NDP52 is an autophagy receptor, which is able to detect the ubiquitin moieties on Salmonella by using its zinc finger domain. Knockdown of NDP52 leads to enhanced proliferation of bacteria in HeLa cells and to an increase of ubiquitin-coated Salmonella. Moreover, NDP52 controls autophagy of Salmonella and recruits autophagosomal marker, microtubule-associated protein 1A/1B-light chain 3 [LC3; (14)]. The p62 protein is a ubiquitin-binding protein associated with ubiquitinated protein aggregates that accumulate, for example, in various neurodegenerative disorders (15). In HeLa cells, p62 binds to ubiquitin through its C-terminal ubiquitin-associated (UBA) domain and it also binds to LC3, facilitating autophagy of cytosolic ubiquitin-coated Salmonella (16). The autophagy receptor OPTN contains the ubiquitin binding in ABIN and NEMO (UBAN) domain to bind ubiquitin (17) and also binds LC3 through its LIR (LC3-interacting region) motif. OPTN knockdown in HeLa cells during Salmonella infection leads to bacterial proliferation. Ubiquitin-binding deficient OPTN mutant or LIR mutant cannot rescue the dysfunction caused by the OPTN knockdown, indicating that both these domains are required to restrict bacterial growth. TANK-binding kinase 1 (Tbk1) phosphorylates OPTN recruited to ubiquitin-coated cytosolic Salmonella, thereby enhancing its binding to LC3 and most likely facilitating clearance of cytosolic bacteria through selective autophagy. As mentioned above, there are several Salmonella-sensing receptors, including p62, NDP52 as well as OPTN, and all of them bind to ubiquitin-coated Salmonella. However, NDP52 and OPTN localize to different microdomains on the surface of ubiquitin-coated Salmonella in comparison with p62 (18). This differential recognition might be caused by diverse affinity of these receptors for various ubiquitin linkages or by secondary interactions with other proteins.

In a study dissecting the function of autophagy cascade in elimination of Salmonella, leucine-rich repeat (LRR) and sterile alpha motif-containing protein 1 (LRSAM1) has been identified as an E3 ubiquitin ligase responsible for recognition and ubiquitination of Salmonella and its subsequent autophagy. LRSAM1 co-localizes with Salmonella, and a knockdown of LRSAM1 results in increased replication of bacteria in the cytoplasm of HeLa cells. Co-localization of LRSAM1 with Salmonella was also observed in infected murine bone marrow-derived macrophages and intra-peritoneal macrophages. LRSAM1 contains a domain commonly found in innate PRRs, LRR, as well as RING domain, which is characteristic of one of the classes of ubiquitin E3 ligases. LRR is required and sufficient for the LRSAM1 localization to Salmonella, but RING domain is essential for its ubiquitination. LRSAM1 and previously mentioned NDP52 localize to intracellular bacteria into spatially separated subdomains, but NDP52 recruitment to Salmonella is dependent on LRSAM1, which is also required for ubiquitin-associated autophagy and most likely can recognize bacteria by itself. Moreover, polyubiquitination directed by LRSAM1 favors K6- and K27-conjugated ubiquitin chains in comparison to other linkages. LRSAM1, therefore, restricts bacterial replication in the cytoplasm and is crucial for ubiquitin-mediated autophagy (19). This study helped to identify mechanisms and specificity of a novel host ubiquitin E3 ligase and define its function in autophagy. Selective autophagy during Salmonella infection is not completely understood; yet, it is clear that it requires ubiquitin pathways to function efficiently and it represents an effective host surveillance mechanism to control Salmonella replication and prevent systemic infection.

SseL is Salmonella’s effector protein that functions as a deubiquitinase. SseL prevents the autophagy machinery from recognizing ubiquitin aggregates and aggresome-like induced structures (ALIS), which are formed in response to bacterial infection or LPS-treatment (32, 33). Infection with SseL-deficient Salmonella strain results in an accumulation of SCV-associated ubiquitinated aggregates in HeLa cells compared to cells infected with wild-type Salmonella. Moreover, by deubiquitination of these ubiquitinated aggregates SseL decreases autophagic flux in macrophages and it favors intracellular replication of Salmonella in bone marrow-derived macrophages (33). Ubiquitin-driven autophagy was identified as one of the host responses to Salmonella and SseL is an effector protein that counteracts this process. Additionally, SseL is necessary for bacterial virulence in mice, required for delayed cytotoxicity by Salmonella in macrophages (34, 35), and it binds to the oxysterol-binding protein [OSBP; (36)]. The deubiquitinating activity of SseL is also related to its role in cell lipid metabolism as SseL prevents lipid droplet accumulation in mouse epithelial cells (37). Since lipid droplet metabolism is regulated in autophagy (38), it could potentially be related to SseL’s function in autophagic flux, although the function of SseL appears to be complex and it might involve several substrates and pathways. From the studies on SseL, some more general host mechanisms could be identified, such as the ubiquitin involvement in the selective autophagy.

NF-κB is a conserved family of transcription factors that regulate diverse processes, such as inflammation, immune response, cell growth, and apoptosis [reviewed in Ref. (39, 40)]. Although induction of this pathway provides immediate immune response and host protection, pathogens utilize the immune cells to replicate and spread to other tissues in the host, resulting in systemic infection. For example, infection with the virulent Salmonella leads to increased inflammatory response by NF-κB pathway activation, while the avirulent strain has an opposite effect (41). Inhibition of the NF-κB pathway is facilitated by AvrA, which is another Salmonella-encoded deubiquitinase (42, 43) that also functions as an acetyltransferase (44). AvrA deubiquitinates and therefore stabilizes IκBα, an inhibitor of NF-κB pathway, thus preventing nuclear translocation of NF-κB p65, which was shown in vivo in mice and in epithelial cells. Infection of mice with AvrA-deficient strain of Salmonella leads to an increased IκBα degradation and secretion of NF-κB dependent cytokine, IL-6. AvrA also stabilizes an inhibitor of the proinflammatory NF-κB pathway, β-catenin, by preventing its proteasomal degradation via removal of ubiquitin moieties from β-catenin (43). Moreover, AvrA was linked to an increased risk of cancer associated with chronic Salmonella infections (45). In summary, AvrA is a bacterial effector protein used to fight the host defense strategies marked by the ubiquitin modification.

Salmonella’s effector, SopA, is a HECT-like E3 ubiquitin ligase that becomes ubiquitinated by a host E3 ligase HsRMA1 (28, 46), although it is also capable of autoubiquitination (47). In an ubiquitination assay to identify the E2 ligases associated with SopA, UbcH5a, UbcH5c, and UbcH7 were preferentially used by SopA, suggesting a regulatory role in inflammation (46). We discuss effects of SopA polyubiquitination in Section “SopA polyubiquitination regulating bacterial escape.”

Salmonella’s SspH2 belongs to the novel ubiquitin E3 ligase (NEL) family. It contains LRR domain, which exerts an inhibitory effect on the NEL domain activity, while the NEL domain expressed alone has a 25-fold increase in E3 ligase activity in comparison to a full-length SspH2. Moreover, in epithelial cells, SspH2 increases the Nod1-mediated IL-8 secretion via monoubiquitination, thereby mediating the innate immune response. This function depends on the E3 ubiquitin ligase activity of SspH2 (48). Apart from the identification of a novel bacterial E3 ligase, these studies bring into light a new modification of the host Nod1 protein and its relevance in the IL-8 secretion.

SspH1 is Salmonella’s effector protein that is a member of the NEL family of ubiquitin E3 ligases (49). It is capable of ubiquitination of protein kinase N1 (PKN1), which functions in androgen receptor (AR) signaling (50, 51). The wild-type SspH1 ubiquitinates PKN1 when co-expressed in HEK293 cells and targets it to the 26S proteasome for degradation, but a catalytic mutant of SspH1 lacks this function. Attenuation of AR activation was observed when wild-type SspH1 was transiently expressed in HEK293 cells in comparison to expression of C492A catalytic mutant or PKN1-interaction mutant. By mediation of ubiquitination and subsequent degradation of PKN1, SspH1 attenuates AR signaling, which might be important in regulation of the cellular immunity during Salmonella infection (52, 53).

Slrp is another Salmonella’s ubiquitin E3 ligase, which belongs to NEL E3 ligase family, and it also contains LRR domains. Mammalian thioredoxin-1 (Trx) is as a binding partner of Slrp (54). Trx proteins regulate redox-related signaling, synthesis of cytokines, growth, and apoptosis [reviewed in Ref. (55, 56)]. Slrp ubiquitinates Trx, while mutation in the cysteine active site of Slrp (C546) disables this activity. In HeLa cells, Slrp expression results in reduction of Trx’s activity in confluent but not in growing cultures. Salmonella infection of HeLa cells decreases Trx activity, which also correlates with an increase in cell death. Collectively, these findings suggest that the E3 ligase activity of Slrp is partially responsible for the cytotoxic effect on HeLa cells during infection (54).

SopA is an ubiquitin E3 ligase that can be ubiquitinated by a host E3 enzyme HsRMA1 (28, 46). SopA ubiquitination by HsRMA1 is important in regulation of the bacterial escape from SCV, as shown by using HsRMA1 knockdown study. Also, compared to the wild-type Salmonella, a sopA Salmonella mutant has an impaired ability to escape from SCV into the cytosol in HeLa cells. This together demonstrates that ubiquitination of this effector protein is important for an effective bacterial escape into the cytosol and that host E3 ligase HsRMA1 contributes to this function (28).

Salmonella’s SopB, is a phosphoinositide phosphatase that regulates several physiological processes owing to its phosphatase activity [reviewed in Ref. (58)]. It is ubiquitinated by host E3 ligase TRAF6 and E2 enzyme UbcH5c (29). Interestingly, SopB ubiquitination does not affect SopB protein stability but it downregulates SopB activity at the plasma membrane. SopB ubiquitination also leads to an internalization of SopB into the host cells, and it causes retention of SopB in the SCV (31). Ubiquitination of SopB is essential for SopB-dependent recruitment of Rab5 to SCV (30), but not for PI(3)P generation on the SCV (31). In summary, SopB ubiquitination by the host ubiquitin machinery is not related to its stability but it does regulate its enzymatic activity at the plasma membrane as well as its intracellular localization.