The SapA Protein Is Involved in Resistance to Antimicrobial Peptide PR-39 and Virulence of Actinobacillus pleuropneumoniae

Antimicrobial peptides are essential to the innate immune defense of the mammal against bacterial infection. However, pathogenic bacteria have evolved multiple strategies to resist and evade antimicrobial peptides, which is vital to bacterial survival and colonization in hosts. PR-39 is a linear porcine antimicrobial peptide containing 39 amino acid residues with a high proline content. Resistance to antimicrobial peptide PR-39 has been observed in Actinobacillus pleuropneumoniae. However, little is known about the factors required for this resistance. In the present study, PR-39 exposure increased the expression of the sapA gene in A. pleuropneumoniae. The sapA gene, which encodes a putative peptide transport periplasmic protein, was deleted from this bacterium. The ΔsapA mutant showed increased sensitivity to PR-39 compared to the wild-type MD12 and complemented PΔsapA strains. However, the ΔsapA mutant did not exhibit any alterations in outer membrane integrity. Scanning electron microscopy showed that the ΔsapA mutant displayed morphological defects, as indicated by a deformed and sunken shape after PR-39 treatment. In addition, disruption of the SapA protein led to reduced colonization and attenuated virulence of A. pleuropneumoniae in the BALB/c mouse model. Collectively, these data suggest that SapA acts as one mechanism for A. pleuropneumoniae to counteract PR-39-mediated killing. To the best of our knowledge, this is the first study to show a mechanism underlying antimicrobial peptide resistance in A. pleuropneumoniae

A Review of Polymer-Based Environment-Induced Nanogenerators: Power Generation Performance and Polymer Material Manipulations

Natural environment hosts a considerable amount of accessible energy, comprising mechanical, thermal, and chemical potentials. Environment-induced nanogenerators are nanomaterial-based electronic chips that capture environmental energy and convert it into electricity in an environmentally friendly way. Polymers, characterized by their superior flexibility, lightweight, and ease of processing, are considered viable materials. In this paper, a thorough review and comparison of various polymer-based nanogenerators were provided, focusing on their power generation principles, key materials, power density and stability, and performance modulation methods. The latest developed nanogenerators mainly include triboelectric nanogenerators (TriboENG), piezoelectric nanogenerators (PENG), thermoelectric nanogenerators (ThermoENG), osmotic power nanogenerator (OPNG), and moist-electric generators (MENG). Potential practical applications of polymer-based nanogenerator were also summarized. The review found that polymer nanogenerators can harness a variety of energy sources, with the basic power generation mechanism centered on displacement/conduction currents induced by dipole/ion polarization, due to the non-uniform distribution of physical fields within the polymers. The performance enhancement should mainly start from strengthening the ion mobility and positive/negative ion separation in polymer materials. The development of ionic hydrogel and hydrogel matrix composites is promising for future nanogenerators and can also enable multi-energy collaborative power generation. In addition, enhancing the uneven distribution of temperature, concentration, and pressure induced by surrounding environment within polymer materials can also effectively improve output performance. Finally, the challenges faced by polymer-based nanogenerators and directions for future development were prospected

Cloning, Expression, Purification, and Characterization of β-Galactosidase from Bifidobacterium longum and Bifidobacterium pseudocatenulatum

Expression and purification of β-galactosidases derived from Bifidobacterium provide a new resource for efficient lactose hydrolysis and lactose intolerance alleviation. Here, we cloned and expressed two β-galactosidases derived from Bifidobacterium. The optimal pH for BLGLB1 was 5.5, and the optimal temperature was 45 °C, at which the enzyme activity of BLGLB1 was higher than that of commercial enzyme E (300 ± 3.6 U/mg) under its optimal conditions, reaching 2200 ± 15 U/mg. The optimal pH and temperature for BPGLB1 were 6.0 and 45 °C, respectively, and the enzyme activity (0.58 ± 0.03 U/mg) under optimum conditions was significantly lower than that of BLGLB1. The structures of the two β-galactosidase were similar, with all known key sites conserved. When o-nitrophenyl-β-D-galactoside (oNPG) was used as an enzyme reaction substrate, the maximum reaction velocity (Vmax) for BLGLB1 and BPGLB1 was 3700 ± 100 U/mg and 1.1 ± 0.1 U/mg, respectively. The kinetic constant (Km) of BLGLB1 and BPGLB1 was 1.9 ± 0.1 and 1.3 ± 0.3 mmol/L, respectively. The respective catalytic constant (kcat) of BLGLB1 and BPGLB1 was 1700 ± 40 s−1 and 0.5 ± 0.02 s−1, respectively; the respective kcat/Km value of BLGLB1 and BPGLB1 was 870 L/(mmol∙s) and 0.36 L/(mmol∙s), respectively. The Km, kcat and Vmax values of BLGLB1 were superior to those of earlier reported β-galactosidase derived from Bifidobacterium. Overall, BLGLB1 has potential application in the food industry

Lysozyme-like Protein Produced by Bifidobacterium longum Regulates Human Gut Microbiota Using In Vitro Models

The extracellular secreted protein of Bifidobacterium longum (B. longum) plays an important role in maintaining the homeostasis of the human intestinal microenvironment. However, the mechanism(s) of interaction remain unclear. Lysozyme is a kind of antibacterial peptide. In this study, the amino acid sequence of a lysozyme-like protein of B. longum based on whole-genome data of an isolate from human gut feces was found. We further predicted functional domains from the amino acid sequence, purified the protein, and verified its bioactivity. The growth of some bacteria were significantly delayed by the 020402_LYZ M1 protein. In addition, the gut microbiota was analyzed via high-throughput sequencing of 16S rRNA genes and an in vitro fermentation model, and the fluctuations in the gut microbiota under the treatment of 020402_LYZ M1 protein were characterized. The 020402_LYZ M1 protein affected the composition of human gut microbiota significantly, implying that the protein is able to communicate with intestinal microbes as a regulatory factor

Chromosomal inactivation of <i>the pdxS</i> and <i>pdxT</i> genes.

<p>(A) Schematic representation of the <i>A</i>. <i>pleuropneumoniae pdxS</i> locus. The figure shows the binding locations for the oligonucleotide primers used to amplify the two flanking regions (1265 bp and 1294 bp, respectively) used in the construction of the pEM△<i>pdxS</i> plasmid and the diagnostic PCR analysis of the S-8Δ<i>pdxS</i> mutant (1106 bp) and WT S-8 strains (1573 bp). The S-8Δ<i>pdxS</i> mutant contains a 487 bp deletion (shadowed domain) in the <i>pdxS</i> gene. (B) (A) Schematic representation of the <i>A</i>. <i>pleuropneumoniae pdxT</i> locus. The figure shows the binding locations for the oligonucleotide primers used to amplify the two flanking regions (1377 bp and 1387 bp, respectively) used in the construction of the pEM△<i>pdxT</i> plasmid and the diagnostic PCR analysis of the S-8Δ<i>pdxT</i> mutant (660 bp) and WT S-8 strains (1151 bp). The S-8Δ<i>pdxT</i> mutant contains a 491 bp deletion (shadowed domain) in the <i>pdxT</i> gene. (C) PCR identification of the S-8Δ<i>pdxS</i> mutant using the primers SCF/SCR. For lane 1, the WT S-8 strain (1573 bp); for lane 2, the S-8Δ<i>pdxS</i> mutant (1106 bp); for lane 3, the complemented S-8Δ<i>pdxS</i>comp strain (1573 and 1106 bp). (D) PCR identification of the S-8Δ<i>pdxT</i> mutant using the primers SCF/TCR. For lane 1, the WT S-8 strain (2151bp); for lane 2, the S-8Δ<i>pdxT</i> mutant (1660 bp); for lane 3, the complemented S-8Δ<i>pdxT</i>comp strain (1660 and 1264 bp). (E) PCR identification of the S-8Δ<i>pdxS</i> mutant using the primers RTSF/RTSR. For lane 1, the WT S-8 strain (214 bp); for lane 2, the S-8Δ<i>pdxS</i> mutant; for lane 3, the complemented S-8Δ<i>pdxS</i>comp strain (214 bp). (F) PCR identification of the S-8Δ<i>pdxT</i> mutant using the primers RTTF/RTTR. For lane 1, the WT S-8 strain (267 bp); for lane 2, the S-8Δ<i>pdxT</i> mutant; for lane 3, the complemented S-8Δ<i>pdxT</i>comp strain (267 bp). For lane M, DL2000 DNA marker (from top to bottom: 2000, 1000, 750, 500, 250, and 100 bp).</p

Response of <i>A</i>. <i>pleuropneumoniae</i> mutants to oxidative and osmotic stresses.

<p>Overnight culture of WT S-8, S-8<i>ΔpdxS</i>, S-8<i>ΔpdxT</i>, S-8Δ<i>pdxS</i>comp strain and S-8Δ<i>pdxT</i>comp strains were diluted into fresh BHI broth and grown to OD₆₀₀ 0.8. Bacteria were then treated with 5 mM H₂O₂ in the absence (A) and presence (B) of PLP for 45 min, 0.4 M NaCl in the absence (C) and presence (D) of PLP for 45 min. Values of stress resistance were calculated as [(stressed sample CFU/ ml)/ (control sample CFU/ ml)]×100. The data shown are the means of three independent assays, and error bars indicate standard deviations. **, <i>p</i> < 0.01; *, <i>p</i> < 0.05.</p

Transmission electron microscopy of <i>A</i>. <i>pleuropneumoniae</i> mutants in the absence of PLP.

<p>TEM of WT S-8, S-8<i>ΔpdxS</i>, S-8<i>ΔpdxT</i>, S-8Δ<i>pdxS</i>comp strain and S-8Δ<i>pdxT</i>comp strains in the mid-log phase in the absence of PLP were carried out. The cell morphology of the S-8Δ<i>pdxS</i> mutant and S-8Δ<i>pdxT</i> mutant exhibited irregular and aberrant shapes, showing holes and deep crater on their surface. S-8Δ<i>pdxS</i> also appeared partially lysed or swollen. Scale bar = 5μm.</p

PLP synthase activity of PdxS in the presence of ammonium.

<p>(A) The growth curves of the WT S-8, S-8Δ<i>pdxS</i>, S-8Δ<i>pdxT</i> mutant, S-8Δ<i>pdxS</i>comp strain and S-8Δ<i>pdxT</i>comp strains in the presence of ammonium supplementation. Overnight culture of each strain was diluted into fresh CDM medium supplemented with 1mM ammonium sulfate. Bacteria were grown at 37°C for 10 h and growth was monitored by OD₆₀₀ at an interval of 2h. (B) PLP synthase activity of PdxS, PdxT, and the mixture of the two proteins using glutamine as substrate in the reaction. (C) PLP synthase activity of PdxS, PdxT, and the mixture of the two proteins using ammonium substitute for glutamine in the reaction. Points indicate the mean values of three independent assays, and error bars indicate standard deviations.</p

Pyridoxal phosphate synthases PdxS/PdxT are required for <i>Actinobacillus pleuropneumoniae</i> viability, stress tolerance and virulence

<div><p>Pyridoxal 5’-phosphate (PLP) is an essential cofactor for numerous enzymes involved in a diversity of cellular processes in living organisms. Previous analysis of the <i>Actinobacillus pleuropneumoniae</i> S-8 genome sequence revealed the presence of <i>pdxS</i> and <i>pdxT</i> genes, which are implicated in deoxyxylulose 5-phosphate (DXP)-independent pathway of PLP biosynthesis; however, little is known about their roles in <i>A</i>. <i>pleuropneumoniae</i> pathogenicity. Our data demonstrated that <i>A</i>. <i>pleuropneumoniae</i> could synthesize PLP by PdxS and PdxT enzymes. Disruption of the <i>pdxS</i> and <i>pdxT</i> genes rendered the pathogen auxotrophic for PLP, and the defective growth as a result of these mutants was chemically compensated by the addition of PLP, suggesting the importance of PLP production for <i>A</i>. <i>pleuropneumoniae</i> growth and viability. Additionally, the <i>pdxS</i> and <i>pdxT</i> deletion mutants displayed morphological defects as indicated by irregular and aberrant shapes in the absence of PLP. The reduced growth of the <i>pdxS</i> and <i>pdxT</i> deletion mutants under osmotic and oxidative stress conditions suggests that the PLP synthases PdxS/PdxT are associated with the stress tolerance of <i>A</i>. <i>pleuropneumoniae</i>. Furthermore, disruption of the PLP biosynthesis pathway led to reduced colonization and attenuated virulence of <i>A</i>. <i>pleuropneumoniae</i> in the BALB/c mouse model. The data presented in this study reveal the critical role of PLP synthases PdxS/PdxT in viability, stress tolerance, and virulence of <i>A</i>. <i>pleuropneumoniae</i>.</p></div

Bacterial loads in organs from BALB/c mice infected with <i>A</i>. <i>pleuropneumoniae</i> mutants.

<p>Mice were infected with WT S-8, S-8Δ<i>pdxS</i>, and S-8Δ<i>pdxT</i> mutant strains, and bacterial loads in (A) lung, (B) liver, (C) kidney examined 3 days post infection. The data shown are the means of bacterial colonies from five mice, and error bars indicate standard deviations. **, <i>p</i> < 0.01; *, <i>p</i> < 0.05.</p