From insect to man: Photorhabdus sheds light on the emergence of human pathogenicity

Photorhabdus are highly effective insect pathogenic bacteria that exist in a mutualistic relationship with Heterorhabditid nematodes. Unlike other members of the genus, Photorhabdus asymbiotica can also infect humans. Most Photorhabdus cannot replicate above 34°C, limiting their host-range to poikilothermic invertebrates. In contrast, P. asymbiotica must necessarily be able to replicate at 37°C or above. Many well-studied mammalian pathogens use the elevated temperature of their host as a signal to regulate the necessary changes in gene expression required for infection. Here we use RNA-seq, proteomics and phenotype microarrays to examine temperature dependent differences in transcription, translation and phenotype of P. asymbiotica at 28°C versus 37°C, relevant to the insect or human hosts respectively. Our findings reveal relatively few temperature dependant differences in gene expression. There is however a striking difference in metabolism at 37°C, with a significant reduction in the range of carbon and nitrogen sources that otherwise support respiration at 28°C. We propose that the key adaptation that enables P. asymbiotica to infect humans is to aggressively acquire amino acids, peptides and other nutrients from the human host, employing a so called “nutritional virulence” strategy. This would simultaneously cripple the host immune response while providing nutrients sufficient for reproduction. This might explain the severity of ulcerated lesions observed in clinical cases of Photorhabdosis. Furthermore, while P. asymbiotica can invade mammalian cells they must also resist immediate killing by humoral immunity components in serum. We observed an increase in the production of the insect Phenol-oxidase inhibitor Rhabduscin normally deployed to inhibit the melanisation immune cascade. Crucially we demonstrated this molecule also facilitates protection against killing by the alternative human complement pathway

Hydrogen bonding interactions in a-substituted cinnamic acid ester derivatives studied by FT–IR spectroscopy and calculations

Intermolecular hydrogen bonding interactions in stereoisomeric cc-substituted cinnamic acid methyl esters (methyl 2,3-diphenylpropenoate, methyl 2-phenyl-3-(2'-methoxyphenyl)-propenoate, methyl 2-(2'-methoxyphenyl)-3-phenylpropenoate and methyl-2,3-bis(2'-methoxyphenyl)-propenoate) were studied by FT-IR spectroscopy and model calculations at the semi-empirical quantum chemical level of theory. Intermolecular hydrogen bonds of C-H...O types were found to be general in the solid stare, but rare in solution. In this hydrogen bond the carbon may be part of either aromatic ring or the olefinic bond. The hydrogen bond acceptor may be the carbonyl oxygen or the oxygen in the methoxy substituent. Modeling helped in determining probable hydrogen bonding sites and their positions and provided with approximate geometric parameters (bond lengths and angles). Pointing out differences between the stereoisomers was also possible

N-(4-Substituted-benzoyl)-N'-beta-(D-glucopyranosyl)ureas as inhibitors of glycogen phosphorylase: Synthesis and evaluation by kinetic, crystallographic, and molecular modelling methods

N-(4-Substituted-benzoyl)-N'-(β-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylated β-d-glucopyranosyl urea as well as in reactions of O-peracetylated or O-unprotected glucopyranosylamines and acyl-isocyanates. O-deprotections were carried out by base or acid catalysed transesterifications where necessary. Kinetic studies revealed that most of these compounds were low micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). The best inhibitor was the 4-methylbenzoyl compound (K(i)=2.3μM). Crystallographic analyses of complexes of several of the compounds with RMGPb showed that the analogues exploited, together with water molecules, the available space at the β-pocket subsite and induced a more extended shift of the 280s loop compared to RMGPb in complex with the unsubstituted benzoyl urea. The results suggest the key role of the water molecules in ligand binding and structure-based ligand design. Molecular docking study of selected inhibitors was done to show the ability of the binding affinity prediction. The binding affinity of the highest scored docked poses was calculated and correlated with experimentally measured K(i) values. Results show that correlation is high with the R-squared (R(2)) coefficient over 0.9

In ovo vitelline duct ligation results in transient changes of bursal microenvironments

The avian bursa of Fabricius has a direct connection to the cloaca via the bursal duct. Using the bursal duct ligation technique, it has been clearly shown that the B cells of the bursal follicles develop under the influence of cloacal antigens. These antigens have been suggested to be present on the bursal secretory dendritic cells in immunoglobulin G (IgG)-containing complexes. We studied the effect of maternal (yolk) antigens on the early development of B cells and the appearance of IgG-containing complexes of the bursal dendritic cells with a novel embryo manipulation technique, in ovo vitelline duct ligation. This operation blocked the direct (intestinal) transport of yolk substances into the intestine, but left the vitelline circulation intact. Vitelline duct ligation performed on embryonic day 17 resulted in serious but transient bursal underdevelopment during the first week of life: (1) IgG and the follicular dendritic cell marker 74·3 were not detectable on the bursal secretory dendritic cells, in spite of a normal serum IgG level and free communication with the cloacal lumen; (2) the number of B cells in the follicles was greatly reduced and they showed an altered phenotype, resembling that of the prebursal B cells. The intracloacal administration of different proteins effectively restored the bursal phenotype. These data suggest that maternal antigens indirectly help the maturation of bursal secretory dendritic cells and concomitantly that of B cells during the first week of life

RNA interference in Lepidoptera: An overview of successful and unsuccessful

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiment