Are there enzymatic treatment options for ocular biofilm-based infections?
Pseudomonas aeruginosa-induced corneal keratitis is a sight-threatening disease. The elevated rise of antibiotic resistance among the Pseudomonas keratitis isolates makes treatment of this disease challenging, justifying the need for alternative therapeutic modalities or treatment strategies that can reinforce antibiotic activities. By comparing the responses of P. aeruginosa infection on an outbred strain of mice (SW) to a susceptible strain of mice (C57BL6/N), we found that the neutrophil killing abilities of these strains segregate with their susceptibilities to infection. Namely, SW-derived neutrophils were significantly more efficient at killing P. aeruginosa in vitro than C57BL6/N-derived neutrophils. Quantitative LC-MS/MS analysis revealed significant differences in proteins with enzymatic activities such as alpha mannosidases. Given that during infection, P. aeruginosa forms mannose-rich biofilms at the ocular surface in the susceptible strain of mice, we compared the therapeutic potency of MoAb, recognizing the mannose-rich extracellular polysaccharide, psl, to that of alpha mannosidase. The topical application of alpha mannosidase reduced in vitro-formed biofilms by P. aeruginosa and consistently reduced the bacterial burden of the infected corneas. Cumulatively, these data illustrate that topical application of enzymes can control bacterial biofilm assembly and improve bacterial clearance.
Sample Processing Protocol
Neutrophils from infected and baseline SW and C57BL6/N mice (in triplicate or quadruplicate) were subjected to in-solution trypsin digestion. Briefly, 200 μl of 8 M Urea containing 40 mM HEPES was added to baseline and infected SW and C57BL6/N samples were sonicated in a rotating water bath at 4°C for 15 min (30 s on, 30 s off). The samples were then reduced with 10 mM dithiothreitol (DTT), alkylated with 55 mM iodoacetamide (IAA), followed by dilution with 50 mM ammonium bicarbonate to 2 M urea, and LysC and trypsin (protein: enzyme ratio 50:1) digestion overnight. Digestion was stopped by addition of 10% v/v Trifluoroacetic acid (TFA) per sample and the acidified peptides were loaded onto StageTips (containing three layers of C18) to desalt and purify according to the standard protocol. Each sample was divided onto two StageTips (one "working" and one "back-up") and stored at 4°C until the LC-MS/MS measurement.
Data Processing Protocol
Raw files were analyzed together using MaxQuant software (version 220.127.116.11). The derived peak list was searched with the built-in Andromeda search engine against the reference Mus musculus proteome downloaded from Uniprot (http://www.uniprot.org/) (April 27, 2016; 53,106 sequences). The parameters were as follows: strict trypsin specificity was required with cleavage at the C-terminal after K or R, allowing for up to two missed cleavages. The minimum required peptide length was set to seven amino acids. Carbamidomethylation of cysteine was set as a fixed modification (57.021464 Da) and N-acetylation of proteins N termini (42.010565 Da) and oxidation of methionine (15.994915 Da) were set as variable modifications. PSM and protein identifications were filtered using a target-decoy approach at a false discovery rate (FDR) of 1%. 'Match between runs' was enabled with a match time window of 0.7 min and an alignment time window of 20 min. Relative, label-free quantification (LFQ) of proteins was done using the MaxLFQ algorithm (24) integrated into MaxQuant using a minimum ratio count of 2, enabled FastLFQ option, LFQ minimum number of neighbors at 3, and the LFQ average number of neighbors at 6.
Jennifer Geddes-McAlister, University of Guelph
Jennifer Geddes-McAlister, Deparment of Molecular and Cellular Biology University of Guelph Guelph, Ontario, Canada Department of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried, Germany ( lab head )
Kugadas A, Geddes-McAlister J, Guy E, DiGiandomenico A, Sykes DB, Mansour MK, Mirchev R, Gadjeva M. Frontline Science: Employing enzymatic treatment options for management of ocular biofilm-based infections. J Leukoc Biol. 2019 PubMed: 30690787