Ellen Yeh

Chemical Rescue of Malaria Parasites Lacking an Apicoplast Defines Organelle Function in Blood-Stage Plasmodium falciparum

The only essential function of a unique plastid organelle, the apicoplast, in blood-stage P. falciparum is the production of isoprenoid precursors.

Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis

Enzymatic incorporation of chlorine, bromine or iodine atoms occurs during the biosynthesis of more than 4,000 natural products. Halogenation can have significant consequences for the bioactivity of these products so there is great interest in understanding the biological catalysts that perform these reactions. Enzymes that halogenate unactivated aliphatic groups have not previously been characterized. Here we report the activity of five proteins—CmaA, CmaB, CmaC, CmaD and CmaE—in the construction of coronamic acid (CMA; 1-amino-1-carboxy-2-ethylcyclopropane), a constituent of the phytotoxin coronatine synthesized by the phytopathogenic bacterium Pseudomonas syringae. CMA derives from l-allo-isoleucine, which is covalently attached to CmaD through the actions of CmaA, a non-ribosomal peptide synthetase module, and CmaE, an unusual acyltransferase. We show that CmaB, a member of the non-haem Fe2+, α-ketoglutarate-dependent enzyme superfamily, is the first of its class to show halogenase activity, chlorinating the γ-position of l-allo-isoleucine. Another previously undescribed enzyme, CmaC, catalyses the formation of the cyclopropyl ring from the γ-Cl-l-allo-isoleucine product of the CmaB reaction. Together, CmaB and CmaC execute γ-halogenation followed by intramolecular γ-elimination, in which biological chlorination is a cryptic strategy for cyclopropyl ring formation.

Type II Thioesterase Restores Activity of a NRPS Module Stalled with an Aminoacyl-S-enzyme that Cannot Be Elongated

Nonribosomal peptide synthetases (NRPSs) carry out the biosynthesis of numerous peptide natural products, including many with important clinical applications. The NRPS, organized into a series of modules, is an efficient, high-fidelity assembly line for the production of a particular peptide. Each module consists of domains, whose activities contribute to the accuracy of these assembly-line systems. The activation (A) domain uses ATP to selectively load an amino acid onto the module through formation of a thioester bond to the pantetheine arm of the thiolation (T) domain. Peptide-bond formation, catalyzed by the condensation (C) domain, is stringent for both sidechain identity and stereochemistry. The C domain accepts an aminoacylor peptidylthioester from the preceding module for nucleophilic addition by the amine of the loaded amino acid; this generates the elongated peptide attached to the downstream module. The peptide product is synthesized one amino acid at a time until it reaches the final module. There, the fully synthesized chain is released by a type I thioesterase (TEI), the terminal domain of the NRPS assembly. Despite the high fidelity of this process, an error in any step of the assembly-line synthesis severely impacts the efficiency of the system and creates a bottleneck that results in a buildup of unprocessed intermediates. For example, an error by the A domain, which can load amino acids other than that normally accepted by the C domain, would prevent peptide-bond formation. The loaded module would be blocked until the incorrect amino acid was hydrolyzed (Figure 1). A type II thioesterase (TEII), whose gene is associated with the gene cluster of many NRPSs and related polyketide synthases (PKSs), improves the efficiency of product formation in these systems and has been proposed to edit modules through hydrolysis of acyl groups. In the surfactin NRPS, TEII was shown to regenerate misacylated modules resulting from priming of the apomodule with acyl-CoA groups. In this study we provide evidence to expand the editing function of TEIIs to include restoring the activity of modules stalled by loaded amino acids that cannot be processed. N-acetylcysteamine (SNAC) thioesters have been used previously to assay NRPS domain activities. 13–15] Hydrolysis of SNAC substrates was used here to explore the specificity of the TEII from the tyrocidine biosynthetic operon, TycF. TycF accepted a broad variety of aminoacyl-SNACs of different sidechain identity and stereochemistry with a 20-fold kcat/Km range between the mostand least-active substrate (Table 1). A series of peptidyl-SNACs derived from the tyrocidine sequence was

Immediate Incubation Reduces Indeterminate Results for QuantiFERON-TB Gold In-Tube Assay

ABSTRACT In vitro gamma interferon release assays (IGRAs) are increasingly used as an alternative to the traditional tuberculin skin test for the diagnosis of latent Mycobacterium tuberculosis infection. Evaluation of the QuantiFERON-TB Gold in-tube assay (QFT-IT) prior to large-scale implementation at the Stanford Hospital and Clinics for a health care worker screening program revealed a critical preanalytical factor affecting the results. We found that incubation delay significantly increased the frequency of indeterminate results. In this study, QFT-IT was performed with samples from healthy volunteers, and replicate tubes were incubated at 37°C either immediately or after a delay at room temperature for 6 and 12 h. No indeterminate results (0/41) were seen when the assay was performed with immediate incubation. Incubation delays of 6 and 12 h yielded indeterminate results at rates of 10% (2/20) (P = 0.10) and 17.1% (7/41) (P = 0.01), respectively. The increased rate of indeterminate results was due to a decrease in the mean values for the mitogen-nil tubes when incubation was delayed for 6 h (P = 0.004) and 12 h (P < 0.001). The rates of concordance of positive or negative results obtained following immediate incubation and following 6- and 12-h delays were 77.8% (14/18) and 79.4% (27/34), respectively. Subsequent implementation of the immediate incubation procedure in our screening program for 14,830 health care workers yielded an indeterminate result rate of 0.36% over a period of 12 months, a significant improvement over the reported rates of 5 to 40% for QFT-IT. We conclude that immediate incubation of QFT-IT tubes is an effective way to minimize indeterminate results. The effect of incubation delay on the accuracy of QFT-IT remains to be determined.

Real-Time PCR Testing for mecA Reduces Vancomycin Usage and Length of Hospitalization for Patients Infected with Methicillin-Sensitive Staphylococci

ABSTRACT Nucleic acid amplification tests (NAATs) have revolutionized infectious disease diagnosis, allowing for the rapid and sensitive identification of pathogens in clinical specimens. Real-time PCR testing for the mecA gene (mecA PCR), which confers methicillin resistance in staphylococci, has the added potential to reduce antibiotic usage, improve clinical outcomes, lower health care costs, and avoid emergence of drug resistance. A retrospective study was performed to identify patients infected with methicillin-sensitive staphylococcal isolates who were receiving vancomycin treatment when susceptibility results became available. Vancomycin treatment and length of hospitalization were compared in these patients for a 6-month period before and after implementation of mecA PCR. Among 65 and 94 patients identified before and after mecA PCR, respectively, vancomycin usage (measured in days on therapy) declined from a median of 3 days (range, 1 to 44 days) in the pre-PCR period to 1 day (range, 0 to 18 days) in the post-PCR period (P < 0.0001). In total, 38.5% (25/65) of patients were switched to β-lactam therapy in the pre-PCR period, compared to 61.7% (58/94) in the post-PCR period (P = 0.004). Patient hospitalization days also declined from a median of 8 days (range, 1 to 47 days) in the pre-PCR period to 5 days (range, 0 to 42 days) in the post-PCR period (P = 0.03). Real-time PCR testing for mecA is an effective tool for reducing vancomycin usage and length of stay of hospitalized patients infected with methicillin-sensitive staphylococci. In the face of ever-rising health care expenditures in the United States, these findings have important implications for improving outcomes and decreasing costs.

A chemical rescue screen identifies a Plasmodium falciparum apicoplast inhibitor targeting MEP isoprenoid precursor biosynthesis

ABSTRACT The apicoplast is an essential plastid organelle found in Plasmodium parasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the “Malaria Box” library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stage Plasmodium falciparum growth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations in P. falciparum IspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activity in vitro with a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action.

Preferential lower respiratory tract infection in swine-origin 2009 A(H1N1) influenza.

We report a case of 2009 influenza A(H1N1) virus infection in which virus was detected predominantly in specimens from the lower respiratory tract but was absent or at very low levels in nasopharyngeal swab samples. This presentation suggests that, in certain hosts or for particular variants of 2009 A(H1N1) virus, the lower respiratory tract may be the preferred site of infection.

The apicoplast: now you see it, now you don’t

Parasites such as Plasmodium and Toxoplasma possess a vestigial plastid homologous to the chloroplasts of algae and plants. The plastid (known as the apicoplast; for apicomplexan plastid) is non-photosynthetic and very much reduced, but has clear endosymbiotic ancestry including a circular genome that encodes RNAs and proteins and a suite of bacterial biosynthetic pathways. Here we review the initial discovery of the apicoplast, and recount the major new insights into apicoplast origin, biogenesis and function. We conclude by examining how the apicoplast can be removed from malaria parasites in vitro, ultimately completing its reduction by chemical supplementation.

Hair Sheep Blood, Citrated or Defibrinated, Fulfills All Requirements of Blood Agar for Diagnostic Microbiology Laboratory Tests

Background Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis [1], [2]. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies. Methods and Findings Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test. Conclusions The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost-prohibitive commercial sheep blood, offers the opportunity to dramatically improve the safety and accuracy of laboratory diagnosis of pathogenic bacteria in resource-poor countries.

The Prenylated Proteome of Plasmodium falciparum Reveals Pathogen-specific Prenylation Activity and Drug Mechanism-of-action

Plasmodium parasites contain several unique membrane compartments in which prenylated proteins may play important roles in pathogenesis. Protein prenylation has also been proposed as an antimalarial drug target because farnesyltransferase inhibitors cause potent growth inhibition of blood-stage Plasmodium. However, the specific prenylated proteins that mediate antimalarial activity have yet to be identified. Given the potential for new parasite biology and elucidating drug mechanism-of-action, we performed a large-scale identification of the prenylated proteome in blood-stage P. falciparum parasites using an alkyne-labeled prenyl analog to specifically enrich parasite prenylated proteins. Twenty high-confidence candidates were identified, including several examples of pathogen-specific prenylation activity. One unique parasite prenylated protein was FYVE-containing coiled-coil protein (FCP), which is only conserved in Plasmodium and related Apicomplexan parasites and localizes to the parasite food vacuole. Targeting of FCP to this parasite-specific compartment was dependent on prenylation of its CaaX motif, as mutation of the prenylation site caused cytosolic mislocalization. We also showed that PfRab5b, which lacks C-terminal cysteines that are the only known site of Rab GTPase modification, is prenylated. Finally, we show that the THQ class of farnesyltransferase inhibitors abolishes FCP prenylation and causes its mislocalization, providing the first demonstration of a specific prenylated protein disrupted by antimalarial farnesyl transferase inhibitors. Altogether, these findings identify prenylated proteins that reveal unique parasite biology and are useful for evaluating prenyltransferase inhibitors for antimalarial drug development.