Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs) of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T. brucei were independently tagged and sequenced. BESs are polymorphic in size and structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs do exist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs). The consequences of duplicated or missing ESAGs, including ESAG9, a newly named ESAG12, and additional variant surface glycoprotein genes (VSGs) were evaluated by functional assays after BESs were tagged with a drug-resistance gene. Phylogenetic analysis of constituent ESAG families suggests that BESs are sequence mosaics and that extensive recombination has shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecular mechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.

Summary Sialylation of the lipopolysaccharide (LPS) is an important mechanism used by the human pathogenHaemophilus influenzae to evade the innate immune response of the host. We have demonstrated that Nacetylneuraminic acid (Neu5Ac or sialic acid) uptake in H. influenzae is essential for the subsequent modification of the LPS and that this uptake is mediated through a single transport system which is a member of the tripartite ATP-independent periplasmic (TRAP) transporter family. Disruption of either the siaP ( HI0146 ) or siaQM ( HI0147 ) genes, that encode the two subunits of this transporter, results in a complete loss of uptake of [ 14 C]-Neu5Ac. Mutant strains lack sialylated glycoforms in their LPS and are more sensitive to killing by human serum than the parent strain. The SiaP protein has been purified and demonstrated to bind a stoichiometric amount of Neu5Ac by electrospray mass spectrometry. This binding was of high affinity with a K d of ∼ ∼ ∼ ∼ 0.1 µ µ µ µ M as determined by protein fluorescence. The inactivation of the SiaPQM TRAP transporter also results in decreased growth of H. influenzae in a chemically defined medium containing Neu5Ac, supporting an additional nutritional role of sialic acid in H. influenzae physiology.

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African trypanosomes show monoallelic expression of one of about 20 telomeric variant surface glycoprotein (VSG) gene-expression sites (ESs) while multiplying in the mammalian bloodstream. We screened for genes involved in ES silencing using flow cytometry and RNA interference (RNAi). We show that a novel member of the ISWI family of SWI2/SNF2-related chromatin-remodelling proteins (TbISWI) is involved in ES downregulation in Trypanosoma brucei. TbISWI has an atypical protein architecture for an ISWI, as it lacks characteristic SANT domains. Depletion of TbISWI by RNAi leads to 30-60-fold derepression of ESs in bloodstream-form T. brucei, and 10-17-fold derepression in insect form T. brucei. We show that although blocking synthesis of TbISWI leads to derepression of silent VSG ES promoters, this does not lead to fully processive transcription of silent ESs, or an increase in ES-activation rates. VSG ES activation in African trypanosomes therefore appears to be a multistep process, whereby an increase in transcription from a silent ES promoter is necessary but not sufficient for full ES activation.

In order to test for human histocompatibility leucocyte antigens (HLA) class II restriction of IgE responses, 431 subjects from 83 families were genotyped at the HLA-DR and HLA-DP loci and serotyped for IgE responses to six major allergens from common aero-allergen sources. A possible excess of HLA-DR1 was found in subjects who were responsive to Fel d I compared with those who were not (Odds Ratio (OR) = 2, P = 0.002), and a possible excess of HLA-DR4 was found in subjects responsive to Alt a I (OR = 1.9, P = 0.006). Increased sharing of HLA-DR/DP haplotypes was seen in sibling pairs responding to both allergens. Der p I, Der p II, Phl p V and Can f I were not associated with any definite excess of HLA-DR alleles. No significant correlations were seen with HLA-DP genotype and reactivity to any of the allergens. The results suggest class II HLA restriction is insufficient to account for individual differences in reactivity to common allergens.

In recent years, there has been an increasing interest in the exploitation of microalgae in industrial biotechnology. Potentially, these phototrophic eukaryotes could be used for the low-cost synthesis of valuable recombinant products such as bioactive metabolites and therapeutic proteins. The algal chloroplast in particular represents an attractive target for such genetic engineering, both because it houses major metabolic pathways and because foreign genes can be targeted to specific loci within the chloroplast genome, resulting in high-level, stable expression. However, routine methods for chloroplast genetic engineering are currently available only for one species—Chlamydomonas reinhardtii—and even here, there are limitations to the existing technology, including the need for an expensive biolistic device for DNA delivery, the lack of robust expression vectors, and the undesirable use of antibiotic resistance markers. Here, we describe a new strain and vectors for targeted insertion of transgenes into a neutral chloroplast locus that (i) allow scar-less fusion of a transgenic coding sequence to the promoter/5′UTR element of the highly expressed endogenous genes psaA or atpA, (ii) employ the endogenous gene psbH as an effective but benign selectable marker, and (iii) ensure the successful integration of the transgene construct in all transformant lines. Transformation is achieved by a simple and cheap method of agitation of a DNA/cell suspension with glass beads, with selection based on the phototrophic rescue of a cell wall-deficient ΔpsbH strain. We demonstrate the utility of these tools in the creation of a transgenic line that produces high levels of functional human growth hormone.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7354-6) contains supplementary material, which is available to authorized users.

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