Unveiling the mechanism of bactericidal activity of a cecropin A-fused endolysin LNT113.

Endolysins are lytic enzymes produced by bacteriophages at the end of their lytic cycle and degrade the peptidoglycan layer of the bacterial cell wall. Thus, they have been extensively explored as a promising antibacterial agent to replace or supplement current antibiotics. Gram-negative bacteria, however, are prone to resist exogenous endolysins owing to their protective outer membrane. We previously engineered endolysin EC340, encoded by the Escherichia coli phage PBEC131, by substituting its seven amino acids and fusing an antimicrobial peptide cecropin A at its N-terminus. The engineered endolysin LNT113 exerted superior activity to its intrinsic form. This study investigated how cecropin A fusion facilitated the bactericidal activity of LNT113 toward Gram-negative bacteria. Cecropin A of LNT113 markedly increased the interaction with lipopolysaccharides, while the E. coli defective in the core oligosaccharide was less susceptible to endolysins, implicating the interaction between the core oligosaccharide and endolysins. In fact, E. coli with compromised lipid A construction was more vulnerable to LNT113 treatment, suggesting that the integrity of the lipid A layer was important to resist the internalization of LNT113 across the outer membrane. Cecropin A fusion further accelerated the inner membrane destabilization, thereby enabling LNT113 to deconstruct it promptly. Owing to the increased membrane permeability, LNT113 could inactivate some Gram-positive bacteria as well. This study demonstrates that cecropin A fusion is a feasible method to improve the membrane permeability of endolysins in both Gram-negative and Gram-positive bacteria.

Corrigendum: Motility increase of Adherent Invasive Escherichia coli (AIEC) induced by a sub-inhibitory concentration of recombinant endolysin LysPA90.

[This corrects the article DOI: 10.3389/fmicb.2022.1093670.].

Corrigendum: Combination effect of engineered endolysin EC340 with antibiotics.

[This corrects the article DOI: 10.3389/fmicb.2022.821936.].

Balancing growth amidst salt stress - lifestyle perspectives from the extremophyte model Schrenkiella parvula.

Schrenkiella parvula, a leading extremophyte model in Brassicaceae, can grow and complete its lifecycle under multiple environmental stresses, including high salinity. Yet, the key physiological and structural traits underlying its stress-adapted lifestyle are unknown along with trade-offs when surviving salt stress at the expense of growth and reproduction. We aimed to identify the influential adaptive trait responses that lead to stress-resilient and uncompromised growth across developmental stages when treated with salt at levels known to inhibit growth in Arabidopsis and most crops. Its resilient growth was promoted by traits that synergistically allowed primary root growth in seedlings, the expansion of xylem vessels across the root-shoot continuum, and a high capacity to maintain tissue water levels by developing thicker succulent leaves while enabling photosynthesis during salt stress. A successful transition from vegetative to reproductive phase was initiated by salt-induced early flowering, resulting in viable seeds. Self-fertilization in salt-induced early flowering was dependent upon filament elongation in flowers otherwise aborted in the absence of salt during comparable plant ages. The maintenance of leaf water status promoting growth, and early flowering to ensure reproductive success in a changing environment, were among the most influential traits that contributed to the extremophytic lifestyle of S. parvula.

Characterization of Three Different Endolysins Effective against Gram-Negative Bacteria.

Genes encoding endolysins were identified and cloned from three different Escherichia coli bacteriophages, 10-24(13), PBEC30, and PBEC56. Putative antimicrobial peptide (AMP)-like C-terminal alpha helix structures with amphipathic natures were predicted from the three endolysins. Each gene was cloned and expressed as hexahistidine-tagged forms, and the products were purified and characterized. The purified endolysins exhibited antibacterial activities against a variety of Gram-negative bacteria including Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumonia. Their antibacterial activities were improved by N-terminal fusion with an antimicrobial peptide, cecropin A. Minimum inhibitory concentrations (MIC) were as low as 4 µg/mL, depending on the targeted strain. The endolysins' enzymatic activities were not affected by changes in pH at ranges from 5 to 10 and were stable at temperatures between 4 and 65 °C. The in vivo efficacies of the three endolysins were also demonstrated using Galleria melonella for infection models.

Combination Effect of Engineered Endolysin EC340 With Antibiotics.

Bacteriophage lysins, also known as endolysins or murein hydrolases, are hydrolytic enzymes produced by bacteriophages during the final stage of the lytic cycle to enable cleavage through the host's cell wall, thus allowing the phages to burst out of their host bacteria after multiplication inside them. When applied externally to Gram-negative bacteria as recombinant proteins, lysins cannot easily reach the cell wall due to the presence of an outer membrane (OM). In this study, endolysin EC340 obtained from phage PBEC131 infecting Escherichia coli was engineered for improved OM permeability and increased activity against Gram-negative bacteria. The engineered endolysin, LNT113, was tested for potential synergistic effects with standard-of-care antibiotics. A synergistic effect was demonstrated with colistin, while an additive effect was seen with meropenem, tigecycline, chloramphenicol, azithromycin, and ciprofloxacin. Neither ceftazidime nor kanamycin showed any synergy or additive effects with the LNT113 endolysin. Moreover, synergy and additive effects could not be generalized by antibiotic class, OM traverse mechanism, molecular weight, or the bactericidal nature of each antibiotic tested.

Motility increase of adherent invasive Escherichia coli (AIEC) induced by a sub-inhibitory concentration of recombinant endolysin LysPA90.

Endolysins are bacteriophage enzymes required for the eruption of phages from inside host bacteria via the degradation of the peptidoglycan cell wall. Recombinant endolysins are increasingly being seen as potential antibacterial candidates, with a number currently undergoing clinical trials. Bacteriophage PBPA90 infecting Pseudomonas aeruginosa harbors a gene encoding an endolysin, lysPA90. Herein, recombinant LysPA90 demonstrated an intrinsic antibacterial activity against Escherichia coli in vitro. It was observed that a sub-inhibitory concentration of the recombinant protein induced the upregulation of genes related to flagella biosynthesis in a commensal E. coli strain. Increases in the number of bacterial flagella, and in motility, were experimentally substantiated. The treatment caused membrane stress, leading to the upregulation of genes rpoE, rpoH, dnaK, dnaJ, and flhC, which are upstream regulators of flagella biosynthesis. When adherent invasive Escherichia coli (AIEC) strains were treated with subinhibitory concentrations of the endolysin, bacterial adhesion and invasion into intestinal epithelial Caco-2 cells was seen to visibly increase under microscopic examination. Bacterial counting further corroborated this adhesion and invasion of AIEC strains into Caco-2 cells, with a resultant slight decrease in the viability of Caco-2 cells then being observed. Additionally, genes related to flagella expression were also upregulated in the AIEC strains. Finally, the enhanced expression of the proinflammatory cytokine genes TNF-α, IL-6, IL-8, and MCP1 in Caco-2 cells was noted after the increased invasion of the AIEC strains. While novel treatments involving endolysins offer great promise, these results highlight the need for the further exploration of possible unanticipated and unintended effects.

A Role for Arabidopsis miR399f in Salt, Drought, and ABA Signaling.

MiR399f plays a crucial role in maintaining phosphate homeostasis in Arabidopsis thaliana. Under phosphate starvation conditions, AtMYB2, which plays a role in plant salt and drought stress responses, directly regulates the expression of miR399f. In this study, we found that miR399f also participates in plant responses to abscisic acid (ABA), and to abiotic stresses including salt and drought. Salt and ABA treatment induced the expression of miR399f, as confirmed by histochemical analysis of promoter-GUS fusions. Transgenic Arabidopsis plants overexpressing miR399f (miR399f-OE) exhibited enhanced tolerance to salt stress and exogenous ABA, but hypersensitivity to drought. Our in silico analysis identified ABF3 and CSP41b as putative target genes of miR399f, and expression analysis revealed that mRNA levels of ABF3 and CSP41b decreased remarkably in miR399f-OE plants under salt stress and in response to treatment with ABA. Moreover, we showed that activation of stress-responsive gene expression in response to salt stress and ABA treatment was impaired in miR399f-OE plants. Thus, these results suggested that in addition to phosphate starvation signaling, miR399f might also modulates plant responses to salt, ABA, and drought, by regulating the expression of newly discovered target genes such as ABF3 and CSP41b.

The Arabidopsis a zinc finger domain protein ARS1 is essential for seed germination and ROS homeostasis in response to ABA and oxidative stress.

The phytohormone abscisic acid (ABA) induces accumulation of reactive oxygen species (ROS), which can disrupt seed dormancy and plant development. Here, we report the isolation and characterization of an Arabidopsis thaliana mutant called ars1 (aba and ros sensitive 1) that showed hypersensitivity to ABA during seed germination and to methyl viologen (MV) at the seedling stage. ARS1 encodes a nuclear protein with one zinc finger domain, two nuclear localization signal (NLS) domains, and one nuclear export signal (NES). The ars1 mutants showed reduced expression of a gene for superoxide dismutase (CSD3) and enhanced accumulation of ROS after ABA treatment. Transient expression of ARS1 in Arabidopsis protoplasts strongly suppressed ABA-mediated ROS production. Interestingly, nuclear-localized ARS1 translocated to the cytoplasm in response to treatment with ABA, H2O2, or MV. Taken together, these results suggest that ARS1 modulates seed germination and ROS homeostasis in response to ABA and oxidative stress in plants.

Genome structures and transcriptomes signify niche adaptation for the multiple-ion-tolerant extremophyte Schrenkiella parvula.

Schrenkiella parvula (formerly Thellungiella parvula), a close relative of Arabidopsis (Arabidopsis thaliana) and Brassica crop species, thrives on the shores of Lake Tuz, Turkey, where soils accumulate high concentrations of multiple-ion salts. Despite the stark differences in adaptations to extreme salt stresses, the genomes of S. parvula and Arabidopsis show extensive synteny. S. parvula completes its life cycle in the presence of Na⁺, K⁺, Mg²âº, Li⁺, and borate at soil concentrations lethal to Arabidopsis. Genome structural variations, including tandem duplications and translocations of genes, interrupt the colinearity observed throughout the S. parvula and Arabidopsis genomes. Structural variations distinguish homologous gene pairs characterized by divergent promoter sequences and basal-level expression strengths. Comparative RNA sequencing reveals the enrichment of ion-transport functions among genes with higher expression in S. parvula, while pathogen defense-related genes show higher expression in Arabidopsis. Key stress-related ion transporter genes in S. parvula showed increased copy number, higher transcript dosage, and evidence for subfunctionalization. This extremophyte offers a framework to identify the requisite adjustments of genomic architecture and expression control for a set of genes found in most plants in a way to support distinct niche adaptation and lifestyles.

Induced degradation of Tat by nucleocapsid (NC) via the proteasome pathway and its effect on HIV transcription.

Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). HIV-1 Tat protein upregulates transcriptional transactivation. The nucleocapsid protein NC of HIV-1 is a component of virion and plays a key role in genome packaging. Herein, we have demonstrated the interaction between NC and Tat by means of a yeast two-hybrid assay, GST pull-down analysis, co-immunoprecipitation and subcellular colocalization analysis. We observed that the level of Tat was significantly reduced in the presence of NC. But NC did not affect mRNA expression level of Tat. The level of Tat in the presence of NC was increased by treating cells with a proteasome inhibitor, MG132. The ubiquitination state of Tat was not seen to increase in the presence of NC, suggesting the proteasomal degradation was independent of ubiquitination. Lowered level of Tat in the presence of NC led to a decrease in Tat-mediated transcriptional transactivation.

Regulation of miR399f transcription by AtMYB2 affects phosphate starvation responses in Arabidopsis.

Although a role for microRNA399 (miR399) in plant responses to phosphate (Pi) starvation has been indicated, the regulatory mechanism underlying miR399 gene expression is not clear. Here, we report that AtMYB2 functions as a direct transcriptional activator for miR399 in Arabidopsis (Arabidopsis thaliana) Pi starvation signaling. Compared with untransformed control plants, transgenic plants constitutively overexpressing AtMYB2 showed increased miR399f expression and tissue Pi contents under high Pi growth and exhibited elevated expression of a subset of Pi starvation-induced genes. Pi starvation-induced root architectural changes were more exaggerated in AtMYB2-overexpressing transgenic plants compared with the wild type. AtMYB2 directly binds to a MYB-binding site in the miR399f promoter in vitro, as well as in vivo, and stimulates miR399f promoter activity in Arabidopsis protoplasts. Transcription of AtMYB2 itself is induced in response to Pi deficiency, and the tissue expression patterns of miR399f and AtMYB2 are similar. Both genes are expressed mainly in vascular tissues of cotyledons and in roots. Our results suggest that AtMYB2 regulates plant responses to Pi starvation by regulating the expression of the miR399 gene.

TsHKT1;2, a HKT1 homolog from the extremophile Arabidopsis relative Thellungiella salsuginea, shows K(+) specificity in the presence of NaCl.

Cellular Na(+)/K(+) ratio is a crucial parameter determining plant salinity stress resistance. We tested the function of plasma membrane Na(+)/K(+) cotransporters in the High-affinity K(+) Transporter (HKT) family from the halophytic Arabidopsis (Arabidopsis thaliana) relative Thellungiella salsuginea. T. salsuginea contains at least two HKT genes. TsHKT1;1 is expressed at very low levels, while the abundant TsHKT1;2 is transcriptionally strongly up-regulated by salt stress. TsHKT-based RNA interference in T. salsuginea resulted in Na(+) sensitivity and K(+) deficiency. The athkt1 mutant lines overexpressing TsHKT1;2 proved less sensitive to Na(+) and showed less K(+) deficiency than lines overexpressing AtHKT1. TsHKT1;2 ectopically expressed in yeast mutants lacking Na(+) or K(+) transporters revealed strong K(+) transporter activity and selectivity for K(+) over Na(+). Altering two amino acid residues in TsHKT1;2 to mimic the AtHKT1 sequence resulted in enhanced sodium uptake and loss of the TsHKT1;2 intrinsic K(+) transporter activity. We consider the maintenance of K(+) uptake through TsHKT1;2 under salt stress an important component supporting the halophytic lifestyle of T. salsuginea.

The genome of the extremophile crucifer Thellungiella parvula.

Thellungiella parvula is related to Arabidopsis thaliana and is endemic to saline, resource-poor habitats, making it a model for the evolution of plant adaptation to extreme environments. Here we present the draft genome for this extremophile species. Exclusively by next generation sequencing, we obtained the de novo assembled genome in 1,496 gap-free contigs, closely approximating the estimated genome size of 140 Mb. We anchored these contigs to seven pseudo chromosomes without the use of maps. We show that short reads can be assembled to a near-complete chromosome level for a eukaryotic species lacking prior genetic information. The sequence identifies a number of tandem duplications that, by the nature of the duplicated genes, suggest a possible basis for T. parvula's extremophile lifestyle. Our results provide essential background for developing genomically influenced testable hypotheses for the evolution of environmental stress tolerance.

NKS1, Na(+)- and K(+)-sensitive 1, regulates ion homeostasis in an SOS-independent pathway in Arabidopsis.

An Arabidopsis thaliana mutant, nks1-1, exhibiting enhanced sensitivity to NaCl was identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated two closely linked mutations in the gene encoded at locus At4g30996. A second allele, nks1-2, was obtained from the Arabidopsis Biological Resource Center. NKS1 mRNA was detected in all parts of wild-type plants but was not detected in plants of either mutant, indicating inactivation by the mutations. Both mutations in NKS1 were associated with increased sensitivity to NaCl and KCl, but not to LiCl or mannitol. NaCl sensitivity was associated with nks1 mutations in Arabidopsis lines expressing either wild type or null alleles of SOS1, SOS2 or SOS3. The NaCl-sensitive phenotype of the nks1-2 mutant was complemented by expression of a full-length NKS1 allele from the CaMV35S promoter. When grown in medium containing NaCl, nks1 mutants accumulated more Na(+) than wild type and K(+)/Na(+) homeostasis was perturbed. It is proposed NKS1, a plant-specific gene encoding a 19kDa endomembrane-localized protein of unknown function, is part of an ion homeostasis regulation pathway that is independent of the SOS pathway.

Genome structures and halophyte-specific gene expression of the extremophile Thellungiella parvula in comparison with Thellungiella salsuginea (Thellungiella halophila) and Arabidopsis.

The genome of Thellungiella parvula, a halophytic relative of Arabidopsis (Arabidopsis thaliana), is being assembled using Roche-454 sequencing. Analyses of a 10-Mb scaffold revealed synteny with Arabidopsis, with recombination and inversion and an uneven distribution of repeat sequences. T. parvula genome structure and DNA sequences were compared with orthologous regions from Arabidopsis and publicly available bacterial artificial chromosome sequences from Thellungiella salsuginea (previously Thellungiella halophila). The three-way comparison of sequences, from one abiotic stress-sensitive species and two tolerant species, revealed extensive sequence conservation and microcolinearity, but grouping Thellungiella species separately from Arabidopsis. However, the T. parvula segments are distinguished from their T. salsuginea counterparts by a pronounced paucity of repeat sequences, resulting in a 30% shorter DNA segment with essentially the same gene content in T. parvula. Among the genes is SALT OVERLY SENSITIVE1 (SOS1), a sodium/proton antiporter, which represents an essential component of plant salinity stress tolerance. Although the SOS1 coding region is highly conserved among all three species, the promoter regions show conservation only between the two Thellungiella species. Comparative transcript analyses revealed higher levels of basal as well as salt-induced SOS1 expression in both Thellungiella species as compared with Arabidopsis. The Thellungiella species and other halophytes share conserved pyrimidine-rich 5' untranslated region proximal regions of SOS1 that are missing in Arabidopsis. Completion of the genome structure of T. parvula is expected to highlight distinctive genetic elements underlying the extremophile lifestyle of this species.

Functional characterization of the SIZ/PIAS-type SUMO E3 ligases, OsSIZ1 and OsSIZ2 in rice.

Sumoylation is a post-translational regulatory process in diverse cellular processes in eukaryotes, involving conjugation/deconjugation of small ubiquitin-like modifier (SUMO) proteins to other proteins thus modifying their function. The PIAS [protein inhibitor of activated signal transducers and activators of transcription (STAT)] and SAP (scaffold attachment factor A/B/acinus/PIAS)/MIZ (SIZ) proteins exhibit SUMO E3-ligase activity that facilitates the conjugation of SUMO proteins to target substrates. Here, we report the isolation and molecular characterization of Oryza sativa SIZ1 (OsSIZ1) and SIZ2 (OsSIZ2), rice homologs of Arabidopsis SIZ1. The rice SIZ proteins are localized to the nucleus and showed sumoylation activities in a tobacco system. Our analysis showed increased amounts of SUMO conjugates associated with environmental stresses such as high and low temperature, NaCl and abscisic acid (ABA) in rice plants. The expression of OsSIZ1 and OsSIZ2 in siz1-2 Arabidopsis plants partially complemented the morphological mutant phenotype and enhanced levels of SUMO conjugates under heat shock conditions. In addition, ABA-hypersensitivity of siz1-2 seed germination was partially suppressed by OsSIZ1 and OsSIZ2. The results suggest that rice SIZ1 and SIZ2 are able to functionally complement Arabidopsis SIZ1 in the SUMO conjugation pathway. Their effects on the Arabidopsis mutant suggest a function for these genes related to stress responses and stress adaptation.

Selection of peptides binding to HCV e2 and inhibiting viral infectivity.

The envelope glycoprotein E2 of hepatitis C virus (HCV) binds to various cell surface receptors for viral infection. We performed biopanning against this protein and selected peptides from phage display peptide libraries. Two short peptides, pep7-1 and pep12-1, were selected and their ability to inhibit the infection process was investigated. When pep7-1 was present, the infectivity of HCV particles in cell culture was notably decreased. This decrease was demonstrated by Western blot analysis, immunofluorescence assay, and reverse transcription PCR assay. However, pep12-1 showed little inhibitory effect on HCV infection.

Comparison of acute responses of mice livers to short-term exposure to nano-sized or micro-sized silver particles.

Mice were fed either 13 nm silver nanoparticles or 2-3.5 mum silver microparticles. The livers were then obtained after 3 days and subjected to a histopathological analysis. The nanoparticle-fed and microparticle-fed livers both exhibited lymphocyte infiltration in the histopathological analysis, suggesting the induction of inflammation. In vitro, a human hepatoma cell line (Huh-7) was treated with the same silver nanoparticles and microparticles. The mitochondrial activity and glutathione production were hardly affected. However, the DNA contents decreased 15% in the nanoparticle-treated cells and 10% in the microparticle-treated cell, suggesting a more potent induction of apoptosis by the nanoparticles. From a microarray analysis of the RNA from the livers of the nano- and micro-particle-fed mice, the expression of genes related to apoptosis and inflammation was found to be altered. These gene expression changes in the nanoparticle-treated livers lead to phenotypical changes, reflecting increased apoptosis and inflammation. The changes in the gene expression were confirmed by using a semi-quantitative RT-PCR.