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The Salmonella transmembrane effector SteD hijacks AP1-mediated vesicular trafficking for delivery to antigen-loading MHCII compartments [1]

['Camilla Godlee', 'Mrc Centre For Molecular Bacteriology', 'Infection', 'Imperial College London', 'London', 'United Kingdom', 'Ondrej Cerny', 'Mei Liu', 'Samkeliso Blundell', 'Alanna E. Gallagher']

Date: 2022-07

SteD is a transmembrane effector of the Salmonella SPI-2 type III secretion system that inhibits T cell activation by reducing the amounts of at least three proteins –major histocompatibility complex II (MHCII), CD86 and CD97 –from the surface of antigen-presenting cells. SteD specifically localises at the trans-Golgi network (TGN) and MHCII compartments; however, the targeting, membrane integration and trafficking of SteD are not understood. Using systematic mutagenesis, we identify distinct regions of SteD that are required for these processes. We show that SteD integrates into membranes of the ER/Golgi through a two-step mechanism of membrane recruitment from the cytoplasm followed by integration. SteD then migrates to and accumulates within the TGN. From here it hijacks the host adaptor protein (AP)1-mediated trafficking pathway from the TGN to MHCII compartments. AP1 binding and post-TGN trafficking require a short sequence in the N-terminal cytoplasmic tail of SteD that resembles the AP1-interacting dileucine sorting signal, but in inverted orientation, suggesting convergent evolution.

Salmonella enterica is an intracellular pathogen that causes a range of diseases from gastroenteritis to systemic typhoid fever. Its pathogenesis relies on virulence proteins known as effectors that are delivered into host cells and modulate host cellular processes. The ability of the Salmonella effector SteD to localise within host MHCII compartment membranes is essential for its function in disrupting the adaptive immune response. Here we show that SteD integrates into membranes of the early secretory pathway through a two-step recruitment and integration mechanism. SteD then behaves like a transmembrane cargo protein and hijacks a post-Golgi vesicular trafficking pathway to reach MHCII compartments. This study highlights the sophistication by which bacterial pathogens interact with host cell biology at the molecular level.

Despite some understanding of the protein-protein interactions required for SteD function, it remains unclear how SteD integrates into membranes, what is required for its localisation, and whether its localisation at the TGN is important for function. Through mutagenesis, we have found three different regions of SteD that are required for its localization, integration, and vesicular trafficking. Our results suggest that following translocation, a cytoplasmic intermediate of SteD is recruited to the ER or Golgi, where it undergoes membrane integration before transport to the TGN. Through interaction with the TGN-associated adaptor protein (AP)1 complex SteD then co-opts a post-TGN vesicular trafficking pathway to MHCII compartments, where it carries out its function.

SteD is 111 amino acids in length, has two transmembrane domains and integrates into host cell membranes such that both the N and C termini are exposed to the cytoplasm, separated by a luminal loop. Interaction with the Salmonella chaperone SrcA is required for SteD solubility in the Salmonella cytoplasm and efficient translocation [ 7 ]. Following bacterial translocation or exogenous expression in host cells, the majority of SteD is at the trans-Golgi network (TGN) [ 4 ]. It also localises to endosomal compartments including MHCII compartments, where mMHCII and CD97 are also found [ 4 , 5 ].

Following uptake into a host cell, Salmonella resides within a membrane-bound compartment known as the Salmonella-containing vacuole (SCV), from which it delivers effectors of the Salmonella pathogenicity Island (SPI)-2 T3SS through the vacuolar membrane. One of these, SteD, reduces mature antigen-loaded major histocompatibility complex (mMHCII) and CD86 from the surface of infected antigen-presenting cells, resulting in a reduction in T cell activation [ 4 ]. It also reduces CD97 cell surface levels, which destablises immunological synapses formed between dendritic cells and T cells [ 5 ]. It thus has an inhibitory effect on the adaptive immune response to Salmonella. mMHCII and CD97 interact with SteD and are ubiquitinated by the NEDD4 family HECT E3 ubiquitin ligase WWP2, generating predominantly K63 linkages and resulting in their lysosomal degradation [ 5 , 6 ]. SteD is also ubiquitinated by WWP2 in a way that augments its activity yet results in its lysosomal degradation [ 6 ]. The mechanism underlying this activity involves an intramembrane interaction between SteD and the transmembrane protein TMEM127, which acts as an adaptor for WWP2 [ 6 ].

The virulence of many bacterial pathogens relies on the delivery of effector proteins into host cells through secretion systems such as the type three secretion system (T3SS). These effectors manipulate immune responses and promote bacterial replication. Many effectors require a specific host cellular localisation for their function [ 1 , 2 ]. A subset of bacterial effectors from diverse pathogens, localise by integrating into specific membranes of host cells. These include Salmonella SteD, SseF and SseG, E. coli Tir and Chlamydia Incs. These transmembrane effectors are often crucial to pathogenesis; however, their targeting, membrane integration and trafficking are poorly understood. It has been proposed that they could integrate into host membranes by either lateral transfer during translocation through the T3SS pore or direct integration following translocation into the cytoplasm [ 3 ].

Amino acids L13 and L14 of SteD, followed by P, P, S and then by two charged residues (E18, R19), resemble an inverted dileucine motif ( Fig 7A ). Alanine substitution of the leucines alone (SteD L13A,L14A -HA) had no noticeable effect on localisation of SteD after translocation from Salmonella ( S7H Fig ). However, further alanine substitution of the two charged residues (E18 and R19) (SteD L13A,L14A,E18A,R19A -HA) significantly increased the proportion of HA signal at the cell surface ( Fig 7B and 7C ) and reduced the level of colocalisation with mMHCII ( Fig 7B and 7D ) when compared to wt SteD-HA. An increase in cell surface signal and decrease in colocalisation with mMHCII was also detected for ectopically expressed GFP-SteD L13A,L14A,E18A,R19A compared to wt GFP-SteD ( S7C–S7E Fig ). Therefore, this quadruple substitution mutation recapitulated the mis-localisation phenotype of the N-terminal truncation of GFP-SteD 37-111 . Furthermore, mMHCII surface levels were significantly higher after translocation of SteD L13A,L14A,E18A,R19A -HA compared to wt SteD-HA ( Fig 7E ). Mis-localisation of SteD L13A,L14A,E18A,R19A did not prevent interaction with TMEM127 ( S7F and S7G Fig ). This suggests that SteD interacts with TMEM127 at the ER, Golgi or TGN through which TMEM127 passes [ 17 ], and is consistent with other work showing that SteD can interact with TMEM127 in the absence of mMHCII [ 6 ]. Finally, interaction between SteD L13A,L14A,E18A,R19A and AP1 was reduced significantly after translocation from Salmonella ( Fig 7F and 7G ) and ectopic expression ( S7F and S7G Fig ). Therefore, these residues are required for interaction with AP1, resulting in trafficking of SteD from the TGN to MHCII compartments, which is required for SteD function.

(A) Amino acid sequence of SteD showing predicted transmembrane domains. The N- and C-terminal residues are highlighted in blue and red respectively. The residues substituted to alanines in SteD L13A,L14A,E18A,R19A are highlighted in green. (B) Representative confocal immunofluorescence microscopy images of Mel Juso cells infected with ΔsteD Salmonella strains carrying a plasmid expressing SteD-HA (wt or mutant). Cells were fixed and processed for immunofluorescence microscopy by labelling for HA (green), MHCII compartments (mMHCII, red), and DNA (DAPI, blue). Arrowheads indicate MHCII compartments. Scale bar– 10 μm. (C) Quantification of HA signal at the surface of cells represented in Fig 7B. The fluorescence intensity of the HA signal at the cell surface was measured in relation to total cellular fluorescence. Data are representative of three independent experiments. Each dot represents the value for one cell. Mean ± SD. The log 10 fold change of the data were analysed by t-test, *** p<0.001. (D) Mander’s overlap coefficient of the fraction of SteD-HA positive pixels that colocalise with mMHCII positive pixels from cells represented in Fig 7B. Data are representative of three independent experiments. Each dot represents the value for one cell. Mean ± SD. Data were analysed by t-test, *** p<0.001. (E) mMHCII surface of Mel Juso cells infected with ΔsteD Salmonella carrying a plasmid expressing SteD-HA (wt or mutant). Cells were analysed by flow cytometry and amounts of surface mMHCII in infected cells are expressed as a percentage of uninfected cells in the same sample. Mean of three independent experiments done in duplicate ± SD. Data were analysed by paired t-test, * p<0.05. (F) Protein immunoblots of whole-cell lysates (Input) and immunoprecipitation with HA beads (HA IP) from Mel Juso cells infected with ΔsteD Salmonella strains carrying a plasmid expressing SteD-HA (wt or mutant) or SseF-HA following crosslinking with DSP. Mutation of charged residues might explain the difference in migration through the SDS gel. AP1 –antibody specific for the γ subunit, AP2 –antibody specific for the α subunit, AP3 –antibody specific for the δ subunit. (G) Levels of immunoprecipitated AP1 were calculated by densitometry from immunoblots as represented in Fig 7F. Protein levels were normalised to wt SteD-HA. Mean of three independent experiments ± SD. The data were analysed by one sample t-test, *** p<0.001, n.s.–not significant.

There are two well-characterised sorting signals that interact with AP complexes: the tyrosine motif, YXXΦ, where Φ is a large hydrophobic residue, and the dileucine motif, [DE]XXXL[LI], with one or more acidic residues upstream from two leucines (X indicates any residue in both motifs) [ 16 ]. No series of residues in SteD match the consensus of either motif, however sequences resembling both motifs are present in inverted orientations in the N terminal tail. F32, N33, A34 and Y35 resemble an inverted tyrosine motif, and are within a region necessary for SteD function, as determined by alanine scanning mutagenesis (Region 7, S1A Fig ) [ 4 ]. A double alanine substitution of F32 and Y35 (SteD F32A,Y35A ) was sufficient to inhibit the effect of SteD on mMHCII surface levels to the same level as SteD ala7 following ectopic expression or translocation from Salmonella ( S7A and S7B Fig ). Alanine substitution of the remaining residues within Region 7, N33 and G36, had no effect on SteD function ( S7A Fig ). However, the F32A, Y35A double mutation had no noticeable effect on the localisation of SteD ( S7C–S7E Fig ) and did not prevent interaction of SteD with AP1 when expressed ectopically ( S7F and S7G Fig ). This rules out the involvement of these residues in AP1-dependent transport and suggests that they contribute to SteD function in other ways.

To test whether the PM localisation was due to traffic from the TGN, we used the photoconvertible fluorescent protein mEos, which converts from green to red fluorescence upon activation with UV light, to specifically activate SteD at the Golgi and track its subsequent fate. As expected, vesicles containing mEos-SteD travelled from the Golgi throughout the cell cytoplasm ( S3 Video and S6C Fig ). In contrast, vesicles containing the N-terminal truncation of SteD trafficked to the periphery of the cell, where the fluorescent signal dissipated ( S3 Video and S6C Fig ), presumably as a result of vesicle fusion and lateral dilution of the fluorescent signal in the PM. These results suggest the presence of a sorting signal in the N-terminal tail of SteD that directs traffic from the TGN to MHCII compartments and whose absence results in mis-trafficking to the PM.

(A) Schematics of SteD showing predicted transmembrane domains and extent of truncation mutations as indicated. (B) Protein immunoblots of membrane fractionation samples from Mel Juso cells expressing GFP-SteD (wt or mutants). Samples were taken from the pellet (P) and supernatant (S) of the total sample, after urea wash and after RIPA wash. (C) Representative confocal immunofluorescence microscopy images of Mel JuSo cells expressing GFP-SteD (wt or mutants). Cells were fixed and processed for immunofluorescence microscopy by labelling for MHCII compartments (mMHCII, red), the TGN (TGN46, grey), and DNA (DAPI, blue). Arrowheads indicate MHCII compartments. Scale bar– 10 μm. (D) Quantification of GFP at the surface of cells represented in Fig 6C. The fluorescence intensity of the GFP signal at the surface of cells was measured in relation to total cellular fluorescence. Data are representative of three independent experiments. Each dot represents the value for one cell. Mean ± SD. The log 10 fold change of the data were analysed by one-way ANOVA followed by Dunnett’s multiple comparison test, *** p<0.001, n.s.–not significant.

We next tested whether there is information in the N- or C-terminal cytoplasmic tails of SteD that is required for its AP1-mediated trafficking. Truncation mutants of GFP-SteD lacking the C-terminal tail or most of the N-terminal tail (Figs 6A and S6A ) underwent membrane integration as assessed by biochemical fractionation ( Fig 6B ). Truncation of the C-terminal tail (GFP-SteD 1-102 ) did not affect localisation at the TGN and MHCII compartments (Figs 6C and S6B ). On the other hand, an SteD truncation lacking most of the N-terminal cytoplasmic tail (GFP-SteD 37-111 ) had reduced localisation at MHCII compartments ( Fig 6C ). Furthermore, and in contrast to AP1 depletion, truncation of the N-terminal tail resulted in a dramatic increase in the proportion of fluorescence signal at the cell surface and a decrease in fluorescence signal at the TGN (Figs 6C and 6D and S6B ). The continuous distribution of fluorescence signal at the cell surface along with the ability to integrate into the membrane implies a PM localisation. Remarkably, a construct lacking most of the N-terminal tail and the C-terminal tail (GFP-SteD 37-102 ), leaving just 65 residues comprising the two transmembrane domains separated by the luminal loop and containing Regions 9 and 13, also integrated efficiently into host cell membranes ( Fig 6A and 6B ) and along with GFP-SteD 37-111 , localised at the TGN as well as the PM ( Fig 6C and 6D ).

AP1 regulates the trafficking of protein cargo between the TGN and endosomes [ 15 ]. To test whether AP1 is involved in trafficking of SteD to MHCII compartments we used siRNA to knock down the β subunit of AP1 in Mel Juso cells expressing GFP-tagged SteD ( Fig 5C ). AP1 knockdown had no noticeable effect on cellular mMHCII signal but caused a significant reduction in colocalisation between GFP-SteD and MHCII compartments when compared to mock-treated cells or knockdown of AP2 and AP3 ( Fig 5D and 5E ). Despite this reduced colocalisation, knockdown of AP1 had no detectable effect on SteD-dependent reduction of mMHCII surface levels ( S5A Fig ). While there was no detectable increase in GFP-SteD at the PM ( S5B Fig ) we conclude that under these conditions a small proportion of SteD is still able to interact with MHCII either intracellularly or at the PM.

(A) Protein immunoblots of whole-cell lysates (Input) and immunoprecipitation with GFP-trap beads (GFP IP) from Mel Juso cells expressing GFP-SteD or GFP-SseG following crosslinking with DSP. AP1 –antibody specific for the γ subunit, AP2 –antibody specific for the α subunit, AP3 –antibody specific for the δ subunit. (B) Levels of immunoprecipitated AP1 were calculated by densitometry from immunoblots as represented in Fig 5A. Protein levels were normalised to GFP-SteD. Mean of three independent experiments ± SD. The data were analysed by one sample t-test, ** p<0.01. (C) Protein immunoblots of Mel JuSo cells treated with scrambled siRNA (SCR) or siRNA specific to the β subunit of AP1, the μ subunit of AP2 or the δ subunit of AP3. AP1 –antibody specific for the β subunit, AP2 –antibody specific for the α subunit AP3 –antibody specific for the δ subunit. (D) Representative confocal immunofluorescence microscopy images of Mel JuSo cells expressing GFP-SteD after treatment with scrambled siRNA (SCR) or siRNA specific to the β subunit of AP1. Cells were fixed and processed for immunofluorescence microscopy by labelling for MHCII compartments (mMHCII, red), and DNA (DAPI, blue). Arrowheads indicate MHCII compartments. Scale bar– 10 μm. (E) Mander’s overlap coefficient of the fraction of GFP-SteD positive pixels that colocalise with mMHCII positive pixels from cells after treatment with siRNA as in Fig 5C and 5D. Data are representative of three independent experiments. Each dot represents the value for one cell. Mean ± SD. Data were analysed by one-way ANOVA followed by Dunnett’s multiple comparison test, *** p<0.001, n.s.–not significant.

The TGN acts as a sorting platform for the anterograde traffic of protein cargo from the Golgi. Transmembrane cargo proteins frequently use short linear motifs known as sorting signals in their cytoplasmic tails. Sorting signals are recognised by cytoplasmic adaptor proteins, including heterotetrameric AP complexes, allowing concentration of cargo into vesicles targeted to specific membrane compartments [ 12 ]. In the absence of a sorting signal, proteins traffic constitutively from the TGN to the PM [ 13 ]. To determine if SteD interacts with an AP complex we used coimmunoprecipitation experiments after chemical crosslinking with dithiobis(succinimidyl propionate) (DSP), which has been shown to be effective for detecting transient interactions between transmembrane proteins and cytoplasmic interaction partners [ 14 ]. Ectopically expressed GFP-SteD was immunoprecipitated from Mel Juso cell lysates using GFP-trap beads and proteins were subjected to immunoblotting with antibodies against specific subunits of the AP1, 2 or 3 complexes. GFP-tagged SseG was used as a negative control. A small amount of AP1 interacted specifically and reproducibly with GFP-SteD ( Fig 5A and 5B ) but not GFP-SseG.

Therefore, rather than integrating into the TGN directly, SteD either interacts with an ER or Golgi cisternae component and integrates into these membranes before accumulating at the TGN. SteD is then trafficked to MHCII compartments, and this is required for its function.

In the presence of BFA, SteD did not cause a significant reduction in mMHCII surface levels (Figs 4D and S4B ) and this correlated with a failure to colocalise with mMHCII ( S4A Fig ). This demonstrates that post-Golgi trafficking is required for SteD to reach MHCII compartments and for SteD function. Indeed, after bleaching the fluorescent signal from GFP-SteD outside of the Golgi area we detected vesicles containing GFP-SteD budding from the Golgi/TGN area by time-lapse microscopy. These vesicles trafficked throughout the cell with some apparently fusing to and budding from other SteD-containing compartments ( S2 Video and S4C Fig ). Since there is a large amount of colocalisation between GFP-SteD and mMHCII in the periphery of the cell [ 4 ], a substantial proportion of these are likely to be MHCII compartments. The stable association of SteD ala13 with the TGN ( Fig 2A and S1 Video ) indicates that post-TGN trafficking of SteD requires membrane integration.

(A) Representative confocal immunofluorescence microscopy images of Mel JuSo cells expressing GFP-SteD (wt or mutant) under a doxycycline-regulated promoter and the ER marker mCherry-ER-3 (red). Cells were either treated with doxycycline plus MG132 (dox) or treated with BFA followed by doxycycline, MG132 and BFA (BFA-dox). Cells were then fixed and processed for immunofluorescence microscopy by labelling for the TGN (TGN46, grey), and DNA (DAPI, blue). Scale bar– 10 μm. (B) Quantification of cells represented in Fig 4A. Pearson’s correlation coefficients for colocalization between GFP-SteD and mCherry-ER-3 or TGN46. Data are representative of three independent experiments. Each dot represents the value for one cell. Mean ± SD. Data were analysed by paired t-test *** p<0.001, ** p<0.01. (C) Protein immunoblots of membrane fractionation samples from Mel Juso cells expressing GFP-SteD (wt or mutant) under a doxycycline-regulated promoter and treated as in Fig 4A. Samples were taken from the pellet (P) and supernatant (S) of the total sample, after urea wash and after RIPA wash. (D) mMHCII surface levels of Mel JuSo cells expressing GFP-SteD under a doxycycline-regulated promoter or GFP and treated as in Fig 4A. Cells were analysed by flow cytometry and amounts of surface mMHCII in GFP-positive cells are expressed as a percentage of GFP-negative cells in the same sample. Mean of three independent experiments done in duplicate ± SD. Data were analysed by one-way ANOVA followed by Dunnett’s multiple comparison test, ** p<0.01, n.s.–not significant.

We investigated whether the membrane interaction and integration steps occur at the TGN or if SteD accumulates there after integrating elsewhere in the secretory pathway. To do this we used a doxycycline-regulated promoter to derepress expression of SteD from a mammalian expression plasmid after blocking Golgi trafficking with brefeldin A (BFA). BFA causes the collapse of Golgi membranes into the ER and fusion of TGN membranes with endosomes, thereby separating the early from the late secretory pathway [ 10 , 11 ]. When SteD expression was induced in the absence of BFA (dox), it integrated into membranes at both the TGN and MHCII compartments, as expected (Figs 4A–4C and S4A ). When expressed after Golgi disruption (BFA-dox) SteD still underwent membrane integration ( Fig 4C ) but localised predominantly at the ER and no longer colocalised with TGN46 ( Fig 4A and 4B ). GFP-SteD ala13 also localised at the ER when expressed after BFA treatment in the presence of MG132 ( Fig 4A and 4B , BFA-dox), demonstrating that the SteD membrane interaction partner re-distributes to the ER after BFA treatment. Therefore, both membrane interaction and integration occur at the ER or Golgi, and not at the TGN.

Collectively, these results show that following translocation from bacteria, SteD integrates into host cell membranes with a two-step mechanism. We propose that SteD is first recruited from the cytoplasm by residues L42 and M43 of Region 9, presumably by interaction with a membrane component(s) and this is followed by integration mediated by residues S68 and G69 of Region 13.

(A) Amino acid sequence of SteD showing predicted transmembrane domains. The N- and C-terminal residues are highlighted in blue and red respectively. The residues substituted to alanines in SteD L42A,M43A and SteD S68A,G69A mutants are highlighted in yellow and orange respectively. (B) mMHCII surface levels of Mel JuSo cells infected with ΔsteD Salmonella carrying a plasmid expressing SteD-HA (wt or mutants) or SPI-2 null ΔssaV Salmonella and treated with MG132. Cells were analysed by flow cytometry and amounts of surface mMHCII in HA-positive cells are expressed as a percentage of HA-negative cells in the same sample. Mean of three independent experiments done in duplicate ± SD. Data were analysed by one-way ANOVA followed by Dunnett’s multiple comparison test, ** p<0.01, n.s.–not significant. (C) Protein immunoblots of membrane fractionation samples from Mel Juso cells infected with ΔsteD Salmonella strains carrying a plasmid expressing SteD-HA (wt or mutants) and treated with MG132. Samples were taken from the pellet (P) and supernatant (S) of the total sample, after urea wash and after RIPA wash. (D) Representative confocal immunofluorescence microscopy images of Mel Juso cells infected with ΔsteD Salmonella strains carrying a plasmid expressing SteD-HA (wt or mutants) and treated with MG132. Cells were fixed and processed for immunofluorescence microscopy by labelling for HA (green), the TGN (TGN46, red), and DNA (DAPI, blue). Arrowheads indicate cellular aggregates. Scale bar– 10 μm. (E) Quantification of HA signal at the TGN of cells represented in Fig 3D . The fluorescence intensity of the HA signal at the TGN was measured in relation to total cellular fluorescence. Data are representative of three independent experiments. Each dot represents the value for one cell. Mean ± SD. The log 10 fold change of the data were analysed by one-way ANOVA followed by Dunnett’s multiple comparison test, *** p<0.001.

We next examined the process of membrane integration after translocation of SteD by the SPI-2 T3SS. However, SteD ala9 and SteD ala13 were not stably expressed or translocated by Salmonella ( S3A Fig ). Therefore, we carried out more specific mutagenesis to identify the residues within Regions 9 and 13, whose loss accounts for the properties of SteD ala9 and SteD ala13 . To do this we first tested the ability of GFP-tagged single and double residue alanine substitutions to reduce mMHCII surface levels. All the single residue substitutions were still functional ( S3B Fig ). The double substitution mutants with the strongest functional impairment (SteD L42A,M43A and SteD S68A,G69A , Figs 3A and S3C ) were tested for expression in Salmonella. Both mutants were translocated into Mel Juso cells and the protein levels were rescued by MG132, demonstrating that they both underwent proteasomal degradation ( S3D Fig ). Mel Juso cells with similar levels of SteD L42A,M43A -HA and SteD S68A,G69A -HA as wt SteD-HA had no reduction in mMHCII surface levels (Figs 3B and S3E ). Biochemical fractionation revealed that both SteD L42A,M43A -HA and SteD S68A,G69A -HA were not solubilised by RIPA indicating that they failed to integrate into host cell membranes ( Fig 3C ). Furthermore, SteD L42A,M43A -HA was found in large puncta throughout the host cell cytoplasm, while SteD S68A,G69A -HA colocalised with TGN46 ( Fig 3D and 3E ). Therefore, following translocation, these mutants recapitulated the non-integration and localisation properties of GFP-SteD ala9 and GFP-SteD ala13 respectively, indicating that translocated SteD uses the same integration mechanism as ectopically expressed GFP-SteD.

To test whether Region 13 promotes membrane integration of other transmembrane sequences, we created a chimeric protein in which the transmembrane domains of SteD were replaced by those of another Salmonella integral membrane effector–SseG, which has a similar membrane topology to SteD. This construct (SteD TMSseG , Fig 2C ) therefore contained SteD Region 13 but lacked Region 9. Fractionation experiments demonstrated that SteD TMSseG underwent membrane integration ( Fig 2D ), and this was dependent on Region 13, as demonstrated by the lack of RIPA solubility following its alanine substitution (SteD TMSseGala13 , Fig 2D ). In contrast to SteD, SseG localised at membranes throughout the cytoplasm ( Fig 2E ). Both GFP-SteD TMSseG and GFP-SteD TMSseGala13 had a similar localisation to mCherry-SseG ( Fig 2E ). This agrees with previous work showing that the transmembrane domains of SseG direct its localisation in the host cell [ 9 ] and demonstrates that this localisation is also independent from membrane integration. Therefore, Region 13 is sufficient to mediate integration of alternative transmembrane domains following targeting to a different membrane compartment.

(A) Representative confocal immunofluorescence microscopy images of Mel JuSo cells expressing GFP-SteD (wt or mutants) after MG132 treatment. Cells were fixed and processed for immunofluorescence microscopy by labelling for the TGN (TGN46, red), and DNA (DAPI, blue). Scale bar– 10 μm. (B) Quantification of GFP at the TGN of cells represented in Fig 2A . The fluorescence intensity of the GFP signal at the TGN was measured in relation to total cellular fluorescence. Data are representative of three independent experiments. Each dot represents the value for one cell. Mean ± SD. The log 10 fold change of the data were analysed by one-way ANOVA followed by Dunnett’s multiple comparison test, *** p<0.001, * p<0.05. (C) Schematic of SteD chimera with transmembrane domains of SseG. (D) Protein immunoblots of membrane fractionation samples from Mel Juso cells expressing GFP-SteD (wt or chimeric mutants) and treated with MG132. Samples were taken from the pellet (P) and supernatant (S) of the total sample, after urea wash and after RIPA wash. (E) Representative confocal immunofluorescence microscopy images of Mel JuSo cells expressing GFP-SteD (wt or chimeric mutants) and mCherry-SseG after MG132 treatment. Cells were fixed and processed for immunofluorescence microscopy by labelling for the TGN (TGN46, grey), and DNA (DAPI, blue). Scale bar– 10 μm.

To determine how these mutations affected SteD localisation in the host cell, we examined GFP-SteD ala9 and GFP-SteD ala13 in the presence of MG132 by fluorescence microscopy. Whereas a large proportion of wt GFP-SteD colocalised with the TGN marker TGN46, GFP-SteD ala9 formed cytoplasmic punctate structures that colocalised with ubiquitin (Figs 2A and S2A ). This, along with its resistance to detergent extraction ( Fig 1D ) indicates that when proteasome activity is inhibited, GFP-SteD ala9 forms cytoplasmic aggregates that resemble aggresomes or inclusion bodies [ 8 ]. In contrast, SteD ala13 colocalised partially with ubiquitin ( S2A Fig ) and predominantly with TGN46, although to a lesser extent than wt GFP-SteD ( Fig 2A and 2B ). Time-lapse microscopy revealed that wt GFP-SteD was present in motile vesicles, whereas GFP-SteD ala13 remained stably associated with the Golgi area ( S1 Video ). Therefore, in the presence of MG132, SteD ala13 remained in a Golgi-associated non-integrating state, which then formed insoluble aggregates after cell lysis ( Fig 1D ). These results show that SteD localisation at the Golgi can be uncoupled from membrane integration, indicating that Region 9 mediates interaction with a membrane component, while Region 13 is required for integration following Golgi association.

To test whether GFP-SteD ala9 or GFP-SteD ala13 integrate into mammalian cell membranes we subjected transfected cells to biochemical fractionation. Cell lysates were pelleted by ultracentrifugation to distinguish cytoplasmic proteins (including actin) from membrane-associated and integral membrane proteins, and then pellets were solubilised with either urea (to extract peripheral membrane proteins, including Golgin-97) or RIPA buffer (to extract integral membrane proteins, including the DRα chain of MHCII). As expected, wt GFP-SteD, along with DRα, was present in the pellet after the initial centrifugation and the urea wash, but was solubilised by RIPA, confirming its integration into host membranes ( Fig 1D ). However, both GFP-SteD ala9 and GFP-SteD ala13 were resistant to solubilisation with RIPA ( Fig 1D ). This indicates that neither mutant underwent membrane integration, but instead formed insoluble aggregates. Therefore, amino acids within the mutated regions of SteD ala9 (hereafter referred to as Region 9) and SteD ala13 (hereafter referred to as Region 13) are required for membrane integration and functionality of SteD.

Next, we analysed whether these regions are important for SteD function by measuring cell surface levels of mMHCII by flow cytometry after expression of both mutants by transfection into Mel Juso cells in the presence of MG132. As expected, wild-type (wt) GFP-SteD decreased surface levels of mMHCII compared to untransfected cells (Figs 1C and S1B ). For cells expressing GFP-SteD the presence of MG132 reduced but did not prevent the decrease in surface mMHCII ( S1C Fig ). However, in cells containing similar levels of either GFP-SteD ala9 , GFP-SteD ala13 or GFP alone, there was no significant reduction in mMHCII surface levels (Figs 1C and S1B ), demonstrating that the mutated regions are required for the function of SteD.

(A) Amino acid sequence of SteD showing predicted transmembrane domains. The N- and C-terminal residues are highlighted in blue and red respectively. The residues substituted to alanines in SteD ala9 and SteD ala13 mutants are highlighted in yellow and orange respectively. (B) Protein immunoblots of whole-cell lysates derived from Mel JuSo cells expressing GFP-SteD (wt or mutants) and treated with MG132 or DMSO carrier. UB–ubiquitin. (C) mMHCII surface levels of Mel JuSo cells expressing GFP or GFP-SteD (wt or mutants) and treated with MG132. Cells were analysed by flow cytometry and amounts of surface mMHCII in GFP-positive cells are expressed as a percentage of GFP-negative cells in the same sample. Mean of three independent experiments done in duplicate ± SD. Data were analysed by one-way ANOVA followed by Dunnett’s multiple comparison test, *** p<0.001, n.s.–not significant. (D) Protein immunoblots of membrane fractionation samples from Mel Juso cells expressing GFP-SteD (wt or mutants) and treated with MG132. Samples were taken from the pellet (P) and supernatant (S) of the total sample, after urea wash and after RIPA wash.

Ectopic expression of GFP-SteD in antigen-presenting cells recapitulates the bacterially translocated protein with respect to subcellular localisation, membrane integration and reduction of mMHCII cell surface levels [ 4 ]. This shows that these processes do not require any other Salmonella factor and that SteD function is not affected by the GFP tag. Therefore, we investigated the requirements for membrane integration and localisation of SteD using this system. In previous work from our group, alanine scanning mutagenesis of sequential blocks of 5–7 amino acids resulted in 20 different mutants ( S1A Fig ), 18 of which localised correctly at the TGN [ 4 ]. The other two mutants (SteD ala9 and SteD ala13 ) were not detectable [ 4 ]. In SteD ala9 , the substituted residues (LMCLG) are in the N-terminal transmembrane domain ( Fig 1A ). In SteD ala13 , the substituted residues (SVSSG) are in the luminal loop ( Fig 1A ). In the presence of MG132 (an inhibitor of proteasome degradation), both mutants were detected by immunoblot ( Fig 1B ), indicating that mutation of either region results in protein degradation.

Discussion

Many bacterial type III secretion system effectors are specifically targeted to organelles and membranes within the infected cell and interference with localisation processes frequently results in their loss of function [18–21]. The targeting mechanisms of transmembrane effectors, which post-translationally integrate into specific host cell membranes, are not well understood. The majority of translocated SteD accumulates at the TGN but its substrates including mature MHCII are located in endosomal compartments and at the PM, raising the question of how SteD reaches these sites. In this work we used site directed mutagenesis of SteD to identify distinct regions of SteD that are required for initial targeting to the ER/Golgi, membrane integration and for AP1-mediated trafficking to MHCII compartments (Fig 8). This demonstrates how a bacterial virulence protein can enter the membrane network of the eukaryotic cell and hijack a vesicular trafficking pathway to regulate its localisation and ultimately its function.

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TIFF original image Download: Fig 8. Model of SteD membrane integration and localisation. Following translocation from Salmonella (green) into the cytoplasm SteD (orange) is recruited to the membranes of the early secretory pathway where it integrates. SteD then migrates to and accumulates within the TGN. Through interaction with the AP1 complex (pink) it is trafficked to MHCII compartments, where it interacts with mMHCII (dark green), which is ubiquitinated through the actions of TMEM127 (blue) and WWP2 (red) causing a reduction in mMHCII surface levels. SteD L42A,M43A mutation prevents membrane recruitment leading to aggregation in the cytoplasm. SteD S68A,G69A mutation prevents membrane integration resulting in a Golgi-associated non-integrative state. SteD L13A,L14A,E18A,R19A mutation prevents AP1 interaction leading to mis-trafficking of SteD to the plasma membrane. https://doi.org/10.1371/journal.ppat.1010252.g008

Apart from SteD, a few other effectors have been reported to undergo integration into membranes of the secretory pathway. Tir, a conserved T3SS effector of pathogenic E. coli with a similar membrane topology to SteD, localises to the PM where it induces actin pedestals and enables tight attachment of extracellular bacteria by binding to the bacterial surface protein, Intimin [22]. Enterohemorrhagic E. coli (EHEC) Tir was also shown to localise at the Golgi network by immunofluorescence and immunoelectron microscopy [23]. Exposure of host cells to BFA prior to infection with EHEC prevented pedestal formation, suggesting that Tir, like SteD must first pass through the Golgi before reaching its site of action at the PM [23]. NleA/EspI, another EHEC effector, contains two putative transmembrane domains and was shown by triton-dependent solubilisation following fractionation of infected host cells to integrate into membranes [24]. NleA/EspI localises to the Golgi through interaction with the COPII component, Sec24 [25]. This interaction stabilises COPII at the Golgi leading to a reduction in general protein secretion [26]. Interestingly, overexpressed NleA interferes with MHCII invariant chain transport and it might thereby also affect antigen presentation [27].

Transmembrane effectors have been suggested to integrate into their target membranes either indirectly, by lateral transfer during translocation into the membrane containing the T3SS translocon, followed by membrane fission and vesicular trafficking to another destination, or directly, following translocation of the effector into the cytoplasm [3]. In the case of SteD, indirect targeting could result from vesicles containing SteD trafficking from the SCV to the Golgi. However, direct integration is more likely for three reasons. First, ectopically expressed GFP-SteD mimics the bacterial translocated effector with respect to Golgi localisation, membrane integration and action on mMHCII [4], (and this work), showing that an SCV membrane and translocon are not required for these processes. Second, Golgi localisation is independent from membrane integration, as demonstrated by SteD mutants incapable of integration, showing that this localisation does not require vesicular trafficking. Third, mutants incapable of either membrane recruitment or integration accumulated within the host cell cytoplasm following translocation.

We propose that SteD is recruited to its target membrane from the cytoplasm through interaction of Region 9 with a protein or lipid, or a combination of components at the cytoplasmic face of the ER and/or the Golgi cisternae. Identification of the host interaction partner(s) and its involvement in membrane integration is needed to better define the process of SteD integration. Following recruitment, SteD undergoes membrane integration by a mechanism involving Region 13, and specifically S68 and G69. The ability of Region 13 to mediate integration of the transmembrane regions of SseG shows that this mechanism is non-specific. As glycine residues have a high propensity to induce a turn between two transmembrane helices [28], G69 could enable the formation of a hairpin-like structure and hence facilitate the conformation required for integration upon interaction with the membrane. A two-step membrane integration process is consistent with the mechanism of integration proposed for Tir, whereby a region close to the transmembrane domain binds peripherally to the membrane, resulting in a conformational change enabling the hydrophobic domains to adopt an orientation that drives membrane insertion [29]. Insertion of purified Tir into reconstituted membrane vesicles does not require host proteins but does depend on sphingomyelin [29], which is made at the ER and Golgi and enriched at the PM [30].

It is possible that aggregation of SteD ala9 and SteD ala13 is due to misfolding and that SteD ala13 forms aggregates within the membrane. However, both mutants were sensitive to proteasomal degradation, which occurs in the cytoplasm. Membrane proteins must be retro-translocated into the cytoplasm before degradation [31], so their protein levels are unlikely to be sensitive to MG132 treatment. In addition, SteD L42A,M43A and SteD S68A,G69A , which fully recapitulated the phenotypes of the full mutants were competent for translocation across the bacterial envelope and vacuole membrane without aggregation. Therefore, the simplest explanation is that the prolonged presence of the exposed hydrophobic transmembrane domains in the host cell cytoplasm causes aggregation and triggers a quality control pathway, leading to ubiquitination of both mutants and their subsequent degradation by the proteasome [32,33].

Microscopic analysis of the non-membrane integrating SteD ala13 mutant showed that it remained confined to the Golgi region, indicating that SteD must be in its membrane-integrated state to undergo onward post-TGN transport. SteD thus resembles transmembrane cargo proteins that are recognised for vesicular traffic by adaptor proteins via sorting signals in their cytoplasmic tails, such as the dileucine motif [DE]XXXL[LI]. It is therefore interesting that the LLPPSER sequence required for transport of SteD to MHCII compartments resembles an inverted dileucine motif. This could represent an example of effector mimicry by convergent evolution. However, individual substitution of either leucine inactivates mammalian [DE]XXXL[LI] sorting signals [12], whereas the localisation of the SteD L13A,L14A -HA double mutant was similar to that of wt SteD. Furthermore, although inverted leucine-containing motifs can mediate substrate recognition in other proteins [34] it is not clear whether SteD’s LLPPSER sequence interacts directly with the AP1 binding site or whether other proteins are involved in the process. Further biochemical and structural studies are required to determine the molecular details of the SteD/AP1 interface.

Other intracellular pathogen proteins have been shown to interact with adaptor proteins through canonical dileucine motifs. The Coxiella burnetii T4SS effector CvpA interacts with AP2 via three dileucine motifs (EESKLL, RHINLL and EIQQLL) and re-routes endocytic compartments to the pathogen-containing vacuole [35]. HIV-1 proteins Nef and Vpu interact with host adaptor protein complexes via ENTSLL and ELSALV sequences respectively, resulting in the redistribution of cell surface proteins involved in cellular immunity [36,37]. By re-routing protein cargo or trafficking pathways these proteins aid pathogen replication. On the other hand, by localising within membranes of the secretory pathway and by virtue of its LLPPSER post-TGN sorting sequence, SteD resembles classical transmembrane protein cargo.

Through interaction with AP1, SteD exploits an established host cell trafficking pathway between the TGN and endosomes [15,38]. Indeed, AP1 mediates delivery of invariant chain-bound MHCII complexes from the TGN to MHCII compartments [39]. Although blocking MHCII compartment localisation by expressing SteD after Golgi disruption totally prevented SteD function, disrupting its post-TGN traffic through mutation of the LLPPSER sequence or AP1 knockdown only partially disrupted or did not affect SteD function, respectively. It is possible that a small but functionally significant amount of SteD is still able to reach MHCII compartments in these conditions. Alternatively, in the case of SteD L13A,L14A,E18A,R19A , this could be due to the partial functionality of SteD at the PM, where it might also come into contact with mMHCII. The differences between AP1 knockdown and mutation of the dileucine sequence in relation to SteD localisation and functionality could be due to incomplete AP1 knockdown or to a redundancy in interaction of wt SteD with other adaptors.

The dependence on AP1 for trafficking to MHCII compartments might well create a rate-limiting step for post TGN SteD traffic, explaining the accumulation of SteD at the TGN. This might also provide a source of SteD to replenish that which is lost by degradation as a result of ubiquitination by the TMEM127/WWP2 machinery [6].

The mutational dissection of SteD described in this and our previous work enables the following functional regions of the protein to be defined: L13, L14, E18 and R19 –an inverted dileucine motif-like sequence involved in post TGN transport to MHCII compartments (this work); K24 –undergoes ubiquitination which contributes to the ability of SteD to induce ubiquitination of mMHCII [6]; L42 and M43 –recruitment to the ER/Golgi (this work); S68 and G69 –integration into membranes (this work); transmembrane regions–intramembrane interaction with TMEM127 [6]; residues in the C-terminal tail–interaction with MHCII [4]. Taken together, this reveals a remarkable level of functional complexity within this small bacterial protein, resulting in the disruption of the adaptive immune response by reducing surface levels of at least three key proteins, CD97 [5], CD86/B7.2 [4] and mMHCII [4].

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010252

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