Production of Lactobacillus plantarum ghosts by conditional expression of a prophage-encoded holin.

Bacterial ghosts (BGs) are nonviable empty bacterial cell envelopes with intact cellular morphology and native surface structure. BGs made from pathogenic bacteria are used for biomedical and pharmaceutical applications. However, incomplete pathogenic cell inactivation during BG preparation raises safety concerns that could limit the intended use. Therefore, safer bacterial cell types are needed for BG production. Here, we produced BGs from the food-grade Gram-positive bacterium Lactobacillus plantarum TBRC 2-4 by conditional expression of a prophage-encoded holin (LpHo). LpHo expression was regulated using the pheromone-inducible pSIP system and LpHo was localized to the cell membrane. Upon LpHo induction, a significant growth retardation and a drastic decrease in cell viability were observed. LpHo-induced cells also showed membrane pores by scanning electron microscopy, membrane depolarization by flow cytometry, and release of nucleic acid contents in the cell culture supernatant, consistent with the role of LpHo as a pore-forming protein and L. plantarum ghost formation. The holin-induced L. plantarum BG platform could be developed as a safer alternative vehicle for the delivery of biomolecules.

Dual role of azo compounds in inhibiting Plasmodium falciparum adenosine deaminase and hemozoin biocrystallization.

Protein-ligand (GOLD) docking of the NCI compounds into the ligand-binding site of Plasmodium falciparum adenosine deaminase (PfADA) identified three most active azo compounds containing 4-[(4-hydroxy-2-oxo-1H-quinolin-3-yl) moiety. These compounds showed IC50 of 3.7-15.4 µM against PfADA, as well as inhibited the growth of P. falciparum strains 3D7 (chloroquine (CQ)-sensitive) and K1 (CQ-resistant) with IC50 of 1.8-3.1 and 1.7-3.6 µM, respectively. The identified compounds have structures similar to the backbone structure (4-N-(7-chloroquinolin-4-yl)) in CQ, and NSC45545 could mimic CQ by inhibiting the bioformation of hemozoin in parasitic food vacuole. The amount of in situ hemozoin in the ring-stage parasite was determined using a combination of synchrotron transmission Fourier transform infrared microspectroscopy and Principal Component Analysis. Stretching of the C-O bond of hemozoin propionate group measured at 1220-1210 cm-1 in untreated intraerythrocytic P. falciparum strains 3D7 and K1 was disappeared following treatment with 1.85 and 1.74 µM NSC45545, similar to those treated with 0.02 and 0.13 µM CQ, respectively. These findings indicate a novel dual function of 4-[(4-hydroxy-2-oxo-1H-quinolin-3-yl) azo compounds in inhibiting both PfADA and in situ hemozoin biocrystallization. These lead compounds hold promise for further development of new antimalarial therapeutics that could delay the onset of parasitic drug resistance.

BeQuIK (Biosensor Engineered Quorum Induced Killing): designer bacteria for destroying recalcitrant biofilms.

This opinion piece describes a new design for the remediation of recalcitrant biofilms. It builds on previous work to develop engineered E. coli that recognize quorum sensing signals from pathogens in a biofilm and secrete toxins in response. To solve the challenge of dilute signalling molecules, we propose to use nanobodies and enzymes displayed on the surface of the cells to localize them to the biofilm and degrade the extracellular polymeric substances, thus creating a solution with better 'seek and destroy' capabilities.

A label-free optical whole-cell Escherichia coli biosensor for the detection of pyrethroid insecticide exposure.

There is a growing need for low-cost, portable technologies for the detection of threats to the environment and human health. Here we propose a label-free, optical whole-cell Escherichia coli biosensor for the detection of 3-phenoxybenzoic acid (3-PBA), a biomarker for monitoring human exposure to synthetic pyrethroid insecticides. The biosensor functions like a competitive ELISA but uses whole-cells surface displaying an anti-3-PBA VHH as the detection element. When the engineered cells are mixed with 3-PBA-protein conjugate crosslinking that can be visually detected occurs. Free 3-PBA in samples competes with these crosslinks, leading to a detectable change in the output. The assay performance was improved by coloring the cells via expression of the purple-blue amilCP chromoprotein and the VHH expression level was reduced to obtain a limit of detection of 3 ng/mL. The optimized biosensor exhibited robust function in complex sample backgrounds such as synthetic urine and plasma. Furthermore, lyophilization enabled storage of biosensor cells for at least 90 days without loss of functionality. Our whole-cell biosensor is simple and low-cost and therefore has potential to be further developed as a screening tool for monitoring exposure to pyrethroids in low-resource environments.

Crystal structure of Plasmodium falciparum adenosine deaminase reveals a novel binding pocket for inosine.

Plasmodium falciparum (Pf), a malarial pathogen, can only synthesize purine nucleotides employing a salvage pathway because it lacks de novo biosynthesis. Adenosine deaminase (ADA), one of the three purine salvage enzymes, catalyzes the irreversible hydrolytic deamination of adenosine to inosine, which is further converted to GMP and AMP for DNA/RNA production. In addition to adenosine conversion, Plasmodium ADA also catalyzes the conversion of 5'-methylthioadenosine, derived from polyamine biosynthesis, into 5'-methylthioinosine whereas the human enzyme is not capable of this function. Here we report the crystal structure of a surface engineered PfADA at a resolution of 2.48 Å, together with results on kinetic studies of PfADA wild-type and active site variants. The structure reveals a novel inosine binding pocket linked to a distinctive PfADA substructure (residues 172-179) derived from a non-conserved gating helix loop (172-188) in Plasmodium spp. and other ADA enzymes. Variants of PfADA and human (h) ADA active site amino acids were generated in order to study their role in catalysis, including PfADA- Phe136, -Thr174, -Asp176, and -Leu179, and hADA-Met155, equivalent to PfADA-Asp176. PfADA-Leu179His showed no effect on kinetic parameters. However, kinetic results of PfADA-Asp176Met/Ala mutants and hADA-Met155Asp/Ala showed that the mutation reduced adenosine and 5'-methylthioadenosine substrate affinity in PfADA and kcat in hADA, thereby reducing catalytic efficiency of the enzyme. Phe136Leu mutant showed increased Km (>10-fold) for both substrates whereas Thr174Ile/Ala only affected 5'-methylthioadenosine binding affinity. Together, the structure with the novel inosine binding pocket and the kinetic data provide insights for rational design of inhibitors against PfADA.

Whole-Cell Biosensor with Tunable Limit of Detection Enables Low-Cost Agglutination Assays for Medical Diagnostic Applications.

Whole-cell biosensors can form the basis of affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing, but to date the detection of analytes such as proteins that cannot easily diffuse across the cell membrane has been challenging. Here we developed a novel biosensing platform based on cell agglutination using an E. coli whole-cell biosensor surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design to detect a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations using straightforward design rules and a mathematical model. Finally, we re-engineer our whole-cell biosensor for the detection of a medically relevant biomarker by the display of two different nanobodies against human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for field-testing in the developing world, emergency or disaster relief sites, as well as routine medical testing and personalized medicine.

Role of Plasmodium vivax Dihydropteroate Synthase Polymorphisms in Sulfa Drug Resistance.

Dihydropteroate synthase (DHPS) is a known sulfa drug target in malaria treatment, existing as a bifunctional enzyme together with hydroxymethyldihydropterin pyrophosphokinase (HPPK). Polymorphisms in key residues of Plasmodium falciparum DHPS (PfDHPS) have been characterized and linked to sulfa drug resistance in malaria. Genetic sequencing of P. vivax dhps (Pvdhps) from clinical isolates has shown several polymorphisms at the positions equivalent to those in the Pfdhps genes conferring sulfa drug resistance, suggesting a mechanism for sulfa drug resistance in P. vivax similar to that seen in P. falciparum To characterize the role of polymorphisms in the PvDHPS in sulfa drug resistance, various mutants of recombinant PvHPPK-DHPS enzymes were expressed and characterized. Moreover, due to the lack of a continuous in vitro culture system for P. vivax parasites, a surrogate P. berghei model expressing Pvhppk-dhps genes was established to demonstrate the relationship between sequence polymorphisms and sulfa drug susceptibility and to test the activities of PvDHPS inhibitors on the transgenic parasites. Both enzyme activity and transgenic parasite growth were sensitive to sulfadoxine to different degrees, depending on the number of mutations that accumulated in DHPS. Ki values and 50% effective doses were higher for mutant PvDHPS enzymes than the wild-type enzymes. Altogether, the study provides the first evidence of sulfa drug resistance at the molecular level in P. vivax Furthermore, the enzyme inhibition assay and the in vivo screening system can be useful tools for screening new compounds for their activities against PvDHPS.

Inhibitors of plasmodial serine hydroxymethyltransferase (SHMT): cocrystal structures of pyrazolopyrans with potent blood- and liver-stage activities.

Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These ligands showed activity (IC50/EC50 values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.

Structures of Plasmodium vivax serine hydroxymethyltransferase: implications for ligand-binding specificity and functional control.

Plasmodium parasites, the causative agent of malaria, rely heavily on de novo folate biosynthesis, and the enzymes in this pathway have therefore been explored extensively for antimalarial development. Serine hydroxymethyltransferase (SHMT) from Plasmodium spp., an enzyme involved in folate recycling and dTMP synthesis, has been shown to catalyze the conversion of L- and D-serine to glycine (Gly) in a THF-dependent reaction, the mechanism of which is not yet fully understood. Here, the crystal structures of P. vivax SHMT (PvSHMT) in a binary complex with L-serine and in a ternary complex with D-serine (D-Ser) and (6R)-5-formyltetrahydrofolate (5FTHF) provide clues to the mechanism underlying the control of enzyme activity. 5FTHF in the ternary-complex structure was found in the 6R form, thus differing from the previously reported structures of SHMT-Gly-(6S)-5FTHF from other organisms. This suggested that the presence of D-Ser in the active site can alter the folate-binding specificity. Investigation of binding in the presence of D-Ser and the (6R)- or (6S)-5FTHF enantiomers indicated that both forms of 5FTHF can bind to the enzyme but that only (6S)-5FTHF gives rise to a quinonoid intermediate. Likewise, a large surface area with a highly positively charged electrostatic potential surrounding the PvSHMT folate pocket suggested a preference for a polyglutamated folate substrate similar to the mammalian SHMTs. Furthermore, as in P. falciparum SHMT, a redox switch created from a cysteine pair (Cys125-Cys364) was observed. Overall, these results assert the importance of features such as stereoselectivity and redox status for control of the activity and specificity of PvSHMT.

Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene.

Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is similar to other plasmodial enzymes, albeit with lower catalytic activity but better tolerance of acidic pH. Importantly, the PoDHFR from Thai isolate EU266602 remains sensitive to the antimalarials pyrimethamine and cycloguanil, in contrast to P. falciparum and P. vivax isolates where resistance to these drugs is widespread.