Although ω3‐ and ω6‐ desaturases have been well studied in terms of substrate preference and regiospecificity, relatively little is known about the membrane‐bound, “front‐end” long chain fatty acid desaturases, such as ∆4, Δ5 or Δ6 desaturases. The first vertebrate ∆4 desaturase was recently identified in the marine teleost fish Siganus canaliculatus (S. canaliculatus), which also possesses a bifunctional Δ5/6 desaturase. These two long chain polyunsaturated fatty acid desaturases are very different in terms of regiospecificity and substrate chain‐length, but share an unusually high degree of amino acid identity (83 %). We took advantage of this similarity by constructing a series of chimeric enzymes, replacing regions of one enzyme with the corresponding sequence of the other. Heterologous expression of the chimeric series of enzymes in yeast indicated that the substitution of a four amino acid region was sufficient to convert a ∆4 desaturase to an enzyme with ∆6 desaturase activity, and convert a ∆5/6 desaturase to an enzyme with a low level of ∆4 desaturase activity. In addition, enzymes having both ∆4 and ∆6 desaturase activities were produced by single or double amino acid substitutions within this four‐amino acid region.

Mycobacterium tuberculosis uses its ESX-1 system to secrete EsxA and EspB into a host to cause disease. We show that EspK, a protein whose role in the ESX-1 machinery was thought to be nonessential, is needed by M. tuberculosis for optimal EsxA and EspB secretion.

Production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plant seed oils has been pursued to improve availability of these omega-3 fatty acids that provide important human health benefits. Canola (Brassica napus), through the introduction of 10 enzymes, can convert oleic acid (OLA) into EPA and ultimately DHA through a pathway consisting of two elongation and five desaturation steps. Herein we present an assessment of the substrate specificity of the seven desaturases and three elongases that were introduced into canola by expressing individual proteins in yeast. In vivo feeding experiments were conducted with 14 potential fatty acid intermediates in an OLA to DHA pathway to determine the fatty acid substrate profiles for each enzyme. Membrane fractions were prepared from yeast expression strains and shown to contain active enzymes. The elongases, as expected, extended acyl-CoA substrates in the presence of malonyl-CoA. To distinguish between enzymes that desaturate CoA- and phosphatidylcholine-linked fatty acid substrates, we developed a novel in vitro method. We show that a delta-12 desaturase from Phytophthora sojae, an omega-3 desaturase from Phytophthora infestans and a delta-4 desaturase from Thraustochytrium sp., all prefer phosphatidylcholine-linked acyl substrates with comparatively low use of acyl-CoA substrates. To further validate our method, a delta-9 desaturase from Saccharomyces cerevisiae was confirmed to use acyl-CoA as substrate, but could not use phosphatidylcholine-linked substrates. The results and the assay methods presented herein will be useful in efforts to improve modeling of fatty acid metabolism and production of EPA and DHA in plants.Electronic supplementary materialThe online version of this article (doi:10.1007/s11745-017-4235-4) contains supplementary material, which is available to authorized users.

No abstract

Mycobacterium tuberculosis (M. tb) uses its type-7 secretion system ESX-1 to translocate key virulence effector proteins. Taking a chemical genetics approach, we demonstrate for the first time the importance of mycobacterial proteostasis to ESX-1. We show that individual treatment with inhibitors of protein synthesis (chloramphenicol and kanamycin) and protein degradation (lassomycin and bortezomib), at concentrations that only reduce M. tb growth by 50% and less, specifically block ESX-1 secretion activity in the tubercle bacillus. In contrast, the mycobacterial cell-wall synthesis inhibitor isoniazid, even at a concentration that reduces M. tb growth by 90% has no effect on ESX-1 secretion activity. We also show that chloramphenicol but not isoniazid at subinhibitory concentrations specifically attenuates ESX-1-mediated M. tb virulence in macrophages. Taken together, the results of our study identify a novel vulnerability in the ESX-1 system and offer new avenues of anti-TB drug research to neutralize this critical virulence-mediating protein secretion apparatus.

No abstract

Domestic pigs share many similarities with humans in their pulmonary anatomy, physiology, and immunology. Accordingly, pigs have been shown to be valuable models to study human tuberculosis (TB). Here we examined the outcome of disease in domestic pigs challenged via two different routes with either the human-adapted TB bacillus Mycobacterium tuberculosis or the zoonotic bovine TB bacillus M. bovis in head-to-head comparisons. We found that pigs challenged intravenously with M. bovis AF2122/97 exhibited severe morbidity and rapid onset of mortality, accompanied by higher tissue bacterial burden and necrosis compared to pigs challenged similarly with M. tb Erdman. Concordantly, pigs challenged with aerosolized M. bovis AF2122/97 exhibited reduced weight gain and more severe pathology than pigs challenged similarly with M. tb Erdman. Moreover, pigs aerosol-challenged with M. bovis AF2122/97 exhibited a spectrum of granulomatous lesions ranging from small well-contained granulomas to caseous-necrotic lesions mimicking active TB disease in humans. In contrast, pigs aerosol-challenged with M. tb Erdman exhibited arrested granuloma development. Irrespective of challenge dose and pathological outcome however, peripheral IFN-γ responses were similar in both M. bovis AF2122/97 and M. tb Erdman challenged pigs. This study demonstrates domestic pigs can support infections with M. bovis and M. tb and develop pathology similar to what is observed in humans. And although M. bovis AF2122/97 appears to be more virulent than M. tb Erdman, both strains can be used to model TB in domestic pigs, depending on whether one wishes to recapitulate either acute and active TB or latent TB infections. Keywords: Tuberculosis, domestic pigs, animal model, active TB, latent TB