BACKGROUND
the secretion of active proteases is one of the most successful strategies used by microbial pathogens to colonize and infect human hosts  <cit> .
by hydrolyzing proteinaceous targets in the host organism, including the proteins in cell membranes and extracellular matrix, these enzymes allow the pathogen to penetrate tissues and acquire nutrients.
these enzymes also play important roles in evading the immune system by cleaving immune regulatory proteins  <cit> , or deregulating the major homeostatic systems of the host that require cascade-activated proteolysis such as the blood coagulation  <cit> , fibrinolysis  <cit> , complement  <cit> , and kallikrein-kinin system  <cit> .
candida albicans is one of the most common fungal pathogens in humans  <cit> .
this yeast-like fungus can release as many as  <dig> aspartic class proteases into the extracellular space  <cit> .
c. albicans is part of the physiologic human microbiota, but, under some circumstances that are generally related to immune system weaknesses in the host, it can convert to a dangerous pathogen that causes diseases of variable severity.
these candidiases can range from relatively mild and easily curable superficial infections of the skin and mucous membranes to life-threatening deep-seated invasions in the inner organs, fungemia, and systemic diseases with high mortality rates  <cit> .
it is assumed that the number and diversity of secreted aspartic proteases  from c. albicans are needed to successfully colonize the variety of niches present in humans  <cit> , but this hypothesis is still unsatisfactorily evidenced by experimental data.
in particular, relatively few studies are devoted to systematically comparing the actions of all  <dig> saps on a single proteinaceous substrate.
bradykinin-related peptides, which are collectively called kinins, are proteolytically released from the serum proteins, kininogens  <cit> .
serine proteases, called kallikreins are primarily devoted to this task  <cit> , but during pathological states, including those associated with microbial infections, other proteases, either from the host or the pathogen, can supplement the actions of the kallikreins  <cit> .
due to the multiple functions of kinins required to regulate various physiological processes, as well as their participation in almost every inflammatory state  <cit> , the kallikrein-kinin system is considered a major system required for biochemical homeostasis in humans.
overactivated kinin production reportedly occurs in infections caused by numerous bacterial species  <cit> .
regarding candidal infections, kaminishi et al.
<cit>  was the first to report that a purified major extracellular protease of c. albicans possesses kinin-releasing potential, albeit indirectly, based on the activation of an upstream-acting zymogen in the kinin-generating cascade .
the direct release of kinins from kininogens was later confirmed for mixtures of proteases that were released into the culture medium by several candida species  <cit>  and the purified c. albicans protease, which was unequivocally identified as sap <dig>  <cit> .
sap <dig> is believed to be the predominant protease secreted by c. albicans when cultured in protein-rich media, but the sap gene expression profile depends on the fungal morphology, and the high expression of different sap genes has been reported in various infection models  <cit> .
this study compares the characteristics of kininogen cleavage and concomitant kinin release due to all  <dig> individual saps, used in recombinant purified forms.
here, we report the exceptionally high kinin-forming activity of sap <dig>  moreover, the active kinins are produced with a high yield by mixtures of sap <dig> and several other saps.
RESULTS
the mechanism responsible for the sap-catalyzed cleavage of domain  <dig> of human kininogen was studied using a synthetic peptide—denoted as hk-d4—because its amino acid sequence  can be obtained from the kinin-containing region of the kininogen molecule.
the cleaved products were separated using high performance liquid chromatography , and the major peaks were collected and analyzed to determine their sequences using tandem mass spectrometry .
comparative chromatograms of the samples, obtained after incubating hk-d <dig> with all individual recombinant saps for long periods of time  at the optimal ph for general proteolytic activity   <cit>  are shown in figure  <dig>  hk-d <dig> was effectively cleaved by all saps, except sap <dig> and sap <dig>  the qualitative distributions of the major formed products are similar for sap1– <dig> and 8; however, sap <dig> demonstrated the ability to release a kinin-like peptide—met-lys-bradykinin —at the highest yield .
moderate-to-small amounts of the same peptide were also detected among the minor products of hk-d <dig> that were cleaved by sap <dig>  which agrees with our recent characterization of the kininogenase activity of the natural, purified sap <dig>  <cit>  and sap <dig>  sap <dig>  and sap <dig>  in contrast, sap <dig> showed a higher preference to hydrolyze the peptide bonds at the carboxyl side of the arginine residues than sap <dig>  and thus could easily cleave the bond between the arginine and proline located at the n-terminal side of the internal bradykinin sequence.
due to this exceptional specificity, the hk-d <dig> cleavage pattern caused by sap <dig> was unique, and a different, kinin-related peptide—des-arg1-bradykinin —was formed at a high yield .figure  <dig>
hplc/ms characteristics of the sap-catalyzed cleavage of the hk-d <dig> peptide.
ten μm of the synthetic peptide hk-d <dig>  were treated with recombinant sap1– <dig> in citrate  or phosphate buffers  at the optimal ph for the general proteolytic activity  <cit>  of each individual sap  at an enzyme:substrate molar ratio of 1: <dig> for 24 hours at 37°c.
the reaction was stopped using hcl, and the samples were analyzed using reversed-phase hplc on an eurosil bioselect 300– <dig> c- <dig> column  in a tfa-water-acn binary gradient system.
the fractions, which were collected at the major absorbance peaks , were evaporated and analyzed using esi-ms/ms in order to determine their amino acid sequence.
the dependence of the kinin yield on the ph for all kinin-forming saps  is illustrated in figure  <dig>  only sap <dig> presented the optimal kinin-forming activity at ph markedly shifted from the optimum with the general protease substrates  towards a more neutral ph.
thus, > 70% of hk-d <dig> was cleaved by sap <dig> towards met-lys-bradykinin production at ph  <dig>  importantly, kinin release was still detectable at ≥ ph  <dig> figure  <dig>
effect of ph on sap-catalyzed kinin release from the hk-d <dig> peptide.
hk-d <dig>  was cleaved using  <dig>  μm saps  in 50 mm citrate buffers  or 25 mm phosphate buffers  for 24 hours at 37°c.
the reaction was stopped using hcl , and the samples were analyzed using hplc as specified in figure  <dig>  the amounts of met-lys-bradykinin or des-arg1-bradykinin  that formed were estimated based on the peak areas and are expressed relative to the maximum possible amount .
data represent mean values from the analysis of three independent samples  ± the standard deviation.
the time course of hk-d <dig> cleavage by the saps reveals the order by which particular peptide bonds are preferably cleaved .
the cleavage preferences of sap <dig> , sap <dig>  sap <dig>  and sap <dig>  are essentially the same as that previously reported for natural, purified sap <dig>  <cit> .
the bonds after lys <dig> and leu <dig> were hydrolyzed at the highest rates.
thus, the n-terminus of the kinin to be formed  was established during the very early stages of sap-catalyzed hk-d <dig> hydrolysis  and persisted on a longer time scale.
a number of additional cleavages occurred downstream from the kinin c-terminus, which ended up with the exposition of ser <dig>  and usually ser <dig> although at a slower rate.
thus, the mkrppgfspfrss and mkrppgfspfrs peptides could be considered the major final products of hk-d <dig> cleavage following treatment with this set of saps.
hydrolyzing the bond after arg <dig>  which completes the formation of met-lys-bradykinin, occurred at very slow rates when treated with sap <dig>  − <dig>  − <dig>  and − <dig> .
due to the high preference of sap <dig> for hydrolyzing the bonds after arg <dig> and arg <dig> , met-lys-bradykinin was one of the major end products of hk-d <dig> cleavage, together with the mkrppgfspfrss and mkrppgfspfrssr peptides.figure  <dig>
time course of hk-d <dig> cleavage by sap <dig>  sap <dig>  sap <dig>  and sap <dig>  hk-d <dig>  was cleaved using  <dig>  μm sap in 50 mm citrate buffer  at 37°c for the specified time.
the reaction was stopped using hcl, and the samples were analyzed using hplc as specified in figure  <dig>  the results from representative kinetic experiments are shown.
the areas under the peaks of the early, major cleavage products, as well as the kinin-related peptides, are expressed relative to the substrate at the beginning of the reaction.
the cleavage of hk-d <dig> by sap <dig> and sap <dig>  was limited to the hydrolysis of the two bonds after lys <dig> and leu <dig>  sap <dig> quickly cleaved the bond after lys <dig> to form the islmkrppgfspfrssrigeik peptide, at the expense of which the mkrppgfspfrssrigeik peptide appeared after a longer period of time at a much lower rate.
these two cleavages alternatively occurred at high and comparable rates due to the action of sap <dig>  which lead to the fast formation of the islmkrppgfspfrssrigeik and mkrppgfspfrssrigeikeet peptides that thereafter were quickly converted to the final major product, mkrppgfspfrssrigeik.
among other very minor products that resulted from the actions of sap <dig> and sap <dig> on hk-d <dig>  no kinin-like peptides were found using the applied chromatographic method .
the unique time course of hk-d <dig> cleavage by sap <dig>  was characterized by the very fast formation of des-arg1-bradykinin, which was only preceded by quickly disappearing precursors such as the islmkrppgfspfr or ppgfspfrssrig peptides.
an analysis using sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis  showed that sap1– <dig>  − <dig>  and − <dig> cleaved native human kininogens, with a high preference for the low-molecular-mass form  in comparison with the high-molecular-mass form  .
using the optimized hplc method, the clearly distinguishable peak of met-lys-bradykinin was visible on the chromatograms obtained after lk digestion by sap1– <dig> and − <dig>  while sap <dig> resulted in the intense peak of des-arg1-bradykinin .
to identify all kinin-like peptides formed by all individual saps , the more sensitive liquid chromatography-coupled tandem mass spectrometry  method was applied.
we found that  in addition to sap1– <dig> and − <dig>  sap <dig> and sap <dig> also exhibited kinin-forming activities toward the lk substrate and  each individual sap produced multiple kinin-like peptides .
these included, in addition to the predominant met-lys-bradykinin , bradykinin itself, and  occasional traces of kinin derivatives without the c-terminal arg residues ,  leu-met-lys-bradykinin , and  versions of kinins with hydroxyproline  at the third position of the bradykinin sequence, as could have been expected from native human kininogens  <cit> , oxidized met residue , or both .figure  <dig>
hplc profiles of the fragmentation of lk by selected saps.
lk samples  were digested with  <dig>  μm of sap <dig>  − <dig>  − <dig>  − <dig>  and − <dig> in the citrate buffer  at 37°c for 24 hours.
the reaction was stopped using pepstatin a , followed by acidification with hcl .
the samples were analyzed using hplc on the luna c <dig> 5 μm  <dig>  × 250 mm column  in a tfa water-acn binary gradient system, as described in the materials and methods section.
arrows indicate the retention times of the met-lys-bradykinin  and des-arg1-bradykinin  standards.
kinin-like peptides in the sap-treated lk samples that were identified using lc-ms/ms
sap
kinin-like peptide
molecular mass
mkrppgfspfr
mkrppgfspfr
mkrppgfspfr
mkrpgfspfr
lmkrppgfspfr
mkrppgfspfr
mkrppgfspfr
ppgfspfr
pgfspfr
lk samples  were digested with  <dig>  μm sap in citrate buffer  at 37°c for 24 hours.
the reaction was stopped using pepstatin a , and the samples were acidified with hcl  and subjected to lc-ms/ms analysis using the bruker hctultra etdii it mass spectrometer that was equipped with an esi ion source and etd ii fragmentation module and was coupled to the dionex ultimate  <dig> uhplc system.
ms/ms data were analyzed using in-house mascot server , and the peptides were identified by searching the swiss-prot database.
the peptides marked in bold generated peaks with the highest intensities , which exceeded by ≥  <dig> order of magnitude in comparison with the other peptides.
the relative distributions of the main kinins were quantitatively determined in the sap-treated lk samples using the hplc analysis, collecting fractions at the retention times for bradykinin, met-lys-bradykinin, and des-arg1-bradykinin, and enzyme-linked immunosorbent assay  for kinins in these fractions .
while detectable, small amounts of bradykinin were released from lk by all studied saps, sap <dig> produced this “main” kinin at the relatively highest yield, which approached 2% of the maximum releasable kinin content in the sample.
all tested saps produced met-lys-bradykinin at the yield, which decreased in the order of sap <dig> > > sap1 ≈ sap8 > sap2 ≈ sap <dig> > > sap5 ≈ sap6 ≈ sap9; however, no sap could favorably compete with sap <dig> over the long term  in terms of the kinin release yield .
at a comparably high yield, sap <dig> could produce des-arg1-bradykinin that would barely be detectable in the lk digests obtained after long incubation with other saps.figure  <dig>
distribution of bradykinin, met-lys-bradykinin, and des-arg
1
-bradykinin in the sap-digested lk samples.
lk samples  were digested using  <dig>  μm sap in the citrate buffer  at 37°c for  <dig> hours.
after sequentially stopping the reaction using pepstatin a and hcl, the obtained peptide mixtures were separated on the luna c <dig> column in a tfa water-acn binary gradient system.
the fractions were collected at the retention times that correspond to the bradykinin, met-lys-bradykinin, and des-arg1-bradykinin standards, evaporated to dryness, and redissolved in the assay buffer of the elisa kit in order to quantitatively determine the kinin concentrations.
the corresponding fractions, obtained from the hplc separation of intact  lk served as the controls, and the kinin concentrations, determined in these fractions are subtracted from those in the sap-digested lk samples.
the corrected amount of each of the three kinins is expressed relative to the maximum possible amount of all kinins .
results, obtained from two independent experiments , with three replicate elisa measurements  for each fraction obtained during each hplc separation, are presented as the mean values ± standard deviation.
asterisks denote the statistical significance of the difference between the kinin levels in the sap-treated and undigested lk samples .
the data plotted in the three panels are for bradykinin , met-lys-bradykinin , and des-arg1-bradykinin .
the sap-treated lk samples were additionally analyzed for their interactions with hek <dig> cells, which were engineered to overexpress b2-subtype kinin receptors on the surface .
this part of our study aimed to assess putative biological activity, typical of kinins, in the peptide mixtures generated by saps from human kininogen.
using this type of ligand-receptor radioassay  <cit> , the highest amount of “bradykinin equivalents” was found in the lk digests obtained using sap <dig>  followed by those generated by sap <dig>  interpreting these results was not straightforward because attempts to calibrate the radioassay with synthetic des-arg1-bradykinin failed to detect any interactions between this peptide and the b <dig> receptors , contrary to met-lys-bradykinin which binds to receptors although at a 10-fold lower affinity than bradykinin  <cit> .
thus, the peak of b <dig> receptor-binding activity, which was found in the sap9-treated lk samples , was probably due to the bradykinin that developed in the largest amount in comparison with all other saps .
the highest peak recorded for sap <dig> was obviously due to the large amount of the true b2-receptor agonist  produced from lk by this protease at a nearly stoichiometric yield .
actually, the apparent amount of kinin in this sample was underestimated by the radioassay, which had been calibrated to the bradykinin standard.figure  <dig>
the amounts of the b <dig> receptor-interacting peptides in the sap-digested lk samples.
lk samples  were digested with  <dig>  μm sap in the citrate buffer  at 37°c for  <dig> or 24 hours, the reaction was stopped using pepstatin a , and the samples were analyzed for kinin content using a competitive radioreceptor assay that used b <dig> receptor-overexpressing hek <dig> cells.
the calibration plot for the assay was prepared using a bradykinin standard.
the results are corrected by subtracting the values, determined in the undigested lk sample.
data represent mean values from three separate radioreceptor binding analyses , with the measurements performed in triplicates within each experiment.
the error bars represent the standard deviations; asterisks denote the statistical significance of the differences between sap-treated and undigested lk samples .
the short peptide—des-arg1-bradykinin —was assumed to be biologically inactive because of its inability to interact with b <dig> receptors.
therefore, we were very interested to determine if sap <dig> can hydrolyze the peptide bond after the third arginine residue in  met-lys-bradykinin, thereby inactivating this biologically active kinin formed from the kininogens by the other saps, and  other peptides with an n-terminal met residue, such as mkrppgfspfrssrigeik, mkrppgfspfrss, mkrppgf-spfrs, and mkrppgfspfrssr—which are the fast-appearing major products of kininogen cleavage by other saps—thereby preventing the subsequent conversion to active kinin.
surprisingly, as shown in figure  <dig>  sap <dig> was essentially unable to process these substrates at the n-terminus, suggesting that this protease requires a long stretch of amino acids upstream from the n-terminal side of the internal bradykinin sequence  to effectively cleave the n-terminal bond between the arg and pro residues.
however, the c-terminal arginine residue of the kinin sequence—which is absolutely required for interactions between the free kinins and b <dig> receptors—was still quickly exposed by sap <dig>  thus, the sap-catalyzed cleavage of all substrates with n-terminal methionine converged to a single product, met-lys-bradykinin, .figure  <dig>
time course of the cleavage of the mkrppgfspfrssr peptide using sap <dig>  mkrppgfspfrssr peptide  was cleaved using  <dig>  μm sap <dig> in 50 mm citrate buffer  at 37°c for the specified time.
the reaction was stopped using hcl, and the sample was analyzed using hplc as specified in figure  <dig>  the results from a representative kinetic experiment are shown.
the areas under the peaks of separated peptides are expressed relative to the substrate at the beginning of the reaction.
the unique specificity of sap <dig>  as determined by our analysis of the synthetic peptides, allows mixtures of sap <dig> and sap <dig>  − <dig>  −4– <dig>  and − <dig> to demonstrate the strikingly enhanced formation of met-lys-bradykinin from kininogen in comparison with the individual actions of these proteases .
at the same time, the production of the biologically inactive peptide, des-arg1-bradykinin, was largely quenched.
this effect was particularly dramatic with sap <dig> and sap <dig>  which individually produced only trace amounts of met-lys-bradykinin, but in the presence of sap <dig> the kinin yield exceeded even that of the best individual kinin producer, sap <dig>  in contrast, the release of met-lys-bradykinin from sap <dig> was not enhanced in the presence of sap <dig>  apparently because both these proteases cleave the bond after the arg residue on the c-side of the internal kinin sequence at a comparably high rate.
however, the production of des-arg1-bradykinin by the sap <dig> and sap <dig> mixture was quenched by about half in comparison with the amount released from the kininogen using sap <dig> alone.figure  <dig>
comparisons of the generation of bradykinin, met-lys-bradykinin, and des-arg
1
-bradykinin in lk samples that were digested using a mixture of sap <dig> and other saps vs digestion with single saps.
lk samples  were digested with either  <dig>  μm individual saps or mixtures of  <dig>  μm sap <dig> with equimolar amounts of other saps in the citrate buffer  at 37°c for 6 hours.
after sequentially stopping the reaction with pepstatin a and hcl, the obtained peptide mixtures were separated on the luna c <dig> column in a tfa water-acn binary gradient system.
fractions were collected at the retention times that correspond to the bradykinin , met-lys-bradykinin , and des-arg1-bradykinin standards , evaporated to dryness, and redissolved in the assay buffer of the elisa kit in order to quantitatively determine the kinin concentrations.
the results are corrected by subtracting the data, obtained for the undigested lk sample as specified in figure  <dig>  the amount of each of the three kinins is expressed relative to the maximum possible amount of all kinins .
for each sap, the light gray bar represents the sum of kinin amounts in two independent samples, treated separately with this sap and sap <dig> while the dark gray bar represents the value for sample of lk that was treated with a mixture of this sap and sap <dig>  all data bars represent the mean values of six determinations  ± standard deviation.
asterisks denote the statistical significance  of the difference between the indicated kinin levels .
discussion
the success of c. albicans as a pathogen is due to its ability to inhabit various niches within the host organism, including the skin, oral and vaginal mucosa, gastrointestinal tract, and, as in systemic infections, all inner organs and the blood  <cit> .
this yeast-like fungus can adapt to different environments due to the development of an impressive array of virulence factors  <cit> , including yeast-to-hyphae transition, morphologic switches, numerous surface-exposed adhesins, and hydrolytic enzymes such as saps.
the multiplicity of the effects that determine candidal infections makes it difficult to reliably dissect the relative role of a single factor in the colonization and invasion of host tissues.
thus, eliminating a single virulence factor—e.g., gene mutations—often unsatisfactorily reduces the strain pathogenicity because of the compensating effects of other factors  <cit> .
regarding the  <dig> different saps of c. albicans, functional redundancy must also exist because numerous attempts to assign specific roles to individual saps have not led to a general consensus  <cit> .
extensive studies on the expression of individual sap genes in various in vitro and in vivo models of candidal infections report contradictory results  <cit> .
regarding the role in microbial infections, the kinin-forming system can be compared to a double-edged sword  <cit> .
primarily, the proinflammatory and vasoactive properties of kinins contribute to the refined multifactorial process that provides host defenses against pathogens.
for instance, kinins recruit defense cells such as neutrophils and monocytes to the infection foci  <cit>  and activate many cell types in order to release other, even more potent proinflammatory mediators  <cit> .
however, at least one activity of kinins—enhancing vascular permeability—is beneficial to pathogens, helping them acquire the necessary nutrients from serum and disseminate within the host organism  <cit> .
the upregulation of kinin production has been frequently reported in association with bacterial infections  <cit> .
relatively recently, the hijacking of the kinin-forming system of the host was suggested to occur as part of fungal infections such as candidiasis.
in vitro studies show that, similar to bacterial pathogens, c. albicans can mimic two mechanisms of kinin production used by the host to defeat infections, although in an uncontrolled manner.
one mechanism depends on the adsorption of hk and the other components of the contact system on the fungal cell wall  <cit> , which resembles the contact activation of kinin release on the surface of numerous host cells  <cit> .
the second mechanism involves saps that activate factor xii  <cit>  or directly release kinins from kininogens  <cit> .
in the current study, for the first time, we compared the kininogenase activities of all  <dig> saps.
eight of these, sap1– <dig>  are soluble secreted proteins, while sap <dig> and sap <dig> are bound to the cell wall via a glycosylphosphatidylinositol  anchor, although some fraction of these proteins can be shed into the extracellular space  <cit> .
we found that all soluble saps  can release kinins from human kininogens and strongly prefer lk to hk as the substrate.
in each case, the same kinin—met-lys-bradykinin—was formed almost exclusively, i.e., in excess over the other identified kinin-like peptides, including bradykinin.
despite its relatively low affinity for b2-type kinin receptors  <cit> , met-lys-bradykinin is equivalent to bradykinin and kallidin  in terms of biological activity  <cit> , most likely because it can be easily converted to the latter “main” kinins in biological fluids and tissues that are abundant with nonspecific aminopeptidases  <cit> .
the current, unequivocal identification of the chemical nature of the formed kinin peptides—together with the findings that the unfractionated peptide mixtures generated by sap1- <dig> and sap <dig> from lk interact with b <dig> receptors —provides strong support for the hypothesis that these soluble saps can release biologically active kinins at infection sites, as they are likely to encounter the appropriate amounts of kininogens to be cleaved  <cit> .
the qualitatively uniform picture observed for all individual soluble saps suggests a high degree of redundancy between these saps with respect to the postulated kinin-forming functions.
quantitatively, however, the kininogenase activity of sap <dig> so strongly exceeded those of the other saps that it should be considered the predominant player in putative sap-dependent kinin production at the sites of candidal infections.
at sufficient sap <dig> levels, sap1– <dig>  sap4– <dig>  and sap <dig> would only comprise a fungal proteolytic reserve for kininogen cleavage.
however, their significance would increase in the rather unlikely case of sap <dig> being absent at the infection site, not only due to their ability to generate a small but persistent stream of kinins but also the quick release of peptides with extended c-terminal sequences .
these peptides have occasionally been reported to exert biological effects that are typical of kinins in animal models  <cit> , but this is most likely due to additional processing by tissue carboxypeptidases.
of the two saps that bind to the fungal cell wall via the gpi anchor, sap <dig> was unable to cleave the kininogens at all; in contrast, sap <dig> rapidly and nearly completely excised from lk des-arg1-bradykinin  that is assumed to be biologically inactive because it does not interact with b2-type kinin receptors.
we also found that its derivative without the c-terminal arginine—i.e., des-arg <dig> -bradykinin —that can potentially form in host tissues due to the actions of carboxypeptidases, is not a high-affinity ligand of b <dig> receptors .
however, a specific sap9-dependent inactivation of the kininogens at the fungal cell wall is unlikely in the light of the previous findings that in virtually any infection model in which sap <dig> gene expression is detected, some soluble saps are also produced  <cit> .
the impressive adaptability of c. albicans to very different environments within the host organism depends on the compatibility of various virulence factors, such as saps, with the broad range of environmental parameters.
ph is one of the most important parameters to consider  <cit> .
indeed, the optimal ph for the enzymatic activity of the  <dig> c. albicans saps ranges between 2– <dig>  which is sufficiently broad to assure that ≥  <dig> sap will effectively hydrolyze proteinaceous targets in each of the major niches that are colonized and infected by this fungus, including the skin , vagina , oral cavity , blood , and different parts of the gastrointestinal tract .
of the two saps that are hypothesized to play critical roles in kinin generation at the sites of candidal infections, sap <dig> optimally acts at an acidic ph.
however, this sap is exceptional among all saps, not only because of its high kinin-releasing activity against lk, but also because the optimal ph for this process markedly shifts toward neutral relative to general proteolytic activity; therefore, remarkable kinin production can be expected even at ph >  <dig>  sap <dig> optimally cleaved lk at ph  <dig>  but, again, the ph optimum of this activity is relatively broad.
a number of soluble saps that exhibit optimal kininogenase activity at a ph as low as  <dig>  and as high as  <dig>  could potentially collaborate with sap <dig>  thus, the extensive generation of active kinins from kininogens can potentially occur in most c. albicans-infected host niches.
the findings of our current study need to be discussed in the light of previous reports on the actual presence of individual saps at infection foci.
by applying multiple infection models and different methodologies, the available data are both diverse and controversial.
moreover, most studies primarily focus on the expression of sap-encoding mrna  <cit>  and only occasionally quantify protein products using western blot analysis  <cit> , but these studies do not reveal anything about the in situ proteolytic activities of individual saps.
however, the hypothesis that soluble sap <dig> alone, or membrane-bound sap <dig> with the assistance of several soluble saps, controls kinins at the sites of candidal infections seems to be relatively insensitive to the discrepancies in the literature because it is difficult to find any studies that definitely exclude the expression of both sap <dig> and sap <dig> from any infection model.
this subject has been extensively reviewed  <cit> , but only a few representative findings can be cited here.
in a study by albrecht et al.
<cit> , the high, constitutive expression of the sap <dig> gene was consistently detected in both reconstituted human epithelium  models and human patient samples.
naglik et al.
<cit>  reported the common expression of the sap <dig>  sap <dig>  sap <dig> and sap <dig> genes in c. albicans isolates obtained from the oral cavity and vagina in human patients.
sap gene expression profiles, as well as their order of appearance during infection, differ between oral and vaginal candidiases.
for instance, sap <dig>  sap <dig>  and sap <dig> are more commonly expressed in vaginal rather than oral infections in humans  <cit> .
in a study on the oral rhe model, sap <dig> and sap <dig> were initially expressed, followed by sap <dig>  and finally sap <dig> and sap <dig> during the late phase of infection  <cit> ; in the vaginal rhe model, the initial expression of the sap <dig>  sap <dig>  and sap <dig> genes was followed by the appearance of the sap <dig>  sap <dig> and sap <dig> transcripts, and finally the expression of sap <dig> and sap <dig>  <cit> .
in both oral and vaginal rhe infected by sap-deficient mutants, sap1– <dig> were found to play predominant roles in mucosal tissue damage during the initial phase of the infection  <cit> .
the results obtained in various animal models are more diverse, but some studies report the expression of all sap genes in murine gastrointestinal infection  <cit>  or the strong, sustained expression of sap <dig> and sap <dig> in murine oropharyngeal candidiasis  <cit> .
CONCLUSIONS
the abilities of most of the  <dig> c. albicans saps to cleave human kininogen and release kinins, and the available literature on sap gene expression during candidal infections strongly support the hypothesis that kinins are effectively produced at the infection foci during many types of candidiasis and contribute to the inflammatory state of this disease in humans.
while small or moderate levels of kinins can be generated by all saps , the highest level of kinin production depends on the actions of soluble sap <dig> or the combined actions of cell wall-bound sap <dig> and other soluble saps such as sap <dig>  sap <dig>  sap <dig>  sap <dig>  sap <dig>  and sap <dig>  the relative contributions of these two mechanisms to potentially generate kinin at infection foci seem to depend on both the type and phase of infection.

 the secretion of active proteases is one of the most successful strategies used by microbial pathogens to colonize and infect human hosts. by hydrolyzing proteinaceous targets in the host organism, including the proteins in cell membranes and extracellular matrix, these enzymes allow the pathogen to penetrate tissues and acquire nutrients. these enzymes also play important roles in evading the immune system by cleaving immune regulatory proteins, or deregulating the major homeostatic systems of the host that require cascade-activated proteolysis such as the blood coagulation, fibrinolysis, complement, and kallikrein-kinin system. candida albicans is one of the most common fungal pathogens in humans. this yeast-like fungus can release as many as aspartic class proteases into the extracellular space. c. albicans is part of the physiologic human microbiota, but, under some circumstances that are generally related to immune system weaknesses in the host, it can convert to a dangerous pathogen that causes diseases of variable severity. these candidiases can range from relatively mild and easily curable superficial infections of the skin and mucous membranes to life-threatening deep-seated invasions in the inner organs, fungemia, and systemic diseases with high mortality rates. it is assumed that the number and diversity of secreted aspartic proteases from c. albicans are needed to successfully colonize the variety of niches present in humans, but this hypothesis is still unsatisfactorily evidenced by experimental data. in particular, relatively few studies are devoted to systematically comparing the actions of all saps on a single proteinaceous substrate. bradykinin-related peptides, which are collectively called kinins, are proteolytically released from the serum proteins, kininogens. serine proteases, called kallikreins are primarily devoted to this task, but during pathological states, including those associated with microbial infections, other proteases, either from the host or the pathogen, can supplement the actions of the kallikreins. due to the multiple functions of kinins required to regulate various physiological processes, as well as their participation in almost every inflammatory state, the kallikrein-kinin system is considered a major system required for biochemical homeostasis in humans. overactivated kinin production reportedly occurs in infections caused by numerous bacterial species. regarding candidal infections, kaminishi et al. was the first to report that a purified major extracellular protease of c. albicans possesses kinin-releasing potential, albeit indirectly, based on the activation of an upstream-acting zymogen in the kinin-generating cascade. the direct release of kinins from kininogens was later confirmed for mixtures of proteases that were released into the culture medium by several candida species and the purified c. albicans protease, which was unequivocally identified as sap. sap is believed to be the predominant protease secreted by c. albicans when cultured in protein-rich media, but the sap gene expression profile depends on the fungal morphology, and the high expression of different sap genes has been reported in various infection models. this study compares the characteristics of kininogen cleavage and concomitant kinin release due to all individual saps, used in recombinant purified forms. here, we report the exceptionally high kinin-forming activity of sap moreover, the active kinins are produced with a high yield by mixtures of sap and several other saps.  the mechanism responsible for the sap-catalyzed cleavage of domain of human kininogen was studied using a synthetic peptide—denoted as hk-d4—because its amino acid sequence can be obtained from the kinin-containing region of the kininogen molecule. the cleaved products were separated using high performance liquid chromatography, and the major peaks were collected and analyzed to determine their sequences using tandem mass spectrometry. comparative chromatograms of the samples, obtained after incubating hk-d with all individual recombinant saps for long periods of time at the optimal ph for general proteolytic activity are shown in figure  hk-d was effectively cleaved by all saps, except sap and sap the qualitative distributions of the major formed products are similar for sap1– and 8; however, sap demonstrated the ability to release a kinin-like peptide—met-lys-bradykinin —at the highest yield. moderate-to-small amounts of the same peptide were also detected among the minor products of hk-d that were cleaved by sap which agrees with our recent characterization of the kininogenase activity of the natural, purified sap and sap sap and sap in contrast, sap showed a higher preference to hydrolyze the peptide bonds at the carboxyl side of the arginine residues than sap and thus could easily cleave the bond between the arginine and proline located at the n-terminal side of the internal bradykinin sequence. due to this exceptional specificity, the hk-d cleavage pattern caused by sap was unique, and a different, kinin-related peptide—des-arg1-bradykinin —was formed at a high yield.figure hplc/ms characteristics of the sap-catalyzed cleavage of the hk-d peptide. ten μm of the synthetic peptide hk-d were treated with recombinant sap1– in citrate or phosphate buffers at the optimal ph for the general proteolytic activity of each individual sap at an enzyme:substrate molar ratio of 1: for 24 hours at 37°c. the reaction was stopped using hcl, and the samples were analyzed using reversed-phase hplc on an eurosil bioselect 300– c- column in a tfa-water-acn binary gradient system. the fractions, which were collected at the major absorbance peaks, were evaporated and analyzed using esi-ms/ms in order to determine their amino acid sequence. the dependence of the kinin yield on the ph for all kinin-forming saps is illustrated in figure  only sap presented the optimal kinin-forming activity at ph markedly shifted from the optimum with the general protease substrates towards a more neutral ph. thus, > 70% of hk-d was cleaved by sap towards met-lys-bradykinin production at ph  importantly, kinin release was still detectable at ≥ ph  figure effect of ph on sap-catalyzed kinin release from the hk-d peptide. hk-d was cleaved using  μm saps in 50 mm citrate buffers or 25 mm phosphate buffers for 24 hours at 37°c. the reaction was stopped using hcl, and the samples were analyzed using hplc as specified in figure  the amounts of met-lys-bradykinin or des-arg1-bradykinin that formed were estimated based on the peak areas and are expressed relative to the maximum possible amount. data represent mean values from the analysis of three independent samples  ± the standard deviation. the time course of hk-d cleavage by the saps reveals the order by which particular peptide bonds are preferably cleaved. the cleavage preferences of sap, sap sap and sap are essentially the same as that previously reported for natural, purified sap. the bonds after lys and leu were hydrolyzed at the highest rates. thus, the n-terminus of the kinin to be formed was established during the very early stages of sap-catalyzed hk-d hydrolysis and persisted on a longer time scale. a number of additional cleavages occurred downstream from the kinin c-terminus, which ended up with the exposition of ser and usually ser although at a slower rate. thus, the mkrppgfspfrss and mkrppgfspfrs peptides could be considered the major final products of hk-d cleavage following treatment with this set of saps. hydrolyzing the bond after arg which completes the formation of met-lys-bradykinin, occurred at very slow rates when treated with sap − − and −. due to the high preference of sap for hydrolyzing the bonds after arg and arg, met-lys-bradykinin was one of the major end products of hk-d cleavage, together with the mkrppgfspfrss and mkrppgfspfrssr peptides.figure time course of hk-d cleavage by sap sap sap and sap hk-d was cleaved using  μm sap in 50 mm citrate buffer at 37°c for the specified time. the reaction was stopped using hcl, and the samples were analyzed using hplc as specified in figure  the results from representative kinetic experiments are shown. the areas under the peaks of the early, major cleavage products, as well as the kinin-related peptides, are expressed relative to the substrate at the beginning of the reaction. the cleavage of hk-d by sap and sap was limited to the hydrolysis of the two bonds after lys and leu sap quickly cleaved the bond after lys to form the islmkrppgfspfrssrigeik peptide, at the expense of which the mkrppgfspfrssrigeik peptide appeared after a longer period of time at a much lower rate. these two cleavages alternatively occurred at high and comparable rates due to the action of sap which lead to the fast formation of the islmkrppgfspfrssrigeik and mkrppgfspfrssrigeikeet peptides that thereafter were quickly converted to the final major product, mkrppgfspfrssrigeik. among other very minor products that resulted from the actions of sap and sap on hk-d no kinin-like peptides were found using the applied chromatographic method. the unique time course of hk-d cleavage by sap was characterized by the very fast formation of des-arg1-bradykinin, which was only preceded by quickly disappearing precursors such as the islmkrppgfspfr or ppgfspfrssrig peptides. an analysis using sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis showed that sap1– − and − cleaved native human kininogens, with a high preference for the low-molecular-mass form in comparison with the high-molecular-mass form. using the optimized hplc method, the clearly distinguishable peak of met-lys-bradykinin was visible on the chromatograms obtained after lk digestion by sap1– and − while sap resulted in the intense peak of des-arg1-bradykinin. to identify all kinin-like peptides formed by all individual saps, the more sensitive liquid chromatography-coupled tandem mass spectrometry method was applied. we found that in addition to sap1– and − sap and sap also exhibited kinin-forming activities toward the lk substrate and each individual sap produced multiple kinin-like peptides. these included, in addition to the predominant met-lys-bradykinin, bradykinin itself, and occasional traces of kinin derivatives without the c-terminal arg residues, leu-met-lys-bradykinin, and versions of kinins with hydroxyproline at the third position of the bradykinin sequence, as could have been expected from native human kininogens, oxidized met residue, or both.figure hplc profiles of the fragmentation of lk by selected saps. lk samples were digested with  μm of sap − − − and − in the citrate buffer at 37°c for 24 hours. the reaction was stopped using pepstatin a, followed by acidification with hcl. the samples were analyzed using hplc on the luna c 5 μm  × 250 mm column in a tfa water-acn binary gradient system, as described in the materials and methods section. arrows indicate the retention times of the met-lys-bradykinin and des-arg1-bradykinin standards. kinin-like peptides in the sap-treated lk samples that were identified using lc-ms/ms sap kinin-like peptide molecular mass mkrppgfspfr mkrppgfspfr mkrppgfspfr mkrpgfspfr lmkrppgfspfr mkrppgfspfr mkrppgfspfr ppgfspfr pgfspfr lk samples were digested with  μm sap in citrate buffer at 37°c for 24 hours. the reaction was stopped using pepstatin a, and the samples were acidified with hcl and subjected to lc-ms/ms analysis using the bruker hctultra etdii it mass spectrometer that was equipped with an esi ion source and etd ii fragmentation module and was coupled to the dionex ultimate uhplc system. ms/ms data were analyzed using in-house mascot server, and the peptides were identified by searching the swiss-prot database. the peptides marked in bold generated peaks with the highest intensities, which exceeded by ≥  order of magnitude in comparison with the other peptides. the relative distributions of the main kinins were quantitatively determined in the sap-treated lk samples using the hplc analysis, collecting fractions at the retention times for bradykinin, met-lys-bradykinin, and des-arg1-bradykinin, and enzyme-linked immunosorbent assay for kinins in these fractions. while detectable, small amounts of bradykinin were released from lk by all studied saps, sap produced this “main” kinin at the relatively highest yield, which approached 2% of the maximum releasable kinin content in the sample. all tested saps produced met-lys-bradykinin at the yield, which decreased in the order of sap > > sap1 ≈ sap8 > sap2 ≈ sap > > sap5 ≈ sap6 ≈ sap9; however, no sap could favorably compete with sap over the long term in terms of the kinin release yield. at a comparably high yield, sap could produce des-arg1-bradykinin that would barely be detectable in the lk digests obtained after long incubation with other saps.figure distribution of bradykinin, met-lys-bradykinin, and des-arg 1 -bradykinin in the sap-digested lk samples. lk samples were digested using μm sap in the citrate buffer at 37°c for hours. after sequentially stopping the reaction using pepstatin a and hcl, the obtained peptide mixtures were separated on the luna c column in a tfa water-acn binary gradient system. the fractions were collected at the retention times that correspond to the bradykinin, met-lys-bradykinin, and des-arg1-bradykinin standards, evaporated to dryness, and redissolved in the assay buffer of the elisa kit in order to quantitatively determine the kinin concentrations. the corresponding fractions, obtained from the hplc separation of intact lk served as the controls, and the kinin concentrations, determined in these fractions are subtracted from those in the sap-digested lk samples. the corrected amount of each of the three kinins is expressed relative to the maximum possible amount of all kinins. results, obtained from two independent experiments, with three replicate elisa measurements for each fraction obtained during each hplc separation, are presented as the mean values ± standard deviation. asterisks denote the statistical significance of the difference between the kinin levels in the sap-treated and undigested lk samples. the data plotted in the three panels are for bradykinin, met-lys-bradykinin, and des-arg1-bradykinin. the sap-treated lk samples were additionally analyzed for their interactions with hek cells, which were engineered to overexpress b2-subtype kinin receptors on the surface. this part of our study aimed to assess putative biological activity, typical of kinins, in the peptide mixtures generated by saps from human kininogen. using this type of ligand-receptor radioassay, the highest amount of “bradykinin equivalents” was found in the lk digests obtained using sap followed by those generated by sap interpreting these results was not straightforward because attempts to calibrate the radioassay with synthetic des-arg1-bradykinin failed to detect any interactions between this peptide and the b receptors, contrary to met-lys-bradykinin which binds to receptors although at a 10-fold lower affinity than bradykinin. thus, the peak of b receptor-binding activity, which was found in the sap9-treated lk samples, was probably due to the bradykinin that developed in the largest amount in comparison with all other saps. the highest peak recorded for sap was obviously due to the large amount of the true b2-receptor agonist produced from lk by this protease at a nearly stoichiometric yield. actually, the apparent amount of kinin in this sample was underestimated by the radioassay, which had been calibrated to the bradykinin standard.figure the amounts of the b receptor-interacting peptides in the sap-digested lk samples. lk samples were digested with  μm sap in the citrate buffer at 37°c for or 24 hours, the reaction was stopped using pepstatin a, and the samples were analyzed for kinin content using a competitive radioreceptor assay that used b receptor-overexpressing hek cells. the calibration plot for the assay was prepared using a bradykinin standard. the results are corrected by subtracting the values, determined in the undigested lk sample. data represent mean values from three separate radioreceptor binding analyses, with the measurements performed in triplicates within each experiment. the error bars represent the standard deviations; asterisks denote the statistical significance of the differences between sap-treated and undigested lk samples. the short peptide—des-arg1-bradykinin —was assumed to be biologically inactive because of its inability to interact with b receptors. therefore, we were very interested to determine if sap can hydrolyze the peptide bond after the third arginine residue in met-lys-bradykinin, thereby inactivating this biologically active kinin formed from the kininogens by the other saps, and other peptides with an n-terminal met residue, such as mkrppgfspfrssrigeik, mkrppgfspfrss, mkrppgf-spfrs, and mkrppgfspfrssr—which are the fast-appearing major products of kininogen cleavage by other saps—thereby preventing the subsequent conversion to active kinin. surprisingly, as shown in figure  sap was essentially unable to process these substrates at the n-terminus, suggesting that this protease requires a long stretch of amino acids upstream from the n-terminal side of the internal bradykinin sequence to effectively cleave the n-terminal bond between the arg and pro residues. however, the c-terminal arginine residue of the kinin sequence—which is absolutely required for interactions between the free kinins and b receptors—was still quickly exposed by sap thus, the sap-catalyzed cleavage of all substrates with n-terminal methionine converged to a single product, met-lys-bradykinin,.figure time course of the cleavage of the mkrppgfspfrssr peptide using sap mkrppgfspfrssr peptide was cleaved using  μm sap in 50 mm citrate buffer at 37°c for the specified time. the reaction was stopped using hcl, and the sample was analyzed using hplc as specified in figure  the results from a representative kinetic experiment are shown. the areas under the peaks of separated peptides are expressed relative to the substrate at the beginning of the reaction. the unique specificity of sap as determined by our analysis of the synthetic peptides, allows mixtures of sap and sap − −4– and − to demonstrate the strikingly enhanced formation of met-lys-bradykinin from kininogen in comparison with the individual actions of these proteases. at the same time, the production of the biologically inactive peptide, des-arg1-bradykinin, was largely quenched. this effect was particularly dramatic with sap and sap which individually produced only trace amounts of met-lys-bradykinin, but in the presence of sap the kinin yield exceeded even that of the best individual kinin producer, sap in contrast, the release of met-lys-bradykinin from sap was not enhanced in the presence of sap apparently because both these proteases cleave the bond after the arg residue on the c-side of the internal kinin sequence at a comparably high rate. however, the production of des-arg1-bradykinin by the sap and sap mixture was quenched by about half in comparison with the amount released from the kininogen using sap alone.figure comparisons of the generation of bradykinin, met-lys-bradykinin, and des-arg 1 -bradykinin in lk samples that were digested using a mixture of sap and other saps vs digestion with single saps. lk samples were digested with either  μm individual saps or mixtures of  μm sap with equimolar amounts of other saps in the citrate buffer at 37°c for 6 hours. after sequentially stopping the reaction with pepstatin a and hcl, the obtained peptide mixtures were separated on the luna c column in a tfa water-acn binary gradient system. fractions were collected at the retention times that correspond to the bradykinin, met-lys-bradykinin, and des-arg1-bradykinin standards, evaporated to dryness, and redissolved in the assay buffer of the elisa kit in order to quantitatively determine the kinin concentrations. the results are corrected by subtracting the data, obtained for the undigested lk sample as specified in figure  the amount of each of the three kinins is expressed relative to the maximum possible amount of all kinins. for each sap, the light gray bar represents the sum of kinin amounts in two independent samples, treated separately with this sap and sap while the dark gray bar represents the value for sample of lk that was treated with a mixture of this sap and sap all data bars represent the mean values of six determinations  ± standard deviation. asterisks denote the statistical significance of the difference between the indicated kinin levels. discussion the success of c. albicans as a pathogen is due to its ability to inhabit various niches within the host organism, including the skin, oral and vaginal mucosa, gastrointestinal tract, and, as in systemic infections, all inner organs and the blood. this yeast-like fungus can adapt to different environments due to the development of an impressive array of virulence factors, including yeast-to-hyphae transition, morphologic switches, numerous surface-exposed adhesins, and hydrolytic enzymes such as saps. the multiplicity of the effects that determine candidal infections makes it difficult to reliably dissect the relative role of a single factor in the colonization and invasion of host tissues. thus, eliminating a single virulence factor—e.g., gene mutations—often unsatisfactorily reduces the strain pathogenicity because of the compensating effects of other factors. regarding the different saps of c. albicans, functional redundancy must also exist because numerous attempts to assign specific roles to individual saps have not led to a general consensus. extensive studies on the expression of individual sap genes in various in vitro and in vivo models of candidal infections report contradictory results. regarding the role in microbial infections, the kinin-forming system can be compared to a double-edged sword. primarily, the proinflammatory and vasoactive properties of kinins contribute to the refined multifactorial process that provides host defenses against pathogens. for instance, kinins recruit defense cells such as neutrophils and monocytes to the infection foci and activate many cell types in order to release other, even more potent proinflammatory mediators. however, at least one activity of kinins—enhancing vascular permeability—is beneficial to pathogens, helping them acquire the necessary nutrients from serum and disseminate within the host organism. the upregulation of kinin production has been frequently reported in association with bacterial infections. relatively recently, the hijacking of the kinin-forming system of the host was suggested to occur as part of fungal infections such as candidiasis. in vitro studies show that, similar to bacterial pathogens, c. albicans can mimic two mechanisms of kinin production used by the host to defeat infections, although in an uncontrolled manner. one mechanism depends on the adsorption of hk and the other components of the contact system on the fungal cell wall, which resembles the contact activation of kinin release on the surface of numerous host cells. the second mechanism involves saps that activate factor xii or directly release kinins from kininogens. in the current study, for the first time, we compared the kininogenase activities of all saps. eight of these, sap1– are soluble secreted proteins, while sap and sap are bound to the cell wall via a glycosylphosphatidylinositol anchor, although some fraction of these proteins can be shed into the extracellular space. we found that all soluble saps can release kinins from human kininogens and strongly prefer lk to hk as the substrate. in each case, the same kinin—met-lys-bradykinin—was formed almost exclusively, i.e., in excess over the other identified kinin-like peptides, including bradykinin. despite its relatively low affinity for b2-type kinin receptors, met-lys-bradykinin is equivalent to bradykinin and kallidin in terms of biological activity, most likely because it can be easily converted to the latter “main” kinins in biological fluids and tissues that are abundant with nonspecific aminopeptidases. the current, unequivocal identification of the chemical nature of the formed kinin peptides—together with the findings that the unfractionated peptide mixtures generated by sap1- and sap from lk interact with b receptors —provides strong support for the hypothesis that these soluble saps can release biologically active kinins at infection sites, as they are likely to encounter the appropriate amounts of kininogens to be cleaved. the qualitatively uniform picture observed for all individual soluble saps suggests a high degree of redundancy between these saps with respect to the postulated kinin-forming functions. quantitatively, however, the kininogenase activity of sap so strongly exceeded those of the other saps that it should be considered the predominant player in putative sap-dependent kinin production at the sites of candidal infections. at sufficient sap levels, sap1– sap4– and sap would only comprise a fungal proteolytic reserve for kininogen cleavage. however, their significance would increase in the rather unlikely case of sap being absent at the infection site, not only due to their ability to generate a small but persistent stream of kinins but also the quick release of peptides with extended c-terminal sequences. these peptides have occasionally been reported to exert biological effects that are typical of kinins in animal models, but this is most likely due to additional processing by tissue carboxypeptidases. of the two saps that bind to the fungal cell wall via the gpi anchor, sap was unable to cleave the kininogens at all; in contrast, sap rapidly and nearly completely excised from lk des-arg1-bradykinin that is assumed to be biologically inactive because it does not interact with b2-type kinin receptors. we also found that its derivative without the c-terminal arginine—i.e., des-arg -bradykinin —that can potentially form in host tissues due to the actions of carboxypeptidases, is not a high-affinity ligand of b receptors. however, a specific sap9-dependent inactivation of the kininogens at the fungal cell wall is unlikely in the light of the previous findings that in virtually any infection model in which sap gene expression is detected, some soluble saps are also produced. the impressive adaptability of c. albicans to very different environments within the host organism depends on the compatibility of various virulence factors, such as saps, with the broad range of environmental parameters. ph is one of the most important parameters to consider. indeed, the optimal ph for the enzymatic activity of the c. albicans saps ranges between 2– which is sufficiently broad to assure that ≥  sap will effectively hydrolyze proteinaceous targets in each of the major niches that are colonized and infected by this fungus, including the skin, vagina, oral cavity, blood, and different parts of the gastrointestinal tract. of the two saps that are hypothesized to play critical roles in kinin generation at the sites of candidal infections, sap optimally acts at an acidic ph. however, this sap is exceptional among all saps, not only because of its high kinin-releasing activity against lk, but also because the optimal ph for this process markedly shifts toward neutral relative to general proteolytic activity; therefore, remarkable kinin production can be expected even at ph >  sap optimally cleaved lk at ph  but, again, the ph optimum of this activity is relatively broad. a number of soluble saps that exhibit optimal kininogenase activity at a ph as low as and as high as could potentially collaborate with sap thus, the extensive generation of active kinins from kininogens can potentially occur in most c. albicans-infected host niches. the findings of our current study need to be discussed in the light of previous reports on the actual presence of individual saps at infection foci. by applying multiple infection models and different methodologies, the available data are both diverse and controversial. moreover, most studies primarily focus on the expression of sap-encoding mrna and only occasionally quantify protein products using western blot analysis, but these studies do not reveal anything about the in situ proteolytic activities of individual saps. however, the hypothesis that soluble sap alone, or membrane-bound sap with the assistance of several soluble saps, controls kinins at the sites of candidal infections seems to be relatively insensitive to the discrepancies in the literature because it is difficult to find any studies that definitely exclude the expression of both sap and sap from any infection model. this subject has been extensively reviewed, but only a few representative findings can be cited here. in a study by albrecht et al., the high, constitutive expression of the sap gene was consistently detected in both reconstituted human epithelium models and human patient samples. naglik et al. reported the common expression of the sap sap sap and sap genes in c. albicans isolates obtained from the oral cavity and vagina in human patients. sap gene expression profiles, as well as their order of appearance during infection, differ between oral and vaginal candidiases. for instance, sap sap and sap are more commonly expressed in vaginal rather than oral infections in humans. in a study on the oral rhe model, sap and sap were initially expressed, followed by sap and finally sap and sap during the late phase of infection; in the vaginal rhe model, the initial expression of the sap sap and sap genes was followed by the appearance of the sap sap and sap transcripts, and finally the expression of sap and sap. in both oral and vaginal rhe infected by sap-deficient mutants, sap1– were found to play predominant roles in mucosal tissue damage during the initial phase of the infection. the results obtained in various animal models are more diverse, but some studies report the expression of all sap genes in murine gastrointestinal infection or the strong, sustained expression of sap and sap in murine oropharyngeal candidiasis.  the abilities of most of the c. albicans saps to cleave human kininogen and release kinins, and the available literature on sap gene expression during candidal infections strongly support the hypothesis that kinins are effectively produced at the infection foci during many types of candidiasis and contribute to the inflammatory state of this disease in humans. while small or moderate levels of kinins can be generated by all saps, the highest level of kinin production depends on the actions of soluble sap or the combined actions of cell wall-bound sap and other soluble saps such as sap sap sap sap sap and sap the relative contributions of these two mechanisms to potentially generate kinin at infection foci seem to depend on both the type and phase of infection. 
this study compared the characteristics of the action of all saps on human kininogens, which results in generating proinflammatory bradykinin-related peptides. our present results, together with the available data on the expression of individual sap genes in candidal infection models, suggest a biological potential of saps to produce kinins at the infection foci. one of the suggested roles of saps is their deregulating effect on various proteolytic cascades that constitute the major homeostatic systems in human hosts, including blood coagulation, fibrinolysis, and kallikrein-kinin systems. the kinin release during candidiasis can involve predominant and complementary contributions of two different sap3- and sap9-dependent mechanisms. however, the collaborative actions of sap and sap − −4– and − on lk rerouted kininogen cleavage toward the high-yield release of the biologically active met-lys-bradykinin. des-arg1-bradykinin was released from lk by sap at a comparably high yield, but this peptide was assumed to be biologically inactive because it was unable to interact with cellular b2-type kinin receptors. except for sap and sap all saps effectively cleaved the kininogens, with the highest hydrolytic activity toward the low-molecular-mass form. candida albicans yeast produces distinct secreted aspartic proteases, which are some of the most important virulence factors of this pathogenic fungus.
 candida albicans yeast produces distinct secreted aspartic proteases, which are some of the most important virulence factors of this pathogenic fungus. one of the suggested roles of saps is their deregulating effect on various proteolytic cascades that constitute the major homeostatic systems in human hosts, including blood coagulation, fibrinolysis, and kallikrein-kinin systems. this study compared the characteristics of the action of all saps on human kininogens, which results in generating proinflammatory bradykinin-related peptides.  recombinant forms of saps, heterologously overexpressed in pichia pastoris were applied. except for sap and sap all saps effectively cleaved the kininogens, with the highest hydrolytic activity toward the low-molecular-mass form. sap1– and produced a biologically active kinin—met-lys-bradykinin—and sap was exceptional in terms of the kinin-releasing yield. des-arg1-bradykinin was released from lk by sap at a comparably high yield, but this peptide was assumed to be biologically inactive because it was unable to interact with cellular b2-type kinin receptors. however, the collaborative actions of sap and sap − −4– and − on lk rerouted kininogen cleavage toward the high-yield release of the biologically active met-lys-bradykinin.  our present results, together with the available data on the expression of individual sap genes in candidal infection models, suggest a biological potential of saps to produce kinins at the infection foci. the kinin release during candidiasis can involve predominant and complementary contributions of two different sap3- and sap9-dependent mechanisms. keywords candidiasishuman kininogenmet-lys-bradykinindes-arg-kininsbradykinin b2-subtype receptorspichia pastorisissue-copyright-statement© the author 2015 