Biotin limitation in the phagosome and Francisella virulence Open Access

Napier, Brooke Ann (2013)

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Cytosolic bacterial pathogens require extensive metabolic adaptations within the host to replicate intracellularly and cause disease. In phagocytic cells such as macrophages, these pathogens must respond rapidly to nutrient-limitation within the harsh environment of the phagosome. Many cytosolic pathogens escape the phagosome quickly (15-60 min) and thereby subvert this host defense, reaching the cytosol where they can replicate. While a great deal of research has focused on strategies used by bacteria to resist antimicrobial phagosomal defenses and transiently pass through this compartment, the metabolic requirements of bacteria within the phagosome are largely uncharacterized. We previously identified a novel Francisella protein, FTN_0818, as an essential factor for intracellular replication and involved in virulence in vivo; however, it was unclear how this new virulence determinant affects bacterial pathogenesis. I demonstrate in this work that FTN_0818, now named BioJ, is involved in biotin biosynthesis and required for rapid escape from the Francisella-containing phagosome (FCP), as well as replication in vitro and in vivo. These results demonstrate that biotin is critical for promoting rapid escape during the short time that the bacteria are in the phagosome, implying biotin may be a limiting factor during infection. Furthermore, functional anaylsis found BioJ works as a carboxyl-esterase that, as a monomer, catalyzes the hydrolysis of Me-pimeloyl-ACP into pimeloyl-ACP, and is necessary for the second step of biotin biosynthesis. Phylogenetic and structural analyses identified BioJ as an evolutionarily distinct Francisella-specific biotin gatekeeper enzyme. Additionally, bioinformatic analysis of BioJ revealed a conserved carboxylesterase catalytic triad, Ser-Asp-His. We found that all three residues within the catalytic triad are required for BioJ function and subsequently Francisella replication in macrophages and survival in mice. Thus, I have demonstrated that biotin is required for phagosomal escape of Francisella and we have characterized BioJ as a novel Francisella-specific biotin gatekeeper enzyme. This is the first demonstration of a bacterial metabolite required for phagosomal escape of an intracellular pathogen, providing new insight into the link between bacterial metabolism and virulence, likely serving as a paradigm for other cytosolic pathogens.

Table of Contents

Chapter 1

Introduction pg. 1

References pg. 19

Chapter 2

A link between intraphagosomal biotin and rapid phagosomal escape in Francisella pg. 30

Abstract pg. 31

Introduction pg. 32

Results pg. 34

Discussion pg. 41

Materials & Methods pg. 45

References pg. 68

Chapter 3

BioJ is a Francisella-specific carboxyl-esterase required for pimelate generation during biotin biosynthesis and virulence pg. 72

Abstract pg. 74

Introduction pg. 75

Results pg. 78

Discussion pg. 85

Materials & Methods pg. 89

References pg. 122

Chapter 4

Discussion pg. 115

References pg. 122

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