Specificities and Redundancies in the NEL Family of Bacterial E3 Ubiquitin Ligases of Salmonella Enterica Serovar Typhimurium

Specificities and redundancies in the NEL family of bacterial E3 ubiquitin ligases of Salmonella enterica serovar Typhimurium Andrea Bullones-Bolaños † , Paula Martı ´n-Muñoz † , Claudia Vallejo-Grijalba † , Joaquı ´n Bernal-Bayard* and Francisco Ramos-Morales* Departamento de Gene ´tica, Facultad de Biologı ´a, Universidad de Sevilla, Sevilla, Spain Salmonella enterica serovar Typhimurium expresses two type III secretion systems, T3SS1 and T3SS2, which are encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. These are essential virulent factors that secrete more than 40 effectors that are translocated into host animal cells. This study focuses on three of these effectors, SlrP, SspH1, and SspH2, which are members of the NEL family of E3 ubiquitin ligases. We compared their expression, regulation, and translocation patterns, their role in cell invasion and intracellular proliferation, their ability to interact and ubiquitinate specific host partners, and their effect on cytokine secretion. We found that transcription of the three genes encoding these effectors depends on the virulence regulator PhoP. Although the three effectors have the potential to be secreted through T3SS1 and T3SS2, the secretion of SspH1 and SspH2 is largely restricted to T3SS2 due to their expression pattern. We detected a role for these effectors in proliferation inside fibroblasts that is masked by redundancy. The generation of chimeric proteins allowed us to demonstrate that the N-terminal part of these proteins, containing the leucine-rich repeat motifs, confers specificity towards ubiquitination targets. Furthermore, the polyubiquitination patterns generated were different for each effector, with Lys48 linkages being predominant for SspH1 and SspH2. Finally, our experiments support an anti-inflammatory role for SspH1 and SspH2. KEYWORDS Salmonella enterica, type III secretion systems, E3 ubiquitin ligase, SlrP, SspH1, SspH2 Frontiers in Immunology frontiersin.org01 OPEN ACCESS EDITED BY Jiri Stulik, University of Defence, Czechia REVIEWED BY Nishanth Gopala Krishna, Hannover Medical School, Germany Susan M Bueno, Pontificia Universidad Cato ´lica de Chile, Chile *CORRESPONDENCE Joaquı ´n Bernal-Bayard [email protected] Francisco Ramos-Morales [email protected] † These authors have contributed equally to this work and share first authorship RECEIVED 27 October 2023 ACCEPTED 17 January 2024 PUBLISHED 01 February 2024 CITATION Bullones-Bolaños A, Martı ´n-Muñoz P, Vallejo-Grijalba C, Bernal-Bayard J and Ramos-Morales F (2024) Specificities and redundancies in the NEL family of bacterial E3 ubiquitin ligases of Salmonella enterica serovar Typhimurium. Front. Immunol. 15:1328707. doi: 10.3389/fimmu.2024.1328707 COPYRIGHT © 2024 Bullones-Bolaños, Martı ´n-Muñoz, Vallejo-Grijalba, Bernal-Bayard and Ramos-Morales. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 01 February 2024 DOI 10.3389/fimmu.2024.1328707 1 Introduction Salmonella enterica serovar Typhimurium is an important bacterial pathogen that is responsible for millions of gastrointestinal infections every year in humans (1). As many other Gram negative pathogens and symbionts, Salmonella relies on type III secretion systems (T3SS) for its interactions with mammalian host cells (2). These systems, also known as injectisomes, are complex molecular devices (3) that allow some bacteriatoinjectspecific proteins, known as effectors (4), directly into host cells. S. enterica possesses two distinct T3SS, T3SS1 and T3SS2, which are encoded by Salmonella pathogenicity island 1 (SPI1) and Salmonella pathogenicity island 2 (SPI2), respectively. T3SS1 is involved in host cell invasion and in initial colonization of the intestine (5). Some of the effectors secreted through this system are responsible for cytoskeletal rearrangements and the formation of membrane ruffles, which facilitate the entry of bacteria into host cells. T3SS2 is activated when Salmonella is inside the host cell and plays a crucial role in intracellular survival and replication of bacteria. The effectors of this system are involved in the generation of a specialized intracellular compartment called the Salmonellacontaining vacuole (SCV), which is important for the evasion of host immune defenses (6). Ubiquitination is a post-translational modification of proteins that involves the covalent attachment of one or more ubiquitins, small proteins consisting of 76 amino acids. It plays a pivotal role in the regulation of cellular processes such as protein degradation, signal transduction, and immune responses (7). Ubiquitin is bound to a protein in a sequential manner involving a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3), which is essential for the selection of the specific target (8). Ubiquitin can be attached to target proteins as single ubiquitin molecules or as polyubiquitin chains. These chains can have different linkage types, with different roles (9). For example, Lys48 (K48)-linked ubiquitin chains are generally associated with protein degradation by the proteasome (10), and K63-linked chains are often involved in signaling processes (11). Although ubiquitination is commonly associated with eukaryotic cells, bacterial pathogens have evolved a variety of virulence factors, including T3SS effectors, to manipulate the host ubiquitination system (12,13). Among them there are effectors with E3 ubiquitin ligase activity that are structurally similar to RING-type eukaryotic E3 ligases, such as NleG from enterohemorrhagic Escherichia coli (14), or HECT-type E3 ligases, such as SopA in S. enterica serovar Typhimurium (15). Interestingly, some effectors belong to a new family of E3 ligases that is not found in eukaryotic cells and is known as the NEL (for novel E3 ligase) family. Members of this family are the IpaH proteins from Shigella flexneri, as well as SlrP, SspH1, and SspH2 from S. enterica serovar Typhimurium (16). In spite of their functional relationship with T3SS1 and T3SS2, the genes encoding these three effectors are located outside of SPI1 and SPI2, in other horizontally acquired regions of the chromosome (16). SlrP is secreted through T3SS1 and T3SS2 (17,18), interacts with mammalian thioredoxin and catalyzes its ubiquitination, reducing its activity and leading to host cell death (19). This effector also binds to ERdj3, a chaperone located in the endoplasmic reticulum, and interferes with its folding activity, which can also contribute to cell death (20). A recently described ubiquitination target for SlrP is SNRPD2, a core component of the spliceosome (21). SspH1 is also secreted through both systems but localizes in the nucleus of host cells where it ubiquitinates PKN1 (22), leading to its proteasome-dependent degradation. SspH2 is specifically secreted through T3SS2 and interacts with the actin binding proteins filamin and profilin (23), the cell cycle regulator and NLR cochaperone SGT1 (24), and several other proteins (25). SspH2 and SGT1 form a trimeric complex with Nod1 leading to monoubiquitination and activation of the latter (24). Nod1 is a canonical NLR involved in the expression of pro-inflammatory chemokines such as IL-8 (26). The presence of several effectors of one family in the same bacteria, displaying the same biochemical activity, as is the case for SlrP, SspH1, and SspH2 in S. enterica serovar Typhimurium, raises the interesting question of the degree of redundancy and specificity of these effectors. Here, we address this issue through a direct comparison of the three proteins in terms of patterns of expression, regulation, and translocation into eukaryotic cells. We also analyze their role in invasion and intracellular proliferation, and their ability to interact and ubiquitinate specific targets. Finally, we compare the ability of these effectors to interfere with the immune response of host cells. 2 Materials and methods 2.1 Bacterial strains and plasmids The bacterial strains and plasmids used in this study are described in Table 1.S. enterica serovar Typhimurium strains derived from the wild-type strain 14028. Transductional crosses using the P22 HT105/1 int-201 phage (37) were used for the construction of Salmonella strains. 2.2 Bacterial culture The standard culture medium for bacteria was LB broth. For SPI1-inducing conditions, S. enterica strains were grown overnight at 37°C in LB-0.3 M NaCl medium without shaking. For SPI2- inducing conditions, the bacteria were inoculated in low magnesium minimal medium (LPM) at pH 5.8, and incubated at 37°C with shaking. LPM contained 80 mM 2-(N-morpholino) ethanesulfonic acid (pH 5.8), 5 mM KCl, 7.5 mM (NH 4 ) 2 SO 4 , 0.5mM K 2 SO 4 , 0.1% casamino acids, 38 mM glycerol, 337.5 mM K 2 HPO 4 -KH 2 PO 4 (pH 7.4), and 8 mM MgCl 2 . The solid media contained 1.5% agar. Antibiotics were used at the following concentrations in LB: kanamycin (Km), 50 mg/ml; chloramphenicol (Cm), 20 mg/ml; ampicillin (Ap), 100 mg/ml. In minimal medium antibiotics were used at these concentrations: Km, 125 mg/ml; Cm, 5 mg/ml; Ap, 50 mg/ml. Bullones-Bolaños et al. 10.3389/fimmu.2024.1328707 Frontiers in Immunology frontiersin.org02 TABLE 1 Bacterial strains and plasmids used in this study. Strain/ Plasmid Relevant characteristics Source/ Reference Escherichia coli BL21(DE3) F - ompT gal dcm lon hsdS B (r - m - ;E. coli B strain), with DE3, a lprophage carrying the T7 RNA pol gene Stratagene DH5asupE44 DlacU169 (Ø80 lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 (27) ER2566 fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11 R (mcr-73::miniTn10–TetS)2 [dcm] R(zgb-210:: Tn10–TetS) endA1 D(mcrC-mrr)114::IS10 New England Biolabs XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 Dlac-pro/F’proAB lacI q lacZDM15 Tn10 (Tet r ) (28) Salmonella enterica serovar Typhimurium a 14028 Wild type ATCC SV4676 trg:: MudJ(29) SV5030 slrP::Cm r Laboratory stock SV5136 ssaV::Cm r Laboratory stock SV5193 slrP::3xFLAG Km r (19) SV5379 prgH Laboratory stock SV5452 ssrB::Cm r (30) SV5604 prgH ssaV::Cm r Laboratory stock SV6016 slrP::mini-Tn5cyaA’(31) SV9213 phoP::Km r Laboratory stock SV9988 sspH2::Km r This work SV9998 sspH1::Km r This work SV10107 sspH1::3xFLAG Km r This work SV10108 sspH2::3xFLAG Km r This work SV10109 slrP::Cm r sspH1::Km r This work SV10110 slrP::Cm r sspH2::Km r This work SV10114 sspH1 sspH2::Km r This work SV10117 slrP sspH1 sspH2::Km r This work SV10239 sspH1::CyaA’This work SV10240 sspH2::CyaA’This work Plasmids pCS2+ Mammalian expression vector F. Romero pGAD1318 Yeast two-hybrid vector, Ap r (32) pGEX- 4T-1 GST fusion vector, Ap r GE Healthcare pIZ1628 pLEX10-SlrP (21) (Continued) TABLE 1 Continued Strain/ Plasmid Relevant characteristics Source/ Reference pIZ1673 pSIF003-R1 DlacI (33) pIZ1749 pQE30-SlrP (21) pIZ1917 pIZ1673-SlrP (1–180) Laboratory stock pIZ2149 pSB377-PslrP This work pIZ2370 pGAD1318-SNRPD2 (21) pIZ3403 pGEX-4T-2-SNRPD2 (21) pIZ3407 pLEX10-SspH1 (21) pIZ3408 pLEX10-SspH2 (21) pIZ3597 pQE80L-SspH1 (21) pIZ3598 pQE80L-SspH1 (21) pIZ3639 pSB377-PsspH2 This work pIZ3640 pIZ1673-SspH1 (1–197) This work pIZ3641 pIZ1673-SspH2 (1–203) This work pIZ3643 pLEX10-SlrP (457–765) This work pIZ3644 pLEX10-SlrP (1–456) This work pIZ3645 pLEX10-SspH1 (399–700) This work pIZ3646 pLEX10-SspH2 (486–788) This work pIZ3649 pSB377-PsspH1 This work pIZ3657 pCS2-HA-UBC This work pIZ3663 pLEX10-SlrP (1–456)/SspH1 (399–700) This work pIZ3668 pLEX10-SspH1 (1–398) This work pIZ3669 pQE80L-SlrP (1–456)/SspH2(486-788) This work pIZ3670 pQE80L-SspH1(1-398)/SlrP(457-765) This work pIZ3671 pQE80L-SspH1(1-398)/SspH2(486-788) This work pIZ3672 pLEX10-SspH2(1-485) This work pIZ3675 pLEX10-SspH1(1-398)-SlrP(457-765) This work pIZ3681 pLEX10-SspH2(1-485)/SspH1(399-700) This work pIZ3682 pCS2-HA-UBC(K48R) This work pIZ3683 pCS2-HA-UBC(K63R) This work pIZ3686 pLEX10-SspH2(1-485)/SlrP(457-765) This work pIZ3687 pQE80L-SspH2(1-485)/SlrP(457-765) This work pIZ3692 pQE80L-SlrP(1-456)/SspH1(399-700) This work pIZ3693 pQE80L-SspH2(1-485)/SspH1(399-700) This work pIZ3695 pTYB21-HA-UBC(K48R) This work pIZ3696 pTYB21-HA-UBC(K63R) This work pIZ3698 pTYB21-HA-UBC This work pIZ3703 pLEX10-SlrP(1-456)-SspH2(486-788) This work pIZ3706 pLEX10-SspH1(1-398)/SspH2(486-788) This work (Continued) Bullones-Bolaños et al. 10.3389/fimmu.2024.1328707 Frontiers in Immunology frontiersin.org03 2.3 Yeast strains, culture, and twohybrid methods The Saccharomyces cerevisiae strain used was L40 (38). The culture media for yeasts were YPD: 1% yeast extract, 2% peptone, 2% glucose; and synthetic drop-out (SD) medium: 0.15% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose; and yeast synthetic drop-out supplements (Formedium) lacking the appropriate components: tryptophan to select for the presence of derivatives of pLEX10 (34), leucine for derivatives of pGAD1318 (32), and histidine to check interactions. Solid medium contained 1.5% agar. For two-hybrid assays, strain L40 was transformed with the appropriate combinations of pLEX10 and pGAD1318 derivatives using the lithium acetate procedure (39). The transformants were selected in SD without tryptophan and leucine. Interactions were analyzed by checking the growth in SD lacking tryptophan, leucine, and histidine. 2.4 Mammalian cell culture, lysis, transfection, and analysis of cytokine secretion HeLa (human epithelial; ECACC no. 93021013), RAW264.7 (murine macrophages; ECACC no. 91062702), and NRK-49F (normal rat kidney fibroblasts; ATCC CRL-1570) cells were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were kept in a humidified atmosphere with 5% CO 2 at 37° C. For cell lysis, 2 × 10 7 to 10 8 cells per ml were incubated at 4°C in NP40 buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1% NP40, 1 mM PMSF, 1% protease inhibitor cocktail P8849 from Sigma-Aldrich) for 20 min. The extract was centrifuged at 20,000× g for 20 min and the supernatant was stored at -80°C. For transient transfection assays, 2–5×10 6 HeLa cells/assay were resuspended in 200 mL of 15 mM HEPES-buffered serum-containing medium, mixed with 50 mL of 210 mM NaCl containing 5–10 mgof plasmid DNA and electroporated using a BTX Electrocell Manipulator 600 set at 240 V, 950 mF, resistance = None. Cells were processed 24 h after electroporation. The supernatants of transfected cells were tested for CCL5 secretion using the human CCL5/RANTES DuoSet ELISA kit (R&D Systems) and for the secretion of IFNg, IL-1b, IL-6 and IL-8 using the appropriate uncoated ELISA kits (Thermo Fisher Scientific). 2.5 Bacterial infection of cultured cells, invasion, proliferation, and protein translocation assays Mammalian cells were plated in 24-well plates at 1.5 x 10 5 cells per well and incubated 24 h at 37°C with 5% CO 2 in media without antibiotics. For infections under SPI1-inducing (invasive) conditions, bacteria grown overnight in LB with 0.3 M NaCl in a tightly closed tube without shaking were added at a multiplicity of infection of 150. For infections of RAW264.7 cells under noninvasive conditions, the bacteria were grown in LB for 24 h at 37°C with shaking. Cell culture was washed twice with phosphatebuffered saline (PBS) at 1 h post-infection (p.i.), overlaid with DMEM containing 100 mg/ml gentamicin, and incubated for another hour. The culture was then washed twice with PBS, covered with DMEM with gentamicin 16 mg/ml, and incubated for 6 h. Following the infections described above, the translocation of CyaA’fusions into eukaryotic cells was monitored by measuring the levels of cyclic AMP (cAMP) 2 h or 8 h p.i. Infected cells were lysed and the level of cAMP in the lysates was determined using a colorimetric direct cAMP enzyme immunoassay kit (Arbor Assays) according to the manufacturer’s instructions. For invasion and proliferation assays, infections were carried out using a 10:1 mix of a mutant strain and a trg::MudJ mutant (wild type for invasion and intracellular proliferation, but Lac + due to the MudJ insertion). Competitive indices for invasion and proliferation were calculated as previously described (29) after plating appropriate dilutions and enumerating white colonies and blue colonies (trg::MudJ) in LB plates supplemented with 40 mg/ml 5-bromo-4-chloro- galactopyranoside (X-Gal). For invasion, the input was the initial mix of bacteria used in the infection and the output, bacteria recovered 2 h p.i. For intracellular proliferation, bacteria were recovered 2 h p.i. (input) and 24 h p.i. (output). 2.6 Luminescence measurements Salmonella strains were grown in triplicate in the appropriate media and samples of 150 ml of each culture were used to measure luminescence and OD 600 , in 96-well white clear bottom plates using a FLUOstar Omega plate reader (BMG LABTECH). To measure the luminescence of intracellular bacteria, RAW264.7 macrophages were plated in 96-well white clear bottom plates at 3 x 10 4 cells per well, and were infected 24 h later with non-invasive bacteria (grown in LB for 24 h at 37°C with shaking) at a multiplicity of infection of 500. The cell culture was washed twice with PBS 30 min p.i., overlaid with DMEM containing 100 mg/ml gentamicin, and incubated for 1.5 h. The culture was then washed twice with PBS, TABLE 1 Continued Strain/ Plasmid Relevant characteristics Source/ Reference pIZ3712 pGEX-4T-1/PKN1(HR1b) This work pIZ3716 pTYB21-HA-UBC(K48R/K63R) This work pLEX10 Yeast two-hybrid vector, Ap r (34) pQE80L 6His fusion vector, Ap r Qiagen pSB377 Parent for luxCDABE transcriptional fusions, Ap r (35) pSIF003- R1 pEX-CyaA (1–412) derivative (36) pTYB21 Vector for generation of intein fusions New England Biolabs a Derivatives of these strains were used as indicated in the text. Bullones-Bolaños et al. 10.3389/fimmu.2024.1328707 Frontiers in Immunology frontiersin.org04 covered with DMEM with 16 mg/ml gentamicin and incubated for 6 additional h. Luminescence was measured at 2, 4 and 8 h p.i. and the colony forming units per well were calculated after incubation with 1% Triton X-100 in PBS for 10 min at 37°C to release bacteria, plating appropriate dilutions in LB with Ap, and counting colonies after 24 h incubation at 37°C. 2.7 DNA amplification with the polymerase chain reaction, cloning, mutagenesis, and sequencing Amplification reactions were carried out on a T100 Thermal Cycler (Bio-Rad) using Q5 High-Fidelity DNA polymerase (New England Biolabs) or MyTaq Red DNA polymerase (Bioline) according to the supplier’s instructions. Oligonucleotides are described in Table 2. Plasmid construction was carried out either using a classical cloning strategy based on enzymatic digestion or by Gibson assembly (40). The plasmids were sequenced with an automated DNA sequencer (Stab Vida). The disruption of sspH1 or sspH2 and replacement in strain 14028 with a gene conferring resistance to Km was carried out as previously described (41) using primers listed in Table 2 and plasmid pKD4. The antibiotic resistance cassette introduced by the gene targeting procedure was eliminated by recombination using the FLP helper plasmid pCP20. The addition of a DNA fragment encoding the 3xFLAG epitope tag at the 3’end of sspH1 or sspH2 was carried out as described (42)usingprimerslistedinTable 2 and the plasmid pSUB11. The protocol to generate chromosomal cyaA’translational fusions was also previously described (43). Overlapping PCR was used to generate point mutations in the gene encoding human ubiquitin, UBC,using pIZ3683 as template and primers indicated in Table 2,andthePCR products were cloned in pCS2 with EcoRI/XbaI restriction endonucleases. The constructs for the purification of HA ubiquitin were made by Gibson assembly using primers P2pTYB21-Fw and P1pTYB21-Rv to amplify the plasmid pTYB21, and primers P1pTYB21-HA-UB-Fw and P2pTYB21-HA-UB-Rv to amplify the different ubiquitins of plasmids pIZ3657, pIZ3682, and pIZ3683. Double mutant ubiquitin was constructed by overlapping PCR using pIZ3683 as a template with the following primers: PCR1: P1-pTYB21- HA-UB-Fw and UB-K48R-Rv; PCR2: UB-K48R-Fw and pCS2-3; PCR3: P1-pTYB21-HA-UB-Fw and P1-pTYB21-HA-UB-Rv and cloning into pTYB21 by Gibson assembly. The presence of the inserts was confirmed with primers pTYB21-ext-Fw and pTYB21- ext-Rv. 2.8 Expression and purification of HA-ubiquitin The plasmids pIZ3698, pIZ3695, pIZ3696, and pIZ3716 were transformed into E. coli ER2566. Overnight cultures of these strains were diluted 50 times in 500 ml of ampicillin-supplemented LB and incubated at 37°C until an OD 600 of 0.5. The expression of target proteins was induced by adding 0.4 mM isopropyl-b-D-thiogalactoside and incubating overnight at 20°C with shaking. Subsequently, the cultures were centrifuged at 5000 g, 15 min, 4°C, the pellets were washed with PBS and centrifuged again to remove PBS. The pellets were lysed by sonication (output 2, duty cycle 30%, 1 min, 5 times) in column buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 0.5% NP40) containing 1 mM PMSF and protease inhibitor cocktail (1:200) (Sigma-Aldrich). The supernatants were separated from the pellets by centrifugation at 15000 g, 20 min, 4°C and loaded into the affinity chromatography column according to the IMPACT E6901 Manual (New England Biolabs), with the following modifications: crystal columns of 1.5 x 20 cm and 5 ml of chitin beads were used. To release target proteins from the column, beads were incubated with cleavage buffer with DTT 100 mM, 72 h at 4°C. The eluted fractions were dialyzed in 10 mM HEPES buffer pH 7.5 with 5% glycerol using the Slide-A-Lyzer®Dialysis cassette 3,500 MWCO, 3-12 ml capacity and incubated overnight at 4°C with slight shaking. To concentrate the proteins centrifugal filter units (Amicon®Ultra-15) were used. The aliquots were stored at -80°C and the protein concentration was quantified using the Coomassie (Bradford) protein assay kit from Thermo Scientific. 2.9 Purification of fusion proteins, electrophoresis, and immunoblotting The expression of GST fusion proteins was induced by the addition of 1 mM isopropyl-b-D-thiogalactoside to E. coli BL21 (DE3) containing pGEX-4T-1, pGEX-4T-2 or their derivatives. Bacteria were sonicated in NP40 buffer and fusion proteins were isolated from bacterial lysates by affinity chromatography with glutathioneagarose beads (Sigma-Aldrich). Proteins tagged with 6His were produced after the addition of 1 mM isopropyl-b-D-thiogalactoside to E. coli XL1-Blue containing derivatives of pQE30 or pQE80L, purified with Ni-NTA agarose beads (Sigma-Aldrich) and eluted with 300 mM imidazole in binding buffer (50 mM NaH 2 PO 4 ,300 mM NaCl). For electrophoresis, bacterial and mammalian lysates were mixed with an equal volume of 4x Laemmli sample buffer and boiled for 5 min. Proteins were separated by SDS-PAGE using mini-protean TGX precast gels, 4-15% gradient (BioRad), and electrophoretically transferred to nitrocellulose filters for western blot analysis. Primary antibodies were: anti-FLAG M2 (mouse,monoclonal,1:5,000,Sigma- Aldrich), anti-HA-peroxidase 3F10 (rat, 1:2000, Roche), anti-GroEL (rabbit, polyclonal, 1:20,000, Sigma-Aldrich), anti-DnaK 8E2/2 (mouse, monoclonal, 1:5,000, Assay Designs). Secondary antibodies were goat anti-mouse IRDye 800CW-conjugated or goat anti-rabbit IRDye 680RD-conjugated antibodies (LI-COR). The bands were detected usingtheOdysseyFcimagingsystem(LI-COR). 2.10 In vitro ubiquitination assays Ubiquitination reactions were carried out in a 20-ml mixture containing buffer A (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM ATP, 10 mM MgCl 2 , 0.1 mM DTT), 2 mg of HA-tagged ubiquitin, 0.25 mg of E1 (Boston Biochem, Cambridge, MA, USA) and 1 mgof E2 (human recombinant UbcH5b from Boston Biochem) in the presence or absence of ubiquitin ligases and substrates. Reactions were incubated at 37°C for 1 h with shaking and stopped by adding Bullones-Bolaños et al. 10.3389/fimmu.2024.1328707 Frontiers in Immunology frontiersin.org05 TABLE 2 Oligonucleotides used in this study. Oligonucleotide / use Sequence 5’-3’ sspH1 deletion sspH1H1P1 TTAATCTCTTTTCATTGTGCTGTAAATTAGGCAGTGGAATGTGTAGGCTGGAGCTGCTTC sspH1H2P2 TTCACCGCACCACATTCGCCTGGTGCGGTGAATATCGTGCCATATGAATATCCTCCTTAG sspH2 deletion sspH2H1P1 CGGACAGATACTATATGTAAATTTATAAAGGTTTTTTGTTGTGTAGGCTGGAGCTGCTTC sspH2H2P2 GGAATATCTTTGTCGCACCGCACCTCATTCACCTGGTGCACATATGAATATCCTCCTTAG Epitope tagging of SspH1 sspH1-3xFLAG-5’CTGGGTAGCTATCTGACAGCCCGGTGGCGTCTTAACGACTACAAAGACCATGACGG sspH1-3xFLAG-3’CCGCACCACATTCGCCTGGTGCGGTGAATATCGTGCCATATGAATATCCTCCTTAG Epitope tagging of SspH2 sspH2-3xFLAG-5’CTGGGGAGCTATCTGAACGTTCAGTGGCGTCGTAACGACTACAAAGACCATGACGG sspH2-3xFLAG-3’TATCTTTGTCGCACCGCACCTCATTCACCTGGTGCACATATGAATATCCTCCTTAG Chromosomal sspH1::cyaA’fusion ssph1-197cyaP1 AGTGGTACAGGAAATGCGTGATTGCCTGAATAACGGCAATCTGCAGCAATCGCATCAGGC ssph1-197cyaP2 GTAAGGTGGTAAGACCTGACGCTCCCACGTTAAGCACTGGTTAGAAAAACTCATCGAGCATC Chromosomal sspH2::cyaA’fusion ssph2-203cyaP1 AGTGGTACAGAAAATGCGTGCCTGCCTGAATAATGGCAATCTGCAGCAATCGCATCAGGC ssph2-203cyaP2 GCAAGGTGGTAAGACCTGATTCTCCCACGTTAAGCACTGCTTAGAAAAACTCATCGAGCATC Construction of pIZ2149 PslrPfwEco CATGGAATTCCGATCGCCAGCGAGTCATCG PslrPrevEco GATCGAATTCATTTTCCCTACCTGATCTG Construction of pIZ3639 psspH2Ecofw ACGTGAATTCAAAGGGTTTATTCGCCGGAAG psspH2Ecorv AGCTGAATTCAACAAAAAACCTTTATAAATTTACATATAG Construction of pIZ3640 ssph1SDbamh1fw GAATGGATCCAGGAGGTGGAATATGTTTAATATCCGC ssph1-197bamh1rv CAAGGGATCCGATTGCCGTTATTCAGGCAATC Construction of pIZ3641 ssph2SDbamh1fw GAATGGATCCAGGAGGTTTGTTATGCCCTTTCATATTGG ssph2-203bamh1rv CAAGGGATCCCATTGCCATTATTCAGGCAGGC Construction of pIZ3643 slrP457bamHIfw AGTCGGATCCTCAATTGTTCGGGTAACTCG slrPsalIrv AGTCGTCGACCTATCGCCAGTAGGCGCTCATG Construction of pIZ3644 slrPecoRIfw AGTCGAATTCATGTTTAATATTACTAATATACAATC slrP456bamHIrv AGTCGGATCCAAAGTCGCCCATGGCAAACAATAC Construction of pIZ3645 sspH1-399bamHIfw AGTCGGATCCTCCGTCCCCCGGGAAGCCCG sspH1xhorv TGACCTCGAGTCAGTTAAGACGCCACCGGG (Continued) Bullones-Bolaños et al. 10.3389/fimmu.2024.1328707 Frontiers in Immunology frontiersin.org06 TABLE 2 Continued Oligonucleotide / use Sequence 5’-3’ Construction of pIZ3646 sspH2-486bamHIfw AGTCGGATCCGCCTCCGCCCCCCGGGAAAC sspH2salrv GATCGTCGACTCAGTTACGACGCCACTGAAC Construction of pIZ3649 PsspH1EcoFw ATGCGAATTCAGCGCTGTTTTGCCTGGCTG psspH1Ecorv ATGCGAATTCATTCCACTGCCTAATTTACAG Construction of pIZ3657 BamHI-kozak-HA-Ub-Fw ACGTGGATCCGCCGCCACCATGTACCCTTATGATGTACCAGACTACGCTGGCCGG ubqXbaIrv GATCTCTAGATCACCCACCTCTGAGACGGAGCAC Construction of pIZ3663 slrPecoRIfw AGTCGAATTCATGTTTAATATTACTAATATACAATC slrP456bamHIrv AGTCGGATCCAAAGTCGCCCATGGCAAACAATAC sspH1-399bamHIfw AGTCGGATCCTCCGTCCCCCGGGAAGCCCG sspH1xhorv TGACCTCGAGTCAGTTAAGACGCCACCGGG Construction of pIZ3668 sspH1bamfw ATGCGGATCCATGTTTAATATCCGCAATAC sspH1-398bamHIrv AGTCGGATCCAGGCCCCGCCATATCGAAGTG Construction of pIZ3669 SlrPBamG5 CTGAGGATCCATGTTTAATATTACTAATATACAATC slrp456sacIsalIrv AGTCGTCGACGAGCTCAAAGTCGCCCATGGCAAACAATAC sspH2-486salIfw AGTCGTCGACGCCTCCGCCCCCCGGGAAAC sspH2hindIIIrv AGTCAAGCTTTCAGTTACGACGCCACTGAAC Construction of pIZ3670 sspH1bamfw ATGCGGATCCATGTTTAATATCCGCAATAC sspH1-398salIsacIrv AGTCGAGCTCGTCGACAGGCCCCGCCATATCGAAGTG slrP457salIfw AGTCGTCGACTCAATTGTTCGGGTAACTCG slrPhindIIIrv AGTCAAGCTTCTATCGCCAGTAGGCGCTCATG Construction of pIZ3671 sspH1bamfw ATGCGGATCCATGTTTAATATCCGCAATAC sspH1-398salIsacIrv AGTCGAGCTCGTCGACAGGCCCCGCCATATCGAAGTG sspH2-486salIfw AGTCGTCGACGCCTCCGCCCCCCGGGAAAC sspH2hindIIIrv AGTCAAGCTTTCAGTTACGACGCCACTGAAC Construction of pIZ3672 sspH2ecofw ATGCGAATTCATGCCCTTTCATATTGGAAG sspH2-485bamHIrv AGTCGGATCCTCCCGCCATATCGAATCGTATTATG Construction of pIZ3675 sspH1bamfw ATGCGGATCCATGTTTAATATCCGCAATAC sspH1-398bamHIrv AGTCGGATCCAGGCCCCGCCATATCGAAGTG slrP457bamHIfw AGTCGGATCCTCAATTGTTCGGGTAACTCG (Continued) Bullones-Bolaños et al. 10.3389/fimmu.2024.1328707 Frontiers in Immunology frontiersin.org07 TABLE 2 Continued Oligonucleotide / use Sequence 5’-3’ slrPsalIrv AGTCGTCGACCTATCGCCAGTAGGCGCTCATG Construction of pIZ3681 sspH2ecofw ATGCGAATTCATGCCCTTTCATATTGGAAG sspH2-485bamHIrv AGTCGGATCCTCCCGCCATATCGAATCGTATTATG sspH1-399bamHIfw AGTCGGATCCTCCGTCCCCCGGGAAGCCCG sspH1xhorv TGACCTCGAGTCAGTTAAGACGCCACCGGG Construction of pIZ3682 UbqEcoFw GATCGAATTCATGCAGATCTTCGTGAAGACC UB-K48R-Rv CCATCTTCCAGCTGTCGCCCAGCAAAGATCAAC Ub-K48R-Fw GTTGATCTTTGCTGGGCGACAGCTGGAAGATGG pCS2-3 TGACCATGATTACGCCAAGC Construction of pIZ3683 UbqEcoFw GATCGAATTCATGCAGATCTTCGTGAAGACC Ub-K63R-Rv GCAGGGTGGACTCTCGCTGGATGTTGTAGTC Ub-K63R-Fw GACTACAACATCCAGCGAGAGTCCACCCTGC pCS2-3 TGACCATGATTACGCCAAGC Construction of pIZ3686 sspH2ecofw ATGCGAATTCATGCCCTTTCATATTGGAAG sspH2-485bamHIrv AGTCGGATCCTCCCGCCATATCGAATCGTATTATG slrP457bamHIfw AGTCGGATCCTCAATTGTTCGGGTAACTCG slrPsalIrv AGTCGTCGACCTATCGCCAGTAGGCGCTCATG Construction of pIZ3687 sspH2Bamfw ATGCGGATCCATGCCCTTTCATATTGGAAG sspH2-485sacIsalIrv AGTCGTCGACGAGCTCTCCCGCCATATCGAATCGTATTATG slrP457salIfw AGTCGTCGACTCAATTGTTCGGGTAACTCG slrPhindIIIrv AGTCAAGCTTCTATCGCCAGTAGGCGCTCATG Construction of pIZ3692 nSlrP-SacI-Fw GTCAGAGCTCATGTTTAATATTACTAATATACAATCTACG sspH1hindIIIrv AGTCAAGCTTTCAGTTAAGACGCCACCGGG Construction of pIZ3693 sspH2Bamfw ATGCGGATCCATGCCCTTTCATATTGGAAG sspH2-485sacIsalIrv AGTCGTCGACGAGCTCTCCCGCCATATCGAATCGTATTATG sspH1-399sacIfw AGTCGAGCTCTCCGTCCCCCGGGAAGCCCG sspH1hindIIIrv AGTCAAGCTTTCAGTTAAGACGCCACCGGG Construction of pIZ3695, pIZ3696, pIZ3698 P2-pTYB21-Fw TAAATAACTAGTTGATCCGGCTGC P1-pTYB21-Rv GTTCTGTACAACAACCTGAGATCC P1pTYB21-HA-UB-Fw GTTGTTGTACAGAACATGTACCCTTATGATGTACCAGAC P2pTYB21-HA-UB-Rv TCAACTAGTTATTTACCCACCTCTGAGACGGAGC (Continued) Bullones-Bolaños et al. 10.3389/fimmu.2024.1328707 Frontiers in Immunology frontiersin.org08 an equal volume of Laemmli sample buffer 4X containing 100 mM DTT and boiling. Some reactions were carried out with GST fusion proteins bound to glutathione-agarose beads, and the beads were washed five times with NP40 buffer before boiling in Laemmli sample buffer with 100 mM DTT. 2.11 Statistical analysis Each competitive index and each cytokine concentration value are the mean of at least three independent infections or transfections. Means and standard deviations were calculated and a Student’st-test was used to evaluate if each competitive index was significantly different from 1 or if every cytokine concentration was significantly different from the concentration in the control assay. P values of 0.05 or less are considered significant. 3 Results 3.1 In vitro expression and regulation of slrP,sspH1, and sspH2 S. enterica serovar Typhimurium has three orthologous genes, slrP, sspH1,andsspH2, which encode E3 ubiquitin ligases belonging to the NEL family of type III secretion effectors. To compare the expression of these genes, we cloned their promoter regions into the plasmid pSB377 to generate luxCDABE transcriptional fusions. Plasmids were introduced into S. enterica serovar Typhimurium strain 14028 and luminescence was measured after growth under conditions inducing SPI1 expression (LB with 0.3 M NaCl without aeration) and conditions inducing SPI2 expression (LPM at pH 5.8 with high aeration). Although the three genes were preferentially expressed under SPI2- inducing conditions (Figure 1A),thereweredramaticdifferencesinthe ratios between SPI2- and SPI1-inducing conditions. While slrP was significantly expressed under SPI1-inducing conditions and the SPI2/ SPI1 ratio was only 3.2, expression of sspH1 and sspH2 was more restricted and the SPI2/SPI1 ratios were 58.8 and 367.3, respectively. Luminescence was also measured using Salmonella strains lacking virulence regulators PhoP or SsrB. This analysis revealed that the twocomponent system PhoQ/PhoP was strictly necessary for the expression of slrP,sspH1,andsspH2 under SPI2-inducing conditions. Furthermore, SsrB, the positive regulator of SPI2, was essential for the expression of sspH2, but has only a partial influence on the expression of slrP and sspH1 (Figure 1B). These results were confirmed at the protein level by immunoblot using 3xFLAG chromosomal fusions (Figure 1C). 3.2 Expression and regulation of slrP, sspH1, and sspH2 in host cells Salmonella carrying transcriptional lux fusions were also used to infect cultures of epithelial HeLa cells, NRK fibroblasts, and TABLE 2 Continued Oligonucleotide / use Sequence 5’-3’ Construction of pIZ3703 slrPecoRIfw AGTCGAATTCATGTTTAATATTACTAATATACAATC slrP456bamHIrv AGTCGGATCCAAAGTCGCCCATGGCAAACAATAC sspH2-486bamHIfw AGTCGGATCCGCCTCCGCCCCCCGGGAAAC sspH2salrv GATCGTCGACTCAGTTACGACGCCACTGAAC Construction of pIZ3706 sspH1bamfw ATGCGGATCCATGTTTAATATCCGCAATAC sspH1-398bamHIrv AGTCGGATCCAGGCCCCGCCATATCGAAGTG sspH2-486bamHIfw AGTCGGATCCGCCTCCGCCCCCCGGGAAAC sspH2salrv GATCGTCGACTCAGTTACGACGCCACTGAAC Construction of pIZ3716 P1pTYB21-HA-UB-Fw GTTGTTGTACAGAACATGTACCCTTATGATGTACCAGAC UB-K48R-Rv CCATCTTCCAGCTGTCGCCCAGCAAAGATCAAC Ub-K48R-Fw GTTGATCTTTGCTGGGCGACAGCTGGAAGATGG pCS2-3 TGACCATGATTACGCCAAGC Bullones-Bolaños et al. 10.3389/fimmu.2024.1328707 Frontiers in Immunology frontiersin.org09 translocation is lost in the ssaV mutant, indicating that T3SS2 is the relevant system under these conditions (Figure 3D). The situation is more complex in fibroblasts, with significant secretion of SlrP through both systems (Figure 3F, the double mutation is needed to prevent translocation). This can be explained because Salmonella invades fibroblasts through multiple routes (52). It should be taken into account that the method used to detect translocation of effectors into host cells has some limitations. In particular, in HeLa and NRK cells the interpretation is more difficult than in RAW264.7 cells because we cannot clearly separate T3SS1-dependent from T3SS2-dependent secretion since T3SS1 expression (invasive conditions) are needed in order to infect these cells. Previous data indicated that half-lives of effectors of this family are longer than several hours (53,54). As a consequence, the high levels of translocation observed for SlrP in HeLa cells (Figure 3B)at8hwiththessaV mutant (lacking T3SS2) could be due to the translocation that occurred previously through T3SS1. The multifactorial nature of bacterial virulence makes it prone to be affected by redundancy phenomena. In fact, the lack of only one of the more than 40 T3SS effectors, which can be obtained with a single null mutant, usually does not lead to a significant decrease in Salmonella pathogenicity. This is what we observe for SlrP, SspH1, and SspH2 in our in vitro cellular infection models (Figure 4). However, a mutant lacking the three effectors is significantly attenuated for intracellular proliferation in NRK cells compared to wild-type bacteria. These results suggest that these three effectors work redundantly to allow Salmonella survival inside fibroblasts. They also show that there are different requirements and mechanisms for the adaptation of this pathogen to different host cell lines since no defects were detected in epithelial cells or macrophages. SlrP, SspH1, and SspH2 share a similar structure and a similar autoinhibition mechanism in which the activity of the NEL domain is repressed by the LRR domain in the absence of substrate (55). B A FIGURE 8 Analysis of the specific type of polyubiquitination catalyzed by SlrP, SspH1, and SspH2. HA-ubiquitin and its derivatives with the indicated mutations replacing lysines by arginines (K48R, K63R, K48R/K63R) were used in ubiquitination assays with 6His fusions of SlrP, SspH1, and SspH2. (A) No additional substrate was added and the whole reaction was analyzed to detect polyubiquitination of ubiquitin. (B) GST-SNRPD2 or GST-PKN1 bound to glutathione-agarose beads were added as substrates and beads were washed prior to immunoblot analysis with anti-HA antibodies. The sizes in kDa of the molecular weight markers are shown on the left. Replicate experiments for this figure are shown in Supplementary Material. Bullones-Bolaños et al. 10.3389/fimmu.2024.1328707 Frontiers in Immunology frontiersin.org16 There are several well defined host substrates for these effectors that include SNRPD2, PKN1, and NOD1, respectively. We have studied the level of specificity in the interaction and ubiquitination of these substrates and the determinants of this specificity. To do that, we have generated all possible combinations of the LRR and NEL domains of the three effectors (Figure 5) and tested their interaction with host proteins using the yeast two-hybrid system. Our previous results showed that SNRPD2 interacts specifically with SlrP but not with SspH1 or SspH2 (21). Here, we show that the N-terminal region of SlrP, including the LRR domain, directs this interaction (Figure 6A). Analogous results were obtained for PKN1, whose Hr1b domain interacts specifically with SspH1. In this case, the interaction with the isolated N-terminal region, containing the LRR domain, could not be tested due to transactivation of the reporter gene in the yeast two-hybrid system. However, the fact that this interaction occurs through this part of the protein was confirmed by the chimeric protein SspH1-N/SspH2-C, which did not show transactivation (Figure 6B). These results confirm the notion that host proteins interact with these effectors through their LRR domains. More interestingly, the chimeric proteins SlrP-N/ SspH1-C and SlrP-N/SspH2-C were able to ubiquitinate SNRPD2 (Figure 7), indicating that the NEL domains of the three effectors can catalyze the ubiquitination of this host target if fused to the interaction determinant provided by the LRR domain of SlrP. In fact, the NEL domains of SspH1 and SspH2 demonstrated greater ubiquitination activity on SNRPD2 than the NEL domain of SlrP under these conditions. Similar results were obtained for PKN1 and SspH1, although in this case ubiquitination was not detected with the chimeric protein SspH1-N/SlrP-C. This may be due to lower activity of the catalytic domain of SlrP or to an improper folding of the chimera. Our ubiquitination experiments showed in all cases the classical ladders that are indicative of polyubiquitination. The type of linkage between ubiquitins within the ubiquitin polymer determines the fate of the marked protein. As mentioned above, K48-linked chains are typically related to proteasomal degradation, while K63-linked chains are generally involved in signaling processes. In order to find out the type of polyubiquitination that was catalyzed by each effector, we generated and used mutant forms of ubiquitin. Assays carried out without a substrate, where polyubiquitination of ubiquitin is observed, revealed a strong dependence on Lys48 for the activity of SspH2 and also SspH1, although in the latter the effect is observed more clearly in very high molecular weight polyubiquitin bands (Figure 8A). The pattern of SlrP-induced polyubiquitination did not change dramatically using K48R or K63R mutant ubiquitins, suggesting that other residues may be involved. In fact, ubiquitin has five additional lysine residues (K6, K11, K27, K29, and K33) that, together with methionine 1 (M1), can be ubiquitinated. Ubiquitination assays using SNRPD2 or PKN1 HR1b as substrates confirm these conclusions (Figure 8B). S. enterica serovar Typhimurium manipulates host inflammatory responses in complex ways (56,57). Stimulation of inflammatory signaling depends on the T3SS effectors SopB, SopE, and SopE2, which act at the beginning of infection to help Salmonella compete B C A FIGURE 9 Effect of SlrP, SspH1 and SspH2 on the secretion of cytokines by mammalian cells. HeLa cells were transfected with a plasmid expressing SlrP, SspH1 or SspH2 or with the empty vector and the concentration of CCL5 (A), IL-6 (B), and IL-8 (C) was measured in the supernatants of cell cultures 24 h after transfection. Data are presented as mean values + standard deviations of at least three biological replicates. Dots represent individual values. * p< 0.05, ** p< 0.01, for a two-tailed Student’st-test comparing cells expressing each effector with cells transfected with the empty vector. Bullones-Bolaños et al. 10.3389/fimmu.2024.1328707 Frontiers in Immunology frontiersin.org17 with the normal intestinal microbiota. However, to prevent an excessive inflammatory response that would be detrimental to the bacteria, Salmonella limits this response by expressing other effectors, including SptP, PipA, GogA, GtgA and AvrA (58–61). SspH1 from S. enterica serovar Typhimurium also down-regulates the production of the pro-inflammatory factor IL-8 (46). In contrast, SspH2 was shown to enhance IL-8 secretion (24), and the effect of SlrP was not previously reported. Here, we studied the effect of the three effectors on several pro-inflammatory cytokines. Although we did not detect significant effects on IL-8 secretion, there was a reduction in CCL5 and IL-6 secretion in the presence of SspH1 or SspH2, but not SlrP (Figure 9). Previous experiments in a mouse model of anorexia suggested that SlrP from S. enterica serovar Typhimurium inhibited inflammasome activation and IL-1bmaturation in the small intestine (62). Furthermore, SspH2 from S. enterica serovar Enteritidis was shown to reduce the expression of pro-inflammatory factors, including IL-1b, IFN-g,andiNOS,inCaco-2BBEcells(47). Together, these results and the results presented here support a predominant anti-inflammatory role for Salmonella NEL effectors that can be expressed in different ways depending on the biological model and context. Interestingly, SlrP and SspH2 are involved in the inhibition of antigen presentation in dendritic cells (63) and in preventing the migration of these cells towardthechemokineCCL19(64). Furthermore, Salmonella has been shown to interfere with MHC class II antigen presentation, inducing polyubiquitination and reduction of cell surface HLA-DR expression (65). This effect depends on T3SS2, so it is tempting to speculate with the involvement of Salmonella NEL effectors. However, previous experiments failed to identify them as potential candidates and showed that the T3SS2 effector SteD decreased surface levels of MHC class II in dendritic cells by recruiting the host E3 ubiquitin ligase Wwp2 (66,67). Further experiments will be required to clarify theroleoftheseeffectorsinthisfunction. In conclusion, despite their similar structure and biochemical activity, the three members of the NEL family of T3SS effectors of S. enterica serovar Typhimurium have different expression and translocation patterns and recognize specific host targets that are modified with different polyubiquitination patterns. Unfortunately, few targets of these effectors have been described so far. Therefore, additional research is needed to obtain a complete list of these targets and a clearer picture of all the effects of these Salmonella proteins on the host. Data availability statement The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors. Author contributions AB-B: Formal analysis, Investigation, Methodology, Visualization, Writing –review & editing. PM-M: Formal analysis, Investigation, Methodology, Visualization, Writing – review & editing. CV-G: Formal analysis, Investigation, Methodology, Visualization, Writing –review & editing. JB-B: Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Writing –review & editing. FR-M: Formal analysis, Funding acquisition, Resources, Supervision, Writing –original draft, Writing –review & editing. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was supported by “Proyecto PID2022-136863NB-I00 financiado por MCIN/AEI /10.13039/501100011033/ y por FEDER Una manera de hacer Europa”. Acknowledgments The authors are grateful to Laura Tomas Gallardo (Servicio de Proteomica y Bioquı  mica, Universidad Pablo de Olavide, Sevilla) for help in the intein-mediated purification of HA-ubiquitin. We also thank the contribution of grant PID2019-106132RB-I00 funded by MCIN/AEI/10.13039/501100011033, grant P20_00576 funded by Fondo Europeo de Desarrollo Regional (FEDER) and Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía, and grant US-1380805 funded by Universidad de Sevilla, FEDER and Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2024.1328707/ full#supplementary-material Bullones-Bolaños et al. 10.3389/fimmu.2024.1328707 Frontiers in Immunology frontiersin.org18 References 1. Teklemariam AD, Al-Hindi RR, Albiheyri RS, Alharbi MG, Alghamdi MA, Filimban AAR, et al. 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