Intracellular pathogens account for a significant majority of infectious disease cases and deaths worldwide. These pathogens enter host cells to hide from the extracellular immune defenses and establish this environment as their replicative niche. Intracellular pathogens have evolved to acquire nutrients from the host cell and use the cell’s machinery to avoid innate immune responses, grow, and disseminate. In the Portnoy lab, we study the pathogenesis of, and host responses to, the model intracellular pathogen Listeria monocytogenes. L. monocytogenes live freely in the soil and decaying plant matter but can become an intracellular pathogen upon ingesting contaminated food. L. monocytogenes affects immunocompromised individuals, including the elderly and pregnant women, where it can infect the placenta and cause miscarriages. L. monocytogenes has relatively few growth requirements and can synthesize most of its nutrients, which allows it to reside in diverse environments. However, unlike most bacteria, L. monocytogenes cannot synthesize riboflavin (vitamin B2). Not much was known about flavin metabolism in L. monocytogenes, the requirements during infection, or why L. monocytogenes are in the minority of bacteria that lack the capacity to synthesize riboflavin de novo. My dissertation aimed to describe flavin metabolism and transport in L. monocytogenes and examine the implications of riboflavin requirement during infection. More broadly, my work explored how intracellular pathogens that cannot produce riboflavin acquire flavins from host cells and why they might have evolved to lack the riboflavin biosynthetic pathway.

The riboflavin-derived molecules, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are essential redox-active cofactors used by all forms of life to perform a myriad of redox reactions, including energy production and catabolism of amino acids. Since L. monocytogenes does not synthesize riboflavin de novo, it must acquire this flavin from the environment. I discovered that L. monocytogenes encodes a flavin transporter (RibU) that is essential exclusively during infection and allows the pathogen to scavenge FMN and FAD, and not riboflavin as previously suggested, directly from the host cytosol. This research was the first report of a pathogen importing FMN and FAD from the host to sustain its intracellular growth. Interestingly, obligate intracellular pathogens in the Rickettsia and Cryptosporidium genera do not produce riboflavin but also lack the enzymes that convert riboflavin to FMN and FAD. I hypothesize that like L. monocytogenes, these obligate intracellular pathogens import FMN and FAD directly from the host to satisfy their flavin requirements. I also found that L. monocytogenes encodes an energy-coupling factor (ECF) transporter that exports FAD and is distributed across the Firmicutes phylum. This ECF exporter is required for the flavinylation of extracytosolic proteins and is essential for extracellular electron transfer, which confers L. monocytogenes a growth advantage in the host's gastrointestinal tract. Importantly, this is the first example of an ECF transporter capable of exporting substrates, as all other characterized ECF complexes are importers.

It is evident that flavins play essential roles in L. monocytogenes physiology and pathogenesis and this led us to question why L. monocytogenes lost the capacity to synthesize riboflavin. We speculated that L. monocytogenes evolved to avoid recognition by mucosal-associated invariant T (MAIT) cells. MAIT cells are innate-like T cells that recognize host cells infected with pathogens that synthesize riboflavin (riboflavin precursors act as MAIT cell activating ligands). Upon encountering host cells that display the riboflavin precursor, MAIT cells kill the infected cells and activate other immune cells by secreting cytokines. To test the hypothesis that L. monocytogenes is avoiding MAIT cells by lacking the capacity to produce riboflavin, I engineered L. monocytogenes to produce riboflavin de novo. We observed that riboflavin-producing L. monocytogenes was highly attenuated in mice and mediated the expansion and activation of MAIT cells. Thus, riboflavin biosynthesis is detrimental to L. monocytogenes pathogenesis and could explain why this pathogen lacks riboflavin biosynthetic genes. The conclusions of my thesis suggest that flavins are essential for L. monocytogenes physiology and pathogenesis but that they lost the capacity to synthesize riboflavin, and instead evolved to import FMN and FAD cofactors from the cytosol of infected cells to avoid activation of MAIT cells.