Clearance Systems in the Brain, From Structure to Function.

As the most metabolically active organ in the body, there is a recognized need for pathways that remove waste proteins and neurotoxins from the brain. Previous research has indicated potential associations between the clearance system in the brain and the pathological conditions of the central nervous system (CNS), due to its importance, which has attracted considerable attention recently. In the last decade, studies of the clearance system have been restricted to the glymphatic system. However, removal of toxic and catabolic waste by-products cannot be completed independently by the glymphatic system, while no known research or article has focused on a comprehensive overview of the structure and function of the clearance system. This thesis addresses a neglected aspect of linkage between the structural composition and main components as well as the role of neural cells throughout the clearance system, which found evidence that the components of CNS including the glymphatic system and the meningeal lymphatic system interact with a neural cell, such as astrocytes and microglia, to carry out vital clearance functions. As a result of this evidence that can contribute to a better understanding of the clearance system, suggestions were identified for further clinical intervention development of severe conditions caused by the accumulation of metabolic waste products and neurotoxins in the brain, such as Alzheimer's disease (AD) and Parkinson's disease (PD).

Overlapping and unique contributions of two conserved polysaccharide loci in governing distinct survival phenotypes in Vibrio vulnificus.

As an aetiological agent of bacterial sepsis and wound infections, Vibrio vulnificus is unique among the Vibrionacea. Its continued environmental persistence and transmission are bolstered by its ability to colonize shellfish and form biofilms on various marine biotic surfaces. We previously identified a polysaccharide locus, brp, which contributes to the survival phenotypes of biofilm formation, rugose colony formation and stress resistance. Here, we describe a second polysaccharide locus, rbd (regulation of biofilm development), which also enhanced biofilm formation when expressed. Despite this functional overlap, the development of stress resistance and rugosity could be uniquely attributed to brp expression, whereas rbd expression augmented aggregate formation. Simultaneous expression of both loci led to the formation of a dramatic pellicle and maximum biofilm formation. Unlike the brp locus, transcription of the rbd locus was regulated not by c-di-GMP, but by a response regulator (RbdG) that was encoded within the locus. We propose that the ability to regulate the expression of polysaccharides with overlapping and unique characteristics in response to different environmental cues enables V. vulnificus to 'fine tune' its biofilm lifestyle to the prevailing environmental conditions and maximally benefit from the characteristics associated with each polysaccharide.

Chitin-induced carbotype conversion in Vibrio vulnificus.

As an etiological agent of bacterial sepsis and wound infections, Vibrio vulnificus is unique among the Vibrionaceae. The most intensely studied of its virulence factors is the capsular polysaccharide (CPS). Over 100 CPS types have been identified, yet little is known about the genetic mechanisms that drive such diversity. Chitin, the second-most-abundant polysaccharide in nature, is known to induce competence in Vibrio species. Here, we show that the frequency of chitin-induced transformation in V. vulnificus varies by strain and that (GlcNAc)(2) is the shortest chitin-derived polymer capable of inducing competence. Transformation frequencies (TFs) increased 8-fold when mixed-culture biofilms were exposed to a strain-specific lytic phage, suggesting that the lysis of dead cells during lytic infection increased the amount of extracellular DNA within the biofilm that was available for transfer. Furthermore, we show that V. vulnificus can undergo chitin-dependent carbotype conversion following the uptake and recombination of complete cps loci from exogenous genomic DNA (gDNA). The acquisition of a partial locus was also demonstrated when internal regions of homology between the endogenous and exogenous loci existed. This suggested that the same mechanism governing the transfer of complete cps loci also contributed to their evolution by generating novel combinations of CPS biosynthesis genes. Since no evidence that cps loci were preferentially acquired during natural transformation (random transposon-tagged DNA was readily taken up in chitin transformation assays) exists, the phenomenon of chitin-induced transformation likely plays an important but general role in the evolution of this genetically promiscuous genus.

Identification of a c-di-GMP-regulated polysaccharide locus governing stress resistance and biofilm and rugose colony formation in Vibrio vulnificus.

As an etiological agent of bacterial sepsis and wound infections, Vibrio vulnificus is unique among the Vibrionaceae. Its continued environmental persistence and transmission are bolstered by its ability to colonize shellfish, form biofilms on various marine biotic surfaces, and generate a morphologically and physiologically distinct rugose (R) variant that yields profuse biofilms. Here, we identify a c-di-GMP-regulated locus (brp, for biofilm and rugose polysaccharide) and two transcription factors (BrpR and BrpT) that regulate these physiological responses. Disruption of glycosyltransferases within the locus or either regulator abated the inducing effect of c-di-GMP on biofilm formation, rugosity, and stress resistance. The same lesions, or depletion of intracellular c-di-GMP levels, abrogated these phenotypes in the R variant. The parental and brp mutant strains formed only scant monolayers on glass surfaces and oyster shells, and although the R variant formed expansive biofilms, these were of limited depth. Dramatic vertical expansion of the biofilm structure was observed in the parental strain and R variant, but not the brp mutants, when intracellular c-di-GMP levels were elevated. Hence, the brp-encoded polysaccharide is important for surface colonization and stress resistance in V. vulnificus, and its expression may control how the bacteria switch from a planktonic lifestyle to colonizing shellfish to invading human tissue.