In this review, we summarize the current knowledge concerning the eukaryotic protozoan parasite Leishmania tarentolae , with a main focus on its potential for biotechnological applications. We will also discuss the genus, subgenus, and species-level classification of this parasite, its life cycle and geographical distribution, and similarities and differences to human-pathogenic species, as these aspects are relevant for the evaluation of biosafety aspects of L . tarentolae as host for recombinant DNA/protein applications. Studies indicate that strain LEM-125 but not strain TARII/UC of L . tarentolae might also be capable of infecting mammals, at least transiently. This could raise the question of whether the current biosafety level of this strain should be reevaluated. In addition, we will summarize the current state of biotechnological research involving L . tarentolae and explain why this eukaryotic parasite is an advantageous and promising human recombinant protein expression host. This summary includes overall biotechnological applications, insights into its protein expression machinery (especially on glycoprotein and antibody fragment expression), available expression vectors, cell culture conditions, and its potential as an immunotherapy agent for human leishmaniasis treatment. Furthermore, we will highlight useful online tools and, finally, discuss possible future applications such as the humanization of the glycosylation profile of L . tarentolae or the expression of mammalian recombinant proteins in amastigote-like cells of this species or in amastigotes of avirulent human-pathogenic Leishmania species.

Competition between viruses and Wolbachia for host lipids is a proposed mechanism of Wolbachia-mediated virus blocking in insects. Yet, the metabolomic interaction between virus and symbiont within the mosquito has not been clearly defined. We compare the lipid profiles of Aedes aegypti mosquitoes bearing mono- or dual-infections of the Wolbachia wMel strain and dengue virus serotype 3 (DENV3). We found metabolic signatures of infection-induced intracellular events but little evidence to support direct competition between Wolbachia and virus for host lipids. Lipid profiles of dual-infected mosquitoes resemble those of DENV3 mono-infected mosquitoes, suggesting virus-driven modulation dominates over that of Wolbachia. Interestingly, knockdown of key metabolic enzymes suggests cardiolipins are host factors for DENV3 and Wolbachia replication. These findings define the Wolbachia-DENV3 metabolic interaction as indirectly antagonistic, rather than directly competitive, and reveal new research avenues with respect to mosquito × virus interactions at the molecular level.

Edited by Dennis R. VoelkerMycobacterium tuberculosis and related Corynebacterineae synthesize a family of lipomannans (LM) and lipoarabinomannans (LAM) that are abundant components of the multilaminate cell wall and essential virulence factors in pathogenic species. Here we describe a new membrane protein, highly conserved in all Corynebacterineae, that is required for synthesis of fulllength LM and LAM. Deletion of the Corynebacterium glutamicum NCgl2760 gene resulted in a complete loss of mature LM/LAM and the appearance of a truncated LM (t-LM). Complementation of the mutant with the NCgl2760 gene fully restored LM/LAM synthesis. Structural studies, including monosaccharide analysis, methylation linkage analysis, and mass spectrometry of native LM species, indicated that the ⌬NCgl2760 t-LM comprised a series of short LM species (8 -27 residues long) containing an ␣1-6-linked mannose backbone with greatly reduced ␣1-2-mannose side chains and no arabinose caps. The structure of the ⌬NCgl2760 t-LM was similar to that of the t-LM produced by a C. glutamicum mutant lacking the mptA gene, encoding a membrane ␣1-6-mannosyltransferase involved in extending the ␣1-6-mannan backbone of LM intermediates. Interestingly, NCgl2760 lacks any motifs or homology to other proteins of known function. Attempts to delete the NCgl2760 orthologue in Mycobacterium smegmatis were unsuccessful, consistent with previous studies indicating that the M. tuberculosis orthologue, Rv0227c, is an essential gene. Together, these data suggest that NCgl2760/ Rv0227c plays a critical role in the elongation of the mannan backbone of mycobacterial and corynebacterial LM, further highlighting the complexity of lipoglycan pathways of Corynebacterineae.

BackgroundSecretory signal peptides (SPs) are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1) of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s). The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins.ResultsWe generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA).ConclusionsThe obtained results demonstrate the importance of SP-sequence optimization for efficient expression-secretion of scFv’s. We could successfully demonstrate that minor modifications in the AA-sequence in the c-region of the natural SP from SAP1, based on in-silico predictions following the (-3, -1) rule, resulted in different expression-secretion rates of the protein of interest. The yield of scFv production could be improved close to one order of magnitude. Therefore, SP-sequence optimization is a viable option to increase the overall yield of recombinant protein production.