dermatophytes are the most common organisms infecting keratinized structures such as skin , hair , and nails , and their ability to degrade keratin is believed to be a major virulence factor . a correlation between keratinolytic activity and pathogenesis has been proposed because dermatophytes secrete a battery of endo- and exo - proteases during infection that degrade keratinized structures into oligopeptides and free amino acids for use as nutrients .
it is likely that proteases with optimal activity at both acidic and alkaline phs are important virulence factors in dermatophytes , and that their regulation during infection is crucial . in the early stages , and in response to the acidic ph of human skin , the pathogen de - represses the synthesis of non - specific keratinases and proteases that have optimal activity at acidic ph .
they act on skin proteins , producing peptides that are hydrolyzed into amino acids , which are then used by the fungus as sources of carbon , nitrogen , and sulfur .
the metabolism of some amino acids promotes the alkalinization of the host microenvironment , making it suitable for the action of keratinases with optimal activity at alkaline ph .
the dermatophyte trichophyton rubrum rapidly responds to ph changes by modulating the expression of genes , allowing the use of skin proteins over a wide ph range , thereby enabling the development of infection and persistence of the dermatophyte in host tissues   moreover , inactivation of the pacc gene , a component of the ph signalling pathway in t. rubrum , reduces the activity of secreted keratinases , indicating that the pacc gene is somehow involved in the regulation of keratinolytic activity , and consequently in the virulence and pathogenicity of this organism .
protein secretion from a eukaryotic cell requires movement through the endoplasmic reticulum ( er ) and the golgi apparatus .
in the course of trafficking , the secreted proteins undergo glycosylation , which is the major post - translational molecular event [ 814 ] . in secreted proteins ,
the glycosyl groups are usually attached to either an amide group ( n - glycosylation ) or a hydroxyl residue ( o - glycosylation ) , which are mainly found on serine and threonine residues . during glycosylation
, the oligosaccharide glcnac2man9glc3 is transferred to an asn residue within the sequence asn - xser / thr by an oligosaccharyltransferase ) , where x represents any amino acid except proline .
the main pathway utilizes sugar nucleotides and is located in the golgi apparatus . in yeasts ,
o - mannosylation begins in the er lumen and , like n - glycosylation , it requires dolichol phosphate - activated sugar residues .
the initial reaction is catalyzed by proteins from the evolutionarily conserved mannosyltransferase ( pmt ) family .
proteins secreted from yeast cells are usually heavily n- and/or o - glycosylated . in proteins that are glycosylated at both sites , it is not known whether n - glycosylation precedes o - mannosylation , or vice versa .
it is also unknown whether the o - mannosylation that takes place in the er prevents n - glycosylation ; however , there is some evidence for the opposite situation .
altered glycosylation may affect the stability and half - life of proteins , thus changing their activities or affinities towards substrates . delineating the mechanisms underlying fungal adaptability to ambient variation
is fundamental to an understanding of the mechanisms of pathogenicity and resistance to inhibitors in pathogenic organisms .
this work was aimed at investigating the expression of genes encoding dolichyl - p - man : man(5)glcnac(2)-pp - dolichyl mannosyltransferase and an o - mannosyltransferase ( referred to as the n - man and o - man genes , respectively ) in the dermatophyte t. rubrum in response to nutrients , ambient ph , and disruption of the pacc gene .
our findings revealed a relationship between the expression of these two mannosyltransferase genes and the pacc gene in response to ambient ph and carbon source .
t. rubrum clinical isolate h6 ( atcc mya-3108 ) and a pacc-1 mutant that carries a disrupted pacc gene , which were used throughout this study , were selected as previously described .
the h6 and pacc-1 strains were cultivated on sabouraud glucose agar for 15 days at 28 c , and pacc-1 cultures were supplemented with 450 g / ml hygromycin .
mycelia were collected with a sterile spatula , vortexed in saline solution [ 0.9% ( w / v ) nacl ] with 0.01% ( v / v ) tween , filtered through fiberglass to remove mycelial debris , and then centrifuged to recover the conidia .
then , 10 conidia were transferred to 50 ml of sabouraud broth , and germination was carried out at 28 c for 72 h on an orbital shaker at 180 rpm ( control ) .
after incubation , the final ph of the culture medium was measured with a ph meter , and the resulting mycelia were harvested by filtration through sterilized whatman paper ( whatman international , maidstone , uk ) , washed with sterilized water , and transferred to minimal medium ( mm )   at ph 5.0 or ph 8.0 , which was , in some cases ( as indicated ) , buffered with 50 mm sodium citrate or 50 mm tris  hcl , respectively .
mm was supplemented with glucose ( 50 mm ) or glucose plus glycine ( 50 mm each ) and sodium nitrate ( 70 mm ) .
all cultures were incubated at 28 c for 3 , 6 , and 24 h with agitation .
mycelia obtained from each culture were harvested by filtration , and total rna was extracted from approximately 100 mg of frozen mycelium using trizol reagent , and treated with rnase - free dnase i ( invitrogen , carlsbad , ca ) , following the manufacturer 's instructions .
one microgram of dnase - treated rna was reverse transcribed into cdna using the high capacity cdna reverse transcription kit ( applied biosystems , foster city , ca ) according to the manufacturer 's instructions .
the cdna from 3 independent biological experiments was quantified spectrophotometrically , and stored at 80 c until pcr amplification . for quantitative real - time pcr analyses ,
the genes encoding dolichyl - p - man : man(5)glcnac(2)-pp - dolichyl mannosyltransferase ( n - man , terg_06338 ; genbank : xm_003233297 ) and o - mannosyltransferase ( o - man , terg_06465 ; genbank : xm_003233430 ) were amplified from cdna using the following primers ( 53 ) : taaacgacagtggtatgccg ( n - manfwd ) and tgtagcctgttgggttctct ( n - manrev ) ; ccatgggacgtgtatactc ( o - manfwd ) and cgtcatcatagcaacattcag ( o - manrev ) .
reactions were performed in three independent experiments using sybr green pcr master mix ( applied biosystems , foster city , ca ) , in the stepone plus real - time pcr system .
a 12.5-l reaction was set up using 50 ng of cdna and 300 nm of each primer , and the pcr cycle was as follows : 95 c for 10 min , followed by 40 cycles of 95 c for 15 s and 60 c for 1 min .
melting curve analyses were performed after each cycling to exclude primer dimers and nonspecific pcr products .
relative transcript quantities were calculated using the ct method using the experimental condition that yielded the lowest ct value ( n - man , non - buffered h6 cultures in keratin , 6 h incubation ) .
the t. rubrum -actin ( terg_06637 ) and -tubulin ( terg_07904 ) genes , which were used as endogenous reference genes , were amplified using the following oligonucleotides ( sequence 53 ) : aacgccatcatgaagtgt ( actinfwd ) and tccttctgcatacggtcaga ( actinrev ) ; ccgtatgatggccacttt ( tubulinfwd ) and ctgacctgggaaacgaagac ( tubulinrev ) .
data normalization and analyses were performed using the genex 5 multid analyses ab ( www.multid.se ) . to confirm the identity of the genes analyzed in this work , as well as the endogenous reference genes , the pcr products were sequenced and analyzed by alignment with sequences retrieved from the broad institute dermatophyte comparative database ( http://www.broadinstitute.org/annotation/genome/dermatophyte_comparative/multihome.html ) .
t. rubrum clinical isolate h6 ( atcc mya-3108 ) and a pacc-1 mutant that carries a disrupted pacc gene , which were used throughout this study , were selected as previously described .
the h6 and pacc-1 strains were cultivated on sabouraud glucose agar for 15 days at 28 c , and pacc-1 cultures were supplemented with 450 g / ml hygromycin .
mycelia were collected with a sterile spatula , vortexed in saline solution [ 0.9% ( w / v ) nacl ] with 0.01% ( v / v ) tween , filtered through fiberglass to remove mycelial debris , and then centrifuged to recover the conidia .
then , 10 conidia were transferred to 50 ml of sabouraud broth , and germination was carried out at 28 c for 72 h on an orbital shaker at 180 rpm ( control ) .
after incubation , the final ph of the culture medium was measured with a ph meter , and the resulting mycelia were harvested by filtration through sterilized whatman paper ( whatman international , maidstone , uk ) , washed with sterilized water , and transferred to minimal medium ( mm )   at ph 5.0 or ph 8.0 , which was , in some cases ( as indicated ) , buffered with 50 mm sodium citrate or 50 mm tris  hcl , respectively .
mm was supplemented with glucose ( 50 mm ) or glucose plus glycine ( 50 mm each ) and sodium nitrate ( 70 mm ) .
all cultures were incubated at 28 c for 3 , 6 , and 24 h with agitation .
mycelia obtained from each culture were harvested by filtration , and total rna was extracted from approximately 100 mg of frozen mycelium using trizol reagent , and treated with rnase - free dnase i ( invitrogen , carlsbad , ca ) , following the manufacturer 's instructions .
one microgram of dnase - treated rna was reverse transcribed into cdna using the high capacity cdna reverse transcription kit ( applied biosystems , foster city , ca ) according to the manufacturer 's instructions .
the cdna from 3 independent biological experiments was quantified spectrophotometrically , and stored at 80 c until pcr amplification .
for quantitative real - time pcr analyses , the genes encoding dolichyl - p - man : man(5)glcnac(2)-pp - dolichyl mannosyltransferase ( n - man , terg_06338 ; genbank : xm_003233297 ) and o - mannosyltransferase ( o - man , terg_06465 ; genbank : xm_003233430 ) were amplified from cdna using the following primers ( 53 ) : taaacgacagtggtatgccg ( n - manfwd ) and tgtagcctgttgggttctct ( n - manrev ) ; ccatgggacgtgtatactc ( o - manfwd ) and cgtcatcatagcaacattcag ( o - manrev ) .
reactions were performed in three independent experiments using sybr green pcr master mix ( applied biosystems , foster city , ca ) , in the stepone plus real - time pcr system .
a 12.5-l reaction was set up using 50 ng of cdna and 300 nm of each primer , and the pcr cycle was as follows : 95 c for 10 min , followed by 40 cycles of 95 c for 15 s and 60 c for 1 min . melting curve analyses were performed after each cycling to exclude primer dimers and nonspecific pcr products .
relative transcript quantities were calculated using the ct method using the experimental condition that yielded the lowest ct value ( n - man , non - buffered h6 cultures in keratin , 6 h incubation ) .
the t. rubrum -actin ( terg_06637 ) and -tubulin ( terg_07904 ) genes , which were used as endogenous reference genes , were amplified using the following oligonucleotides ( sequence 53 ) : aacgccatcatgaagtgt ( actinfwd ) and tccttctgcatacggtcaga ( actinrev ) ; ccgtatgatggccacttt ( tubulinfwd ) and ctgacctgggaaacgaagac ( tubulinrev ) .
data normalization and analyses were performed using the genex 5 multid analyses ab ( www.multid.se ) . to confirm the identity of the genes analyzed in this work , as well as the endogenous reference genes , the pcr products were sequenced and analyzed by alignment with sequences retrieved from the broad institute dermatophyte comparative database ( http://www.broadinstitute.org/annotation/genome/dermatophyte_comparative/multihome.html ) .
in vitro growth of the dermatophyte t. rubrum is dependent on the initial culture ph , with apparent optimal growth at ph 4.05.0 , irrespective of carbon source ( glucose , glycine , or protein ) .
the initial ph of the t. rubrum cultures increased from 5.0 to a ph that ranged from 8.3 to 8.9 after 7296 h of incubation in glycine or keratin .
this effect was not observed when the fungus was cultivated with glucose as the carbon source , and the ph was maintained at approximately 5.0 .
hydrolysis of keratin and other proteins releases amino acids , such as glycine , whose metabolism leads to the secretion of ammonia , thereby shifting the ph of the culture from acidic to alkaline .
therefore , the metabolism of both glycine and keratin at ph 5.0 were alkalinizing events , even though the culture ph was still acidic after 24 h of incubation in glycine or keratin ( fig .
disruption of the pacc gene in t. rubrum neither affects this ph shift ( fig .
however , a marked decrease in the conidiation on sabouraud solid medium and in the secretion of keratinolytic activity by the pacc-1 mutant , which correlated with its reduced capacity to infect human nails in vitro , were observed as compared to the control strain .
no ambient ph changes were observed when the fungus was cultured in buffered media ( data not shown ) .
t. rubrum grows poorly in culture medium with a starting ph of 8.0 , even though the culture becomes acidified after incubation with glucose as a carbon source ( fig .
1 ) . growth in glycine or keratin dropped the ph of the culture to approximately 7 after the first 36 h of incubation , which increased to approximately ph 7.5 after 24 h of incubation ( fig .
an attractive hypothesis is that t. rubrum senses the alkaline environment and then acidifies the culture medium to an ambient ph at which its growth is stimulated .
metabolism of glycine or keratin leads to the secretion of ammonia , which shifts the culture ph to alkaline ph values .
therefore , to better understand the transcription of both the o - man and n - man genes during the first 24 h of incubation , when the ph of the medium was still acidic in non - buffered cultures ( fig .
1 ) , we estimated the expression of both genes by qrt - pcr in different culture conditions .
transcription of the o - man and n - man genes in both the h6 and pacc-1 mutant strains was affected differently by the carbon source , the culture ph , and the time of incubation ( fig .
o - man was preferentially transcribed in both buffered and non - buffered keratin cultures , whereas n - man was apparently preferentially transcribed in non - buffered glucose cultures .
the expression of n - man decreased as incubation time increased at ph 5.0 , but the expression decreased more consistently in buffered glucose cultures ( fig .
disruption of the pacc gene decreased transcription of o - man in both glucose and keratin at ph 5.0 ; however , o - man expression was enhanced in glucose plus glycine .
interestingly , the transcription of n - man in keratin cultures was enhanced in the pacc-1 mutant at ph 5.0 .
therefore , at acidic ph , transcription of the o - man and n - man genes was positively and negatively affected , respectively , by the pacc gene in keratine cultures ( fig .
2 ) . moreover , transcription of the o - man and n - man genes in buffered cultures indicated that the o - man gene was preferentially transcribed at acidic ph in the presence of keratin compared to glucose or glucose plus glycine as the carbon sources , whereas transcription of the n - man gene was almost the same in both acidic and alkaline ph in the presence of keratin , and these properties were affected by disruption of the pacc gene ( fig .
it is also worth noting that while the disruption of the pacc gene resulted in changed transcription of the o - man gene in the different conditions at ph 5.0 , it also resulted in the opposite changes for growth in glucose and glucose plus glycine at ph 8.0 .
thus , our results suggest that the product of the pacc gene negatively affect both the transcription of the n - man gene in keratin at ph 5.0 and the o - man gene at ph 8.0 in the presence of different nutrients ( fig .
2 ) i.e. , the pacc gene is functional irrespective of the culture conditions assayed .
it has been well documented that the dermatophyte t. rubrum , as well as other filamentous fungi , acidifies the culture medium and represses the secretion of proteases during growth in glucose as the sole carbon source . moreover , during growth in glycine or keratin , the culture medium is alkalinized ; however , this change is dependent on the initial ph of the culture , with an apparent optimum at ph 4.05.0 .
interestingly , glycine utilization is apparently not repressed by glucose , because alkalinization of the culture medium occurs with glucose and glycine as carbon sources .
however , it is worth noting that during the first 24 h of cultivation , the ph of the medium is still acidic ( fig .
1 ) , implying that the metabolism of glycine or keratin , an alkalinizing event , occurs during the first 24 h of cultivation , exclusively in an acidic environment . in the model fungi
neurospora crassa and aspergillus nidulans , one of the metabolic responses to the ph of the culture medium is the ph - dependent glycosylation of secreted enzymes [ 3437 ] . for example , the level of glycosylation of the pho-2 alkaline phosphatase synthesized by n. crassa at alkaline ph differs from that synthesized at acidic ph , which is approximately 13% and 21% for the pho-2 enzyme purified from mycelium grown at ph 5.4 and 7.8 , respectively .
the loss of enzymatic activity observed for the pi - repressible alkaline phosphatase secreted at acidic ph is probably because the glycosylation of this enzyme is lower than that secreted at alkaline ph .
we have also provided evidence that glycosylation of secreted enzymes , as documented for the pi - repressible phosphatases in n. crassa and a. nidulans , is pacc - dependent . in t.
rubrum , disruption of the pacc gene , as in the pacc-1 mutant strain , resulted in decreased growth on human nails and decreased secretion of keratinolytic proteases in liquid medium when supplemented with keratin , which suggests that the keratinases secreted by t. rubrum are somehow regulated by the pacc protein .
pacc might be involved in the glycosylation of these keratinases through transcriptional modulation of o- and n - linked mannosyltransferases , a hypothesis supported by the results described here .
transcriptional profiling of both the o - man and n - man genes revealed a high level of complexity , because transcription of these genes was affected by nutrients , culture ph , and the functioning of the pacc gene .
disruption of the pacc gene increased the expression of n - man at ph 5.0 in keratin cultures .
moreover , if o - mannosylation precedes n - glycosylation in t. rubrum , as demonstrated in yeast , this physiological effect is dependent on the function of the pacc gene at acidic ph . in conclusion ,
the genes encoding the o- and n - mannosyltransferases had different expression profiles , and the o - man gene was preferentially expressed at acidic ph when t. rubrum was grown on medium containing keratin .
the balance between n - man and o - man expression levels in cultures at acidic ph may be under the control of the pacc transcription factor ( in response to different carbon sources ) .
therefore , the product of the pacc gene of t. rubrum is functional at acidic ph .
moreover , transcription of the n - man and o - man genes might be required at different culture phs for the glycosylation of transported proteins , according to the stage of infection , which suggests a possible role in cell adhesion and activation of signalling pathways regulating the production of enzymes that enable nutrient uptake for fungal development and maintenance in the host [ 3941 ] .
 in fungi , ambient ph sensing involves the activation of the pal / pacc signalling pathway . in the dermatophyte trichophyton rubrum , 
 ph - dependent secretion of keratinases , which are major virulence determinants , is affected by disruption of the pacc gene . here , 
 the transcription profiling of the genes coding for n- and o - linked mannosyltransferases , enzymes involved in protein glycosylation , was evaluated in t. rubrum in response to disruption of the pacc gene and growth in keratin , glucose , and glucose plus glycine . 
 we show that transcription of these mannosyltransferase genes is affected by nutrients at acidic ph and by pacc . 
Introduction
Materials and methods
Strains and growth conditions
RNA extraction and cDNA synthesis
Real-time PCR
Results
Discussion