Involvement of TIP60 acetyltransferase in intracellular Salmonellareplication
© Wang et al; licensee BioMed Central Ltd. 2010
Received: 11 May 2010
Accepted: 26 August 2010
Published: 26 August 2010
Salmonella enterica is a facultative intracellular pathogen that replicates within a membrane-bound compartment termed Salmonella containing vacuole (SCV). The biogenesis of SCV requires Salmonella type III protein secretion/translocation system and their effector proteins which are translocated into host cells to exploit the vesicle trafficking pathways. SseF is one of these effectors required for SCV formation and Intracellular Salmonella replication through unknown mechanisms.
In an attempt to identify host proteins that interact with SseF, we conduct a yeast two-hybrid screening of human cell cDNA library using SseF as the bait. We identified that TIP60, an acetyltransferase, interacts with SseF. We showed that the TIP60 acetylation activity was increased in the presence of SseF, and TIP60 was upregulated upon Salmonella infection. In addition, TIP60 is required for efficient intracellular Salmonella replication in macrophages.
Taken together, our data suggest that Salmonella may use SseF to exploit the host TIP60 acetyltransferase activity to promote efficient Salmonella replication inside host cells.
Protein acetylation adds the acetyl group on either the amino-terminal residues or on the epsilon-amino group of lysine residues. Lysine acetylation affects many protein functions, including DNA binding, protein-protein interactions, and protein stability. TIP60 catalyzes histone acetylation [1, 2]. It was originally identified as a cellular acetyltransferase protein that interacts with HIV-1 Tat . Over-expression of TIP60 increased Tat transactivation of the HIV-1 promoter . Recent studies found that TIP60 has diverse functions involved in transcription, cellular signaling, DNA damage repair, cell cycle checkpoint control and apoptosis [2, 4, 5].
Salmonella enterica serovar Typhimurium (S. typhimurium) causes gastrointestinal diseases in humans and typhoid-like fever in the mouse. S. typhimurium encodes two Type III secretion systems within the Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that are required for Salmonella entry and subsequent survival inside the host cells, respectively [6–10]. Following entry into the host cells, S. typhimurium replicates within a membrane-bound compartment termed S almonella-containing vacuole (SCV). Previous studies have shown that SifA, SseF and SseG are involved in the formation of S almonella induced filaments (Sifs) that are required for maintaining the SCV [11–13].
SseF, working together with SseG, has been shown to be involved in the aggregation of host endosomes and may help to position the Salmonella-containing vacuoles in close association with the Golgi network [14–19]. In the absence of SseF, the vacuolar compartments containing Salmonella were discontinuous and intracellular Salmonella replication was reduced [10, 14, 15, 20–22]. SseG was shown to be co-localized with the trans-Golgi network and only bacteria closely associated with the Golgi network were able to multiply . It has been shown that SseF interacts functionally and physically with SseG but not SifA and is also required for the perinuclear localization of Salmonella vacuoles . The molecular mechanism on how SseF and SseG function remains unknown. In the present study, we set out to search the host target that interacts with SseF. We presented evidence indicating that Salmonella SseF interacts with TIP60 to potentiate its histone acetylation activity to promote intracellular replication.
Bacterial strains and plasmids
Strains and plasmids
S. typhimurium and E. coli
Wild-type S. typhimurium, Strr
SseF in-frame deletions
thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu (Kanr) λpir
SsaV in-frame deletions in pSB890; Tcr
SseF in-frame deletions in pSB890; Tcr
SseFΔ67-106, 161-174, 186-205 in pGBT9, Apr
His-SseF in pET28a; Kanr
His-SseG in pET28a; Kanr
GAL4AD-iTIP60164-546 in pGAD-GH; Apr
GAL4AD-TIP60α in pGAD-GH; Apr
GAL4AD-TIP60β in pGAD-GH; Apr
HA-TIP60α in pcDNA3; Apr
MBP-TIP60α in pIADL16; Apr
GAL4-BD-SseF1-66 in pGBT9; Apr
GAL4-BD-SseF50-66 in pGBT9; Apr
GST-SseF1-66 in pGEX-KG; Apr
GST-SseF50-66 in pGEX-KG; Apr
GAL4-BD-SseF1-56 in pGBT9; Apr
GAL4-BD-SseF50-260 in pGBT9; Apr
GAL4-BD-SseF1-228 in pGBT9; Apr
Mammalian cell lines and bacterial infection assay
The murine macrophage RAW264.7 (TIB-71, ATCC) and the human epithelial cell line HeLa (CCL-2, ATCC) were from the ATCC (Manassas, VA) and were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% FBS. Bacterial infection of RAW264.7 and survival assays were carried out using opsonized bacteria in DMEM containing 10% normal mouse serum as described before [10, 20, 27]. The extent of replication was then determined by dividing the number of intracellular bacteria at twenty-four hours by the number at two hours.
Yeast Two-hybrid Screening
The GAL4-based yeast two-hybrid system was used following standard procedures . The bait plasmid (pZP784) was constructed by deleting the putative three trans-membrane regions (67-106, 161-174, 186-205 a.a.) of SseF and fusing it to the yeast GAL4 binding domain in pGBT9.m . A human cell cDNA library was constructed by oligo(dT) priming in pACT2 (Clontech Laboratories, Palo Alto, CA). A total of 5 × 105 transformants were screened in the yeast indicator strain AH109, using the sequential transformation protocol as described (Clontech Laboratories). Clones that grow on the yeast synthetic drop-out media lacking histidine and exhibited positive galactosidase on the X-Gal plates were chosen for further analysis.
Protein purification and biochemical pull down assay
GST, His and MBP-tagged recombinant proteins were expressed and purified in Escherichia coli BL21 (DE3) using the pGEX-KG, pET28a, or the pMAL-c2x expression systems, respectively. The purification of the GST-tagged proteins was performed according to the manufacturer's instructions (Amersham, Pittsburgh, PA). Purified proteins were concentrated and buffer exchanged in PBS, using a 10 K and 30 K molecular weight cut-off dialysis cassette (Sartorius, Elk Grove, IL). Purified proteins were snap-frozen in liquid nitrogen and stored at -80°C in PBS/20% glycerol. Proteins were pre-clarified at 120,000 Xg, and their concentration was determined by Bradford assay (Bio-Rad) using bovine serum albumin as standard. Pulled-down proteins were analyzed by SDS-PAGE and Western blotting using appropriate antibodies. Western blots were developed with using the SuperSignal West Pico detection reagent according to the manufacturer's instructions (Thermo Fisher, Rockford, IL).
HAT assays were performed using recombinant MBP-TIP60 protein (100 ng) as acetyltransferase and histone (2 μg, Sigma, St. Louis, MO) as the substrate in 20 μl HAT buffer (50 mM Tris, pH 8.0, 10% glycerol, 1 mM dithiothreitol, 0.1 mM EDTA, 10 mM sodium butyrate) containing Acetyl-CoA (100 μM, Sigma, St. Louis, MO) for 30 min at 30°C. Acetylated histones were detected by Western blot, using the pan-acetyl antibody (Santa Cruz Biotech, Santa Cruz, CA).
TIP60 siRNA expression plasmids were constructed in pSilencer 2.1 (Ambion, Austin, TX) with a pair of 63-bp oligonucleotides, each containing a unique 19-bp TIP60 sequence. For use in human cell lines: 5'-GATCCGAACAAGAGTTAATTCCCAGTTC AAGAGACTGGGAATAACTCTTGTTCTTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAA GAACAAGAGTTATTCCCAGTCTCTTGAACTGGGAATAACTCTTGTTCG-3'. For use in mouse cell lines: 5'-GATCCAGACTGGAGCAAGAGAGGATTCAAGAGATCCTCTCTTGC TCCAGTCTTTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAAAGACTGGAGCAAGAG AGGATCTCTTGAATCCTCTCTTGCTCCAGTCTG-3'. For negative control, a scrambled siRNA hairpin was placed into the same sites in pSilencer 2.1. These plasmids were transfected into cells using the siPORT XP-1 provided by Ambion. Transfected cells were maintained for 24 hours without selection; cultures were then subjected to G418 selection before infection.
SalmonellaSPI2 effector protein SseF interacts with TIP60 histone acetylase
SseF increases the histone acetylation activity of TIP60
TIP60 is a multifunctional acetyltransferase involved in many transcriptional regulations by serving as a co-regulator . The interaction of SseF with TIP60 suggested that SseF may serve as the substrate for TIP60-mediated acetylation. To test whether SseF serves as the substrate for TIP60, an in vitro HAT assay was conducted, using purified recombinant MBP-TIP60 as acetyltransferase and GST-SseF1-66 as the substrate [2, 4, 5]. When probed with antibodies specific for acetylated species, adducts were detected when histone was added to the reaction in the presence of MBP-TIP60 (data not shown). No SseF acetylation was observed when GST-SseF1-66 was used in the reaction. Similar results were obtained when partially enriched full-length SseF was used in the reaction (data not shown). Thus, SseF is not likely the substrate for TIP60.
TIP60 protein level is increased upon Salmonellainfection
TIP60 is required for efficient intracellular Salmonellareplication
We do not know yet the molecular mechanism of how SseF and TIP60 interaction affects the SCV and intracellular Salmonella replication. Ideally, a mutant SseF lacking the TIP60-binding domain can be used to assess the requirement for SseF-TIP60 interaction for its function, however such a mutant is defective in secretion and thus not translocated, making it impossible to assess its effect during infection. Definitive identification of the acetylation site and subsequent characterization of proper mutants lacking TIP60-mediated acetylation will be required to validate this hypothesis. Alternatively, SseF-TIP60 interaction may alter the acetylation activity of TIP60, thus affecting TIP60 related functions. Supporting this hypothesis, our preliminary in vitro acetylation assays suggest that SseF increased the histone acetylation activity of TIP60, especially for histone H2. Histone is the only known substrate for Tip60. Total histone acetylation was not increased in infected cells (data not shown). This is consistent with the low amount of SseF translocated. It is possible that local SseF concentration may be higher in infected cells.
Although TIP60 is not known to be directly involved in vesicle trafficking, it is possible that TIP60 affected histone acetylation leading to altered expression of trafficking-related proteins. Interestingly, our preliminary data showed that knock down of TIP60 reduced continuous Sif formation, a phenotype similar to that of the sseF null mutant (Additional file 1: Fig. S1). Future experiments are required to determine whether the increase in histone acetylation leads to increases in TIP60-mediated downstream functions. This may ultimately help us to understand how SseF interact with TIP60 to promote Salmonella replication inside the host cells.
We found that TIP60, an acetyltransferase, interacts with Salmonella SseF. We further showed that the TIP60 acetylation activity was increased in the presence of SseF, and TIP60 was upregulated upon Salmonella infection. More importantly, TIP60 is required for efficient intracellular Salmonella replication in macrophages. Our study demonstrated that Salmonella may use SseF to exploit the host TIP60 acetyltransferase activity to promote efficient Salmonella replication inside host cells.
Research was supported by NSFC grant 30628001 to D. Z., and by "863" grant 2006AA02A253 to D.Q.
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