Isolation and characterization of duck sewage source Salmonella phage P6 and antibacterial activity for recombinant endolysin LysP6.

Salmonella is a globally prevalent foodborne pathogen, and adverse events caused by S. Enteritidis and S. Typhimurium are extremely common. With the emergence of drug resistance, there is an urgent need for efficient and specific lytic bacteriophages as alternative to antibiotics in clinical practice. In this study, phage P6 was isolated and screened from effluent and fecal samples from duck farm environments to specifically lyse the duck sources S. Typhimurium and S. Enteritidis. Phage P6 belongs to the genus Lederbergvirus, unclassified Lederbergvirus species. The phage P6 genome did not contained non-coding RNA, virulence genes and drug resistance genes, indicating that phage P6 was biologically safe for clinical applications. Phage P6 lysed 77.78% (28/36) of multidrug-resistant Salmonella and reduced biofilms formed by S. Enteritidis CVCC 3377, 4, and 24, and S. Typhimurium 44 by 44% to 75% within 3 h, and decreased Salmonella in duckling feces by up to 1.64 orders of magnitude. Prokaryotic expression of endolysin LysP6 lysed the chloroform-treated bacterial outer membrane from different serotypes of duck-derived Salmonella and E. coli standard strain ATCC 25922. The host range was expanded compared to phage P6, and the growth of Salmonella was effectively inhibited by LysP6 in conjunction with the membrane permeabilizer EDTA within 24 h. Therefore, phage P6 and phage-derived endolysins LysP6 are suitable for application as potent biocontrol agents to improve poultry health and food safety.

Thermosensitive Hydrogel Wound Dressing Loaded with Bacteriophage Lysin LysP53.

Wound infections are prone to attacks from infectious pathogens, including multidrug resistant bacteria that render conventional antimicrobials ineffective. Recently, lysins have been proposed as alternatives to conventional antimicrobials to tackle the menace of multidrug resistance pathogens. The coupling of lysins with a material that will cover the wound may prove beneficial in both protecting and treating wound infections. Hence, in this study, a Gram-negative lysin, LysP53, was coupled with a thermosensitive hydrogel, poloxamer P407, and its efficacy to treat wound infection was tested. In vitro, the addition of LysP53 to the poloxamer did not affect its thermosensitive characteristics, nor did it affect the hydrogel structure. Moreover, the lysin hydrogel could hydrolyze the peptidoglycan, demonstrating that it may have bactericidal activity. Up to 10.4% of LysP53 was released from the hydrogel gradually within 24 h, which led to a 4-log reduction of stationary phase Acinetobacter baumannii. Lastly, the lysin hydrogel was found safe with no cytotoxic effects observed in cells. Ex vivo, LysP53 hydrogel could inhibit bacterial growth on a pig skin decolonization model, with 3-log differences compared to non-treated groups. Overall, our results suggest that lysin-loaded hydrogels may provide a novel solution to treat wound infections caused by resistant bacteria.

Phage Endolysin LysP108 Showed Promising Antibacterial Potential Against Methicillin-resistant Staphylococcus aureus.

As a potential antibacterial agent, endolysin can directly lyse Gram-positive bacteria from the outside and does not lead to drug resistance. Considering that XN108 is the first reported methicillin-resistant Staphylococcus aureus (MRSA) strain in mainland China with a vancomycin MIC that exceeds 8 µg mL-1, we conducted a systematic study on its phage-encoded endolysin LysP108. Standard plate counting method revealed that LysP108 could lyse S. aureus and Pseudomonas aeruginosa with damaged outer membrane, resulting in a significant reduction in the number of live bacteria. Scanning electron microscopy results showed that S. aureus cells could be lysed directly from the outside by LysP108. Live/dead bacteria staining results indicated that LysP108 possessed strong bactericidal ability, with an anti-bacterial rate of approximately 90%. Crystal violet staining results implied that LysP108 could also inhibit and destroy bacterial biofilms. In vivo animal experiments suggested that the area of subcutaneous abscess of mice infected with MRSA was significantly reduced after the combined injection of LysP108 and vancomycin in comparison with monotherapy. The synergistic antibacterial effects of LysP108 and vancomycin were confirmed. Therefore, the present data strongly support the idea that endolysin LysP108 exhibits promising antibacterial potential to be used as a candidate for the treatment of infections caused by MRSA.

Increasing L-lysine production in Corynebacterium glutamicum by engineering amino acid transporters.

Corynebacterium glutamicum has a long and successful history in the biotechnological production of L-lysine. Besides the adjustment of metabolic pathways, intracellular and extracellular transport systems are critical for the cellular metabolism of L-lysine or its by-products. Here, three amino acid transmembrane transporters, namely, GluE, BrnE/BrnF, and LysP, which are widely present in C. glutamicum strains, were each investigated by gene knockout. In comparison with that in the wild-type strain, the yield of L-lysine increased by 9.0%, 12.3%, and 10.0% after the deletion of the gluE, brnE/brnF, and lysP genes, respectively, in C. glutamicum 23,604. Moreover, the amount of by-product amino acids decreased significantly when the gluE and brnE/brnF genes were deleted. It was also demonstrated that there was no effect on the growth of the strain when the gluE or lysP gene was deleted, whereas the biomass of C. glutamicum WL1702 (ΔbrnE/ΔbrnF) in the fermentation medium was significantly reduced in comparison with that of the wild type. These results also provide useful information for enhancing the production of L-lysine or other amino acids by C. glutamicum.

Production of bacteriophage-encoded endolysin, LysP11, in Nicotiana benthamiana and its activity as a potent antimicrobial agent against Erysipelothrix rhusiopathiae.

KEY MESSAGE: We produced a biologically active phage-encoded endolysin, LysP11, in N. benthamiana. Plant-produced LysP11 exhibited robust antimicrobial activity against E. rhusiopathiae, and C-terminal domain of LysP11 bound specifically to E. rhusiopathiae. Bacterial resistance to antibiotics, a serious issue in terms of global public health, is one of the leading causes of death today. Thus, new antimicrobial agents are needed to combat pathogens. Recent research suggests that bacteriophages and endolysins derived from bacteriophages are potential alternatives to traditional antibiotics. Here, we examined the antimicrobial activity of LysP11, which is encoded by Propionibacterium phage P1.1 and comprises an N-terminal amidase-2 domain and a C-terminal domain with no homology to other bacteriophage endolysins. LysP11 was produced in Nicotiana benthamiana (N. benthamiana) using an Agrobacterium-mediated transient expression strategy. LysP11 was purified on microcrystalline cellulose-binding resin after attachment of the Clostridium thermocellum-derived family 3 cellulose-binding domain as an affinity tag. The affinity tag was removed using the small ubiquitin-related modifier (SUMO) domain and SUMO-specific protease. Plant-produced LysP11 showed strong antimicrobial activity toward Erysipelothrix rhusiopathiae (E. rhusiopathiae), mediated via lysis of the cell wall. Lytic activity was optimal at pH 8.0-9.0 (37 °C) and increased at higher concentrations of NaCl up to 400 mM. Furthermore, the C-terminal domain of LysP11 bound specifically to the E. rhusiopathiae cell wall. Based on these results, we propose that LysP11 is a potential candidate antimicrobial agent against E. rhusiopathiae.

Differential protein-DNA contacts for activation and repression by ArgP, a LysR-type (LTTR) transcriptional regulator in Escherichia coli.

ArgP is a LysR-type transcriptional regulator (LTTR) that operates with two effector molecules, lysine and arginine, to differentially regulate gene expression. Effector-free ArgP stimulates transcription of all investigated regulon members, except argO, whereas lysine abolishes this effect. Activation of argO, encoding an exporter for arginine and canavanine, is strictly dependent on arginine-bound ArgP. Lysine counteracts this effect and even though lysine-bound ArgP stimulates RNA polymerase recruitment at the argO promoter, the complex is non-productive. It is presently unclear what distinguishes argO from other ArgP targets and how binding of arginine and lysine translates in antagonistic effects on promoter activity. Here we generate high resolution contact maps of effector-free and effector-bound ArgP-DNA interactions and identify the sequence 5'-CTTAT as the consensus recognition motif for ArgP binding. argO is the only operator at which ArgP binding overlaps the -35 promoter element and binding of arginine results in a repositioning of the promoter proximal bound ArgP-arg subunits. This effect was mimicked by the generation of a 10bp insertion mutant (ins-10) in the argO operator that renders its activation by ArgP arginine-independent. ArgP-induced DNA bending of the argO operator by approximately 60° was found to be effector independent. An ArgP:DNA binding stoichiometry of 4:1 indicates binding of four ArgP subunits even to DNA constructs that are truncated for one binding subsite (ΔABS). These results provide insight into the molecular mechanisms of ArgP-mediated regulation and a molecular explanation for the unique arginine-dependence of argO activation that distinguishes this particular ArgP target from all others.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease from Pseudomonas aeruginosa.

The prokaryotic lysine-specific permease (LysP) belongs to the amino acid-polyamine-organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP from Pseudomonas aeruginosa was recombinantly expressed in Escherichia coli and purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Šresolution. The diffraction data were indexed in space group P21, with unit-cell parameters a = 169.53, b = 169.53, c = 290.13 Å, γ = 120°.