Elevated tyrosine results in the cytosolic retention of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in Arabidopsis thaliana.

The shikimate pathway plays a central role in the biosynthesis of aromatic amino acids and specialized metabolites in plants. The first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) serves as a key regulatory point for the pathway in various organisms. These enzymes are important in regulating the shikimate pathway in multiple microbial systems. The mechanism of regulation of DAHPS is poorly understood in plants, and the role of tyrosine (Tyr) with respect to the three DAHPS isozymes from Arabidopsis thaliana was investigated. In vitro enzymatic analyses established that Tyr does not function as an allosteric regulator for the A. thaliana DAHPS isozymes. In contrast, Arabidopsis T-DNA insertional mutants for the DAHPS1 locus, dahps1, are hypersensitive to elevated Tyr. Tyr hypersensitivity can be reversed with tryptophan and phenylalanine supplementation, indicating that Tyr is affecting the shikimate pathway flux in the dahps1 mutant. Tyr treatment of Arabidopsis seedlings showed reduced accumulation of overexpressed DAHPS2 in the chloroplast. Further, bimolecular fluorescence complementation studies revealed that DAHPS2 interacts with a 14-3-3 protein in the cytosol, and this interaction is enhanced with Tyr treatment. This interaction with 14-3-3 may retain DAHPS2 in the cytosol, which prevents its ability to function in the chloroplast with elevated Tyr.

Role of Hsp90 in Plasmodium falciparum Malaria.

Studies in mammalian and yeast chaperone systems have significantly advanced our understanding of the biochemistry of heat shock protein 90 (Hsp90), particularly as it pertains to structural details, interaction regulation, and function. Harnessing this body of knowledge, parasitologists have made tremendous steps in understanding the role of the Hsp90 chaperone machine in protozoal parasites, especially in malaria. Heat shock proteins are a crucial part of the signaling events involved in malaria parasite acclimatization in the host. Even though studies of the role of Hsp90 in malaria have been thorough and discovered some of the most intricate details of the utility of the Hsp90 interactome for pathogen-host interactions, the full and elaborate extent of the role of Hsp90 homologs in malaria remains to be uncovered. This chapter discusses the current knowledge about the biochemistry and cellular role of Hsp90 in Plasmodium falciparum malaria. In addition, the involvement of both parasitic and host Hsp90s by the parasite in the infected cell is discussed.

Characterization of an Enterococcus gallinarum Isolate Carrying a Dual vanA and vanB Cassette.

The ability of vancomycin resistance determinants to be horizontally transferred within enterococci species is a concern. Identification and characterization of vancomycin-resistant enterococci (VRE) in a clinical isolate have a significant impact on infection control practices. In this study, we describe a clinical isolate of Enterococcus gallinarum exhibiting high-level resistance to vancomycin and teicoplanin. The genetic characterization of this isolate showed the presence of vanA and vanB genes in addition to the naturally carried vanC gene. vanA was identified on pA6981, a 35,608-bp circular plasmid with significant homology to plasmid pS177. The vanB operon was integrated into the bacterial chromosome and showed a high level of homology to previously reported Tn1549 and Tn5382. To the best of our knowledge, this is the first report of E. gallinarum carrying both vanA and vanB operons, indicating the importance of identifying the vancomycin resistance mechanism in non-E. faecium and non-E. faecalis enterococcal species.

Hsp90 inhibitors as new leads to target parasitic diarrheal diseases.

Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 µM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.

Outbreak of vancomycin-susceptible Enterococcus faecium containing the wild-type vanA gene.

Accurate detection of vancomycin-resistant enterococci (VRE) is essential in preventing transmission in health care settings. Chromogenic media are widely used for screening VRE because of fast turnaround times (TAT) and high sensitivity. We report an outbreak of Enterococcus faecium bearing vanA yet susceptible to vancomycin (vancomycin-variable Enterococcus [VVE]). Between October 2009 to March 2011, clinical and screening specimens (n=14,747) were screened for VRE using VRE-selective medium and/or PCR. VVE isolates were genotyped to determine relatedness. Plasmids from these isolates were characterized by sequencing. Overall, 52 VVE isolates were identified, comprising 15% of all VRE isolates identified. Isolates demonstrated growth on Brilliance VRE agar (Oxoid) at 24 h of incubation but did not grow on brain heart infusion agar with 6 µg/ml vancomycin (Oxoid) or bile esculin azide agar with 6 µg/ml vancomycin (Oxoid) and were susceptible to vancomycin. Genotyping of 20 randomly selected VVE isolates revealed that 15/20 were identical, while 5 were highly related. PCR of the VVE transposon confirmed the presence of vanHAXY gene cluster; however, vanS (sensor) and vanR (regulator) genes were absent. The outbreak was controlled through routine infection control measures. We report an emergence of a fit strain of E. faecium containing vanA yet susceptible to vancomycin. Whether this new strain represents VRE has yet to be determined; however, unique testing procedures are required for reliable identification of VVE.

Vancomycin-variable Enterococcus faecium: in vivo emergence of vancomycin resistance in a vancomycin-susceptible isolate.

We report the emergence of vancomycin resistance in a patient colonized with a vanA-containing, vanRS-negative isolate of Enterococcus faecium which was initially vancomycin susceptible. This is a previously undescribed mechanism of drug resistance with diagnostic and therapeutic implications.

A suppressor screen of the chimeric AtCNGC11/12 reveals residues important for intersubunit interactions of cyclic nucleotide-gated ion channels.

To investigate the structure-function relationship of plant cyclic nucleotide-gated ion channels (CNGCs), we identified a total of 29 mutant alleles of the chimeric AtCNGC11/12 gene that induces multiple defense responses in the Arabidopsis (Arabidopsis thaliana) mutant, constitutive expresser of PR genes22 (cpr22). Based on computational modeling, two new alleles, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), were identified as counterparts of human CNGA3 (a human CNGC) mutants. Both mutants lost all cpr22-mediated phenotypes. Transient expression in Nicotiana benthamiana as well as functional complementation in yeast (Saccharomyces cerevisiae) showed that both AtCNGC11/12:G459R and AtCNGC11/12:R381H have alterations in their channel function. Site-directed mutagenesis coupled with fast-protein liquid chromatography using recombinantly expressed C-terminal peptides indicated that both mutations significantly influence subunit stoichiometry to form multimeric channels. This observation was confirmed by bimolecular fluorescence complementation in planta. Taken together, we have identified two residues that are likely important for subunit interaction for plant CNGCs and likely for animal CNGCs as well.

Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009-2013).

BACKGROUND: Bangladesh is a malaria hypo-endemic country sharing borders with India and Myanmar. Artemisinin combination therapy (ACT) remains successful in Bangladesh. An increase of artemisinin-resistant malaria parasites on the Thai-Cambodia and Thai-Myanmar borders is worrisome. K13 propeller gene (PF3D7_1343700 or PF13_0238) mutations have been linked to both in vitro artemisinin resistance and in vivo slow parasite clearance rates. This group undertook to evaluate if mutations seen in Cambodia have emerged in Bangladesh where ACT use is now standard for a decade. METHODS: Samples were obtained from Plasmodium falciparum-infected malaria patients from Upazila health complexes (UHC) between 2009 and 2013 in seven endemic districts of Bangladesh. These districts included Khagrachari (Matiranga UHC), Rangamati (Rajasthali UHC), Cox's Bazar (Ramu and Ukhia UHC), Bandarban (Lama UHC), Mymensingh (Haluaghat UHC), Netrokona (Durgapur and Kalmakanda UHC), and Moulvibazar (Sreemangal and Kamalganj UHC). RESULTS: Out of 296 microscopically positive P. falciparum samples, 271 (91.6%) were confirmed as mono-infections by both real-time PCR and nested PCR. The K13 propeller gene from 253 (93.4%) samples was sequenced bi-directionally. One non-synonymous mutation (A578S) was found in Bangladeshi clinical isolates. The A578S mutation was confirmed and lies adjacent to the C580Y mutation, the major mutation causing delayed parasite clearance in Cambodia. Based on computational modeling A578S should have a significant effect on tertiary structure of the protein. CONCLUSION: The data suggest that P. falciparum in Bangladesh remains free of the C580Y mutation linked to delayed parasite clearance. However, the mutation A578S is present and based on structural analysis could affect K13 gene function. Further in vivo clinical studies are required to validate the effect of this mutation.

Harmine is a potent antimalarial targeting Hsp90 and synergizes with chloroquine and artemisinin.

Previous studies have shown an antimalarial effect of total alkaloids extracted from leaves of Guiera senegalensis from Mali in West Africa. We independently observed that the beta-carboline alkaloid harmine obtained from a natural product library screen inhibited Plasmodium falciparum heat shock protein 90 (PfHsp90) ATP-binding domain. In this study, we confirmed harmine-PfHsp90-specific affinity using surface plasmon resonance analysis (dissociation constant [K(d)] of 40 µM). In contrast, the related compound harmalol bound human Hsp90 (HsHsp90) (K(d) of 224 µM) more tightly than PfHsp90 (K(d) of 7,010 µM). Site-directed mutagenesis revealed that Arg98 in PfHsp90 is essential for harmine selectivity. In keeping with our model indicating that Hsp90 inhibition affords synergistic combinations with existing antimalarials, we demonstrated that harmine potentiates the effect of chloroquine and artemisinin in vitro and in the Plasmodium berghei mouse model. These findings have implications for the development of novel therapeutic combinations that are synergistic with existing antimalarials.

Whole-genome sequence of Streptococcus pseudopneumoniae isolate IS7493.

Streptococcus pseudopneumoniae is a member of the viridans group streptococci (VGS) whose pathogenic significance is unclear. We announce the complete genome sequence of S. pseudopneumoniae IS7493. The genome sequence will assist in the characterization of this new organism and facilitate the development of accurate diagnostic assays to distinguish it from Streptococcus pneumoniae and Streptococcus mitis.

Novel mutations in a patient isolate of Streptococcus agalactiae with reduced penicillin susceptibility emerging after long-term oral suppressive therapy.

Penicillin nonsusceptibility has been demonstrated in group B streptococci (GBS), but there is limited information regarding mechanisms of resistance. We report a case of GBS with reduced susceptibility to penicillin emerging after long-term suppressive oral penicillin therapy for a prosthetic joint infection. Molecular characterization of the isolate before and after long-term penicillin therapy revealed 5 mutations in the ligand-binding regions of PBP1a, -2a, and -2x not previously reported in GBS.

Artesunate misuse and Plasmodium falciparum malaria in traveler returning from Africa.

Plasmodium falciparum malaria developed in an African-born traveler who returned to Canada after visiting Nigeria. While there, she took artesunate prophylactically. Isolates had an elevated 50% inhibitory concentration to artemisinin, artesunate, and artemether, compared with that of other African isolates. Inappropriate use of artemisinin derivatives can reduce P. falciparum susceptibility.

Importance of the alphaC-helix in the cyclic nucleotide binding domain for the stable channel regulation and function of cyclic nucleotide gated ion channels in Arabidopsis.

The involvement of cyclic nucleotide gated ion channels (CNGCs) in the signal transduction of animal light and odorant perception is well documented. Although plant CNGCs have recently been revealed to mediate multiple stress responses and developmental pathways, studies that aim to elucidate their structural and regulatory properties are still very much in their infancy. The structure-function relationship of plant CNGCs was investigated here by using the chimeric Arabidopsis AtCNGC11/12 gene that induces multiple defence responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22) for the identification of functionally essential residues. A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12. One of these mutants, suppressor S58 possesses a single amino acid substitution, arginine 557 to cysteine, in the alphaC-helix of the cyclic nucleotide-binding domain (CNBD). The suppressor S58 lost all cpr22 related phenotypes, such as spontaneous cell death formation under ambient temperature conditions. However, these phenotypes were recovered at 16 degrees C suggesting that the stability of channel function is affected by temperature. In silico modelling and site-directed mutagenesis analyses suggest that arginine 557 in the alphaC-helix of the CNBD is important for channel regulation, but not for basic function. Furthermore, another suppressor mutant, S136 that lacks the entire alphaC-helix due to a premature stop codon, lost channel function completely. Our data presented here indicate that the alphaC-helix is functionally important in plant CNGCs.

Identification of a functionally essential amino acid for Arabidopsis cyclic nucleotide gated ion channels using the chimeric AtCNGC11/12 gene.

We used the chimeric Arabidopsis cyclic nucleotide-gated ion channel AtCNGC11/12 to conduct a structure-function study of plant cyclic nucleotide-gated ion channels (CNGCs). AtCNGC11/12 induces multiple pathogen resistance responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22). A genetic screen for mutants that suppress cpr22-conferred phenotypes identified an intragenic mutant, #73, which has a glutamate to lysine substitution (E519K) at the beginning of the eighth beta-sheet of the cyclic nucleotide-binding domain in AtCNGC11/12. The #73 mutant is morphologically identical to wild-type plants and has lost cpr22-related phenotypes including spontaneous cell death and enhanced pathogen resistance. Heterologous expression analysis using a K(+)-uptake-deficient yeast mutant revealed that this Glu519 is important for AtCNGC11/12 channel function, proving that the occurrence of cpr22 phenotypes requires active channel function of AtCNGC11/12. Additionally, Glu519 was also found to be important for the function of the wild-type channel AtCNGC12. Computational structural modeling and in vitro cAMP-binding assays suggest that Glu519 is a key residue for the structural stability of AtCNGCs and contributes to the interaction of the cyclic nucleotide-binding domain and the C-linker domain, rather than the binding of cAMP. Furthermore, a mutation in the alpha-subunit of the human cone receptor CNGA3 that causes total color blindness aligned well to the position of Glu519 in AtCNGC11/12. This suggests that AtCNGC11/12 suppressors could be a useful tool for discovering important residues not only for plant CNGCs but also for CNGCs in general.

Genome-wide dissection of globally emergent multi-drug resistant serotype 19A Streptococcus pneumoniae.

BACKGROUND: Emergence of multi-drug resistant (MDR) serotype 19A Streptococcus pneumoniae (SPN) is well-documented but causal factors remain unclear. Canadian SPN isolates (1993-2008, n = 11,083) were serotyped and in vitro susceptibility tested. A subset of MDR 19A were multi-locus sequence typed (MLST) and representative isolates' whole genomes sequenced. RESULTS: MDR 19A increased in the post-PCV7 era while 19F, 6B, and 23F concurrently declined. MLST of MDR 19A (n = 97) revealed that sequence type (ST) 320 predominated. ST320 was unique amongst MDR 19A in that its minimum inhibitory concentration (MIC) values for penicillin, amoxicillin, ceftriaxone, and erythromycin were higher than for other ST present amongst post-PCV7 MDR 19A. DNA sequencing revealed that alleles at key drug resistance loci pbp2a, pbp2x, pbp2b, ermB, mefA/E, and tetM were conserved between pre-PCV7 ST 320 19F and post-PCV7 ST 320 19A most likely due to a capsule switch recombination event. A genome wide comparison of MDR 19A ST320 with MDR 19F ST320 identified 822 unique SNPs in 19A, 61 of which were present in antimicrobial resistance genes and 100 in virulence factors. CONCLUSIONS: Our results suggest a complex genetic picture where high-level drug resistance, vaccine selection pressure, and SPN mutational events have created a "perfect storm" for the emergence of MDR 19A.

A Novel Single Domain Antibody Targeting FliC Flagellin of Salmonella enterica for Effective Inhibition of Host Cell Invasion.

The enteric pathogen, Salmonella enterica is a major cause of human gastroenteritis globally and with increasing bacterial resistance to antibiotics, alternative solutions are urgently needed. Single domain antibodies (sdAbs), the smallest antibody fragments that retain antigen binding specificity and affinity, are derived from variable heavy-chain only fragments (VHH) of camelid heavy-chain-only immunoglobulins. SdAbs typically contain a single disulfide bond simplifying recombinant protein production in microbial systems. These factors make sdAbs ideally suited for the development of effective anti-bacterial therapeutics. To this end, we generated an anti-Salmonella VHH library from which we screened for high affinity sdAbs. We present a novel sdAb (Abi-Se07) that targets the Salmonella virulence factor, FliC, required for bacterial motility and invasion of host cells. We demonstrate that Abi-Se07 bound FliC with a K D of 16.2 ± 0.1 nM. In addition, Abi-Se07 exhibited cross-serovar binding to whole cells of S. enterica serovar Typhimurium, Heidelberg, and Hadar. Abi-Se07 significantly inhibited bacterial motility and significantly reduced S. enterica colonization in a more native environment of chicken jejunum epithelium. Taken together, we have identified a novel anti-Salmonella sdAb and discuss future efforts toward therapeutic development.

First draft genome sequence of Aureimonas altamirensis, isolated from patient blood culture.

Aureimonas altamirensis (A. altamirensis) is a recently described aerobic Gram-negative bacillus related to Brucella species, which is a potential opportunistic pathogen of humans. Aureimonas altamirensis ON-56566 was isolated from the blood culture of a patient who presented with cellulitis. This brief report describes a short case report and the first draft genome (13 contigs) of A. altamirensis ON-56566 which consists of 4,202,944 nucleotides with G+C content of 65.2%.

Heat shock protein 90 inhibitors repurposed against Entamoeba histolytica.

Hsp90 is an essential chaperone responsible for trafficking a vast array of client proteins, which are substrates that Hsp90 regulates in eukaryotic cells under stress conditions. The ATP-binding N-terminal domain of Hsp90 (also known as a GHKL type ATPase domain) can serve as a specific drug target, because sufficient structural diversity in the ATP-binding pocket of Hsp90 allows for ortholog selectivity of Hsp90 inhibitors. The primary objective of this study is to identify inhibitors specific for the ATP-binding domain of Entamoeba histolytica Hsp90 (EhHsp90). An additional aim, using a combination of site-directed mutagenesis and a protein in vitro assay, is to show that the antiparasitic activity of Hsp90 inhibitors is dependent on specific residues within the ATP-binding domain. Here, we tested the activity of 43 inhibitors of Hsp90 that we previously identified using a high-throughput screen. Of the 43 compounds tested, 19 competed for binding of the EhHsp90 ATP-binding domain. Five out of the 19 EhHsp90 protein hits demonstrated activity against E. histolytica in vitro culture: rifabutin, rutilantin, cetylpyridinium chloride, pararosaniline pamoate and gentian violet. These five top E. histolytica Hsp90 inhibitors showed 30-100% inhibition of E. histolytica in culture in the micromolar range. These data suggest that E. histolytica-specific Hsp90 inhibitors are possible to identify and provide important lead compounds for the development of novel antiamebic drugs.

Targeting Plasmodium falciparum Hsp90: Towards Reversing Antimalarial Resistance.

Malaria continues to exact a great human toll in tropical settings. Antimalarial resistance is rife and the parasite inexorably develops mechanisms to outwit our best drugs, including the now first-line choice, artesunate. Novel strategies to circumvent resistance are needed. Here we detail drug development focusing on heat shock protein 90 and its central role as a chaperone. A growing body of evidence supports the role for Hsp90 inhibitors as adjunctive drugs able to restore susceptibility to traditionally efficacious compounds like chloroquine.

A purine analog synergizes with chloroquine (CQ) by targeting Plasmodium falciparum Hsp90 (PfHsp90).

BACKGROUND: Drug resistance, absence of an effective vaccine, and inadequate public health measures are major impediments to controlling Plasmodium falciparum malaria worldwide. The development of antimalarials to which resistance is less likely is paramount. To this end, we have exploited the chaperone function of P. falciparum Hsp90 (PfHsp90) that serves to facilitate the expression of resistance determinants. METHODS: The affinity and activity of a purine analogue Hsp90 inhibitor (PU-H71) on PfHsp90 was determined using surface plasmon resonance (SPR) studies and an ATPase activity assay, respectively. In vitro, antimalarial activity was quantified using flow cytometry. Interactors of PfHsp90 were determined by LC-MS/MS. In vivo studies were conducted using the Plasmodium berghei infection mouse model. RESULTS: PU-H71 exhibited antimalarial activity in the nanomolar range, displayed synergistic activity with chloroquine in vitro. Affinity studies reveal that the PfHsp90 interacts either directly or indirectly with the P. falciparum chloroquine resistance transporter (PfCRT) responsible for chloroquine resistance. PU-H71 synergized with chloroquine in the P.berghei mouse model of malaria to reduce parasitemia and improve survival. CONCLUSIONS: We propose that the interaction of PfHsp90 with PfCRT may account for the observed antimalarial synergy and that PU-H71 is an effective adjunct for combination therapy.