UC Berkeley

Listeria monocytogenes is a Gram-positive bacterial pathogen that cycles between life as a common environmental saprophyte and an intracellular pathogen of animals, including humans. It is a common model organism to study intracellular pathogenesis and mammalian immunity due to its amenability to genetic manipulation and undemanding growth requirements. Its diverse repertoire of lifestyles requires L. monocytogenes to be able to sense and respond to changing conditions quickly to maximize its fitness in all niches. Upon infection, bacteria must initiate a regulatory response during which its encoded virulence factors must be precisely coordinated in order to maximize survival. Listeriolysin O (LLO) is a pore-forming cytolysin and an essential determinant of virulence and its regulation through the pathogenic lifecycle is absolutely necessary. The synthesis of LLO protein is known to be regulated at the level of translation but the specific mechanism is unknown. This thesis will detail two individual projects that are linked by their common connection to translation.

LLO is responsible for freeing bacteria from host phagosomes and allowing them entry into the cytosol. In contrast to related toxins that are encoded by other bacterial pathogens, the regulation of LLO must be tightly regulated to allow for proper expression, which maximizes phagosome escape and prevents destruction of host plasma membrane integrity. For this reason, the regulation of LLO is believed to specifically promote the intracellular lifecycle of L. monocytogenes. Here we report that LLO synthesis is regulated by the formation of an extensive secondary structure of its mRNA (hly). This structure forms base-pair interactions between the ribosome binding site and a region in the coding region of the gene and affects translation. Genetic mutants that disrupt the secondary structure but maintain the amino acid sequence of the protein result in a virulence defect of as much as 10,000-fold in mice, and compensatory mutations almost completely restored virulence to WT levels. The nucleotide sequence of hly is selective to maintain maximum virulence while minimizing host cell death. We show that translational inhibition of LLO is growth phase dependent and that non-growing bacteria secrete proportionally more toxin than growing bacteria. Dependence on growth phase corresponds with a destabilization of the mRNA secondary structure in non-growing bacteria.

Cyclic di-AMP (c-di-AMP) is a nucleotide second messenger molecule in L. monocytogenes that is essential on rich media and coordinates the regulation of carbon flux through central metabolism, osmotic turgor pressure and sensitivity to cell wall antibiotics. Its depletion is known to trigger the stringent response, which is characterized by the production of the alarmone (p)ppGpp and a re-structuring of protein synthesis at the translational and transcriptional level. Here we show that c-di-AMP binding protein B (CbpB), is an activator of the stringent response during low c-di-AMP conditions. Deletion of cbpB in the absence of c-di-AMP restores normal (p)ppGpp levels and its expression during low c-di-AMP levels raises alarmone levels. CbpB directly binds the stringent response-activating enzyme RelA and directs the accumulation of (p)ppGpp in vitro. These observations describe a novel non-canonical activation pathway of the stringent response and highlight the crosstalk between nucleotide signaling molecules in bacteria.