The Salmonella SPI2 TTSS Effector Protein SseF Interacts With Host Histone Acetyltransferase TIP60

Salmonella enterica is a facultative intracellular pathogen that can replicate 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 the host cells to exploit host cell vesicle trafficking pathways. The biogenesis of SCV requires a panel of secreted effectors: SifA, SseF and SseG, which contribute to the formation of specific structure induced by salmonella infection, named salmonella induced filaments (SIFs).which also maintain the vacuolar membrane enclosing the pathogen. Results from recent studies demonstrated that SifA play this role is mediated by interaction with SKIP, its cellular host target, which in turn down-regulates recruitment of kinesin-1 onto the vacuolar membrane. But most targets of SPI2 effectors are unknown. SseF is one of these effectors involved in SCV formation and the formation of Salmonella-induced continuous filaments (SIFs) in epithelial cells through unknown mechanisms. In this study, we propose to investigation the host target which interacts with SseF and further research the mechanism of the interaction between them.In an attempt to identify host proteins that interact with SseF, we conduct a yeast two-hybrid screening of human kidney cell cDNA library using full length of SseF as the bait and didn't obtained positive clone. However, when using the truncated SseF (deleted three trans-membrane sequence regions) as bait, we identified that TIP60, an acetyltransferase, interacts specifically with SseF. This interaction was further confirmed by a MBP pull-down assay. To determine the region of SseF that is responsible for interacting with TIP60, a series of SseF deletions was constructed. We found that amino acids 50-66 of SseF protein were sufficient for mediating the interaction between SseF and TIP60.To study the mechanism of interaction between SseF and TIP60, SseF truncated version, GST-SseF1-66 polypeptide was expressed in BL21(DE3) using a pGEX-KG construct obtained by insertion of a EcoRI/Sal fragment. Meanwhile MBP-TIP60 polypeptide was expressed in BL21 (DE3) using a pMAL-c2x construct obtained by insertion of a EcoRI/XbaI fragment. An in vitro HAT assay was conducted using purified recombinant MBP-TIP60 as acetyltransferase and GST-SseF1-66 as the substrate. The result is shown that SseF was acetylated by TIP60. Further HAT assay was conducted when GST-SseF Lys2Arg and GST-SseF Lys25Arg mutation were made, respectively. Acetylated GST-SseF Lys2Arg was detected. To confirm the SseF acetylation by TIP60, we construct a histone acetylase-deficient mutant of TIP60, the Gln-377 and Gly-380 of TIP60 was mutated to glutamic acid. Purified recombinant mutant MBP-TIP60 was used in place of recombinant wild type MBP-TIP60 in identical conditions, and no SseF1-66 acetylization was observed.To further analyze effect of SseF acetylization on its phenotype, we constructâ–³sseF (deleted full sseF length in Salmonella chromosome), sseF Lys2Arg and sseF Lys25Arg mutant strains. RAW264.7 macrophages were infected with AsseF, sseF Lys2Arg and sseF Lys25Arg mutant strains, Meanwhile RAW264.7 macrophages were infected with wild-type serovar Typhimurium. Extracellular bacteria were removed by washing and gentamycin treatment. At 2 and 12h post bacterial invasion, cells were lysed and the number of intracellular bacteria was determined by plating on LB medium-streptomycin plates. The result showed that sseF Lys25Arg mutant strain replicated less efficiently inside macrophage than wild type and sseF Lys2Arg mutant strain, The replicate efficiency of sseF Lys25Arg mutant was similar to that of the AsseF mutant strain. To study the role of Tip60 in Salmonella-induced filament formation, we used siRNA to suppress TIP60 endogenous expression of HeLa cells. To assess the effectiveness of our TIP60 siRNA, we transfected a plasmid encoding TIP60 siRNA or a control vector encoding the scrambled siRNA into HeLa cells (invitrogen). The expression of TIP60 was examined under the fluorescent microscope or by Western blotting. Our data showed that the TIP60 siRNA effectively inhibited TIP60 expression. We then transfected HeLa cells with the plasmid encoding TIP60 siRNA or the control vector encoding the scrambled siRNA, transfected cells were then infected with wild type or the sseF mutant Salmonella. As previously reported, sseF mutant strain induced aggregation of LAMP-2-containing filamentous structures, which was in contrast to the continuous distribution of LAMP-2 along Sifs induced by the wild-type Salmonella in infected Hela cells. But wild-type Salmonella induced Sifs displaying punctate distribution of LAMP-2 in TIP60 siRNA treated Hela cells. This discontinuous distribution pattern of LAMP-2 is similar to the "pseudo-Sif" induced by the sseF mutant strain.Then we used wild-type serovar Typhimurium to infect RAW264.7 macrophages treated with TIP60 siRNA or control RNAi. Extracellular bacteria were removed by washing and gentamicin treatment. At 2 and 12 h post bacterial invasion, cells were lysed and the number of intracellular bacteria was determined by plating them on LB medium-streptomycin plates. The result suggested that Salmonella Typhymurium replicated less efficiently in TIP60 knock-down cells than control ones. And the replication efficiency of Salmonella Typhymurium in TIP60 knock-down cells was similar to that of the sseF mutant strain in macrophage cells. The above results further confirmed that TIP60-mediated SseF acetylization is required for SseF function, as to modulate host cell vesicle trafficking and SCV biogenesis, which may be able to affect the phenotype of SseF.In conclusion, Salmonella strains secrete and translocate a panel of effectors by the SPI-2 encoded type m secretion system to facilitate their successful survival and replications inside host cells. SseF is one of those effectors that are required for the formation of Sifs and bacteria replication in host cells. We report here that SseF interacts with TIP60, the host acetyltransferase. Our data show that SseF is acetylated by the TIP60 and that the acetylated amino acid of SseF is 25-lysinine. TIP60 may modulate SseF phenotype and SCV biogenesis through acetylating SseF and further affect Salmonella SCV biogenesis. Our discovery may help to elucidate pathogenesis of Salmonella and provide vital insights into basic host cell biology.