The interplay between Salmonella and host: Mechanisms and strategies for bacterial survival

Abstract

Salmonella, an intracellular pathogen, infects both humans and animals, causing diverse diseases such as gastroenteritis and enteric fever. The Salmonella type III secretion system (T3SS), encoded within its pathogenicity islands (SPIs), is critical for bacterial virulence by directly delivering multiple effectors into eukaryotic host cells. Salmonella utilizes these effectors to facilitate its survival and replication within the host through modulating cytoskeletal dynamics, inflammatory responses, the biogenesis of Salmonella-containing vacuole (SCV), and host cell survival. Moreover, these effectors also interfere with immune responses via inhibiting innate immunity or antigen presentation. In this review, we summarize the current progress in the survival strategies employed by Salmonella and the molecular mechanisms underlying its interactions with the host. Understanding the interplay between Salmonella and host can enhance our knowledge of the bacterium's pathogenic processes and provide new insights into how it manipulates host cellular physiological activities to ensure its survival.

1. Introduction

Salmonella enterica is a Gram-negative pathogenic bacterium and the leading cause of bacterial foodborne illness globally (Pires et al., 2021). The species Salmonella enterica consists of over 2600 different serovars, which are classified into two main groups based on their surface antigenic composition: typhoidal serovars and non-typhoidal serovars (Gal-Mor et al., 2014). Salmonella enterica serovar Typhi and serovar Paratyphi, collectively known as typhoidal serovars, are human-restricted pathogens that cause systemic diseases: typhoid fever and paratyphoid fever, respectively. These diseases are still major global health challenges, especially in developing countries (Tadesse et al., 2023). Non-typhoidal serovars such as Salmonella enterica serovar Enteritidis and Typhimurium, have a broad host range and primarily cause self-limiting gastroenteritis in humans. These two serovars are significant contributors to foodborne illness (Authority et al., 2023). Most of the studies discussed in this review used Salmonella Typhimurium (S. Typhimurium) as a model organism.

As an intracellular pathogen, Salmonella can invade and survive within various host cell types, particularly epithelial cells and macrophages. The pathogenesis of Salmonella is intricately linked to its ability to manipulate host cellular processes through its two distinct type III secretion systems (T3SSs) encoded within its pathogenicity islands (SPI) 1 and 2. The SPI-1-encoded T3SS1 and SPI-2-encoded T3SS2 inject more than 40 proteins, known as effectors, into host cells to manipulate various cellular processes, thereby facilitating bacterial invasion, survival, and replication (Shanker & Sun, 2023; Srikanth et al., 2011).

Indeed, the effectors delivered by the two distinct T3SSs play crucial roles in bacterium's ability to invade, survive, and replicate within host cells. T3SS1 effectors are essential during the early stages of bacterial infection, particularly for the invasion of Salmonella into host cells. Salmonella uses these effectors to manipulate host cell skeleton proteins, leading to the induction of membrane ruffling and subsequent facilitation of bacterial uptake (Clark et al., 2011). In addition to bacterial invasion, T3SS1 effectors can also manipulate different inflammatory signaling pathways. By targeting pathways such as the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription 3 (STAT3), these effectors can effectively modulate host inflammatory responses (Fattinger et al., 2021; Jaslow et al., 2018; Jones et al., 2008). Remarkably, unlike T3SS1, T3SS2 is exclusively expressed within the Salmonella-containing vacuole (SCV), a unique intracellular membrane-bound niche of Salmonella (Steele-Mortimer, 2008). This system plays a pivotal role in the systemic infection and intracellular pathogenesis of Salmonella by delivering effectors across the membrane of the SCV into the host cell cytoplasm. These effectors can modulate SCV biogenesis, movement, and maturation, as well as interfere with immune responses, ultimately facilitating bacterial survival and replication within the SCV in host cells. Additionally, many effectors delivered by the T3SS1 are also involved in SCV biogenesis and immune evasion (Chandrasekhar et al., 2023). In this review, we summarize the survival strategies utilized by Salmonella and the molecular mechanisms underlying its interactions with host cells. By exploring these processes, we aim to enhance our understanding of Salmonella's pathogenicity and clarify how this bacterium manipulates host cellular functions to ensure its survival. Furthermore, this review offers new insights into the complex interplay between Salmonella and host cells, with the goal of advancing therapeutic strategies against Salmonella infection.

2. Cellular processes manipulated by Salmonella effectors

2.1. Entry of Salmonella into nonphagocytic epithelia

Pathogenic bacteria, including Salmonella, utilize sophisticated mechanisms to invade nonphagocytic epithelial cells. A key aspect of this process involves altering phosphatidylinositol metabolism and regulating actin rearrangements. By modifying the signaling pathways associated with phosphoinositides and manipulating the actin cytoskeleton, these bacteria induce membrane ruffling and create structures that facilitate their internalization (Cossart et al., 2003). It is well demonstrated that the invasion of Salmonella into non-phagocytic epithelial cells is a complex process that largely relies on the T3SS-1 and its effectors. These effectors play crucial roles in manipulating host cell signaling pathways and the actin cytoskeleton, thereby facilitating bacterial entry (Raffatellu et al., 2005). Notably, the mechanisms by which T3SS1 effectors mediate Salmonella invasion into non-phagocytic cells are also required for its entry into phagocytes, such as macrophages, although this entry occurs via phagocytosis. Fig. 1 summarizes the T3SS1 effectors and their targets involved in bacterial internalization.

Fig. 1.

Entry of Salmonella into nonphagocytic epithelia.

After interacting with host epithelial cells, Salmonella internalizes by modifying phosphatidylinositol metabolism and regulating actin dynamics. Effectors secreted by SPI-1 play central roles in Salmonella invasion to epithelial cells. For instance, SipC organizes actin into bundles and enhances polymerization while SipA stabilizes actin filaments and prevents their disassembly. The Salmonella guanine nucleotide exchange factor (GEF) SopE and SopE2 activate Rho GTPases, including Rac1, Cdc42, and RhoG, thereby recruiting WAVE, N-WASP, Arp2/3 and other nucleator proteins. This activation prompts actin rearrangement and membrane ruffling. SopE forms a complex with Caveolin-1 and Rac1, further promoting membrane ruffling. SopB activates RhoG and recruits SNX9 and SNX18, aiding in the formation of membrane ruffles for Salmonella uptake. SopB also pulls EXOC2 from Rab10 membranes to invasion sites and works with SopE2 to recruit EXOC3 from the Golgi apparatus, which are crucial for the membrane delivery necessary for bacterial uptake. Meanwhile, SopD inhibits Rab10, leading to its removal and the subsequent recruitment of Dynamin-2, which initiates membrane scission to form Salmonella-containing vacuoles (SCVs) and promote bacterial invasion. While the effector SptP exerts its GAP function towards Rho GTPase to reverse actin rearrangements, thus helping to restore the host cell's cytoskeletal architecture. Created in https://BioRender.com.

2.1.1. SipC/SipA-dependent invasion

SipC is a translocon component of T3SS that integrates into the host cell plasma membrane to form a translocation pore (Park et al., 2018). SipC is involved in both the assembly of the T3SS needle complex and the translocation of additional effectors into the host cell. Furthermore, SipC also contributes to the actin rearrangements necessary for invasion. The N-terminal region of SipC is primarily involved in actin bundling. This domain promotes the organization of actin filaments into parallel bundles, which is important for forming stable structures that support the membrane ruffling necessary for bacterial uptake. While the C-terminal region of SipC exhibits actin-binding activity that specifically promotes actin polymerization. This domain interacts directly with actin monomers (G-actin) and enhances their conversion into filamentous actin (F-actin), which is also necessary for membrane ruffling and the formation of the bacterial uptake structure (HAYWARD & KORONAKIS, 1999). SipA is another important effector protein secreted by the T3SS1 that plays a critical role in the invasion of Salmonella into host cells. It contains amino acid motifs that are recognized by caspase-3, a key enzyme involved in apoptosis, which cleaves SipA into an active form. This cleavage activates SipA's capacity to interact with the actin cytoskeleton of the host cell. The C-terminal region of SipA possesses an actin-binding site that allows it to bind to F-actin. By binding to F-actin, SipA stabilizes the actin filaments and prevents their disassembly, thereby promoting actin polymerization and rearrangement (Niedzialkowska et al., 2024; Srikanth et al., 2010). Furthermore, SipA enhances the action of SipC by preventing the disassembly of actin filaments, which is typically mediated by actin-depolymerization factors such as ADF (Actin Depolymerizing Factor) and cofilin, as well as by inhibiting the severing of F-actin mediated by gelsolin (McGhie et al., 2004). Therefore, the collaboration between SipA and SipC exemplifies how Salmonella utilizes its effector proteins to orchestrate actin dynamics in host cells. By promoting actin polymerization, inhibiting depolymerization, and preventing severing of F-actin, these effectors create a stable and dynamic actin network that is essential for bacterial entry (McGhie et al., 2001).

2.1.2. SopE/SopE2/SopB/SopD-dependent invasion

Previous studies have shown that Salmonella activates Rho-family GTPases to regulate the actin cytoskeletal network during invasion (Brown et al., 2007). The guanine nucleotide exchange factors (GEFs) SopE and SopE2, secreted by T3SS1, activate the small Rho GTPases Rac1, Cdc42, and RhoG (Hänisch et al., 2011; Hardt et al., 1998; Stender et al., 2000). Activated Rac1 recruits and activates nucleation-promoting factors of WASP family (WAVE and N-WASP), Arp2/3, and other nucleator proteins. This process induces rearrangement of the actin cytoskeleton and stimulates cell membrane ruffling, thus facilitating the invasion of Salmonella into host cells (Chen et al., 1996; CRISS & CASANOVA, 2003; Ridley et al., 1992). Additionally, SopE enhances membrane ruffling and bacterial entry by forming a complex with Caveolin-1 and Rac1 (Lim et al., 2014). It has also been reported that the activation of phospholipase C gamma (PLCγ) and villin by SopE can depolymerize F-actin in polarized epithelial cells (PEC), which is essential for the dynamic remodeling of the cytoskeleton (Felipe-López et al., 2023). The dual functions of SopE-promoting actin polymerization through Rac1 activation and inducing depolymerization via PLCγ and villin-allow for a finely tuned response during bacterial invasion. This dual functionality underscores the complexity of bacterial effector proteins like SopE and their ability to exploit host cell signaling pathways to their advantage. Contrary to previous studies, Dr. Koronakis and colleagues recently reported that the activation of SopE2-Cdc42-N-WASP axis can block Salmonella invasion into host cells under certain conditions. Another effector SopA can interfere with Cdc42-N-WASP signaling. By modifying the signaling cascade through its ubiquitin ligase activity, SopA inhibits the activation of Cdc42, thereby preventing excessive actin polymerization (Davidson et al., 2023). This action of SopA creates a balance, allowing for efficient bacterial invasion while also ensuring that the process is self-limited. Notably, this self-regulation is crucial for the bacteria to avoid overstimulating the host immune response and causing excessive cellular damage.

SopB, also known as SigD, is an inositol phosphatase secreted by Salmonella T3SS1, playing a significant role in altering the host cell membrane's lipid composition. This modification is essential for recruiting various host proteins that facilitate bacterial invasion (Burkinshaw et al., 2012). SopB indirectly stimulates the endogenous transducing factor SH3-containing GEF (SGEF) through its phosphatase activity, which then activates the Rho-family GTPase RhoG, resulting in actin rearrangement (PATEL & GALáN, 2006). In addition, SopB recruits Rho GTPases, like RhoB, RhoD, RhoH, and RhoJ, to the invasion site through its catalytic activity, which then activates downstream signaling pathways. For instance, the activation of RhoB and RhoD leads to actin polymerization, contributing to the formation of membrane ruffles and facilitating the engulfment of Salmonella by host cells (Truong et al., 2018). Moreover, SopB also recruits SNX18, a SH3-PX-BAR structural domain-sorting nexin protein, to the plasma membrane via its inositol phosphatase activity, which alters the phosphoinositide landscape of the membrane. This recruitment occurs independently of the activation of Rac1 and Cdc42, indicating a direct effect of SopB on membrane dynamics. Once localized to the plasma membrane, SNX18, acts as a scaffold, recruits Dynamin-2 and N-WASP to the plasma membrane, which is essential for the formation of SCVs and the invasion of host cells by Salmonella (Liebl et al., 2017). Additionally, SopB recruits the sorting nexin 9 (SNX9) to the plasma membrane, where SNX9 plays a critical role in actin rearrangement and vesicle transport regulation. This recruitment leads to SNX9-mediated ruffling, which is essential for facilitating Salmonella invasion into host cells (Piscatelli et al., 2016).

However, the cytoskeletal changes can be reversed by another T3SS1 effector, SptP, a tyrosine phosphatase that exerts GTPase-activating protein (GAP) activity during Salmonella infection (Stebbins & GaláN, 2000). SptP specifically targets Rho family GTPases, including Cdc42 and Rac1, to exert its GAP function. By inactivating these GTPases, SptP inhibits the actin rearrangements typically induced by other Salmonella effectors, such as SopE, SopE2, and SopB, leading to decreased membrane ruffling and reduced efficiency of Salmonella invasion into host cells (Fu & GaláN, 1999). Interestingly, SopE's shorter half-life, resulting from host-mediated ubiquitination compared to SptP, suggests that the activation of Rho GTPases by SopE can be reversed following the secretion of SptP into host cells, which explains the transient nature of F-actin-mediated membrane ruffles formation and the self-limiting nature of Salmonella invasion in another way (Kubori & GaláN, 2003). Therefore, the balance between the pro-invasive effects of other effectors and the inhibitory effects of SptP is crucial for the success of Salmonella in invading and surviving within host cells. However, the mechanisms underlying different degradation rates of SopE and SptP via the ubiquitin-mediated proteasomal degradation pathway remain to be investigated.

In the context of bacterial pathogens like Salmonella, the exocyst complex is recruited to sites of invasion to facilitate membrane ruffling. The membrane source for the formation of these ruffles is derived from pre-existing tubular compartments associated with the plasma membrane. This process requires small GTPase Rab10, which stabilizes membrane reservoirs in its GTP-bound state through interactions with its effectors MICAL-L1, EHBP1, PACSIN2, and PACSIN3 (Farmer et al., 2021; Nichols & Casanova, 2010; Wang et al., 2016). It has been demonstrated that SopB, SopE2, and SipC recruit exocyst subunits from membrane reservoirs and cellular compartments, facilitating the assembly of the exocyst complex at the plasma membrane. Specifically, SopB facilitates the mobilization of membrane reservoirs and the recruitment of EXOC2 from Rab10 membranes to the invasion sites via its catalytic activity. Simultaneously, SopB and SopE2 work together to recruit EXOC3 from the Golgi apparatus, an interaction that requires SopB to bind to Cdc42 and its catalytic activity. Additionally, EXOC7 is recruited to the invasion sites, where it binds directly to SipC. These recruitments are crucial for delivering the membrane needed for bacterial uptake, as they contribute to the formation of membrane ruffles that engulf the bacteria (Boddy et al., 2021; Zhu, Sydor, et al., 2024). Interestingly, another effector SopD, secreted by both T3SS1 and T3SS2, inhibits SopB-mediated recruitment of Rab10 to invasion sites through its GAP activity, resulting in the removal of Rab10 and the recruitment of Dynamin-2, which initiates membrane scission to generate SCVs and ultimately facilitates bacterial invasion (Boddy et al., 2021). Therefore, SopD acts cooperatively with SopB by targeting Rab10 to induce plasma membrane scission, allowing Salmonella to effectively invade into host cells.

2.2. Effector-mediated inflammation

In general, inflammation is considered as a crucial host defense mechanism to eliminate invading pathogens. Upon microbial infection, like virus or bacteria, inflammatory responses are one of the outcomes of the recognition of pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) (Lian et al., 2018a, 2018b; Wei et al., 2017, 2018; Zang et al., 2020). However, this is not the case for non-typhoidal Salmonella, such as S. Typhimurium, for which the inflammatory response is critical for the pathogen's colonization of the intestinal tract (GaláN, 2021; Yu et al., 2023). It is well established that the endogenous protective barriers in the intestine, formed by metabolites of either host or resident microbial origin, typically serve to resist the invasion of bacterial pathogens into intestinal epithelial cells (Litvak & Bäumler, 2019; Rangan & Hang, 2017; Rogers et al., 2021). Previous studies have reported that intestinal inflammation leads to the disruption of protective barriers and alters nutrient availability. Consequently, the induction of inflammation in the intestinal tract allows S. Typhimurium to compete with the endogenous microbiota for carbon sources and electron acceptors, which are essential for bacterial metabolism and replication within the intestinal environment (Winter et al., 2010; Zeng et al., 2017). Thus, the stimulation of inflammatory responses is a survival strategy employed by Salmonella, rather than a host defense mechanism against Salmonella infection (Zeng et al., 2017).

It has been reported that the stimulation of inflammatory responses by Salmonella is strictly dependent on its functional T3SS (Bruno et al., 2009). On one hand, Salmonella utilizes T3SS to deliver a battery of effectors into host cells that stimulate inflammatory signaling (see below). On the other hand, the secreted effectors can also interfere with pro-inflammatory signaling or induce anti-inflammatory signaling, resulting in the inhibition of inflammation (see below). However, excessive inflammation leads to tissue damage. Therefore, decreasing inflammatory responses not only enable bacterial survival and replication but also preserve the host's homeostasis (Sun et al., 2016). The ‘Yin and Yang’ of T3SS effectors in modulating inflammatory responses is a remarkable example of the complex adaptations that arise from long-standing host-pathogen interactions (Fig. 2).

Fig. 2.

Effector-mediated inflammation.Salmonella uses T3SS effectors to directly stimulate innate immune responses in epithelial cells without activating actual innate immune receptors (depicted in green). SopE, SopE2, and SopB activate Cdc42, which in turn activates PAK1, leading to the formation of a signaling complex with TRAF6 and TAK1, ultimately resulting in the production of pro-inflammatory cytokines. SopA stimulates pro-inflammatory signaling by ubiquitinating the host E3 ubiquitin ligases TRIM56 and TRIM65, resulting in the production of pro-inflammatory cytokines and the expression of ΙFN-β. SopD amplifies inflammatory responses by antagonizing Rab8-dependent anti-inflammatory signaling pathways through its GAP activity toward Rab8. To maintain host cell homeostasis, Salmonella has evolved specific mechanisms to reduce inflammatory responses via its T3SS effectors (depicted in red). Some effectors antagonize pro-inflammatory pathways, including SptP, which utilizes its GAP activity to inactivate Cdc42 and Rac1; PipA, GtgA, and GogA, which cleave the NF-κB transcription factors RELA and RELB; SseK1, SseK2, and SseK3, which inhibit NF-κB signaling by transferring N-acetylglucosamine to specific arginine residues in the death structural domains; GogB, which prevents host SCF E3 ubiquitin ligase from degrading IκB to inhibit NF-κB signaling; SpvD, which impairs nuclear translocation of p65, thereby inhibiting the inflammatory response; IpaJ, which inhibits the activation of the MAPK signaling pathway; AvrA, which inhibits JNK signaling through acetylation of MAPKKs MKK4 and MKK7 and also exerts its inhibitory function on the NF-κB signaling pathway and SpvC, which inhibits the activation of ERK1, ERK2, and p38, leading to the cessation of downstream pro-inflammatory signaling and cytokine productions. Effectors can also inhibit inflammation by directly activating anti-inflammatory signaling pathways. These include SopB, which utilizes its phosphatidylinositol phosphatase activity to exert anti-inflammatory effects, triggering a signaling cascade involving PI3K, PDK1, and mTORC2. SopD activates Rab8-dependent PI3K-PKB-mTOR anti-inflammatory signaling cascades by stimulating the dissociation of Rab8 from its cognate GDP-dissociation inhibitor (GDI), thereby suppressing inflammation. SteE targets signal transducer and activator of transcription 3 (STAT3) to positively regulate anti-inflammatory signaling. Interestingly, SopB and SopD are two effectors that exhibit both anti-inflammatory and pro-inflammatory activities. Created in https://BioRender.com.

2.2.1. Effector-mediated promotion of inflammatory responses

Salmonella uses T3SS to directly stimulate innate immune responses in epithelial cells without activating actual innate immune receptors (Miao et al., 2010; Zhao & Shao, 2015). By activating the Rho GTPase Cdc42, SPI-1 T3SS effectors SopE, SopE2, and SopB not only facilitate actin cytoskeleton rearrangements for bacterial invasion into host cells but also stimulate pro-inflammatory responses that lead to intestinal inflammation (Hobbie et al., 1997; Patel & Galán, 2006). Upon activation by these effectors, Cdc42 promotes the p21-activated kinase (PAK1), a member of the PAK family, leading to the formation of a non-canonical signaling complex that includes PAK1, tumor necrosis factor-associated factor 6 (TRAF6), and transforming growth factor-beta (TGFβ)-activated kinase 1 (TAK1). Notably, TRAF6 and TAK1 are recognized as crucial components of signaling hubs downstream of multiple Toll-like receptors (Sun et al., 2018). This activation of Cdc42 subsequently stimulates the MAPK and NF-κB signaling pathways, ultimately resulting in the production of pro-inflammatory cytokines such as interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α). The addition of PAK inhibitors significantly reduced the production of IL-8 and TNF-α in the intestines infected with Salmonella (Chen et al., 1996; Sun et al., 2018). Therefore, by passing innate immune receptors, Salmonella employs effectors SopE, SopE2, and SopB to engage in innate immune signaling pathways downstream of the actual receptors, eliciting responses similar to those triggered by canonical innate immune receptors.

In addition to SopE, SopE2, and SopB, the T3SS effector SopD and SopA also help amplify the inflammatory responses. SopD functions to both activate and inhibit the inflammatory response by targeting the small GTPase Rab8 (see below). Another effector SopA is a HECT-type E3 ubiquitin ligase that has been reported to utilize the host's ubiquitylation machinery to exert its functions after delivery into host cells (Kamanova et al., 2016a; Zhang et al., 2006). It has been shown that SopA stimulates pro-inflammatory signaling by ubiquitinating the host E3 ubiquitin ligases TRIM56 and TRIM65, resulting in the production of pro-inflammatory cytokines and the expression of interferon-beta (IFN-β) (Fiskin et al., 2017; Kamanova et al., 2016b; Versteeg et al., 2013). TRIM proteins constitute a large family of E3 ubiquitin ligases with diverse functions (Hatakeyama, 2017). Specifically, TRIM56 and TRIM65 enhance the capacity of innate immune receptors, such as retinoic acid-inducible gene I protein (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), to stimulate IFN-β expression. Additionally, TRIM56 modulates innate immune responses by ubiquitinating and activating stimulator of interferon genes (STING), also known as MITA, leading to the induction of inflammation (Dixit & Kagan, 2013; Kamanova et al., 2016a; Zhang & Zhong, 2022; Zhong et al., 2008).

2.2.2. Effector-mediated inhibition of inflammatory responses

To maintain host cell homeostasis for their survival and replication, Salmonella has evolved specific mechanisms to reduce inflammatory responses via utilizing their T3SS effectors. As previously mentioned, the effector SptP employs its GAP activity to inactivate Cdc42 and Rac1, thereby counteracting the pro-inflammatory effects induced by SopE and SopE2 (Johnson et al., 2017). In addition to SptP, there are other effectors that possess the ability to negatively regulate pro-inflammatory signaling pathways. For example, PipA, GtgA, and GogA are three highly homologous Salmonella T3SS effectors (Jennings et al., 2017). They cleave the NF-κB transcription factors RELA and RELB, leading to the inhibition of pro-inflammatory responses (Baruch et al., 2011; Cerdà-Costa & Gomis-Rüth, 2014; Sun et al., 2016). Similarly, the T3SS secreted effectors SseK1, SseK2, and SseK3 share a high degree of homology with NleB1 secreted by E. coli T3SS, which is known to inhibit NF-κB signaling (Choy et al., 2004). These three effectors inhibit NF-κB signaling by transferring N-acetylglucosamine to specific arginine residues in the death domains of several key proteins, such as FAS-associated death domain (FADD) and TNF receptor 1-associated death domain (TRADD), within this signaling pathway (Günster et al., 2017; Newson et al., 2019). Another effector GogB inhibits NF-κB signaling by preventing the degradation of IκB, a protein that normally sequesters NF-κB in the cytoplasm. GogB exerts this function by interacting with the host's SCF E3 ubiquitin ligase complex, specifically with components such as Skp1 and FBXO22. By interfering with this pathway, GogB effectively down-regulates the host's inflammatory response during chronic infections, which helps to limit tissue damage and mitigate host pathology (Pilar et al., 2012, 2013). The effector SpvD interacts specifically with the export protein Xpo2. This interaction disrupts the nuclear-cytoplasmic recycling of importins which leads to defective nuclear translocation of p65, thereby inhibiting inflammatory responses and promoting systemic bacterial multiplication in mice (Rolhion et al., 2016). It has been demonstrated that Salmonella enterica serovar Pullorum utilizes the effector IpaJ to inhibit the ubiquitination of IκBα for degradation, which suppresses the NF-κB signaling pathway and the subsequent inflammatory response. Additionally, IpaJ inhibits the activation of the MAPK signaling pathway (Yin et al., 2022). AvrA, another effector secreted by T3SS1, is recognized as an acetyltransferase (Pruneda et al., 2016). It effectively inhibits JUN amino-terminal kinase (JNK) signaling by acetylating upstream kinases, specifically MAPKKs MKK4 and MKK7 (Jones et al., 2008; Labriola et al., 2018). In addition, AvrA exerts its inhibitory function on the NF-κB signaling pathway by suppressing the phosphorylation of both JNK and Beclin-1 (Jiao et al., 2020; Yin et al., 2020). This multifaceted approach allows AvrA to modulate key signaling pathways involved in the inflammatory response, contributing to the virulence of Salmonella. SpvC is a phosphothreonine lyase that plays a critical role in modulating host inflammatory responses during Salmonella infection. By directly inhibiting the activation of extracellular signal-regulated kinase1 and 2 (ERK1 and ERK2) as well as p38 MAPK, SpvC effectively halts downstream pro-inflammatory signaling pathways. This inhibition reduces the production of pro-inflammatory cytokines, such as IL-8 and TNF-α (Haneda et al., 2012; Zhu et al., 2007). In studies using mouse models, it has been observed that S. Typhimurium strains lacking either AvrA or SpvC resulted in more severe intestinal inflammation compared to wild-type strains, suggesting that both effectors play significant roles in modulating the host's immune response, allowing the bacteria to evade immune detection and establish infection (Wu et al., 2012; Zhou et al., 2024). It is well demonstrated that effectors can also inhibit inflammation by directly activating anti-inflammatory signaling pathways. Both SopB and SopD activate PI3K-mediated anti-inflammatory signaling, promoting the production of the anti-inflammatory cytokine IL-10, which helps to preserve host homeostasis (see below). The Salmonella SPI-2 T3SS effector SteE (also known as GogC, SarA, or PagJ) targets the signal transducer and activator of transcription 3 (STAT3) to positively regulate anti-inflammatory signaling. Interestingly, SteE converts the serine-threonine kinase GSK3 into a tyrosine kinase, and it can be phosphorylated by GSK3, which is essential for its activity (Gibbs et al., 2020). Moreover, phosphorylated SteE recruits GSK3 to STAT3 and promotes STAT3 phosphorylation by GSK3, leading to the activation of STAT3 and the up-regulation of the anti-inflammatory markers of IL-10 and IL-4Rα (Gaggioli et al., 2024; Jaslow et al., 2018; Liu et al., 2023). Remarkably, the up-regulation of IL-4Rα drives the polarization of M2 macrophages, enabling bacterial virulence proteins to reorganize innate immune signals and establish an anti-inflammatory environment (Stapels et al., 2018).

2.2.3. Effector-mediated dual-directional regulation of inflammation

Here are some interesting effectors that have both anti-inflammatory and pro-inflammatory activities. Specifically, SopD amplifies inflammatory responses by antagonizing Rab8-dependent anti-inflammatory signaling pathways (Lian et al., 2021). It has been demonstrated that Rab8 is associated with an anti-inflammatory pathway downstream of the Toll-like receptors, which leads to the recruitment of phosphoinositide 3-kinase (PI3K), activation of Akt, and a shift in cytokine production toward anti-inflammatory programs (Luo et al., 2018; Tong et al., 2021; Wall et al., 2017). Recently, Dr. Galan and colleagues reported that SopD exerts its pro-inflammatory function through its GAP activity toward Rab8, resulting in increasing production of the pro-inflammatory cytokines IL-1β and TNF-α, and decreasing the expression of anti-inflammatory factor IL-10 by antagonizing Rab8-mediated anti-inflammatory programs (Lian et al., 2021; Savitskiy & Itzen, 2021). Furthermore, the molecular interface between SopD and Rab8 suggests the existence of a potential additional function in SopD that is independent of its GAP activity. Interestingly, SopD activates Rab8-dependent PI3K-PKB-mTOR anti-inflammatory signaling cascades by stimulating the dissociation of Rab8 from its cognate GDP-dissociation inhibitor (GDI), thereby suppressing inflammation (Lian et al., 2021). Notably, in this process, the activity of SopD is comparable to that of eukaryotic GDI-displacement factors (GDF), as it activates Rab GTPase by displacing GDI (Sivars et al., 2003). Therefore, SopD is a representative example of dual-functional group effectors that target a single cellular component. The opposing regulatory effects of SopD on inflammation suggest that its roles during bacterial infection may vary over time. Because pro-inflammatory signaling precedes the generation of an anti-inflammatory response, the GAP activity of SopD should precede its Rab8-stimulating activity. Indeed, the pro-inflammatory response mediated by T3SS effectors occurs immediately after infection. To maintain host cell homeostasis, SopD exerts its Rab8-stimulating activity to initiate anti-inflammatory response along with other anti-inflammatory effectors. Nevertheless, the exact mechanism that determines which activity of SopD exerts at a given time during infection remains to be investigated.

Another dual-functional effector is SopB. SopB activates Cdc42 and stimulates downstream pro-inflammatory signaling pathways as previously described (Burkinshaw et al., 2012; Zhou et al., 2001). Meanwhile, SopB utilizes its phosphatidylinositol phosphatase activity to exert anti-inflammatory effects. SopB triggers a signaling cascade involving PI3K, PDK1, and mTORC2, which activates Akt. This activation leads to the phosphorylation of Yes-associated protein (YAP), which in turn inhibits the B-cell NLRC4 inflammasome, thus transforming the host environment into one that promotes survival (García-Gil et al., 2018).

2.3. Biogenesis of the Salmonella-containing vacuole

As previously mentioned, Salmonella employs multiple effectors to modulate the host cytoskeleton and complete membrane scission at the invasion sites for internalization. After internalization into the host cell, Salmonella establishes a unique replicative ecological niche known as the Salmonella-containing vacuole (SCV) (Cossart et al., 2003; Eswarappa et al., 2010; Raffatellu et al., 2005; STEELE-MORTIMER, 2008; ZHU, SYDOR, et al., 2024). The biogenesis and maturation of the SCV depend on the formation of a network of membranous tubules known as Salmonella Induced Tubules (SIT), as well as the movement of vacuole from the plasma membrane to the perinuclear region. The process of SCV biogenesis is accompanied by the recruitment of several small Rab GTPases and the participation of T3SS1 and T3SS2 effectors (Brumell & Scidmore, 2007; Tuli & Sharma, 2019; Vaughn & Abu kwaik, 2021). SCV biogenesis can be divided into three phases: early (10 minutes-1 hour post-infection), intermediate (1–4 h) and late (>4 h) (Ramos-Morales, 2012). To understand the molecular mechanisms underlying SCV biogenesis and maturation, it is essential to identify all the molecules involved. Fig. 3 summarizes the roles of T3SS effectors in SCV biogenesis.

Fig. 3.

Biogenesis of the Salmonella-containing vacuole.

After internalization into the host cell, Salmonella establishes a unique replicative ecological niche known as the Salmonella-containing vacuole (SCV). The biogenesis and maturation of SCV are dependent on the formation of a network of membranous tubules known as Salmonella Induced Tubules (SIT) and the movement of vacuole from the plasma membrane to the perinuclear region. SCV biogenesis can be divided into three phases: early (10 min-1 h post-infection), intermediate (1–4 h) and late (>4 h). Within a few minutes of early maturation, the SCV interacts with early endosomes. The T3SS effectors SopB and SopE recruit the small GTPase Rab5 to the SCV. The phosphatase activity of SopB leads to the accumulation of PI3P on SCV membranes, which in turn recruits EEA1, SNX1, and SNX3. Over the next 30–60 min, the SCV matures into late endosomes, marked by the loss of Rab5 and the recruitment of Rab7, LAMP1/LAMP2, and v-ATPase to the SCV membrane. SopF helps maintain the integrity of nascent SCVs. While SipA, mimicking an R-SNARE, recruits Syn8 to the SCV. In the intermediate phase, the SCV moves to the perinuclear microtubule organizing center (MTOC). This process is mediated by cytoplasmic dynein and Rab7, along with its downstream effector RILP, which position the SCV near the Golgi and MTOC. SPI-2 effectors SseF and SseG interact with the Golgi network-associated protein ACBD3 to anchor SCV to the Golgi network. In the later stage of infection, as the SPI-2 effectors translocate into the host cytosol, highly dynamic tubules known as Salmonella-induced filaments (SIFs) emerge from the perinuclear-localized SCVs. The SPI-2 effectors SifA, SopD2, PipB2, SteA, SseJ, SseF, SseG, and Ssek3 contribute to SIFs formation, whereas SpvB appears to have a negative effect on SIFs formation. In addition to these SPI-2 effectors, the SPI-1 effector SopB binds to and activates Cdc42 GTPase, which in turn recruits vimentin around SCVs to concrete SCVs, thereby promoting Salmonella replication. Created in https://BioRender.com.

Within a few minutes of early maturation, SCV undergoes interaction with early endosomes. The T3SS effectors SopB and SopE recruit the small GTPase Rab5 to the SCV membrane to promote the fusion of SCV with early endosomes. This interaction subsequently recruits the Rab5 downstream effector VPS34/p150, which is a phosphatidylinositol 3-kinase (PI(3)K) complex that generates the phosphoinositide (PI) PtdIns(3)P (Huotari & Helenius, 2011; Mallo et al., 2008; Mukherjee et al., 2001). Notably, the phosphatase activity of SopB leads to the accumulation of PI3P on the early SCV membranes, which maintains the binding of SCV membranes to Rabs and early endosome antigen 1 (EEA1). This accumulation inhibits the fusion of SCVs with lysosomes and prompts the recruitment of the sorting nexins SNX1 and SNX3, which are central for the formation of dynamic tubules known as spacious vacuole associated tubules (SVATs) (Bakowski et al., 2010; Braun et al., 2010; Hernandez et al., 2004; Terebiznik et al., 2002). Over the next 30–60 min, the maturation of SCV progresses to late endosomes, characterized by the loss of Rab5 and the recruitment of Rab7, lysosomal associated membrane protein 1 and 2 (LAMP1/LAMP2), as well as v-ATPase to the SCV membranes. This process requires the recruitment of SNX1 and SNX3 (Braun et al., 2010; Lahiri et al., 2010; Méresse et al., 1999; Scott et al., 2002). In addition to SopB, other T3SS1 effectors also play pivotal roles in SCV biogenesis and maturation. For example, the integrity of SCV is essential for its biogenesis. The T3SS effector SopF, known as a phosphoinositide binding effector protein, has been reported to bind to multiple phosphoinositides in protein-lipid overlays. It has been demonstrated that SopF associates with the host cell membrane by binding to phosphoinositides, thereby maintaining the integrity of nascent SCVs. Consequently, Salmonella has evolved to inject this unique T3SS effector to promote the stability of nascent SCVs (Lau et al., 2019). As previously mentioned, the effector SipA is essential for the invasion of Salmonella into host cells. It has also been reported that SipA, by mimicking an R-SNARE, recruits Syntaxin8 (Syn8) to the SCV and promotes the fusion of SCVs with early endosomes. This process prevents the maturation of SCVs into lysosomes, thereby promoting Salmonella survival (Singh et al., 2018).

In the intermediate phase, SCV moves from the invasion site to the perinuclear microtubule organizing center (MTOC). This process is mediated by cytoplasmic dynein and Rab7. GTP-bound Rab7 recruits its downstream effector RILP to position SCV in a dynein-dependent manner near the perinuclear region, bringing the SCV in close proximity to the Golgi apparatus and the MTOC (Guignot et al., 2004). The majority of SCVs reside in this region during the initial rounds of bacterial replication, forming a clustered microcolony of vacuoles. It has been demonstrated that the recruited v-ATPase in the SCV membrane acidifies SCV, which promotes the binding of T3SS-2 to the SCV membrane. This interaction subsequently leads to the secretion of SPI-2 effectors into the host cytoplasm (Rathman et al., 1996).

In the later stage of infection, as the SPI-2 effectors translocate into the host cytosol, highly dynamic tubules emerge from the perinuclear-localized SCVs. These tubules are known as Salmonella-induced filaments (SIFs). The SPI-2 effectors SifA, SopD2, PipB2, SseJ, SseF, SseG, SteA, and Ssek3 contribute to the formation of SIFs, whereas SpvB appears to have a negative effect on SIFs formation. (i) SifA plays important roles in SIFs formation. More data suggest that SifA functions as GEF to stimulate small G protein signaling events. It has been demonstrated that SifA has two distinct domains: the N-terminal domain binds to kinesin-interacting protein (SKIP), which links the SCV to the microtubular network; the C-terminal domain, which has a fold similar to SopE, exhibits GEF activity towards the small GTPase RhoA. RhoA subsequently binds to and activates another effector SseJ (Ohlson et al., 2008; Rosa-Ferreira & Munro, 2011). SseJ, which exhibits phospholipase A1, deacylase, and glycerophospholipid: cholesterol acyltransferase activity, has been reported to specifically bind to the GTP-bound form of the RhoA GTPase. This interaction activates the lipase activity of SseJ, which contributes to the esterification of cholesterol (Lossi et al., 2008; Nawabi et al., 2008). Cholesterol plays a crucial role in endocytic trafficking events and accumulates in the SCV. Notably, these four proteins-SifA, SKIP, RhoA, and SseJ-form a complex at regions of SseJ-induced membrane alterations, likely facilitating membrane tubulation through movement along microtubules. This process contributes to endosomal membrane tubulation (ET), indicating that Salmonella effectors manipulate host membranes to promote bacterial intracellular replication (Ohlson et al., 2008). Additionally, SifA inhibits the interaction between SKIP and the small GTPase Rab9 via its PH domain, thereby impairing Rab9-dependent mannose-6-phosphate receptor (M6PR) recruitment to the SCV membrane. M6PR is a marker of late endosomes that plays a crucial role in the transport of soluble lysosomal enzymes to lysosomes. Therefore, decreased M6PR recruitment to the SCV membrane dampens the recruitment of lysosomal enzymes to the SCV and inhibits the fusion of SCV with lysosomes, ultimately protecting intracellular Salmonella from host defenses (McGourty et al., 2012). As previously mentioned, SIFs formation requires kinesin instead of RILP and dynein. Interestingly, it has been demonstrated that SifA can interact with Rab7 to disrupt the Rab7-RILP-dynein motor complex, facilitating the centrifugal extension of tubules from the SCVs. This process creates additional protected spaces for bacterial replication (Harrison et al., 2004). (ii) The T3SS-2 effector SopD2 acts as an inhibitor of vesicle transport from the vacuole. On one hand, SopD2 inhibits RILP- and FYCO1-mediated microtubule-based trafficking, thereby preventing the delivery of SCV to lysosomes (D'Costa et al., 2015). On the other hand, SopD2 and SifA functionally interact with each other to antagonistically regulate SCV stability and the formation of SIFs. SopD2 impairs vesicle transport and the formation of tubules extending outward from SCVs by inhibiting the peripheral transport of kinesin-1-positive vesicles (Schroeder et al., 2010). This inhibition disrupts normal trafficking pathways, allowing the bacteria to evade lysosomal degradation and maintain a niche for replication within host cells. Additionally, recent studies have reported that both SopD2 and another effector, PipB2, bind to host protein annexin A2 (AnxA2) to promote the positioning of SCVs within host cells. This interaction is important for the formation of the actin nest around the SCV and leads to alteration in the host's endosomal system, which helps stabilize the SCV and create a more favorable environment for bacterial replication (Knuff-Janzen et al., 2021). PipB2 is translocated into host cells through T3SS-2, where it localizes to SCVs and SIFs. PipB2, along with the host GTPase ARL8B, has been shown to be important for recruiting kinesin 1 to the SCV and endosomal tubules, leading to the elongation of these tubles (Rosa-Ferreira & Munro, 2011). Importantly, the SifA/SKIP complex is essential for activation of the kinesin-1 recruited by PipB2. This functional interaction is vital for the formation of tubules involved in membrane exchange and the movement of SCVs within host cells (Alberdi et al., 2020). By facilitating these processes, PipB2 enhances the ability of Salmonella to manipulate the host's cytoskeletal dynamics, thereby promoting bacterial replication and survival in the intracellular environment. (iii) T3SS-2 effectors SseF and SseG have been reported to associate with SCV membrane and are necessary for SIFs formation. These two effectors play a crucial role in manipulating the host cell's cytoskeletal dynamics and membrane trafficking processes (Krieger et al., 2014; Moest et al., 2018). SseF and SseG not only colocalize with microtubules but also induce significant bundling of these structures, which serve as a scaffold for the formation of SIF. This bundling is crucial for the structural integrity and stability of SIFs (Domingues et al., 2014). In addition to their role in SIFs formation, SseF and SseG interact with the mammalian Golgi network-associated protein ACBD3 to anchor SCV to the Golgi network (Yu et al., 2016). Understanding these interactions could provide deeper insights into the mechanisms by which Salmonella manipulates host cell processes to establish and maintain its intracellular niche. The effector SteA also plays an important role in the SIF biogenesis (Knuff-Janzen et al., 2020). SteA can be secreted into epithelial cells and macrophages through both T3SS1 and T3SS2 (Geddes et al., 2005). It has been demonstrated that a virulence function of SteA could be related to its delivery into host cells by the SPI-2 T3SS. Secreted SteA specifically binds to PI(4)P, and this binding is necessary for its localization on SCV membrane and Salmonella-induced tubules. SteA can regulate the activity of microtubule motors, such as dynein and kinesin-1, which leads to decreased SIFs formation and increased clustering of SCVs. As previously mentioned, the clustering and positioning of SCVs are also managed by the effectors SseF and SseG, suggesting that these three effectors work together to maintain a balance in the activity of microtubule motors to control SCV membrane dynamics (Domingues et al., 2014). By coordinating their functions, these three effectors ensure proper SCV membrane dynamics, which is critical for the successful establishment and maintenance of Salmonella infection. (iv) The SPI-2 effector SseK3 inhibits SNARE pairing through Arg-GlcNAcylation of SNARE proteins, such as SNAP25, VAMP8, and Syntaxin, which facilitates the formation of SIFs. In response to the presence of SseK3, host cells activate the E3 ubiquitin ligase TRIM32, which leads to K48-linked ubiquitination of SseK3 and the subsequent degradation of its membrane-associated segment (Meng et al., 2023). A recent screening identified potential interactions between other SPI-2 effectors, including SseG, SopD2, PipB2, and SifA, with proteins in the SNARE complex. This finding suggests that multiple SPI-2 effectors may collaboratively regulate SNARE pairing dynamics, further facilitating SIF biogenesis during Salmonella infection (D'Costa et al., 2019). Future research should focus on elucidating the interplay among these various effectors and their roles in modulating SNARE dynamics. Understanding the temporal and spatial regulation of these interactions will provide deeper insights into the mechanisms that Salmonella employs to successfully infect host cells and evade immune responses. In addition to these SPI-2 effectors, a recent study has reported that SopB binds to and activates Cdc42 GTPase, which in turn recruits vimentin around SCVs to concrete SCVs, thereby promoting Salmonella replication (Zhao et al., 2023).

2.4. Effector-mediated cell death

Cell death pathways play a crucial role in the host's immune response to bacterial infection. Based on molecular mechanisms and morphological characteristics, cell death is classified into two main categories: programmed cell death (PCD), such as apoptosis and pyroptosis, and non-programmed cell death (non-PCD), known as necrosis (Nisa et al., 2022). It has been demonstrated that Salmonella can manipulate signaling pathways associated with cell death in host cells, allowing it not only to evade host immunity but also to promote its dissemination through both T3SS-dependent and T3SS-independent mechanisms (Guiney, 2005). In this section, we primarily discussed Salmonella T3SS effectors-modulated cell death pathways (see below).

2.4.1. Effector-mediated apoptosis

Apoptosis is a form of programmed cell death that can be initiated by extrinsic or intrinsic stresses. This process involves the activation of initiator caspases, which activate executioner caspases, such as caspase-3 and caspase-7, leading to the formation of apoptotic bodies. These apoptotic bodies express “eat me” signals and therefore are recognized and phagocytosed by phagocytic cells without causing inflammatory responses (Elmore, 2007). During bacterial infection, certain secreted effectors can trigger cell death responses by initiating extrinsic apoptosis. For instance, SpvB, an ADP-ribosylase, promotes apoptosis in human monocyte-derived macrophages (HMDMs), possibly due to the loss of polymerized F-actin and the activation of caspase-3. However, the precise mechanisms by which SpvB promotes apoptosis remain unclear (Libby et al., 2000; Stepien Taylor et al., 2024; Wemyss & pearson, 2019). Additionally, Salmonella utilizes its secreted effectors to prevent and delay apoptosis, which is beneficial for the pathogen to establish a stable intracellular niche for proliferation. The effector SopB interacts with TRAF6 to block its recruitment to mitochondria and inhibit the accumulation of reactive oxygen species (ROS) within mitochondria, thus preventing endogenous apoptosis (Ruan et al., 2014, 2016). Akt kinase is a central regulator of cell survival. Interestingly, SopB facilitates Akt activation by activating Rho GTPase, such as RhoB and RhoH, to promote cell survival and inhibit downstream apoptotic responses (Chang et al., 2003; Ruan et al., 2016). Another effector, AvrA, has been reported to prevent apoptosis induced by exogenous stimuli through inhibiting JNK MAPK apoptotic pathway, allowing pathogen to establish a stable intracellular niche for replication (Haraga & Miller, 2006; Wu et al., 2012).

2.4.2. Effector-mediated pyroptosis

Pyroptosis is another form of programmed cell death characterized by the activation of inflammatory caspases, which leads to cell lysis and the release of pro-inflammatory cytokines. This process plays a crucial role in the immune response to infections, particularly those caused by intracellular pathogens, such as Salmonella enterica. The canonical cell death mechanism depends on caspase-1 and downstream gasdermin. The activated caspase-1 cleaves pro-IL-1β and pro-IL-18 to produce mature IL-1β and IL-18. Meanwhile, it can also cleave gasdermin and release a 31 kDa N-terminal domain of this protein that has a pore-forming activity, which is the final and direct executor of pyroptotic cell death (Yu et al., 2021). Salmonella infection can induce pyroptosis in host cells, particularly in macrophages. This response is part of the host's immune defense mechanism aimed at eliminating the bacteria. The recognition of Salmonella by intracellular pattern recognition receptors (PRRs) triggers inflammasome activation, resulting in subsequent pyroptosis (Broz & Monack, 2011; Fattinger et al., 2023). However, Salmonella has evolved various strategies to evade pyroptosis, allowing it to survive and replicate within host cells. Indeed, several Salmonella T3SS effectors enable to modulate the cellular pyroptosis pathway. Upon Salmonella infection, the secreted effector SopB triggers the PI3K-Akt-YAP pathway to down-regulate the transcription of NLRC4 inflammasome in B cells, leading to the inhibition of IL-1β secretion and promotion of cell survival (García-Gil et al., 2018). Another effector, SpvC, has been reported to suppress pyroptosis in macrophage and NETosis in neutrophil through its phosphothreonine lyase activity. This mechanism involves the subversion of GSDMD activity via both canonical and non-canonical inflammasomes, as well as the suppression of NLRC4 through an ASC-independent manner (Zhou et al., 2024; Zuo et al., 2020). However, Salmonella secreted effectors can also induce pyroptosis. For example, SopE not only activates host cellular Rho GTPases to trigger bacterial invasion but also activates caspase-1 to produce mature IL-1β and IL-18. SopE-mediated caspase-1 activation mostly depends on its guanine nucleotide exchange factor (GEF) activity (Hoffmann et al., 2010). SipB interacts with SipC to form a translocator pore, which is sufficient to facilitate translocation of SPI-1 effectors into the host cells. Besides that, SipB directly interacts with and activates caspase-1, which in turn promotes the maturation and secretion of IL-1β and IL-18 in macrophages (Hersh et al., 1999).

2.4.3. Effector-mediated necroptosis

Necroptosis, which serves as a link between host cell death and inflammation, is mediated by various cytokines and pattern recognition receptors (PRRs). This process involves the activation of receptor-interacting protein kinase 3 (RIPK3), which phosphorylates mixed-lineage kinase domain-like protein (MLKL), ultimately leading to the formation of necrosome complex. Necroptosis is characterized by the swelling of cells, loss of membrane integrity, and release of cellular contents, which can lead to inflammation (Seo et al., 2021). Salmonella infection can induce necroptosis in host cells, which serves as a defense mechanism to eliminate infected cells and promote inflammation; however, it can also lead to tissue damage and contribute to the pathology of the infection. Indeed, several Salmonella T3SS effectors have been reported to modulate necroptosis in host cells. The effectors SseK1 and SseK3, known as arginine glycosyltransferases, inhibit NF-κB activation and necroptotic cell death in infected macrophages by mediating the GlcNAcylation of the death domain-containing proteins FADD and TRADD (Günster et al., 2017; Pan et al., 2014). Another effector, SpvB, has recently been reported to increase the protein levels of RIPK3 by suppressing its degradation. The accumulation of RIPK3 leads to the up-regulation of MLKL phosphorylation, which contributes to necroptosis in epithelia cells (Dong et al., 2022). Recent studies have demonstrated that caspase-8 can cleave RIPK1 and RIPK3, thereby preventing necroptotic cell death. Additionally, caspase-8 plays a crucial role in mediating both extrinsic apoptosis and pyroptosis. Interestingly, a recent study showed that the Salmonella effector SopF activates the PDK1-RSK signaling pathway to inhibit caspase-8 activation in intestinal epithelial cells, leading to the inhibition of apoptosis and pyroptosis while promoting necroptosis. This mechanism contributes to the systemic infection caused by Salmonella (Yuan et al., 2023).

2.5. Effector-mediated innate immune evasion

The innate immune system is the first line of host defense against pathogen infections, playing a central role in inhibiting and eliminating invading pathogens (Chen & Gao, 2024; Lian et al., 2018a, 2018b; Wei et al., 2017, 2018). The first immune cells to respond to intracellular pathogen, such as Salmonella, are macrophages and dendritic cells, which eliminate intracellular bacteria through a series of mechanisms such as xenophagy and cell-intrinsic defense (Randow et al., 2013). These cells are necessary for the early local control of infection and, subsequently, for the induction of acquired adaptive immunity. However, as a successful intracellular pathogen, Salmonella has responded to these host defense strategies by evolving virulence factors, such as secreted effectors, to evade or inhibit host antibacterial mechanisms and promote successful intracellular survival (Reddick & Alto, 2014). Herein, we mainly focus on the mechanisms by which Salmonella secreted effectors manipulate bacteria-induced autophagy and cell-intrinsic defense (see below).

2.5.1. Inhibition of autophagy

Autophagy is a metabolic adaptation triggered by various stresses in host cells. It is characterized by the lysosomal degradation of cytosolic or vesicular components, including bacteria. Autophagy is an essential component of the innate immune system, contributing significantly to the intracellular clearance of pathogens (Yang et al., 2024; Zhu, Liu, et al., 2024). In the case of Salmonella-infected host cells, both non-selective autophagy and selective autophagy can be triggered to clear bacteria (Wang et al., 2022). However, Salmonella has developed sophisticated strategies to manipulate the autophagic process, allowing it to evade host immune responses. Indeed, Salmonella can secrete certain effectors to inhibit autophagy or prevent the fusion of autophagosomes with lysosomes, allowing bacterial survival within host cells. By modifying phosphoinositide levels, the SPI-1 effector SopB activates mTORC1, a well-known inhibitor of autophagy. This activation promotes mTORC1-mediated phosphorylation of ULK1, resulting in decreased levels of LC3-II in infected B cells. Consequently, this suppresses autophagy and promotes Salmonella survival (Luis et al., 2022). It has been shown that vacuolar ATPase (v-ATPase)-ATG16L1 axis can initiate bacteria-induced autophagy. Notably, the T3SS1 effector SopF, an ADP-ribosyltransferase, specifically modifies Gln124 of ATP6V0C in v-ATPase for ADP-ribosylation, which blocks the recruitment of ATG16L1 to bacteria-containing vacuole by v-ATPase, thereby preventing the degradation of Salmonella and other intracellular pathogens. This allows Salmonella to evade autophagic clearance and persist within host cells (Xu et al., 2019, 2022). Additionally, another effector, AvrA, suppresses autophagy by reducing Beclin-1 protein levels through the c-Jun N-terminal kinase (JNK) pathway (Jiao et al., 2020).

It should be noted that Salmonella not only inhibits autophagy initiation but also disrupts autophagosome formation via its T3SS effectors. For example, both SseF and SseG inhibit autophagosome formation by interfering with Rab1A signaling. By interacting with Rab1A, a GTPase, these two effectors disrupt the association between Rab1A and the transport protein particle III complex, which acts as a guanine nucleotide exchange factor (GEF) for Rab1A. This disruption blocks the activation of Rab1A and then subsequently inhibits the recruitment and activation of Unc-51-like autophagy-activating kinase-1, ultimately suppressing autophagosome formation (Feng et al., 2018).

2.5.2. Modulation of cell autonomous immunity

Cell-autonomous immunity is an intrinsic and ubiquitous form of host defense against intracellular pathogen infection, playing a crucial role in inhibiting and eliminating invading pathogens into primary macrophages and dendritic cells (DCs). Recently, Dr. Galan and colleagues reported that the small Rab GTPase Rab32 facilitates the delivery of an antimicrobial metabolite itaconate, produced in mitochondria, to SCV by associating with IRG1, impairing intracellular Salmonella Typhi replication (Chen et al., 2020; Spanò & Galán, 2012). Furthermore, Parkinson's disease-associated leucine-rich repeat kinase 2 (LRRK2) orchestrates this itaconate-dependent cell-intrinsic defense by scaffolding a complex between Rab32 and IRG1, facilitating itaconate local synthesis and subsequent delivery to SCV. Interestingly, this study demonstrated a robust association of mitochondria with SCV in Salmonella-infected cells, which requires LRRK2 but not Rab32 guanine nucleotide exchange factor BLOC-3. This finding suggests that Rab32 is dispensable for the tethering of the SCV to the mitochondria, although it is essential for the delivery of itaconate to the SCV. However, the mechanisms by which LRRK2 is involved in this association remain unclear (Lian et al., 2023) (Fig. 4). Meanwhile, other groups also reported that IRG1-dependent itaconate production promotes lysosomal biogenesis by activating the transcription factor TFEB to promote antibacterial innate immunity (Zhang et al., 2022). TFEB activation, in response to bacterial stimuli, promotes the transcription of IRG1 and the synthesis of its product itaconate. Activation of the TFEB-IRG1-itaconate signaling axis reduces the survival of the intravacuolar pathogen Salmonella Typhimurium. This group also demonstrated that TFEB-driven itaconate is subsequently transferred into the SCV via the IRG1-Rab32–BLOC3 system, thereby exposing the pathogen to elevated itaconate levels (Schuster et al., 2022). Although itaconate has regulatory activities in multicellular responses, including inflammation, the Rab32-BLOC3-mediated pathogen restriction mechanisms are not related to these processes. Instead, they involve the direct delivery of itaconate to bacterial-containing vacuoles. Notably, S. Typhimurium can neutralize LRRK2-Rab32-itaconate-dependent restriction mechanism by delivering two effectors SopD2 and GtgE, but S. Typhi does not express these two effectors. GtgE is a cysteine protease that specifically cleaves three Rab GTPases, including Rab32, Rab38, and Rab29, cleavage of G59 in Rab32 leads to conformational inactivation of this GTPase. SopD2 acts as a GAP towards Rab32 to deactivate Rab32 via the catalytic arginine residue in its C-terminus (Chen et al., 2020; Kohler et al., 2014; Lian et al., 2023; Savitskiy et al., 2021; Spanò, Gao, Hannemann, Lara-Tejero, & GaláN, 2016; Spanò & Galán, 2012). In addition to these two effectors, whether other Salmonella secreted effectors regulate the itaconate-dependent cell-intrinsic defense still needs to be investigated. As previously mentioned, following Salmonella invasion into host cells, SCV develops a tight association with mitochondria, while interestingly, Salmonella was observed to deploy a type III secretion system at the SCV-mitochondrial interface, suggesting that Salmonella has the ability to modulate the mitochondria-SCV interactions through the delivery of T3SS effectors. So far, host and bacterial proteins mediating or regulating the interaction between SCV and mitochondria remain unclear, which need further investigation (Fig. 4).

Fig. 4.

Modulation of cell autonomous immunity.

Cell-autonomous immunity is an intrinsic and ubiquitous form of host defense against intracellular pathogen infection. Parkinson's disease-associated leucine-rich repeat kinase 2 (LRRK2) orchestrates Rab32-IRG1-itaconate-dependent cell-intrinsic defense by scaffolding a complex between Rab32 and IRG1, facilitating itaconate local synthesis and subsequent delivery to SCV, ultimately impairing intracellular Salmonella Typhi replication. Notably, S. enterica serovar Typhimurium (S. Typhimurium) can neutralize LRRK2-Rab32-itaconate-dependent restriction mechanism by delivering two effectors SopD2 and GtgE. These two effectors exert their functions by targeting Rab32 with different biochemical activities. Remarkably, upon Salmonella infection, the Salmonella-containing vacuole (SCV) and the mitochondria establish a very close association, this association requires LRRK2 but not Rab32 guanine nucleotide exchange factor BLOC-3 (not depicted in this Figure). Created in https://BioRender.com.

2.6. Effector-mediated DCs migration and antigen presentation

Phagocytes bridge innate immunity and acquired immunity by presenting antigens to T cells, thus promoting the formation of long-term immunity. The activation of CD4⁺ T cells is central for the suppression of Salmonella systemic infection during persistence phase. Therefore, to establish a long-term systemic infection, Salmonella must disrupt the normal functions of DCs (Cerny & Holden, 2019; Cheminay et al., 2005; Halici et al., 2008). Studies have shown that the T3SS2 effector SseI blocks the migration of primary macrophages and DCs by associating with the host factor IQ motif containing GTPase activating protein1(IQGAP1), an important regulator of cell migration, which is required for Salmonella to maintain a long-term systemic infection (McLaughlin et al., 2009). Other effectors, including SseF, SifA, SspH2, SlrP, and PipB2, may also inhibit DC migration, however, the exact mechanisms remain unclear (McLaughlin et al., 2014). Studies have reported that the Salmonella SPI-2 secreted effector SteD plays important roles in inhibiting of DC-mediated antigen presentation to CD4⁺T cells (Yrlid et al., 2001). Mechanistically, SteD recruits the E3 ligase MARCH8 to membrane-bound MHC class II (mMHC II), leading to its ubiquitination. This process targets mMHC II for degradation, resulting in decreased levels on the cell surface and subsequently diminishing T cell activation (Bayer-Santos et al., 2016). In addition to inhibiting T cell activation by reducing the levels of cell surface proteins such as MHC II, SteD also degrade CD97, which stabilizes the immune synapse between DCs and T cells, through ubiquitination mediated by Tmem127/Wwp2. By degrading CD97, SteD disrupts the stability of the immune synapse between DCs and T cells. This leads to impaired T cell activation and a reduced immune response, allowing the bacteria to evade detection and destruction by the host immune system. Overall, SteD plays a significant role in modulating host immune responses by suppressing T cell immunity via two distinct mechanisms (Cerny et al., 2021; Godlee et al., 2022).

3. Conclusions and future perspectives

The ability of Salmonella to invade and survive in various mammalian cells, and to establish systemic infections, is closely linked to the function of effectors secreted into host cells via its T3SS. These secreted effectors target different host proteins to alter various host cellular processes, such as actin rearrangements for internalization, inflammatory responses, the biogenesis of SCV, and host cell survival. Moreover, Salmonella also uses these effectors to resist immune responses of the host through blockage of innate immunity and disruption of antigen presentation. These mechanisms enable Salmonella to manipulate host cellular physiological activities, promoting its survival within the host.

Considerable progress has been made in our understanding of how Salmonella utilizes its secreted effectors to make intracellular survival and to establish systemic infection. So far, more than 40 T3SS effectors have been identified, and the host targets and the biological functions of some of them have been extensively studied in the last years. However, the biochemical activities and functions of numerous effectors remain poorly characterized, and further identification of novel effectors that are critical for bacterial pathogenesis is needed. Notably, bacterial effectors are delivered in low concentrations, thereby targeting to precise subcellular locations not only increase the concentration of these effectors but also enable the engagement of the accurate targets of them for their biological functions. Therefore, it is central to understand the accurate subcellular localization of translocated effectors following delivery into host cells. However, systemic identification of the subcellular localization of bacterial effectors is less studied, and visualization of the secreted effectors in live cells during infection is still challenging. Additionally, fully understanding the accurate subcellular localization of bacterial effectors is also important for studying the molecular details of the associations between different organelles and SCV. Moreover, it has been shown that SPI-1 and SPI-2 seem to be more inclined to function at specific temporal phases during Salmonella infection, perhaps there are hidden mechanisms regulating the temporal variability of SPI-1 and SPI-2 expressions. Overall, this review summarizes the survival strategies employed by Salmonella to manipulate host cellular processes and the pathogenic mechanisms. Further studies are needed to help us understand the mechanisms of the interactions between Salmonella and hosts in bacterial infection and pathogenesis. Such studies are expected to enhance our understanding of the pathogenic processes of Salmonella as well as provide new insights into the development of novel therapeutic strategies for infectious diseases.

CRediT authorship contribution statement

Hongyu Zhao: Writing – original draft, Formal analysis, Conceptualization. Xinyue Zhang: Writing – original draft, Formal analysis. Ningning Zhang: Writing – review & editing. Li Zhu: Writing – review & editing. Huan Lian: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Conflict of interest

The authors state that the manuscript was conducted without any commercial or financial relationships that could be considered as a potential conflict of interest.