Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.

The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P2 substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5–7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P2 position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P2 Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite’s life cycle, make it an excellent target for therapeutic inhibitors or vaccination.

SummaryThe availability of complete genome sequences for a large number of pathogenic organisms has opened the door for large-scale proteomic studies to dissect both protein expression/regulation and function. This review highlights key proteomic methods including two-dimensional gel electrophoresis, reference mapping, protein expression profiling and recent advances in gel-free separation techniques that have made a significant impact on the resolution of complex proteomes. In addition, we highlight recent developments in the field of chemical proteomics, a branch of proteomics aimed at functionally profiling a proteome. These techniques include the development of activity-based probes and activity-based protein profiling methods as well as the use of synthetic small molecule libraries to screen for pharmacological tools to perturb basic biological processes. This review will focus on the applications of these technologies to the field of microbiology.

These include diabetes, hypertension, heart disease, kidney disease, immune deficiency, and malignancy. Forty-six percent of the patients had bilateral endophthalmitis. The majority of cases were fungal (46%), while Staphylococcus aureaus accounted for 15% of the cases. Sources of infection were infected hospital tubing in 31% of cases, endocarditis in 15% of cases, and GI abscesses in 15% of cases. The majority of cases were managed with intravitreal antibiotics and vitrectomies with disappointing visual outcomes. Only three of the eyes had final visual acuities of 20/100 or better. Conclusion: These results represent a trend of increasing incidence of fungal endogenous endophthalmitis. They also indicate predisposing conditions and infection sources of this disease. Prognosis is poor in the majority of patients and is most likely determined by the extent of the infection and how soon the infection is treated.

Unlike most protozoa, T. gondii has a limited repertoire of clan CA cysteine proteinases, with only one cathepsin B (TgCPB), one L (TgCPL), and three cathepsin C's (TgCPC1, 2, and 3) identified in the genome project. These enzymes are attractive drug targets, as specific inhibitors of TgCPB blocked invasion in vitro and in vivo. Consequently, we cloned the cathepsin L from a cDNA library. Amino acid sequence homology was closest to the cathepsins of Sarcocystis muris (54%) and Cryptosporidium parvum (36%). The sequence contains the highly conserved ERFNIN motif of cathepsin L's with a transmembrane span. The recombinant protein was expressed as a promature enzyme in Pichia pastoris and then self-processed into the active enzyme. We have identified the preferred peptide substrates of TgCPL: Z-Phe-Arg and Z-Arg-Arg and based on these findings have begun to test biological substrates. Testing a range of pH, cathepsin L cleaves peptide substrates optimally at a pH of 6.0. E-64 inhibits > 99% of the activity of cathepsin L, consistent with its specific inhibition of cysteine proteinases. Antibodies have been developed to help localize this enzyme's site of action. We have also successfully screened an epoxide library and specific cathepsin L inhibitors are currently being used to help in further characterizing TgCPL's biological activity. Determining the crystalline structure of cathepsin L may eventually lead to targeted drug design.