ClpP-deletion impairs the virulence of Legionella pneumophila and the optimal translocation of effector proteins
© The Author(s). 2016
Received: 16 March 2016
Accepted: 27 July 2016
Published: 2 August 2016
The opportunistic bacterial pathogen Legionella pneumophila uses substrate effectors of Dot/Icm type IVB secretion system (T4BSS) to accomplish survival and replication in amoebae cells and mammalian alveolar macrophages. During the conversion between its highly resistant, infectious dormant form and vigorously growing, uninfectious replicative form, L. pneumophila utilizes a complicated regulatory network in which proteolysis may play a significant role. As a highly conserved core protease, ClpP is involved in various cellular processes as well as virulence in bacteria, and has been proved to be required for the expression of transmission traits and cell division of L. pneumophila.
The clpP-deficient L. pneumophila strain failed to replicate and was digested in the first 3 h post-infection in mammalian cells J774A.1. Further investigation demonstrates that the clpP deficient mutant strain was unable to escape the endosome-lysosomal pathway in host cells. We also found that the clpP deficient mutant strain still expresses T4BSS components, induces contact-dependent cytotoxicity and translocate effector proteins RalF and LegK2, indicating that its T4BSS was overall functional. Interestingly, we further found that the translocation of several effector proteins is significantly reduced without ClpP.
The data indicate that ClpP plays an important role in regulating the virulence and effector translocation of Legionella pneumophila.
First isolated in 1977, Legionella pneumophila, a Gram-negative, intracellular bacterial pathogen is the agent causing the severe form of pneumonia named Legionnaires’ disease, as well as the less severe flu-like Pontiac fever . It has drawn much attention for its capability of intracellular replication in both protozoa and human beings. After the endocytosis by protozoan hosts like amoebae or human alveolar macrophages, the Legionella-containing vacuole (LCV) inhibits phagolysosomal fusion and recruits mitochondria followed by the association of ribosome-studded membranes that later disguise LCV as endoplasmic reticulum (ER). Within this ER-like compartment, the bacterium replicates to high numbers and eventually is released through lysing the host cell for the next invasion .
During this process, L. pneumophila requires most protein products of 27 dot/icm (defect in organelle trafficking/intracellular multiplication) genes to constitute a type IVB secretion system (T4BSS) . Although neither the composition nor the function of T4BSS has been fully understood in L. pneumophila, progress has been achieved in identifying and characterizing the Dot/Icm proteins. DotC, DotD, DotF, DotG and DotH comprise the core of the secretion complex which spans across the bacterial membrane. DotC and DotD are outer-membrane lipoproteins and required for DotH to target the outer membrane . DotH may be the out-membrane channel through which substrates get delivered following the transit from the DotF-DotG inner-membrane proteins with the assistance of the DotL–DotM ATPase . DotB, also an ATPase, interacts with DotL and may play a role in various functions such as the assembly of T4BSS, retraction of pili and/or export of substrates [4, 5]. IcmQ participates in the membrane pore formation , and IcmT is crucial for pore formation-mediated escape of L. pneumophila from protozoan or mammalian cells . DotL is proposed to be a type IV coupling protein (T4CP) of T4BSS and interacts with other inner-membrane proteins including DotN, DotM and IcmS/W, a heterodimer complex functions as T4BSS adaptor, to constitute the T4CP subcomplex, a very important complex for T4BSS to facilitate substrate secretion [8, 9].
Through the T4BSS, L. pneumophila secretes a large number of substrate proteins called effectors that interfere with the host pathways to help bacteria evade the endosome-lysosomal pathway and replicate in host cells . The effector RalF, which has guanine nucleotide exchange activity and mediates the exchange of GDP for GTP, disturbs vesicle traffic between the ER and Golgi and further promotes the biogenesis of LCV by modulating the activity and localization of the key intracellular regulator Arf1 . The effector AnkB is important for the moorage of K48-linked polyubiquitinated proteins when it is anchored into the phagosome membrane by host-mediated farnesylation and interacts with the SCF1 E3 ubiquitin ligase complex. Then the K48-linked proteins are degraded and the amino acids are utilized for bacterial intracellular proliferation [11, 12]. LegK2, whose deletion causes reduced cytotoxicity, and adversely affect the intracellular survival and replication of L. pneumophila, acts as a protein kinase . So far more than 300 effectors have been identified but many of them are considered functionally redundant, only a few are indispensable for the intracellular proliferation of L. pneumophila, such as MavN and SdhA [14–16].
During the shift between extracellular and intracellular environments, L. pneumophila encounters different growth conditions and has to respond accordingly to survive. To make the appropriate responses, L. pneumophila has developed a complex network to modulate the transition at different phases. Proteolysis has been regarded as an important and precise regulatory mechanism for both eukaryote and prokaryote to adapt to a variety of growth conditions by removing short-lived regulatory proteins, as well as misfolded and damaged proteins . It is now clear that cellular proteolysis is carried out by the energy-dependent proteases such as the Lon and Clp proteases and the eukaryotic 26S proteasome . To date, Clp protease is the most characterized protease in prokaryotes. It consists of two functional subunits: a cylinder-like proteolytic core named ClpP which is widely distributed and highly conserved, and two chaperone rings with ATPase activity such as ClpA, ClpC, ClpE or ClpX [17, 18]. The protease core consists of 14 ClpP serine peptidase subunits stacked in two heptameric rings, forming an internal chamber in which the active sites are sequestered from the cytoplasm . The Clp ATPases are responsible for the recognition, unfolding and translocation of substrates into the degradation chamber .
It is widely accepted that Clp proteases are involved in many physiological processes of bacteria. In a range of low GC Gram-positive bacteria including Bacillus subtilis, Listeria monocytogenes and Lactococcus lactis, ClpP-deficient mutants suffer restricted growth at high temperatures [21–24]. ClpP is also considered as the major determinant for the turnover of bulk proteins in B. subtilis at the transition from exponential stage to competence and further sporulation stages [24, 25]. Moreover, both ClpP and its ATPase chaperones play significant roles in virulence expression and regulation in various bacterial pathogens. For instance, S. aureus cells lacking the ClpB chaperone are unable to replicate intracellularly in bovine cells . The absence of ClpP in L. monocytogenes results in the lack of listeriolysin O, a major virulence factor implicated in phagosome lysis [23, 27]. Our recent research has shown that ClpP is required for the transmission traits of Legionella pneumophila during the transition in its biphasic life cycle, including some traits associated with virulence such as cytotoxicity and intracellular proliferation in the amoebae host Acanthamoeba castellanii . In this report, studies were focused on the function of Legionella pneumophila ClpP in the mammalian cell J774A.1 and results revealed that the deletion of clpP severely impaired the bacterial virulence and the translocation of several T4BSS effectors though the functional integrity of T4BSS was not fully neutralize.
ClpP is essential for intracellular proliferation of L. pneumophila in macrophages
clpP mutant is degraded rapidly upon entry into the host cells
The clpP mutant fails to evade the late endosome-lysosomal pathway
Loss of ClpP does not affect the expression of dot/icm components
The T4BSS secretion apparatus is still functional in the L. pneumophila clpP deficient mutant
ClpP controls the translocation efficiency of some T4BSS effectors
In the current study, we found that the L. pneumophila clpP deficient mutant exhibited poor survival and intracellular multiplication in J774A.1 cells (Figs. 1 and 2). Furthermore, the mutant strain could not escape the late endosome-lysosomal pathway (Fig. 3). Thus, consistent with our previous results obtained in the amoebae host A. castellanii , ClpP may be required for the expression of virulence in L. pneumophila. To investigate how ClpP regulates the virulence, we tested whether the deficiency of clpP could affect the component expression and the function of T4BSS complex. The 27 proteins of dot/icm components may not be significantly affected based on the results of transcriptional activity assay together with immunoblotting analysis (Fig. 4). Although we did not examine the expression of all 27 proteins, the findings that the clpP mutant could still induce contact-dependent cytotoxicity against host cells (Fig. 5), together with that both RalF and LegK2 could be translocated into host cells, indicated that the T4BSS function was not compromised seriously in the absence of ClpP. However, the fact that the secretion of some effectors was impaired in the clpP-deficient strain suggested that one of the strategies for ClpP to affect the virulence of Legionella pneumophila is via regulating the translocation of effectors.
The phenotype of clpP-deficient L. pneumophila resembles that of the IcmS/W mutants, the T4BSS chaperone. They all exhibit significantly impaired intracellular replication, but still maintain fair contact-dependent cytotoxicity against host cells (Figs. 1 and 5) [35, 36]. Moreover, the translocation of three IcmS/W-mediated effectors (SidA, SidB and SidD) and LegU1 was impaired obviously in the absence of ClpP, and the translocation of non-IcmS/W-mediated RalF was unchanged without ClpP (Fig. 6) . More interestingly, the non-IcmS/W-mediated effector SidF showed reduced translocation efficiency in clpP-deletion mutant (Fig. 6), which was similar in icmS/icmW double mutant, and the translocation of SidA, SidB and SidD in clpP-deletion mutant was more neutralized than that in icmS or icmW single mutant . Taken together, a hypothesis that clpP-deletion mutant might resemble an absolutely IcmSW-abolishing mutant could be proposed. Currently, how ClpP affects IcmS/W subcomplex is difficult to be clarified because the expression level of each protein in clpP-deletion mutant is unchanged (Fig. 4e). Previous studies have shown that ClpP affects the virulence expression in some gram-positive pathogens such as Staphylococcus aureus, Streptococcus pneumoniae and L. monocytogenes [23, 26, 38, 39]. More details about the virulence regulation by ClpP have been revealed in Salmonella enterica serovar Typhimurium and Yersinia pestis, where ClpP governs the protein levels of important virulence-regulating factors for type III secretory systems (T3SS), RpoS and YomA [40, 41]. For pathogens containing T4BSS, little information about the association between Clp proteases and other virulence-related factors is available. Recently studies on the DotL-IcmSW coupling subcomplex of L. pneumophila T4BSS have revealed that ClpAP protease is responsible for the degradation of DotL in the absence of IcmS. Briefly, in the absence of IcmS/W mediation, abundant effector proteins could be trapped within DotL, and subsequently the jamming complex is subjected to specific degradation by ClpAP [8, 42]. Interestingly, although DotL degradation by ClpP requires specific recognition by ClpA, the single deletion of clpA does not affect the intracellular replication or the protein level of DotL . Thus, it is possible that ClpP may affect DotL-IcmSW-mediated effector translocation through other processes bypassing ClpA. On the one hand, ClpP may influence the optimal assembly of T4BSS rather than protein expression, especially in DotL-IcmSW coupling subcomplex. On the other hand, successful effector translocation mediated by IcmS/W might need essential cleavage or modification by ClpP protease. In the second situation, translocation signal peptides may be involved. Up to now, a C-terminal signal peptide has been proven to be essential for the translocation of nearly all T4BSS effectors and an internal signal sequence has been found to be important in IcmS/W-mediated translocation [43, 44]. Thus, the latter signal peptide may be the possible target of ClpP modification or regulation. In our future proposal, the association of ClpP with the internal signal peptide in IcmS/W-mediated effectors needs to be explored, and we also hope to examine the assembly status of the DotL-IcmSW subcomplex in clpP-deletion mutant.
It is also possible that the neutralized intracellular replication is partially due to the impaired stress tolerance of clpP-deficient L. pneumophila. In both natural aquatic and intracellular environment, L. pneumophilla would encounter various stresses [45, 46]. Legitimately, a rapid responding system involving proteolytic procedures would be beneficial to the stress tolerance of L. pneumophilla. Studies have revealed that ClpP protease plays significant roles in DNA repair and other stress responses and helps bacteria adapt to many harsh conditions [38, 47–49]. Likewise, L. pneumophila ClpP is required for the stationary-phase resistance against various stresses such as oxidation, acidity, osmotic stress and inappropriate temperatures . Among these stresses, acid resistance might be the most critical in Legionella’s intravacuolar survival. L. pneumophila could neutralize the acidic environment of phagosome and subsequently maintain a nearly neutral-pH vacuole during the first hours of uptake . However, about 18 h later L. pneumophila would still encounter the acidic environment when the LCV mature into low-pH and endocytic compartments . Considering our previous finding that the clpP mutant strain exhibited reduced acid resistance and vulnerable cell surface compared to the wild type strain , we wonder whether and how much the “weakness” the clpP mutant displays under unfavorable environments contributes to the disability of virulence expression, this assumption needs deeper exploration.
In this study, our data show that the loss of clpP prevents L. pneumophila from evading the endocytic pathway and replicating in host cells. Our results also revealed that the clpP-deficiency affects the translocation of some T4BSS effectors without impairing the integrity of T4BSS. Taken together, L. pneumophila ClpP is an indispensable factor for the virulence and the translocation of some T4BSS effectors.
Bacterial strains, cells and reagents
Bacterial strains used in this study
Reference or source
host strain used for cloning
Virulent L. pneumophila serogroup 1, strain Philadelphia, rpsL, HsdR − , Thy −
Virulent L. pneumophila serogroup 1, strain Philadelphia, dot03, rpsL, HsdR − , Thy −
Lp02 with clpP deletion
Xp02 containing pXL901 for complementation
Lp02 containing pJB908
Lp03 containing pJB908
Xp02 containing pJB908
Lp02 containing plasmids pGB908X*
with icm promoter-gfp fusions
Xp02 containing plasmids pGB908X*
with icm promoter-gfp fusions
Lp03 containing plasmid pGB9089
with icmV promoter-gfp fusion
Plasmids used in this study
Suicide delivery vector, rdxA sacB Cm
pBRDX::clpP for clpP deletion
Insert thy gene, mutate mob into pkB5
ColE1 ori Cm Pmip gfpmut2
Insert SacI/PstI fragment encoding GFP, Pmip into pJB908
Complementation plasmid, derived from pZL01, with gfpmut2 changed for clpP
Insert BamHI/SphI fragment encoding GFP, flaA promoter into pTLP6
Insert XbaI/SphI fragment of gfpmut3 into pJB908
Insert 9 icm promoters of operon areas into pGB908
Insert EcoRI/SalI fragment of CyaA catalytic domain and SidJ into pKB5
Primers used in this study
5’ → 3’ sequence
DNA manipulation, chromosomal in-frame deletion, complementation assay
All DNA manipulations were performed according to standard protocols and the in-frame deletion was carried out as described before  except that the suicide vector was substituted for pBRDX and the L. pneumophila strain for Lp02. Briefly, the upstream and downstream flanking sequences of clpP were amplified by PCR using PXP-F1/PXP-R1 and PXP-F2/PXP-R2 primer pairs, respectively. The PCR products were mixed as templates for the subsequent fusion PCR using PXC-F1/PXC-R2 as the primer pair. The Fusion PCR product was digested with BamHI and BglII, and sub-cloned into the pBRDX vector , yielding pBRΔclpP. Then, pBRΔclpP was introduced into the wild-type Lp02 strain by electroporation and chloramphenicol-resistant colonies were selected on BCYE-Cm plates. Transformants were patched and inoculated into AYE broth and then spread on BCYE plates containing 20 μg/ ml metronidazole. Bacteria were cultured for about 3 days at 37 °C to screen for strains without the suicide vector. Positive colonies were verified by PCR and sequencing.
In the complementation assay, a RSF1010 pKB5-derived vector pJB908 was utilized as the cloning backbone . The ColE1-type plasmid pBC(gfp)Pmip, which had been used as a complementation vector previously , was digested with SacI and PstI. The fragment was ligated with pJB908, yielding pZL01. Then, the sequence of clpP was amplified using PXL091F/PXL091R and digested with XbaI and SphI. Finally, the digestion product was ligated with pZL01 to construct pXL091, in which Pmip controlled the transcription of clpP gene constitutively. Because the resulting plasmid pXL091 contains the thymidylate synthetase gene originating from pJB908 backbone, transformants harboring pXL091 could be easily selected on BCYE plates devoid of thymidine, without addition of any antibiotics.
Phagocytosis assay and intracellular growth assay
For phagocytosis, J774A.1 cells were seeded into 24-well tissue culture plates (2.5 × 105 cells per well) and allowed to adhere overnight. L. pneumophila strains harboring pJB908 were grown to stationary phase, which were predominantly motile (OD600 3.7–4.5), and used for infection at a multiplicity of infection (MOI) of 1 after suspension in culture medium. The infection was synchronized by centrifugation at 500 g for 10 min and incubating for 30 min. The extracellular bacteria were removed by washing 3 times. After another 3 h of incubation, cells were lysed with 0.05 % saponin for plating dilutions onto BCYE plates and colony forming unit (CFU) counting. The percentages of bacteria resided within host cells were calculated as below: Phagocytosis percentage (%) = 100 x (CFU of cell lysate after incubating for 3 h)/(CFU of bacterial suspensions added at the initiation of infection).
The intracellular growth assay in J774A.1 cells was performed using a similar protocol as the assay in A. castellanii , except that the cell culture medium and the washing buffer were replaced with pre-warmed RPMI1640, the MOI was reduced from 10 to 1, and the lysing reagent was changed to 0.05 % saponin.
Contact-dependent cytotoxicity assay
J774A.1 cells were seeded into 96-well plates (1.0 × 105 cells/well) 24 h prior to infection. L. pneumophila strains harboring pJB908 were grown to post-exponential phase (OD600 3.0–3.7) in AYE broth, then harvested and used to infect J774A.1 cells at an MOI of 50 and 100. The culture plates were centrifuged at 400 g for 10 min and incubated for 2 h at 37 °C. After incubation, the cytosolic enzyme lactate dehydrogenase (LDH) release of supernatants was determined using the CytoTox-ONE 96 cytotoxicity assay kit (Promega), according to the instructions provided by the manufacturer. The level of Legionella-induced contact-dependent cytotoxicity was calculated as below: LDH release (%) = 100 x (experimental – culture medium background)/(maximum LDH release – culture medium background). Maximum LDH release was the LDH release from cells treated with 0.9 % Triton X-100, used as a positive control.
Cya translocation assays
To construct the CyaA fusions, the sequences of ralF, legK2, sidA, sidB, sidD, sidF and legU1 were amplified using the primers PRAc-F/PRAc-R, PLK2c-F/PLK2c-R, PSDAc-F/PSDAc-R, PSDBc-F/PSDBc-R, PSDDc-F/PSDDc-R, PSDFc-F/PSDFc-R and PLU1c-F/PLU1c-R respectively. Then the DNA products were digested with BamHI and SalI and ligated into the BamHI/SalI site of a pJB2581-derived plasmid with cyaA-sidJ fusion [34, 50], yielding pCA1-pCA7.
Cya translocation assay was carried out as described before . Briefly, J774A.1 cells were seeded into 96-well plates (2.5 × 105 cells/well) and infected with L. pneumophila strains expressing CyaA fusion proteins at an MOI of 30. After incubation for 1 h at 37 °C, cells were washed and lysed, and total cAMP was extracted and determined using cAMP Enzyme Immunoassay Kit (Sigma-Aldrich). Creation of the fusion proteins was assessed by Western blotting using a monoclonal antibody to CyaA (CyaA (3D1), Santa Cruz Biotechnology).
L. pneumophila bacteria were harvested by centrifugation and washed in pre-cooled sterile water for 3 times. Then the samples were resuspended in 1 × Laemmli Sample Buffer, boiled for 10 min and centrifuged at 13,000 rpm for 10 min at 4 °C. Supernatants were then collected and loaded on SDS-polyacrylamide gels for analysis. The primary antibodies used and their dilutions were as follows: rabbit Anti-DotH, DotG and DotI (1:750; gifts from Dr. Luo ZQ), rabbit Anti-IcmS, IcmW and ICDH (1:200, 1:1000 and 1:2000; gifts from Dr. Vogel JP). The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit (1:10000; Sigma). SuperSignal West Pico (Pierce) was used for signal detection.
Interaction of phagosomes with the endocytic pathway
The fusion of Legionella-containing phagosomes with the late endosome was assessed by detecting the co-localization of phagosomes with the lysosome-associated membrane protein 1 (LAMP-1) . J774A.1 cells were seeded into 24-well tissue culture plates with 12-mm coverslips (1 × 105 cells/well) and incubated overnight at 37 °C. Then the cells were infected with L. pneumophila harboring pJB908 (OD600 3.7–4.5) at an MOI = 5. After incubation for 1 h, extracellular bacteria were eliminated by treating with 100 μg/ml gentamicin sulfate for 0.5 h. Then the coverslips were washed, transferred to clean wells, and fixed with 4 % paraformaldehyde solution for 10 min. To label extracellular bacteria, cells blocked with PBSSG (PBS containing 5 % sucrose and 2 % goat serum) buffer for 1 h were loaded with mouse anti-Legionella primary antibody (1:150; Santa Cruz Biotechnology), followed by Alex Fluo 350 conjugated goat anti-mouse IgG secondary antibody (1:2000; Molecular Probes). After being permeabilized with cold methanol for 20 s, intracellular bacteria were also blocked and labeled as above, with secondary antibody changed to the Alex Fluo 488 conjugated goat anti-mouse IgG. Finally, cells were loaded with rat anti-mouse Lamp-1 primary antibody (1:150; Santa Cruz Biotechnology) and Alex Fluo 594 conjugated goat anti-rat IgG secondary antibody (1:2000; Molecular Probes).
To assess the fusion with lysosomes, Texas-red conjugated ovalbumin (TroV) was used to label the lysosomes as described previously [51, 52]. Before the infection, 500 μl of pre-warmed TroV (100 μg/ml; Molecular Probes) was added to each well and incubated with cells for 0.5 h at 37 °C. Then the cells were washed 3 times for later use. The following procedures including infection, fixation, blocking, permeabilization and bacteria labeling were performed as in the Lamp-1 co-localization assay.
Construction of icm:gfp fusion plasmids and fluorescence analysis
To analyze the activities of icm promoters, a GFP coding gene gfpmut3, the product of which emits intense fluorescence , was employed. The coding sequence of gfpmut3 was cut out from pTLpflaG  with XbaI and SphI, and ligated with pJB908, yielding pGB908. Then the DNA sequences containing icm promoters of the 9 operon areas were amplified respectively and cloned into the multiple cloning site (MCS) upstream of gfpmut3, yielding pGB908X (from pGB9081 to pGB9089). The resulting plasmids were transformed into L. pneumophila strains by electroporation and the positive transformants were screened on thymidine-free BCYE plates.
For fluorescence analysis, L. pneumophila strains harboring pGB908X were inoculated into 5 ml AYE broth, and grown at 37 °C, 220 rpm for 20 h. Then the cultures were expanded into 25 ml AYE in flasks with initial optical densities (OD600) at approximate 0.2–0.3, and incubated to a stationary phase (OD600 3.7–4.5). Subsequently 100 μl of bacteria culture was transferred into 96-well fluorometer plates for fluorescence quantification on a microplate reader (TECAN INFINITE M200) with excitation at 488 nm and emission at 540 nm. Bacteria with pGB908X were also streak inoculated on thymidine-free BCYE plates and the GFP fluorescence intensity was estimated by direct observation.
Basic statistical analyses were performed using Excel, and one-way ANOVA was performed using SPSS followed by a post hoc Student-Newman-Keul’ s test.
CFU, Colony-forming units; dot, defect in organelle trafficking; GFP, green fluorescent protein; icm, intracellular multiplication; Lamp-1, lysosome associated membrane protein 1; LCV, Legionella containing vacuole; MOI, multiplicity of infection; T4BSS, type IVB secretion system; TroV, texas-red conjugated ovalbumin.
We thank Dr. Zhao-Qing Luo, Dr. Paul Hoffman and Dr. Joseph Vogel for their kindly gifts of strains, antibodies and vectors.
This work was supported by the National Natural Science Foundation of China (No. 30970123) and the Natural Science Foundation of Guangdong province (No. 2016A030311036) to Yong-jun Lu.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
The authors declare that they have no competing interests.
BBZ, LXH and YJL designed the experiments and drafted the manuscript. BBZ and LXH performed the experiments. YLZ participated in the study design and performed the phagocytosis analysis. All authors read and approved the final manuscript.
Consent for publication
Ethics approval and consent to participate
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