Klebsiella pneumoniae NdpA suppresses ERK pathway-mediated host early inflammatory responses and is degraded through the ubiquitin-proteasome pathway

Klebsiella pneumoniae (KP) is an important opportunistic pathogen causing community-acquired and nosocomial infections. When the host is immunocompromised, the pathogen would infect the host and cause diseases, such as pneumoniae, sepsis, liver abscess, meningitis, urinary tract inflammation and wound infection (Karaiskos et al., 2016; Park et al., 2015). The phenomenon that K. pneumoniae has a preference to infect immunocompromised populations, especially seniors suggests that the outcomes of K. pneumoniae infection depend on the pathogen-host interactions, but up to now, the molecular mechanisms underlying K. pneumoniae-host interactions remain largely unknown. Previous studies reported that K. pneumoniae suppresses inflammatory cytokine production during early period of infection (Lawlor et al., 2006), and this bacteria can block the activation of inflammatory responses by antagonizing NF-κB and MAPK signaling pathways (Frank et al., 2013; Regueiro et al., 2011). We discovered that the K. pneumoniae nucleoid-associated protein (NdpA) is highly conserved among gram-negative bacteria. Transient expression of NdpA in human embryonic kidney HEK239T cells inhibited the Elk activation induced by RasV12, as well as V-Raf (constitutive active Raf) and MEK1-ED (constitutive active MEK1), respectively (Fig. 1A–C). K. pneumoniae NdpA also promoted tumor necrosis factor (TNF) α-stimulated NF-κB activation (Fig. S1A), and had little, if any, inhibitory effect on JNK and p38 signaling pathways (Fig. S1B). Given the lack of inflammation at the early stage of K. pneumoniae infection (Lawlor et al., 2006), we thus focused on the elucidation of the suppressive effects of K. pneumoniae NdpA on ERK signaling pathway-mediated host early inflammatory responses. Because many pathogenic bacteria have secretion systems to inject their virulence factors into host cells to interfere their functions. Thus, we sought to examine whether K. pneumoniae NdpA could be secreted into host cells during infection. Immunoblot analysis showed that NdpA entered into the cytosol of the human alveolar epithelial cells A549 during K. pneumoniae infection (Fig. S2A). In addition, we found that the phosphorylation of ERK1/2 activated by MEK1-ED was largely reduced by NdpA (Fig. 1D). Consistently, NdpA also abolished extracellular stimuli epidermal growth factor (EGF)-activated ERK1/2 phosphorylation (Fig. 1E). To further confirm the role of NdpA in the suppression of ERK signaling during K. pneumoniae infection, we tried to knockout the gene encoding NdpA with several methods available, but after many attempts we failed to obtain the expected mutant strain. We thus adopted the alternative strategy to investigate the host immune-regulatory function of NdpA by overexpressing it in K. pneumoniae. We found that overexpression of NdpA in K. pneumoniae and E. coli resulted in down-regulation of phospho-ERK1/2 (p-ERK1/2) in A549 cells during K. pneumoniae infection (Figs. 1F and S2B). With a central role in recruiting and infiltrating neutrophils into inflammatory sites, IL8 is known as a main inflammatory molecule involved in host defense against K. pneumoniae infection (Harada et al., 1994). We thus next explored whether NdpA regulates the expression of IL8 in human alveolar epithelial cells during K. pneumoniae infection. The data from quantitative real-time PCR showed that compared with the wild-type (WT) K. pneumoniae strain, the NdpA-overexpressing K. pneumoniae strain significantly down-regulated the mRNA of IL8 in A549 cells (Fig. 1G). Accordingly, the secretion of IL8 was apparently attenuated by the overexpression of NdpA in K. pneumoniae as analyzed by enzyme-linked immunosorbent assay (ELISA) (Fig. 1H). To determine whether the inhibitory effects of NdpA on IL8 production is dependent on ERK signaling pathway, we pretreated A549 cells with U0126, a specific inhibitor of ERK pathway, before the infection assay, and we found that the WT K. pneumoniae strain and the NdpA-overexpressing K. pneumoniae strain showed similar amount of IL8 production during infection of A549 cells (Fig. S3A and S3B).

2 Ⅱ Fast Mutagenesis Kit V2 (C214-01; Vazyme). The detailed information on strains, plasmids, and oligonucleotides used in this study are list in Supplementary Table 1.

Cell culture and infection
A549, HEK293T and Hela cells were grown in DMEM tissue culture medium supplemented with 10% heated-inactivated fetal bovine serum and 1% antibiotics (penicillin and streptomycin) in 10 cm cell culture dishes at 37°C in a water-saturated atmosphere containing 5% CO 2 . For infection, A549 cells were seeded to about 90% confluence (1.4 × 10 6 per well) in 6-well tissue culture plates. Before infection, cells 3 were serum starved for 12 to 16 h. The infection was performed at a multiplicity of infection (M.O.I) of 150 K. pneumoniae or 100 E. coli ATCC25922 per cell.

Cell transfection, immunoblotting and immunoprecipitation
HEK293T cells were transfected using (PEI) or standard calcium phosphate method.
A549 or HeLa cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacture's instruction. For immunoblotting, HEK293T or A549 cells were seeded in 6-well plates and grown to about 70% confluence, then vectors were transfected, four to six hours after transfection, cells were fed with fresh tissue culture medium. After 24-48 h, cells were harvested and lysed with the Cell Lysis Buffer for Western and IP (P0013, Beyotime). Total cell lysates were separated by SDS-PAGE and transferred to polyvinylidenedifluoride (PVDF) membrane (Millipore). The membrane was block with 5% skimmed milk in Tris-buffered saline (TBS) for 1 h at room temperature and subsequently incubated with appropriate primary antibodies in TBS with 1% (V/V) twwen-20 (TBST) and 5% (W/V) skimmed milk at 4°C overnight. Following three washes of 10 min each with TBST, the PVDF membranes were incubated with goat anti-mouse or goat anti-rabbit IgG conjugated to HRP at a dilution of 1:10000 in TBST for 1 h at room temperature. After three washes with TBST, the membrane was subjected to Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500; Millipore) and exposed to X-ray film. For immunoprecipitation, transfected HEK293T cells were lysed with buffer containing 4 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, and a protease inhibitor mixture (P8340; Sigma). Cell lysates were incubated with Flag M2 beads (A2220; Sigma) or anti-Myc beads (sc-40AC; Santa CruZ) at 4°C for 4 h, followed by extensive wash with the lysis buffer. Both lysates and immunoprecipitates were examined by immunoblotting.

Dual-luciferase assay
Dual luciferase assay was performed using the Promega luciferase reporter system.

Protein purification and His pull-down
All proteins were purified from E. coli BL21 (DE3). His-NdpA, His-NdpA (L21E) and GST-ERK2 were induced at 30°C overnight with 100 μM isopropyl-β-D-thiogalactopyranoside (IPTG) upon OD 600 reached 0.6-0.8. His tagged proteins were purified by affinity chromatography using Ni-NTA Agarose, GST-ERK2 was purified by Glutathione Sepharose 4B. For His pull-down, the His fusions of proteins were immobilized onto Ni-NTA Agarose followed by incubation with prey proteins in lysis buffer containing 50 mM NaH 2 PO 4 (pH 8), 300 mM NaCl, 20 mM imidazole and 1% Triton X-100, or HEK293T cell lysate supplemented with protease inhibitor mixture at 4˚C for 4 h. The beads were extensively washes four or five times. The bound proteins were analyzed by immunoblotting.

Cell fractionation
Cells were lysed on ice for 5 min with buffer containing 10 mM HEPES (pH7.9), 1.5 mM MgCl 2 , 10 mM KCl, 0.34 M sucrose, 10% glycerol, protease inhibitors and 0.1% Triton X-100, followed by centrifugation at 13,000 g for 10 min. The supernatant was collected as cytosolic fraction. The pellet was resuspended with 1 × PBS plus 0.1% SDS and 0.1% Triton X-100, followed by centrifugation in a 15% sucrose cushion twice at 2,500 g for 15 min at 4°C.The supernatant was the nuclear fraction.
6 Quantitative PCR and enzyme-linked immunosorbent assay A549 cells seeded into 6-well plates were infected as described above. After infection, the cells culture media were collected for ELISA, the cells were washed with 1 × PBS and extracted total RNA using commercial kit (R1061). Subsequently the RNA was reverse transcribed into cDNA using commercially available kits follows the manufacturers' instructions. The cDNA was then analyzed by quantitative PCR with KAPASYBR FAST qPCR Kit (KAPABiosystems) on ABI 7300 system (Applied