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 β-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 α-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. © 2008 The Authors

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

<p>Abstract</p> <p>Background</p> <p>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 <it>in vitro </it>susceptibility tested. A subset of MDR 19A were multi-locus sequence typed (MLST) and representative isolates' whole genomes sequenced.</p> <p>Results</p> <p>MDR 19A increased in the post-PCV7 era while 19F, 6B, and 23F concurrently declined. MLST of MDR 19A (<it>n </it>= 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 <it>pbp2a</it>, <it>pbp2x</it>, <it>pbp2b</it>, <it>ermB</it>, <it>mefA/E</it>, and <it>tetM </it>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.</p> <p>Conclusions</p> <p>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.</p

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 KD 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.Fil: Huen, Jennifer. AbCelex Technologies Inc.; Canadá. University of Toronto; CanadáFil: Yan, Zhun. AbCelex Technologies Inc.; CanadáFil: Iwashkiw, Jeremy. AbCelex Technologies Inc.; CanadáFil: Dubey, Shraddha. AbCelex Technologies Inc.; CanadáFil: Gimenez, Maria C.. University of Toronto; CanadáFil: Ortiz, María Eugenia. University of Toronto; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; ArgentinaFil: Patel, Saumil V.. AbCelex Technologies Inc.; CanadáFil: Jones, Michael D.. AbCelex Technologies Inc.; CanadáFil: Riazi, Ali. AbCelex Technologies Inc.; CanadáFil: Terebiznik, Mauricio. University of Toronto; CanadáFil: Babaei, Saeid. AbCelex Technologies Inc.; CanadáFil: Shahinas, Dea. AbCelex Technologies Inc.; Canad

Importance of the αC-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 αC-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 °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 αC-helix of the CNBD is important for channel regulation, but not for basic function. Furthermore, another suppressor mutant, S136 that lacks the entire αC-helix due to a premature stop codon, lost channel function completely. Our data presented here indicate that the αC-helix is functionally important in plant CNGCs

Structural and Functional Insights on Regulation by Phenolic Compounds

The shikimate pathway is a primary metabolic pathway involved in the synthesis of aromatic compounds in plants, fungi, apicomplexan parasites and microbes. The absence of this pathway in animals makes it ideal for the synthesis of antimicrobial compounds and herbicides. Additionally, its branching into indole hormone synthesis and phenylpropanoid secondary metabolism makes this pathway attractive for metabolic engineering. Here, the focus is on the first step of the shikimate pathway catalyzed by DAHP synthase. This step consists of the condensation of phosphoenol pyruvate and erythrose-4-phosphate to make DAHP, which undergoes another six catalytic steps to synthesize chorismate, the precursor of the aromatic amino acids. Arabidopsis thaliana contains three DAHP synthase isozymes, which are known to indirectly regulate downstream pathways in response to wounding and pathogen stress. The model presented here proposes that DAHP synthase isozymes are regulated by the end products tyrosine, tryptophan and phenylalanine.MAS

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

Abstract 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