Mutant Constructs Research Articles

Construction of VcrV-deleted mutant of Vibrio alginolyticus and its biological characteristics

A mutant strain ΔVcrV was constructed by using homologous recombination method for investigating the function of the VcrV gene in Vibrio alginolyticus type Ⅲ secretion system. The genetic stability of ΔVcrV was detected by PCR, and the biological characteristics between the mutant and the wild type strains were compared. ΔVcrV muntat had no significant changes in growth rate and autoagglutination compared with the wild type strain, but the ability to form biofilms was reduced, and the LD50 was increased by 16.5 times. The swimming and swarming motility of the mutant strain ΔVcrV were significantly enhanced, while cell adhesion was significantly reduced than the wild strain (P < 0.01). The tolerance of ΔVcrV mutant to H2O2 and NaCl was decreased. Compared with that of the wild type strain, the sensitivity of ΔVcrV mutant to cefuroxime, medimycin and clindamycin was increased, but to amikacin and polymxin B was decreased. The reactive oxygen species (ROS) content of ΔVcrV mutant was significantly decreased (P < 0.01), and the indexes of proline, peptidoglycan, β-lactamase, catalase, superoxide dismutase and glutathione peroxidase of ΔVcrV mutant were significantly increased than that of the wild type strain (P < 0.01). The biological characteristics of ΔVcrV mutant indicated that VcrV gene was involved in pathogenicity and various biological functions of V. alginolyticus type Ⅲ secretion system.

Biochemical Characterizations of the Putative Endolysin Ecd09610 Catalytic Domain from Clostridioides difficile.

Clostridioides difficile is the major pathogen of pseudomembranous colitis, and novel antimicrobial agents are sought after for its treatment. Phage-derived endolysins with species-specific lytic activity have potential as novel antimicrobial agents. We surveyed the genome of C. difficile strain 630 and identified an endolysin gene, Ecd09610, which has an uncharacterized domain at the N-terminus and two catalytic domains that are homologous to glucosaminidase and endopeptidase at the C-terminus. Genes containing the two catalytic domains, the glucosaminidase domain and the endopeptidase domain, were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. The purified domain variants showed lytic activity almost specifically for C. difficile, which has a unique peptide bridge in its peptidoglycan. This species specificity is thought to depend on substrate cleavage activity rather than binding. The domain variants were thermostable, and, notably, the glucosaminidase domain remained active up to 100 °C. In addition, we determined the optimal pH and salt concentrations of these domain variants. Their properties are suitable for formulating a bacteriolytic enzyme as an antimicrobial agent. This lytic enzyme can serve as a scaffold for the construction of high lytic activity mutants with enhanced properties.

A Novel NADP(H)-Dependent 7alpha-HSDH: Discovery and Construction of Substrate Selectivity Mutant by C-Terminal Truncation

7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays an important role in the biosynthesis of tauroursodeoxycholic acid (TUDCA) using complex substrate chicken bile powder as raw material. However, chicken bile powder contains 4.74% taurocholic acid (TCA), and a new by-product tauroursocholic acid (TUCA) will be produced, having the risk of causing colorectal cancer. Here, we obtained a novel NADP(H)-dependent 7α-HSDH with good thermostability from Ursus thibetanus gut microbiota (named St-2-2). St-2-2 could catalyze taurochenodeoxycholic acid (TCDCA) and TCA with the catalytic activity of 128.13 and 269.39 U/mg, respectively. Interestingly, by a structure-based C-terminal truncation strategy, St-2-2△C10 only remained catalytic activity on TCDCA (14.19 U/mg) and had no activity on TCA. As a result, it can selectively catalyze TCDCA in waste chicken bile powder. MD simulation and structural analysis indicated that enhanced surface hydrophilicity and improved C-terminal rigidity affected the entry and exit of substrates. Hydrogen bond interactions between different subunits and interaction changes in Phe249 of the C-terminal loop inverted the substrate catalytic activity. This is the first report on substrate selectivity of 7α-HSDH by C-terminal truncation strategy and it can be extended to other 7α-HSDHs (J-1-1, S1-a-1).

Combinational Deletions of MGF360-9L and MGF505-7R Attenuated Highly Virulent African Swine Fever Virus and Conferred Protection against Homologous Challenge.

ABSTRACTMultigene family (MGF) gene products are increasingly reported to be implicated in African swine fever virus (ASFV) virulence and attenuation of host defenses, among which the MGF360-9L and MGF505-7R gene products are characterized by convergent but distinct mechanisms of immune evasion. Herein, a recombinant ASFV mutant, ASFV-Δ9L/Δ7R, bearing combinational deletions of MGF360-9L and MGF505-7R, was constructed from the highly virulent ASFV strain CN/GS/2018 of genotype II that is currently circulating in China. Pigs inoculated intramuscularly with 104 50% hemadsorption doses (HAD50) of the mutant remained clinically healthy without any serious side effects. Importantly, in a virulence challenge, all four within-pen contact pigs demonstrated clinical signs and pathological findings consistent with ASF. In contrast, vaccinated pigs (5/6) were protected and clinical indicators tended to be normal, accompanied by extensive tissue repairs. Similar to most viral infections, innate immunity and both humoral and cellular immune responses appeared to be vital for protection. Notably, transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR) analysis revealed a regulatory function of the mutant in dramatic and sustained expression of type I/III interferons and inflammatory and innate immune genes in vitro. Furthermore, infection with the mutant elicited an early and robust p30-specific IgG response, which coincided and was strongly correlated with the protective efficacy. Analysis of the cellular response revealed a strong ASFV-specific interferon gamma (IFN-γ) response and immunostaining of CD4+ T cells coupled with a high level of CD163+ macrophage infiltration in spleens of vaccinated pigs. Our study identifies a new mechanism of immunological regulation by ASFV MGFs that rationalizes the design of live attenuated vaccine for implementation of improved control strategies to eradicate ASFV.IMPORTANCE Currently, the deficiency in commercially available vaccines or therapeutic options against African swine fever constitutes a matter of major concern in the swine industry globally. Here, we report the design and construction of a recombinant ASFV mutant harboring combinational deletions of interferon inhibitors MGF360-9L and MGF505-7R based on a genotype II ASFV CN/GS/2018 strain currently circulating in China. The mutant was completely attenuated when inoculated at a high dose of 104 HAD50. In the virulence challenge with homologous virus, sterile immunity was achieved, demonstrating the mutant’s potential as a promising vaccine candidate. This sufficiency of effectiveness supports the claim that this live attenuated virus may be a viable vaccine option with which to fight ASF.

Identification and characterization of TGF-β1-responsive Runx2 acetylation sites for matrix Metalloproteinase-13 expression in osteoblastic cells

In skeletal tissues, transforming growth factor-beta 1 (TGF-β1) serves a number of activities. For example, in osteoblastic cells, TGF-β1 stimulates the expression of matrix metalloproteinase-13 (MMP-13, a bone remodeling gene), which requires the bone transcription factor Runx2. Although TGF-β1 is known to stimulate Runx2 acetylation, the sites involved in MMP-13 gene activation remain unknown. Mass spectrometry analysis revealed that Runx2 was acetylated at one site (K134) and three sites (K24, K134, and K169) following control and TGF-β1-treatment, respectively, in osteoblastic cells. In addition, we mutated the lysine residues in the Runx2 construct into arginine and transfected the construct into mouse mesenchymal stem cells (C3H10T1/2). Wild-type Runx2 expression and acetylation were significantly increased by TGF-β1-treatment, whereas this effect was decreased in the presence of the Runx2 double mutant construct (K24 + K169) in C3H10T1/2 cells. TGF-β1 enhanced MMP-13 promoter activity in cells transfected with the wild-type Runx2 construct, but this effect was considerably reduced in cells transfected with the Runx2 double mutant construct (K24 + K169), according to a luciferase reporter test. Hence, the stability of Runx2 may be mediated by TGF-β1-induced acetylation at K24 and K169 and is required for MMP-13 expression in osteoblastic cells. These findings add to our knowledge of TGF-β1, Runx2, and MMP-13's physiological roles in bone metabolism.

Identification of the Novel G250R Variant Indicates a Role for Thrombomodulin in Modulating the Risk for Venous Thromboembolism

Thrombomodulin displays anticoagulant properties through thrombin binding and protein C activation. In close relatives who had a history of recurrent VTE, a missense variant (G250R) in the EGF-1 domain was identified. In an asymptomatic 250R carrier, higher thrombin generation plasma values were found. Evaluation of cells transfected with the wild-type or the mutant construct using imaging technologies and flow cytometry provided evidences that the 250R variant significantly decreased protein localization on cell membrane. The different pattern of cell distribution suggested that the 250R variant is not able to sufficiently target the plasma membrane. Present findings add information to characterize protein structure-function relationships, further suggesting that misfolding of the protein modulates the expression of mature protein. Functional analyses of the 250R variant increase our understanding of the role of Thrombomodulin in coagulation and support the hypothesis that gene variants reducing protein functionality cause deficiency and behave as a thrombophilic risk factor.

Solution Structural Studies of Pre-amyloid Oligomer States of the Biofilm Protein Aap

Staphylococcus epidermidis is a commensal bacterium on human skin that is also the leading cause of medical device-related infections. The accumulation-associated protein (Aap) from S. epidermidis is a critical factor for infection via its ability to mediate biofilm formation. The B-repeat superdomain of Aap is composed of 5 to 17 Zn2+-binding B-repeats, which undergo rapid, reversible assembly to form dimer and tetramer species. The tetramer can then undergo a conformational change and nucleate highly stable functional amyloid fibrils. In this study, multiple techniques including analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) are used to probe a panel of B-repeat mutant constructs that assemble to distinct oligomeric states to define the structural characteristics of B-repeat dimer and tetramer species. The B-repeat region from Aap forms an extremely elongated conformation that presents several challenges for standard SAXS analyses. Specialized approaches, such as cross-sectional analyses, allowed for in-depth interpretation of data, while explicit-solvent calculations via WAXSiS allowed for accurate evaluation of atomistic models. The resulting models suggest mechanisms by which Aap functional amyloid fibrils form, illuminating an important contributing factor to recurrent staphylococcal infections.

Abstract 2947: Surveying the tumor suppressive genetic network underlying chr4p deletion in TNBC

Abstract Triple negative breast cancer (TNBC) is a breast cancer subtype lacking targetable biomarkers, resulting in the worst prognosis compared to other breast cancer subtypes. TNBC is characterized by many large copy number variants that result in the deletion and amplifications of many genes, with TP53 being the only common oncogenic driver. Using TCGA data and in-depth functional genomic analysis of TNBC patient-derived xenografts (PDX), our group showed that chr4p is a recurrently deleted region in basal breast cancer, which TNBC is an enriched subtype. This correlated with poor prognosis and a highly proliferative state. Here, we set out to survey the tumor suppressive genetic network underlying the TNBC-specific chr4pdeletion. Using an arrayed CRISPR-enCas12 screening approach, I will generate a panel of mutant cell lines deleted for all protein-coding genes residing within chr4p. MCF10A series of cell lines will be used for mutant cell line construction, because it is an established normal human breast epithelial model system with a normal karyotype to ensure the diploid state ofchr4p and includes other derivatives (MCF10A(-E7-Bcl2)) that show basal anchorage independent growth in 3D to assess cell transformation. The resulting panel of single gene deletion mutant cell lines will be characterized for their effects on proliferation, apoptosis, cell transformation and senescence. Additionally, the tumor suppressive genetic interaction network of chr4p will be mapped using a multiplexed CRISPR-enCas12 screening methodology. A dual guide-RNA library will be generated for all protein-coding genes to test all pairwise combinations for tumor suppressive genetic interactions. The proliferation due to double gene deletions will be monitored and compared to single gene deletions to identify tumor suppressive interactions. This study will be the first to systematically identify tumor suppressor genetic network underlying chr4p. Ultimately, it will provide an in-depth understanding of the genetic network of large copy number variants in TNBC and insight into new avenues for precision oncology. Citation Format: Joseph Del Corpo, Rohan Dandage, Lea Harrington, Elena Kuzmin. Surveying the tumor suppressive genetic network underlying chr4p deletion in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2947.

Abstract 899: Effects of innate immune deaminase deregulation on initiation and progression of myeloproliferative neoplasms

Abstract Inflammatory cytokine responsive APOBEC3 cytidine deaminases have been studied extensively with regard to innate immunity and more recently during cancer evolution. However, the mechanisms by which the APOBEC3 enzymes promote cancer initiation and progression in the malignant microenvironment remains to be investigated, especially in hematopoietic malignancies. Through whole genome and whole transcriptome sequencing analyses of MPN patient samples FACS sorted into stem and progenitor populations, we have found a cell type and context specific nature of these enzymes, notably the upregulation of APOBEC3C (A3C) in the high-risk Myelofibrosis (MF) stem cell population as compared to normal aged counterparts. Through lentiviral overexpression of each APOBEC3 enzyme, we can now study the effects of changes in APOBEC3 transcript level in relation to the known changes in expression seen in many cancers, focusing on the upregulation of A3C. Using these techniques, we have identified novel RNA and DNA editing targets, as well as differential gene expression patterns of each APOBEC3 in normal CD34+ cord blood and aged normal bone marrow. Gene set enrichment analysis (GSEA) performed on this dataset has exposed numerous deregulated pathways brought on by exaggerated levels of APOBEC3, including changes in splicing pathways. In addition, novel identification of the relationship between A3C and ADAR1, another innate immune deaminase, has important implications in initiation and prognosis of MPNs. Both A3C and ADAR1 transcript levels are elevated in high risk MF stem cells, and co-immunoprecipitation studies reveal a direct binding of the enzymes. Furthermore, we are able to study the detailed effects of editing by both A3C and ADAR1 using editase-deficient mutant constructs, allowing for a mechanistic look into the role of these deaminases and their deregulation in vitro and in vivo using patient samples and humanized mouse models. This novel connection, as well as the role of A3C in initiation and progression of hematopoietic malignancies will continued to be studied using this system to elucidate effects on proliferation, differentiation, self-renewal, and changes to the cell cycle, in hopes of creating both a marker of early detection and potentially a druggable target. Citation Format: Jane Marie Isquith, Adam Mark, Jessica Pham, Mary Donohoe, Luisa Ladel, Catriona Jamieson. Effects of innate immune deaminase deregulation on initiation and progression of myeloproliferative neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 899.

Abstract 1472: Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer

Abstract Nuclear factor E2-related factor 2 (NRF2) is a master regulator of cellular redox homeostasis. The sustained overactivation of NRF2 has been observed in many different types of human malignancy, conferring an advantage for growth and survival of cancer cells. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) recognizes phosphorylated serine or threonine of a target protein and isomerizes the adjacent proline residue, thereby altering the structure, stability, and function of target proteins. We identified the three specific serine residues (Ser215, 408, and 577) present in NRF2, each of which precedes proline and undergoes phosphorylation. This prompted us to investigate whether PIN1 could bind to NRF2 and thereby influence its stability. For this purpose, we initially co-transfected HEK293T cells with expression vectors for GST-PIN1 and HA-tagged wild-type Nrf2 or non-phosphorylatable mutant constructs (NRF2S215A, NRF2S408A, and NRF2S577A). Notably, there was the most pronounced ubiquitination with concomitant degradation of NRF2 in cells harbouring NRF2S215A which was associated with abrogation of its interaction with PIN1. We then investigated the clinical relevance of NRF2 and PIN1 interaction to breast cancer progression. Immunofluorescence analysis of tissue microarrays (90 breast cancer tissues and 10 adjacent normal ones) revealed much higher (P &amp;lt; 0.001) expression of both NRF2 and PIN1 in the invasive ductal carcinomas than in normal tissues, which correlated with disease stages. The overexpression of NRF2 and PIN1 in breast cancer patients was also confirmed by immunohistochemical and immunoblot analyses. Next, we compared the co-localization as well as expression of NRF2 and PIN1 in the two molecular subtype tissues (140 each). Both expression and co-localization of NRF2 and PIN1 were significantly higher (P &amp;lt; 0.001) in more aggressive TNBC than in the luminal type tissues. We also detected the significantly elevated levels of the Pin1-Nrf2 complex formed in human breast cancer specimens, compared with those in adjacent normal tissues. Silencing of Pin1 reduced the nuclear accumulation of NRF2 which was attributable to enhanced ubiquitination. The cycloheximide pulse-chase assay unravelled a significantly faster degradation of pre-existing NRF2 in PIN1-silenced MDA-MB-231 cells, corroborating the stabilization of NRF2 protein by PIN1. To explore the regulation of NRF2 by PIN1 and their interaction in vivo, we inoculated MDA-MB-231 cells into male BALB/c nude mice. Tumors derived from PIN1 knockdown cells displayed markedly reduced levels of NRF2. In conclusion, stabilization of NRF2 through direct interaction with PIN1 may provide a novel mechanism underlying their cooperative role in breast cancer growth and progression. Citation Format: Soma Saeidi, Su-Jung Kim, Yanymee N Guillen-Quispe, Achanta Sri Venkata Jagadeesh, Young-Joon Surh. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1472.

Evaluating the Contribution of the Predicted Toxin-Antitoxin System HigBA to Persistence, Biofilm Formation, and Virulence in Burkholderia pseudomallei.

Melioidosis is an underreported human disease caused by the Gram-negative intracellular pathogen Burkholderia pseudomallei (Bpm). Both the treatment and the clearance of the pathogen are challenging, with high relapse rates leading to latent infections. This has been linked to the bacterial persistence phenomenon, a growth arrest strategy that allows bacteria to survive under stressful conditions, as in the case of antibiotic treatment, within a susceptible clonal population. At a molecular level, this phenomenon has been associated with the presence of toxin-antitoxin (TA) systems. We annotated the Bpm K96243 genome and selected 11 pairs of genes encoding for these TA systems, and their expression was evaluated under different conditions (supralethal antibiotic conditions; intracellular survival bacteria). The predicted HigB toxin (BPSL3343) and its predicted antitoxin HigA (BPS_RS18025) were further studied using mutant construction. The phenotypes of two mutants (ΔhigB and ΔhigB ΔhigA) were evaluated under different conditions compared to the wild-type (WT) strain. The ΔhigB toxin mutant showed a defect in intracellular survival on macrophages, a phenotype that was eliminated after levofloxacin treatment. We found that the absence of the toxin provides an advantage over the WT strain, in both in vitro and in vivo models, during persister conditions induced by levofloxacin. The lack of the antitoxin also resulted in differential responses to the conditions evaluated, and under some conditions, it restored the WT phenotype, overall suggesting that both toxin and antitoxin components play a role in the persister-induced phenotype in Bpm.

Involvement of the hemP-hemA-smlt0796-smlt0797 Operon in Hemin Acquisition by Stenotrophomonas maltophilia.

ABSTRACTThe hemin acquisition system of Stenotrophomonas maltophilia was elucidated in this study. To identify the TonB-dependent outer membrane receptor for hemin in S. maltophilia, the hemin acquisition systems of Pseudomonas aeruginosa were referenced. PhuR, HasA, and HxuA are three known TonB-dependent outer membrane receptors involved in hemin acquisition by P. aeruginosa. Thus, HemA (Smlt0795) and Smlt2937, the orthologs of PhuR and HasA/HxuA in S. maltophilia, were first considered. KJΔEnt, a stenobactin-null strain, was used as the parental strain for the hemin utilization assay. Deletion of hemA, but not Smlt2937, of KJΔEnt impaired hemin acquisition under iron-depleted conditions, indicating that HemA is the TonB-dependent receptor for hemin uptake. The hemA gene is a member of the hemP-hemA-smlt0796-smlt0797 operon, whose expression was upregulated in a fur mutant and under iron-depleted conditions. The contribution of the hemP-hemA-smlt0796-smlt0797 operon to hemin acquisition was investigated by in-frame deletion mutant construction and hemin utilization assays. Inactivation of hemP, smlt0796, and smlt0797 of KJΔEnt insignificantly affected hemin acquisition under iron-depleted conditions. However, hemP deletion in a fur mutant increased hemin acquisition under iron-depleted conditions. Collectively, we revealed that (i) HemA likely functions as the outer membrane receptor for hemin uptake; (ii) HemP, a predicted transcriptional factor, apparently functions as a repressor of the expression of the hemA transcript; and (iii) in a fur mutant, HemP has a negative impact on hemin acquisition under iron-depleted conditions.IMPORTANCE Stenotrophomonas maltophilia is an emerging multidrug-resistant opportunistic pathogen, increasing the difficulty of treatment of this infection. Iron is a critical element for bacterial viability. Heme is the most abundant iron source in the human host; thus, heme is the major iron source for a pathogen in the infection niche. Blocking iron acquisition from heme can be an alternative strategy to control S. maltophilia infection. Although several hemin acquisition systems have been reported in various pathogens, very little is known about the hemin acquisition systems of S. maltophilia. By in-frame deletion mutant construction and hemin utilization assays, we demonstrated that HemA (Smlt0795) is the TonB-dependent outer membrane receptor for hemin uptake and that HemP (Smlt0794), a predicted transcriptional factor, had a negative impact on hemin acquisition in a fur mutant. The negative regulatory role of HemP in hemin acquisition is first reported.

Electrophysiological characterization of schizophrenia-associated variants in NaV1.2 sodium channel

IntroductionA major pathophysiological hypothesis of schizophrenia states an increased activity of glutamatergic neurons leading to an imbalance of neural excitation and inhibition (E/I-imbalance). One potential molecular mechanism of E/I-imbalance is a dysfunction of voltage-gated sodium channels, which are crucial for the generation of action potentials, the fundamental event of neuronal excitation. Indeed, patients with schizophrenia exhibit an increased burden of rare exonic variants of sodium channel genes, but the literature describing their electrophysiological effect is scarce.ObjectivesThe aim of this project is to assess the functional impact of three mutations of the Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A) gene / NaV1.2 channel which were identified in four patients with schizophrenia, using a heterologous expression system.MethodsThree variants of the human SCN2A gene (R850P, V1282F and S1656P) were created using site-directed mutagenesis. HEK293T cells transfected with either the mutant or wild type constructs are being investigated by voltage-clamp technique, applying activation, steady-state fast inactivation, use dependency and ramp protocols.ResultsAll three mutated constructs were successfully created. Preliminary recordings from the V1282F mutant indicate a shift of both the activation and steady-state fast inactivation to the hyperpolarized direction.ConclusionsIn a subgroup of patients, E/I imbalance may be a consequence of Nav1.2 mutations leading to increased excitability of glutamatergic neurons. By integrating insights from different mutations we aim to identify traits of a potentially shared disease pathway which may provide a basis for the development of novel therapeutics.DisclosureNo significant relationships.

Rapid Hypothesis Testing in Candida albicans Clinical Isolates Using a Cloning-Free, Modular, and Recyclable System for CRISPR-Cas9 Mediated Mutant and Revertant Construction.

ABSTRACTAs increasing evidence emerges that interstrain genetic diversity among Candida albicans clinical isolates underpins phenotypic variation compared to the reference isolate SC5314, new genetic tools are required to interrogate gene function across strain backgrounds. Here, the SAT1-flipper plasmid was reengineered to contain a C. albicans codon optimized hygromycin B resistance gene (CaHygB). Cassettes were PCR-amplified from both SAT1-flipper and CaHygB-flipper plasmids using primers with homologous sequences flanking target genes of interest to serve as repair templates. Ribonucleoprotein (RNP) complexes containing proprietary CRISPR RNAs (crRNAs), universal transactivating CRISPR RNA (tracrRNA), and Cas9 protein were assembled in vitro and transformed, along with both repair templates, by electroporation into C. albicans. Homozygous deletion of the ADE2 gene results in red-pigmented colonies and this gene was used to validate our approach. Both in SC5314 and a variety of clinical isolates (529L, JS15, SJCA1, TW1), homozygous gene targeting was nearly 100% when plating on media containing nourseothricin and hygromycin B with transformation efficiencies exceeding 104 homozygous deletion mutants per μg of DNA. A gene reversion system was also employed with plasmids pDUP3 and pDIS3 engineered to contain the ADH1 terminator and an overlap extension PCR-mediated approach combined with CRISPR-Cas9 targeting at the NEUT5 neutral locus. A variety of single or compound mutants (Δ/Δals3, Δ/Δcph1 Δ/Δefg1, Δ/Δece1) and their revertant strains were constructed and phenotypically validated by a variety of assays, including biofilm formation, hyphal growth, and macrophage IL-1β response. Thus, we have established a cloning-free, modular system for highly efficient homozygous gene deletion and reversion in diverse isolates.IMPORTANCE Recently, phenotypic heterogeneity in Candida albicans isolates has been recognized as an underappreciated factor contributing to gene diversification and broadly impacts strain-to-strain antifungal resistance, fitness, and pathogenicity. We have designed a cloning-free genetic system for rapid gene deletion and reversion in C. albicans clinical isolates that interlaces established recyclable genetic systems with CRISPR-Cas9 technology. The SAT1-flipper was reengineered to contain CaHygB encoding resistance to hygromycin B. Using a modular PCR-mediated approach coupled with in vitro ribonucleoprotein assembly with commercial reagents, both SAT1- and CaHygB-flipper cassettes were simultaneously integrated at loci with high efficiency (104 transformants per μg DNA) and upward of 99% homozygous gene targeting across a collection of diverse isolates of various anatomical origin. Revertant strains were constructed by overlap extension PCR with CRISPR-Cas9 targeted integration at the NEUT5 locus. Thus, this facile system will aid in unraveling the genetic factors contributing to the complexity of intraspecies diversity.

Toxic SOD1 trimers are off-pathway in the formation of amyloid-like fibrils in ALS

Accumulation of insoluble amyloid fibrils is widely studied as a critical factor in the pathology of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Misfolded Cu, Zn superoxide dismutase (SOD1) was the first protein linked to ALS, and non-native SOD1 trimeric oligomers were recently linked to cytotoxicity, while larger oligomers were protective to cells. The balance between trimers and larger aggregates in the process of SOD1 aggregation is, thus, a critical determinant of potential therapeutic approaches to treat ALS. However, it is unknown whether these trimeric oligomers are a necessary intermediate for larger aggregate formation or a distinct off-pathway species competing with fibril formation. Depending on the on- or off-pathway scenario of trimer formation, we expect drastically different therapeutic approaches. Here, we show that the toxic SOD1 trimer is an off-pathway intermediate competing with protective fibril formation. We design mutant SOD1 constructs that remain in a trimeric state (super-stable trimers) and show that stabilizing the trimeric SOD1 prevents formation of fibrils in vitro and in a motor neuron-like cell model (NSC-34). Using size exclusion chromatography, we track the aggregation kinetics of purified SOD1 and show direct competition of trimeric SOD1 with larger oligomer and fibril formation. Finally, we show the trimer is structurally independent of both larger soluble oligomers and insoluble fibrils using circular dichroism spectroscopy and limited proteolysis.

The Impact of Profilin‐1 Mutations on Protein Homeostasis in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease characterized by the dysfunction and degeneration of motor neurons in the central nervous system. ALS has been linked to a number of genetic mutations, including point mutations in the actin‐binding protein profilin‐1 (Pfn1). While the prevalence of Pfn1 mutations is low, around 1% of inherited forms of the disease, an understanding of their impact on motor neuron cell death can provide insight into the mechanisms underlying ALS onset and progression. As Pfn1 is an important regulator of the actin cytoskeleton, we hypothesize that ALS‐linked mutations of Pfn1 promote defects in protein homeostasis and synaptic communication. To test this hypothesis, neuronal cells were transfected with Pfn1 constructs (including wild‐type, C71G, and M118V) and subjected to cellular stressors (including heat shock and sodium arsenite treatments). Immunostaining was then used to visualize and quantify differences in protein aggregation and stress granule formation. Our results have established the baseline response with the wild‐type construct and indicate that there is an increase in protein aggregation as well as in the number and size of stress granules with the mutant constructs. Our ongoing studies are aimed at validating these preliminary results, and our goal is to provide mechanistic insight pertaining to Pfn1’s role in ALS disease pathogenesis.

LL-37 transports immunoreactive cGAMP to activate STING signaling and enhance interferon-mediated host antiviral immunity.

Cyclic 2',3'-GMP-AMP (cGAMP) binds to and activates stimulator of interferon genes (STING), which then induces interferons to drive immune responses against tumors and pathogens. Exogenous cGAMP produced by infected and malignant cells and synthetic cGAMP used in immunotherapy must traverse the cell membrane to activate STING in target cells. However, as an anionic hydrophilic molecule, cGAMP is not inherently membrane permeable. Here, we show that LL-37, a human host defense peptide, can function as a transporter of cGAMP. LL-37 specifically binds cGAMP and efficiently delivers cGAMP into target cells. cGAMP transferred by LL-37 activates robust interferon responses and host antiviral immunity in a STING-dependent manner. Furthermore, we report that LL-37 inducers vitamin D3 and sodium butyrate promote host immunity by enhancing endogenous LL-37 expression and its mediated cGAMP immune response. Collectively, our data uncover an essential role of LL-37 in innate immune activation and suggest new strategies for immunotherapy.

The fciTABC and feoABI systems contribute to ferric citrate acquisition in Stenotrophomonas maltophilia

BackgroundStenotrophomonas maltophilia, a member of γ-proteobacteria, is a ubiquitous environmental bacterium that is recognized as an opportunistic nosocomial pathogen. FecABCD system contributes to ferric citrate acquisition in Escherichia coli. FeoABC system, consisting of an inner membrane transporter (FeoB) and two cytoplasmic proteins (FeoA and FeoC), is a well-known ferrous iron transporter system in γ-proteobacteria. As revealed by the sequenced genome, S. maltophilia appears to be equipped with several iron acquisition systems; however, the understanding of these systems is limited. In this study, we aimed to elucidate the ferric citrate acquisition system of S. maltophilia.MethodsCandidate genes searching and function validation are the strategy for elucidating the genes involved in ferric citrate acquisition. The candidate genes responsible for ferric citrate acquisition were firstly selected using FecABCD of E. coli as a reference, and then revealed by transcriptome analysis of S. maltophilia KJ with and without 2,2′-dipyridyl (DIP) treatment. Function validation was carried out by deletion mutant construction and ferric citrate utilization assay. The bacterial adenylate cyclase two-hybrid system was used to verify intra-membrane protein–protein interaction.ResultsSmlt2858 and Smlt2356, the homologues of FecA and FecC/D of E. coli, were first considered; however, deletion mutant construction and functional validation ruled out their involvement in ferric citrate acquisition. FciA (Smlt1148), revealed by its upregulation in DIP-treated KJ cells, was the outer membrane receptor for ferric citrate uptake. The fciA gene is a member of the fciTABC operon, in which fciT, fciA, and fciC participated in ferric citrate acquisition. Uniquely, the Feo system of S. maltophilia is composed of a cytoplasmic protein FeoA, an inner membrane transporter FeoB, and a predicted inner membrane protein FeoI. The intra-membrane protein–protein interaction between FeoB and FeoI may extend the substrate profile of FeoB to ferric citrate. FeoABI system functioned as an inner membrane transporter of ferric citrate.ConclusionsThe FciTABC and FeoABI systems contribute to ferric citrate acquisition in S. maltophilia.

Construction of a phosphodiesterase mutant and evaluation of its potential as an effective live attenuated vaccine in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus)

Vibrio alginolyticus is a dominant pathogen that causes vibriosis of fish and shellfish. VAGM003125 is a specific phosphodiesterase bearing HD-GYP domain, which extensively regulates multicellular behavior and physiological processes in bacteria. In this study, an in-frame deleted ΔVAGM003125 mutant was constructed and changes of ΔVAGM003125 mutant in physiology and pathogenicity were examined. The potential application of ΔVAGM003125 mutant as a live attenuated vaccine was also assessed. The ΔVAGM003125 mutant displayed no significant differences in the growth rate and morphology in comparison to the wild type strain. However, the ΔVAGM003125 mutant significantly enhanced biofilm formation compared to the wild type strain. Also, the ΔVAGM003125 mutant was noted as being able to attenuate swarming motility, ECPase, and adherence compared to the wild type strain. Moreover, the ΔVAGM003125 mutant induced high antibody titers and provided effective immune protection, which was evidenced with a relative survival rate of 81% without histopathological abnormality. Following ΔVAGM003125 mutant vaccination, immune-related genes of pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus) including IgM, MHC-Iα, IL-16, IL-1, and TNF-α was up-regulated. Taken together, the present data suggested that the ΔVAGM003125 mutant might be applied as an attenuated live vaccination against V. alginolyticus during fish culture.

Construction of spvD gene deletion mutant and compensation strains in Salmonella enteritidis and its effects on Caco-2 cells

Objective: To analyze the effects of spvD gene on invasion and intracellular proliferation of Caco-2 cells and in order to provide insight into the function of that gene and the underlying mechanism of Salmonella caused infection. Methods: Functional verification of spvD gene deletion mutant and compensation strain. The deletion mutant strain was constructed through a suicide plasmid-mediated homologous recombination. The compensation plasmid constructed by cloning the coding sequence of spvD by PCR into plasmid pBAD33 was mobilized into the deletion mutant by conjugation and the pBAD33 was introduced into wild strains and deleted mutant strains as control. The relative expression of spvD mRNA was detected by quantitative reverse transcription PCR. In order to analyze the virulence of spvD against Caco-2 cells, Caco-2 cells was cocultured with wild type Salmonella enteritidis carrying spvD gene, the deletion mutant strain and compensation strain respectively. The expression level of spvD mRNA and the the number of Salmonella enteritidis after Caco-2 cells intervention were compared between the three groups by LSD-t test, and the invasion rate was compared by χ2 test. Results: The expression level of spvD mRNA in wild type Salmonella enteritidis was set as unit "1", the deletion mutant strain was "0.00", and the compensation strain was "2.60" (LSD-twild, deleted=1.11, P=0.31; LSD-twild, compensation=-1.77, P=0.13; LSD-t deleted, compensation=-2.88, P=0.03), which confirmed the successful construction of the deletion mutant strain and the compensation strain. The invasion experiment results of the above three Salmonella enteritidis strains on Caco-2 cells showed that the invasion rate of wild strain was 0.23%, the invasion rate of deleted mutant strain was 0.16%, and the invasion rate of compensation strain was 0.16%, with no statistical significance (χ2=1.13, P=0.570). By comparing the number of Salmonella enteritidis at different time points after Caco-2 cells intervention, it was discovered that the number of Salmonella enteritidis in wild strains (6.50×106 CFU/ml) and compensation strains (7.25×106 CFU/ml) was significantly increased than that in deletion mutant strain (1.90×106 CFU/ml) after 16 h coculture (LSD-twild, deleted=7.95, P=0.00; LSD-twild, compensation=-1.27, P=0.25; LSD-t deleted, compensation=-9.22, P=0.00). Conclusion: It is not considered that spvD gene can affect the invasion of Salmonella enteritidis on Caco-2 cells, but the gene can promote the reproduction of Salmonella enteritidis in Caco-2 cells.