJ274156 was down-regulated
in HL. This suggests that adaptation to HL may include
alterations in cell wall composition.
Of key importance to understanding the mechanisms
behind adaptation to cuticles is the identification of com-
ponents of signal transduction that will allow M. anisopliae
to screen its surroundings to regulate protein synthesis and
secretion. PacC-mediated pH signalling is crucial to the
pathogenicity of the human pathogen Candida albicans and
the plant pathogen Fusarium oxysporum (Caracuel et al.,
2003; Davis et al., 2000). Consistent with a crucial role for
PacC in M. anisopliae, extrac ellular pH rises during cuticle
degradation and acts as a key signal for the production of
alkaline-active enzymes such as subtilisins (St Leger et al.,
1998). Significant for their absence of response were
adenylate cyclase and protein kinase A, as transcriptional
regulation in response to the cAMP signalling pathways
seems central to infection-related development in M. aniso-
pliae (St Leger, 1993). Constitutive expression may be a
feature of some primary initiators of physiological pro-
cesses, so their importance will not be detected in micro-
array analyses. A downstream activity, MAP kinase kinase 2
(AJ273356) was upregulated in GC- and BC-containing
media. This enzyme, and other transcription factors, may
constitute downstream ‘ground-level’ components which
are immediately concerned with recognizing and respond-
ing to specific host features, and which do not control fungal
metabolism as a whole. As such, they may be useful for strain
improvement purposes.
Microarray technology has made it possible to decipher
the transcriptional programmes of organisms by studying
gene expression en masse while assessing individual gene
function in a detailed manner (Brown & Botstein, 1999).
Thus, knowing when and where a gene is expressed ofte n
provides a strong clue as to its function (DeRisi et al.,
1997). Almost 50 % of the arrayed ESTs upregulated in
cuticle-containing media have undiscovered biological acti-
vities (Table 1 and Supplementary Table S1), and 25 % of
these are not upregulated in MM or HL. These genes have
never been recognized to have a role in pathogenicity, but
are now implicated by co-regulation with known virulence
factors. They thus pro vide an additional rich resource for
future research.
Evolutionary theory has long held that the process of
adaptation is driven by competition for limited resources.
Among heterotrophic micro-organisms, the availability of
carbon limits the ability of these organisms to multiply. As a
result, the machinery of central metabolism is tuned to
exploit reduced carbon resources in natural environments,
where they vary greatly in both form and abundance (Ferea
et al., 1999 ). Comparisons between M. anisopliae, T. reesei
and S. cerevisiae suggest that the three fungi will respond
differently to environmental changes, presumably reflecting
their adaptation to predictable differences in the composi-
tion of thes e environments. The similarities between M.
anisopliae and T. reesei may reflect their close relationship
as clavicipitaceous pyrenomycetes. However, the alterna-
tively regulated forms of catabolic enzymes in M. anisopliae
and T. reesei suggest they will differ in how they coordinate
the regulation of key parts of metabolism, such as fermen-
tation at different levels of glucose. This could affect the
extent to which aerobic respiration prevails in glucose-rich
Evidently, gene-duplication events and altered patterns
of regulation could provide mechanisms for evolution to 369
Microarray analysis of Metarhizium anisopliae
fine-tune ATP-producing pathways, allowing these organ-
isms to adapt to their different environments and nutri-
tional requirements. It is tempting to speculate that
fermentation may play a more pivotal role in the life of
M. aniso pliae, compared to that of T. reesei, to enable it to
exploit sugars in the anaerobic environment of the dead
host. However, complicating interpretation of these results,
ATP-producing pathways can be co-opted to other func-
tions. Thus, some fungal acetyl coenzyme A synthetases are
involved in the biosynthesis of secondary metabolites such
as penicillin, as well as in primary metabolism (Martinez-
Blanco et al., 1993). It is axiomatic that as more is learned
about the function of each gene, comparative studies on
transcriptomes will become an increasingly powerful tool
allowing predictive insights into the behavioural plasticity of
each saprophyte or pathogen.
This work was supported by the USDA (grant 2003-35302-13588).
F. M. F. acknowledges support from the Swiss National Science Founda-
tion. We would like to thank A. Wilson and S. Grundschober for
computer support.
Bagga, S., Hu, G., Screen, S. E. & St Leger, R. J. (2004).
Reconstructing the diversification of subtilisins in the pathogenic
fungus Metarhizium anisopliae. Gene 324, 159–169.
Brown, P. O. & Botstein, D. (1999). Exploring the new world of the
genome with DNA microarrays. Nat Genet 21, 33–37.
Caracuel, Z., Roncero, M. I., Espeso, E. A., Gonzalez-Verdejo, C. I.,
Garcia-Maceira, F. I. & Di Pietro, A. (2003). The pH signalling
transcription factor PacC controls virulence in the plant pathogen
Fusarium oxysporum. Mol Microbiol 48, 765–779.
Chambergo, F. S., Bonaccorsi, E. D., Ferreira, A. J., Ramos, A. S.,
Ferreira, J. R. J. J. R., Abrahao-Neto, J., Farah, J. P. & El-Dorry, H.
(2002). Elucidation of the metabolic fate of glucose in the
filamentous fungus Trichoderma reesei using expressed sequence
tag (EST) analysis and cDNA microarrays. J Biol Chem 277,
Davis, D., Wilson, R. B. & Mitchell, A. P. (2000). RIM101-dependent
and-independent pathways govern pH responses in Candida albicans.
Mol Cell Biol 20, 971–978.
DeRisi, J. L., Iyer, V. R. & Brown, P. O. (1997). Exploring the
metabolic and genetic control of gene expression on a genomic scale.
Science 278, 680–686.
Donzelli, B. G., Lorito, M., Scala, F. & Harman, G. E. (2001). Cloning,
sequence and structure of a gene encoding an antifungal glucan
b-glucosidase from Trichoderma atroviride (T. harzianum). Gene
277, 199–208.
Doseff, A. I. & Arndt, K. T. (1995). LAS1 is an essential nuclear
protein involved in cell morphogenesis and cell surface growth.
Genetics 141, 857–871.
Dysvik, B. & Jonassen, I. (2001). J-Express: exploring gene
expression data using Java. Bioinformatics 17, 369–370.
Ferea, T. L., Botstein, D., Brown, P. O. & Rosenzweig, R. F. (1999).
Systematic changes in gene expression pattern following adaptive
evolution in yeast. Proc Natl Acad Sci U S A 96, 9721–9726.
Freimoser, F. M., Screen, S., Bagga, S., Hu, G. & St Leger, R. J.
Expressed sequence tag (EST) analysis of two subspecies of
Metarhizium anisopliae reveals a plethora of secreted proteins with
potential activity in insect hosts. Microbiology 149, 239–247.
Goffeau, A., Barrell, B. G., Bussey, H. & 13 other authors (1996).
Life with 6000 genes. Science 274, 563–567.
Grundschober, A., Tuor, U. & Aebi, M. (1998). In vitro cultivation
and sporulation of Neozygites parvispora (Zygomycetes: Entomo-
phthorales). Syst Appl Microbiol 21, 461–469.
Holtzman, D. A., Yang, S. & Drubin, D. G. (1993). Synthetic–lethal
interactions identify two novel genes, SLA1 and SLA2, that control
membrane cytoskeleton assembly in Saccharomyces cerevisiae. J Cell
Biol 122, 635–644.
Hu, G. & St Leger, R. J. (2002). Field studies using a recombinant
mycoinsecticide (Metarhizium anisopliae) reveal that it is rhizosphere
competent. Appl Environ Microbiol 68, 6383–6387.
Hu, G. & St Leger, R. J. (2004). A phylogenomic approach to
reconstructing the diversification of serine proteases in fungi.
J Evolution Biol 17, 1204–1214.
Joshi, L. & St Leger, R. J. (1999). Cloning, expression, and substrate
specificity of MeCPA, a zinc carboxypeptidase that is secreted
into infected tissues by the fungal entomopathogen Metarhizium
anisopliae. J Biol Chem 274, 9803–9811.
Joshi, L., St Leger, R. J. & Roberts, D. W. (1997). Isolation of a
cDNA encoding a novel subtilisin-like protease (Pr1B) from the
entomopathogenic fungus, Metarhizium anisopliae using differential
display-RT-PCR. Gene 197, 1–8.
Martinez-Blanco, H., Orejas, M., Reglero, A., Luengo, J. M. &
Penalva, M. A. (1993).
Characterisation of the gene encoding acetyl-
CoA synthetase in Penicillium chrysogenum: conservation of intron
position in plectomycetes. Gene 130, 265–270.
Parsley, T. B., Chen, B., Geletka, M. & Nuss, D. L. (2002).
Differential modulation of cellular signaling pathways by mild and
severe hypovirus strains. Eukaryot Cell 1, 401–413.
Paterson, I. C., Charnley, A. K., Cooper, R. M. & Clarkson, J. M.
Partial characterization of specific inducers of a cuticle-
degrading protease from the insect pathogenic fungus Metarhizium
anisopliae. Microbiology 140, 3153–3159.
Samuels, K. D. Z., Pinnock, D. E. & Allsopp, P. G. (1989).
The potential of Metarhizium anisopliae (Metschnikoff) Sorokin
(Deutermycotina, Hyphomycetes) as a biological control-agent of
Inopus rubriceps (Macquart) (Diptera, Stratiomyidae). J Aust Entomol
Soc 28, 69–74.
Screen, S. E. & St Leger, R. J. (2000). Cloning, expression, and
substrate specificity of a fungal chymotrypsin. Evidence for lateral
gene transfer from an actinomycete bacterium. J Biol Chem 275,
St Leger, R. J. (1993). Biology and mechanisms of insect cuticle
invasion by deuteromycete fungal pathogens. In Parasites and
Pathogens of Insects, pp. 211–229. Edited by N. C. Beckage, S. N.
Thompson & B. A. Federici. San Diego, CA: Academic Press.
St Leger, R. J., Charnley, A. K. & Cooper, R. M. (1986a). Cuticle-
degrading enzymes of entomopathogenic fungi: mechanisms of
interaction between pathogen enzymes and insect cuticle. J Invertebr
Pathol 47, 295–302.
St Leger, R. J., Charnley, A. K. & Cooper, R. M. (1986b). Cuticle-
degrading enzymes of entomopathogenic fungi: synthesis in culture
on cuticle. J Invertebr Pathol 48, 85–95.
St Leger, R. J., Charnley, A. K. & Cooper, R. M. (1987).
Characterization of cuticle-degrading proteases produced by the
entomopathogen Metarhizium anisopliae. Arch Biochem Biophys 253,
370 Microbiology 151
F. M. Freimoser, G. Hu and R. J. St Leger
St Leger, R. J., May, B., Allee, L. L., Frank, D. C., Staples, R. C. &
Roberts, D. W. (1992).
Genetic differences in allozymes and in
formation of infection structures among isolates of the entomo-
pathogenic fungus Metarhizium anisopliae. J Invertebr Pathol 60,
St Leger, R. J., Bidochka, M. J. & Roberts, D. W. (1994). Isoforms of
the cuticle-degrading Pr1 proteinase and production of a metallo-
proteinase by Metarhizium anisopliae. Arch Biochem Biophys 313,
St Leger, R. J., Joshi, L., Bidochka, M. J., Rizzo, N. W. & Roberts,
D. W. (1996).
Biochemical characterization and ultrastructural locali-
zation of two extracellular trypsins produced by Metarhizium aniso-
pliae in infected insect cuticles. Appl Environ Microbiol 62, 1257–1264.
St Leger, R. J., Joshi, L. & Roberts, D. (1998). Ambient pH is a major
determinant in the expression of cuticle-degrading enzymes and
hydrophobin by Metarhizium anisopliae. Appl Environ Microbiol 64,
Warner, J. R. (1999). The economics of ribosome biosynthesis in
yeast. Trends Biochem Sci 24, 437–440.
Watanabe, R., Ohishi, K., Maeda, Y., Nakamura, N. & Kinoshita, T.
(1999). Mammalian PIG-L and its yeast homologue Gpi12p are
N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential
in glycosylphosphatidylinositol biosynthesis. Biochem J 339, 185–192.
Yuen, T., Wurmbach, E., Pfeffer, R. L., Ebersole, B. J. & Sealfon,
S. C. (2002). Accuracy and calibration of commercial oligonucleotide
and custom cDNA microarrays. Nucleic Acids Res 30, 48. 371
Microarray analysis of Metarhizium anisopliae
... Two trypsin-related proteases, previously reported to be less efficient in degrading insect cuticles compared to Pr1 (Rosas-García et al., 2014), were noticed and might contribute in the early stages of colonization thus helping invasion (Manalil et al., 2010;St Leger et al., 1987). Taken as a pattern of evolution (Gao et al., 2020) and a potential virulence marker for R. microplus (Perinotto et al., 2014a), the rise in Pr1 expression (Freimoser et al., 2005;Staats et al., 2014), as well as its enzymatic activity over time (Perinotto et al., 2014b), have been demonstrated in in vitro studies. Although Pr1 isoforms are well-known (Andreis et al., 2019), the specific up-regulation of Pr1D and Pr1K observed here endorses their importance during growth and corroborates with the superior Pr1 activities found in our protease assays. ...
... Although Pr1 isoforms are well-known (Andreis et al., 2019), the specific up-regulation of Pr1D and Pr1K observed here endorses their importance during growth and corroborates with the superior Pr1 activities found in our protease assays. These isoforms were also highly expressed in the presence of Manduca sexta cuticle (Freimoser et al., 2005) and, we speculate that MND somehow modulated their increased differential expression. Accordingly, MND might potentially evoke a positive regulation of some specific isoforms, Table 3 Differentially expressed proteins identified in Metarhizium anisopliae s.l. ...
Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC50 by evaluating conidia germination (38µM). To ensure high cell integrity, a sublethal dose of MND (half of LC50) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC50 and LT50, particularly at 10⁸ conidia mL⁻¹. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC50), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.
... Likewise, the limitation of nutrients in the fungus was decisive to induce conidiation, and affect its virulence. In fact, the nutrient limitation has been described as a strong inducer of the expression of genes involved in sporulation [23,24], in the expression of enzymes involved in virulence, and in nutrient metabolism [7,10,25]. ...
... The results obtained in the present study show that culture media formulations based on insect cuticle (Sph®) and a reduction of nutrients (¼ SDAY) were more convenient than commercial culture media to enhance the pathogenicity of B. bassiana, C. javanica, and M. robertsii; they also turn out to be cheaper, easy to prepare, and with multiple advantages in various fungal applications. Our findings suggest that the culture media provide the nutrients (proteins, chitin, lipids, sugars, and amino acids) in the concentrations required (Sph® 1%) for the development of fungi and the induction of the enzymes (i.e., a twofold increase in Pr1 activity; Supporting Information) involved in their pathogenicity [25]. On the contrary, to make a convincing and reliable choice of the nutrient source, we suggest the use of PCA as an exploratory tool to find a correlation of variables, with which strong selection criteria can be established. ...
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Media formulated with insect cuticle (0.5% and 1%; Sphequit Sph®), with a reduction in nutrients (¼ Sabouraud dextrose agar + yeast [SDAY]) and commercial media (potato dextrose agar, Sabouraud dextrose agar) were evaluated for the cultivation of Beauveria bassiana, Cordyceps javanica (Isaria javanica [Bally] Samson & Hywel‐Jones), and Metarhizium robertsii. By using principal component analysis, it was determined that the ¼ SDAY and Sph formulations have greater advantages than commercial media for the development of fungi. The ¼ SDAY and Sph (0.5% and 1%) improved hydrophobicity, radial growth rate, germination, conidia yield, and virulence in B. bassiana; in M. robertsii, they favored conidia yield, germination, and virulence, and in C. javanica, the ¼ SDAY and Sph 0.5% media enhanced conidia yield, germination, radial growth rate, and virulence. We suggest that these formulations are an alternative to commercial culture media as they are cheaper and appropriate to improve the growth characteristics and virulence of the three strains evaluated. Some applications of culture media are suggested, and the importance of multivariate analysis as an exploratory tool to carry out the choice of culture media in a suitable way for the development of mycoinsecticides is also discussed.
... Comparing the virulence results of the A. fijiensis GDIZM-1 strain to the nymphs and adults of D. citri, it was found that its insecticidal ability against younger nymphs of D. citri was higher than that against older nymphs and adults, which may be related to the nutrition and structure of the integument, defense mechanism and microflora composition on the body surface at different developmental stages of D. citri [67][68][69]. The nymphs of D. citri have weak activity on twigs or buds and are easily infected by fungal spores. ...
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The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is the most widespread and devastating pest species in citrus orchards and is the natural vector of the phloem-limited bacterium that causes Huanglongbing (HLB) disease. Thus, reducing the population of D. citri is an important means to prevent the spread of HLB disease. Due to the long-term use of chemical control, biological control has become the most promising strategy. In this study, a novel highly pathogenic fungal strain was isolated from naturally infected cadavers of adult D. citri. The species was identified as Aspergillus fijiensis using morphological identification and phylogenetic analysis and assigned the strain name GDIZM-1. Tests to detect aflatoxin B1 demonstrated that A. fijiensis GDIZM-1 is a non-aflatoxin B1 producer. The pathogenicity of the strain against D. citri was determined under laboratory and greenhouse conditions. The results of the laboratory study indicated that nymphs from the 1st to 5th instar and adults of D. citri were infected by A. fijiensis GDIZM-1. The mortality of nymphs and adults of D. citri caused by infection with A. fijiensis increased with the concentration of the conidial suspension and exposure time, and the median lethal concentration (LC50) and median lethal time (LT50) values gradually decreased. The mortality of D. citri for all instars was higher than 70%, with high pathogenicity at the 7th day post treatment with 1×108 conidia/mL. The results of the greenhouse pathogenicity tests showed that the survival of D. citri adults was 3.33% on the 14th day post-treatment with 1 × 108 conidia/mL, which was significantly lower than that after treatment with the Metarhizium anisopliae GDIZMMa-3 strain and sterile water. The results of the present study revealed that the isolate of A. fijiensis GDIZM-1 was effective against D. citri and it provides a basis for the development of a new microbial pesticide against D. citri after validation of these results in the field.
... Recent research found that high concentrations of Butyl Benzyl Phthalate (BBP) significantly downregulated ribosomal RNA transcription on Chironomus riparius [68]; therefore, ribosomal gene expression in TF6 was inhibited, most likely due to some plant allelochemicals in S. tuberosum. Other studies have reported many insect ribosomal genes to be downregulated, including Metarhizium anisopliae [69], C. suppressalis [50], and Anopheles gambiae [70], in response to unsuitable diets or virus infection. Paracoccus marginatus fed C. papaya grew faster than those fed S. tuberosum (Table 1) [15], indicating that C. papaya is a more suitable plant for P. marginatus. ...
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Paracoccus marginatus (Hemiptera: Pseudococcidae) is an invasive pest with a diverse host range, strong diffusion, and high fecundity. It has been observed that P. marginatus feeding on Carica papaya have a higher survival rate, fecundity, and longer lifespan than P. marginatus feeding on Solanum tuberosum, indicating their successful adaptation to C. papaya; however, the mechanisms underlying host plant adaptation remain unclear. Therefore, RNA-seq was performed to study the transcriptional responses of P. marginatus feeding on C. papaya and S. tuberosum plants. A total of 408 genes with significant differential expression were defined; most of them were downregulated in S. tuberosum, including those of digestive enzymes, detoxifying enzymes, ribosomes, and reproductive-related genes, which may result from the adaptation of the host to nutritional needs and changes in toxic chemical levels. Enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes showed that lysosome and longevity regulating pathways related to digestion, detoxification, and longevity were enriched. We suggest that C. papaya is a more suitable host than S. tuberosum, and downregulated target genes may have important effects on the adaptation of P. marginatus to host transfer.
... Lipolytic enzymes including lipases, act primarily on the epicuticle, followed by proteases and chitinases, according to the presence of polymeric substrates in the different portions of the cuticle (Beys da Silva et al., 2010a,b). Among the proteases that can act at this level are subtilisins, trypsins, chymotrypsins, metallopeptidases, aspartyl peptidases, and exopeptidases (Semenova et al., 2020); where their expression from fungi such as M. anisopliae will depend specifically on the composition of the cuticle and hemolymph (Freimoser et al., 2005). EPF such as M. anisopliae and B. bassiana can express up to 11 different subtilisins, one of the most important being the Pr1 subtilisin-like peptidases, which intervene in the arthropod pathogenesis, causing hydrolysis of the cuticle and providing nutrients to the fungus (Gao et al., 2020;Semenova et al., 2020). ...
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Ticks are one of the main economic threats to the cattle industry worldwide affecting productivity, health and welfare. The need for alternative methods to control tick populations is prompted by the high prevalence of multiresistant tick strains to the main chemical acaricides and their ecological consequences. Biological control using entomopathogenic fungi (EPF) is one of the most promising alternative options. The objective of this paper is to review the use of EPF as an alternative control method against cattle ticks in Mexico. Metarhizium anisopliae sensu lato (s.l.) and Beauveria bassiana s.l. are the most studied EPF for the biological control of ticks in the laboratory and in the field, mainly against Rhipicephalus microplus ; however, evaluations against other important cattle ticks such as Amblyomma mixtum and R . annulatus , are needed. A transdisciplinary approach is required to incorporate different types of tools, such as genomics, transcriptomics and proteomics in order to better understand the pathogenicity/virulence mechanism in EPF against ticks. Laboratory tests have demonstrated the EPF efficacy to control susceptible and resistant/multiresistant tick populations; whereas, field tests have shown satisfactory control efficiency of M . anisopliae s.l. against different stages of R . microplus when applied both on pasture and on cattle. Epidemiological aspects of ticks and environmental factors are considered as components that influence the acaricidal behavior of the EPF. Finally, considering all these aspects, some recommendations are proposed for the use of EPF in integrated control schemes for livestock ticks.
... Leger et al. 1987, 1988, 1992. The other subtilisins showed differences in regulation that could probably allow these virulence determinants to target different hosts and stages (Freimoser et al. 2005). Using liquid chromatography/tandem mass spectrometry (LC-MS/MS), Santi and co-workers detected carboxypeptidase and Pr1A protease produced by M. anisopliae and induced by the Rhipicephalus microplus cuticle. ...
During its history, humankind has been affected by three factors: food deficiency, health problems, and environmental issues. With world’s population increasing at a high rate, our requirement for food is increasing. Consequently, agricultural practices that maximize crop productivity are necessary. These include the development of new agronomic technologies and new plant varieties, the use of fertilizers, pesticides and herbicides, in order to minimize losses due to plant predators and weeds, respectively. Thus, a continued need for pest management in agriculture became evident, with pressure to efficiently produce more food using less land. To solve this issue, conventional chemical pesticides have been widely used in agriculture despite presenting risks to human health, hazards to the environment as well as affecting non-target species. Therefore, the use of biopesticides is desired due to their target specificity and low environmental damage. They encompass different types of molecules, usually produced by microbial biosynthesis, and are widely used for pest control. Biocontrol, which depends on microorganisms or their products such as hydrolytic enzymes, became a promising alternative to conventional pest control. Microbial hydrolytic enzymes such as proteases, chitinases, lipases, and glucanases are attractive for this purpose, since they present toxic properties, acting synergistically to control pest attacks. Proteases act on the insect cuticles, since proteins constitute the majority of this structure. These enzymes also can act in the insect midgut and hemocoel. Proteases can also be used in the biological control of other noxious agents, such as bacteria, fungi, and nematodes. Chitinases can degrade the peritrophic matrix and cuticle of insects, as well as the fungal cell wall. Lipases hydrolyze lipoproteins, waxes and fats present in the insect integument, causing its disruption. Glucanases affect fungal cell wall development, differentiation, and mycoparasitism, because glucan is a major cell wall component. In this chapter we cover details about enzymes structure, biochemistry, mechanisms of action, applications, and perspectives in this field.
Phakopsora pachyrhizi causes Asian soybean rust, resulting in severe losses of soybean yield. This study assessed the biocontrol efficacy of cell-free culture filtrates and conidial suspensions of Metarhizium anisopliae (MABR-01) and M. humberi (MHBR-03) against P. pachyrhizi in vitro and in planta. A 50% concentration of culture filtrates of M. anisopliae and M. humberi inhibited the germination of P. pachyrhizi urediniospores by 85 and 96%, respectively, compared to the 50% potato-dextrose broth (PDB) control. The conidial suspensions caused no inhibition of P. pachyrhizi in vitro, but the conidial suspension of M. anisopliae controlled the rust disease in plants with a 51% efficiency. Soybean plants colonised by M. anisopliae MABR-01 and exposed to P. pachyrhizi showed less severe disease in the shoots compared to plants not colonised by M. anisopliae; the disease was reduced by 40%. Spraying plants with 50 and 75% culture filtrates of M. humberi and a 75% concentration of M. anisopliae significantly reduced rust disease, by an average of 86%. Plants pretreated with Metarhizium culture filtrates showed a 30% reduction in P. pachyrhizi colonisation based on qPCR quantification of the pathogen genomic DNA. These results indicate a high biotechnological and sustainable potential of these two Metarhizium species for control of Asian soybean rust.
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Metarhizium anisopliae (Hypocreales: Clavicipitaceae, M. anisopliae), as an invertebrate fungal pathogen, has played a significant role in the control of many agricultural pests and human disease vectors. M. anisopliae is typically used as a chemical in dry or liquid formulations of large numbers of aerial conidia. The conidia can directly infect arthropod pests by penetrating their cuticular layer. The availability of the complete genome of M. anisopliae and capable techniques for its transformation have brought several developments in its use as a pest controller. For prospects, more in-depth research to improve fermentation and formulation technologies is needed, while multistress-tolerant and engineering entomopathogenic strains are selected to promote the widespread acceptance and usefulness of M. anisopliae as a cost-effective fungal biopesticide. Here, we provide an overview of the pathogenesis and formulation strategies, as well as the host ranges and molecular approaches of increasing virulence and efficacy. Several studies of this fungus pathogen demonstrated that M. anisopliae can be an efficient biocontrol agent and has great potential for further research and development.
Cordyceps militaris, an entomopathogenic Cordyceps mushroom, is a crucial ethnopharmacological agricultural product with applications in traditional oriental remedies in East Asia. Since lipases are reported to serve as key enzymatic equipment for entomopathogenic fungi during the host infection, the presence of various lipases with different biochemical features in C. militaris was elucidated. Three lipases from C. militaris (CML) of 60–70 kDa were isolated according to protein hydrophobicity; isoform relationships were identified by peptide mapping using liquid chromatography–electrospray ionization–tandem mass spectrometry. The CML isoforms exhibited distinct substrate specificities, which were related to the hydrophobicity of each isoform. Furthermore, the integral stereoselectivity of each lipase towards trioleoylglycerol diverged into two classes (sn-1,3 and sn-2 regioselectivity) that are rare in canonical fungal lipases. Overall, our results demonstrate that C. militaris secretes lipase isoforms with cocktail-like enzyme functions that may contribute to the entomopathogenic life cycle of C. militaris. Each CML isoform has distinct advantages for biocatalyst applications in the food and oleochemical industries.
Metarhizium anisopliae is an entomopathogenic fungus used as a biological control agent due to its capacity to infect more than three hundred species of arthropods. It is broadly used as a model to the development of host-pathogen interaction studies, including the study of the description of the involved genes in the infection process by the construction of functional mutants. This process may be facilitated by the use of the CRISPR/Cas9 methodology to genomic manipulation, which was derived from the immune adaptive system in prokaryotes and has demonstrated technological potential in genetic of eukaryotes edition through the activity of incorporation of protospacers (sequences arising from bacteriophages or plasmids invaders) in their loci. CRISPR and CRISPR associated protein (Cas) code a guided nuclease (Cas9) that targets a specific site of the invading DNA leading to breakage of double-stranded target DNA in a specific sequence. Multiple gene editing studies have been developed in different organisms including its adaptation to eukaryotes. In order to improve the efficiency of the generation of mutants in M. anisopliae, CRISPR/Cas9 system and a RNAi system were used in order to develop new tools. Both CRISPR/Cas9 and RNAi methodologies were developed having as a target the gfp gene from M. anisopliae E6 GFP+ strain. To perform genome editing in M. anisopliae, stable transgenic fungi that express fungal codon-optimized Cas9 were generated by Agrobacterium tumefaciens-mediated transformation. Oligonucleotides were designed to target four different regions along the gfp gene by using the CRISPR RGEN tool in order to avoid off-targets. The binary vector was the pPZP plasmid with the U6-1 to RNAi expression. For RNAi analysis, gene knockdown were developed by agrotransformation with a suitable plasmid (pPZP::SUR::DP) containing dual promoters with opposite directions (Pdgp and Ptrpc) and a cassette for the expression of the selective marker (gene sur, conferring sulfonylurea resistance) for the selection of transformants. The 420 bp gfp sequence was cloned between promoters, obtaining thus the plasmid pPZP::SUR::DP::GFP. The development of these two techniques proves itself viable for functional analysis of M. anisopliae genes.
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The insect pathogenic fungus Metarhizium anisopliae produces several extracellular cuticle-degrading proteases and evidence is consistent with one of these, PR1, which is a chymoelastase, being a determinant of pathogenicity. We have shown previously that PR1 production is regulated by both carbon catabolite and nitrogen metabolite repression and also by specific induction under derepressed conditions by insect cuticle. In the present work we have established that an enzymically released proteinaceous component(s) of insect cuticle is capable of inducing PR1 (based on appearance of extracellular activity). Cuticle of the desert locust Schistocerca gregaria treated with KOH to remove protein failed to induce PR1 production, whereas cuticle treated with either chloroform or ether to remove lipids still induced PR1. Cuticle digested with either PR1 or the trypsin-like PR2 of M. anisopliae released peptides mainly in the range 150-2000 Da; addition of these peptides generated by PR1 or PR2 at 3 micrograms alanine equivalents ml-1 induced PR1 production to a level similar (75%) to that obtained with untreated insect cuticle. Several amino acids and peptides which are abundant in insect cuticular protein (Ala, Gly, Ala-Ala, Ala-Ala-Ala, Ala-Pro and Pro-Ala) were tested at a range of concentrations and in restricted cultures for their ability to induce PR1. None induced the protease to the levels seen with cuticle or peptides enzymically released from cuticle, although some dimers and notably the monomers Ala and Gly gave 2-2.7-fold enhanced PR1 activity above depressed basal levels (up to 48-57% of that achieved with induced synthesis on cuticle).(ABSTRACT TRUNCATED AT 250 WORDS)
DNA microarrays containing virtually every gene of Saccharomyces cerevisiae were used to carry out a comprehensive investigation of the temporal program of gene expression accompanying the metabolic shift from fermentation to respiration. The expression profiles observed for genes with known metabolic functions pointed to features of the metabolic reprogramming that occur during the diauxic shift, and the expression patterns of many previously uncharacterized genes provided clues to their possible functions. The same DNA microarrays were also used to identify genes whose expression was affected by deletion of the transcriptional co-repressor TUP1 or overexpression of the transcriptional activator YAP1. These results demonstrate the feasibility and utility of this approach to genomewide exploration of gene expression patterns.
Abplp is a yeast cortical actin-binding protein that contains an SH3 domain similar to those found in signal transduction proteins that function at the membrane/cytoskeleton interface. Although no detectable phenotypes are associated with a disruption allele of ABP1, mutations that create a requirement for this protein have now been isolated in the previously identified gene SAC6 and in two new genes, SLA1 and SLA2. The SAC6 gene encodes yeast fimbrin, an actin filament-bundling protein. Null mutations in SLA1 and SLA2 cause temperature-sensitive growth defects. Sla1p contains three SH3 domains and is essential for the proper formation of the cortical actin cytoskeleton. The COOH terminus of Sla2p contains a 200 amino acid region with homology to the COOH terminus of talin, a membrane cytoskeletal protein which is a component of fibroblast focal adhesions. Sla2p is required for cellular morphogenesis and polarization of the cortical cytoskeleton. In addition, synthetic-lethal interactions were observed for double-mutants containing null alleles of SLA2 and SAC6. In total, the mutant phenotypes, sequences, and genetic interactions indicate that we have identified novel proteins that cooperate to control the dynamic cytoskeletal rearrangements that are required for the development of cell polarity in budding yeast.
Neozygites parvispora was isolated from Tbrips tabaci infesting leek plants. In vitro cultivation of two isolates was achieved using supplemented Grace's insect tissue culture medium complemented with fetal bovine serum (FBS) and pre-treated lepidopteran hemolymph. This medium composition enabled exponential cell growth as rod-shaped hyphal bodies with a length over diameter ratio of 4.5-5.0. Both FBS and hemolymph were mandatory for sustained growth. The growth requirements indicate the presence of one or several growth factors in both the hemolymph and FBS. Absence of FBS resulted in growth arrest with subsequent cell lysis. Omission of hemolymph led to rounding and size decrease of the hyphal bodies, some of which formed septated germ tubes in a later stage. Eventually, cell death occurred under such conditions. Hyphal bodies from in vitro culture were capable of capilliconidia formation after entrapment in alginate pellets. This suggests the requirement of a liquid/air interface, which triggers primary sporulation and subsequent capilliconidium formation.
In this study, we examined variation among isolates of the entomopathogenic fungus Metarhizium anisopliae in their ability to germinate and produce infection structures (appressoria) in different nutrient media. We also examined allozyme variation within and among 120 isolates of Metarhizium spp. Electromorph differences were noted and genotypic designations were assigned for eight loci encoding the following enzymes: glucose phosphate isomerase, glutamate dehydrogenase, glutamic pyruvic transaminase, glutathione reductase, malate dehydrogenase, phosphogluconate dehydrogenase, peptidase with glycyl leucine, and triosephosphate isomerase. Forty-eight distinct genotypic classes were found. Only the thirteen M. anisopliae var. majus isolates and an isolate of Metarhizium flavoviride possessed multibanded phenotypes characteristic of heterozygous genotypes. Three isolates were heterozygous at one locus, seven isolates at two loci, and four isolates at four loci. Based on the level of genetic similarities between the three recognized species (M. anisopliae, Metarhizium album, and M. flavoviriae) we report that isolates currently assigned as M. anisopliae are a strongly heterogenous array of multilocus genotypes that includes five varieties and two undescribed cryptic species. Genetic similarities provided evidence for geographical clustering. For example, Brazil and Colombia contain a common, widely dispersed genotypic class; eight additional classes are more locally distributed. Nutrient requirements for germination and appessorium formation were frequently host related, irrespective of genetic and geographical distance. Most of those lines of M. anisopliae var. anisopliae and var. majus isolated from scarabaeids germinated readily only in the presence of a crude protein/chitin product. Lines isolated from other coleopterans were much more adaptable and produced appressoria in yeast extract media. Unlike the majority of isolates from other hosts, lines isolated from Hemiptera usually germinated well in media containing glucose as the sole carbon source, and glucose allowed formation of appressoria. The role of selectable strain variations in the development of pathogen strategies is discussed.
Extracellular enzymes of Metarhizium anisopliae had considerable affinity for insect cuticle. Binding of proteases immobilized over 70% of soluble enzyme activity, which in vivo could have a significant influence on the extent and nature of cuticle degradation. Adsorbed protease, carboxypeptidase, and N-acetylglucosaminidase activities were recoverable with 0.2 m buffer suggesting nonspecific ionic binding. Chitinase bound irreversibly as a specific enzyme-substrate complex. Cuticle degradation by an alkaline (optimum pH 9) basic (pI 9.5) protease was inhibited by increasing salt concentrations while anilide hydrolysis was unaffected. Inhibition arose from interference with essential electrostatic adsorption of the enzyme on to the cuticle. An anionic detergent enhanced enzymic solubilization of cuticle proteins (probably due to increased electronegativity of cuticle) at the expense of continued proteolysis of released peptides, clearly distinguishing between the two processes. A cationic detergent inhibited cuticle degradation, indicating that salt labile bonds form between the negative (probably carboxyl) groups of cuticle and the positively charged groups of the protease. The significance of these results in understanding the mechanism of cuticle degradation are discussed.
Reverse transcription differential display PCR (RT-DD-PCR) was used to identify genes that are specifically expressed by Metarhizium anisopliae when it contacts the host insect cuticle. Using a homology-based subtilisin-like protease primer we identified a hitherto unsuspected differentially expressed subtilisin-like protease (Pr1B) encoding gene. The deduced amino acid sequence shows 54% similarity to the well characterized Pr1A subtilisin of M. anisopliae and karyotype analysis revealed that Pr1A and Pr1B are located on separate chromosomes. Like Pr1A, Pr1B is synthesized as a large precursor (1158 nucleotides; deduced molecular mass=40 031 Da) containing a signal peptide, a propeptide and the mature protease (283 aa; deduced molecular mass=28 714 Da). However, Pr1B possesses several substitutions in the highly conserved sequences comprising the active sites of subtilisins. In particular, the substitution of Thr220 by serine is unique to Pr1B. Substitution of Asn155 by glycine is also very unusual, and we discuss the likely effects these changes will have on the catalytic efficiency of Pr1B.
Several pathogenic isolates of Metarhizium anisopliae, Beauveria bassiana, and Verticillium lecanii when grown in buffered liquid cultures containing comminuted locust cuticle as composite carbon source (good growth occurred on most monomeric and polymeric cuticular constituents), produced a variety of extracellular enzymes corresponding to the major components of insect cuticle, e.g., endoproteases, aminopeptidase, carboxypeptidase A, lipase, esterase, chitinase, and N-acetylglucosaminidase. Considerable variations occurred in levels of production between species and even within a species, but endoproteases were exceptional as production of them was high with all the isolates. Cuticle-degrading enzymes were produced rapidly and sequentially in culture. The first activities to appear (<24 hr) were those of the proteolytic complex; chitinases were always produced substantially later. Chitin was probably masked by protein as a fluorescent chitin-specific lectin and calcofluor (binds to β-glucans) stained mainly cuticles from which protein had been removed (by protease or KOH).
Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.
The entomopathogenic fungus, Metarhizium anisopliae, produces three distinct types of proteinases during growth on cockroach cuticle. These were separated by analytical isoelectric focusing and characterized according to their substrate specificity and inhibition patterns as Pr1 subtilisin-like proteinases (four isoforms pI range approximately 9.3-10.2), a thermolysin-like metalloproteinase (pI approximately 7.3), and trypsin-like serine Pr2 proteinases (two major isoforms, pI approximately 4.4 and 4.9 and two minor isoforms, pI approximately 5.2). Preparative isoelectric focusing was used to separate the four Pr1(2) components produced during growth on cockroach cuticle with isoelectric points of 10.2 (m = 30.2 kDa), 9.8 (m = 28.5 kDa), 9.3 (m = 29.5 kDa), and 9.0 (m = 31.5 kDa). Two of the isoforms were also produced, at diminished levels, during growth on elastin or cellulose presumably as a result of carbon and nitrogen derepression. The pI 10.2 Pr1 differed from the other isoforms in preferring alanine over bulky hydrophobic groups at P2 and P3, in discriminating against proline at P2 and in its lack of sensitivity to tetra-butyl-oxycarbonyl-Gly-Leu-Phe-chloromethyl ketone. Differences in the N-terminal amino acid sequences confirmed that the four isoforms are related products of at least two distinct genes. The isoforms showed similar primary specificities, with the aromatic P1 phenylalanine being 10- to 16-fold more reactive than a P1 leucine residue reflected principally in Kcat. However, methionine (containing a long unsubstituted side chain) was also a good substrate for each isoform confirming the low selectivity of their S1 subsites. The isoforms all degraded a variety of solubilized cuticle proteins, with high-molecular-weight acidic proteins being preferentially hydrolyzed. The metalloproteinase is active against the Pr1 substrate succinyl-(Ala)2-Pro-Phe-7-amino-4-coumarin trifluoromethyl, but differs from the Pr1 isoforms in being inhibited by 1,10-phenanthroline and phosphoramidon. The potential role of the metalloproteinase in pathogenicity is discussed.