Null Strain Research Articles

A2A Receptor Contributes to Tumor Progression in P2X7 Null Mice.

ATP and adenosine are key constituents of the tumor niche where they exert opposite and complementary roles. ATP can be released in response to cell damage or actively released by tumor cells and subsequently degraded into adenosine, which accumulates within the tumor microenvironment. Notably, while ATP promotes immune eradicating responses mainly via the P2X7 receptor (P2X7R), extracellular adenosine acts as a potent immune suppressor and facilitates neovascularization thanks to the A2A receptor (A2AR). To date, studies exploring the interplay between P2X7R and A2AR in the tumor microenvironment are as yet missing. Here, we show that, in C57/bl6 P2X7 null mice inoculated with B16-F10 melanoma cells, several pro-inflammatory cytokines, including interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 17 (IL-17), interferon gamma (IFN-γ) were significantly decreased, while the immune suppressant transforming growth factor beta (TGF-β) was almost three-fold increased. Interestingly, tumors growing in P2X7-null mice upregulated tumor-associated and splenic A2AR, suggesting that immunosuppression linked to lack of the P2X7R might depend upon A2AR overexpression. Immunohistochemical analysis showed that tumor cells’ A2AR expression was increased, especially around necrotic areas, and that vascular endothelial growth factor (VEGF) and the endothelial marker CD31 were upregulated. A2AR antagonist SCH58261 treatment reduced tumor growth similarly in the P2X7 wild type or null mice strain. However, SCH58261 reduced VEGF only in the P2X7 knock out mice, thus supporting the hypothesis of an A2AR-mediated increase in vascularization observed in the P2X7-null host. SCH58261 administration also significantly reduced intratumor TGF-β levels, thus supporting a key immune suppressive role of A2AR in our model. Altogether, these results indicate that in the absence of host P2X7R, the A2AR favors tumor growth via immune suppression and neovascularization. This study shows a novel direct correlation between P2X7R and A2AR in oncogenesis and paves the way for new combined therapies promoting anti-cancer immune responses and reducing tumor vascularization.

Peering Into Candida albicans Pir Protein Function and Comparative Genomics of the Pir Family.

The fungal cell wall, comprised primarily of protein and polymeric carbohydrate, maintains cell structure, provides protection from the environment, and is an important antifungal drug target. Pir proteins (proteins with internal repeats) are linked to cell wall β-1,3-glucan and are best studied in Saccharomyces cerevisiae. Sequential deletion of S. cerevisiae PIR genes produces strains with increasingly notable cell wall damage. However, a true null mutant lacking all five S. cerevisiae PIR genes was never constructed. Because only two PIR genes (PIR1, PIR32) were annotated in the Candida albicans genome, the initial goal of this work was to construct a true Δpir/Δpir null strain in this species. Unexpectedly, the phenotype of the null strain was almost indistinguishable from its parent, leading to the search for other proteins with Pir function. Bioinformatic approaches revealed nine additional C. albicans proteins that share a conserved Pir functional motif (minimally DGQ). Examination of the protein sequences revealed another conserved motif (QFQFD) toward the C-terminal end of each protein. Sequence similarities and presence of the conserved motif(s) were used to identify a set of 75 proteins across 16 fungal species that are proposed here as Pir proteins. The Pir family is greatly expanded in C. albicans and C. dubliniensis compared to other species and the orthologs are known to have specialized function during chlamydospore formation. Predicted Pir structures showed a conserved core of antiparallel beta-sheets and sometimes-extensive loops that contain amino acids with the potential to form linkages to cell wall components. Pir phylogeny demonstrated emergence of specific ortholog groups among the fungal species. Variation in gene expression patterns was noted among the ortholog groups during growth in rich medium. PIR allelic variation was quite limited despite the presence of a repeated sequence in many loci. Results presented here demonstrate that the Pir family is larger than previously recognized and lead to new hypotheses to test to better understand Pir proteins and their role in the fungal cell wall.

Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway

Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in Saccharomyces cerevisiae. Here, we explore how two of these strategies, pathway re-localization and redox cofactor-balancing, affect the performance and physiology of isobutanol producing strains. We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced (NADPH-dependent) or redox-balanced (NADH-dependent) ketol-acid reductoisomerase enzyme. We then conducted transcriptomic, proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains. Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes. Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version, albeit at low overall pathway flux. Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe–S clusters, which are required cofactors for the dihydroxyacid dehydratase enzyme. We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation, thereby increasing cellular iron levels. The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold, demonstrating that both localizations can support flux to isobutanol.

Filament Negative Regulator CDC4 Suppresses Glycogen Phosphorylase Encoded GPH1 That Impacts the Cell Wall-Associated Features in Candida albicans.

We have previously identified Candida albicans GPH1 (orf19.7021) whose protein product was associated with C. albicans Cdc4. The GPH1 gene is a putative glycogen phosphorylase because its Saccharomyces cerevisiae homolog participates in glycogen catabolism, which involves the synthesis of β-glucan of the fungal cell wall. We made a strain whose CaCDC4 expression is repressed, and GPH1 is constitutively expressed. We established a GPH1 null mutant strain and used it to conduct the in vitro virulence assays that detect cell wall function. The in vitro virulence assay is centered on biofilm formation in which analytic procedures are implemented to evaluate cell surface hydrophobicity; competence, either in stress resistance, germ tube formation, or fibronection association; and the XTT-based adhesion and biofilm formation. We showed that the constitutively expressed GPH1 partially suppresses filamentation when the CaCDC4 expression is repressed. The C. albicans Gph1 protein is reduced in the presence of CaCdc4 in comparison with the absence of CaCdc4. Compared with the wild-type strain, the gph1Δ/gph1Δ mutant displayed a reduction in the capability to form germ tubes and the cell surface hydrophobicity but an increase in binding with fibronectin. Compared with the wild-type strain, the gph1Δ/gph1Δ mutant showed a rise in adhesion, the initial stage of biofilm formation, but displayed a similar capacity to form a mature biofilm. There was no major impact on the gph1Δ/gph1Δ mutant regarding the conditions of cell wall damaging and TOR pathway-associated nutrient depletion. We conclude that GPH1, adversely regulated by the filament suppressor CDC4, contributes to cell wall function in C. albicans.

FLO8 deletion leads to decreased adhesion and virulence with downregulated expression of EPA1, EPA6, and EPA7 in Candida glabrata.

The Candida glabrata does not develop into a pathogenic hiphal form; however, it has become the second most common pathogen of fungal infections in humans, partly because of its adhesion ability and virulence. The present study aimed to determine whether Flo8, a transcription factor that plays an important role in the virulence and drug resistance in Candida albicans, has a similar role in C. glabrata. We constructed FLO8 null strains of a C. glabrata standard strain and eight clinical strains from different sources, and a FLO8 complemented strain. Real-time quantitative PCR, biofilm formation assays, hydrophobicity tests, adhesion tests, Caenorhabditis elegans survival assay, and drug-susceptibility were then performed. Compared with the wild-type strains, the biofilm formation, hydrophobicity, adhesion, and virulence of the FLO8-deficient strains decreased, accompanied by decreased expression of EPA1, EPA6, and EPA7. On the other hand, it showed no changes in antifungal drug resistance, although the expression levels of CDR1, CDR2, and SNQ2 increased after FLO8 deletion. These results indicated that Flo8 is involved in the adhesion and virulence of C. glabrata, with FLO8 deletion leading to decreased expression of EPA1, EPA6, and EPA7 and decreased biofilm formation, hydrophobicity, adhesion, and virulence.

Toll-like receptors change morphine-induced antinociception, tolerance and dependence: Studies using male and female TLR and signalling gene KO mice

Toll-like receptors (TLR) have been proposed as a site of action that alters opioid pharmacodynamics. However, a comprehensive assessment of acute opioid antinociception, tolerance and withdrawal behaviours in genetic null mutant strains with altered innate immune signalling has not been performed. Nor has the impact of genetic deletion of TLR2/4 on high-affinity opioid receptor binding. Here we show that diminished TLR signalling potentiates acute morphine antinociception equally in male and female mice. However, only male TIR8 null mutant mice showed reduced morphine analgesia. Analgesic tolerance was prevented in TLR2 and TLR4 null mutants, but not MyD88 animals. Withdrawal behaviours were only protected in TLR2−/− mice. In silico docking simulations revealed opioid ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. There was no binding of [3H](-)-naloxone or [3H]diprenorphine to TLR4 in the concentrations explored. These data confirm that opioids have high efficacy activity at innate immune pattern recognition binding sites but do not bind to TLR4 and identify critical pathway and sex-specific effects of the complex innate immune signalling contributions to opioid pharmacodynamics. These data further support the behavioural importance of the TLR-opioid interaction but fail to demonstrate direct evidence for high-affinity binding of the TLR4 signalling complex to ligands.

Smooth Muscle Cell Notch2 Is Not Required for Atherosclerotic Plaque Formation in ApoE Null Mice.

We previously identified Notch2 in smooth muscle cells (SMC) in human atherosclerosis and found that signaling via Notch2 suppressed human SMC proliferation. Thus, we tested whether loss of Notch2 in SMC would alter atherosclerotic plaque progression using a mouse model. Atherogenesis was examined at the brachiocephalic artery and aortic root in a vascular SMC null (inducible smooth muscle myosin heavy chain Cre) Notch2 strain on the ApoE-/- background. We measured plaque morphology and size, as well as lipid, inflammation, and smooth muscle actin content after Western diet. We generated an inducible SMC Notch2 null on the ApoE-/- background. We observed ∼90% recombination efficiency with no detectable Notch2 in the SMC. Loss of SMC Notch2 did not significantly change plaque size, lipid content, necrotic core, or medial area. However, loss of SMC Notch2 reduced the contractile SMC in brachiocephalic artery lesions and increased inflammatory content in aortic root lesions after 6 weeks of Western diet. These changes were not present with loss of SMC Notch2 after 14 weeks of Western diet. Our data show that loss of SMC Notch2 does not significantly reduce atherosclerotic lesion formation, although in early stages of plaque formation there are changes in SMC and inflammation.

The bZIP Ap1 transcription factor is a negative regulator of virulence attributes of the anthropophilic dermatophyte Trichophyton rubrum.

Trichophyton rubrum is a fungus that causes chronic skin and nail infections in healthy individuals and immunocompromised patients. During infection, T. rubrum invades host cutaneous tissues by adapting to the acidic pH and the innate immune response of the host. Several genes are upregulated during the growth of T. rubrum in substrates found in human tissue, including the ap1 gene, which codes for the transcription factor Ap1. Here, we generated a null mutant strain by deleting the T. rubrum ap1 gene and performed a functional analysis of this gene. Our results showed that the Δap1mutant increased its growth in nail fragments and co-cultures with keratinocytes compared to the wild type. Furthermore, the mutant displayed hyperpigmentation, thickening of the conidia cell wall, increased conidia susceptibility to calcofluor-white compared to the wild type, and loss of control of the keratinolytic activity. Although the ap1 gene was upregulated during exposure to the antifungal drugs amphotericin B, nystatin, and terbinafine, its deletion did not alter the fungal susceptibility to these drugs, revealing the role of the ap1 gene in the physiological response to the stress caused by these drugs, but not in their resistance. Moreover, ap1 was also involved in the oxidative stress response caused by menadione, but not paraquat or hydrogen peroxide. These findings indicate that the ap1 gene plays a role in the negative control of virulence-related attributes and may contribute to the chronicity of nail infection caused by T. rubrum.

Use of Mutagenesis and Functional Screens to Characterize Essential Genes Involved in Lipopolysaccharide Transport.

Structure-function analysis is a powerful strategy to characterize the contribution of specific residues to the biogenesis and function of a protein. This approach requires the characterization of strains that express mutant alleles in the absence of the wild-type protein. When studying nonessential bacterial genes, collections of mutants can be easily constructed by introducing plasmid-encoded alleles of interest into a strain that already lacks the wild-type gene. However, this high-throughput approach is not applicable to studying essential genes since their respective null strains are not viable. While there are several tools currently available to modify essential genes, they can be greatly limited by the amount of effort it takes to build and analyze each mutant strain. Here, we describe a high-throughput system for the rapid structure-function analysis of essential genes involved in lipopolysaccharide transport in Escherichia coli. This method, which can be applied to study any essential gene, relies on the initial construction of a single bacterial strain that can be used to generate and functionally characterize multiple plasmid-encoded alleles in under 24h. We will discuss the advantages and possible shortcomings of our protocol in comparison to other commonly used methods.

The inositol pyrophosphate metabolism of Dictyostelium discoideum does not regulate inorganic polyphosphate (polyP) synthesis.

Initial studies on the inositol phosphates metabolism were enabled by the social amoeba Dictyostelium discoideum. The abundant amount of inositol hexakisphosphate (IP6 also known as Phytic acid) present in the amoeba allowed the discovery of the more polar inositol pyrophosphates, IP7 and IP8, possessing one or two high energy phosphoanhydride bonds, respectively. Considering the contemporary growing interest in inositol pyrophosphates, it is surprising that in recent years D. discoideum, has contributed little to our understanding of their metabolism and function. This work fulfils this lacuna, by analysing the ip6k, ppip5k and ip6k-ppip5K amoeba null strains using PAGE, 13C-NMR and CE-MS analysis. Our study reveals an inositol pyrophosphate metabolism more complex than previously thought. The amoeba Ip6k synthesizes the 4/6-IP7 in contrast to the 5-IP7 isomer synthesized by the mammalian homologue. The amoeba Ppip5k synthesizes the same 1/3-IP7 as the mammalian enzyme. In D. discoideum, the ip6k strain possesses residual amounts of IP7. The residual IP7 is also present in the ip6k-ppip5K strain, while the ppip5k single mutant shows a decrease in both IP7 and IP8 levels. This phenotype is in contrast to the increase in IP7 observable in the yeast vip1Δ strain. The presence of IP8 in ppip5k and the presence of IP7 in ip6k-ppip5K indicate the existence of an additional inositol pyrophosphate synthesizing enzyme. Additionally, we investigated the existence of a metabolic relationship between inositol pyrophosphate synthesis and inorganic polyphosphate (polyP) metabolism as observed in yeast. These studies reveal that contrary to the yeast, Ip6k and Ppip5k do not control polyP cellular level in amoeba.

Development of Humanized Diffuse Large B-Cell Lymphoma Mouse Models

IntroductionDLBCL is a commonly diagnosed, aggressive non-Hodgkin's lymphoma with ~40% of patients experiencing refractory or relapsed disease. Development of alternative therapies that target molecular features defining these unresponsive tumors is an active area of research to significantly advance the field and improve these patient's clinical management. However, few DLBCL animal models exist to test the efficacy of newly developed treatments, and are restricted to transgenic or xenograft mice that often fail to recapitulate the heterogeneous sub-classifications of this complex disease. While transgenic mice allow for spontaneous tumor formation, these models rely on inducing expression of specific oncogenes that drive a select group of DLBCL. The xenograft model offers several advantages, such as reproducing late-stage disease and shortening the model development time, but consist of implanting the tumor cells in a localized region or subcutaneously into immune-deficient mice. Despite some benefits of the transplant approach, these models are limited by their engraftment reproducibility and interactions with host micro-environments. Here, we explored the utility of humanizing Nod-Scid-IL2Rg null (NSG) mouse strains with factors associated with enhancing myeloid and lymphoma cell growth to establish a pipeline for rapid, reliable generation of in vivo DLBCL models.MethodsWe transduced the well-established human DLBCL cells, U2932, with the luciferase (Luc)-EGFP gene. The Luc-expressing (U2932-Luc) tumor cells were sorted for GFP positivity (GFP +) and expanded. The U2932-Luc cells (1 x 10 6/100µl PBS) were injected IV via tail vein into 8~12-week-old mice of various humanized NSG strains (representing equal numbers of each sex). NSG mice were humanized by transgenic expression of human cytokines (either human IL6 alone or IL6 plus SCF, GM-CSF, and IL-3) with the CMV promoter. Each experiment included of U2932-Luc cell transplanted group and control groups. We assessed U2932-Luc cell engraftment and growth by weekly in vivo imaging (IVIS 200 Imager, Perkin Elmer). To evaluate the organ specific engraftment/progression, we confirmed engraftment by bioluminescence imaging at the 2 nd week, then euthanized one mouse per week. At 15 minutes before euthansia, luciferin was injected via intraperitoneal injection. Following euthanasia, the organs were excised and underwent ex vivo IVIS imaging. The spleen, lungs, and liver were then fixed with 10% formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin, and an anti-CD20 antibody to evaluate the tumor morphology using a Zeiss AXIO Imager M2 microscope (Zeiss, Nashville, TN). All other mice were monitored for survival and the median survival between the IL6 and IL6/SGM3 mice were compared using the Log-rank test.ResultsSimilar to previously reported DLBCL humanized strain (MISTRG) (Hashwah, 2019), we used the IL6/SGM3 expressing strain. However, our studies also included the IL6 only humanized strain. We found that both the IL6 and IL6/SGM3 strains were highly permissive to DLBCL growth. The IL-6 strain exhibited a heightened growth of U2932 cells relative to the IL-6/SGM3 mice. As shown in Figure 1, the IL6 mice survived longer than IL6/SGM3 mice. Significant difference between the median survival of IL6 and IL6/SGM3 mice i.e. 48 days vs 42 days was observed (p < 0.0482). The organ specific evaluation demonstrated that U2932-Luc cells were initially engrafted and grew in the lung, liver, and spleen. Subsequently, U2932 cells were found in the skeleton, ovary, and brain. Of note, we detected significantly enlargements of the kidney, spleen, and ovary at the terminal stage.ConclusionsOur humanized mouse model approach of using U2932 human DLBCL cells transduced with the Luc gene in the NSG-IL6 and NSG-IL6/SGM3 mice reproduced the clinical features of an aggressive DLBCL that paralleled the original patient. This model will provide a new tool to enable expansion of patient samples while overcoming the current limitations of DLBCL xenografts and transgenic mice. The ability to maintain growth of patient-derived samples within clinically relevant locations has great potential to more accurately test patient-specific, personalized treatment strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An exocyst component, Sec5, is essential for ascospore formation in Bipolaris maydis.

In this study, we identified Sec5 in Bipolaris maydis, a homologue of Sec5 in Saccharomyces cerevisiae and a possible exocyst component of the fungus. To examine how Sec5 affects the life cycle of B. maydis, we generated null mutant strains of the gene (Δsec5). The Δsec5 strains showed a strong reduction in hyphal growth and a slight reduction in pathogenicity. In sexual reproduction, they possessed the ability to develop pseudothecia. However, all ascospores were aborted in any of the asci obtained from crosses between Δsec5 and the wild-type. Our cytological study revealed that the abortion was caused by impairments of the post-meiotic stages in ascospore development, where ascospore delimitation and young spore elongation occur.

MrGcn5 is required for the mycotoxin production, sexual and asexual development in Monascus ruber

Hongqu is a fermented product of Monascus species, which is widely used as food or food additives in Southeast Asian countries. The contamination of citrinin in Hongqu has caused controversy about its safety. So control of citrinin has always been an important issue of Hongqu products. In current study, we isolated a putative histone acetyltransferase named MrGcn5 and explored its regulatory function on citrinin production. The results showed the citrinin content of MrGcn5 null strain dramatically reduced in the late growth period. Even the citrinin content was as low as 21 % of the wild-type strain, and RT-qPCR experiment suggested that the expression level of genes involving in citrinin synthesis was significantly attenuated in MrGcn5 null strain. With regard to the asexual and sexual development, the conidia germination rate was significantly accelerated in MrGcn5 null strain in the vegetative growth phase, but the number of ascospores was 41.9 % less than the wild-type strain. RT-qPCR revealed the expression level of the homologs to flbA-flbE, fluG and wetA in Aspergillus had changed to some extent in MrGcn5 null strain. Meanwhile, there was no significant effects on the growth, nutrition adaption and Monascus azaphilone pigments production. Taken together, these characteristics of MrGcn5 make it very suitable for targeted improvement to reduce citrinin production in fermentation process.

Role of agglutinin-like sequence protein 3 (Als3) in the structure and antifungal resistance of Candida albicans biofilms.

Agglutinin-like sequence protein 3 (Als3) is a cell surface glycoprotein of Candida albicans that plays essential roles in the processes of adherence and biofilm formation in vitro. In this study, we focused on the contribution of Als3 to the structure and drug susceptibility of biofilms. The C. albicans wild-type (WT) strain DAY185, the als3Δ/Δ null strain and the als3Δ/Δ + pALS3 complemented strain were used. Colony-forming unit enumeration, crystal violet and cell surface hydrophobicity assays, scanning electron microscopy and confocal laser scanning microscopy coupled with analyses using COMSTAT software were performed to evaluate the biomass and architecture of the biofilms. The detailed architectural analysis showed a significant variation in the biofilm parameters of the als3Δ/Δ biofilms compared with those of the WT biofilms. Fluconazole, miconazole and amphotericin B were selected as the antifungal agents for the antimycotic susceptibility test, and increased susceptibility was found with the ALS3 deletion biofilms. A quantitative real-time polymerase chain reaction analysis showed downregulation of biofilm formation-related genes (ALS1, EFG1, HWP1 and CSH1) and drug resistance-related genes (ERG11, CDR1, CDR2 and MDR1) in the als3Δ/Δ biofilms. We concluded that Als3 contributes to biofilm formation by changing the biofilm architecture and is involved in the antifungal resistance of C. albicans biofilms.

An efficient i-GONAD method for creating and maintaining lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain.

As the efficiency of the clustered regularly interspaced short palindromic repeats/Cas system is extremely high, creation and maintenance of homozygous lethal mutants are often difficult. Here, we present an efficient in vivo electroporation method called improved genome editing via oviductal nucleic acid delivery (i-GONAD), wherein one of two alleles in the lethal gene was selectively edited in the presence of a non-targeted B6.C3H-In(6)1J inversion identified from the C3H/HeJJcl strain. This method did not require isolation, culture, transfer, or other in vitro handling of mouse embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. Using this strategy, we successfully generated the first Tprkb null knockout strain with an embryonic lethal mutation and showed that B6.C3H-In(6)1J can efficiently suppress recombination. As B6.C3H-In(6)1J was tagged with a gene encoding the visible coat color marker, Mitf, the Tprkb mutation could be visually recognized. We listed the stock balancer strains currently available as public bioresources to create these lethal gene knockouts. This method will allow for more efficient experiments for further analysis of lethal mutants.

Inhibition of Escherichia coli ATP synthase by dietary ginger phenolics

For centuries, dietary ginger has been known for its antioxidant, anticancer, and antibacterial properties. In the current study, we examined the link between antibacterial properties of 7 dietary ginger phenolics (DGPs)—gingerenone A, 6-gingerol, 8-gingerol, 10-gingerol, paradol, 6-shogaol, and zingerone—and inhibition of bacterial ATP synthase. DGPs caused complete (100%) inhibition of wild-type Escherichia coli membrane-bound F1Fo ATP synthase, but partial and variable (0%–87%) inhibition of phytochemical binding site mutant enzymes αR283D, αE284R, βV265Q, and γT273A. The mutant enzyme ATPase activity was 16-fold to 100-fold lower than that of the wild-type enzyme. The growth of wild-type, null, and mutant strains in the presence of the 7 DGPs were abrogated to variable degrees on limiting glucose and succinate media. DGPs-caused variable inhibitory profiles of wild-type and mutant ATP synthase confirm that residues of α-, β-, and γ-subunits are involved in the formation of phytochemical binding site. The variable degree of growth in the presence of DGPs also indicates the possibility of molecular targets other than ATP synthase. Our results establish that antibacterial properties of DGPs can be linked to the binding and inhibition of bacterial ATP synthase. Therefore, bacterial ATP synthase is a valuable molecular target for DGPs.

Atp23p and Atp10p coordinate to regulate the assembly of yeast mitochondrial ATP synthase.

Two chaperones, Atp23p and Atp10p, were previously shown to regulate the assembly of yeast mitochondrial ATP synthase, and extra expression of ATP23 was found to partially rescue an atp10 deletion mutant, by an unknown mechanism. Here, we identified that the residues 112-115 (LRDK) of Atp23p were required for its function in assisting assembly of the synthase, and demonstrated both functions of Atp23p, processing subunit 6 precursor and assisting assembly of the synthase, were required for the partial rescue of atp10 deletion mutant. By chasing labeling with isotope 35 S-methionine, we found the stability of subunit 6 of the synthase increased in atp10 null strain upon overexpression of ATP23. Further co-immunoprecipitation (Co-IP) and blue native PAGE experiments showed that Atp23p and Atp10p were physically associated with each other in wild type. Moreover, we revealed the expression level of Atp23p increased in atp10 null mutant compared with the wild type. Furthermore, we found that, after 72hours growth, atp10 null mutant showed leaky growth on respiratory substrates, presence of low level of subunit 6 and partial recovery of oligomycin sensitivity of mitochondrial ATPase activity. Further characterization revealed the expression of Atp23p increased after 24hours growth in the mutant. These results indicated, in atp10 null mutant, ATP10 deficiency could be partially complemented with increased expression of Atp23p by stabilizing some subunit 6 of the synthase. Taken together, this study revealed the two chaperones Atp23p and Atp10p coordinated to regulate the assembly of mitochondrial ATP synthase, which advanced our understanding of mechanism of assembly of yeast mitochondrial ATP synthase.

Molecular Characterization of Xyloglucanase cel74a from Trichoderma reesei.

Background: The filamentous fungus Trichoderma reesei is used on an industrial scale to produce enzymes of biotechnological interest. This fungus has a complex cellulolytic system involved in the degradation of lignocellulosic biomass. However, several aspects related to the regulation of the expression of holocellulolytic genes and the production of cellulases by this fungus are still understood. Methods: Here, we constructed a null mutant strain for the xyloglucanase cel74a gene and performed the characterization of the Δcel74a strain to evaluate the genetic regulation of the holocellulases during sugarcane bagasse (SCB) cultivation. Results: Our results demonstrate that the deletion of xyloglucanase cel74a may impact the regulation of holocellulase expression during SCB cultivation. The expression of cellulases cel7a, cel7b, and cel6a was reduced in Δcel74a strain, while the hemicellulases xyn1 and xyn2 were increased in the presence of SCB. The cel74a mutation also affected the xyloglucan hydrolysis patterns. In addition, CEL74A activity was modulated in the presence of calcium, suggesting that this ion may be required for efficient degradation of xyloglucan. Conclusions: CEL74A affects the regulation of holocellulolytic genes and the efficient degradation of SCB in T. reesei. This data makes a significant contribution to our understanding of the carbon utilization of fungal strains as a whole.

Identification of novel pentose transporters in Kluyveromyces marxianus using a new screening platform.

ABSTRACTThe capacity of yeasts to assimilate xylose or arabinose is strongly dependent on plasma membrane transport proteins. Because pentoses comprise a substantial proportion of available sugars in lignocellulosic hydrolysates, their utilisation is centrally important for the development of second generation biorefineries. Relatively few native pentose transporters have been studied and there is intense interest in expanding the repertoire. To aid the identification of novel transporters, we developed a screening platform in the native pentose-utilising yeast Kluyveromyces marxianus. This involved the targeted deletion of twelve transporters of the major facilitator superfamily (MFS) and application of a synthetic biology pipeline for rapid testing of candidate pentose transporters. Using this K. marxianus ΔPT platform, we identified several K. marxianus putative xylose or arabinose transporter proteins that recovered a null strain's ability to growth on these pentoses. Four proteins of the HGT-family were able to support growth in media with high or low concentrations of either xylose or arabinose, while six HXT-like proteins displayed growth only at high xylose concentrations, indicating solely low affinity transport activity. The study offers new insights into the evolution of sugar transporters in yeast and expands the set of native pentose transporters for future functional and biotechnological studies.

Role of stathmin microtubule-destabilizing activity in Shigella flexneri motility and tunneling

The infection of the intestinal mucosa by Shigella bacteria is a global health issue resulting in a variety of potentially life-threatening gastrointestinal complications. Their unique method of intracellular motility depends on microtubule destabilization to clear the dense host cytoskeletal network in a process called tunneling. It is hypothesized that the host protein stathmin may play a role in this process, due to its tubulin-sequestering capability. This proposal aims to provide potential methodologies to elucidate the function of stathmin with respect to Shigella flexneri motility. Three experiments are proposed, involving comparisons between human intestinal epithelial cell strains under varying levels of stathmin expression, each infected with S. flexneri. Respectively, the experiments examine tunnel widths via electron microscopy, microtubule densities via imaging fluorescence correlation spectroscopy, and bacterial movement patterns via live fluorescence microscopy. If microtubule destabilization and movement is impaired in null stathmin strains, as predicted, such findings may inform a novel therapeutic target for Shigellosis by preventing internal spreading. This is particularly significant in our current landscape, as antibiotic-resistant strains of Shigella are growing increasingly prevalent.