Integrated Activity and Genetic Profiling of Secreted Peptidases in Cryptococcus neoformans Reveals an Aspartyl Peptidase Required for Low pH Survival and Virulence
[['Cryptococcus neoformans is an opportunistic fungal pathogen responsible for 40% of all AIDS-related deaths [1 , 2] .', 'Of the one million new infections occurring worldwide annually , greater than 60% result in death due to the limited efficacy and availability of therapeutics [3] .', 'Only three classes of drugs are currently approved for treatment of fungal infections , thus there is a significant need for development of new antifungal compounds [3–5] .', 'Peptidases are secreted by many types of pathogens including bacteria , fungi and parasites and often serve critical roles related to survival and virulence [6–11] .', 'Direct targeting of peptidases expressed by pathogenic organisms has proven to be a successful therapeutic strategy , notably in the development of Hepatitis C Virus ( HCV ) and Human Immunodeficiency Virus ( HIV ) protease inhibitors [12 , 13] .', 'Additionally , the identification and characterization of peptidases secreted by pathogens have contributed to the formulation of new diagnostic approaches based on detection of these proteolytic activities [14–16] .', 'Pathogenic fungi express extracellular peptidases for wide-ranging functions including host tissue invasion , nutrient acquisition and regulation of mating [17–19] .', 'A single organism may simultaneously secrete multiple peptidases with divergent substrate specificities and requirements for activity that are tailored to their biological functions .', 'In addition , peptidase secretion and activation are often stimulated by extracellular conditions , as distinct proteolytic functions can be important for different environments .', 'Candida albicans and Aspergillus fumigatus , two prominent fungal pathogens , each secrete several peptidases with defined roles in virulence , while dermatophytes and the causative agent of white-nose syndrome Pseudogymnoascus destructans use extracellular peptidases to degrade host tissues [20–26] .', 'Multiple peptidases have been identified in the secreted proteome of C . neoformans , including a metallopeptidase that is required for dissemination to the central nervous system ( CNS ) in a mouse infection model [27–34] .', 'Interestingly , the level of peptidase secretion has been shown to vary between isolates in Cryptococcus species and in many cases higher secretion has been correlated with increased virulence [35–38] .', 'Although these findings suggest that extracellular peptidases are involved in C . neoformans pathogenicity , the delineation of their functions and their validation as therapeutic targets is limited by poor understanding of their activity , specificity and regulation .', 'In this work , we used a comprehensive activity-based approach to characterize secreted peptidases in C . neoformans culture supernatants .', 'This strategy , termed Multiplex Substrate Profiling by Mass Spectrometry ( MSP-MS ) , relies on mass spectrometry to identify cleavage events within a defined 228-member library comprising physiochemically diverse tetradecapeptides [39] .', 'The scope and design of the library allows detection of cleavage events from multiple peptidases simultaneously , and the resulting data are informative for understanding activity on both a global and individual enzyme level .', 'Activity-based profiling stands in contrast to traditional proteomics methods that catalog which peptidases are present but do not provide information on how each enzyme contributes to the overall proteolytic activity [11 , 27] .', 'Likewise , candidate-based approaches focusing on single proteolytic activities isolated from cultures may not accurately represent how these enzymes function within the secreted peptidase milieu [31 , 32] .', 'To investigate the secreted peptidases of C . neoformans and test the influence of environment on global proteolytic activity , we cultured fungal cells under two different conditions and then isolated the cell-free supernatants for substrate specificity profiling .', 'These experiments revealed that overall peptidase specificity differs greatly in response to extracellular conditions .', 'To uncover the contribution of individual enzymes to the total proteolytic activity , ten candidate peptidases were individually deleted and conditioned media generated from each mutant strain was compared to the parental strain .', 'Through this approach , we identified and defined the putative substrate preferences of three peptidases , including a previously uncharacterized secreted aspartyl peptidase .', 'We found that this enzyme is the dominant contributor to extracellular endopeptidase activity at acidic pH and determined that this activity is required for tolerance to low pH environments .', 'Analysis of its substrate specificity enabled us to screen an appropriately focused library of aspartyl peptidase inhibitors , which led to the identification of potent in vitro antagonists .', 'Finally , we found that deletion strains of this enzyme are attenuated for virulence in a mouse inhalation model of C . neoformans infection .', 'Our in-depth characterization of extracellular peptidases in C . neoformans establishes a framework for uncovering the biological functions of these enzymes .', 'As demonstrated by our identification of a peptidase required for virulence , examining the roles of these enzymes is critical to understanding the pathogenicity of C . neoformans .', 'Furthermore , the methods described here are applicable to the discovery and characterization of secreted peptidases from other pathogenic organisms .'], ['C . neoformans was cultured in either microbial minimal media ( yeast nitrogen base [YNB] pH 5 . 0 ) or mammalian tissue-culture media ( DMEM pH 7 . 4 ) , and supernatants from each condition were assayed using a panel of internally quenched ( IQ ) fluorogenic peptides ( Fig 1A , S1 Table for sequences ) .', 'These substrates were previously developed to detect a broad range of microbial peptidases from diverse peptidase families [40–42] .', 'The speed and flexibility of this assay allowed us to optimize the conditions for peptidase activity and to determine which class-specific inhibitors affect it .', 'Although peptidase activity was evident under both culture conditions , differential substrate cleavage reflected differences in specificity .', 'Notably , IQ-2 and IQ-6 were cleaved more efficiently by peptidases in YNB media , while proportionally higher activity was observed against IQ-3 and IQ-4 in DMEM media ( Fig 1A ) .', 'These differences suggested that alternate peptidases were active in each culture condition , which was further confirmed by assaying the substrates in the presence of class-specific peptidase inhibitors .', 'This analysis revealed that aspartyl peptidase activity was present in YNB conditioned media while metallopeptidase activity could be detected in DMEM media ( S1 Fig ) .', 'Adjustment of YNB supernatants from pH 5 . 0 to 7 . 4 ( the pH of DMEM media ) yielded only very low levels of any peptidase activity , while lowering the pH of DMEM supernatants from 7 . 4 to pH 5 . 0 produced a peptidase activity pattern similar to YNB media ( S1 Fig ) .', 'This result suggests that growth in DMEM media using mammalian cell culture conditions induces peptidases optimized for neutral pH in addition to the acidic pH activities detected after growth in YNB media .', 'To investigate global peptidase substrate specificity , MSP-MS was conducted on YNB and DMEM supernatants at the optimal pH for activity observed for each condition , pH 5 . 0 and 7 . 4 , respectively .', 'In the MSP-MS assay , peptide sequencing by LC-MS/MS is used to identify all peptide cleavage products within the 228-member library , revealing peptidase substrate specificity preferences ( Fig 1B ) .', 'The reproducibility of these substrate specificity preferences , and of the cleavage events from which they derive , was confirmed by assessing three technical replicate samples ( S2 Fig and S2 Table ) .', 'Importantly , since there are no modifications to either the N- or C-termini in the peptide library , both exo- and endo-peptidases can be identified in an unbiased manner .', 'Using MSP-MS we observed that peptidases in YNB media cleaved at 423 total sites , whereas peptidases in DMEM media cleaved at 283 total sites ( Fig 1C ) .', 'Only 107 of these sites were cleaved by enzymes in both samples .', 'This difference in cleavage site preference indicated that peptidase activity and specificity differs between the two culture conditions .', 'Positional analysis of all bonds cleaved within the tetradecapeptides of the MSP-MS library illustrates the proportion of endo- and exo-peptidase activity in each sample ( Fig 1D ) .', 'In YNB supernatants , the most frequently cleaved bond was the carboxyl terminal bond between amino acids thirteen and fourteen , representing 32% of total proteolysis events .', 'In fact , 137 of the 228 peptides had their carboxyl-terminal amino acid cleaved .', 'Moreover , we observed that single amino acids were often sequentially hydrolyzed from the carboxyl termini of substrates until an unfavored residue was reached , consistent with the presence of abundant carboxypeptidase activity .', 'This cleavage preference was not observed for proteases in the DMEM media .', 'These studies indicate that carboxypeptidase activity is more abundant in conditioned media from C . neoformans cultures grown in YNB .', 'To further illustrate the differences in proteolytic activity between the two conditions , representative examples of peptides cleaved in both samples are shown ( Fig 1E ) .', 'To identify which C . neoformans peptidases may be contributing to the global substrate specificity profile , we conducted a proteomic analysis of secreted proteins .', 'We observed 199 and 131 proteins in YNB and DMEM conditioned media respectively , with 52 proteins common to both conditions ( S3 Table ) .', 'Recently , Geddes and colleagues identified 61 proteins in the secretions of C . neoformans grown for 16 to 120 hours in minimal media [43] , while Campell and coworkers identified 22 secreted proteins after 168 hours growth in RPMI media [29] .', 'In total , 24 of the proteins detected in our study contained predicted secretion signals ( SignalP 4 . 0 ) [44] , 127 were predicted to be non-classically secreted ( SecretomeP 2 . 0 ) [45] , and 17 have been associated with extracellular microvesicles [46] .', 'The remaining proteins identified had no known mechanism of secretion ( S3 Fig , S3 Table ) .', 'Seven of the proteins with predicted signal sequences were peptidases and included members of the aspartyl , metallo and serine peptidase families .', 'Both endopeptidases and carboxypeptidases were identified , consistent with our predictions from analysis of C . neoformans extracellular proteolytic activity ( Fig 1 ) .', 'Five of these enzymes have been detected in studies of the C . neoformans secretome by other groups; however Prc1 and CNAG_05872 have not been observed previously .', 'To determine which enzymes are responsible for the proteolytic activity present in C . neoformans conditioned media , we performed targeted gene deletions on ten candidate secreted peptidases ( Table 1 , S4 Table ) .', 'Of the seven aforementioned peptidases with predicted signal sequences that were identified by our secretome proteomics , one was predicted to be GPI-anchored ( CNAG_04380 ) [27 , 47] , and thus excluded from further analysis , as our study was focused on non-cell wall anchored enzymes .', 'One other peptidase could not be mapped unambiguously to a single gene , as three paralogs of this enzyme exist in the C . neoformans var grubii genome [48] .', 'Therefore , all three genes were individually targeted for deletion ( CNAG_00919 , CNAG_01040 and CNAG_02966 ) .', 'Because these genes are unnamed and lack orthologs in Saccharomyces cerevisiae , we propose naming them Carboxypeptidase D 1 , 2 and 3 ( CXD1-3 ) , respectively .', 'This resulted in eight genes deleted based on our proteomics results ( Table 1 , S4 Table ) .', 'We additionally targeted two secreted peptidases that were not identified here but have been reported in previous proteomics studies [27] .', 'Two independent isolates of each of the ten deletion strains were generated and are indicated in the text and figures by the gene name or CNAG number followed by “-1” or “-2” ( S4 Table ) .', 'Based on our characterization of secreted peptidase activity present in wild type C . neoformans , we selected deletion strains for in-depth substrate profiling analysis by MSP-MS under either DMEM or YNB culture conditions .', 'Subsequently , by comparing the secreted peptidase activity in conditioned media from the wild type and mutant strains , we were able to correlate extracellular proteolytic activities to specific candidate enzymes .', 'To analyze the peptidase substrate specificity of DMEM media conditioned by wild-type cells , we generated a frequency plot from the 283 cleavage events detected by MSP-MS ( Fig 2A , S5 Table ) [50] .', "The amino acid preferences are shown for four positions on either side of the cleaved bond ( P4-P4' ) , as the majority of substrate specificity is determined by residues closest to the scissile bond .", "This analysis revealed that peptidases in DMEM supernatants prefer positively charged residues on either side of the cleaved bond , as well as hydrophobic residues in the P1' position .", "Negatively charged amino acids are disfavored at the majority of positions , and proline and glycine are both highly disfavored in most positions from P2-P2' ( Fig 2A ) .", 'To identify the enzymes responsible for this activity , we examined proteolytic activity in peptidase deletion strains .', 'Because DMEM conditioned media contained metallopeptidase activity ( S1 Fig ) and a single metallopeptidase ( Mpr1 ) was identified by proteomics ( Table 1 , S3 Table ) , we began by investigating the contribution of this enzyme to the global specificity profile .', 'Mpr1 had previously been characterized as a secreted factor that is important for C . neoformans invasion of the CNS [28] .', "Matched comparison of the substrate specificity profiles obtained from DMEM media conditioned by wild type or mpr1Δ cells revealed that Mpr1 deficiency caused a loss of the P1' preference for hydrophobic amino acids seen in wild type ( Fig 2A and 2B ) .", 'However , the selection for positively charged residues on either side of the cleaved bond remained unaltered and the same amino acids were disfavored in most positions .', 'To further analyze the impact of MPR1 deletion , a Venn diagram was used to compare the overlap of cleavage events between wild type and mpr1Δ ( Fig 2C ) .', 'A majority of cleaved peptides were detected in both samples; however 107 cleavage events were detected in wild type but not media conditioned by mpr1Δ .', 'These cleavages , presumed to be absent due to the loss of this enzyme , were used to generate a frequency plot representing the putative specificity of Mpr1 ( Fig 2C ) .', "A prominent feature of this substrate specificity profile is enrichment for phenylalanine , leucine and norleucine ( a replacement for methionine in the MSP-MS library ) at the P1' position , a result that is consistent with the specificity of other peptidases predicted to be related to this enzyme ( members of the M36 peptidase family ) [51] .", "It is also notable that the P1' site exhibits the greatest degree of selectivity of any position from P4-P4' .", 'To further illustrate the changes in substrate specificity observed in the mpr1Δ deletion strain , a representative example of a peptide cleaved by enzymes in both wild type and mpr1Δ supernatants is shown ( Fig 2D ) .', 'An additional activity in DMEM media conditioned by wild type displays a trypsin-like preference for proteolysis between two positively charged residues , indicating the presence of serine peptidase activity [51] .', 'This specificity is particularly evident in the mpr1Δ culture media ( Fig 2B ) .', 'Two serine endopeptidases were present in the deletion collection and DMEM conditioned media was analyzed from both strains ( prb1Δ and CNAG_00150Δ ) .', 'Deletion of either gene did not substantially impact the extracellular peptidase activity profile , suggesting functional redundancy or the existence of additional , unidentified peptidases ( S4 Fig ) .', 'One predicted serine peptidase with a secretion signal , KEX2 , was identified in a genome search .', 'However , our attempts to delete this gene were unsuccessful , indicating it may be essential for C . neoformans survival .', 'In some cases , media conditioned by knockout cells produced additional peptide cleavage sites as compared to wild type conditioned media , despite similar overall peptidase specificity profiles ( e . g . , S4C Fig ) .', 'This observation is consistent with the fact that iterative cleavage of an MSP-MS substrate peptide can hinder identification of a given cleavage event due to loss of the cleavage’s reaction product .', 'In this way , the loss of a minor peptidase activity can result in the appearance of new cleavage sites [25] .', 'A substrate specificity profile constructed from the 423 cleavages observed in YNB media conditioned by wild-type cells indicated a preference for hydrolysis between hydrophobic residues , while positively charged residues , proline and glycine are disfavored ( Fig 3A ) .', 'From positional analysis of these cleavage sites , we identified carboxypeptidase activity as the dominant proteolytic activity in this media ( Fig 1D ) .', "Since carboxypeptidases cleave the carboxyl-terminal bond , no enrichment of amino acids is evident in the P2' to P4' positions in these substrates ( Fig 3A ) .", 'To determine whether any of the three carboxypeptidase D paralogs we identified in our proteomics analysis were responsible for the observed carboxypeptidase activity , the gene for each enzyme was deleted and conditioned media from the resulting mutant strains ( cxd1Δ , cxd2Δ and cxd3Δ ) was profiled by MSP-MS .', 'After comparison of the specificity profiles , it was clear that the carboxypeptidase specificity was most dependent on Cxd1 .', 'In media conditioned by this deletion strain only 17 primary carboxyl-termini were cleaved , as compared to 137 in wild type ( Fig 3B , 3D and 3E ) .', 'By comparison , 134 and 123 primary carboxyl-termini were cleaved in media conditioned by cxd2Δ and cxd3Δ , respectively , suggesting that these enzymes do not contribute substantially to the extracellular carboxypeptidase activity ( S5 Fig , S5 Table ) .', 'As anticipated , endopeptidase cleavages were not affected in any of the three carboxypeptidase deletion strains ( Fig 3B and 3D , S5 Fig ) .', 'Fluorogenic assays demonstrated aspartyl endopeptidase activity in wild type YNB supernatants ( S1 Fig ) .', 'To assign activity to the candidate aspartyl peptidases , conditioned YNB media was profiled from the two aspartyl peptidase deletion strains listed in Table 1 ( CNAG_05872Δ and pep4Δ ) .', 'Proteolytic activity remained unchanged relative to wild type in the pep4Δ strain ( S5 Fig ) .', 'In contrast , CNAG_05872Δ conditioned media exhibited a near-total loss of endopeptidase cleavage events as well as substantially decreased carboxypeptidase activity as evidenced by proteolysis of only 55 primary carboxyl termini ( Fig 3C–3E ) .', 'This result suggests that CNAG_05872 is the dominant endopeptidase under these culture conditions .', 'This finding is consistent with fluorogenic assays , where deletion of CNAG_05872 led to a loss of endopeptidase activity in conditioned YNB media , while all other strains exhibited activity levels similar to wild type ( Fig 3F , S6 Fig ) .', 'As the putative dominant endopeptidase , we propose renaming CNAG_05872 to Major Aspartyl peptidase 1 ( MAY1 ) .', 'Due to its substantial contribution to peptidase activity in YNB supernatants , we performed an in-depth biochemical characterization of May1 .', 'This enzyme consists of a 16 residue secretory signal ( SignalP 4 . 0 ) [44] followed by an 82 residue prodomain ( Fig 4A ) .', 'The prodomain is positively charged ( pI = 9 . 97 ) , which likely facilitates interaction with the negatively charged catalytic domain ( pI = 4 . 03 ) at neutral or slightly acidic pH [52] .', 'The pepsin-like aspartyl peptidase domain includes residues 100–434 and is expected to auto-activate in acidic environments , causing release of the pro-domain .', 'The N-terminal region ( position 101–223 ) is also an N-terminal xylanase inhibitor domain , TAXi_N [53] .', 'Homology between xylanase inhibitors and fungal aspartyl peptidases has been noted previously and likely indicates an evolutionary relationship [54] .', 'May1 readily cleaves IQ-2 between phenylalanine and leucine ( S1 Table , S6 Fig ) , which allowed us to use fluorogenic assays to monitor enrichment of this enzyme from YNB supernatants and investigate the impact of pH on its activity .', 'Ion exchange chromatography was used to enrich May1 from conditioned YNB media , resulting in a 292 nM peptidase stock solution .', 'May1 was diluted from this stock into buffers ranging from pH 1 . 5 to 7 . 0 and activity against IQ-2 was tested .', 'Optimal activity was observed between pH 3 . 5–4 . 5 , a range that is consistent with other members of the aspartyl peptidase family of enzymes ( Fig 4B ) [55] .', 'The aspartyl peptidase antagonist pepstatin A fully inhibited proteolysis of IQ-2 in this assay , providing further verification that May1 is the predominant endopeptidase activity under these conditions .', 'To investigate the time- and growth-dependent secretion of May1 , we added a CBP-2xFLAG tag to the carboxyl-terminus through homologous recombination .', 'By monitoring activity using IQ-2 , we confirmed that the addition of this tag did not diminish May1 activity in YNB conditioned media .', 'Interestingly , although recombinant May1 activity could be detected in the culture supernatant after overnight growth , it could not be detected by immunoblot even after three days of growth .', 'We hypothesized that the enzyme rapidly hydrolyses the C-terminal tag; therefore pepstatin A was added to the culture to inhibit this processing .', 'This inhibitor also prevented activation of pro-May1 to mature May1 , resulting in the observation of only the pro form of recombinant May1 .', 'Under these conditions the protein was detected by immunoblot after 48 hours of growth ( Fig 4C ) .', 'The apparent molecular weight of pro-May1 was approximately 13 kDa greater than the predicted 56 . 8 kDa for the tagged enzyme , suggesting that this protein could contain post-translational modifications .', 'When C . neoformans was cultured in YNB media buffered to pH 6 . 5 , the May1 activity detectable in supernatants using IQ-2 was approximately 10-fold lower than in unbuffered YNB , and no signal could be seen by immunoblot after 48 hours of growth ( Fig 4D ) .', 'This result suggests that low extracellular pH could stimulate May1 secretion and activation .', 'We observed that may1Δ strains grown in YNB had a lower cell density at saturation than wild type or any of the other nine peptidase deletion strains ( Fig 5A , S7 Fig , S6 Table ) .', 'Higher cell density could not be achieved by the may1Δ strains even after culturing for 96 hours .', 'These data suggested that may1Δ strains were not merely slow to replicate but were incapable of growth at high density .', 'In fact , during the exponential growth phase , may1Δ strains had an average doubling time of 2 . 36 hours , which was on par with the other nine peptidase deletion strains .', 'However , all other deletion strains exhibited wild-type saturation densities ( S6 Table ) .', 'Studies of deletion strains did not clarify whether May1 activity , as opposed to simply the presence of the May1 protein , was required to reach a saturation density equivalent to wild type .', 'Therefore , we assessed growth of wild type C . neoformans in the presence and absence of pepstatin A . Treatment of wild type cultures with this aspartyl peptidase inhibitor resulted in a saturation density defect equivalent to that observed for the MAY1 deletion strains ( Fig 5B ) .', 'Importantly , the inhibitor had no effect on may1Δ cells , suggesting that the growth defect observed in wild-type cells treated with pepstatin A was mediated through inhibition of May1 .', 'Plating assays on various stress conditions were conducted with two independent isolates of each of the ten peptidase deletion strains ( S8 and S9 Figs ) .', 'After 48 hours may1Δ strains exhibited a growth defect at pH 3 . 5 but not at pH 5 . 0 or 6 . 5 ( Fig 5C ) .', 'Longer growth periods at pH 3 . 5 did not allow may1Δ colonies to overcome this sensitivity and after three days of growth , it became apparent that may1Δ colonies also had a slight defect at pH 5 . 0 but not pH 6 . 5 ( S8 Fig ) .', 'None of the other peptidase deletion strains displayed sensitivity to acidic pH , although pep4Δ was sensitive to hydrogen peroxide and SDS , and prb1Δ had a slight sensitivity to hydrogen peroxide ( S8 and S9 Figs ) .', 'Based on this result , we hypothesized that the inability of may1Δ strains to reach wild-type saturation densities in YNB was a result of acidification of the media and would be rescued by buffering the media to pH 6 . 5 .', 'These culture conditions fully rescued the saturation density of may1Δ ( Fig 5D ) .', 'Surprisingly , it also allowed the final saturation densities of both wild type and may1Δ cultures to approximately double , revealing that low pH is a condition limiting growth even for wild-type C . neoformans .', 'We also assessed melanization , an established virulence factor , for each of the ten peptidase deletion strains .', 'Because melanin production occurs extracellularly , we hypothesized that this process could be influenced by secreted peptidase activity .', 'Only the serine endopeptidase deletion strain prb1Δ exhibited a hypomelanization phenotype ( S9 Fig ) .', 'While pepstatin A inhibits May1 with an IC50 of 1 . 4 nM , it is a broad acting antagonist of many members of the aspartyl class of peptidases , thereby limiting its utility .', 'To determine whether additional inhibitors targeting May1 could be obtained , we conducted an in vitro screen using knowledge of May1 substrate specificity derived from MSP-MS analysis .', "We screened a panel of 21 peptidomimetic molecules with similarities to May1 substrate preferences but with a non-cleavable bond between the P1 and P1' position ( S7 Table ) .", 'Compounds 1 to 11 are linear peptidomimetics , while compounds 12 through 21 are macrocycles ( S7 Table ) [56–59] .', 'We also screened ten HIV protease inhibitors because some of these molecules have been reported to inhibit C . neoformans peptidase activity [60 , 61] .', 'May1 was incubated with 100 μM , 10 μM and 1 μM of each inhibitor and activity was detected using IQ-2 ( Fig 6A , S10 Fig ) .', 'IC50 values were then calculated for the most potent compounds .', 'The best inhibition by an HIV protease inhibitor was observed with Brecanavir , which reduced activity by 80% at 1 μM and had an IC50 of approximately 352 nM ( S10 Fig ) .', "Among the peptidomimetic molecules , the macrocycles were the most potent , with the best compounds ( 15 to 21 ) containing P2 –P3' tethered side chains , statines in P1 and an α-amino acid in P2' ( Fig 6 , S7 Table ) .", 'Compounds 16 , 21 , and 18 all exhibited nanomolar IC50 values of 1 . 6 nM , 9 . 4 nM and 41 nM , respectively ( S10 Fig ) .', 'Among the linear peptidomimetic inhibitors , those with a phenylstatine or hydroxyethylamine scissile bond isoster ( compounds 4 , 7 , 8 and 11 ) were superior to compounds with a reduced bond ( 1 , 2 , 5 , 6 and 9 ) or a homo-amide ( 2 ) .', 'Compound 4 was the most potent May1 antagonist out of this group of inhibitors , with an IC50 of 3 . 1 nM ( S10 Fig ) .', 'From analysis of the four most effective inhibitors identified in our screen ( compounds 4 , 16 , 18 and 21 ) , it is clear that a phenylalanine side chain , either unsubstituted ( 4 and 16 ) or with a small substituent ( 18 and 21 ) , is preferred in P1 while a bulkier P1 side chain leads to decreased potency , for example compounds 17 , 19 and 20 .', 'These results match the P1 substrate preference for phenylalanine that we had predicted for May1 and fit our expectations that bulkier residues such as tryptophan are not well tolerated in this position ( Figs 3 and 6B ) .', 'Next , we selected the two best in vitro hits to test their potency in culture relative to pepstatin A by measuring inhibition of May1 and restriction of culture growth using fluorogenic assays and OD600 respectively .', 'Wild-type C . neoformans was grown in YNB treated with 5 μM , 1 μM or 0 . 1 μM of compound 4 , 16 or pepstatin A and the culture density and May1 activity were measured at saturation .', 'While compound 16 exhibited an in vitro IC50 comparable to pepstatin A , it was not as effective at inhibiting May1 activity or restricting culture growth ( Fig 6D , S11 Fig ) .', 'Curiously , despite having an in vitro IC50 approximately twice that of compound 16 , compound 4 was better at inhibiting culture growth .', 'None of the three compounds affected the culture density of a may1Δ strain , consistent with the idea that May1 is the compounds’ relevant target in this context ( S11 Fig ) .', 'These results demonstrate that May1 can be targeted by small molecule inhibitors and provide a discovery framework for further inhibitor development .', 'However , additional medicinal chemistry efforts are necessary for in vivo applications .', 'Therefore , subsequent studies investigating the role of May1 in virulence were carried out using deletion strains of this enzyme .', 'Because may1Δ strains exhibit phenotypes in both peptidase activity assays and growth at low pH , we examined the role of this protein in virulence using an established mouse inhalation model of Cryptococcal infection [81] .', 'Wild-type cells were mixed with an equivalent number of may1Δ cells and used to infect mice intranasally ( Fig 7A ) .', 'These experiments were conducted using two independent isolates of may1Δ as well as a negative control known not to affect fungal replication in this assay ( sxi1Δ ) [63] .', 'In each isolate , genes neighboring may1Δ were tested by RT-qPCR to confirm expression ( S12 Fig ) .', 'Ten days after infection , mouse lungs were harvested and plated for colony forming units ( CFUs ) , at which point may1Δ strains contributed only 22 ± 3% of the colonies recovered from the lungs , a substantial decrease from the approximately 50% present upon infection .', 'This result reveals that may1Δ cells have a growth defect within a mammalian host because the ratio between deletion strain and wild type cells was reduced after host infection .', 'Given the defect in may1Δ cell accumulation in the lung , we evaluated the role of May1 during Cryptococcus interaction with macrophages .', 'Opsonized wild type and may1Δ strains were phagocytosed with equal efficiency by bone marrow-derived macrophages ( S13A Fig ) .', 'We next tested the ability of may1Δ cells to accumulate within macrophages after phagocytosis , since the phagolysosome environment may represent a low-pH setting in which May1 is active .', 'Indeed , cells lacking May1 accumulated significantly more slowly within macrophages than did wild type cells ( S13B Fig ) , consistent with a role for May1 within host cells .', 'The defect in accumulation of may1Δ cells during intranasal infection and within macrophages suggested that these strains would be attenuated for virulence .', 'We directly investigated the virulence of may1Δ strains by performing monotypic infections [62] .', 'Ten mice per group were infected intranasally with wild type , may1Δ-1 or may1Δ-2 cells ( Fig 7B ) .', 'Loss of May1 caused significant attenuation of virulence , with mice infected by may1Δ-1 or may1Δ-2 exhibiting a mean survival time of 60 . 1 and 60 . 7 days respectively , whereas those infected by wild type had a mean survival time of 25 days .', 'The results from these in vivo experiments indicate an important role for May1 during mammalian infection .'], ['In this work , we investigated secreted proteolytic activity in C . neoformans var .', 'grubii culture media using an unbiased approach that can detect both endo- and exo-peptidase activity .', 'In combination with proteomic methods and single gene deletion approaches , this strategy allowed us to characterize peptidase activity from a global perspective as well as interrogat
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals , resulting in more than 600 , 000 deaths per year .', 'Many human fungal pathogens secrete peptidases that influence virulence , but in most cases the substrate specificity and regulation of these enzymes remains poorly understood .', 'The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets .', 'We report here an unbiased analysis of secreted peptidase activity and specificity in C . neoformans using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations .', 'Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions .', 'To uncover the substrate preferences of individual enzymes and interrogate their biological functions , we constructed and profiled a ten-member gene deletion collection of candidate secreted peptidases .', 'Through this deletion approach , we characterized the substrate specificity of three peptidases within the context of the C . neoformans secretome , including an enzyme known to be important for fungal entry into the brain .', 'We selected a previously uncharacterized peptidase , which we term Major aspartyl peptidase 1 ( May1 ) , for detailed study due to its substantial contribution to extracellular proteolytic activity .', 'Based on the preference of May1 for proteolysis between hydrophobic amino acids , we screened a focused library of aspartyl peptidase inhibitors and identified four high-affinity antagonists .', 'Finally , we tested may1Δ strains in a mouse model of C . neoformans infection and found that strains lacking this enzyme are significantly attenuated for virulence .', 'Our study reveals the secreted peptidase activity and specificity of an important human fungal pathogen , identifies responsible enzymes through genetic tests of their function , and demonstrates how this information can guide the development of high affinity small molecule inhibitors .']
['Many pathogenic organisms secrete peptidases .', 'The activity of these enzymes often contributes to virulence , making their study crucial for understanding host-pathogen biology and developing therapeutics .', 'In this report , we employed an unbiased , activity-based profiling assay to examine the secreted peptidases of a fungal pathogen , Cryptococcus neoformans , which is responsible for 40% of AIDS-related deaths .', 'We discovered which peptidases are secreted , identified their substrate specificity , and interrogated their biological functions .', 'Through this analysis , we identified a principal enzyme responsible for the extracellular peptidase activity of C . neoformans , May1 , and demonstrated its importance for growth in acidic environments .', 'Characterization of its substrate preferences allowed us to identify compounds that are potent substrate-based inhibitors of May1 activity .', 'Finally , we found that the presence of this enzyme promotes virulence in a mouse model of infection .', 'Our comprehensive study reveals the expression , regulation and function of C . neoformans secreted peptidases , including evidence for the role of a novel aspartyl peptidase in virulence .']
['blood cells', 'cryptococcus neoformans', 'medicine and health sciences', 'cryptococcus', 'immune cells', 'pathology and laboratory medicine', 'enzymes', 'pathogens', 'animal models of disease', 'immunology', 'enzymology', 'microbiology', 'fungi', 'peptide libraries', 'fungal pathogens', 'research and analysis methods', 'animal models of infection', 'mycology', 'white blood cells', 'animal cells', 'proteins', 'medical microbiology', 'animal studies', 'microbial pathogens', 'proteomics', 'biochemistry', 'peptides', 'cell biology', 'biology and life sciences', 'proteases', 'cellular types', 'macrophages', 'organisms']