An in vitro tissue model for screening sustained release of phosphate-based therapeutic attenuation of pathogen-induced proteolytic matrix degradation.

Tissue response to intestinal injury or disease releases pro-inflammatory host stress signals triggering microbial shift to pathogenic phenotypes. One such phenotype is increased protease production resulting in collagen degradation and activation of host matrix metalloproteinases contributing to tissue breakdown. We have shown that surgical injury depletes local intestinal phosphate concentration triggering bacterial virulence and that polyphosphate replenishment attenuates virulence and collagenolytic activity. Mechanistic studies of bacterial and host protease expression contributing to tissue breakdown are difficult to achieve in vivo necessitating the development of novel in vitro tissue models. Common techniques for screening in vitro protease activity, including gelatin zymography or fluorogenic protease-sensitive substrate kits, do not readily translate to 3D matrix degradation. Here, we report the application of an in vitro assay in which collagenolytic pathogens are cultured in the presence of a proteolytically degradable poly(ethylene) glycol scaffold and a non-degradable phosphate and/or polyphosphate nanocomposite hydrogel matrix. This in vitro platform enables quantification of pathogen-induced matrix degradation and screening of sustained release of phosphate-based therapeutic efficacy in attenuating protease expression. To evaluate matrix degradation as a function of bacterial enzyme levels secreted, we also present a novel method to quantify hydrogel degradation. This method involves staining protease-sensitive hydrogels with Sirius red dye to correlate absorbance of the degraded gel solution with hydrogel weight. This assay enables continuous monitoring and greater accuracy of hydrogel degradation kinetics compared to gravimetric measurements. Combined, the proposed in vitro platform and the presented degradation assay provide a novel strategy for screening efficacy of therapeutics in attenuating bacterial protease-induced matrix degradation.

Controlling biofilms using synthetic biology approaches.

Bacterial biofilms are formed by the complex but ordered regulation of intra- or inter-cellular communication, environmentally responsive gene expression, and secretion of extracellular polymeric substances. Given the robust nature of biofilms due to the non-growing nature of biofilm bacteria and the physical barrier provided by the extracellular matrix, eradicating biofilms is a very difficult task to accomplish with conventional antibiotic or disinfectant treatments. Synthetic biology holds substantial promise for controlling biofilms by improving and expanding existing biological tools, introducing novel functions to the system, and re-conceptualizing gene regulation. This review summarizes synthetic biology approaches used to eradicate biofilms via protein engineering of biofilm-related enzymes, utilization of synthetic genetic circuits, and the development of functional living agents. Synthetic biology also enables beneficial applications of biofilms through the production of biomaterials and patterning biofilms with specific temporal and spatial structures. Advances in synthetic biology will add novel biofilm functionalities for future therapeutic, biomanufacturing, and environmental applications.

Medium chain unsaturated fatty acid ethyl esters inhibit persister formation of Escherichia coli via antitoxin HipB.

Persisters represent a small bacterial population that is dormant and that survives under antibiotic treatment without experiencing genetic adaptation. Persisters are also considered one of the major reasons for recalcitrant chronic bacterial infections. Although several mechanisms of persister formation have been proposed, it is not clear how cells enter the dormant state in the presence of antibiotics or how persister cell formation can be effectively controlled. A fatty acid compound, cis-2-decenoic acid, was reported to decrease persister formation as well as revert the dormant cells to a metabolically active state. We reasoned that some fatty acid compounds may be effective in controlling bacterial persistence because they are known to benefit host immune systems. This study investigated persister cell formation by pathogens that were exposed to nine fatty acid compounds during antibiotic treatment. We found that three medium chain unsaturated fatty acid ethyl esters (ethyl trans-2-decenoate, ethyl trans-2-octenoate, and ethyl cis-4-decenoate) decreased the level of Escherichia coli persister formation up to 110-fold when cells were exposed to ciprofloxacin or ampicillin antibiotics. RNA sequencing analysis and gene deletion persister studies elucidated that these fatty acids inhibit bacterial persistence by regulating antitoxin HipB. A similar persister cell reduction was observed for pathogenic E. coli EDL933, Pseudomonas aeruginosa PAO1, and Serratia marcescens ICU2-4 strains. This study demonstrates that fatty acid ethyl esters can be used to disrupt bacterial dormancy to combat persistent infectious diseases.

Probiotic Escherichia coli inhibits biofilm formation of pathogenic E. coli via extracellular activity of DegP.

Many chronic infections involve bacterial biofilms, which are difficult to eliminate using conventional antibiotic treatments. Biofilm formation is a result of dynamic intra- or inter-species interactions. However, the nature of molecular interactions between bacteria in multi-species biofilms are not well understood compared to those in single-species biofilms. This study investigated the ability of probiotic Escherichia coli Nissle 1917 (EcN) to outcompete the biofilm formation of pathogens including enterohemorrhagic E. coli (EHEC), Pseudomonas aeruginosa, Staphylococcus aureus, and S. epidermidis. When dual-species biofilms were formed, EcN inhibited the EHEC biofilm population by 14-fold compared to EHEC single-species biofilms. This figure was 1,100-fold for S. aureus and 8,300-fold for S. epidermidis; however, EcN did not inhibit P. aeruginosa biofilms. In contrast, commensal E. coli did not exhibit any inhibitory effect toward other bacterial biofilms. We identified that EcN secretes DegP, a bifunctional (protease and chaperone) periplasmic protein, outside the cells and controls other biofilms. Although three E. coli strains tested in this study expressed degP, only the EcN strain secreted DegP outside the cells. The deletion of degP disabled the activity of EcN in inhibiting EHEC biofilms, and purified DegP directly repressed EHEC biofilm formation. Hence, probiotic E. coli outcompetes pathogenic biofilms via extracellular DegP activity during dual-species biofilm formation.

Generation of inhibitory monoclonal antibodies targeting matrix metalloproteinase-14 by motif grafting and CDR optimization.

Matrix metalloproteinase-14 (MMP-14) plays important roles in cancer metastasis, and the failures of broad-spectrum MMP compound inhibitors in clinical trials suggested selectivity is critical. By grafting an MMP-14 specific inhibition motif into complementarity determining region (CDR)-H3 of antibody scaffolds and optimizing other CDRs and the sequences that flank CDR-H3, we isolated a Fab 1F8 showing a binding affinity of 8.3 nM with >1000-fold enhancement on inhibition potency compared to the peptide inhibitor. Yeast surface display and fluorescence-activated cell sorting results indicated that 1F8 was highly selective to MMP-14 and competed with TIMP-2 on binding to the catalytic domain of MMP-14. Converting a low-affinity peptide inhibitor into a high potency antibody, the described methods can be used to develop other inhibitory antibodies of therapeutic significance.