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A screen for Listeria monocytogenes hemolytic activity mutants reveals HPF, a homolog of Hibernation Promoting Factor, which is essential for ribosomal hibernation and important for competitive fitness in vitro and in vivo.


Listeria monocytogenes is an opportunistic, potentially deadly, foodborne pathogen of humans and other vertebrates. Resistance to stressful conditions such as low pH, high salt, and refrigeration temperatures makes L. monocytogenes ideally suited to thrive on food sources, promoting transmission. A set of conserved virulence factors promotes establishment of infection once L. monocytogenes enters the host. In this work, we sought to better understand both the pathogenesis and the biology of L. monocytogenes by investigating factors involved both in virulence and in tolerance to sub-optimal growth conditions.

Listeriolysin O (LLO) is an essential virulence factor that allows L. monocytogenes to escape from the host phagosome into the cytosol. LLO is regulated on multiple levels to compartmentalize its function and prevent loss of virulence caused by host cell toxicity. To gain new information on the various mechanisms of LLO regulation, we performed a transposon mutagenesis screen for L. monocytogenes mutants in LLO production and activity. This screen identified PrsA2, a peptidyl-prolyl isomerase that we showed was important for L. monocytogenes virulence based on its contribution to re-folding of virulence factors following secretion. PrsA2 was shown to be important for folding and activity of LLO, and its deletion reduced bacterial virulence by 105-fold in a mouse model of infection. This work was published in the Journal of Bacteriology.

A L. monocytogenes homolog of Hibernation Promoting Factor (HPF) was also isolated from this hemolytic activity screen. Ribosomal hibernation is characterized by dimerization of two 70S ribosomes into a 100S complex. These 100S dimers are translationally inactive and important for survival during post-exponential growth in certain bacteria. HPF is conserved in long and short forms, which are present in different sets of bacteria and appear to be functionally distinct. Long HPF, which is present in L. monocytogenes, is also conserved in S. aureus, where it is necessary for 100S formation. In this work, we report the existence of a functional HPF homolog that is necessary for formation of 100S ribosomes in L. monocytogenes. HPF was observed to bind 100S ribosomes based on its co-migration through sucrose density gradients in the 100S fractions. We observed that the hpf transcript was upregulated by glucose starvation in stationary phase media, and that several σB-inducing stresses also stimulated hpf expression. Bacteria lacking HPF showed dramatically reduced survival in stationary phase competitive co-culture with wild-type bacteria. Finally, HPF-deficient bacteria displayed a > 300-fold survival defect in the mesenteric lymph nodes of orally-infected mice. We conclude that the L. monocytogenes HPF is functional for formation of 100S ribosomes and is critical for optimal survival in competitive environments, both in vitro and in vivo.

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