Vaccination against trypanosomiasis: Can it be done or is the trypanosome truly the ultimate immune destroyer and escape artist?

To date, human African trypanosomiasis (HAT) still threatens millions of people throughout sub-Sahara Africa, and new approaches to disease prevention and treatment remain a priority. It is commonly accepted that HAT is fatal unless treatment is provided. However, despite the well-described general symptoms of disease progression during distinct stages of the infection, leading to encephalitic complications, coma and death, a substantial body of evidence has been reported suggesting that natural acquired immunity could occur. Hence, if under favorable conditions natural infections can lead to correct immune activation and immune protection against HAT, the development of an effective anti-HAT vaccine should remain a central goal in the fight against this disease.<br /> In this review, we will (1) discuss the vaccine candidates that have been proposed over the past years, (2) highlight the main obstacles that an efficient anti-trypanosomiasis vaccine needs to overcome and (3) critically reflect on the validity of the widely used murine model for HAT

Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6‑Deoxyerythronolide B Synthase

Assembly line polyketide synthases (PKSs) are large multimodular enzymes responsible for the biosynthesis of diverse antibiotics in bacteria. Structural and mechanistic analysis of these megasynthases can benefit from the discovery of reagents that recognize individual domains or linkers in a site-specific manner. Monoclonal antibodies not only have proven themselves as premier tools in analogous applications but also have the added benefit of constraining the conformational flexibility of their targets in unpredictable but often useful ways. Here we have exploited a library based on the naïve human antibody repertoire to discover a Fab (3A6) that recognizes the terminal thioesterase (TE) domain of the 6-deoxyerythronolide B synthase with high specificity. Biochemical assays were used to verify that 3A6 binding does not inhibit enzyme turnover. The co-crystal structure of the TE-3A6 complex was determined at 2.45 Å resolution, resulting in atomic characterization of this protein-protein recognition mechanism. Fab binding had minimal effects on the structural integrity of the TE. In turn, these insights were used to interrogate via small-angle X-ray scattering the solution-phase conformation of 3A6 complexed to a catalytically competent PKS module and bimodule. Altogether, we have developed a high-affinity monoclonal antibody tool that recognizes the TE domain of the 6-deoxyerythronolide B synthase while maintaining its native function

Integrated Activity and Genetic Profiling of Secreted Peptidases in Cryptococcus neoformans Reveals an Aspartyl Peptidase Required for Low pH Survival and Virulence.

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

Immunoproteasome functions explained by divergence in cleavage specificity and regulation.

The immunoproteasome (iP) has been proposed to perform specialized roles in MHC class I antigen presentation, cytokine modulation, and T cell differentiation and has emerged as a promising therapeutic target for autoimmune disorders and cancer. However, divergence in function between the iP and the constitutive proteasome (cP) has been unclear. A global peptide library-based screening strategy revealed that the proteasomes have overlapping but distinct substrate specificities. Differing iP specificity alters the quantity of production of certain MHC I epitopes but does not appear to be preferentially suited for antigen presentation. Furthermore, iP specificity was found to have likely arisen through genetic drift from the ancestral cP. Specificity differences were exploited to develop isoform-selective substrates. Cellular profiling using these substrates revealed that divergence in regulation of the iP balances its relative contribution to proteasome capacity in immune cells, resulting in selective recovery from inhibition. These findings have implications for iP-targeted therapeutic development

Integrated Activity and Genetic Profiling of Secreted Peptidases in <i>Cryptococcus neoformans</i> Reveals an Aspartyl Peptidase Required for Low pH Survival and Virulence

<div><p>The opportunistic fungal pathogen <i>Cryptococcus neoformans</i> 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 <i>C</i>. <i>neoformans</i> 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 <i>C</i>. <i>neoformans</i> secretome, including an enzyme known to be important for fungal entry into the brain. We selected a previously uncharacterized peptidase, which we term <b>M</b>ajor <b>a</b>spart<b>y</b>l 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 <i>may1Δ</i> strains in a mouse model of <i>C</i>. <i>neoformans</i> 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.</p></div

Profiling of <i>C</i>. <i>neoformans</i> conditioned media reveals abundant secreted peptidase activity with environment-specific regulation.

<p>(A) Profiling of secreted peptidase activity present in YNB or DMEM media conditioned by wild-type cells using a panel of internally quenched (IQ) fluorescent peptides. Columns represent mean ± S.D. (B) Schematic of Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS). Conditioned media is combined with a 228-member peptide library and mass spectrometry analysis is run before and after incubation to identify cleavage events. Norleucine replaces methionine in the MSP-MS library and is indicated by an “n” in representations of the peptides. (C) Several hundred cleavage events were detected in both YNB and DMEM conditioned media profiled by MSP-MS. (D) Positional profiling of all cleaved bonds detected by MSP-MS in either media type. (E) Two representative examples of peptides cleaved in the MSP-MS assay by both media conditions. Arrows indicate the location of cleavage sites.</p

DMEM conditioned media contains a metallopeptidase and trypsin-like endopeptidase activity.

<p>(A) The peptidase substrate specificity profile of DMEM media conditioned by wild type. Residues are significantly favored or disfavored as determined by the frequency of detection in substrates versus the frequency of the residue in the peptide library, p < 0.05. (B) Substrate specificity profile of peptidase activity from <i>mpr1Δ</i> conditioned media, p < 0.05. (C) Peptidase substrate specificity profile constructed from cleavage events detected in wild type but not <i>mpr1Δ</i>, p < 0.05. (D) A representative peptide cleaved by peptidases in both wild type and <i>mpr1Δ</i> conditioned media.</p

May1 is a member of the pepsin-like aspartyl peptidase family with optimal expression and activity at acidic pH.

<p>(A) The domain architecture of May1. The catalytic aspartic acids are indicated by stars. (B) pH titration of May1 activity using IQ-2. Averages and standard deviation (S.D.) of triplicates are shown. (C) Immunoblot detection of May1 tagged with a CBP-2xFLAG tag in supernatants after culturing in YNB for different lengths of time. (D) Activity against IQ-2 in conditioned media from wild type <i>C</i>. <i>neoformans</i> grown in unbuffered or pH 6.5 buffered YNB. All samples were adjusted to pH 4.5 prior to being assayed. Columns represent mean ± S.D.</p