In the current study, LpΔclpP was shown to exhibit reduced growth rate at high temperatures (Figure 2D) and impaired resistance to heat shock (Figure 3C) compared to the wild type. The LpΔclpP mutant also displayed impaired resistance to oxidative and low-pH conditions in stationary phase. As oxidative and acid stress are generally considered as harsh and detrimental to DNA [48, 49], ClpP homologue may play an important role in L. pneumophila DNA repair, consistent with its demonstrated function in E. coli , S. aureus  and Lactococcus lactis . However, while several previous studies have demonstrated growth defect as a result of ClpP deficiency over a broad temperature range [34, 35, 51], deletion of clpP appeared to compromise the growth of L. pneumophila only at higher temperatures (Figure 2A to 2C), suggestive of a more restricted role independent of cold response.
Attenuation of ClpP or Clp ATPase activities has been shown to lead to abnormal bacterial morphology such as filamentation, aberrant cell wall structure and irregular cell division [29, 32, 53–55]. Likewise, results from SEM and cyro-TEM revealed that the LpΔclpP mutant cells were elongated and defective in cell division (Figure 4). Furthermore, SEM results also implicated a role of clpP in stress tolerance in L. pneumophila. In contrast to the defective cell surface observed in SEM (Figure 4D and 4E), largely normal cell surface were found by cyro-TEM in LpΔclpP mutant cells grown under normal conditions (Figure 4A to 4C), suggesting that the chemical treatment during SEM sample preparation, not clpP deletion, may have resulted in the abnormal cell surface.
How ClpP affects cell division is not fully understood. In C. crescentus, degradation of the cell cycle repressor CtrA by the ClpXP complex has been shown to contribute to G1-S transition, and deletion of clpP blocked cell division . In B. subtilis, cells overproducing MurAA, an enzyme in peptidoglycan biosynthesis and a substrate of the Clp protease, displayed a filamentous, undivided morphology reminiscent of the clpP mutant cells, suggesting that degradation of MurAA by ClpP might contribute to normal cell segregation . Furthermore, through a ClpP-independent pathway, the B. subtilis ClpX appeared to modulate the assembly of the tubulin-like protein FtsZ , which is known to be a key process in the replication and division of Gram-negative bacteria . Identification of the substrate(s) for ClpP may shed light on the regulatory mechanism of cell division in L. pneumophila.
ClpP proteolytic complexes play pivotal roles in protein degradation or modification [26, 31, 32]. During the transition of B. subtilis cells to stationary phase, ClpP degrades massive amounts of proteins previously produced in exponential growth phase . Notably, L. pneumophila also undergoes a biphasic life cycle with mutually exclusive gene expression for replication or transmission [10, 11]. While transiting from replication (exponential phase in vitro) to transmission (stationary phase in vitro), L. pneumophila activates an intricate network of regulators such as LetA/S, RpoS, PmrA, CpxR, rsmYZ, CsrA and LqsR [11, 13, 20, 21, 59]. As shown in our results, unlike the stationary-phase wild type which exhibits transmission traits, LpΔclpP mutant cells in stationary phase exhibit replicative forms such as reduced stress tolerance (Figure 2 and 3), cell elongation (Figure 4), enhanced sodium resistance (Figure 5), impaired cytotoxicity and growth on amoebae plates (Figure 6) and severely compromised intracellular multiplication in amoebae host (Figure 7). Thus, ClpP may play an important role in the transition from replication to transmission in L. pneumophila. On the other hand, several transmission traits are not affected by clpP-deletion such as pigment accumulation and transcription from the flaA (legionella flagellin coding) gene (our unpublished data), suggesting that the impact of ClpP on the transition to transmissive form in L. pneumophila is somewhat limited. Considering that ClpP always executes the post-transcriptional feedback regulation, and moreover, degrades the same substrates by cooperating with other proteases [26, 31], one explanation to such a limitation is that the degradation of ClpP substrates could be compensated by other proteases in clpP-deletion mutant, thus ClpP cannot govern the transition just as the global regulators such as RpoS, CsrA or LetA/S in L. pneumophila.
ClpP plays prominent roles in virulence of various Gram-positive pathogens such as S. aureus, S. pneumoniae and L. monocytogenes [34–36, 60]. Furthermore, ClpP was reported to control the levels of key virulence factors of type III secretory systems (T3SS) in certain pathogens such as S. typhimurium and Yersinia pestis [61, 62]. Recently, it was reported that loss of ClpP attenuated the virulence of Helicobacter pylori, a pathogen owning type IV secretory system (T4SS) . It is interesting that clpP-deletion severely compromised the L. pneumophila infection against amoebae host (Figure 6 and 7). In our results, the sodium resistance exhibited by LpΔclpP mutant (Figure 5), which is a phenotype shared by the mutants without functional Dot/Icm T4SS [48, 64], together with the comparable decline in intracellular multiplication observed in LpΔclpP and ΔdotA mutants (Figure 7), suggest a role of ClpP in T4SS-dependent virulence through degrading a repressor or activating an up-regulator of the substrate(s) of ClpP. One possibility is that the ClpP protease has a major impact on the expression or function of Dot/Icm T4SS in L. pneumophila. Another possibility is that ClpP might be required for the expression of some T4SS substrates. In this case, loss of ClpP would also severely attenuate the intracellular growth even if the T4SS is intact, just as the case of L. pneumophila Sigma S factor (RpoS) . Thus, identification of the substrate(s) of ClpP, which is currently underway in our laboratory, would help to discern the underlying relationship between ClpP and T4SS-dependent virulence in L. pneumophila.