RESUMO
Bacteriophages ("phages") infect and multiply within specific bacterial strains, causing lysis of their target. Due to the specific nature of these interactions, phages allow a high-precision approach for therapy which can also be exploited for the detection of phage-sensitive pathogens associated with chronic diseases due to gut microbiome imbalance. As rapid phage-mediated detection assays becoming standard-of-care diagnostic tools, they will advance the more widespread application of phage therapy in a precision approach. Using a conventional method and a new cloning approach to develop luminescent phages, we engineered two phages that specifically detect a disease-associated microbial strain. We performed phage sensitivity assays in liquid culture and in fecal matrices and tested the stability of spiked fecal samples stored under different conditions. Different reporter gene structures and genome insertion sites were required to successfully develop the two nluc-reporter phages. The reporter phages detected spiked bacteria in five fecal samples with high specificity. Fecal samples stored under different conditions for up to 30 days did not display major losses in reporter-phage-based detection. Luminescent phage-based diagnostics can provide a rapid co-diagnostic tool to guide the growing field of phage therapy, particularly for a precision-based approach to chronic diseases treatment.
RESUMO
BACKGROUND: (Pseudo)Bacteroides cellulosolvens is a cellulolytic bacterium that produces the most extensive and intricate cellulosomal system known in nature. Recently, the elaborate architecture of the B. cellulosolvens cellulosomal system was revealed from analysis of its genome sequence, and the first evidence regarding the interactions between its structural and enzymatic components were detected in vitro. Yet, the understanding of the cellulolytic potential of the bacterium in carbohydrate deconstruction is inextricably linked to its high-molecular-weight protein complexes, which are secreted from the bacterium. RESULTS: The current proteome-wide work reveals patterns of protein expression of the various cellulosomal components, and explores the signature of differential expression upon growth of the bacterium on two major carbon sources-cellobiose and microcrystalline cellulose. Mass spectrometry analysis of the bacterial secretome revealed the expression of 24 scaffoldin structural units and 166 dockerin-bearing components (mainly enzymes), in addition to free enzymatic subunits. The dockerin-bearing components comprise cell-free and cell-bound cellulosomes for more efficient carbohydrate degradation. Various glycoside hydrolase (GH) family members were represented among 102 carbohydrate-degrading enzymes, including the omnipresent, most abundant GH48 exoglucanase. Specific cellulosomal components were found in different molecular-weight fractions associated with cell growth on different carbon sources. Overall, microcrystalline cellulose-derived cellulosomes showed markedly higher expression levels of the structural and enzymatic components, and exhibited the highest degradation activity on five different cellulosic and/or hemicellulosic carbohydrates. The cellulosomal activity of B. cellulosolvens showed high degradation rates that are very promising in biotechnological terms and were compatible with the activity levels exhibited by Clostridium thermocellum purified cellulosomes. CONCLUSIONS: The current research demonstrates the involvement of key cellulosomal factors that participate in the mechanism of carbohydrate degradation by B. cellulosolvens. The powerful ability of the bacterium to exhibit different degradation strategies on various carbon sources was revealed. The novel reservoir of cellulolytic components of the cellulosomal degradation machineries may serve as a pool for designing new cellulolytic cocktails for biotechnological purposes.
