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: Combination effect of engineered endolysin EC340 with antibiotics.

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

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.

Comparison of Enterococcus faecalis Biofilm Removal Efficiency among Bacteriophage PBEF129, Its Endolysin, and Cefotaxime.

Enterococcus faecalis is a Gram-positive pathogen which colonizes human intestinal surfaces, forming biofilms, and demonstrates a high resistance to many antibiotics. Especially, antibiotics are less effective for eradicating biofilms and better alternatives are needed. In this study, we have isolated and characterized a bacteriophage, PBEF129, infecting E. faecalis. PBEF129 infected a variety of strains of E. faecalis, including those exhibiting antibiotic resistance. Its genome is a linear double-stranded DNA, 144,230 base pairs in length. Its GC content is 35.9%. The closest genomic DNA sequence was found in Enterococcus phage vB_EfaM_Ef2.3, with a sequence identity of 99.06% over 95% query coverage. Furthermore, 75 open reading frames (ORFs) were functionally annotated and five tRNA-encoding genes were found. ORF 6 was annotated as a phage endolysin having an L-acetylmuramoyl-l-alanine amidase activity. We purified the enzyme as a recombinant protein and confirmed its enzymatic activity. The endolysin's host range was observed to be wider than its parent phage PBEF129. When applied to bacterial biofilm on the surface of in vitro cultured human intestinal cells, it demonstrated a removal efficacy of the same degree as cefotaxime, but much lower than its parent bacteriophage.

Phage-Antibiotic Synergy via Delayed Lysis.

When phages infect bacteria cultured in the presence of sublethal doses of antibiotics, the sizes of the phage plaques are significantly increased. This phenomenon is known as phage-antibiotic synergy (PAS). In this study, the observation of PAS was extended to a wide variety of bacterium-phage pairs using different classes of antibiotics. PAS was shown in both Gram-positive and Gram-negative bacteria. Cells stressed with ß-lactam antibiotics filamented or swelled extensively, resulting in an increase in phage production. PAS was also sometimes observed in the presence of other classes of antibiotics with or without bacterial filamentation. The addition of antibiotics induced recA expression in various bacteria, but a recA deletion mutant strain of Escherichia coli also showed filamentation and PAS in the presence of quinolone antibiotics. The phage adsorption efficiency did not change in the presence of the antibiotics when the cell surfaces were enlarged as they filamented. Increases in the production of phage DNA and mRNAs encoding phage proteins were observed in these cells, with only a limited increase in protein production. The data suggest that PAS is the product of a prolonged period of particle assembly due to delayed lysis. The increase in the cell surface area far exceeded the increase in phage holin production in the filamented host cells, leading to a relatively limited availability of intracellular holins for aggregating and forming holes in the host membrane. Reactive oxygen species (ROS) stress also led to an increased production of phages, while heat stress resulted in only a limited increase in phage production.IMPORTANCE Phage-antibiotic synergy (PAS) has been reported for a decade, but the underlying mechanism has never been vigorously investigated. This study shows the presence of PAS from a variety of phage-bacterium-antibiotic pairings. We show that increased phage production resulted directly from a lysis delay caused by the relative shortage of holin in filamented bacterial hosts in the presence of sublethal concentrations of stress-inducing substances, such as antibiotics and reactive oxygen species (ROS).

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.

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.

Nanosized polyamidoamine (PAMAM) dendrimer-induced apoptosis mediated by mitochondrial dysfunction.

Nano-materials are currently being used in a variety of fields. One of the concerns associated with their use is their potential to harm human health. In an attempt to identify genes expressed differently in human lung cells (WI-26 VA4) exposed to nanosized (45 nm in diameter) PAMAM (polyamidoamine) dendrimers, we observed down-regulation of mitochondrial DNA-encoded genes involved in the maintenance of mitochondrial membrane potential. Down-regulation of gene expression was confirmed by semi-quantitative RT-PCR. Dendrimers were shown to colocalize with mitochondria and cause the release of cytochrome C. Mitochondrial membrane potential was disrupted and the viability of cells was decreased in the presence of dendrimers. Activation of caspases 3 and 9 was increased. Apoptosis was observed by annexin V/propidium iodide staining and DNA fragmentation. In summary, nanosized dendrimers damaged mitochondria resulting in apoptosis.

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.

Onset of Voluntary Communication: Smiling Looks to Mother.

Playing infants often direct smiling looks toward social partners. In some cases the smile begins before the look, so it cannot be a response to the sight or behavior of the social partner. In this study we asked whether smiles that anticipate social contact are used by 8- to 12-month-old infants as voluntary social signals. Eighty infants-20 at each of 8, 9, 10, and 12 months of age-completed 5 tasks. The tasks assessed anticipatory smiling during toy play, means-end understanding (2 tasks), intentional communication via gesture and vocalizations, and memory for mother's location. Across all ages, anticipatory smiling was strongly predicted by intentional gestural and vocal communication and by means-end understanding. The findings are discussed in terms of the nature and origins of infants' voluntary communications.