Oral streptococci subvert the host innate immune response through hydrogen peroxide.

In periodontal health, oral streptococci constitute up to 80% of the plaque biofilm. Yet, destructive inflammatory events of the periodontium are rare. This observation suggests that oral streptococci may possess mechanisms to co-exist with the host. However, the mechanisms employed by oral streptococci to modulate the innate immune response have not been well studied. One of the key virulence factors produced by oral streptococci is hydrogen peroxide (H2O2). In mammalian cells, H2O2 triggers the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a key pathway mediating antioxidant defence. This study aimed to determine (1) if H2O2 producing oral streptococci activated the Nrf2 pathway in macrophages, and (2) if the activation of Nrf2 influenced the innate immune response. We found that oral streptococci downregulated the innate immune response in a H2O2 dependent manner through the activation of the Nrf2. The activation of the Nrf2 signalling pathway led to the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB), the key transcription factor regulating pro-inflammatory response. This study showed for the first time that oral streptococci are unlikely passive bystanders but could play an active role in the maintenance of periodontal health by preventing overt inflammation.

Features of the Streptomyces hygroscopicus HtpG reveal how partial geldanamycin resistance can arise with mutation to the ATP binding pocket of a eukaryotic Hsp90.

Much attention is focused on the benzoquinone ansamycins as anticancer agents, with several derivatives of the natural product geldanamycin (GdA) now in clinical trials. These drugs are selective inhibitors of Hsp90, a molecular chaperone vital for many of the activities that drive cancer progression. Mutational changes to their interaction site, the extremely conserved ATP binding site of Hsp90, would mostly be predicted to inactivate the chaperone. As a result, drug resistance should not arise readily this way. Nevertheless, Streptomyces hygroscopicus, the actinomycete that produces GdA, has evolved an Hsp90 family protein (HtpG) that lacks GdA binding. It is altered in certain of the highly conserved amino acids making contacts to this antibiotic in crystal structures of GdA bound to eukaryotic forms of Hsp90. Two of these amino acid changes, located on one side of the nucleotide-binding cleft, weakened GdA/Hsp90 binding and conferred partial GdA resistance when inserted into the endogenous Hsp90 of yeast cells. Crystal structures revealed their main effect to be a weakening of interactions with the C-12 methoxy group of the GdA ansamycin ring. This is the first study to demonstrate that partial GdA resistance is possible by mutation within the ATP binding pocket of Hsp90.

Atypical caseinolytic protease homolog from Plasmodium falciparum possesses unusual substrate preference and a functional nuclear localization signal.

Although ATP-dependent caseinolytic protease (Clp) complexes are important for regulating the pathogenicity, survival, and development of many pathogens, their physiological roles in the pathogenicity of malarial parasites remain unknown. This study reports the cloning, authentication, and characterization of a putative Clp protease subunit from Plasmodium falciparum (PfClpP). Heterologous expression studies showed that signal peptide hindered the soluble expression of the full-length PfClpP. Biochemical analyses of the recombinant PfClpP showed that it did not cleave the known ClpP substrate, succinyl-leucine-tyrosine-7-amido-4-methylcoumarin hydrochloride (AMC). Instead, PfClpP readily hydrolyzed a different substrate, glycine-arginine-AMC. The distinctive substrate preference of PfClpP suggests structural uniqueness in its substrate-binding sites that might be exploitable in anti-malarial drug development. Whether PfClpP resembles most eukaryotic ClpPs in being localized to the mitochondria and chloroplasts was also investigated using a mammalian surrogate host system. The results observed showed that green-fluorescence protein tagged PfClpP proteins were localized to the nucleus. PfClpP may have a unique and specialized role in the plasmodial nucleus. Taken together, this study has shown that PfClpP has a unique peptide cleavage function that is localized at the plasmodial nucleus, probably positioned to elicit a regulatory role in the parasite's pathogenicity.

Structural basis of the radicicol resistance displayed by a fungal hsp90.

Heat shock protein 90 (Hsp90) is a promising cancer drug target, as multiple oncogenic proteins are destabilized simultaneously when it loses its activity in tumor cells. Highly selective Hsp90 inhibitors, including the natural antibiotics geldanamycin (GdA) and radicicol (RAD), inactivate this essential molecular chaperone by occupying its nucleotide binding site. Often cancer drug therapy is compromised by the development of resistance, but a resistance to these Hsp90 inhibitors should not arise readily by mutation of those amino acids within Hsp90 that facilitate inhibitor binding, as these are required for the essential ATP binding/ATPase steps of the chaperone cycle and are tightly conserved. Despite this, the Hsp90 of a RAD-producing fungus is shown to possess an unusually low binding affinity for RAD but not GdA. Within its nucleotide binding site a normally conserved leucine is replaced by isoleucine, though the chaperone ATPase activity is not severely affected. Inserted into the Hsp90 of yeast, this conservative leucine to isoleucine substitution recreated this lowered affinity for RAD in vitro. It also generated a substantially enhanced resistance to RAD in vivo. Co-crystal structures reveal that the change to isoleucine is associated with a localized increase in the hydration of an Hsp90-bound RAD but not GdA. To the best of our knowledge, this is the first demonstration that it is possible for Hsp90 inhibitor resistance to arise by subtle alteration to the structure of Hsp90 itself.

Pfnek3 functions as an atypical MAPKK in Plasmodium falciparum.

Eukaryotes generally rely on signal transduction by mitogen-activated protein kinases (MAPKs) for activating their regulatory pathways. However, the presence of a complete MAPK cascade in Plasmodium falciparum is debatable because a search of the entire genome did not portray known MAPK kinase (MAPKK) sequences. Via homology PCR experiments, only two copies of plasmodial MAPK homologues (Pfmap1 and Pfmap2) have been identified but their upstream activators remain unknown. In an earlier experiment, Pfnek3 was found to be an unusual activator of Pfmap2 in in vitro experiments, despite its molecular identity as a malarial protein kinase from the NIMA (Never in Mitosis, Aspergillus) family. In this study, the role of Pfnek3 as a likely upstream MAPKK is defined through molecular and biochemical characterization. Since a previous report proposes a TSH motif as an activation site of Pfmap2, its site-directed mutants, T290A, S291A, and H292K were constructed to elucidate the involvement of Pfnek3 in phosphorylating and activating Pfmap2 in a battery of kinase assays. The results suggested that residue T290 is the site of phosphorylation by Pfnek3. This supposition was further supported by liquid chromatography mass spectrometry. Although P. falciparum does not appear to possess a conventional MAPK cascade, they may rely on other kinases such as Pfnek3 to carry out similar phosphorylation to activate its signaling pathways.

A relevant in vitro eukaryotic live-cell system for the evaluation of plasmodial protein localization.

Understanding the functional genomics and proteomics of plasmodia underpins the development of new approaches to antimalarial chemotherapy. Although genome databanks (e.g. PlasmoDB) and biocomputing tools (e.g. PlasMit, PlasmoAP, PATS) are useful in providing a global albeit predictive view of the myriad of about 5000 genes, only 40% are annotated, with few cases of endorsed subcellular localizations of the corresponding proteins in animal models. Progress in plasmodial protein trafficking has been hampered by the lack of a simple yet reliable method for studying subcellular localization of plasmodial proteins. In this study, we have used a combination of fluorescent markers, organelle-specific probes, phase contrast microscopy, and confocal microscopy to locate a selection of signal peptides from 10 plasmodial proteins in CHO-K1 cells. These eukaryotic cells serve as an in vitro living system for studying the cellular destinations of four mitochondrial-targeted TCA cycle proteins (citrate synthase, CS; isocitrate dehydrogenase, ICDH; branched chain alpha-keto-acid dehydrogenase E1alpha subunit, BCKDH; succinate dehydrogenase flavoprotein-subunit, SDH), two nuclear-targeted proteins (histone deacetylase, HDAC; RNA polymerase, RPOL), two apicoplast-targeted proteins (pyruvate kinase 2, PK2; glutamate dehydrogenase, GDH), and two cytoplasmic resident proteins (malate dehydrogenase, MDH; glycerol kinase, GK). The respective localizations of these malarial proteins have complied with the selected molecular targets, viz. mitochondrial, nuclear and cytoplasmic. Interestingly, MDH that is widely known to be resident in eukaryotic mitochondria was found to be cytoplasmic, probably due to the absence of molecular target sequences. Since the localization of plasmodial proteins is central to the authentication of their pathophysiological roles, this experimental system will serve as a useful a priori approach.

Characterization of amino acid variation at strategic positions in parasite and human proteases for selective inhibition of falcipains in Plasmodium falciparum.

Falcipains (FP) of Plasmodium falciparum are important virulence factors marked as potential targets for antimalarial drug discovery. In this study, the previously uncharacterized fp2B (PF11_0161) was shown to be highly expressed as an active enzyme during the erythrocytic stage. With three related proteases in the FP family and the existence of human homologues, it is prudent to identify clusters of residues unique to the parasite proteases that can be targeted selectively for drug design. Using bioinformatic tools, we have carefully mapped out a highly conserved and unique region constituted by I85, S149, and A151 in the plasmodial proteases that can influence the development of compounds capable of inhibiting the entire FP family. Taking drug interactions with the human homologues into consideration, these residues in FP2B were replaced with the cognate residues found in human cathepsin L (catL) for evaluation. Despite the high sequence similarity between the FP2 isozymes (97.5%), FP2B is found to be more tolerant to amino acid substitution at position 149 than FP2A. This structural disparity implied that residues mediating peptide substrate interactions are not fully conserved across the FP family and warrant attention in the design and evaluation of protease inhibitors focused on the FPs. The simultaneous substitution of the neighboring residues (I85 or A151) rendered the double mutants (S149A/I85M and S149A/A151D) completely inactive. Significantly, the mutations did not result in 'catL-like' specificity, suggesting that substrate-based inhibitors could be rationally designed against these important parasite-specific structural determinants.

Differences in biochemical properties of the Plasmodial falcipain-2 and berghepain-2 orthologues: implications for in vivo screens of inhibitors.

Falcipain-2A, the cysteine protease of Plasmodium falciparum has been proposed as a good drug target. This study evaluated the suitability of Plasmodium berghei as the animal model and reports the first functional expression and characterization of the falcipain-2A orthologue, berghepain-2. Comparative studies revealed that the orthologues exhibited different biochemical properties. Berghepain-2 demonstrated optimal activity at a narrower pH optima of 5.5-6 and a lack of preference for substrates with leucine at position 2. Mutagenesis studies revealed roles for residues Val63 and Arg230 of berghepain-2 in contributing to its distinctive biochemical properties. This warrants re-evaluation of employing P. berghei as the murine model for the in vivo screening of falcipain-2A inhibitors. More importantly, these findings stress the underlying importance of establishing the functionality of relevant genes of P. falciparum with concomitant relevance to its murine counterpart prior to its use as the animal model for the screening of potential antimalarials.

Homology modeling and mutagenesis analyses of Plasmodium falciparum falcipain 2A: implications for rational drug design.

The hemoglobin-degrading cysteine proteases falcipains of the malaria parasite Plasmodium falciparum are regarded as potential drug targets. Despite their obvious importance in the virulence of malaria, these proteases remain poorly characterized at the structural levels. Using a bioinformatic and site-directed mutagenesis approach, residues essential for the structure and function of FP2A are elucidated in this study. In total, nine mutants of FP2A were constructed to test the proposed importance of seven different amino acid residues. These recombinant protease mutants were solubly expressed in Escherichia coli and purified by affinity chromatography for enzymatic assessments. Notably, substitutions at positions C99 and C119 induce structural alterations and led to significant reduction in enzyme activity (>97%). The analyses also validated the role of the active triad comprising of C42, H174, and N204 in catalysis and identified a serine at position 149 which is required for specific peptide substrate interactions. The parasite-specific residues, C99, C119, and S149, represent potential sites for differential targeting, since the corresponding residues are absent in the human host's isozymes.

Deacetoxycephalosporin C synthase isozymes exhibit diverse catalytic activity and substrate specificity.

The biosynthesis of cephalosporins involving a thiozolidine ring expansion is catalyzed by deacetoxycephalosporin C synthase (DAOCS). In this study, three DAOCS isozymes were cloned and expressed as active enzymes together with Streptomyces jumonjinensis DAOCS that was newly isolated and partially characterized. The enzymes showed excellent substrate conversion for penicillin G, phenethicillin, ampicillin and carbenicillin, but they were less effective in the ring expansion of penicillin V, amoxicillin and metampicillin. Streptomyces clavuligerus DAOCS was the most active among the recombinant enzymes. The results also showed that truncation of 20 amino acids at the C-terminus of the Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase polypeptide did not affect penicillin ring expansion.

Soluble expression of a functionally active Plasmodium falciparum falcipain-2 fused to maltose-binding protein in Escherichia coli.

Falcipain-2 (fp2) is a hemoglobinase required for supplying peptides and amino acids for the proliferation of Plasmodium falciparum in blood. The prospect of circumventing its activity thereby serves as a potential strategy for mining drugs for anti-malarial therapy. However, to date, efforts to express soluble and active fp2 in Escherichia coli have been futile. To overcome this problem, fp2 was expressed under an array of conditions including the exploitation of multiple gene constructs in eukaryotic and prokaryotic hosts. A series of experiments led to the finding that the placement of maltose-binding protein (MBP) before the fp2 mature domain was best in availing the soluble expression of the protease. The results also indicate that the prodomain impaired the bacterial expression of the protease and the amino acid residues at the N-terminal segment of mature fp2 can have a significant effect on the folding and solubility of the enzyme. The overexpressed MBP-fp2 fusion protein was purified and shown to be functionally active, providing a very useful alternative to the use of resolubilized enzyme for future study of structure and function of fp2.