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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.

Background Secretory 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. Results We 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). Conclusions The 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.

G-protein-coupled receptors (GPCRs), especially chemokine receptors, play a central role in the regulation of T cell migration. Various GPCRs are upregulated in activated CD4 T cells, including P2Y10, a putative lysophospholipid receptor that is officially still considered an orphan GPCR, i.e., a receptor with unknown endogenous ligand. Here we show that in mice lacking P2Y10 in the CD4 T cell compartment, the severity of experimental autoimmune encephalomyelitis and cutaneous contact hypersensitivity is reduced. P2Y10-deficient CD4 T cells show normal activation, proliferation and differentiation, but reduced chemokine-induced migration, polarization, and RhoA activation upon in vitro stimulation. Mechanistically, CD4 T cells release the putative P2Y10 ligands lysophosphatidylserine and ATP upon chemokine exposure, and these mediators induce P2Y10-dependent RhoA activation in an autocrine/paracrine fashion. ATP degradation impairs RhoA activation and migration in control CD4 T cells, but not in P2Y10-deficient CD4 T cells. Importantly, the P2Y10 pathway appears to be conserved in human T cells. Taken together, P2Y10 mediates RhoA activation in CD4 T cells in response to auto-/paracrine-acting mediators such as LysoPS and ATP, thereby facilitating chemokine-induced migration and, consecutively, T cell-mediated diseases. P2Y10 is a G-protein-coupled receptor that is expressed in CD4 T cells. Here authors show that its ligands, lysophosphatidylserine and ATP, are induced in T cells upon chemokine stimulation and regulate RhoA activation and migration through an autocrine/paracrine loop.

Acinetobacter baumannii is a pathogenic and multidrug-resistant Gram-negative bacterium that causes severe nosocomial infections. To better understand the mechanism of pathogenesis, we compare the proteomes of uninfected and infected human cells, revealing that transcription factor FOS is the host protein most strongly induced by A. baumannii infection. Pharmacological inhibition of FOS reduces the cytotoxicity of A. baumannii in cell-based models, and similar results are also observed in a mouse infection model. A. baumannii outer membrane vesicles (OMVs) are shown to activate the aryl hydrocarbon receptor (AHR) of host cells by inducing the host enzyme tryptophan-2,3-dioxygenase (TDO), producing the ligand kynurenine, which binds AHR. Following ligand binding, AHR is a direct transcriptional activator of the FOS gene. We propose that A. baumannii infection impacts the host tryptophan metabolism and promotes AHR- and FOS-mediated cytotoxicity of infected cells. In this work, authors study the proteins in uninfected and Acinetobacter baumannii infected human cells to understand how this bacterium causes disease.

Reconstructing the evolutionary origins of Mycobacterium tuberculosis , the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M . tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens , M . spongiae is the most closely related species to the M . tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M . tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M . tuberculosis , together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M . tuberculosis challenge compared to BCG vaccination alone. The discovery of M . spongiae adds to our understanding of the emergence of the M . tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M . tuberculosis .

Among the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus , only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus , the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis ( ltaS ): translation ( rplK ), DNA compaction ( hup ), initiation of DNA replication ( dnaA , hup ) and purine nucleotide metabolism ( prs ). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics. The opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.

Characterisation and diagnosis of idiopathic Parkinson’s disease (iPD) is a current challenge that hampers both clinical assessment and clinical trial development with the potential inclusion of non-PD cases. Here, we used a targeted mass spectrometry approach to quantify 38 metabolites extracted from the serum of 231 individuals. This cohort is currently one of the largest metabolomic studies including iPD patients, drug-naïve iPD, healthy controls and patients with Alzheimer’s disease as a disease-specific control group. We identified six metabolites (3-hydroxykynurenine, aspartate, beta-alanine, homoserine, ornithine (Orn) and tyrosine) that are significantly altered between iPD patients and control participants. A multivariate model to predict iPD from controls had an area under the curve (AUC) of 0.905, with an accuracy of 86.2%. This panel of metabolites may serve as a potential prognostic or diagnostic assay for clinical trial prescreening, or for aiding in diagnosing pathological disease in the clinic.

The pericyte coverage of microvessels is altered in metabolic diseases, but the mechanisms regulating pericyte–endothelial cell communication remain unclear. This study investigated the formation and function of pericyte tunneling nanotubes (TNTs) and their impact on endothelial cell metabolism. TNTs were analyzed in vitro in retinas and co-cultures of pericytes and endothelial cells. Using mass spectrometry, the influence of pericytes on endothelial cell metabolism was examined. TNTs were present in the murine retina, and although diabetes was associated with a decrease in pericyte coverage, TNTs were longer. In vitro, pericytes formed TNTs in the presence of PDGF, extending toward endothelial cells and facilitating mitochondrial transport from pericytes to endothelial cells. In experiments with mitochondria-depleted endothelial cells displaying defective TCA cycle metabolism, pericytes restored the mitochondrial network and metabolism. 19,20-Dihydroxydocosapentaenoic acid (19,20-DHDP), known to disrupt pericyte–endothelial cell junctions, prevented TNT formation and metabolic rescue in mitochondria-depleted endothelial cells. 19,20-DHDP also caused significant changes in the protein composition of pericyte-endothelial cell junctions and involved pathways related to phosphatidylinositol 3-kinase, PDGF receptor, and RhoA signaling. Pericyte TNTs contact endothelial cells and support mitochondrial transfer, influencing metabolism. This protective mechanism is disrupted by 19,20-DHDP, a fatty acid mediator linked to diabetic retinopathy.

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 w Mel 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. Koh, Islam, Ye et al. describe lipid profiles of Aedes aegypti mosquitoes bearing mono- or dual-infections of Wolbachia ( w Mel) and dengue virus serotype 3 (DENV3), finding that virus modulation dominates the dual-infection lipid profile and that cardiolipins support DENV3 and Wolbachia replication. This study suggests that direct competition for lipids do not underlie Wolbachia -mediated virus blocking.

Omega‐6 fatty acids are the primary polyunsaturated fatty acids in most Western diets, while their role in diabetes remains controversial. Exposure of omega‐6 fatty acids to an oxidative environment results in the generation of a highly reactive carbonyl species known as trans, trans‐2,4‐decadienal (tt‐DDE). The timely and efficient detoxification of this metabolite, which has actions comparable to other reactive carbonyl species, such as 4‐hydroxynonenal, acrolein, acetaldehyde, and methylglyoxal, is essential for disease prevention. However, the detoxification mechanism for tt‐DDE remains elusive. In this study, the enzyme Aldh9a1b is identified as having a key role in the detoxification of tt‐DDE. Loss of Aldh9a1b increased tt‐DDE levels and resulted in an abnormal retinal vasculature and glucose intolerance in aldh9a1b−/− zebrafish. Transcriptomic and metabolomic analyses revealed that tt‐DDE and aldh9a1b deficiency in larval and adult zebrafish induced insulin resistance and impaired glucose homeostasis. Moreover, alterations in hyaloid vasculature is induced by aldh9a1b knockout or by tt‐DDE treatment can be rescued by the insulin receptor sensitizers metformin and rosiglitazone. Collectively, these results demonstrated that tt‐DDE is the substrate of Aldh9a1b which causes microvascular damage and impaired glucose metabolism through insulin resistance. Omega‐6 fatty acids, prevalent in Western diets, are oxidized to form trans, trans‐2,4‐decadienal (tt‐DDE). This study reveals ALDH9a1b as a novel detoxifying system for tt‐DDE, confirmed by a CRISPR KO line. Analysis of transcriptome, metabolomics, and molecular experiments shows that aldh9a1b mutation not only alters tt‐DDE metabolism but induces diabetes and diabetic retinopathy, offering new insights into the omega‐6‐diabetes link.