Expression, Purification, and Characterization of a Polysaccharide Depolymerase from Acinetobacter baumannii Bacteriophage AbauYa1

The use of bacteriophages offers an appealing alternative to antibiotics for the control of pathogenic bacteria. Recently, bacteriophage AbauYa1 was isolated as part of an effort to find phages to combat Acinetobacter baumannii infections. In addition to causing lysis of the host cell, AbauYa1 depolymerizes the polysaccharides of the A. baumannii capsules. The A. baumannii capsule is an important virulence factor, and phage depolymerases have been shown to disrupt biofilms of other pathogenic bacterial strains. The gene encoding the protein responsible for this activity was cloned, and the protein was expressed, purified, and enzymatically assayed. The protein was found to degrade the polysaccharide capsule, as shown by an increase in reducing ends upon incubating the capsule with the depolymerase. Finally, the protein removes A. baumannii biofilms from a polystyrene surface. The protein degrades A. baumannii capsule and biofilms and therefore carries high therapeutic potential for treating A. baumannii infections

Doctor of Philosophy

dissertationBacteriophages as the most abundant biological entities on the planet play a significant role in microbial population dynamics in various ecosystems. The potential of bacteriophages as a driving force in evolution of microbial communities through controlling the bacterial population, naturally selecting phage-resistant bacteria, and facilitating horizontal gene transfer have been studied. However, few studies have demonstrated the effect of phages on the microbial communities in different natural ecosystems, biological nutrient removal reactors, and hypersaline environment. In this study, the role of bacteriophages in functional gene transfer and how they affect different nutrient cycles and bacterial diversity and population in lab-scale and natural ecosystems were investigated. The research was accomplished through studying the bacteriophage population, diversity, and their role in bacterial infection and subsequent alteration in bacterial population and diversity. The ecosystems studied in this study included lab-scale biological phosphorus removal and hypersaline Great Salt Lake as engineered and natural models for understanding the phage-host interaction. The biomass and sediment samples were collected from the lab-scale bioreactor and deep brine layer in Great Salt Lake and subjected to various environmental stress factors to understand the role of bacteriophage and prophage induction in bacterial diversity. In addition, the sediment sample from the Great Salt Lake was analyzed with metagenomics approach. The evaluation of prophage induction showed that various environmental stress factors including nutrients, heavy metals, toxic chemical, and antibiotic can induce phages integrated onto bacterial genomes (i.e. prophages), resulting in a decrease of the bacterial population involved in different nutrient cycles. Analyzing the viral and bacterial metagenomes explored the GC content, oligonucleotide and k-mer profile, genetic homology, CRISPRs, and prophage network. Our in-depth metagenomics analysis identified phage and bacteria communities comprehensively and proved the role of bacteriophages in defining the bacterial community population, diversity, and their effects on various nutrient cycles. Identification of bacteriophage diversity, population, and their functional genes using metagenomics approach in this study will shed light on the bacterial and viral diversity in Great Salt Lake and this information will be helpful in constructing metabolic models to better study the microbial interaction in various hypersaline ecosystems

Controlling Microbial Multicellular Behaviors With Saccharide Derivatives

Microbial multicellular behaviors like biofilm formation and swarming motility are known to increase their tolerance against antimicrobials. From microbial standpoint, nonmicrobicidal agents that do not impede growth are tolerable and therefore, there is a lower propensity to develop resistance against such agents as compared to microbicidal ones (antibiotics). This study describes a new antibiofilm approach of using nonmicrobicidal saccharide derivatives for controlling the multicellular behaviors of gram-negative bacteria, Pseudomonas aeruginosa and fungus, Candida albicans. Pseudomonas aeruginosa is known to secrete rhamnolipids, a class of biosurfactants that plays an important role in maintaining the architecture of its biofilm and promoting its swarming motility. Here we show the ability of certain synthetic nonmicrobicidal disaccharide derivatives (DSDs) to mimic the biofunctions of rhamnolipids. The rhlA mutant of P. aeruginosa is incapable of synthesizing rhamnolipids and also unable to swarm on semi-solid agar gel. When the natural ligands, rhamnolipids were externally added into the semi-solid agar gel in a concentration dependent manner, the swarming of the rhlA mutant reactivated at lower concentrations (10 μM) and then at relatively higher concentrations (15 μM), the swarming reactivation was reversed. When some active synthetic DSDs were tested on the rhlA mutant, the bacterial swarming first reactivated and then the activation reversed at higher DSD concentrations, similar to the effect of externally added rhamnolipids. Previously, a known bacterial signalling molecule has been shown to exhibit a similar concentration dependent activation and then activation reversal for light simulation by Vibrio fischeri. Some DSDs having disaccharide stereochemistries (cellobiose or maltose) and a bulky aliphatic tail (3, 7, 11-trimethyl-dodecanyl) caused swarming reactivation of the rhlA mutant at concentrations lower than that caused by the externally added rhamnolipids. The synthetic nonmicrobicidal DSDs were also very effective at inhibiting the adhesion of P. aeruginosa to polystyrene surface, and at inhibiting the bacterial biofilm formation. These DSDs were also potent dispersers of pre-formed biofilm of P. aeruginosa. The potent antibiofilm (inhibition and dispersion) activities were observed for those DSDs that possessed a disaccharide (cellobiose or maltose) stereochemistry and a bulky aliphatic chain such as 3, 7, 11-trimethyl-dodecanyl. These potent DSDs had half-maximal inhibitory concentrations for biofilm inhibition (IC50) and dispersion (DC50) comparable to those of known potent antibiofilm agents against P. aeruginosa. Gene-reporting assays indicate that the mechanism of action of such DSDs is not via the known las or rhl quorum sensing systems of P. aeruginosa but that the adhesin potein, pilin maybe a likely target of such molecules. Biofilms formed under natural settings are usually formed by both bacteria and fungus that co-reside in the same microenvironment. Therefore, agents that can prevent mixed biofilms are desirable from a therapeutic standpoint. Despite being nonmicrobicidal to both fungal blastospores and hyphae, the synthetic DSDs were able to inhibit the biofilm formation of fungus Candida albicans. Microscopic evaluation showed that most DSDs did not prevent the blastospores-to-hyphae morphogenesis. The DSDs were effective at inhibiting biofilm formation of Candida albicans when applied within five minutes of seeding the test surface with fungal cells. Using a surface based assay it was shown that one DSD dramatically reduced the surface adhesion of Candida albicans hyphae. The antibiofilm activity of such DSDs against Candida albicans is probably due to their ability to prevent hyphae surface adhesion