The Circadian Clock Protein Timeless Regulates Phagocytosis of Bacteria in Drosophila

Survival of bacterial infection is the result of complex host-pathogen interactions. An often-overlooked aspect of these interactions is the circadian state of the host. Previously, we demonstrated that Drosophila mutants lacking the circadian regulatory proteins Timeless (Tim) and Period (Per) are sensitive to infection by S. pneumoniae. Sensitivity to infection can be mediated either by changes in resistance (control of microbial load) or tolerance (endurance of the pathogenic effects of infection). Here we show that Tim regulates resistance against both S. pneumoniae and S. marcescens. We set out to characterize and identify the underlying mechanism of resistance that is circadian-regulated. Using S. pneumoniae, we found that resistance oscillates daily in adult wild-type flies and that these oscillations are absent in Tim mutants. Drosophila have at least three main resistance mechanisms to kill high levels of bacteria in their hemolymph: melanization, antimicrobial peptides, and phagocytosis. We found that melanization is not circadian-regulated. We further found that basal levels of AMP gene expression exhibit time-of-day oscillations but that these are Tim-independent; moreover, infection-induced AMP gene expression is not circadian-regulated. We then show that phagocytosis is circadian-regulated. Wild-type flies exhibit up-regulated phagocytic activity at night; Tim mutants have normal phagocytic activity during the day but lack this night-time peak. Tim appears to regulate an upstream event in phagocytosis, such as bacterial recognition or activation of phagocytic hemocytes. Interestingly, inhibition of phagocytosis in wild type flies results in survival kinetics similar to Tim mutants after infection with S. pneumoniae. Taken together, these results suggest that loss of circadian oscillation of a specific immune function (phagocytosis) can have significant effects on long-term survival of infection

Tim regulates resistance to infection by <i>S. pneumoniae</i> and <i>S. marcescens</i>.

<p>Mutants lacking resistance, or bacterial killing, have shorter survival times and greater bacterial loads than wild-type flies. <i>Tim</i> null mutants lack resistance to (A) <i>S. pneumoniae</i> and (B) <i>S. marcescens</i>. <i>Tim</i> mutants have bacterial loads similar to wild-type flies when infected with (C) <i>B. cepacia</i> and (D) <i>S. typhimurium</i>. p-values were obtained by two-tailed, unpaired t-test; all experiments were performed with day-time injections.</p

Bead inhibition of phagocytosis in wild type flies eliminates the difference in survival kinetics with Tim mutants after <i>S. pneumoniae</i> infection.

<p>Shown here are Kaplan-Meier survival curves comparing wild-type flies and <i>Tim</i> mutants infected with <i>S. pneumoniae</i> with and without pre-injection of beads to inhibit phagocytosis. Infected wild-type flies pre-injected with beads were more sensitive than wild-type flies without pre-injection (p<0.0001) and had similar survival kinetics as <i>Tim</i> mutants (p = 0.0502). Infected <i>Tim</i> mutants had identical survival kinetics with or without bead pre-injection (p = 0.1327). In this experiment infected, bead-injected wild-type flies were more sensitive than infected, bead-injected Tim mutants (p<0.0001); however, this result varied from experiment to experiment.</p

Tim protein regulates an early stage of phagocytosis of bacteria by hemocytes.

<p>(A) Wild-type flies in the night phase of the circadian cycle have more phagocytic activity than wild-type flies in the day phase. Shown here are images of the dorsal surface of representative flies after injection of dead <i>S. aureus</i> labeled with the fluorophore, pHrodo, which emits fluorescence in acidic environments such as the lysosomes. Hemocytes that have phagocytosed these bacteria become fluorescent. Dashed lines indicate the region enlarged in the inset. (B) Phagocytic activity for wild type and <i>Tim</i> mutant flies was quantified by measuring areas of fluorescence. Wild-type flies exhibited significant circadian differences in phagocytosis of <i>S. aureus</i> (p<0.001) but not <i>E. coli</i> (p = 0.74). This circadian difference is not present in <i>Tim</i> mutant flies with either <i>S. aureus</i> (p = 0.51) or <i>E. coli</i> (p = 0.63). p-values were obtained by unpaired, two-tailed t-test.</p

Tim activity has significant consequences for immunity.

<p>Shown here are Kaplan-Meier survival curves comparing <i>Tim</i> null mutants (orange circles) and wild-type control flies (green squares). <i>Tim</i> mutants die more quickly after infection with (A) <i>S. pneumoniae</i> (p<0.0001) or (B) <i>S. marcescens</i> (p<0.0001). <i>Tim</i> mutants die at the same rate as wild-type flies after infection with (C) <i>B. cepacia</i> (p = 0.1001), (D) <i>S. typhimurium</i> (p = 0.1707), or (E) medium alone (p = 0.9836). p-values were obtained by log-rank analysis.</p

Resistance against <i>S. pneumonia</i> correlates with Tim activity.

<p>Shown here are Kaplan-Meier survival curves comparing wild-type flies and <i>Tim</i> mutants infected with <i>S. pneumoniae</i> and subjected to different light conditions as detailed in the schematic diagrams. (A) Light-induced degradation of Tim protein in wild-type flies results in increased sensitivity to <i>S. pneumoniae</i>. Infected wild-type flies incubated in constant light were more sensitive than wild-type flies incubated in darkness (p<0.0001). <i>Tim</i> mutants incubated in constant light died as rapidly as did <i>Tim</i> mutants incubated in darkness (p = 0.3404). (B) Wild-type flies were more sensitive to <i>S. pneumoniae</i> when infected at a time of day when Tim protein is low. Wild-type flies were less sensitive when infected 6 hours after lights off (DARK) than 6 hours after lights on, when Tim protein is minimally and maximally expressed, respectively (p<0.0001). <i>Tim</i> mutant sensitivity was not rescued by infection 6 hours after lights off (DARK) (p = 0.4134). (C) Light-induced sleep-wake rhythms (a startle response called “masking”) do not rescue <i>Tim</i> mutant sensitivity to infection. <i>Tim</i> mutant sensitivity was not rescued by incubation in cycling light:dark conditions compared to incubation in the dark. In fact, both <i>Tim</i> and wild-type flies were more sensitive to infection in cycling light:dark conditions than in the dark (p = 0.0125 and p<0.0001 respectively).</p