Citation Details

Journal Article

Xen5, Xen 5, Pseudomonas aeruginosa Xen 5

Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.

Journal Article

Xen14, Xen 14, E. coli Xen14, IVIS, Animals; Cell Line, Tumor; Female; Histocompatibility Antigens Class I/genetics/*immunology; Humans; Klebsiella Infections/*immunology/prevention & control; Klebsiella pneumoniae/*immunology; Male; Mice; Mice, Inbred C57BL; Mice, Knockout

As opposed to the well established role of MHC-linked, polymorphic, class I (MHC-I) genes in adaptive immunity, a universal role for non-conventional MHC-I is unknown, thus requiring a case-by-case study. The MHC unlinked, monomorphic, but beta(2)microglobulin (beta(2)m)-associated “MHC class I related” MR1 molecule interacts with a semi-invariant TCR. The pathophysiology of this interaction or more importantly of this peculiar MHC-I remains mostly unknown. Recently it was shown that beta(2)m deficient mice were more susceptible to infection by Klebsiella pneumoniae, a widely spread Gram-negative bacteria that causes diverse and often severe ailments in man. Here we demonstrate, using both an in vivo imaging system and survival tests, the increased susceptibility to K. pneumoniae (but not to several other Gram negative bacteria) of MR1 deficient mice. This is accompanied by a consequent change in body temperature and systemic cytokine profile. Hence MR1 controls K. pneumoniae infection in vivo.

Journal Article

Xen14, Xen 14, E. coli Xen14, IVIS

Biofilm-associated infections are a major complication of implanted and indwelling medical devices like urological and venous catheters. They commonly persist even in the presence of an oral or intravenous antibiotic regimen, often resulting in chronic illness. We have developed a new approach to inhibiting biofilm growth on synthetic materials through controlled release of salicylic acid from a polymeric coating. Herein we report the synthesis and testing of a ultraviolet-cured polyurethane acrylate polymer composed, in part, of salicyl acrylate, which hydrolyzes upon exposure to aqueous conditions, releasing salicylic acid while leaving the polymer backbone intact. The salicylic acid release rate was tuned by adjusting the polymer composition. Anti-biofilm performance of the coatings was assessed under several biofilm forming conditions using a novel combination of the MBEC Assay biofilm multi-peg growth system and bioluminescence monitoring for live cell quantification. Films of the salicylic acid-releasing polymers were found to inhibit biofilm formation, as shown by bioluminescent and GFP reporter strains of Pseudomonas aeruginosa and Escherichia coli. Urinary catheters coated on their inner lumens with the salicylic acid-releasing polymer significantly reduced biofilm formation by E. coli for up to 5 days under conditions that simulated physiological urine flow.

Journal Article

Xen14, Xen 14, E. coli Xen14, IVIS, Animals; Bacteroides fragilis/*isolation & purification; Diagnostic Imaging/methods; Disease Progression; Escherichia coli/*isolation & purification; Luciferases, Bacterial/*diagnostic use; Luminescent Agents/*diagnostic use; Male; Peritoneal Lavage; Peritonitis/*microbiology/pathology/therapy; Rats; Rats, Wistar

BACKGROUND: Bacterial peritonitis is a life-threatening abdominal infection associated with high morbidity and mortality. The rat is a popular animal model for studying peritonitis and its treatment, but longitudinal monitoring of the progression of peritonitis in live animals has been impossible until now and thus required a large number of animals. Our objective was to develop a noninvasive in vivo imaging technique to monitor the spatiotemporal spread of bacterial peritonitis. METHODS: Peritonitis was induced in 8 immunocompetent male Wistar rats by placing fibrin clots containing 5x10(8) cells of both Bacteroides fragilis (American Type Tissue Culture [ATCC)] 25,285 and bioluminescent Escherichia coli Xen14. After 1 or 2 days, infected clots were removed and open abdomen lavage was performed. In vivo bioluminescent imaging was used to monitor the spread of peritonitis. RESULTS: Bioluminescent in vivo imaging showed an increase in the area of spread, and the number of E. coli tripled into the rat's abdominal cavity on day 1 after clot insertion; however, on day 2, encapsulation of the clot confined bacterial spread. Bioluminescent E. coli respread over the peritoneal cavity after lavage; within 10 days, however, in vivo imaging showed a decrease of 3-4 orders of magnitude in bacterial load. CONCLUSION: Bioluminescent in vivo imaging can be effectively used to monitor the spatiotemporal behavior of the peritonitis during 3 different stages of the disease process: initiation, treatment, and follow-up. Imaging allows researchers to repeatedly image the same animal, thereby reducing variability and providing greater confidence in determining treatment efficacies for therapeutic interventions using a small number of animals.

Journal Article

Animals; Escherichia coli/immunology; Escherichia coli Infections/enzymology/immunology/*prevention & control; Extracellular Space/genetics/*immunology/metabolism; Glutathione Reductase/deficiency/genetics/*physiology; Humans; Mice; Mice, Inbred C3H; Mice, Knockout; Neutrophils/*immunology/*metabolism/microbiology; Oxidative Stress/genetics/*immunology; Phagocytosis/genetics/*immunology; Staphylococcal Infections/enzymology/immunology/*prevention & control; Staphylococcus aureus/immunology

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in the bactericidal function of phagocytes. Because Gsr has been implicated in the oxidative burst in human neutrophils and is abundantly expressed in the lymphoid system, we hypothesized that Gsr-deficient mice would exhibit marked defects during the immune response against bacterial challenge. We report in this study that Gsr-null mice exhibited enhanced susceptibility to Escherichia coli challenge, indicated by dramatically increased bacterial burden, cytokine storm, striking histological abnormalities, and substantially elevated mortality. Additionally, Gsr-null mice exhibited elevated sensitivity to Staphylococcus aureus. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed impaired phagocytosis and defective bacterial killing activities. Although Gsr catalyzes the regeneration of glutathione, a major cellular antioxidant, Gsr-deficient neutrophils paradoxically produced far less reactive oxygen species upon activation both ex vivo and in vivo. Unlike wild-type neutrophils that exhibited a sustained oxidative burst upon stimulation with phorbol ester and fMLP, Gsr-deficient neutrophils displayed a very transient oxidative burst that abruptly ceased shortly after stimulation. Likewise, Gsr-deficient neutrophils also exhibited an attenuated oxidative burst upon encountering E. coli. Biochemical analysis revealed that the hexose monophosphate shunt was compromised in Gsr-deficient neutrophils. Moreover, Gsr-deficient neutrophils displayed a marked impairment in the formation of neutrophil extracellular traps, a bactericidal mechanism that operates after neutrophil death. Thus, Gsr-mediated redox regulation is crucial for bacterial clearance during host defense against massive bacterial challenge.