What is the significance of the lytic enzyme

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Sommer, M. Each point represents the CFU recovered from an individual rat. Data were compiled from five independent experiments. Statistical comparisons were made with the Mann-Whitney test. We previously described a head-to-tail fusion of LysK-lysostaphin that included two CBDs and showed weak staphylolytic activity Improvements were made to create two triple-acting constructs, each with a single CBD the SH3b domain from lysostaphin.

Parental and triple-acting fusion proteins were expressed in E. The triple-acting PGHs demonstrated intermediate activity compared to the parental PGHs against Coagulase negative strains as well as antibiotic-sensitive and antibiotic-resistant S. Importantly, all three lytic domains were active in each of the fusion constructs examined, as illustrated in the representative electron spray ionization mass spectrometry results of PG digestion products created by K-L Supplementary Fig.

The fusions were also as effective as the parental enzymes at eradicating static biofilms. Increased biofilm eradication was obtained at the highest concentrations tested when the parental enzymes were added together Supplementary Fig.

Development of resistance to the triple-acting fusion enzymes was tested in vitro on S. After 10 rounds of sub-lethal exposure in liquid culture, parental LysK two lytic domains and lysostaphin one lytic domain yielded cultures with fold and fold increases in MICs, respectively. When S. These passaged bacteria retained their elevated MIC, suggesting that resistance was not physiologically induced e.

We compared our triple-acting fusions to small molecule antibiotics in similar assays. Resistance development during 10 rounds of sub-lethal exposure in plate lysis assays showed an 8-fold increase in resistance to lysostaphin, a 2-fold increase for LysK, and no detectable increase in resistance for triple fusions K-L or L-K. To determine whether our triple-acting fusion enzymes showed in vivo potency, we tested the parental and chimeric PGHs in a robust nasal colonization model.

Rats were inoculated on day 0, treated twice daily on days 3, 4 and 5, and euthanized for quantitative cultures of their nasal tissue on day In contrast, equimolar concentrations of recombinant LysK or triple-acting fusion K-L were unable to significantly reduce the bacterial load. Neither commercial lysostaphin 1-Sigma; Sigma-Aldrich, St. However, the addition of PTDs to the C-terminus of His 6 -tagged lysostaphin resulted in significant reductions in intracellular S.

In MAC-T cells, virtually all tested constructs displayed this effect, despite the fact that modification with PTDs often reduced the enzymatic activity of most constructs tested Supplementary Fig. Cultured cells were infected, treated with gentamicin to kill extracellular S.

All results are standardized to the gentamicin GENT only control. Nomenclature of the PGH constructs is as in Fig. PTDs are listed in Table 1. Error bars represent SEM. C Murine primary osteoblasts mOB infected with S. D Single plane of confocal microscopy z-stack overlaid on bright field exposure of live cultured MAC-T cells exposed to both S. Live S. Yellow staining in the combined panel represents intracellular co-localization in a single plane as determined by z-stack analysis of both S.

Many, but not all, S. A similar result was apparent in the maximum intensity projections with all z-planes visualized Fig. To examine intracellular uptake and eradication in murine osteoblast containing tissue samples, both an ex vivo calvaria skull cap and in vivo femur injury model were tested.

Surviving S. These two animal models correlate well with the results of cultured osteoblasts in Fig. Calvaria were embedded in freeze media, sectioned, and subjected to fluorescence imaging. The image shown is a representative figure for triplicate sections of three separate calvaria.

B Fluorescence intensity from these sections was measured in ImageJ and defined as arbitrary fluorescence standardized to the untreated control.

Error bars represent SEM of four replicate experiments. C Ex-vivo intracellular S. Infected calvaria were homogenized and CFU were counted post treatment. D Murine model of staphylococcal osteomyelitis. The femurs were removed, homogenized, and plated to quantify the bacterial load. Despite a much reduced molar concentration compared to vancomycin [K-L 1. Compiling mean fluorescent intensities from the entire biofilm z-stack i. B Bacterial viability.

All values were found to be significantly different from PBS and each other using a two-tailed, unpaired, t-test; K-L vs. To determine if addition of a PTD impacts the ability of a PGH to reduce the bacterial load in a murine model of mastitis, four constructs were tested. In turbidity reduction assays triple-acting fusion K-L was slightly more effective against S.

Mice were challenged with CFU S. In vivo, lysostaphin was effective in reducing the bacterial load approximately 4 logs compared to controls Fig. In contrast to our ex vivo data, triple-acting fusion K-L was not able to clear the mammary gland bacterial infection more than buffer alone.

However, S. A Specific activity of PGH fusion constructs in vitro. Turbidity reduction assay with S. The maximum specific activity for three experiments is represented as an average with SEM error bars. B Bacterial burden in mice with mastitis treated with recombinant PGHs. Each data point represents the average of duplicate bacterial platings.

Data represent a minimum of 7 measurements in duplicate. In response to the critical need for novel antimicrobials with reduced resistance development for treating MDR S. These constructs simultaneously degrade S. A primary advantage conferred by the PGH attack at the pathogen cell wall is the avoidance of most intracellular resistance mechanisms e. PGHs derived from bacteriophage endolysins have the added advantage of co-evolution with bacteria, allowing them to target bonds that the host cell cannot readily modify, yielding enzymes that are, in theory, inherently refractory to resistance development resistance data for endolysins is reviewed in With multiple PG-degrading domains glycosidase, endopeptidase, and amidase in one molecule, there is considerable diversity and often near species-specificity of these activities, ensuring low selective pressure on unrelated, co-resident commensal strains, further reducing the potential for resistance development in non-targeted species.

The PGHs have additional favorable qualities. These qualities include non-toxic 32 , biodegradable, effective on both biofilms 33 and MDR strains 24 , are synergistic with antibiotics 34 , and thus hold great potential for treating MDR strains. LysK was not as refractory to resistance development measured by its MIC as previously reported for Streptococcus and Bacillus endolysins 31 and a staphylolytic fusion construct 35 in serial dilution plating assays.

However, our strategy to reduce resistant strain development by creating triple-acting staphylolytic fusions was successful with very little resistance development for triple-acting fusion L-K in vitro when tested against the lab strain S. Triple-acting fusion L-K also reduced the bacterial load 5—10 fold better than either parental enzyme in a rat nasal decolonization model, with no in vivo resistance development detected.

Despite these successes, as proteins the PGHs must overcome a unique set of therapeutic hurdles. A separate therapeutic hurdle is created in systemic infections where S. Intracellular invasion has been reported for bovine mastitis [where S. Toxic levels of conventional antibiotics are often required to treat classic intracellular pathogens To address this concern, we engineered our PGH constructs with 11 different PTDs and identified the optimal PTD domain s to facilitate import into multiple cultured cells.

Our Initial data with lysostaphin indicated that a PTD was essential for eradication of intracellular S. In contrast, triple-acting fusion K-L did not require a PTD to invade cultured bovine mammary cells or murine osteoblasts, and showed a similar efficacy with or without a PTD in bone infection models. Despite equivalent intracellular efficacy with either triple fusion K-L or construct K-L-PTD1 in cultured mammary cells, the latter showed almost 3 orders of magnitude greater CFU reduction in vivo in the mastitis model.

The inconsistencies in these results between ex vivo and in vivo assays underline the complexity of cellular uptake mechanisms including the poorly defined role of PTD sequences, cargo protein sequences, and the variety of cellular uptake mechanisms that can be employed to achieve intracellular localization. Triple-acting fusions K-L which showed highest efficacy in intracellular S.

PGHs ability to reduce or eradicate static biofilms 33 is an important advantage over conventional antibiotics, since biofilms are proposed to play a critical role in infectious disease Again, the exact role of the PTD is unknown in this capacity. Another therapeutic hurdle is the fact that as protein, PGHs are potentially antigenic and might engender host immune responses in a manner similar to that seen in phage-based therapies However, PGHs show minimal immunogenicity in mammals, and adverse responses have not been reported.

Bovine intramammary infusions of lysostaphin resulted in detectable levels of specific antibodies only after 18—21 treatments. The antibodies were not neutralizing, nor did they elicit observable effects on the host animal or eliminate the antimicrobial properties of lysostaphin Serum antibodies raised to phage endolysins specific to Bacillus anthracis, Streptococcus pyogenes, or Streptococcus pneumoniae slowed, but did not inhibit microbial killing in vitro 31 , There is also a concern for proinflammatory components released from lysed bacteria However, adverse immune responses have not been observed in mouse models for an array of systemically delivered staphylolytic enzyme constructs.

The hurdles to commercialization of PGH antimicrobials are non-trivial. Still to be addressed are the physicochemical and pharmacokinetic aspects of PGH treatments [which are likely to be significant, considering the potential for intracellular transport]. Production hurdles are also expected when producing protein therapeutics. Despite these hurdles, the ability to engineer a PGH antimicrobial with qualities not readily achievable with conventional antibiotics refractory to resistance development, biofilm eradication, treatment of intracellular and MDR pathogens begs the question: What other desirable traits could be engineered into PGH antimicrobials?

In the absence of an approved PGH therapeutic, we hope that a continued demonstration of the versatility of engineered PGHs will lower the threshold to commercialization for this new and much needed class of antibacterials. All bacterial strains, culture conditions, details of E. Resistance development assays liquid- and plate lysis-based are essentially as described previously 43 Supplementary Information Materials and Methods.

Static and dynamic biofilm reduction assays are as previously described 44 SI Materials and Methods. Intracellular S. Confocal microscopy to demonstrate intracellular colocalization of both fusion enzymes and S.

Four previously described animal models in vivo rat nasal colonization 46 , ex vivo murine cavalaria, in vivo femur osteomyelitis 30 and in vivo murine mastitis 47 were used to demonstrate efficacy of the fusion constructs Supplementary Information Materials and Methods. All animal experiments were conducted in accordance with protocols approved by the appropriate Institutional Animal Care and Use Committees. How to cite this article : Becker, S.

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Certain Listeria monocytogenes lysins have cell wall binding domains that specifically recognize and bind to teichoic acids before degradation of the peptidoglycan can occur [ 40 ]. For the lysins tested here, it is not known if and how the cell wall binding domains are interacting with the cell wall or the prevalence of potential cell wall binding epitopes on the cell surface of the LAB tested in this study.

In support of this possibility is the fact that LysA2 endopeptidase activity [hydrolyzes the bond between the terminal D-alanine of the PG tetrapeptide and the D-aspartic acid residue that forms the bridge with the L-lysine of a neighboring PG chain] was reported to function on species that harbor either an L-lysine or an L-ornithine at position three in the neighboring tetrapeptide [ 28 ]. Although not a definitive or even a direct comparison, this suggests a degree of flexibility in the recognition sequences surrounding the cut site of these PG hydrolase lytic domains.

It is interesting to speculate on whether or not sensitivity to ethanol will be a significant factor in the efficacy of the lytic enzymes we have tested. At this initial aerobic stage in the fermentation there is no significant ethanol concentration that would be ideal for lysin treatment therefore ethanol levels might not be a factor. Certainly enzymes that have a broad range of activity regardless of ethanol concentration might provide a longer-lived protection during the fermentation process, especially if they are designed to be secreted from recombinant fermentative yeast throughout the fermentation process.

Yet to be determined is whether a broader species-target range or biochemical resilience under ethanolic fermentation conditions is more important for the optimal antimicrobial when considering the complex environment of a lignocellulosic fermentation.

By homology screening of these lysins to other known PG hydrolase lytic and cell wall binding domains there is no shortage of phage lysins for future consideration as public datasets contain numerous putative lytic PG hydrolases from both bacterial prophage and phage genome origins.

Due to the high interest in phages that impinge on yogurt production, there are hundreds of known lactobacilli phage, with nine complete Lactobacillus phage genomes and 11 Lactobacillus poly-lysogenic bacterial genomes with sequence available on the NCBI website [ 42 ]. There is also a report of an amidase domain from the PL-1 that infects L. However, due to limitations in the species range of lytic activity each candidate will need to be tested empirically against target LAB.

However promising these results may seem, these initial trials do not necessarily reflect normal fermentation conditions. In order to easily detect changes in target contaminant profiles, the mock trial was performed under pre-sterilization conditions, such that the only contaminant was L.

Also, neither the yeast nor the LAB were adapted for growth on hydrolysate, rather, they were both grown in rich broth and then added to the hydrolysate. This assay was designed to test the enzymes for activity under hydrolysate conditions, the results of which are encouraging, but activity on the contaminant might be affected by the cell wall of the contaminant during hydrolysate growth conditions. Thus, the usefulness of these enzymes in large scale fermentations remains to be determined. Therefore, lysins appear to share qualities worth considering for future works to protect fuel ethanol fermentations from LAB contaminants.

Phage lytic enzymes are strong candidate antimicrobials to control LAB contamination in fuel ethanol fermentations. These qualities make phage lytic enzymes excellent candidate antimicrobials for testing in biofuel fermentations as either additives or engineered to be expressed by the fermentative yeast. Pritchard [ 30 ]. All lysin proteins were over expressed in E. Purified pET21a harboring the lysin genes of interest, were transformed into E.

Mid log phase OD nm of 0. Protein elutes were filter sterilized 0. The turbidity assays were performed in a Molecular Devices Spectra Max plate reader. Immediately, absorbance OD nm readings were taken every 30 s for 30 min and specific activities were determined on a sliding scale as described by Becker et al.

Corn fiber, obtained from a commercial wet-mill ethanol facility, was hydrolyzed by dilute acid treatment as previously described [ 50 ]. The hydrolysate was cleared of particulate matter by centrifugation. Sterility was confirmed by plating aliquots of hydrolysate on MRS agar. The S. Cells were harvested by centrifugation and resuspended in a volume of phosphate buffered saline PBS; Fisher Scientific pH7. The aliquots were titered by plating on 1.

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