Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector

Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val¹⁰⁹Ile, Asp³³⁵Glu and Asn³⁸⁴Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies.     

High Frequency of Transmitted HIV-1 Gag HLA Class I-Driven Immune Escape Variants but Minimal Immune Selection over the First Year of Clade C Infection

In chronic HIV infection, CD8+ T cell responses to Gag are associated with lower viral loads, but longitudinal studies of HLA-restricted CD8+ T cell-driven selection pressure in Gag from the time of acute infection are limited. In this study we examined Gag sequence evolution over the first year of infection in 22 patients identified prior to seroconversion. A total of 310 and 337 full-length Gag sequences from the earliest available samples (median = 14 days after infection [Fiebig stage I/II]) and at one-year post infection respectively were generated. Six of 22 (27%) individuals were infected with multiple variants. There was a trend towards early intra-patient viral sequence diversity correlating with viral load set point (p = 0.07, r = 0.39). At 14 days post infection, 59.7% of Gag CTL epitopes contained non-consensus polymorphisms and over half of these (35.3%) comprised of previously described CTL escape variants. Consensus and variant CTL epitope proportions were equally distributed irrespective of the selecting host HLA allele and most epitopes remained unchanged over 12 months post infection. These data suggest that intrapatient diversity during acute infection is an indicator of disease outcome. In this setting, there is a high rate of transmitted CTL escape variants and limited immune selection in Gag during the first year of infection. These data have relevance for vaccine strategies designed to elicit effective CD8+ T cell immune responses.     

δ15N and δ13C diet–tissue discrimination factors for large sharks under semi-controlled conditions

Stable isotopes (δ¹⁵N and δ¹³C) are being widely applied in ecological research but there has been a call for ecologists to determine species- and tissue-specific diet discrimination factors (?¹³C and ?¹⁵N) for their study animals. For large sharks stable isotopes may provide an important tool to elucidate aspects of their ecological roles in marine systems, but laboratory based controlled feeding experiments are impractical. By utilizing commercial aquaria, we estimated ?¹⁵N and ?¹³C of muscle, liver, vertebral cartilage and a number of organs of three large sand tiger (Carcharias taurus) and one large lemon shark (Negaprion brevirostris) under a controlled feeding regime. For all sharks mean ± SD for ?¹⁵N and ?¹³C in lipid extracted muscle using lipid extracted prey data were 2.29‰ ± 0.22 and 0.90‰ ± 0.33, respectively. The use of non-lipid extracted muscle and prey resulted in very similar ?¹⁵N and ?¹³C values but mixing of lipid and non-lipid extracted data produced variable estimates. Values of ?¹⁵N and ?¹³C in lipid extracted liver and prey were 1.50‰ ± 0.54 and 0.22‰ ± 1.18, respectively. Non-lipid extracted diet discrimination factors in liver were highly influenced by lipid content and studies that examine stable isotopes in shark liver, and likely any high lipid tissue, should strive to remove lipid effects through standardising C:N ratios, prior to isotope analysis. Mean vertebral cartilage ?¹⁵N and ?¹³C values were 1.45‰ ± 0.61 and 3.75‰ ± 0.44, respectively. Organ ?¹⁵N and ?¹³C values were more variable among individual sharks but heart tissue was consistently enriched by ~ 1–2.5‰. Minimal variability in muscle and liver δ¹⁵N and δ¹³C sampled at different intervals along the length of individual sharks and between liver lobes suggests that stable isotope values are consistent within tissues of individual animals. To our knowledge, these are the first reported diet–tissue discrimination factors for large sharks under semi-controlled conditions, and are lower than those reported for teleost fish.     

Molecular basis for chloronium-mediated meroterpene cyclization: cloning, sequencing, and heterologous expression of the napyradiomycin biosynthetic gene cluster

Structural inspection of the bacterial meroterpenoid antibiotics belonging to the napyradiomycin family of chlorinated dihydroquinones suggests that the biosynthetic cyclization of their terpenoid subunits is initiated via a chloronium ion. The vanadium-dependent haloperoxidases that catalyze such reactions are distributed in fungi and marine algae and have yet to be characterized from bacteria. The cloning and sequence analysis of the 43-kb napyradiomycin biosynthetic cluster (nap) from Streptomyces aculeolatus NRRL 18422 and from the undescribed marine sediment-derived Streptomyces sp. CNQ-525 revealed 33 open reading frames, three of which putatively encode vanadium-dependent chloroperoxidases. Heterologous expression of the CNQ-525-based nap biosynthetic cluster in Streptomyces albus produced at least seven napyradiomycins, including the new analog 2-deschloro-2-hydroxy-A80915C. These data not only revealed the molecular basis behind the biosynthesis of these novel meroterpenoid natural products but also resulted in the first in vivo verification of vanadium-dependent haloperoxidases.     

Effect of vitamin B6 availability on serine hydroxymethyltransferase in MCF-7 cells

Folate-activated one-carbon units are derived from serine through the activity of the pyridoxal-phosphate (PLP)-dependent isozymes of serine hydroxymethyltransferase (SHMT). The effect of vitamin B(6) availability on the activity and expression of the human mitochondrial and cytoplasmic SHMT isozymes was investigated in human MCF-7 cells. Cells were cultured for 6 months in vitamin B(6) replete (4.9 microM pyridoxine) minimal essential medium (alphaMEM) or vitamin B(6)-deficient medium containing 49, 4.9 or 0.49 nM pyridoxine. Total cellular PLP levels and SHMT activity were reduced 72% and 7%, respectively, when medium pyridoxine was decreased from 4.9 microM to 49 nM. Cells cultured in medium containing 4.9 nM pyridoxine exhibited 75%, 27% and 60% reduced levels of PLP, SHMT activity and S-adenosylmethionine, respectively, compared to cells cultured in alphaMEM. Cytoplasmic SHMT activity and protein levels, but not mRNA levels, were decreased in cells cultured in vitamin B(6) deficient medium, whereas mitochondrial SHMT activity and protein levels were less sensitive to vitamin B(6) availability. PLP bound to cytoplasmic SHMT with a K(d)=850 nM, a value two orders of magnitude lower than previously reported for the bovine cytoplasmic SHMT isozyme. Collectively, these data indicate that vitamin B(6) restriction decreases the activity and stability of SHMT, and that the cytoplasmic isozyme is more sensitive to vitamin B(6) deficiency than the mitochondrial isozyme in MCF-7 cells.     

Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.     

Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements

The human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) share a unique mechanism of colonization that results from the concerted action of effector proteins translocated into the host cell by a type III secretion system (T3SS). EPEC and EHEC not only induce characteristic attaching and effacing (A/E) lesions, but also subvert multiple host cell signalling pathways during infection. Our understanding of the mechanisms by which A/E pathogens hijack host cell signalling has advanced dramatically in recent months with the identification of novel activities for many effectors. In addition to further characterization of established effectors (Tir, EspH and Map), new effectors have emerged as important mediators of virulence through activities such as mimicry of Rho guanine nucleotide exchange factors (Map and EspM), inhibition of apoptosis (NleH and NleD), interference with inflammatory signalling pathways (NleB, NleC, NleE and NleH) and phagocytosis (EspF, EspH and EspJ). The findings have highlighted the multifunctional nature of the effectors and their ability to participate in redundant, synergistic or antagonistic relationships, acting in a co-ordinated spatial and temporal manner on different host organelles and cellular pathways during infection.     

GABARAPL1 antibodies: target one protein, get one free!

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.     

The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel

The type II secretion system (T2SS) is a large macromolecular complex spanning the inner and outer membranes of many Gram-negative bacteria. The T2SS is responsible for the secretion of virulence factors such as cholera toxin (CT) and heat-labile enterotoxin (LT) from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. CT and LT are closely related AB₅ heterohexamers, composed of one A subunit and a B-pentamer. Both CT and LT are translocated, as folded protein complexes, from the periplasm across the outer membrane through the type II secretion channel, the secretin GspD. We recently published the 19 Å structure of the V. cholerae secretin (VcGspD) in its closed state and showed by SPR measurements that the periplasmic domain of GspD interacts with the B-pentamer complex. Here we extend these studies by characterizing the binding of the cholera toxin B-pentamer to VcGspD using electron microscopy of negatively stained preparations. Our studies indicate that the pentamer is captured within the large periplasmic vestibule of VcGspD. These new results agree well with our previously published studies and are in accord with a piston-driven type II secretion mechanism.Key words: secretin, GspD, electron cryomicroscopy, type II secretion system (T2SS), cholera toxin