Use of the mCherry fluorescent protein to optimize the expression of class I lanthipeptides in Escherichia coli.

BACKGROUND: Lanthipeptides are a rapidly expanding family of ribosomally synthesized and post-translationally modified natural compounds with diverse biological functions. Lanthipeptide structural and biosynthetic genes can readily be identified in genomic datasets, which provides a substantial repository for unique peptides with a wide range of potentially novel bioactivities. To realize this potential efficiently optimized heterologous production systems are required. However, only a few class I lanthipeptides have been successfully expressed using Escherichia coli as heterologous producer. This may be attributed to difficulties experienced in the co-expression of structural genes and multiple processing genes as well as complex optimization experiments. RESULTS: Here, an optimized modular plasmid system is presented for the complete biosynthesis for each of the class I lanthipeptides nisin and clausin, in E. coli. Genes encoding precursor lanthipeptides were fused to the gene encoding the mCherry red fluorescent protein and co-expressed along with the required synthetases from the respective operons. Antimicrobially active nisin and clausin were proteolytically liberated from the expressed mCherry fusions. The mCherry-NisA expression system combined with in vivo fluorescence monitoring was used to elucidate the effect of culture media composition, promoter arrangement, and culture conditions including choice of growth media and inducer agents on the heterologous expression of the class I lanthipeptides. To evaluate the promiscuity of the clausin biosynthetic enzymes, the optimized clausin expression system was used for the heterologous expression of epidermin. CONCLUSION: We succeeded in developing novel mCherry-fusion based plug and play heterologous expression systems to produce two different subgroups of class I lanthipeptides. Fully modified Pre-NisA, Pre-ClausA and Pre-EpiA fused to the mCherry fluorescence gene was purified from the Gram-negative host E. coli BL21 (DE3). Our study demonstrates the potential of using in vivo fluorescence as a platform to evaluate the expression of mCherry-fused lanthipeptides in E. coli. This allowed a substantial reduction in optimization time, since expression could be monitored in real-time, without the need for extensive and laborious purification steps or the use of in vitro activity assays. The optimized heterologous expression systems developed in this study may be employed in future studies for the scalable expression of novel NisA derivatives, or novel genome mined derivatives of ClausA and other class I lanthipeptides in E. coli.

Heterologous Expression of Plantaricin 423 and Mundticin ST4SA in Saccharomyces cerevisiae.

Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX). The native and codon-optimised variants of the plaA and munST4SA genes were cloned into episomal expression vectors containing either the S. cerevisiae alpha mating factor (MFα1) or the Trichoderma reesei xylanase 2 (XYNSEC) secretion signal sequences. The recombinant peptides retained their activity and stability, with the MFα1 secretion signal superior to the XYNSEC secretion signal for both bacteriocins. An eight-fold increase in activity against Listeria monocytogenes was observed for MunX after codon optimisation, but not for PlaX-producing strains. After HPLC-purification, the codon-optimised genes yielded 20.9 mg/L of MunX and 18.4 mg/L of PlaX, which displayed minimum inhibitory concentrations (MICs) of 108.52 nM and 1.18 µM, respectively, against L. monocytogenes. The yields represent a marked improvement relative to an Escherichia coli expression system previously reported for PlaX and MunX. The results demonstrated that S. cerevisiae is a promising host for recombinant bacteriocin production that requires a simple purification process, but the efficacy is sensitive to codon usage and secretion signals.

Migration of Bacteriocins Across Gastrointestinal Epithelial and Vascular Endothelial Cells, as Determined Using In Vitro Simulations.

Little is known about the migration of bacteriocins across human cells. In this study, we report on migration of three bacteriocins nisin, plantaricin 423 and bacST4SA across colonic adenocarcinoma (Caco-2) cells and human umbilical vein endothelial cells (HUVECs). Bacteriocins were fluorescently labelled while still maintaining antimicrobial activity. Migration of fluorescently labelled bacteriocins across monolayers was assessed in vitro using transmigration well inserts. After 3 h, 75% of nisin, 85% of plantaricin 423 and 82% of bacST4SA migrated across the Caco-2 cell monolayer. Over the same time span, 88% nisin, 93% plantaricin 423 and 91% bacST4SA migrated across the HUVEC monolayer. The viability of both cell types remained unchanged when exposed to 50 µM of nisin, plantaricin 423 or bacST4SA. The effect of human plasma on bacteriocin activity was also assessed. Activity loss was dependent on bacteriocin type and concentration, with the class-IIa bacteriocins retaining more activity compared to nisin. This is the first report of bacteriocins migrating across simulated gastrointestinal- and vascular-barriers. This study provides some of the first evidence that bacteriocins are capable of crossing the gut-blood-barrier. However, in vivo studies need to be performed to confirm these findings and expand on the role of bacteriocin migration across cell barriers.

Corrigendum: A Review: The Fate of Bacteriocins in the Human Gastro-Intestinal Tract: Do They Cross the Gut-Blood Barrier?

[This corrects the article DOI: 10.3389/fmicb.2018.02297.].

A Review: The Fate of Bacteriocins in the Human Gastro-Intestinal Tract: Do They Cross the Gut-Blood Barrier?

The intestinal barrier, consisting of the vascular endothelium, epithelial cell lining, and mucus layer, covers a surface of about 400 m2. The integrity of the gut wall is sustained by transcellular proteins forming tight junctions between the epithelial cells. Protected by three layers of mucin, the gut wall forms a non-permeable barrier, keeping digestive enzymes and microorganisms within the luminal space, separate from the blood stream. Microorganisms colonizing the gut may produce bacteriocins in an attempt to outcompete pathogens. Production of bacteriocins in a harsh and complex environment such as the gastro-intestinal tract (GIT) may be below minimal inhibitory concentration (MIC) levels. At such low levels, the stability of bacteriocins may be compromised. Despite this, most bacteria in the gut have the ability to produce bacteriocins, distributed throughout the GIT. With most antimicrobial studies being performed in vitro, we know little about the migration of bacteriocins across epithelial barriers. The behavior of bacteriocins in the GIT is studied ex vivo, using models, flow cells, or membranes resembling the gut wall. Furthermore, little is known about the effect bacteriocins have on the immune system. It is generally believed that the peptides will be destroyed by macrophages once they cross the gut wall. Studies done on the survival of neurotherapeutic peptides and their crossing of the brain-blood barrier, along with other studies on small peptides intravenously injected, may provide some answers. In this review, the stability of bacteriocins in the GIT, their effect on gut epithelial cells, and their ability to cross epithelial cells are discussed. These are important questions to address in the light of recent papers advocating the use of bacteriocins as possible alternatives to, or used in combination with, antibiotics.

Selective Laser Melting of Integrated Ti6Al4V ELI Permeable Walls for Controlled Drug Delivery of Vancomycin.

Bacteria colonizing the surface of orthopedic implants are responsible for most postoperative periprosthetic joint infections. A possible alternative route for drug delivery is described in this study by utilizing the bulk of the implant itself as a reservoir. Drug release is enabled by manufacturing of integrated permeable structures possessing high porosity through application of selective laser melting technology. The concept was evaluated in two paths, with 400 µm permeable thin walls and with dense reservoirs containing an integrated 950 µm permeable wall. Components were designed and preprocessed as separate parts, allowing for allocation of different settings of laser power and scanning speed. Lowering the energy input into the selective laser melting process to induce intermittent melting of the Ti6Al4V ELI powder produced porous components through which vancomycin was released with differing profiles. Static water contact angle measurements demonstrated a significant effect on the hydrophilicity by permeable wall thickness. Relative porosities of the 400 µm structures were determined with microcomputed tomography analyses. A transition zone of 21.17% porosity was identified where release profiles change from porosity-dependent to near free diffusion. Antimicrobial activity of released vancomycin was confirmed through evaluation against Staphylococcus aureus Xen 36 in two separate agar diffusion assays. The approach is promising for incorporation into the design and manufacturing of next-generation prosthetic implants with controlled release of antibiotics in situ and the subsequent prevention of periprosthetic joint infections.

Titanium-Based Hip Stems with Drug Delivery Functionality through Additive Manufacturing.

Postoperative infections are a major concern in patients that receive implants. These infections generally occur in areas with poor blood flow and pathogens do not always respond to antibiotic treatment. With the latest developments in nanotechnology, the incorporation of antibiotics into prosthetic implants may soon become a standard procedure. The success will, however, depend on the ability to control the release of antibiotics at concentrations high enough to prevent the development of antibiotic-resistant strains. Through additive manufacturing, antibiotics can be incorporated into cementless femoral stems to produce prosthetic devices with antimicrobial properties. With the emerging increase in resistance to antibiotics, the incorporation of antimicrobial compounds other than antibiotics, preferably drugs with a broader spectrum of antimicrobial activity, will have to be explored. This review highlights the microorganisms associated with total hip arthroplasty (THA), discusses the advantages and disadvantages of the latest materials used in hip implants, compares different antimicrobial agents that could be incorporated, and addresses novel ideas for future research.

Delivery of antibiotics from cementless titanium-alloy cubes may be a novel way to control postoperative infections.

Bacterial colonisation and biofilm formation onto orthopaedic devices are difficult to eradicate. In most cases infection is treated by surgical removal of the implant and cleaning of the infected area, followed by extensive treatment with broad-spectrum antibiotics. Such treatment causes great discomfort, is expensive, and is not always successful. In this study we report on the release of vancomycin through polyethersulfone membranes from channels in cementless titanium-alloy cubes. The cubes were constructed with LaserCUSING from Ti6Al4V ELI powder. Vancomycin was released by non-Fickian anomalous (constraint) diffusion. Approximately 50% of the vancomycin was released within the first 17 h. However, sustained delivery of vancomycin for 100 h was possible by reinjecting the channels. Refillable implants may be a novel way to control postoperative infections.

Calcium orthophosphate-based bone cements (CPCs): Applications, antibiotic release and alternatives to antibiotics.

Calcium orthophosphate bone cements (CPCs) are widely used in orthopedic surgery. Implants are highly susceptible to infection and often lead to the formation of microbial biofilms. Antibiotics are often incorporated into bone cement to prevent infection. The increase in the number of microorganisms acquiring or developing resistance to antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), is a major concern. Bacteriocins (antimicrobial peptides) offer an alternative to antibiotics. Their mode of activity involves permanent destabilization of the plasma membrane of target cells. A number of broad-spectrum bacteriocins produced by lactic acid bacteria and Bacillus spp. have recently been reported. In this REVIEW the major characteristics of calcium phosphate bone cements, prosthetic joint-associated infections, and treatment of these infections is discussed. The role of antimicrobial agents in CPCs is discussed and the possibility of incorporating bacteriocins in prosthetic devices is investigated.

Release of Enterococcus mundtii Bacteriocin ST4SA from Self-Setting Brushite Bone Cement.

Maxillofacial and craniofacial surgery is on the increase, which exposes more patients at risk of acquiring microbial infections. The use of antibiotic-loaded calcium phosphate bone cements has been shown to reduce the incidence of infection. A marked increase in antibiotic-resistant pathogens, including multidrug-resistant pathogens, has been reported. This has led to the investigation of various compounds as alternatives to conventional treatments. In this paper, we report on the incorporation and release of a broad-spectrum class II antimicrobial peptide, bacteriocin ST4SA produced by Enterococcus mundtii, into a calcium orthophosphate-based bone cement. Our results suggest class II bacteriocins may be incorporated into self-setting bone cements to produce implants with antimicrobial activity over extended periods of time.