Investigating the genetic determinants for Escherichia coli capsular polysaccharide biosynthesis

Abstract

Bacterial capsules are protective layers of high molecular weight polysaccharides that surround the cell surfaces of many Gram-negative bacterial pathogens. Capsular polysaccharides (CPSs) show remarkable structural diversity and have classically been identified by their distinct immunochemistries. In Escherichia coli, this results in approximately 80 K- (capsular) antigen serotypes. In many pathogens, CPSs are often essential for cell survival and virulence within the host because they allow evasion of immune defenses. The documented attenuation of unencapsulated mutants in animal models of infection makes CPS biosynthesis and export proteins candidate therapeutic targets. Proof-of principle is provided in the literature by small-molecule inhibitors of E. coli capsule assembly, which offer protection in a murine model of sepsis. However, such “antivirulence” applications require a fundamental understanding of the underpinning cellular processes. Despite their structural diversity, CPSs are formed by one of two conserved assembly strategies. The E. coli “group 1” capsules are assembled via a Wzy-dependent pathway. The E. coli “group 2” capsule systems are defined by an assembly strategy with polymer export dependent on an ATP-binding cassette (ABC) transporter. This system is shared by extraintestinal pathogenic E. coli isolates, Neisseria meningitidis, Haemophilus influenzae, and other pathogens featured on the World Health Organization’s global priority list of antibiotic-resistant bacteria. The dedicated proteins involved in the synthesis and export of E. coli group 2 CPSs are encoded by the kps locus, and the protein functions are known, to various degrees of detail. In contrast, other supporting cellular components involved in CPS expression, and the global connectivity of this pathway with the other cellular processes, have not been established. Filling the information gap concerning the involvement of other cellular processes in CPS assembly requires a different experimental approach than the targeted strategies used to date. Here, I used genome-wide phenotypic screening as an unbiased approach to identify “housekeeping” genes (genes required for maintenance of basal cellular functions) necessary for capsule assembly. Surprisingly, the list of newly discovered genes required for group 2 CPS biosynthesis is brief and is often dependent on growth conditions, indicating kps functionality is mostly independent of genetic background, and yet is highly responsive to environmental cues. This may contribute to the ease of horizontal transfer of capsule production to new isolates, as there are few additional genetic requirements for the receiving bacterium. The conserved requirements of housekeeping elements across the two principle CPS assembly strategies has yet to be well studied. The requirement for Braun’s lipoprotein (Lpp; a key protein in the maintenance of outer membrane integrity and essential in the assembly of the E. coli group 2 CPS export machinery) in capsule assembly by different strategies is investigated in this thesis. The results show that the importance of Lpp varies depending on the structure of the outer membrane CPS translocon. These studies offer important new insight into CPS assembly in Gram-negative bacteria and the relationship between the CPS biosynthesis pathway and other cellular processes.