Phosphorylation of caspases by a bacterial kinase inhibits host programmed cell death

Ge, Jinli; Wang, Ying; Li, Xueyu; Lu, Qian; Yu, Hangqian; Liu, Hongtao; Ma, Kelong; Deng, Xuming; Luo, Zhao-Qing; Liu, Xiaoyun; Qiu, Jiazhang

doi:10.1038/s41467-024-52817-1

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Published: 30 September 2024

Phosphorylation of caspases by a bacterial kinase inhibits host programmed cell death

Jinli Ge¹^na1,
Ying Wang^2,3^na1,
Xueyu Li¹,
Qian Lu¹,
Hangqian Yu¹,
Hongtao Liu¹,
Kelong Ma¹,
Xuming Deng¹,
Zhao-Qing Luo ORCID: orcid.org/0000-0001-8890-6621⁴,
Xiaoyun Liu ORCID: orcid.org/0000-0001-7083-5263^2,3 &
…
Jiazhang Qiu ORCID: orcid.org/0000-0002-7723-5073¹

Nature Communications volume 15, Article number: 8464 (2024) Cite this article

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Abstract

The intracellular bacterial pathogen Legionella pneumophila utilizes the Dot/Icm system to translocate over 330 effectors into the host cytosol. These virulence factors modify a variety of cell processes, including pathways involved in cell death and survival, to promote bacterial proliferation. Here, we show that the effector LegK3 is a eukaryotic-like Ser/Thr kinase that functions to suppress host apoptosis. Mechanistically, LegK3 directly phosphorylates multiple caspases involved in apoptosis signaling, including Caspase-3, Caspase-7, and Caspase-9. LegK3-induced phosphorylation of these caspases occurs at serine (Ser29 in Caspase-3 and Ser199 in Caspase-7) or threonine (Thr102 in Caspase-9) residues located in the prodomain or interdomain linkers. These modifications interfere with the suitability of the caspases as the substrates of initiator caspases or upstream regulators without impacting their proteolytic activity. Collectively, our study reveals a novel strategy used by L. pneumophila to maintain the integrity of infected cells for its intracellular growth.

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Introduction

Successful pathogens have acquired various strategies to manipulate cellular functions to evade immune clearance and promote infection¹. During the complex interactions with their hosts, many intracellular bacterial pathogens utilize specialized protein translocation apparatuses, such as type III and IV secretion systems (T3SS and T4SS)², to deliver effector proteins into the cytosol of host cells, where they modulate signaling pathways to construct environments permissive for bacterial survival and proliferation¹. These effectors regulate host processes via distinct mechanisms, among which induction of post-translational modification (PTM) of critical host signaling proteins represents one of the most effective and widely used mechanisms³. Protein phosphorylation conducted by kinases is arguably the most prevalent and important PTM in cells, which controls virtually every cellular process^4,5. This PTM often alters the activity, localization, or stability of the involved proteins, leading to changes in signaling transduction and subsequent physiological responses⁶. Given the critical roles of protein phosphorylation in cell signaling, it is not unexpected that many bacterial pathogens encode effectors with kinase activity to target important signaling hubs to promote their virulence. For example, the Salmonella SPI-1 substrate SteC is a eukaryotic-like Ser/Thr protein kinase (eSTPK) capable of phosphorylating the actin sequestering protein Hsp27 and the mitogen-activated protein (MAP) kinase MEK, whose activity causes alterations in the structure of the host actin cytoskeleton^7,8. Similarly, the T3SS effectors NleH1 and NleH2 of enteropathogenic Escherichia coli are kinases that phosphorylate microvillus protein Eps8 to alter the cytoskeletal composition of attaching and effacing (AE) lesions⁹.

The facultative intracellular bacterium Legionella pneumophila is the causative agent of Legionnaires’ disease, a potentially fatal pneumonia¹⁰. This bacterium utilizes its Dot/Icm T4SS to inject over 330 effector proteins directly into the host to construct an intracellular niche that supports its survival and growth¹¹. These effectors interfere with a wide range of host cell processes to promote the establishment of Legionella-containing vacuole (LCV), a membrane-enclosed compartment that avoids lysosomal degradation and supports bacterial proliferation¹². To successfully hijack host functions, many L. pneumophila effectors adopt structural domains typical for proteins of eukaryotes that are involved in protein-protein interactions or in catalyzing distinct activities (e.g., kinases, ubiquitin ligases, guanine nucleotide exchange factors, deubiquitinases, phospholipases). Among these, at least six L. pneumophila effectors, named LegK1-4, LegK7, and Lem28 had been identified as potential protein kinases due to the presence of structural elements similar to kinase domains of eukaryotic proteins and such activity has been detected by biochemical reactions using self-phosphorylation as a readout^13,14,15. Of these kinases, LegK1 functions to mimic the host IKK kinase to phosphorylate IκBα on Ser32 and Ser36, thereby activating NF-κB signaling¹⁶. LegK2 targets subunits of the ARP2/3 complex to suppress actin nucleation on the LCV, thus contributing to the evasion of the late endosome/lysosome trafficking pathway by the bacterium¹⁷. LegK4 induces threonine phosphorylation of HSP70 to inhibit protein synthesis in host cells¹⁸ and LegK7 is a mimicry of the host Hippo kinase that phosphorylates the scaffolding protein MOB1, thus altering host gene expression to promote infection¹⁴.

Programmed cell death (PCD) pathways, including apoptosis, necroptosis, and pyroptosis are essential in determining the outcome of bacterial infections. The death of infected cells will eliminate the replication niche of the pathogen and thus is disastrous for the pathogen. As a result, intracellular bacterial pathogens have evolved various mechanisms to manipulate host PCDs to accommodate their replication¹⁹. Earlier studies suggest that infection by L. pneumophila causes apoptosis via the activation of Caspase-3^20,21. Later studies reveal that L. pneumophila inhibits infected cells from undergoing apoptosis by either targeting pro-death proteins²² or by activating signaling cascades that induce expression of anti-apoptotic proteins²³.

Caspases are a family of cysteine-dependent aspartate-specific proteases that function as central mediators of inflammatory responses and/or apoptosis²⁴. Caspases are synthesized as inactive zymogens consisting of prodomain of variable size followed by large (p20) and small (p10) catalytic subunits²⁵. Apoptotic caspases are functionally sub-categorized into initiator caspases (Caspases-8/−9/−10) and effector (or executioner) caspases (Caspases-3/−6/−7)²⁶. Initiator caspases undergo autoactivation by dimerization-induced conformational changes to release the large and small catalytic subunits, which can cleave and activate executioner caspases²⁶. In this study, we found that executioner Caspase-3 and Caspase-7 are not activated in cells infected by L. pneumophila, and subsequent screening identified the Dot/Icm effector LegK3 as the factor responsible for such inhibition. Further study revealed that this kinase induces phosphorylation on multiple caspases including Caspase-3, Caspase-7, and Caspase-9. Although this modification did not affect the enzymatic activity of these caspases, it impacts their suitability as substrates for upstream initiator caspases. Our results reveal a novel strategy employed by L. pneumophila to ensure the integrity of infected cells for its intracellular replication.

Results

Identification of Dot/Icm effectors involved in inhibition of host cell death

To determine the mechanism of host cell death manipulation by L. pneumophila, we infected FcγRII-expressing HeLa cells with wild-type L. pneumophila for the indicated durations and detected the active forms of Caspase-3 and Caspase-7 and found that these caspases were not activated in infected cells, which was in sharp contrast to cells treated with the cell death inducer staurosporine (STS) (Fig. S1). This observation suggests that, in line with earlier studies^22,23, L. pneumophila inhibits host apoptosis.

To identify the Dot/Icm effector responsible for the suppression of caspase activation, we utilized several cluster deletion strains of L. pneumophila, each lacking distinct chromosomal regions²⁷, for similar infection experiments. Notably, only the Δ7 mutant exhibited significantly higher levels of caspase activation than the wild-type strain (Figs. 1A–E and S2). We thus individually examined the ability of effectors carried by the deleted region of strain ∆7 by expressing their GFP fusions in HeLa cells and examined caspase activation²⁸. After STS treatment, expression of LegK3 (Lpg2556) markedly blocked the activation of Caspase-3 and Caspase-7, leading to a marked reduction of cell death (Fig. 1F–H). Other Dot/Icm effectors carried by the missed region in strain ∆7 did not detectably inhibit cell death induced by STS (Fig. 1F–H). These results indicate that LegK3 functions as an apoptosis inhibitor during L. pneumophila infection.

**Fig. 1: The Dot/Icm effector LegK3 inhibits caspase activation in eukaryotic cells.**

The S/T kinase motif of LegK3 is required for its ability to inhibit apoptosis

LegK3 was previously established as a protein kinase owing to the presence of an eSTPK domain^13,29,30, but its cellular target remains elusive (Fig. 2A). To investigate whether LegK3’s kinase activity is required to block apoptosis, we constructed a kinase-dead mutant of LegK3 via mutation of D187 in its catalytic loop (HRD₁₈₇IKPEN) to alanine (referred to as LegK3_D/A hereafter). Although expressed at levels similar to the wild-type allele in both HeLa and HEK293 cells, LegK3_D/A has lost the ability to inhibit STS- or etoposide (also known as VP-16)-triggered production of mature Caspase-3/−7 (Figs. 2B, C and S3A–C). In line with this, the production of cleaved poly-ADP-ribose polymerase (PARP), one of the cellular targets of activated caspases and a hallmark of apoptosis³¹, was remarkably reduced in cells expressing wild-type LegK3 but not LegK3_D/A (Figs. 2B and S3B). Similarly, LegK3 also suppressed the maturation of Caspase-3/−7 induced by overexpression of pro-death members of the Bcl-2 family, such as BAX or BimEL (Fig. S3E, F). In line with LegK3-mediated Caspase-3/−7 inactivation, the ratio of STS-, VP-16-, or BAX/BimEL-induced apoptotic cell death significantly decreased in samples transfected to express wild-type but not the kinase-dead LegK3 mutant when cell death was determined by measuring the ATP levels, flow cytometry after staining with propidium iodide (PI) and DEVD-GreenNuc^TM or by TUNEL staining (Figs. 2D, E, S3D, S3G, and S4A–C).

**Fig. 2: The kinase activity of LegK3 is required for the inhibition of host apoptosis.**

Next, we investigated the role of LegK3 in apoptotic status of cells infected by L. pneumophila. To this end, we first measured the gene expression of intracellular L. pneumophila by quantitative real-time PCR (qRT-PCR). Remarkably, transcription of legK3 was detected in bacteria retrieved from wild-type and dotA^- L. pneumophila-infected cells (Fig. S4D). In addition, we employed the TEM (β-lactamase) reporter system to visualize Dot/Icm-dependent translocation of LegK3²⁸. At 3 h post-infection, approximately 50% of the cells infected with the wild-type L. pneumophila strain expressing TEM-LegK3 exhibited blue fluorescence, which was generated via the cleavage of CCF4/AM by TEM (Fig. S4E, F). By contrast, only green fluorescence was observed in cells similarly infected with the dotA^- mutant strain producing TEM-LegK3. These analyses establish that LegK3 is delivered into host cells during L. pneumophila infection in a Dot/Icm-dependent fashion (Fig. S4E, F). We then infected FcγRII-expressing HeLa cells with each of the following L. pneumophila strains: wild-type, dotA^-, Lp02ΔlegK3, Lp02ΔlegK3 (pLegK3), and Lp02ΔlegK3 (pLegK3_D/A). The level of active p17 of Caspase-3/−7 was higher in cells infected with strain Lp02ΔlegK3 than those infected with the wild-type strain (Figs. 2F, G and S5). Expression of LegK3, but not the LegK3_D/A mutant from a plasmid fully restored the capacity of ΔlegK3 strain to suppress Caspase-3/−7 activation (Figs. 2F, G and S5). In addition, cells infected by strain Lp02ΔlegK3 (pLegK3) exhibited resistance to STS-induced Caspase-3/−7 activation (Fig. 2H, I). Consistent with the defect in Caspase-3/−7 activation, the apoptotic rates were significantly higher in cells infected with the Lp02ΔlegK3 mutant than those infected with the wild-type strain (Fig. S4G–K). Similarly, the elevation in apoptosis exhibited by cells infected with the Lp02ΔlegK3 mutant can be completely complemented to levels of wild-type infected samples by expressing LegK3 but not the LegK3_D/A mutant (Fig. S4G–K). Thus, the kinase activity of LegK3 is required for its ability to inhibit apoptosis.

LegK3 directly phosphorylates Caspase-3

The requirement of kinase activity for LegK3 to inhibit Caspase-3 activation suggested that LegK3 targets key components of the apoptotic pathway by phosphorylation. When co-expressed in mammalian cells, GFP-LegK3 extensively colocalized with HA-Caspase-3 (Fig. S6). In addition, HA-Caspase-3 co-transfected with GFP-LegK3 in HEK293T cells consistently migrated slower in SDS-PAGE gels. Such retardation did not occur in samples that were co-expressed with LegK3_D/A (Fig. 3A). This observation suggests Caspase-3 as a substrate of LegK3. Indeed, probing HA-Caspase-3 isolated from cells co-expressing LegK3 with anti-Phospho-(Ser) antibodies detected phosphorylation signals (Fig. 3A). We determined the LegK3-induced phosphorylation site on Caspase-3 using LC-MS/MS and found that Ser29 is the sole modified residue (Figs. 3B and S7). This site lies between the prodomain and the large subunit of Caspase-3. To confirm phosphorylation on Ser29 by LegK3, we created and co-expressed the Caspase-3_S29A mutant with GFP-LegK3 and probed phosphorylation by immunoblotting with phospho-specific antibodies. LegK3-induced phosphorylation no longer occurred in this mutant when co-expressed with LegK3 (Fig. 3A).

Next, we attempted to use biochemical reactions with recombinant proteins to prove that LegK3 directly phosphorylates Caspase-3. However, we were unable to obtain recombinant LegK3 of high purity owing to extensive degradation in E. coli expression systems. We thus co-expressed Caspase-3_C/A in which the catalytic cysteine (C163) was mutated into alanine with LegK3 or LegK3_D/A in E. coli, which revealed that His₆-Caspase-3_C/A co-expressed with LegK3 exhibited a marked molecular weight shift in native PAGE gels and such shift did not occur for samples co-expressed with the kinase-defective mutant (Fig. 3C). Similarly, probing by a phosphoprotein staining agent or immunoblotting with phospho-specific antibodies detected robust phosphorylation signals associated with His₆-Caspase-3_C/A co-expressed with wild-type but not LegK3_D/A (Fig. 3C). Furthermore, Caspase-3_S29A can no longer be modified by LegK3 in these assays (Fig. 3C).

We next investigated LegK3-induced phosphorylation of Caspase-3 in cells infected with L. pneumophila. To this end, we transfected HEK293 cells to express the FcγRII receptor and 4xFlag-Caspase-3 and infected these cells with relevant L. pneumophila strains. Flag-Caspase-3 isolated by immunoprecipitation was analyzed for phosphorylation by immunoblotting. Flag-Caspase-3 purified from cells infected with the wild-type L. pneumophila strain displayed phosphorylation signals, but not from cells infected with the dotA^- or the Lp02ΔlegK3 mutant (Fig. 3D). Introduction of a plasmid expressing LegK3 into strain Lp02ΔlegK3 allowed the mutant to cause Caspase-3 phosphorylation and similarly expressed LegK3_D/A failed to induce the modification (Fig. 3D, E). Finally, mass spectrometric analysis indicated that Ser29 was phosphorylated in Caspase-3 isolated from cells infected with wild-type L. pneumophila (Fig. 3F). No phosphorylation was detected in Caspase-3_S29A isolated from cells infected with wild-type L. pneumophila (Fig. 3D). Taken together, these results demonstrate that LegK3 directly induces Caspase-3 phosphorylation on Ser29 during L. pneumophila infection.

Phosphorylation of Caspase-3 at Ser29 by LegK3 blocks its cleavage by initiator caspases

To determine the impact of Ser29 phosphorylation on the function of Caspase-3, we purified the active p17/12 complex of Caspase-3 from the E. coli strain harboring a plasmid carrying Caspase-3s_29-C under different conditions. Co-expression of this complex with LegK3 resulted in phosphorylation of p17 as indicated by its retarded migration in acrylamide gels electrophoresis and positive signals in phosphoprotein staining (Fig. S8A). Phosphorylated p17/p12 (P-p17/p12) complex purified by an ion exchange column was used to cleave several established Caspase-3 substrates (Fig. S8B). P-p17/p12 cleaved GSDMD, GSDME, and PARP indistinguishably to its native unmodified counterpart (Fig. S8C–I). Thus, LegK3-catalyzed phosphorylation of Caspase-3 does not affect its enzymatic activity.

Activation of Caspase-3 involves two steps: initiator caspases (−8/−9/−10) cleave procaspase-3 at the interdomain linker (IETD₁₇₅-S₁₇₆) to generate an immature p20 subunit with an exposed active Cys163 and a p12 subunit; the p20 subunit subsequently underdoes self-processing at the prodomain linker (ESMD₂₈-S₂₉) to remove the N-terminal fragment, yielding a mature p17 large subunit that forms a complex with the p12 small subunit capable of executing proteolytic activity³². To determine whether LegK3-induced Ser29 phosphorylation of Caspase-3 affects its processing by upstream caspases, we purified phosphorylated Caspase-3_C/A co-expressed with wild-type LegK3 from E. coli (Fig. S9) and examined its cleavage by Caspase-8 and Caspase-9. Both initiator caspases cleaved P-Caspase-3_C/A at rates significantly lower than those of native Caspase-3_C/A (Fig. 4A–D). Similar results were observed when 4xFlag-Caspase-3_C/A co-expressed with GFP-LegK3 in HEK293T cells was used as the substrate for recombinant active Caspase-9 (Fig. 4E). Moreover, phosphomimetic mutants of Caspase-3 (S29D, S29Q, and S29E) exhibited markedly lower sensitivity to Caspase-8/-9 than the native form (Figs. 4F–I and S10A–D). Taken together, these results indicate that LegK3-mediated phosphorylation of Caspase-3 at Ser29 made it less cleavable by upstream caspases without detectably impacting its enzymatic activity.

**Fig. 4: Phosphorylation of Caspase-3 at Ser29 by LegK3 inhibits its cleavage by initiator caspases.**

LegK3 targets multiple caspases involved in the apoptosis pathway by phosphorylation

To study whether LegK3 targets other caspases involved in apoptosis, we first co-expressed cleavage-deficient mutants of Caspase-7/-8-9 with LegK3 in E. coli. Detection by phosphoprotein staining or immunoblotting with phospho-specific antibodies revealed that Caspase-7/-9 but not Caspase-8 can be modified by this bacterial kinase (Fig. 5A). Mass spectrometric analysis identified Ser199 within the interdomain linker (IQAD₁₉₈-S₁₉₉) of Caspase-7 and Thr102 within the prodomain linker (KLSKPT₁₀₂) of Caspase-9 as the sole phosphorylation site, respectively (Figs. 5B, C and S11A, B). Indeed, mutants of Caspase-7 and Caspase-9 with a substitution in Ser199 or Thr102 can no longer be modified by LegK3 (Fig. 5D, E). When co-expressed in mammalian cells, both HA-Caspase-7 and HA-Caspase-9 extensively colocalized with GFP-LegK3 (Fig. S12), and phosphorylation of these caspases by LegK3 was similarly observed in a kinase activity-dependent manner (Fig. 5F, G). By contrast, LegK3-induced phosphorylation was not detected for Caspase-7_S199A and Caspase-9_T102A when co-expressed with LegK3 in HEK293T cells (Fig. 5F, G).

**Fig. 5: LegK3 targets multiple caspases.**

We next investigated whether modification of Caspase-7/-9 by LegK3 occurs under infection conditions. To this end, HEK293 cells transfected to produce FcγRII and HA-Caspase-7/-9 were challenged with relevant L. pneumophila strains and phosphorylation was determined in proteins isolated by immunoprecipitation. Phosphorylation of Caspase-7/-9 occurred in samples isolated from cells infected with the wild-type but not the dotA^- or the ΔlegK3 mutant L. pneumophila (Fig. S13A, B). Importantly, complementation of the ΔlegK3 strain with wild-type LegK3 but not LegK3_D/A by a plasmid led to robust Caspase-7/-9 phosphorylation (Fig. S13A, B). Additionally, mass spectrometric analysis indicated that, similar to samples obtained by co-expression in E. coli, Ser199 and Thr102 of Caspase-7 and Caspase-9 were phosphorylated, respectively (Fig. S13C, D). Taken together, these results demonstrate that, in addition to Caspase-3, Caspase-7 and Caspase-9 are also the physiological targets of LegK3.

The Asp198-Ser199 located within the interdomain linker of Caspase-7 is the cleavage site of upstream initiator Caspases, we thus hypothesized that Ser199 phosphorylation may affect its recognition by Caspase-8 and Caspase-9. To test this, we purified active cysteine-deficient phosphomimetic mutants of Caspase-7 (C186 AS199D, C186AS199E, and C186AS199Q) and determined their cleavage by Caspase-8/-9. The cleavage efficiency of all Caspase-7 phosphomimetic mutants was markedly lower than the native protein (Fig. 6A–H). Caspase-9 is activated by the apoptosis protease-activating factor 1 (Apaf-1), which oligomerizes in response to cytochrome c release and forms a large complex^33,34. To determine whether phosphorylation at Thr102 affects Caspase-9 processing, a constitutively active mutant of Apaf-1 (Apaf-1_1-541)³⁵ was purified from mammalian cells and incubated with either His₆-Caspase-9 or its phosphomimetic mutant His₆-Caspase-9_T102E. Whereas native Caspase-9 was readily cleaved by Apaf-1, as evidenced by the production of mature p35 fragment, little cleavage occurred in reactions containing His₆-Caspase-9_T102E (Fig. 6I). Corresponding to this observation, expression of wild-type LegK3 but not LegK3_D/A in HeLa cells suppressed the maturation of Caspase-9 induced by overexpression of BimEL (Fig. S14). Taken together, these results indicate that phosphorylation of Caspase-7/-9 by LegK3 inhibits their activation by upstream regulators.

**Fig. 6: Phosphorylation of Caspase-7 and Caspase−9 by LegK3 inhibits their activation.**

LegK3 blocks pyroptosis initiated by the Caspase-3-GSDME axis

Caspase-3 has long been considered the hallmark of activated apoptosis³⁶. In addition, recent studies have showed that Caspase-3 also cleaves GSDME to activate pyroptosis³⁷. Given the inhibitory effect of LegK3 on Caspase-3 activation, we investigated whether LegK3 is able to block pyroptosis induced by GSDME activation. To test this, we treated HeLa cells that had been transfected to express GFP, GFP-LegK3 or GFP-LegK3_D/A with STS or VP-16. Cleavage of GSDME occurred at significantly lower rates in cells expressing LegK3, which is accompanied with lower rates of pyroptosis (Fig. 7A–F). Consistent with this observation, infection of HeLa cells with wild-type L. pneumophila led to very limited GSDME cleavage and pyroptosis, which is in contrast to cells infected with the Lp02ΔlegK3 mutant (Fig. 7G–I). The observed inhibition can be restored by expressing LegK3 but not its enzymatically inactive mutant in strain Lp02ΔlegK3 (Fig. 7G–I).

**Fig. 7: LegK3 inhibits pyroptosis induced by Caspase-3 and GSDME.**

LegK3 is required for optimal bacterial growth in VP-16-treated host cells

To investigate the role of LegK3 in intracellular bacterial replication, we infected FcγRII-expressing HeLa cells with the wild-type strain and the Lp02ΔlegK3 mutant and evaluated their growth. The growth of the mutant was indistinguishable from that of the wild-type strain (Fig. S15A), indicating that LegK3 is dispensable for intracellular proliferation in this cell line under standard experimental conditions.

Because of its role in suppressing host cell death, we determined the role of LegK3 in bacterial intracellular growth under apoptosis/pyropotosis-inducing conditions. To this end, we treated infected cells with the cell death induction agent VP-16 and evaluated bacterial growth, which revealed that LegK3 is required for optimal replication (Fig. S15A). VP-16 did not detectably affect L. pneumophila growth in bacteriological medium (Fig. S15B). Thus, inhibition of host cell death is important for intracellular growth of L. pneumophila and LegK3 contributes importantly to such inhibition.

Discussion

The balance between host cell death and survival is critical for pathogenic organisms to establish infection³⁸. Consequently, during intimate interactions with their hosts, intracellular bacterial pathogens, such as Shigella, Salmonella, Coxiella, and Legionella, have developed a variety of intricate strategies to manipulate PCD pathways to promote infection^19,39. Such manipulation can be accomplished by transcriptional regulation or by directly targeting critical regulatory hubs involved in cell death signaling by secreted virulence factors^22,40. L. pneumophila infection had been previously suggested to trigger apoptosis via activation of Caspase-3 in a manner that is dependent upon its Dot/Icm secretion system^20,21. These observations are counterintuitive to the concept that cell integrity is essential for productive intracellular replication. Later studies demonstrated that cells harboring actively replicating L. pneumophila are resistant to cell death stimuli⁴¹. Consistent with this notion, induction of apoptosis by pharmacological inhibition of the host cell pro-survival protein BCL-XL or activation of Caspase-3 abrogates Legionella replication^41,42. One rational explanation for these observations is that host apoptotic signaling is triggered in response to virulent L. pneumophila infection, but the bacteria are capable of suppressing or reversing this process, possibly via the actions of Dot/Icm substrates.

Indeed, L. pneumophila infection robustly elevates the transcription of a large number of anti-apoptotic genes through activation of the NF-κB pathway in a Dot/Icm-dependent manner²³, which might be attributed to the functions of effectors LnaB and LegK1^16,43. Whereas the biochemical mechanism of LnaB remains to be investigated, LegK1 was proven to be a host IKK mimic capable of directly phosphorylating IκBα and other IκB family inhibitors, including p100 in the noncanonical NF-κB pathway¹⁶. In addition, effector SidF suppresses apoptosis of L. pneumophila-infected cells at least partially via directly interacting with and neutralizing the functions of BNIP3 and Bcl-rambo, two noncanonical pro-death members of the Bcl2 protein family²². Moreover, effector SdhA appears to be required for protecting host cells from death, as cells infected with the sdhA-null mutant strain exhibit elevated nuclear degradation, mitochondrial disruption, membrane permeability, and caspase activation⁴⁴. Although its mechanism of action remains elusive, SdhA appears to suppress host innate immune defenses that attack the integrity of LCV membranes^45,46. Our finding that LegK3 functions to inhibit host apoptosis by phosphorylating several caspases has expanded the mechanisms used by L. pneumophila to maintain the integrity of infected cells (Fig. S16).

Caspase cascades are central for host cells to execute PCD, including apoptosis, pyroptosis, and necroptosis²⁵. Among these, Caspase−3/−7/−8/−9 play pivotal roles in controlling apoptosis²⁵. In addition, Caspase-3 was also depicted as a regulatory knot for pyroptosis upon stimulation by chemotherapy drugs via the cleavage of GSDME³⁷. Therefore, direct targeting of caspases represents a highly efficient strategy to manipulate PCD, which was best exemplified by OspC-like arginine ADP-riboxanase⁴⁷, which utilizes NAD to modify arginine residues in Caspase-4/-11 by adenosine diphosphate riboxanation (ADP-riboxanation), thereby blocking their autoprocessing activity as well as recognition and cleavage of GSDMD⁴⁷. OspC3 of Chromobacterium violaceum attacks Caspase-3/-7/-8/-9 by a similar mechanism to dysregulate apoptosis, necroptosis, and pyroptosis^38,48. In addition, the E. coli effector NleF and the Yersinia pestis effector YopM can inactivate caspases via direct interactions, thus inhibiting PCDs^49,50. Interestingly, phosphorylation by LegK3 does not affect the proteolytic activity of these caspases (Fig. S16). Instead, the modification thwarts their cleavage by initiator caspases or upstream regulators (Fig. S16). To the best of our knowledge, LegK3 is the first bacterial virulence factor known to hijack caspases via phosphorylation. In mammalian cells, the activities of several caspases, including Caspase-2, Caspase-3, Caspase-6, Caspase-7, Caspase-8, and Caspase-9, are regulated by phosphorylation catalyzed by various kinases^{51,52,53,54,55,56}. For instance, p38-MAPK can directly phosphorylate Caspase-3 at Ser150, thereby inhibiting its activity and further hindering apoptosis⁵⁵; PAK2 targets Caspase-7 by phosphorylation at multiple sites, in which Ser30 phosphorylation allosterically obstructs Caspase-7 activation by Caspase-9, whereas Ser239 phosphorylation renders active Caspase-7 incapable of binding substrates, thus blocking subsequent events in apoptosis⁵⁶. Similarly, Caspase-9 was shown to be phosphorylated at Thr125 by multiple kinases, including ERK1/2, CDK1-cyclin B and DYRK1A, depending upon the stimulus and cell cycle^57,58,59. Phosphorylation on Thr125 prevents Caspase-9 from being processed by Apaf-1, thus inhibiting apoptosis⁵⁷. The fact that the catalytic motif of LegK3 is structurally similar to those found in eukaryotic proteins suggests that gene coding for this effector may be acquired by the pathogen via horizontal gene transfer⁶⁰.

Protein phosphorylation is reversed by phosphatases which downregulate the signaling in response to environmental cues⁶¹. Manipulation of host targets by L. pneumophila effectors in many cases is specifically regulated by effectors with opposite biochemical activities, which impose sophisticated spatial-temporal regulation of host signaling. For example, Rab1 activity is tightly controlled by reversible AMPylation executed by SidM/DrrA⁶² and SidD^63,64, reversible phosphorylcholination carried out by AnkX and Lem3^65,66, reversible phosphoribosyl-linked serine ubiquitination conducted by SidEs^67,68,69 and DupA/DupB^70,71. Hence, it is possible that L. pneumophila also encodes effectors to counteract the function of LegK3, thus imposing temporal regulation of PCD to promote host cell death in the final phase of L. pneumophila infection⁷². Dot/Icm effectors with phosphatase activity have been identified, including WipA⁷³, WipB⁷⁴, and Ceg4⁷⁵. It will be of interest to investigate whether any of these enzymes or other effectors with phosphatase activity counteract the activity of LegK3 to fine-tune PCD in cells infected by L. pneumophila to ensure maximal bacterial replication.

Although we have demonstrated that several caspases, including Caspase-3, Caspase-7, and Caspase-9, are bona fide substrates of LegK3, this bacterial S/T kinase might target diverse host proteins besides caspases to facilitate bacterial infection. A further unbiased proteome-wide phosphoproteomics study will potentially uncover additional phosphorylation sites and phosphorylated proteins targeted by LegK3 during L. pneumophila infection.

Methods

Bacterial strains, plasmids, cell lines, and culture methods

The bacterial strains used in this study are listed in Table S1. L. pneumophila strains were grown at 37 °C either on buffered charcoal yeast extract (CYE) agar plates or in AYE liquid media. L. pneumophila ΔlegK3 was constructed by allelic exchange using pSR47S as described previously⁷⁶, and complementation of the mutant strain was done by electronic transformation of pZL507⁷⁷ carrying LegK3. E. coli strains were cultured on LB agar plates or in broth supplemented with appropriate antibiotics (100 µg/ml ampicillin, 50 µg/ml kanamycin). E. coli strains DH5α and BL21(DE3) were used to maintain plasmids and express proteins, respectively. For expression of proteins in E. coli, genes were inserted into pET28a or pETDuet. For expression of proteins in mammalian cells, PCR products were ligated into pEGFP, pCDNA3.1-HA, or pCMV-4xFlag⁷⁷. Site-directed mutants were constructed using the QuikChange II site-directed mutagenesis kit according to the recommended protocols. All plasmids and primers used in this study are presented in Tables S2 and S3.

HeLa, HEK293, HEK293T, and Raw264.7 cells were obtained from the American Type Culture Collection (ATCC) and were grown at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS).

Protein expression and purification

Twenty milliliters of overnight cultures of E. coli BL21 (DE3) strains harboring relevant plasmids were transferred into 1 liter of fresh LB broth supplemented with appropriate antibiotics. The cultures were grown at 37 °C to an OD_600nm of 0.6–0.8. Protein expression was induced for 12 h at 18 °C with constant shaking (200 rpm) in the presence of 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Then, bacteria were harvested by centrifugation and lysed by a homogenizer (JN-mini, JNBIO, Guangzhou, China). The cell debris was pelleted by centrifugation at 14,000×g for 1 h at 4 °C. The resulting supernatants were subsequently passed through the Ni-NTA beads (QIAGEN) for three times to retain His₆-tagged recombinant proteins. After extensive washing of the beads with a buffer containing 20 mM imidazole, the proteins on the beads were eluted with 250 mM imidazole. When necessary, phosphorylated recombinant proteins were further separated by ion exchange chromatography. All purified proteins were dialyzed twice against a buffer containing 300 mM NaCl, 20 mM Tris-HCl (pH 7.5), and 10% glycerol at 4 °C.

Mammalian cell transfection, immunoprecipitation, western-blot, and immunostaining

Mammalian cells seeded on 6-well plates were grown to a confluence of 80%. Transfection of cells was performed using Lipofectamine 3000 transfection reagent (Invitrogen). At 24 h post-transfection, cells were either left untreated or treated with 1 μM of STS or 40 μM of VP-16 for 4 h and 24 h. Cells were collected by centrifugation (100×g for 10 min) at 4 °C and lysed with RIPA cell lysis reagent containing 1x protease inhibitors (Sigma) at 4 °C for 10 min. Cell debris and unbroken cells were removed by centrifugation (14,000×g) for 10 min at 4 °C. Immunoprecipitation was performed by incubating the cell lysates with anti-HA (Sigma) or anti-Flag (Sigma) agarose for 2 h at 4 °C on an end-to-end rotator. The agarose-bound proteins were either eluted by adding 1xSDS sample buffer followed by boiling at 100 °C for 5 min or by Flag/HA peptides (Sigma). For western-blot analysis, protein samples were resolved by SDS-PAGE and subsequently transferred to nitrocellulose membranes (Pall Life Sciences). After a blocking step using 5% w/v nonfat dry milk, the membranes were sequentially incubated with appropriate primary and secondary antibodies for 1 h at room temperature. The blot images were captured under the Odyssey® CLx Infrared Imaging System (LI-COR Biosciences). The antibodies used in the study are described in Table S4.

For immunostaining, HEK293 cells transfected with the indicated plasmids were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X−100 in PBS. After a blocking step using 4% goat serum (Sigma) in PBS for 30 min, the cells were immunostained with the anti-HA antibodies (ABclonal, cat# AE008, 1:1000) and Texas Red-conjugated secondary antibodies (Thermo Fisher Scientific, cat# T-6390, 1:500). The nuclei were stained by Hoechst 33342 (Beyotime, cat# C1022). Immunofluorescent signals were visualized via an Olympus IX-83 fluorescence microscope.

L. pneumophila infection

Wild-type, dotA^-, ΔlegK3, ΔlegK3 (pLegK3), and ΔlegK3 (pLegK3_D/A) L. pneumophila strains were grown in AYE liquid medium at 37 °C to the post-exponential phase as judged by an OD_600nm of 3.3–3.8. The bacteria were opsonized with an anti-Legionella antibody (1: 5000 dilution) at 37 °C for 30 min before infection. HEK293 or HeLa cells transfected with FcγRII⁷⁸ and relevant plasmids were then infected with opsonized L. pneumophila for 3 h at an MOI of 10. Infected cells were collected and lysed using the RIPA lysis buffer. The cell lysates were immunoprecipitated with anti-HA or anti-Flag beads. Proteins were further detected by western-blot analysis using the indicated antibodies, as listed in Table S4.

TEM translocation assay

Raw264.7 cells were seeded on 24-well plates at a density of 1 × 10⁴/well. L. pneumophila strains expressing relevant TEM fusion proteins were used to infect the cells at an MOI of 20. At 3 h post-infection, the infection samples were loaded with the 6×CCF4/AM reaction mixture (K1095; Thermo Fisher) and reacted in the dark at room temperature for 1 h. Then, translocation of the TEM fusion proteins by L. pneumophila was visualized by a fluorescence microscope (IX-83; Olympus) equipped with a β-lactamase FL-Cube (U-N41031; Chroma Technology Corp., Bellows Falls, VT). The wild-type L. pneumophila strain producing TEM-RalF or TEM-Fab1⁷⁹ was used as the positive and negative controls, respectively.

Quantitative real-time PCR

Raw264.7 cells seeded on six-well plates (1×10⁶/well) were infected with wild-type, dotA^- or ΔlegK3 L. pneumophila strains for 3 h at an MOI of 10. Infected cells were washed with PBS for five times to remove extracellular bacteria. Then, cells were collected and RNA was extracted using TRIzol (TIANGEN). After removing DNA with RNase-free DNaseI (TIANGEN), mRNA was reverse-transcribed into cDNA using a cDNA synthesis kit (Applied Biological Materials) according to the manufacturer’s instructions. Detection of the legK3 transcription levels was performed with the BlasTaq™ 2×qPCR MasterMix (Applied Biological Materials) and the QuantStudio™ Real-Time PCR system (Thermo Fisher). The 16S rRNA gene was used as an internal control. Relative gene expression was calculated by the 2^-ΔΔCT method. The primer sequences used for qRT-PCR analysis were listed in Table S3.

Detection of phosphorylated proteins

Recombinant proteins purified from E. coli strains co-expressing wild-type LegK3 or D187A mutant LegK3 were separated by either SDS-PAGE or native PAGE. SDS-PAGE gels were subjected to western-blot analysis using antibodies recognizing phosphorylated serine/threonine or by staining the gel with Pro-Q™ Diamond Phosphoprotein Gel Stain (ThermoFisher Scientific). For native PAGE analysis, protein samples were mixed with 5x native gel loading buffer (pH 8.8) and loaded onto 10% native gels. After running the gels in the Tris-glycine buffer (pH 8.8), the protein bands were observed by Coomassie brilliant blue staining (CBB).

Proteins retrieved from transfected cells or infected cells were dissolved by SDS-PAGE and subsequently analyzed by western-blot using phospho serine/threonine-specific antibodies.

Identification of phosphorylation sites by mass spectrometry

To identify phosphorylation sites, Caspase-3, Caspase-7 or Caspase-9 purified from E. coli or HEK293 cells co-expressing LegK3, or cells infected with L. pneumophila strains, were separated by SDS-PAGE. The excised protein bands of interest were further digested with trypsin and analyzed by LC-MS/MS as described below. LC-MS experiments were performed on a hybrid linear ion trap-Orbitrap mass spectrometer (ThermoFisher Scientific). A 40-min gradient was employed that ranged from 10% to 40% solvent B (100% ACN, 0.1% FA). Raw MS files were processed by Mascot for peptide assignments. The following settings were used for database search: 20 ppm for precursor ion mass tolerance, 0.8 Da for fragment ion mass tolerance, cysteine carbamidomethylation as a fixed modification, and methionine oxidation, serine/threonine/tyrosine phosphorylation as variable modifications.

Measurement of Caspase-3/-7 activity, ATP cell viability, and LDH release

HeLa or HEK293 cells plated in 24- or 6-well plates were stimulated by distinct treatments. To measure LDH levels, the culture media were collected and LDH release was detected by the LDH Cytotoxicity Assay Kit (Beyotime). For measurement of Caspase-3/-7 activity and ATP cell viability, cells were lysed in RIPA buffer. Then, the Caspase-3/-7 assay kit (Beyotime) and Enhanced ATP Assay Kit (Beyotime) were used to quantify the Caspase-3/-7 activity and ATP cell viability in the cell lysates, respectively.

Detection of cell apoptosis by TUNEL staining

HeLa cells were transfected with plasmids to express GFP, GFP-LegK3, or GFP-LegK3_D/A for 24 h. These cells were either left untreated or treated with 1 μM STS for 4 h prior to fixation by 4% paraformaldehyde. The fixed cells were further stained for DNA fragmentation using the Colorimetric TUNEL Apoptosis Assay Kit (Beyotime) following the manufacturer’s instructions.

HeLa cells transfected with FcγRII were either uninfected or infected with wild-type, dotA^-, ΔlegK3, ΔlegK3 (pLegK3), and ΔlegK3 (pLegK3_D/A) L. pneumophila strains for 3 h at an MOI of 10. The cells were fixed by 4% paraformaldehyde in PBS for 30 min followed by permeabilization with 0.2% Triton for 5 min. Immunostaining was subsequently performed using anti-L. pneumophila antibodies and a Colorimetric TUNEL Apoptosis Assay Kit (Beyotime). Immunofluorescence was observed under an Olympus IX-83 fluorescence microscope.

Detection of cell apoptosis by flow cytometry

HeLa cells were transfected with BFP, BFP-LegK3, or BFP-LegK3_D/A. At 24 h post-transfection, cells were either left untreated or treated with 1 μM STS for 4 h. Then, the cells were resuspended in PBS and incubated with propidium iodide (PI) (Beyotime) and GreenNuc™ Caspase-3 Substrate (Beyotime) for 10 min in the dark. Flow cytometry was performed on FACSCelesta (BD Biosciences) using 488 nm and 530 nm lasers.

FcγRII-producing HeLa cells were either uninfected or infected with relevant L. pneumophila strains for 3 h (MOI = 10). Infected cells were washed and resuspended in PBS for three times. After incubation of the cells with PI and GreenNuc™ Caspase-3 Substrate, apoptosis was determined via flow cytometry on FACSCelesta.

Biochemical cleavage assays

To determine the impact of LegK3-catalyzed phosphorylation on the enzymatic activity of Caspase-3, 3 μg of recombinant GSDMD or GSDME was reacted with 0.1 μg of p17/p12 or P-p17/p12 complex of Caspase-3 at 37 °C for 1 h. For cleavage of PARP, cell lysates of HEK293 cells were incubated with p17/p12 or P-p17/p12 complex of Caspase-3 at 37 °C for 20 min. Reactions were stopped by the addition of 5xSDS sample buffer and boiling for 5 min. Cleavage of the substrates was visualized by CBB staining (GSDMD and GSDME) or western-blot analysis using anti-PARP antibodies after separation by SDS-PAGE. For determination of the cleavage dynamics, aliquots of the reaction mixtures were withdrawn at the indicated time points and added with 5xSDS sample buffer. After separation by SDS-PAGE and staining by CBB, cleavage of the substrates was calculated by measuring the band intensities using ImageJ.

To evaluate the influence of Caspase-3/-7 phosphorylation on their hydrolysis by initiator caspases, cleavage reaction mixtures containing recombinant active Caspase-9 or Caspase-8 and Caspase-3_C/A/Caspase-7_C/A, phosphorylated Caspase-3_C/A, or Caspase-3_C/A/Caspase-7_C/A phosphomimetics were incubated at 37 °C. At the indicated durations, aliquots of the samples were stopped by the addition of 5xSDS sample buffer and boiling for 5 min. Proteins dissolved by SDS-PAGE were observed by CBB staining. Protein intensities were measured by ImageJ.

To investigate the effect of LegK3-mediated phosphorylation on Apaf-1-induced Caspase-9 cleavage, HEK293T cells were transfected with Flag-Apaf-1_1-541 for 24 h. Flag-Apaf-1_1-541 eluted by 3xFlag peptides were reacted with recombinant wild-type Caspase-9 or Caspase-9_T102E in a buffer contain 20 mM Hepes (pH 7.5), 10 mM KCl, 1.5 mM MgCl, 1 mM EDTA, 1 mM DTT and 0.1 mM PMSF for 1 h at 30 °C. The reaction mixtures were terminated by 5xSDS sample buffer. Cleaved Caspase-9 was detected by western-blot analysis using anti-Caspase-9 antibodies.

L. pneumophila intracellular replication assay

FcγRII-expressing HeLa cells were infected with anti-Legionella antibody-opsonized wild-type, ΔlegK3, ΔlegK3 (pLegK3), or ΔlegK3 (pLegK3_D/A) bacteria for 2 h at an MOI of 0.05. Extracellular bacteria were removed by washing the plates three times with prewarmed PBS. The infection samples were added with fresh culture media supplemented with or without 40 μM VP-16. At 2, 24, 48, and 72 h post-infection, cells were lysed with 0.2% saponin for 30 min. Serial dilutions of the lysates were spotted onto CYE agar plates and grown at 37 °C for 4-5 days before counting the colony-forming units.

Quantification and statistical analysis

The band intensities shown in the cleavage assays were quantified by the ImageJ software. To determine the percentage of apoptotic cells by TUNEL staining, 100 transfected or L. pneumophila-positive cells were calculated. The data are presented as the mean ± SD as indicated in the figure legends. Statistical analyses were performed using unpaired two-tailed Student’s t tests, and p < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD054358. The link to access the data: https://www.iprox.cn//page/project.html?id=IPX0009364000. The data that support the conclusions of this study are included in this published article along with its Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

We thank Dr. Ralph Isberg (Tufts School of Medicine, Boston, MA, USA) for L. pneumophila cluster deletion strains. This work was supported by the National Natural Science Foundation of China (grants 32170182 and 82372268 to J.Z.Q, 22174003 and 21974002 to X.Y.L) and the National Key Research and Development Program of China (2022YFA1304500 to X.Y.L); the Fundamental Research Funds for the Central Universities to J.Z.Q.

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These authors contributed equally: Jinli Ge, Ying Wang.

Authors and Affiliations

State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
Jinli Ge, Xueyu Li, Qian Lu, Hangqian Yu, Hongtao Liu, Kelong Ma, Xuming Deng & Jiazhang Qiu
Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
Ying Wang & Xiaoyun Liu
NHC Key Laboratory of Medical Immunology, Peking University, Beijing, China
Ying Wang & Xiaoyun Liu
Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
Zhao-Qing Luo

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Jinli Ge
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Ying Wang
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Xueyu Li
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Qian Lu
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Hangqian Yu
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Hongtao Liu
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Kelong Ma
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Xuming Deng
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Zhao-Qing Luo
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Xiaoyun Liu
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Jiazhang Qiu
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Contributions

J.Z.Q., X.Y.L., and Z.Q.L. conceived the study and provided critical revisions to the manuscript. J.Z.Q. and J.L.G. were responsible for the experimental design, data analysis, and composition of the manuscript. J.L.G., Y.W., X.Y.L., and Q.L. performed biochemical, cell biological, and infection assays. J.L.G. and Y.W. performed mass spectrometry analyses. H.Q.Y., H.T.L., K.L.M., and X.M.D. contributed to the experimental design and data analysis.

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Correspondence to Xiaoyun Liu or Jiazhang Qiu.

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Ge, J., Wang, Y., Li, X. et al. Phosphorylation of caspases by a bacterial kinase inhibits host programmed cell death. Nat Commun 15, 8464 (2024). https://doi.org/10.1038/s41467-024-52817-1

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Received: 25 March 2024
Accepted: 20 September 2024
Published: 30 September 2024
DOI: https://doi.org/10.1038/s41467-024-52817-1

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