Modeling the growth of multicellular cancer spheroids in a\ud bioengineered 3D microenvironment and their treatment with an\ud anti-cancer drug

A critical step in the dissemination of ovarian cancer cells is the formation of multicellular spheroids from cells shed from the primary tumor. The objectives of this study were to establish and validate bioengineered three-dimensional (3D) microenvironments for culturing ovarian cancer cells in vitro and simultaneously to develop computational models describing the growth of multicellular spheroids in these bioengineered matrices. Cancer cells derived from human epithelial ovarian carcinoma were embedded within biomimetic hydrogels of varying stiffness and cultured for up to 4 weeks. Immunohistochemistry was used to quantify the dependence of cell proliferation and apoptosis on matrix stiffness, long-term culture and treatment with the anti-cancer drug paclitaxel.\ud \ud Two computational models were developed. In the first model, each spheroid was treated as an incompressible porous medium, whereas in the second model the concept of morphoelasticity was used to incorporate details about internal stresses and strains. Each model was formulated as a free boundary problem. Functional forms for cell proliferation and apoptosis motivated by the experimental work were applied and the predictions of both models compared with the output from the experiments. Both models simulated how the growth of cancer spheroids was influenced by mechanical and biochemical stimuli including matrix stiffness, culture time and treatment with paclitaxel. Our mathematical models provide new perspectives on previous experimental results and have informed the design of new 3D studies of multicellular cancer spheroids

Growth of confined cancer spheroids: a combined experimental and mathematical modelling approach

We have integrated a bioengineered three-dimensional platform by generating multicellular cancer spheroids in a controlled microenvironment with a mathematical model to investigate\ud confined tumour growth and to model its impact on cellular processes

Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail

Enhancing bacteriophage therapeutics through in situ production and release of heterologous antimicrobial effectors

Bacteriophages operate via pathogen-specific mechanisms of action distinct from conventional, broad-spectrum antibiotics and are emerging as promising alternative antimicrobials. However, phage-mediated killing is often limited by bacterial resistance development. Here, we engineer phages for target-specific effector gene delivery and host-dependent production of colicin-like bacteriocins and cell wall hydrolases. Using urinary tract infection (UTI) as a model, we show how heterologous effector phage therapeutics (HEPTs) suppress resistance and improve uropathogen killing by dual phage- and effector-mediated targeting. Moreover, we designed HEPTs to control polymicrobial uropathogen communities through production of effectors with cross-genus activity. Using phage-based companion diagnostics, we identified potential HEPT responder patients and treated their urine ex vivo. Compared to wildtype phage, a colicin E7-producing HEPT demonstrated superior control of patient E. coli bacteriuria. Arming phages with heterologous effectors paves the way for successful UTI treatment and represents a versatile tool to enhance and adapt phage-based precision antimicrobials

Rapid Analysis of Listeria monocytogenes Cell Wall Teichoic Acid Carbohydrates by ESI-MS/MS

We report the application of electrospray ionization (ESI) mass spectrometry for compositional characterization of wall teichoic acids (WTA), a major component of Gram-positive bacterial cell walls. Tandem mass spectrometry (ESI-MS/MS) of purified and chemically hydrolyzed monomeric WTA components provided sufficient information to identify WTA monomers and their specific carbohydrate constituents. A lithium matrix was used for ionization of uncharged WTA monomers, and successfully applied to analyze the WTA molecules of four Listeria strains differing in carbohydrate substitution on a conserved polyribitol-phosphate backbone structure. Carbohydrate residues such as N-acetylglucosamine or rhamnose linked to the WTA could directly be identified by ESI-MS/MS, circumventing the need for quantitative analysis by gas chromatography. The presence of a terminal N-acetylglucosamine residue tethered to the ribitol was confirmed using fluorescently labeled wheat-germ agglutinin. In conclusion, the mass spectrometry method described here will greatly facilitate compositional analysis and characterization of teichoic acids and similar macromolecules from diverse bacterial species, and represents a significant advance in the identification of serovar-specific carbohydrates and sugar molecules on bacteria

Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these molecular syringes for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells

Improved Biodistribution and Extended Serum Half-Life of a Bacteriophage Endolysin by Albumin Binding Domain Fusion

The increasing number of multidrug-resistant bacteria intensifies the need to develop new antimicrobial agents. Endolysins are bacteriophage-derived enzymes that degrade the bacterial cell wall and hold promise as a new class of highly specific and versatile antimicrobials. One major limitation to the therapeutic use of endolysins is their often short serum circulation half-life, mostly due to kidney excretion and lysosomal degradation. One strategy to increase the half-life of protein drugs is fusion to the albumin-binding domain (ABD). By high-affinity binding to serum albumin, ABD creates a complex with large hydrodynamic volume, reducing kidney excretion and lysosomal degradation. The aim of this study was to investigate the in vitro antibacterial activity and in vivo biodistribution and half-life of an engineered variant of the Staphylococcus aureus phage endolysin LysK. The ABD sequence was introduced at different positions within the enzyme, and lytic activity of each variant was determined in vitro and ex vivo in human serum. Half-life and biodistribution were assessed in vivo by intravenous injection of europium-labeled proteins into C57BL/6 wild-type mice. Our data demonstrates that fusion of the endolysin to ABD improves its serum circulation half-life and reduces its deposition in the kidneys in vivo. The most active construct reduced S. aureus counts in human serum ex vivo by 3 logs within 60 min. We conclude that ABD fusions provide an effective strategy to extend the half-life of antibacterial enzymes, supporting their therapeutic potential for treatment of systemic bacterial infections

Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection

Objectives In the light of increasing drug resistance in Staphylococcus aureus, bacteriophage endolysins [peptidoglycan hydrolases (PGHs)] have been suggested as promising antimicrobial agents. The aim of this study was to determine the antimicrobial activity of nine enzymes representing unique homology groups within a diverse class of staphylococcal PGHs. Methods PGHs were recombinantly expressed, purified and tested for staphylolytic activity in multiple in vitro assays (zymogram, turbidity reduction assay and plate lysis) and against a comprehensive set of strains (S. aureus and CoNS). PGH cut sites in the staphylococcal peptidoglycan were determined by biochemical assays (Park-Johnson and Ghuysen procedures) and MS analysis. The enzymes were tested for their ability to eradicate static S. aureus biofilms and compared for their efficacy against systemic MRSA infection in a mouse model. Results Despite similar modular architectures and unexpectedly conserved cleavage sites in the peptidoglycan (conferred by evolutionarily divergent catalytic domains), the enzymes displayed varying degrees of in vitro lytic activity against numerous staphylococcal strains, including cell surface mutants and drug-resistant strains, and proved effective against static biofilms. In a mouse model of systemic MRSA infection, six PGHs provided 100% protection from death, with animals being free of clinical signs at the end of the experiment. Conclusions Our results corroborate the high potential of PGHs for treatment of S. aureus infections and reveal unique antimicrobial and biochemical properties of the different enzymes, suggesting a high diversity of potential applications despite highly conserved peptidoglycan target site

Spontaneous resistance of Erwinia amylovora against bacteriophage Y2 affects infectivity of multiple phages

Broad application of antibiotics gave rise to increasing numbers of antibiotic resistant bacteria. Therefore, effective alternatives are currently investigated. Bacteriophages, natural predators of bacteria, could work as such an alternative. Although phages can be highly effective at eliminating specific bacteria, phage resistance can be observed after application. The nature of this resistance, however, can differ depending on the phage. Exposing Erwinia amylovora CFBP 1430, the causative agent of fire blight, to the different phages Bue1, L1, S2, S6, or M7 led to transient resistance. The bacteria reversed to a phage sensitive state after the phage was eliminated. When wild type bacteria were incubated with Y2, permanently resistant colonies (1430 Y2R ) formed spontaneously. In addition, 1430 Y2R revealed cross-resistance against other phages (Bue1) or lowered the efficiency of plating (L1, S2, and S6). Pull down experiments revealed that Y2 is no longer able to bind to the mutant suggesting mutation or masking of the Y2 receptor. Other phages tested were still able to bind to 1430 Y2R . Bue1 was observed to still adsorb to the mutant, but no host lysis was found. These findings indicated that, in addition to the alterations of the Y2 receptor, the 1430 Y2R mutant might block phage attack at different stage of infection. Whole genome sequencing of 1430 Y2R revealed a deletion in the gene with the locus tag EAMY_2231. The gene, which encodes a putative galactosyltransferase, was truncated due to the resulting frameshift. The mutant 1430 Y2R was monitored for potential defects or fitness loss. Weaker growth was observed in LB medium compared to the wild type but not in minimal medium. Strain 1430 Y2R was still highly virulent in blossoms even though amylovoran production was observed to be reduced. Additionally, LPS structures were analyzed and were clearly shown to be altered in the mutant. Complementation of the truncated EAMY_2231 in trans restored the wild type phenotype. The truncation of EAMY_2231 can therefore be associated with manifold modifications in 1430 Y2R , which can affect different phages simultaneously

Systemic application of bone-targeting peptidoglycan hydrolases as a novel treatment approach for staphylococcal bone infection

The rising prevalence of antimicrobial resistance in S. aureus has rendered treatment of staphylococcal infections increasingly difficult, making the discovery of alternative treatment options a high priority. Peptidoglycan hydrolases, a diverse group of bacteriolytic enzymes, show high promise as such alternatives due to their rapid and specific lysis of bacterial cells, independent of antibiotic resistance profiles. However, using these enzymes for the systemic treatment of local infections, such as osteomyelitis foci, needs improvement, as the therapeutic distributes throughout the whole host, resulting in low concentrations at the actual infection site. In addition, the occurrence of intracellularly persisting bacteria can lead to relapsing infections. Here, we describe an approach using tissue-targeting to increase the local concentration of therapeutic enzymes in the infected bone. The enzymes were modified with a short targeting moiety that mediated accumulation of the therapeutic in osteoblasts and additionally enables targeting of intracellularly surviving bacteria