Search

Summary Bacteriophage endolysins are peptidoglycan hydrolases employed by the virus to lyse the host at the end of its multiplication phase. They have found many uses in biotechnology; not only as antimicrobials, but also for the development of novel diagnostic tools for rapid detection of pathogenic bacteria. These enzymes generally show a modular organization, consisting of N‐terminal enzymatically active domains (EADs) and C‐terminal cell wall‐binding domains (CBDs) which specifically target the enzymes to their substrate in the bacterial cell envelope. In this work, we used individual functional modules of Listeria phage endolysins to create fusion proteins with novel and optimized properties for labelling and lysis of Listeria cells. Chimaeras consisting of individual EAD and CBD modules from PlyPSA and Ply118 endolysins with different binding specificity and catalytic activity showed swapped properties. EAD118–CBDPSA fusion proteins exhibited up to threefold higher lytic activity than the parental endolysins. Recombineering different CBD domains targeting various Listeria cell surfaces into novel heterologous tandem proteins provided them with extended recognition and binding properties, as demonstrated by fluorescent GFP‐tagged CBD fusions. It was also possible to combine the binding specificities of different single CBDs in heterologous tandem CBD constructs such as CBD500‐P35 and CBDP35‐500, which were then able to recognize the majority of Listeria strains. Duplication of CBD500 increased the equilibrium cell wall binding affinity by approximately 50‐fold, and the enzyme featuring tandem CBD modules showed increased activity at higher ionic strength. Our results demonstrate that modular engineering of endolysins is a powerful approach for the rational design and optimization of desired functional properties of these proteins.

The Gram-positive pathogen Listeria monocytogenes can be subdivided into at least 12 different serovars, based on the differential expression of a set of somatic and flagellar antigens. Of note, strains belonging to serovars 1/2a, 1/2b, and 4b cause the vast majority of foodborne listeriosis cases and outbreaks. The standard protocol for serovar determination involves an agglutination method using a set of sera containing cell surface-recognizing antibodies. However, this procedure is imperfect in both precision and practicality, due to discrepancies resulting from subjective interpretation. Furthermore, the exact antigenic epitopes remain unclear, due to the preparation of the absorbed sera and the complex nature of polyvalent antibody binding. Here, we present a novel method for quantitative somatic antigen differentiation using a set of recombinant affinity proteins (cell wall-binding domains and receptor-binding proteins) derived from a collection of Listeria bacteriophages. These proteins enable rapid, objective, and precise identification of the different teichoic acid glycopolymer structures, which represent the O-antigens, and allow a near-complete differentiation. This glycotyping approach confirmed serovar designations of over 60 previously characterized Listeria strains. Using select phage receptor-binding proteins coupled to paramagnetic beads, we also demonstrate the ability to specifically isolate serovar 1/2 or 4b cells from a mixed culture. In addition, glycotyping led to the discovery that strains designated serovar 4e actually possess an intermediate 4b-4d teichoic acid glycosylation pattern, underpinning the high discerning power and precision of this novel technique., Applied and Environmental Microbiology, 86 (13), ISSN:0099-2240, ISSN:1098-5336

Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections., mBio, 11 (5), ISSN:2150-7511, ISSN:2161-2129

Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureus., mBio, 11 (2), ISSN:2150-7511, ISSN:2161-2129

Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells., Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections.

The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time. Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus. Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections., Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureus.

Bacteria are an integral part of human life on earth and surround us from the beginning to the end of our existence. In this thesis, we present two projects that permit better understanding and control of two major pathogens from the bacterial order Bacillales: Staphylococcus aureus and Listeria monocytogenes. Both bacteria are Gram-positive and foodborne pathogens, have various virulence factors and can invade human cells. S. aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistant strains. In addition, intracellular localization of S. aureus plays a key role in recurrent infections, as intracellular pathogens are protected from antibiotics and immune responses. Bacteriophage encoded endolysins and other peptidoglycan hydrolases (PGHs) are alternative therapeutics, which are currently under clinical investigation. They offer several advantages such as effective and specific killing by means of an active mechanism, which eliminates metabolically inactive and drug resistant S. aureus. However, PGHs are limited in their ability to target intracellular bacteria and in Manuscript 1 of this thesis, we describe the preclinical development of PGHs for effective targeting of intracellular S. aureus. To this end, we screened 322 PGHs for staphylolytic activity simulating intracellular and other relevant physiological conditions. The most active constructs were extensively characterized in vitro and six enzymes were fused to selected cell penetrating peptides (CPPs) to enhance cellular uptake, as demonstrated by time-resolved fluorescence of Europium labeled enzymes. Cellular infection models with different S. aureus strains (including methicillin resistant S. aureus) were employed in combination with fluorescence microscopy to identify the most suitable combinations of PGHs and CPPs. We demonstrate killing of intracellular S. aureus by up to 4.5 log units. PGHs fused to the CCP "TAT", yielded the best results, and efficacy could be further enhanced by combining synergistically active PGHs. Finally, synergistically active PGH TAT cocktails and their individual components with and without TAT were evaluated in a murine abscess model. The synergistic PGH-TAT cocktail showed significantly improved killing compared to all other tested treatments, with a total S. aureus reduction of 2.2 log units. The PGH TAT cocktail also significantly enhanced killing of intracellular Staphylococci, additionally abscess size and body weight loss were most reduced in animals treated with this cocktail. Altogether, the superior performance of the PGH TAT cocktail is based on the careful selection of appropriate enzymes by activity screening, the synergistic action of its components and their increased penetration due to CPP fusion. In conclusion, CPP fused PGHs represent promising novel protein therapeutics against intracellular, drug tolerant and drug resistant S. aureus. In the future, their safety should be assessed and a clinical trial should be designed for the treatment of diseases with reported intracellular localization of S. aureus, such as atopic dermatitis. Manuscript 2 focuses on Listeria monocytogenes, a bacterium, which can be ubiquitous found in the environment, particularly in soil. It is a major concern in the food industry, as it frequently causes listeriosis outbreaks due to contamination of fresh produce, dairy products and pre cut meat or fish. Listeriosis can lead to abortion and infected individuals have high mortality rates of 20-30%. Listeria monocytogenes can be divided into 12 different serovars, which diverge in their wall-teichoic acid glycosylation. The current standard procedure for serovar determination is based on an agglutination method using antisera. The differential binding patterns of those antisera to the somatic and flagellar antigens of Listeria allow distinction of different serovars. Only strains designated as serovars 1/2a, 1/2b and 4b are regarded as highly virulent and are responsible for 98% of all listeriosis cases. Unfortunately, serotyping is imperfect in both, precision and practicality, due to wide discrepancies in the subjective interpretation of results. Additionally, the exact antigen epitopes remain unclear. We used structural predictions to find proteins derived from bacteriophage endolysins and receptor binding proteins to develop of a novel method for somatic antigen differentiation. The six selected proteins recognize differentially glycosylated wall teichoic acids on the Listeria surface. Their fusion to green fluorescent protein permits quantification of surface glycosylation, which is directly linked to different Listeria serovars. This objective glycotyping approach enables clear, non-biased determination of all somatic antigen structures with the ability to quantify the relative amount of surface receptor present on each strain. As such, the developed glycotyping system facilitated near complete and quantitative serovar differentiation of over 60 Listeria strains from a cohort of previously characterized Listeria. In addition, we used the selected proteins to functionalize paramagnetic beads. These functionalized beads can now specifically isolate strains belonging to the pathogenic serovars 1/2 and 4b from a mixture of Listeria strains. Furthermore our serovar-specific profiling method led to the discovery that strains designated as SV 4e possess an intermediate 4b-4d glycosylation phenotype, revealing the high discriminative power of this novel technique. In the future, our system may prove valuable for enrichment from foods, prior to detection with highly sensitive systems, such as novel luciferase reporter phages constructed in our laboratory. In summary, in this work we use bacteriophage proteins to meet the demand for novel diagnostic tools and ways to combat drug-resistant bacterial pathogens.