Evolution of a cis-acting SNP that controls Type VI Secretion in Vibrio cholerae

Mutations in regulatory mechanisms that control gene expression contribute to phenotypic diversity and thus facilitate the adaptation of microbes to new niches. Regulatory architecture is often inferred from transcription factor identification and genome analysis using purely computational approaches. However, there are few examples of phenotypic divergence that arise from the rewiring of bacterial regulatory circuity by mutations in intergenic regions, because locating regulatory elements within regions of DNA that do not code for protein requires genomic and experimental data. We identify a single cis-acting single nucleotide polymorphism (SNP) dramatically alters control of the type VI secretion system (T6), a common weapon for inter-bacterial competition. Tight T6 regulatory control is necessary for adaptation of the waterborne pathogen Vibrio cholerae to in vivo conditions within the human gut, which we show can be altered by this single non-coding SNP that results in constitutive expression in vitro. Our results support a model of pathogen evolution through cis-regulatory mutation and preexisting, active transcription factors, thus conferring different fitness advantages to tightly regulated strains inside a human host and unfettered strains adapted to environmental niches.

High Dimensional Analyses of Circulating Immune Cells in Psoriatic Arthritis Detects Elevated Phosphorylated STAT3

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis, affecting up to 40% of patients with psoriasis. Constitutive expression by CD4+ T cells of an active form of STAT3, a signal transducer and transcription factor, has been shown to induce many of the major features of PsA in an animal model. We used high dimensional mass cytometry (CyTOF) to probe ex-vivo levels of phosphorylated STAT3 (pSTAT3) in circulating immune cell subpopulations from PsA patients during active and inactive states. We evaluated the frequency of 16 immune cell populations and the levels of the activated forms of STAT3 (pSTAT3) and, for comparison, STAT1 (pSTAT1) and Src (pSrc) in whole blood fixed shortly after collection. In addition to PsA patients, we studied active rheumatoid arthritis (RA) patients. Increased levels of pSTAT3 were found in all the CD4+ T cell subsets analyzed, specifically, Th1, Th2, Th17, T follicular helper (Tfh) and T regulatory (Treg) as well as in CD14+CD16- (classical) monocytes from active PsA patients compared to inactive patients. After correcting for body mass index (BMI), smoking and conventional disease modifying antirheumatic drugs (c-DMARDs), levels of pSTAT3 levels remained increased in Th1 and Tfh CD4+ T cells, and in CD14+CD16- monocytes from active patients compared to inactive patients. No differences between the patient groups were observed for pSTAT1 or pSrc. No differences were found between the active PsA and active RA groups after correction for multiple testing. During active PsA, circulating Th1 and Tfh CD4+ T cells, and CD14+CD16- monocytes expressing high levels of pSTAT3 may play a role in PsA pathophysiology, perhaps by migration to inflamed sites.

Characterisation of Candida parapsilosis CYP51 as a Drug Target Using Saccharomyces cerevisiae as Host

The fungal cytochrome P450 lanosterol 14α-demethylase (CYP51) is required for the biosynthesis of fungal-specific ergosterol and is the target of azole antifungal drugs. Despite proven success as a clinical target for azole antifungals, there is an urgent need to develop next-generation antifungals that target CYP51 to overcome the resistance of pathogenic fungi to existing azole drugs, toxic adverse reactions and drug interactions due to human drug-metabolizing CYPs. Candida parapsilosis is a readily transmitted opportunistic fungal pathogen that causes candidiasis in health care environments. In this study, we have characterised wild type C. parapsilosis CYP51 and its clinically significant, resistance-causing point mutation Y132F by expressing these enzymes in a Saccharomyces cerevisiae host system. In some cases, the enzymes were co-expressed with their cognate NADPH-cytochrome P450 reductase (CPR). Constitutive expression of CpCYP51 Y132F conferred a 10- to 12-fold resistance to fluconazole and voriconazole, reduced to ~6-fold resistance for the tetrazoles VT-1161 and VT-1129, but did not confer resistance to the long-tailed triazoles. Susceptibilities were unchanged in the case of CpCPR co-expression. Type II binding spectra showed tight triazole and tetrazole binding by affinity-purified recombinant CpCYP51. We report the X-ray crystal structure of ScCYP51 in complex with VT-1129 obtained at a resolution of 2.1 Å. Structural analysis of azole—enzyme interactions and functional studies of recombinant CYP51 from C. parapsilosis have improved understanding of their susceptibility to azole drugs and will help advance structure-directed antifungal discovery.

Polymer-directed inhibition of reversible to irreversible attachment prevents Pseudomonas aeruginosa biofilm formation

Non-toxic, biocompatible materials that inhibit bacterial biofilm formation on implanted medical devices and so prevent infection are urgently required. Weakly amphiphilic acrylate polymers with rigid hydrocarbon pendant groups resist bacterial biofilm formation in vitro and in vivo but the biological mechanism involved is not known. By comparing biofilm formation on polymers with the same acrylate backbone but with different pendant groups, we show that poly(ethylene glycol dicyclopentenyl ether acrylate; pEGdPEA) but not neopentyl glycol propoxylate diacrylate (pNGPDA) inhibited the transition from reversible to irreversible attachment. By using single-cell tracking algorithms and controlled flow microscopy we observed that fewer Pseudomonas aeruginosa PAO1 cells accumulated on pEGdPEA compared with pNGPDA. Bacteria reaching the pEGdPEA surface exhibited shorter residence times and greater asymmetric division with more cells departing from the surface post-cell division, characteristic of reversible attachment. Migrating cells on pEGdPEA deposited fewer exopolysaccharide trails and were unable top adhere strongly. Discrimination between the polymers required type IV pili and flagella. On pEGdPEA, the lack of accumulation of cyclic diguanylate or expression of sadB were consistent with the failure to transit from reversible to irreversible attachment. Constitutive expression of sadB increased surface adhesion sufficient to enable P. aeruginosa to form biofilms in a Mot flagellar stator dependent manner. These findings were extendable to other biofilm resistant acrylates highlighting their unique ability to inhibit reversible to irreversible attachment as a mechanism for preventing biofilm-associated infections.

Construction of Baculovirus-Inducible CRISPR/Cas9 Antiviral System Targeting BmNPV in Bombyx mori

The silkworm Bombyx mori is an economically important insect. The sericulture industry is seriously affected by pathogen infections. Of these pathogens, Bombyx mori nucleopolyhedrovirus (BmNPV) causes approximately 80% of the total economic losses due to pathogen infections. We previously constructed a BmNPV-specific CRISPR/Cas9 silkworm line with significantly enhanced resistance to BmNPV. In order to optimize the resistance properties and minimize its impact on economic traits, we constructed an inducible CRISPR/Cas9 system for use in transgenic silkworms. We used the 39k promoter, which is induced by viral infection, to express Cas9 and the U6 promoter to express four small guide RNA targeting the genes encoding BmNPV late expression factors 1 and 3 (lef-1 and lef-3, respectively), which are essential for viral DNA replication. The system was rapidly activated when the silkworm was infected and showed considerably higher resistance to BmNPV infection than the wild-type silkworm. The inducible system significantly reduced the development effects due to the constitutive expression of Cas9. No obvious differences in developmental processes or economically important characteristics were observed between the resulting transgenic silkworms and wild-type silkworms. Adoption of this accurate and highly efficient inducible CRISPR/Cas9 system targeting BmNPV DNA replication will result in enhanced antivirus measures during sericulture, and our work also provides insights into the broader application of the CRISPR/Cas9 system in the control of infectious diseases and insect pests.

Expression of OsDREB2A in Transgenic Tomato Improves Drought Tolerance

Dehydration responsive element binding (DREB) are important regulatory molecules which have a crucial role in abiotic stress tolerance. The productivity of tomato, as a drought-sensitive crop, is highly restricted by drought stress. The current study aimed at introducing the OsDERB2A gene into two tomato genotypes via Agrobacterium-mediated transformation system. Cotyledonary explants were pre-cultured for two days with Agrobacterium strain LBA4404 harboring pCAMBIA1301 with OsDREB2A driven by the constitutive promoter CaMV35S for transformation. Shoots were directly regenerated on MS medium containing 1 mg l-1 zeatin and 1 mg l-1 BAP, and in presence of 30 mg l-1 hygromycin as selective agent. Only eight weeks were needed to regenerate transgenic tomato using this protocol. An OD600 of 0.4 resulted in 64.3-76.9% transformation efficiency. Stable integration and expression of the OsDREB2A gene were confirmed in transgenic tomato using PCR and RT-PCR analyses, and drought tolerance of T0 transgenic lines was confirmed by leaf disc assay in 300 mM mannitol. The superior biomass, photosynthetic pigments, free soluble sugars and proline accumulation of OsDREB2A transgenic lines over wild type in response to mannitol-stress revealed their enhanced drought tolerance and indicated that the constitutive expression of OsDREB2A might modulate the expression of other drought responsive genes.

Inducer-Free Cellulase Production System Based on the Constitutive Expression of Mutated Xyr1 and Ace3 in the Industrial Fungus Trichoderma Reesei

Background: Trichoderma reesei (Hypocrea jecorina) is a filamentous fungus that can produce extremely high levels of protein; consequently, it is utilized as a host for the production of cellulase and hemicellulase cocktails for lignocellulosic biomass degradation. Several hyper-producer strains of T. reesei have been bred for use in industrial production, but they generally require inducers to achieve high production capacities. The most commonly used inducers are soluble sugars produced by the degradation of cellulose; however, the dependence on cellulose degradation is problematic because cellulose is insoluble and has poor handling properties as a carbon source. Furthermore, once cellulose is decomposed, little cellulase is produced, making it difficult to produce the enzyme continuously and efficiently. The aim of this study was to establish a simple, inducer-free, cellulase production system using glucose as the sole carbon source.Results: Here, we focused on transcription factors that regulate both cellulase and hemicellulase genes. First, we verified that the previously reported Xylanase regulator 1 (Xyr1) mutation had a glucose-blind phenotype in T. reesei, and confirmed that constitutive expression of the V821F mutation in Xyr1 produced high levels of proteins, especially hemicellulase and cellulase, even in inducer-free conditions. However, the majority of proteins were hemicellulases. To reproduce cellulase/hemicellulase production similar to those observed under induced conditions, an activator of cellulase expression 3 (Ace3) was expressed in Xyr1V821F expressed strain additionally. As a result, the T. reesei strain constitutively expressing Xyr1V821F and Ace3 exhibited a 1.5-fold increase than Xyr1V821F expressed only in protein productivity under inducer-free conditions. Notably, the enzyme composition significantly improved for cellulases ratio and similar to that induced by cellulose. Furthermore, the enzymes exhibited a high saccharification efficiency when compared to that of produced by the strain expressing only the mutated Xyr1.Conclusions: This work shows that the constitutive expression of mutated Xyr1 and Ace3 can increase cellulase and hemicellulase production in T. reesei without inducers. This inducer-free enzyme production method could provide an effective system to reduce costs and simplify production processes, and is expected to be applied in the production of various proteins.

Therapeutic IGF-I receptor inhibition alters fibrocyte immune phenotype in thyroid-associated ophthalmopathy

Thyroid-associated ophthalmopathy (TAO) represents a disfiguring and potentially blinding autoimmune component of Graves’ disease. It appears to be driven, at least in part, by autoantibodies targeting the thyrotropin receptor (TSHR)/insulin-like growth factor I receptor (IGF-IR) complex. Actions mediated through either TSHR or IGF-IR are dependent on IGF-IR activity. CD34+ fibrocytes, monocyte lineage cells, reside uniquely in the TAO orbit, where they masquerade as CD34+ orbital fibroblasts. Fibrocytes present antigens to T cells through their display of the major histocompatibility complex class II (MHC II) while providing costimulation through B7 proteins (CD80, CD86, and programmed death-ligand 1 [PD-L1]). Here, we demonstrate that teprotumumab, an anti-IGF-IR inhibitor, attenuates constitutive expression and induction by the thyroid-stimulating hormone of MHC II and these B7 members in CD34+ fibrocytes. These actions are mediated through reduction of respective gene transcriptional activity. Other IGF-IR inhibitors (1H7 and linsitinib) and knocking down IGF-IR gene expression had similar effects. Interrogation of circulating fibrocytes collected from patients with TAO, prior to and following teprotumumab treatment in vivo during a phase 2 clinical trial, demonstrated reductions in cell-surface MHC II and B7 proteins similar to those found following IGF-IR inhibitor treatment in vitro. Teprotumumab therapy reduces levels of interferon-γ and IL-17A expression in circulating CD4+ T cells, effects that may be indirect and mediated through actions of the drug on fibrocytes. Teprotumumab was approved by the US Food and Drug Administration for TAO. Our current findings identify potential mechanisms through which teprotumumab might be eliciting its clinical response systemically in patients with TAO, potentially by restoring immune tolerance.

Improved Production of Streptomyces sp. FA1 Xylanase in a Dual-Plasmid Pichia pastoris System

Methanol is considered as a potential hazard in the methanol-induced yeast expression of food-related enzymes. To increase the production efficiency of recombinant proteins in Pichia pastoris without methanol induction, a novel dual-plasmid system was constructed, for the first time, by a combining the strategies of genomic integration and episomal expression. To obtain a high copy number of the target gene, the autonomously replicating sequence derived from Kluyveromyces lactis (PARS) was used to construct episomal vectors carrying the constitutive promoters PGAP and PGCW14. In addition, an integrative vector carrying the PGCW14 promoter was constructed by replacing the PGAP promoter sequence with a partial PGCW14 promoter. Next, using xylanase XynA from Streptomyces sp. FA1 as the model enzyme, recombination strains were transformed with different combinations of integrating and episomal vectors that were constructed to investigate the changes in the protein yield. Results in shake flasks indicated that the highest enzyme yield was achieved when integrated PGAP and episomal PGCW14 were simultaneously transformed into the host strain. Meanwhile, the copy number of xynA increased from 1.14 ± 0.46 to 3.06 ± 0.35. The yield of XynA was successfully increased to 3925 U·mL−1 after 102 h of fermentation in a 3.6 L fermenter, which was 16.7-fold and 2.86-fold of the yields that were previously reported for the constitutive expression and methanol-induced expression of the identical protein, respectively. Furthermore, the high-cell-density fermentation period was shortened from 132 h to 102 h compared to that of methanol-induced system. Since the risk of methanol toxicity is removed, this novel expression system would be suitable for the production of proteins related to the food and pharmaceutical industries.