Identifying Pantoea Genetic Factors Involved in Host Association by Integrating Comparative Genomics with a Dictyostelium discoideum Grazing Resistance Model

Abstract

Pantoea is a genus of Gram-negative bacteria within the Enterobacterales. Much of what is known about Pantoea has been determined through studies focused on its phytopathogenicity and epiphytic associations. Limited research has been conducted on Pantoea to characterize its capacity for opportunism in humans, despite numerous reports of clinical infections. This thesis aims to identify genetic factors that could contribute to host association and thus, opportunistic infections by Pantoea. Chapter 1 provides a broad literature review of Pantoea, opportunism and virulence, model systems to assess virulence, and the field of genomics. Chapter 2 describes the adaptation of a model system using an amoebal predator, Dictyostelium discoideum, to screen bacteria for candidate virulence factors. Ten strains of Pantoea ananatis were screened for resistance to amoebal grazing, which was used as a potential indicator for virulence. A highthroughput genetic screen was developed and performed with grazing-resistant P. ananatis BRT175. This screen identified genes involved in quorum sensing, periplasmic protein redox maintenance, anaerobic metabolism regulation, and nucleotide biosynthesis. A novel hexose-based glycolipid biosurfactant, produced by the rhlA and rhlB genes in P. ananatis BRT175, was identified as being key for both swarming motility and grazing resistance. However, the rhlA and rhlB genes were present in both grazing-resistant and -susceptible strains. Chapter 3 addresses the differential regulation of rhlA in Pantoea. Variants within the putative promoter region of rhlA were identified in the 10 P. ananatis strains with an allele being correlated with grazing resistance. The promoters of rhlA from both P. ananatis BRT175 and P. stewartii DC283 were cloned into a lux reporter plasmid. The P. ananatis BRT175 rhlA promoter showed higher expression at 21°C compared to 30°C and 37°C. The P. stewartii DC283 rhlA promoter was less active, both in P. stewartii DC283 and P. ananatis BRT175, in hrp inducing medium than the native rhlA promoter from P. ananatis BRT175. Chapter 4 explores the genes associated with grazing resistance in the 10 P. ananatis strains using multiple comparative genomic techniques. This included kmer analysis with Neptune and gene presence/absence analyses with Roary and OrthoMCL to identify additional virulence factors to explain the spectrum of grazing resistance within P. ananatis. Multiple candidate genes were identified in subsets of resistant strains including formate dehydrogenase-N, which may increase fitness when dealing with phagocytes. P. ananatis virulence factors that differentiate it from P. stewartii were also determined. All 10 P. ananatis strains have the genes, yhcF and yhcA, which are part of putative fimbrial biogenesis cluster that may aid in host-specific adhesion. The pagC gene, a homologue of the Salmonella Resistance to Complement Killing (rck) virulence factor, was also present in P. ananatis but not in P. stewartii. SNP and indel analysis identified variation in the upstream region of the quorum sensing regulator eanI that was common to grazingsusceptible strains. This thesis identified multiple virulence factors in P. ananatis that have the potential to facilitate opportunistic infections of humans. This work also describes the genetic versatility of P. ananatis and highlights that its ability to colonize and persist in multiple host niches lies within both the conserved core and open pangenome.