In the present study, we provide the first evidence that hTERT is important for Listeria infection. In addition, we show that L. monocytogenes is able to induce a decrease in hTERT levels through the formation of LLO pores in the plasma membrane prior to host cell invasion. The pores formed by LLO induce a decrease of the hTERT level that is proteasome independent but requires Ca2+ influx.
induced an hTERT decrease without affecting transcription of the hTERT gene. Because the half-life of hTERT is around 6 h (28
), the decrease of the hTERT level observed 20 min after LLO treatment probably does not result from a decrease in hTERT translation but, rather, from posttranslational regulation of the hTERT protein. Several studies have reported that hTERT is degraded through the ubiquitin-proteasome pathway (27
). Here, we observed a decrease in hTERT levels that was proteasome independent. Similarly, a proteasome-independent pathway was suggested previously for the degradation of UBC9 upon treatment with LLO (43
). However, the degradation of UBC9 involves an aspartyl protease and is calcium independent (43
). In contrast, we observed a contribution of calcium to the pathway leading to the decrease of hTERT levels. Calcium is known to contribute to the activation of cysteine proteases, such as calpains (46
). However, other proteins belonging to serine proteases, aspartyl proteases, or metalloproteases can also be activated by the presence of calcium (16
). We blocked these classes of proteases using well-characterized inhibitors, but under the conditions tested, none of the protease inhibitors impaired the decrease in hTERT levels. Two possible explanations exist for the inability to prevent the reduction in hTERT levels induced by LLO: (i) the doses of protease inhibitors and the duration of the treatment were not sufficient to prevent the decrease in hTERT levels, and (ii) proteolytic cleavage can be very specific. For example, identification of the protease responsible for the cleavage of paxillin upon treatment with the pore-forming toxin α-hemolysin from uropathogenic Escherichia coli
relied on a highly specific trypsin-like serine protease inhibitor (tosyl-l
-lysine-chloromethyl ketone), while it was insensitive to the chymotrypsin-like serine protease inhibitor tosyl-l
-phenylalanine-chloromethyl ketone (12
). Our current work is focusing on identifying the molecular basis of the degradative pathways activated by LLO via proteomic analysis and genome-wide siRNA screening approaches.
We found that hTERT was important for Listeria infection. Our siRNA experiments show that reduced hTERT expression early during infection does not impair bacterial adhesion and entry, yet it results in a decrease of the intracellular bacterial load at later time points. The degradation of hTERT could therefore represent an event that protects host cells at a specific stage of the Listeria infectious cycle. In the absence of siRNA treatment, hTERT levels start recovering 5 h after infection, allowing for the full intracellular replication of L. monocytogenes, further suggesting that this antibacterial effect is time restricted. In addition, the recovery of hTERT levels may contribute to cell survival, given the proposed antiapoptotic role of hTERT.
A decrease in hTERT levels could have important consequences during an in vivo
infection. Telomerase activity has been detected in human adult stem cells, including hematopoietic and nonhematopoietic stem cells (25
). Secreted LLO could diffuse to such progenitor cells or stem hematopoietic cells in colonized organs. An LLO-induced decrease in hTERT levels in LLO-targeted cells would lower their self-renewal capacity and therefore impair the immune response and promote L. monocytogenes
). It remains to be tested whether hTERT levels are affected in vivo
The first characterized role of hTERT concerns telomere elongation that contributes to the extension of cellular life span (5
). To detect this effect, cells have to be followed through several generations. However, given that long-term infections (for more than 48 h) are toxic in HeLa cells and the observed L. monocytogenes
-induced reduction of hTERT levels was followed by a recovery process, we did not expect to provoke detectable effects on telomere length in HeLa cells. In agreement with this, all of our attempts to detect a change in telomere length were unsuccessful.
Most virus-induced tumor cells possess high telomerase activity but short telomeres (3
). Indeed, hTERT can extend the cellular life span without inducing net telomere lengthening (37
). It is possible that L. monocytogenes
affects the recently described noncanonical functions of hTERT (31
). As mentioned, hTERT was shown to play roles in processes as diverse as DNA damage, Wnt signaling, and the decrease of the RNA component of a mitochondrial RNA-processing endoribonuclease (RMRP). These roles of hTERT seem to be at least partially independent of each other (37
). Indeed, while hTERT induced cell proliferation independently of an increase in Wnt signaling, it was associated with a decrease in RMRP levels (37
). The next challenge will thus be to determine whether noncanonical functions of hTERT are necessary for bacterial infection and which specific function of hTERT is targeted by bacterial infection.