Peroxiredoxins in Parasites

Significance: Parasite survival and virulence relies on effective defenses against reactive oxygen and nitrogen species produced by the host immune system. Peroxiredoxins (Prxs) are ubiquitous enzymes now thought to be central to such defenses and, as such, have potential value as drug targets and vaccine antigens. Recent Advances: Plasmodial and kinetoplastid Prx systems are the most extensively studied, yet remain inadequately understood. For many other parasites our knowledge is even less well developed. Through parasite genome sequencing efforts, however, the key players are being discovered and characterized. Here we describe what is known about the biochemistry, regulation, and cell biology of Prxs in parasitic protozoa, helminths, and fungi. At least one Prx is found in each parasite with a sequenced genome, and a notable theme is the common patterns of expression, localization, and functionality among sequence-similar Prxs in related species. Critical Issues: The nomenclature of Prxs from parasites is in a state of disarray, causing confusion and making comparative inferences difficult. Here we introduce a systematic Prx naming convention that is consistent between organisms and informative about structural and evolutionary relationships. Future Directions: The new nomenclature should stimulate the crossfertilization of ideas among parasitologists and with the broader redox research community. The diverse parasite developmental stages and host environments present complex systems in which to explore the variety of roles played by Prxs, with a view toward parlaying what is learned into novel therapies and vaccines that are urgently needed. Antioxid. Redox Signal. 17, 608-633.Keywords: Entamoeba histolytica, Plasmodium falciparum, Alkyl hydroperoxide reductase, Falciparum 1 Cys Peroxiredoxin, Thiol specific antioxidant, One conserved cysteine, Protozoan trichomonas vaginalis, Amebic liver abscess, Schistosoma mansoni thioredoxin, Redox sensitive oligomerizatio

Fiction Fix 10

https://digitalcommons.unf.edu/fiction_fix/1002/thumbnail.jp

First Universities Allied for Essential Medicines (UAEM) Neglected Diseases and Innovation Symposium

Universities Allied for Essential Medicines organized its first Neglected Diseases and Innovation Symposium to address expanding roles of public sector research institutions in innovation in research and development of biomedical technologies for treatment of diseases, particularly neglected tropical diseases. Universities and other public research institutions are increasingly integrated into the pharmaceutical innovation system. Academic entities now routinely undertake robust high-throughput screening and medicinal chemistry research programs to identify lead compounds for small molecule drugs and novel drug targets. Furthermore, product development partnerships are emerging between academic institutions, non-profit entities, and biotechnology and pharmaceutical companies to create diagnostics, therapies, and vaccines for diseases of the poor. With not for profit mission statements, open access publishing standards, open source platforms for data sharing and collaboration, and a shift in focus to more translational research, universities and other public research institutions are well-placed to accelerate development of medical technologies, particularly for neglected tropical diseases

Structural analysis of beta-lactamase and resistant transpeptidase inhibition

Beta-lactam antibiotics have achieved phenomenal success in the treatment of infections by inhibiting the transpeptidase enzymes that cross-link the bacterial cell wall. Beta-lactamase-producing pathogenic bacteria and multi-drug-resistant “superbugs” such as methicillin-resistant Staphylococcus aureus (MRSA) have emerged, however. Overcoming resistance factors is thus a research priority. BLIP (Beta-Lactamase Inhibitory Protein) from Streptomyces clavuligerus binds a variety of beta-lactamase enzymes with widely ranging specificity. Its interaction with Escherichia coli beta-lactamase TEM-1 is a well-established model system for protein-protein interaction studies. Presented in Chapter 2 are crystal structures of two BLIP relatives: BLIP-I (a highaffinity inhibitor, alone and in complex with TEM-1) and BLP (which appears not to inhibit beta-lactamases). Substantial variation appears possible in the sub-nanomolar binding of TEM-1 by two homologous proteinaceous inhibitors and such favorable interactions can be negated by a few, strongly unfavorable interactions. OXA-10 is a Pseudomonas aeruginosa beta-lactamase that is resistant to inhibitors in clinical use. Cyclobutanone beta-lactam mimics could be used instead. Chapter 3 reports the crystal structure of OXA-10 covalently modified at its catalytic serine nucleophile with a cyclobutanone inhibitor to form a hemiketal. Favorable and unfavorable contacts made at the active site are examined with a view to improved inhibitor design. PBP2a is the resistant transpeptidase that allows MRSA to maintain the bacterial cell wall in the presence of beta-lactam antibiotics. Ceftobiprole is the most clinically-advanced among a new generation of beta-lactams designed to treat MRSA by targeting PBP2a itself. Chapter 4 uses the crystal structure of a truncated, soluble form of PBP2a solved in complex iii with ceftobiprole to explain its inhibitory power and evaluate current anti-MRSA drug design hypotheses. Its efficacy appears to arise from improved binding affinity that overcomes the disfavored energetics of acylation. Ceftobiprole clinical trials reported no bacterial resistance, yet fully ceftobiproleresistant MRSA (MIC 128 !g/ml) were generated by passage through subinhibitory concentrations of ceftobiprole, discussed in Chapter 5. Resistance emerges in most cases via mutations to the gene encoding PBP2a. Computational modeling predicts that ceftobiprole resistance may be mediated in PBP2a by alteration of binding affinity, acylation efficiency, or by influencing interactions with other proteins.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

In Vitro Selection and Characterization of Ceftobiprole-Resistant Methicillin-Resistant Staphylococcus aureus▿ †

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to β-lactam antibiotics because it expresses penicillin-binding protein 2a (PBP2a), a low-affinity penicillin-binding protein. An investigational broad-spectrum cephalosporin, ceftobiprole (BPR), binds PBP2a with high affinity and is active against MRSA. We hypothesized that BPR resistance could be mediated by mutations in mecA, the gene encoding PBP2a. We selected BPR-resistant mutants by passage in high-volume broth cultures containing subinhibitory concentrations of BPR. We used strain COLnex (which lacks chromosomal mecA) transformed with pAW8 (a plasmid vector only), pYK20 (a plasmid carrying wild-type mecA), or pYK21 (a plasmid carrying a mutant mecA gene corresponding to five PBP2a mutations). All strains became resistant to BPR by day 9 of passaging, but MICs continued to increase until day 21. MICs increased 256-fold (from 1 to 256 μg/ml) for pAW8, 32-fold (from 4 to 128 μg/ml) for pYK20, and 8-fold (from 16 to 128 μg/ml) for pYK21. Strains carrying wild-type or mutant mecA developed six (pYK20 transformants) or four (pYK21 transformants) new mutations in mecA. The transformation of COLnex with a mecA mutant plasmid conferred BPR resistance, and the loss of mecA converted resistant strains into susceptible ones. Modeling studies predicted that several of the mecA mutations altered BPR binding; other mutations may have mediated resistance by influencing interactions with other proteins. Multiple mecA mutations were associated with BPR resistance in MRSA. BPR resistance also developed in the strain lacking mecA, suggesting a role for chromosomal genes

A mecA-Negative Strain of Methicillin-Resistant Staphylococcus aureus with High-Level β-Lactam Resistance Contains Mutations in Three Genes▿

We previously generated a ceftobiprole-resistant Staphylococcus aureus strain after high inoculum serial passage of a mecA-negative variant of strain COL (R. Banerjee, M. Gretes, L. Basuino, N. Strynadka, and H. F. Chambers, Antimicrob. Agents Chemother. 52:2089-2096, 2008). Genome resequencing of this strain, CRB, revealed that it differs from its parent by five single-nucleotide polymorphisms in three genes, specifically, those encoding PBP4, a low-molecular-weight penicillin-binding protein, GdpP, a predicted signaling protein, and AcrB, a cation multidrug efflux transporter. CRB displayed resistance to a variety of β-lactams but was hypersusceptible to cefoxitin

Mapping the Active Site Helix-to-Strand Conversion of CxxxxC Peroxiredoxin Q Enzymes

Peroxiredoxins (Prx) make up a family of enzymes that reduce peroxides using a peroxidatic cysteine residue; among these, members of the PrxQ subfamily are proposed to be the most ancestral-like yet are among the least characterized. In many PrxQ enzymes, a second “resolving” cysteine is located five residues downstream from the peroxidatic Cys, and these residues form a disulfide during the catalytic cycle. Here, we describe three hyperthermophilic PrxQ crystal structures originally determined by the RIKEN structural genomics group. We reprocessed the diffraction data and conducted further refinement to yield models with <i>R</i>_free values lowered by 2.3–7.2% and resolution extended by 0.2–0.3 Å, making one, at 1.4 Å, one of the best resolved peroxiredoxins to date. Comparisons of two matched thiol and disulfide forms reveal that the active site conformational change required for disulfide formation involves a transition of ∼20 residues from a pair of α-helices to a β-hairpin and 3₁₀-helix. Each conformation has ∼10 residues with a high level of disorder providing slack that allows the dramatic shift, and the two conformations are anchored to the protein core by distinct nonpolar side chains that fill three hydrophobic pockets. Sequence conservation patterns confirm the importance of these and a few additional residues for function. From a broader perspective, this study raises the provocative question of how to make use of the valuable information in the Protein Data Bank generated by structural genomics projects but not described in the literature, perhaps remaining unrecognized and certainly underutilized