pathogens Article Hsp90 Interacts with the Bacterial Effector NleH1 Miaomiao Wu and Philip R. Hardwidge * Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS 66506, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-785-532-2506 Received: 26 September 2018; Accepted: 11 November 2018; Published: 13 November 2018 Abstract: Enterohemorrhagic Escherichia coli (EHEC) utilizes a type III secretion system (T3SS) to inject effector proteins into host cells. The EHEC NleH1 effector inhibits the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway by reducing the nuclear translocation of the ribosomal protein S3 (RPS3). NleH1 prevents RPS3 phosphorylation by the IκB kinase-β (IKKβ). IKKβ is a central kinase in the NF-κB pathway, yet NleH1 only restricts the phosphorylation of a subset of the IKKβ substrates. We hypothesized that a protein cofactor might dictate this inhibitory specificity. We determined that heat shock protein 90 (Hsp90) interacts with both IKKβ and NleH1 and that inhibiting Hsp90 activity reduces RPS3 nuclear translocation. Keywords: E. coli; Hsp90; NleH1; RPS3 1. Introduction The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) family of transcription factors regulates innate and adaptive immune responses. In addition to the well-characterized Rel family proteins [1], ribosomal protein S3 (RPS3) is a key non-Rel subunit and was identified as a “specifier” NF-κB component. RPS3 guides NF-κB to specific κB sites by increasing the affinity of the NF-κB p65 subunit for target gene promoters [2]. Activation of NF-κB signaling is initiated by external stimuli that activate the IκB kinase (IKK) complex. This complex consists of two catalytic components, IKKα (IKK1) and IKKβ (IKK2), in addition to a regulatory subunit, the NF-κB Essential Modulator (NEMO; IKKγ). The canonical IKK complex plays a central role in regulating many cellular processes in response to a variety of physiological and pathological stimuli, among which NF-κB is the best known. Activated IKKβ phosphorylates IκBα, resulting in its subsequent ubiquitination and degradation, which allows for p65 and p50 nuclear translocation [3]. IKKβ also phosphorylates RPS3 on Ser209, enhancing its association with importin-α and mediating RPS3 nuclear translocation [4]. Bacteria have evolved secretion systems to transport virulence proteins, termed ‘effectors’, to counteract host innate immunity. Enterohemorrhagic Escherichia coli (EHEC) encodes numerous type three secretion system (T3SS) effectors that subvert cellular processes to create an environment conductive to bacterial survival. EHEC encodes two forms of the NleH effector, NleH1 and NleH2 [5,6]. Both effectors bind RPS3, but only NleH1 inhibits RPS3 nuclear translocation by preventing RPS3 phosphorylation on S209 by IKKβ [4,5]. NleH1 activity only restricts the phosphorylation of a subset of the IKKβ substrates, suggesting that an additional host co-factor might dictate this inhibitory specificity. Here we identified heat shock protein 90 (Hsp90) as an NleH1 binding partner and found that inhibiting Hsp90 activity reduced RPS3 nuclear translocation. Pathogens 2018, 7, 87; doi:10.3390/pathogens7040087 www.mdpi.com/journal/pathogens Pathogens 2018, 7, 87 2 of 9 Pathogens 2018, 7, x FOR PEER REVIEW 2 of 9 2. Results 2.1. NleH1 and NleH2 Bind to IKKβb To determine whether EHEC NleH1 binds directly to IKK β wewe conducted conducted GST GST pulldown pulldown assays. assays. His-IKKβ, GST, GST, GST-NleH1 GST-NleH1 and GST-NleH2 GST-NleH2 were were purified purified using using nickel-nitrilotriacetic nickel-nitrilotriacetic acid acid (Ni-NTA) (Ni-NTA) agarose beads.beads. PurifiedPurified GST GST (negative (negative control), control), GST-NleH1 GST-NleH1 and and GST-NleH2 GST-NleH2 were were immobilized immobilized on GST on beadsGST beads and incubated and incubated with His-IKKwith His-IKKβ. IKKβ. wasIKKβ enriched was enriched in the NleH1 in the andNleH1 NleH2 and pulldown NleH2 pulldown samples, assamples, compared as compared to the negative to the control negative (Figure control1A). (Figure To determine 1A). To whether determine NleH1 whether and NleH2 NleH1 interact and NleH2 with IKKinteractβ in with mammalian IKKβ in cells, mammalian we performed cells, we co-immunoprecipitation performed co-immunoprecipitation experiments. After experiments. co-transfecting After eitherco-transfecting NleB1-HA either (as a NleB1-HA negative control), (as a negative NleH1-HA control), or NleH2-HA NleH1-HA with or NleH2-HA FLAG-IKKβ with, cell FLAG-IKK lysates wereβ, immunoprecipitatedcell lysates were immunoprecipitated with anti-FLAG M2with beads anti-FLAG and subsequently M2 beads and immunoblotted. subsequently immunoblotted. NleH1-HA and NleH2-HA,NleH1-HA and but notNleH2-HA, NleB1-HA, but interacted not NleB1-HA, with FLAG-IKKinteractedβ with(Figure FLAG-IKK1B). Thus,β (Figure NleH1 1B). and Thus, NleH2 NleH1 bind directlyand NleH2 to IKK bindβ indirectly vitro, to and IKK interactβ in vitro, with and IKK interactβ in mammalian with IKKβ cells. in mammalian cells. Figure 1. NleH1 and NleH2 bind to IKKβ..( (AA)) Pulldown Pulldown assay assay to to detect binding between NleH1, NleH2, and IKK IKKββ.. His6-IKK His6-IKKββ waswas incubated incubated with with GST, GST, GST-NleH1 GST-NleH1 and and GST-NleH2 GST-NleH2 and and subjected subjected to toa GST a GST pulldown pulldown assay assay using using glutathione-sepharose glutathione-sepharose beads. beads. Protein Protein complexes complexes were were eluted eluted with with 10 10mM mM reduced reduced glutathione glutathione followed followed by by 10% 10% SDS-PAGE SDS-PAGE analysis. analysis. GST GST was was used used as as a a negative negative control. control. β (B) ImmunoprecipitationImmunoprecipitation ofof NleB1-HA,NleB1-HA, NleH1-HANleH1-HA andand NleH2-HANleH2-HA withwith FLAG-IKKFLAG-IKKβ.. HEK293 cells were transfectedtransfected and and cell lysatescell lysates were immunoprecipitatedwere immunoprecipitated using anti-FLAG using M2anti-FLAG gel and immunoblottedM2 gel and forimmunoblotted FLAG and HA. for NleB1-HAFLAG and wasHA. used NleB1-H as aA negative was used control. as a negative control. To identify other proteins that are enriched with IKKβ as a function of co-expression of NleH1, To identify other proteins that are enriched with IKKβ as a function of co-expression of NleH1, we repeated the transfection experiments described above and then processed the immunoprecipitated we repeated the transfection experiments described above and then processed the proteins samples for mass spectrometry analysis. We identified Hsp90, Cdc37 and RPS3 in these samples. immunoprecipitated proteins samples for mass spectrometry analysis. We identified Hsp90, Cdc37 Hsp90 is an important chaperone protein that regulates the folding, stability and trafficking and RPS3 in these samples. of many client proteins [7]. Cell division cycle protein 37 (Cdc37) is one of the best-studied Hsp90 Hsp90 is an important chaperone protein that regulates the folding, stability and trafficking of co-chaperones [8,9]. Hsp90 interacts with Cdc37 and the Hsp90-Cdc37 complex can form a stable many client proteins [7]. Cell division cycle protein 37 (Cdc37) is one of the best-studied Hsp90 co- chaperones [8,9]. Hsp90 interacts with Cdc37 and the Hsp90-Cdc37 complex can form a stable Pathogens 2018, 7, 87 3 of 9 complexPathogens with 2018 IKK,, 7, x FOR which PEERplays REVIEW a critical role in its activation and regulation [10,11]. Due3 of 9 to the important association between Hsp90 and IKK, we focused on the potential interactions between Hsp90,complex IKKβ, andwith NleH1.IKK, which plays a critical role in its activation and regulation [10,11]. Due to the important association between Hsp90 and IKK, we focused on the potential interactions between 2.2. Hsp90Hsp90, Interacts IKKβ, and with NleH1. NleH1, NleH2 and IKKb To2.2. assess Hsp90 the Interacts association with NleH1, of Hsp90 NleH2 and IKK IKKβ β in mammalian cells, we performed co-transfection and co-immunoprecipitationTo assess the association experiments of Hsp90 and with IKK Hsp90-HAβ in mammalian and FLAG-IKK cells, we performedβ. HEK293 co-transfection cell lysates were immunoprecipitatedand co-immunoprecipitation with anti-FLAG experime M2nts beads with andHsp90-HA subsequently and FLAG-IKK immunoblottedβ. HEK293 withcell lysates anti-FLAG and anti-HAwere immunoprecipitated antibodies. Hsp90-HA with anti-FLAG interacted M2 beads with FLAG-IKKand subsequentlyβ (Figure immunoblotted2A), which with is consistent anti- with aFLAG previous and studyanti-HA showing antibodies. that endogenousHsp90-HA interacted Hsp90 co-precipitatedwith FLAG-IKKβ from (Figure HeLa 2A), cell which extracts is with IKKβ [consistent10]. We consideredwith a previous whether study IKK showingβ might thatinteract endogenous with Hsp90 both NleH1/NleH2co-precipitated from and HeLa Hsp90. cell To test extracts with IKKβ [10]. We considered whether IKKβ might interact with both NleH1/NleH2 and this idea, we co-transfected HEK293 cells with FLAG-IKKβ Hsp90-HA and NleH-HA. We observed Hsp90. To test this idea, we co-transfected HEK293 cells with FLAG-IKKβ Hsp90-HA and NleH-HA. β that IKKWe observedinteracted that with IKK Hsp90,β interacted NleH1 with and Hsp90, NleH2 NleH1 (Figure and2B) NleH2 and that(Figure Hsp90 2B) interactedand that Hsp90 with both NleH1interacted and NleH2 with (Figure both NleH12C). and NleH2 (Figure 2C). FigureFigure 2. 2.Interaction