Mechanisms and strategies of host xenophagy manipulation by intracellular bacteria

Abstract

Xenophagy, a specialized form of autophagy, serves as a critical cellular defense mechanism, targeting and degrading intracellular bacterial pathogens. This review explores the intricate interplay between bacteria and host xenophagy, focusing on the mechanisms that underlie xenophagic recognition, encapsulation, and degradation of bacterial invaders. Despite xenophagy’s essential role in host immunity, many bacteria have evolved sophisticated strategies to evade, inhibit, or even subvert this process, ensuring their survival within host cells. We provide an in-depth analysis of the bacterial mechanisms that counteract xenophagy, detailing how specific pathogens manipulate autophagosome formation, acidification, and autophagosome-lysosome fusion. Furthermore, we highlight outstanding research questions, including the mechanisms of autophagosome formation at the bacterial surface, the interaction between xenophagy and the immune system, and the impact of xenophagy on bacterial antibiotic resistance. By examining the evolving understanding of xenophagy and bacterial countermeasures, this review underscores the potential for therapeutic strategies targeting xenophagy to enhance host defense against persistent intracellular bacterial infections.

Introduction

Autophagy is a highly conserved cellular process that mediates the degradation and recycling of cytoplasmic materials. It can be broadly divided into non-selective and selective autophagy. Non-selective autophagy is often induced by nutrient deprivation and degrades cytosolic components in bulk, whereas selective autophagy targets specific cargoes for degradation. Selective autophagy pathways are commonly classified according to their cargo, such as mitophagy (mitochondria), lysophagy (lysosomes), aggrephagy (aggregated proteins and RNA), xenophagy (intracellular pathogens), ER-phagy (endoplasmic reticulum), and lipophagy (lipid droplets) [1]. Dysregulation of selective autophagy has been linked to diverse diseases, including neurodegeneration, infection, metabolic disorders, cancer, and aging, making these pathways attractive targets for therapeutic intervention [2].

Xenophagy is a specialized form of selective autophagy that targets intracellular pathogens, including bacteria, viruses, and parasites, for lysosomal degradation [3, 4]. Although the term “xenophagy” can be traced back to the 1980 s [5], its molecular mechanisms began to be systematically investigated only in the early 21 st century [6]. Xenophagy functions in both phagocytic and non-phagocytic cells, including epithelial cells, providing a cell-autonomous defense against infection [7]. In general, host recognition of intracellular pathogens leads to the recruitment of selective autophagy receptors and autophagy-related proteins to pathogen-associated sites, thereby initiating xenophagy and promoting lysosomal degradation.

Intracellular bacterial pathogens pose a major challenge to global health. Mycobacterium tuberculosis (Mtb), Brucella spp., and Salmonella spp. cause tuberculosis, brucellosis, and foodborne infections, respectively, resulting in substantial morbidity and mortality worldwide [8–10]. The increasing prevalence of antimicrobial resistance further limits the effectiveness of conventional antibiotic-based therapies. In this context, host-directed therapy (HDT) has emerged as a complementary strategy that aims to enhance host antimicrobial pathways rather than directly target the pathogen. Because xenophagy represents a central cell-autonomous mechanism for restricting intracellular bacteria, a deeper understanding of its regulation may help identify host-derived therapeutic targets for controlling bacterial persistence, chronic infection, and drug resistance.

In this review, we discuss the interplay between intracellular bacteria and host xenophagy. We focus on how host cells recognize and target bacteria for autophagic degradation, and how bacterial pathogens evade, inhibit, or exploit xenophagy to promote intracellular survival.

The mechanism of xenophagy

Xenophagy utilizes core autophagy machinery and proceeds through several coordinated steps, including signal initiation, membrane elongation, cargo targeting, autophagosome formation and fusion with lysosomes, and cargo degradation or recycling [1]. Although xenophagy is a crucial defense mechanism against intracellular pathogens, its molecular regulation remains incompletely understood [11]. Intracellular bacteria, such as Mtb, Brucella spp., Salmonella spp., and Listeria monocytogenes (L. monocytogenes), can survive within host cells after invasion. Many of these bacteria initially reside within phagosomes or pathogen-containing vacuoles. However, some pathogens can damage the limiting membrane of these compartments or gain access to the cytosol, thereby exposing themselves to cytosolic immune surveillance. In response, host cells activate xenophagy-related pathways to target cytosol-exposed bacteria, damaged bacteria-containing vacuoles, or pathogen-associated signals. The following sections will describe these processes in detail.

Xenophagy targeting bacteria-containing phagosomes

After entering host cells, many intracellular bacterial pathogens are initially enclosed within phagosomes or bacteria-containing vacuoles. These bacteria may follow several possible fates: (1) They may be degraded through phagosomal acidification, maturation, and fusion with lysosomes, collectively referred to as the phagolysosomal pathway. (2) They may be targeted by LC3-associated phagocytosis (LAP), a phagocytosis-coupled form of non-canonical autophagy and a phagosome-associated example of conjugation of ATG8 to single membranes (CASM). During LAP, components of the autophagy machinery conjugate LC3/ATG8-family proteins to the single limiting membrane of bacteria-containing phagosomes, rather than to newly formed double-membrane autophagosomes [12, 13]. In classical LAP, LC3 recruitment to phagosomes is strongly linked to NADPH oxidase/NOX2-derived ROS production and involves LAP-associated regulators such as Rubicon, which distinguishes LAP from canonical autophagy [13, 14]. This LC3 decoration promotes phagosome maturation, fusion with lysosomes, and degradation of the engulfed bacteria. More broadly, LAP can be regarded as a specific phagosome-associated subset of CASM, whereas CASM also includes ATG8 lipidation on other endolysosomal single membranes triggered by lysosomal stress, ion perturbation, STING activation, or pharmacological disruption of endosomal homeostasis. These CASM pathways often involve the ATG16L1 WD40 domain and the V-ATPase–ATG16L1 axis but are not necessarily dependent on phagocytosis or NADPH oxidase [15, 16]. (3) Some bacteria may escape into the cytosol by secreting virulence factors that disrupt the phagosomal membrane, and a subset of these cytosolic bacteria can be eliminated by xenophagy [17]. (4) Upon sensing infection or phagosomal damage, host cells may recruit ubiquitin to bacteria-containing phagosomes or exposed bacterial surfaces. This ubiquitin labeling promotes the recruitment of selective autophagy receptors, such as p62, NDP52, and OPTN, which further recruit the autophagy machinery and facilitate LC3 lipidation and autophagosome formation. The resulting autophagosomes subsequently fuse with lysosomes, leading to degradation of the bacterial cargo. However, the mechanisms by which host cells sense bacteria-containing phagosomes and initiate ubiquitin recruitment remain incompletely understood. In particular, how different host cell types detect and respond to damaged bacteria-containing phagosomes remains unclear. Further studies are needed to clarify these processes.

Phagosomal membrane disruption is an important danger signal for the initiation of xenophagy. When the limiting membrane of bacteria-containing vacuoles is damaged, luminal host glycans become exposed to the cytosol and are rapidly recognized by cytosolic galectins. The accumulation of galectins at damaged vacuoles not only marks membrane injury but also provides a platform for recruiting downstream autophagy machinery. A well-established example is provided by Salmonella-containing vacuoles, where galectin-8 binds exposed host glycans and recruits the autophagy receptor NDP52 to activate antibacterial xenophagy [18–20].

This galectin-based damage-sensing model has also been extended to Mtb infection. Work from the Deretic laboratory showed that galectin-8 is not merely a marker of damaged endomembranes, but can also act as a signaling molecule. Upon lysosomal injury, galectin-8 associates with the lysosomal mTOR machinery and suppresses mTOR activity through the Ragulator–Rag pathway, thereby promoting autophagic responses involved in host defense against Mtb [21]. Work from the Gutierrez laboratory further demonstrated that galectin-8 marks Mtb-induced phagosomal membrane damage in human lymphatic endothelial cells [22]. Building on these findings, Bell et al. showed that galectin-8 recognizes damaged Mtb-containing phagosomes in macrophages and promotes anti-Mtb xenophagy through its interaction with the selective autophagy adaptor TAX1BP1 [23]. Although galectin-3, galectin-8, and galectin-9 can all be recruited to Mtb-containing phagosomes that colocalize with ubiquitin, p62, and LC3, galectin-8 plays a particularly important role in TAX1BP1 recruitment, xenophagic targeting, and restriction of Mtb growth [23]. Other galectins also participate in antibacterial responses. For example, galectin 1 and galectin 7 are recruited to Group A Streptococcus (GAS)-containing vacuoles in a Tollip­dependent manner and inhibit GAS intracellular survival [24]. Thus, galectins function not only as danger signals but also as multistep regulators of autophagosome formation during xenophagy.

The V-ATPase–ATG16L1 axis has been implicated in LC3 recruitment to Salmonella-containing vacuoles. During Salmonella infection, the T3SS effector SopF blocks this pathway by ADP-ribosylating Gln124 of ATP6V0C, thereby disrupting the interaction between V-ATPase and the WD40 domain of ATG16L1. This prevents recruitment of the ATG16L1–ATG5–ATG12 LC3-conjugation machinery to bacteria-containing vacuoles and promotes intracellular proliferation of Salmonella enterica serovar Typhimurium (S. Typhimurium) [25, 26]. Although this pathway was previously discussed in the context of Salmonella xenophagy, it is now more accurately placed within the frame work of CASM and LAP. In these non-canonical autophagy pathways, LC3/ATG8 is conjugated directly to pre-existing single-membrane compartments, such as endosomes or phagosomes, rather than to double-membrane autophagosomes [14, 16]. Thus, SopF-sensitive LC3 decoration of Salmonella-containing vacuoles likely represents a CASM- or LAP-like response to bacteria-containing compartments. This pathway may intersect with canonical xenophagy after vacuolar damage or bacterial exposure to the cytosol, but it should not be considered identical to canonical xenophagy. Whether similar mechanisms occur during infection by Mtb, Brucella spp., or L. monocytogenes remains unclear.

Xenophagy targeting cytosolic bacteria

A growing body of evidence suggests that several important intracellular bacteria, including Mtb, Salmonella, and L. monocytogenes, can damage pathogen-containing vacuoles or phagosomal membranes and thereby become exposed to cytosolic surveillance pathways, including xenophagy. For Mtb, vacuolar damage and cytosolic access depend largely on the region of difference 1 (RD1) locus, which encodes the type VII secretion system ESX-1 [27]. Quigley and colleagues further showed that the cell-wall lipid phthiocerol dimycocerosate (PDIM) facilitates Mtb phagosomal escape. Using a loss-of-function genetic approach, they demonstrated a correlation between PDIM levels and the efficiency of Mtb access to the cytosol [28]. Salmonella is also capable of becoming exposed to the cytosol, although this occurs only in a subset of intracellular bacteria and is not considered an obligatory stage of the Salmonella life cycle. After entry into the host cell, Salmonella primarily resides within a unique vacuole known as the Salmonella-containing vacuole (SCV), which serves as its main replication niche [29]. The stability of the SCV is regulated by both bacterial effectors and host membrane-remodeling pathways. SPI-2 effectors such as SifA and SseJ modulate SCV membrane dynamics and Salmonella-induced filament formation. SifA is particularly important for maintaining SCV integrity, whereas SseJ, through its effects on host lipids and SCV membrane composition, can influence vacuolar stability. Disruption of this balance may compromise SCV integrity and contribute to the cytosolic exposure of a subset of intracellular Salmonella [30, 31].

Mechanistic studies have clarified how L. monocytogenes escapes from the phagosome. The pore-forming toxin listeriolysin O (LLO) is a major virulence factor that enables L. monocytogenes to disrupt the phagosomal membrane and establish cytosolic replication. LLO belongs to the cholesterol-dependent cytolysin family and preferentially binds to cholesterol-containing host membranes, where it oligomerizes and forms large pores that destabilize the vacuolar membrane [32, 33]. Importantly, LLO is adapted to the acidic environment of the phagosome: its pore-forming activity is maximal at low pH but markedly reduced at neutral cytosolic pH, which helps confine membrane damage to the vacuole and prevents excessive injury to the host plasma membrane [34, 35]. Phagosomal escape is further supported by bacterial phospholipases, including PlcA and PlcB, which cooperate with LLO to disrupt vacuolar membranes and may increase the accessibility of membrane cholesterol for LLO-mediated pore formation [33]. In addition to these bacterial virulence factors, host determinants can modulate vacuolar rupture. For example, cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed to transiently increase phagosomal chloride concentrations, thereby potentiating LLO pore formation, vacuole lysis, and Listeria infection [36].

Together, these studies show that phagosomal escape is controlled by coordinated bacterial and host factors. However, the relative contribution of each factor may vary with bacterial strain, host cell type, and infection context. Important questions remain regarding how frequently phagosomal escape occurs, how it is regulated during infection, and how cytosolic access affects bacterial replication, immune detection, and virulence. Addressing these questions will further clarify the role of vacuolar rupture in intracellular bacterial pathogenesis.

Upon entering the host cytosol, certain bacteria are marked by host ubiquitin, which promotes the recruitment of selective autophagy receptors, such as OPTN, NDP52, and p62, together with the autophagy-related protein LC3. This process leads to autophagosome formation, lysosomal fusion, and bacterial degradation [37]. In Mtb, the protein Rv1468c contains a conserved eukaryotic-like ubiquitin-associated (UBA) domain that enables direct binding to ubiquitin through hydrophobic interactions, independently of E3 ligase-mediated ubiquitination. This interaction promotes the recruitment of p62 and LC3, thereby facilitating autophagosome formation and bacterial clearance. Deletion of Rv1468c or mutation of its UBA domain increases the intracellular Mtb burden and exacerbates host inflammatory responses, suggesting that Rv1468c may help modulate bacterial load and host inflammation to support long-term intracellular persistence [38]. Other mechanisms of bacterial recognition have also been described. Yamada and co-workers showed that FBXO2, a substrate-recognition component of the SKP1–CUL1–F-box protein (SCF) ubiquitin ligase complex, recognizes GlcNAc side chains on the surface carbohydrate structure of cytosolic Group A Streptococcus (GAS) and promotes ubiquitin-mediated xenophagy [39]. In addition, the ubiquitin coat on cytosolic Salmonella can be initiated through RNF213-mediated ubiquitylation of lipid A, a non-proteinaceous component of bacterial lipopolysaccharide (LPS), revealing an unconventional mechanism of bacterial ubiquitination [40]. However, not all cytosolic bacteria are eliminated by xenophagy. Many bacterial pathogens have evolved strategies to evade or suppress this pathway, as discussed in the following sections. How xenophagy-evading bacteria subsequently exploit host nutrients for replication and dissemination remains an important question.

Together, these studies highlight the complexity of bacteria-targeted xenophagy and indicate that only part of this process has been defined so far (Fig. 1). Because many studies have been performed in vitro, our understanding of how xenophagy is initiated, regulated, and executed in vivo remains incomplete. Further in vivo studies will be important for clarifying the physiological relevance of xenophagy and for evaluating whether key components of this pathway can be targeted for therapeutic or vaccine development.

Fig. 1.

Mechanism of xenophagy targeting intracellular bacteria. After host cell entry, bacteria in phagosomes face four outcomes: (1) Phagosomal-lysosomal degradation: phagosome maturation and fusion with lysosomes. (2) LC3-associated phagocytosis (LAP): LC3 is conjugated to single-membrane phagosomes (LAPosomes) in a Rubicon-dependent manner, promoting lysosomal fusion. (3) Xenophagy: ubiquitin tags bacteria-containing phagosomes, recruiting autophagy receptors (e.g., p62, NDP52, OPTN) that facilitate LC3 lipidation and autophagosome formation, leading to lysosomal degradation. (4) Cytosolic escape: bacteria disrupt the phagosome and enter the cytosol; some are eliminated by ubiquitin-mediated xenophagy

The initiation and progression of xenophagy involve several key molecules, including autophagy-related (ATG) proteins, E3 ubiquitin ligases, selective autophagy receptors, and galectins. Defects in these molecules can impair pathogen recognition, autophagosome formation, or lysosomal degradation. The following sections summarize the roles of these key factors in xenophagy.

The role of ATG proteins in autophagosome formation

ATG proteins are essential for autophagosome biogenesis, which involves the initiation, expansion, and closure of the phagophore membrane. Key ATG proteins involved in autophagosome biogenesis include [41]: (1) The ATG1/ULK1 complex, which initiates autophagy in response to cellular stress signals. In mammals, this complex consists of ULK1/2, ATG13, FIP200/RB1CC1, and ATG101, whereas in yeast it includes Atg1, Atg13, Atg17, Atg29, and Atg31. (2) ATG9A/Atg9, the transmembrane ATG protein, critical for delivering lipids to the expanding autophagosomal membrane. (3) ATG2, which transfers lipids between membranes and supports phagophore expansion. (4) The class III phosphatidylinositol 3-kinase (PI3K) complex, also known as the Vps34 complex, contains Vps34/VPS34, Vps15/p150, Vps30/BECN1, and Atg14/ATG14L, and generates phosphatidylinositol 3-phosphate (PI3P) at the phagophore assembly site to recruit downstream PI3P-binding effectors, including Atg18/WIPI proteins. (5) ATG8/LC3 and GABARAP family proteins, which undergo essential post-translational modifications for membrane expansion and closure. (6) The ATG12–ATG5-ATG16L1 complex, which facilitates the lipidation of ATG8/LC3 and GABARAPs, which is required for phagophore expansion, closure, and autophagosome maturation.

These proteins not only drive the membrane remodeling required for autophagosome formation but also contribute to cargo selection, particularly through interactions with selective autophagy receptors and adaptor proteins. The coordinated regulation of these processes ensures the specificity and efficiency of autophagy under different physiological conditions. The roles of ATG proteins in autophagosome biogenesis and their functions in this process have been comprehensively reviewed by Nishimura et al. [41].

The role of E3 ubiquitin ligase in xenophagy

In 2004, Perrin et al. [42] first reported the ubiquitin system plays a major role in the recognition of cytosolic bacteria, such as S. Typhimurium, by mammalian cells. This landmark finding spurred two decades of research into ubiquitin-dependent mechanisms of bacterial recognition, xenophagy, and innate immunity. In this section, we discuss key xenophagy-related E3 ubiquitin ligases, including Parkin, LUBAC, SMURF1, ARIH1, RNF213, and LRSAM1, with a focus on their domain organization, bacterial targets, activation mechanisms, ubiquitin-linkage specificity, and roles in antibacterial host defense (Fig. 2).

Fig. 2.

Schematic view of E3 ubiquitin ligase domains. Diagram of Parkin showing the Ub-like (UBL) domain, the zinc-coordinating unique parkin domain (RING0), and the repressor element (REP), and the catalytic part comprising the RBR module at the C terminus. LUBAC contains three subunits: HOIP (RNF31), HOIL-1 L (RBCK1), and SHARPIN. The interaction region of RBCK1 with RNF31 is amino acids 69–131, and the interaction region of SHARPIN with RNF31 is amino acids 206–309. Smurf1 comprises an N-terminal C2 domain, two central WW domains, and a C-terminal HECT domain, which possesses E3 ligase activity and represents the catalytic module. ARIH1 contains an acidic/glycine-rich region, an UBA-like domain (UBAL), an RBR E3 ligase module, and a regulatory Ariadne domain at the C terminus. RNF213 comprises a C3HC4-type RING finger domain, an E3 ubiquitin ligase domain, and six AAA+ ATPase domains. LRSAM1 contains a leucine-rich repeat (LRR) domain at the N-terminus, a sterile alpha motif (SAM) domain, and two PTAP motifs (PTAP1 and PTAP2) proximal to its C-terminal RING domain, which possesses E3 ligase activity. Parkin, LUBAC, and ARIH1 belong to the RBR family of E3 ligases. Smurf1 is a member of the HECT-type E3 ubiquitin ligase family. RNF213 and LRSAM1 are both RING-type E3 ligases

Parkin

Parkin is a member of the RBR family of E3 ubiquitin ligases and is best known for its role in PINK1-dependent mitophagy and its association with autosomal recessive juvenile Parkinson’s disease. Structurally, Parkin contains an N-terminal ubiquitin-like (UBL) domain, a catalytic RBR module, and additional zinc-coordinating regulatory domains, including the unique Parkin domain (UPD). These domains together maintain Parkin in an autoinhibited state and regulate its E3 ligase activity [43, 44]. During mitophagy, Parkin activation is controlled by PINK1-dependent phosphorylation of ubiquitin and Parkin itself, leading to ubiquitination of mitochondrial outer-membrane proteins and recruitment of the autophagy machinery. Although Parkin can generate multiple ubiquitin chain types in mitochondrial quality control, its antibacterial function has been most closely associated with the deposition of K63-linked ubiquitin chains around intracellular bacteria-containing compartments.

Beyond mitochondrial quality control, Parkin also contributes to antibacterial xenophagy, as demonstrated in Mtb infection [45]. Following entry into host cells, Mtb initially resides within a phagosome. However, the ESX-1 secretion system can compromise phagosomal membrane integrity and expose Mtb-containing compartments to cytosolic ubiquitin-dependent surveillance pathways [46]. In Mtb-infected murine bone marrow-derived macrophages, Parkin is recruited to Mtb-containing compartments and promotes the accumulation of K63-linked ubiquitin chains around damaged phagosomes. These ubiquitin signals then recruit selective autophagy receptors, including p62 and NDP52, through their ubiquitin-binding domains, thereby facilitating LC3 recruitment, autophagosome formation, and delivery of Mtb to lysosomes for degradation [45]. Thus, Parkin-mediated ubiquitination provides a link between phagosomal damage, ubiquitin marking, and autophagic restriction of intracellular Mtb. The importance of Parkin in host defense is further supported by in vivo studies. Parkin-deficient mice and flies show increased susceptibility to Mtb and L. monocytogenes infection, indicating that Parkin has a conserved role in metazoan innate immunity [45].

While Parkin-mediated xenophagy and mitophagy share mechanistic similarities, fundamental questions persist regarding how Parkin is specifically recruited to intracellular pathogens and which cellular signals relieve its autoinhibition to enable antibacterial activity. Addressing these questions will help clarify how Parkin contributes to antimicrobial cellular immunity and how its antibacterial functions are regulated during infection.

LUBAC

The Linear Ubiquitin Chain Assembly Complex (LUBAC) is the only E3 ubiquitin ligase complex identified to date that generates linear (Met1-linked) ubiquitin chains. LUBAC belongs to the RBR-family E3 ligase system and consists of three core subunits: E3 ubiquitin ligases HOIL-1 interacting protein (HOIP/RNF31), heme-oxidized IRP2 Ub ligase 1 L (HOIL-1 L/RBCK1), and Shank-associated RH domain interacting protein (SHARPIN). HOIP is the catalytic subunit responsible for Met1-linked ubiquitin chain formation, whereas HOIL-1 L and SHARPIN stabilize the complex and regulate its recruitment and activity [47]. Through the assembly of linear ubiquitin chains, LUBAC acts as a signaling scaffold in several innate immune pathways, including NF-κB activation, inflammatory signaling, cell-death regulation, antiviral responses, and antibacterial autophagy. In contrast to E3 ligases that mainly generate K48- or K63-linked ubiquitin chains, LUBAC has a highly specialized linkage specificity for Met1-linked ubiquitination, making it particularly important for organizing ubiquitin-dependent signaling platforms.

LUBAC has been implicated in the host response to cytosol-exposed S. Typhimurium. Rather than initiating bacterial ubiquitination de novo, LUBAC is recruited to pre-existing ubiquitin coats surrounding cytosolic bacteria, mainly through its HOIP subunit. Once recruited, LUBAC deposits Met1-linked ubiquitin chains within the bacterial ubiquitin coat, generating discrete linear-ubiquitin-enriched patches [39]. These Met1-linked ubiquitin patches do not simply mark bacteria for degradation and also function as signaling platforms that coordinate antibacterial autophagy and inflammatory responses. In particular, LUBAC-dependent linear ubiquitination promotes the recruitment of OPTN, which contributes to xenophagic targeting of Salmonella, and NEMO, which supports local NF-κB activation. These two outputs appear to act through separable mechanisms but together restrict intracellular bacterial proliferation and enhance host defense [48].

The activity of LUBAC is counterbalanced by deubiquitinases that specifically regulate Met1-linked ubiquitin chains, most notably OTULIN and CYLD [49]. This reversible control allows linear ubiquitin signaling to be spatially and temporally restricted during infection. Thus, LUBAC should be viewed not simply as another bacterial ubiquitin ligase, but as a linkage-specific amplifier and organizer of pre-existing ubiquitin signals on cytosolic bacteria.

Important questions remain regarding how LUBAC is recruited to different pathogen-associated ubiquitin coats, how its activity is coordinated with other E3 ligases such as RNF213 and LRSAM1, and whether LUBAC-dependent Met1-linked ubiquitination contributes similarly to host defense against other intracellular bacteria beyond Salmonella.

SMURF1

SMAD ubiquitin regulatory factor 1 (SMURF1) is a HECT-type E3 ubiquitin ligase with broad roles in cell signaling, DNA damage responses, chromatin organization, and selective autophagy [50]. Structurally, SMURF1 contains an N-terminal C2 domain, two central WW domains, and a C-terminal HECT catalytic domain. The C2 domain contributes to membrane and bacterial targeting, the WW domains mediate interactions with substrates or adaptor proteins, and the HECT domain provides E3 ubiquitin ligase activity [51]. In the context of antibacterial autophagy, SMURF1 has been linked mainly to K48-linked, rather than K63-linked, ubiquitination, indicating that it may promote xenophagy through a ubiquitin signal distinct from the K63-linked chains commonly associated with other antibacterial E3 ligases.

The connection between SMURF1 and xenophagy was initially suggested by a genome-wide siRNA screen that identified host genes required for selective autophagy of viruses and damaged mitochondria [52]. Subsequent work demonstrated a direct role for SMURF1 in the xenophagy of Mtb. In Mtb-infected macrophages, SMURF1 colocalizes with bacteria-containing compartments and promotes bacterial degradation while limiting intracellular replication [53]. Mechanistically, SMURF1-deficient macrophages show impaired recruitment of polyubiquitin, the proteasome, the ubiquitin-binding autophagy receptor NBR1, LC3, and the lysosomal marker LAMP1 to Mtb-associated sites. These defects indicate that SMURF1 helps organize the ubiquitin-dependent autophagy machinery around Mtb-containing compartments and promotes their delivery to lysosomes. Both the C2 domain and the HECT catalytic activity are required for this function, supporting the idea that SMURF1-mediated bacterial targeting depends on membrane association as well as E3 ligase activity [53].

The physiological relevance of SMURF1-mediated xenophagy has been supported by animal and human studies. In a murine model of tuberculosis, SMURF1-deficient mice exhibit higher bacterial burdens, more severe lung inflammation, and accelerated mortality during the chronic phase of infection, although the acute phase is less affected [53]. SMURF1 also contributes to the control of Mtb replication in human macrophages and has been detected in association with bacteria in lung tissues from patients with pulmonary tuberculosis. Together, these findings identify SMURF1 as an important host factor in Mtb-selective autophagy and tuberculosis defense.

However, several questions remain unresolved, including how SMURF1 is recruited to Mtb-containing compartments, which bacterial or host substrates are directly ubiquitinated by SMURF1, and how K48-linked ubiquitination cooperates with other ubiquitin signals during antibacterial xenophagy.

ARIH1

ARIH1, also known as human homolog of Ariadne (HHARI), is an RBR-family E3 ubiquitin ligase. It contains an N-terminal acidic/glycine-rich region, a UBA-like domain (UBAL), a catalytic RBR module composed of RING1, IBR, and Rcat domains, and a C-terminal Ariadne domain. Structural studies have shown that the Ariadne domain contributes to ARIH1 autoinhibition by masking the catalytic Rcat/RING2 site, thereby regulating its E3 ligase activity [54, 55].

ARIH1 was identified by Polajnar et al. through an image-based RNAi screen as one of several RBR E3 ligases involved in ubiquitin coating of cytosolic Salmonella [56]. After Salmonella damages the Salmonella-containing vacuole and becomes exposed to the cytosol, ARIH1 is rapidly recruited to bacteria, with kinetics similar to galectin-8 recruitment to damaged vacuoles. Together with LRSAM1 and HOIP, ARIH1 forms part of a broader E3 ligase network that contributes to the formation and diversification of the ubiquitin coat on cytosolic Salmonella. Through K48-linked ubiquitination, ARIH1 increases the heterogeneity of ubiquitin signals surrounding bacteria and may help shape downstream antibacterial responses.

Compared with K63- and Met1-linked ubiquitin chains, which have well-established roles in recruiting selective autophagy receptors and inflammatory signaling factors to ubiquitin-coated bacteria, the functional contribution of ARIH1-dependent K48-linked ubiquitination appears to be distinct. Under the conditions examined during Salmonella infection, ARIH1 depletion did not markedly impair the recruitment of the selective autophagy receptors p62, OPTN, or NDP52 to cytosolic bacteria [56].This suggests that ARIH1-mediated K48-linked ubiquitination is not a major determinant of autophagy-receptor recruitment, but may instead contribute to the linkage diversity, organization, or signaling properties of the bacterial ubiquitin coat.

However, the upstream signals that activate ARIH1 during bacterial infection, the direct substrates modified by ARIH1, and the precise functional consequences of ARIH1-dependent K48-linked ubiquitination remain unclear. Clarifying these issues will be important for understanding how ARIH1 cooperates with other E3 ligases, such as LRSAM1 and HOIP, to organize ubiquitin-dependent host defense against cytosol-invading bacteria.

RNF213

Ring finger protein 213 (RNF213) is a large cytosolic E3 ubiquitin ligase of approximately 591 kDa and is also known as a major genetic risk factor for Moyamoya disease. Structurally, RNF213 is unusual because it combines a C3HC4-type RING finger domain, an E3 ubiquitin ligase domain, and six AAA+ ATPase domains, giving it both ubiquitin-ligase-related and ATPase activities [57]. Unlike several antibacterial E3 ligases that mainly diversify pre-existing ubiquitin signals, RNF213 has emerged as an upstream factor in the initiation of ubiquitin coating on cytosol-exposed bacteria. A key feature of RNF213 is that its substrates can extend beyond proteins. In particular, RNF213 has been shown to ubiquitinate lipopolysaccharide (LPS), especially lipid A, on Gram-negative bacterial surfaces, thereby challenging the traditional view that ubiquitination is restricted to protein substrates [40].

The role of RNF213 has been clearly demonstrated during S. Typhimurium infection. Otten et al. showed that RNF213 recognizes cytosolic Salmonella and promotes the formation of the bacterial ubiquitin coat by directly ubiquitinating LPS. This early ubiquitination event is required for the efficient recruitment of downstream ubiquitin-dependent defense factors, including selective autophagy receptors and LUBAC, which subsequently deposits Met1-linked ubiquitin chains on pre-existing ubiquitin coats [40]. In this model, RNF213 occupies an upstream position in the antibacterial ubiquitination hierarchy, whereas E3 ligases such as LUBAC, LRSAM1, and others may contribute to subsequent signal amplification, linkage diversification, and downstream effector recruitment. Consistent with this upstream role, loss of RNF213 impairs ubiquitin-coat formation, reduces the recruitment of ubiquitin-dependent autophagy receptors, compromises antibacterial autophagy, and enhances proliferation of cytosolic Salmonella. The LPS-directed ubiquitination activity of RNF213 has been reported to require its dynein-like ATPase core rather than the canonical RING domain, although the precise molecular mechanism by which RNF213 recognizes bacterial LPS and catalyzes its ubiquitination remains incompletely understood [40].

RNF213-dependent antibacterial defense is not limited to Salmonella. RNF213 has also been implicated in host restriction of L. monocytogenes, a Gram-positive bacterium that lacks LPS, suggesting that RNF213 may recognize additional bacterial or host-associated signals during infection [58, 59]. For example, knockdown of RNF213 increases the intracellular burden of L. monocytogenes, indicating a broader role for RNF213 in cell-autonomous antibacterial immunity [59].

Nevertheless, several key questions remain unresolved. It is still unclear how RNF213 distinguishes cytosol-exposed bacteria from intact vacuolar bacteria, how its AAA+ ATPase core and RING domain cooperate during infection, and what substrates are targeted during infections with Gram-positive bacteria that lack LPS. Clarifying these mechanisms will be important for understanding how RNF213 initiates antibacterial ubiquitin coating and how this early signal is integrated with downstream E3 ligases to promote xenophagy and other ubiquitin-dependent host-defense pathways.

LRSAM1

LRSAM1 is a RING-type E3 ubiquitin ligase implicated in the recognition and ubiquitination of cytosol-exposed bacteria. Structurally, LRSAM1 contains an N-terminal leucine-rich repeat (LRR) domain, a sterile alpha motif (SAM) domain, two PTAP motifs, and a C-terminal RING domain [60]. The LRR domain is required for bacterial localization and has been proposed to mediate recognition of bacterial surface-associated patterns, whereas the RING domain provides the catalytic activity required for ubiquitin signal generation. In vitro studies further suggest that LRSAM1 preferentially catalyzes atypical K6- and K27-linked polyubiquitin chains, indicating that LRSAM1 may contribute to linkage diversification within the ubiquitin coat surrounding cytosolic bacteria [61, 62].

A prior study identified LRSAM1 as an E3 ubiquitin ligase involved in the xenophagy-associated ubiquitination that targets cytosolic Salmonella [61]. However, later study indicates that LRSAM1 is unlikely to act as the sole initiating E3 ligase for bacterial ubiquitin-coat formation. When reconstituted in vitro, the direct ubiquitination of bacteria by LRSAM1 proved to be relatively inefficient, which may in part reflect the absence of RNF213-dependent priming and other cellular factors that promote ubiquitin-coat formation in intact cells. Thus, LRSAM1 may contribute to the diversification or amplification of pre-existing ubiquitin coats rather than independently initiating bacterial ubiquitination [40].

The relationship between LRSAM1 and different bacterial pathogens appears to be context-dependent. Under in vitro conditions, LRSAM1 can ubiquitinate several bacterial species, including L. monocytogenes and S. flexneri, even though these bacteria may evade efficient LRSAM1 recruitment, ubiquitination, or autophagic detection during infection. This discrepancy suggests that bacterial evasion strategies and host-cell context strongly influence LRSAM1-dependent recognition. For example, the limited association of LRSAM1 with intracellular Shigella raises the possibility that Shigella possesses mechanisms to avoid LRSAM1-mediated detection, in addition to its known ability to interfere with autophagy through the effector IcsB [63].

The identification of LRSAM1 as a bacterial recognition protein and ubiquitin ligase has advanced our understanding of how the innate immune system detects bacteria within the host cytosol. However, this discovery also raises several pivotal questions: (1) Which pattern(s) on cytosolic bacteria are identified by the LRR domain of LRSAM1? (2) What mechanisms allow for the detection of membrane-enclosed bacteria by autophagic processes? (3) Are there additional innate sensors that identify these intracellular bacteria by focusing on pathogen-specific molecules on the vacuolar membrane or host molecules that signal the presence of pathogenic vacuoles? Future research is poised to reveal further complexities in both how pathogens are sensed within the cytosol and the evasion tactics employed by microbes adapted to cytosolic environments [64].

As described above, studies have identified several E3 ligases, including SMURF1, Parkin, ARIH1, LRSAM1, RNF213, and LUBAC, that are capable of ubiquitinating multiple types of bacteria. In some cases, the same bacterium can be targeted by multiple E3 ligases. For example, ARIH1, LRSAM1, RNF213, and LUBAC have been observed to colocalize with damaged Salmonella-containing vacuoles (SCVs), thereby facilitating the formation of a ubiquitin-coat around cytosolic S. Typhimurium. In addition, a distinct group of E3 ligases, such as RNF166 [65], MARCH8 [66], TRIM21 [67], TRIM22 [68], and TRIM16 [69], may not directly recognize bacteria but instead respond to infection-associated signals, such as exposed glycans. Together, these findings highlight the diversity and specificity of E3 ligase functions in ubiquitin-mediated antibacterial responses. Further investigation of these E3 ligases in xenophagy may help identify host pathways that can be targeted for therapeutic intervention in infectious diseases.

Manipulation of xenophagy by bacteria

Although xenophagy provides an important host-defense mechanism against intracellular pathogens, many bacteria, including Mtb, Salmonella enterica, and Legionella pneumophila, have evolved strategies to subvert this pathway and persist within host cells. This section summarizes current understanding of how bacterial pathogens inhibit xenophagy, with a focus on their interference with autophagosome formation, vesicle acidification, and autophagosome–lysosome fusion (Fig. 3).

Fig. 3.

Unique strategies by different bacteria for inhibition of host autophagy. Mycobacterium tuberculosis utilizes its effector proteins (red text) to dampen autophagy by inhibiting phagosome formation, phagosome maturation, LAPosome and autophagosome formation, autophagosome fusion with the lysosome, and autolysosome acidification. Salmonella Typhimurium uses its effectors (blue text) to dampen autophagy by inhibiting autophagosome formation and autophagosome fusion with the lysosome. The protein RavZ (green) of Legionella pneumophila inhibits autophagosome formation to subvert autophagy. Listeria monocytogenes employs InlB (purple) to prevent autophagy by inhibit phagosome maturation. Chlamydia trachomatis GarD (brown) prevents ubiquitin deposition on inclusions. Burkholderia employs the deubiquitylase TssM (magenta), a deubiquitylase, removes ubiquitin from bacterial surfaces. S. flexneri IpaH1.4 (orange) targets and degrades host E3 ligases, thereby dampening ubiquitin-dependent “eat me” signals

Regulation of autophagosome formation by bacteria

Autophagosome formation, a critical stage in xenophagy, involves the recruitment of autophagy-related (ATG) proteins and the assembly of a double-membraned vesicle around the pathogen. To establish intracellular niches, various bacterial pathogens have evolved sophisticated strategies to evade this process by targeting distinct stages of autophagosome biogenesis.

Mtb employs multiple effectors to inhibit autophagy initiation. The protein SapM exerts a dual function: it not only suppresses autophagy initiation by dephosphorylating Raptor to sustain mTORC1 activity [70], but also suppresses the autophagic flux by blocking RAB7 through its CT domain [71]. Similarly, the virulence lipid PDIM specifically blocks LAP by preventing the recruitment of the NADPH oxidase complex to the mycobacterial phagosome. This disruption of LAP also extends to impairing classical autophagy [72, 73]. Furthermore, the Mtb protein Rv0790c suppresses host autophagy at the initiation stage by directly interacting with and activating mTOR, which subsequently inhibits the autophagy initiation cascade, as shown by increased inhibitory phosphorylation of its downstream substrates, ULK1 at Ser757 and P70S6K at Thr389 [74]. In addition, PE_PGRS20 and PE_PGRS47 proteins directly bind to the small GTPase Rab1A, a key initiator of autophagy, to suppress phagophore formation [75]. Finally, Mtb virulence effector PPE51 inhibits host autophagy and phagocytosis by antagonizing Toll-like receptor 2 (TLR2) signaling. By interacting with TLR2, PPE51 dampens activation of the downstream ERK1/2 pathway, which is an important stimulator of autophagy [76].

Legionella pneumophila secretes the effector RavZ, which subverts autophagy through a unique irreversible mechanism. RavZ acts as a protease that directly hydrolyzes the critical C-terminal glycine of the autophagy protein Atg8 (the LC3 homolog), rendering it permanently inactive and incapable of reconjugation [77]. Structural and biochemical analyses have revealed that RavZ recognizes LC3 through flexible N- and C-terminal LC3-interacting region (LIR) motifs in a 1:2 stoichiometric complex, ensuring precise substrate targeting [78]. Beyond deconjugating LC3, RavZ also exhibits deubiquitinase activity, removing ubiquitin from bacterial vacuoles to evade ubiquitin-mediated autophagic recognition independently of the LC3 system [79].

S. Typhimurium also uses multiple effectors to block autophagosome formation. The effector SpvB has been reported to suppress autophagosome formation through its function in depolymerizing actin [80, 81]; however, whether SpvB acts as a direct autophagy inhibitor remains to be fully established. The SPI-2 T3SS effectors SseF and SseG directly interact with host Rab1A to inhibit Rab1A-mediated autophagy initiation [82]. In B cells, SopB promotes bacterial survival by activating mTORC1, leading to the inhibitory phosphorylation of ULK1 and reduced LC3-II levels, thereby suppressing autophagy [83]. Finally, the deubiquitinase SseL counteracts host selective autophagy by removing ubiquitin signals from Salmonella-containing vacuoles and cytosolic aggregates, thereby preventing their recognition by autophagy receptors such as p62 [84].

Other intracellular pathogens also interfere with ubiquitin-dependent antibacterial autophagy. Chlamydia trachomatis GarD acts as a cis-acting stealth factor that directly antagonizes the ubiquitin E3 ligase RNF213, thereby preventing ubiquitin-mediated destruction of the bacterial inclusion [85]. Burkholderia employs the deubiquitylase TssM, which possesses a previously unrecognized esterase activity, to directly reverse RNF213-mediated non-canonical ubiquitylation of bacterial lipopolysaccharide, thereby blocking the initiation of antibacterial autophagy and enabling intracellular replication [86]. S. flexneri employs the type III secretion system effector IpaH1.4 to directly target and degrade host E3 ligases RNF213 and LUBAC, thereby dampening ubiquitin-dependent “eat me” signals and evading cell-autonomous immunity to promote intracellular bacterial proliferation [87–89].

In summary, bacteria can inhibit xenophagy by interfering with autophagosome formation and ubiquitin-dependent pathogen recognition. In addition, many pathogens disrupt later stages of the pathway, including phagosome and autophagosome maturation as well as fusion with lysosomes. These mechanisms are discussed in the following section.

Regulation of phagosome and autophagosome maturation by bacteria

The acidification and maturation of phagosomes and autophagosomes are essential for the activation of degradative enzymes and the clearance of intracellular pathogens. However, many intracellular bacteria have evolved sophisticated mechanisms to disrupt these processes and maintain a non-degradative intracellular niche.

Mtb employs multiple effectors to inhibit phagosomal and autophagosomal maturation. The effector PknG exerts a dual regulatory effect on host autophagy to promote intracellular bacterial survival. Although PknG can induce autophagy initiation by competitively binding to the PH domain of AKT, it concurrently blocks autophagosome maturation by targeting the small GTPase RAB14. Specifically, PknG inhibits RAB14-GTP hydrolysis through direct interaction and by phosphorylating TBC1D4/AS160 to suppress its GAP activity [90]. Meanwhile, the Mtb protein tyrosine phosphatase (PtpA) binds to subunit H of the macrophage vacuolar H+-ATPase (V-ATPase) and excludes V-ATPase from the phagosome, thereby inhibiting phagosomal acidification. Since V-ATPase is required for lowering luminal pH, this process prevents proper activation of lysosomal degradative enzymes [91]. Moreover, mannose-capped lipoarabinomannan (ManLAM), a glycosylated Mtb phosphatidylinositol, arrests phagosome maturation by interfering with the acquisition of the lysosomal cargo and syntaxin 6 from the trans-Golgi network [92]. The Mtb type VII secretion system effector Rv2347c inhibits both phagosome maturation and autophagy by suppressing the early endosomal marker RAB5 [93]. Additionally, the mycobacterial glycolipid trehalose dimycolate (TDM) inhibits phagosome maturation by binding host SNARE proteins (VTI1B and STX8), and redirecting them to form a non-canonical complex with VAMP2 rather than VAMP8, thereby promoting intracellular bacterial survival [94]. The bacterial effector Ndk disrupts phagosome maturation by inactivating the small GTPases Rab5 and Rab7, preventing the recruitment of their effectors, early endosome antigen 1(EEA1) and Rab7-interacting lysosomal protein (RILP) [95]. The Mtb-secreted phosphatase SapM contributes to the arrest of phagosomal maturation by hydrolyzing phosphatidylinositol 3-phosphate (PI3P), thereby inhibiting phagosome–late endosome fusion [96].

Similar strategies are also observed in other intracellular bacteria. In Mycobacterium bovis, the lipoprotein P27 (LprG) is responsible for arresting phagosome maturation during early infection, as shown by the full restoration of the wild-type phenotype only upon complementation with functional p27 [97]. In contrast, L. monocytogenes adopts a different strategy: its surface protein InlB recruits Rab7 to the bacterial vacuole by subverting the host class III PI3K/Vps34 pathway, thereby accelerating vacuole maturation into a Rab7-positive compartment, which facilitates more efficient phagosomal escape mediated by LLO [98].

Collectively, these findings illustrate the diverse and highly specialized molecular strategies employed by bacteria to manipulate host membrane trafficking and establish intracellular replication niches.

Regulation of autophagosome-lysosome fusion by bacteria

The fusion of autophagosomes with lysosomes is a critical step in xenophagy and is required for bacterial degradation. Multiple intracellular pathogens have evolved virulence factors to disrupt this final stage of autophagy, thereby evading destruction and promoting their intracellular survival.

Mtb employs several distinct effectors to interfere with the process of autophagosome-lysosome fusion. The ESX-1-secreted protein ESAT-6 impairs autophagic flux by blocking autophagosome-lysosome fusion in human dendritic cells [99]. The lipoprotein LprG inhibits phagosome-lysosome fusion by promoting the transfer of LAM to the cell surface [100]. Furthermore, the phosphatase SapM directly binds to the small GTPase Rab7 via its C-terminal domain, thereby preventing autophagosome-lysosome fusion [71]. Another key effector, PtpA, acts by dephosphorylating the host vacuolar sorting protein VPS33B, thereby disrupting the membrane fusion machinery required for phagolysosome formation [101].

Similarly, S. Typhimurium also disrupts this degradative pathway through the SPI-1 effector SopB. SopB prevents the fusion of Salmonella-containing vacuoles (SCVs) with lysosomes and autophagosomes, and also suppresses lysosomal biogenesis by inhibiting the nuclear translocation of the master regulator TFEB through the Akt–TFEB axis [102]. This coordinated strategy effectively reduces the degradative capacity of the host cell while protecting the bacterial niche.

In addition to blocking autophagosome-lysosome fusion, intracellular bacteria can also subvert autophagy by masking virulence factors or hijacking autophagy-related proteins to evade degradation. Shigella evades host autophagy through a unique “camouflage” mechanism mediated by the type III secretion effector IcsB. The bacterial surface protein VirG, which is essential for actin-based motility, is recognized by the host autophagy protein ATG5, thereby targeting the bacterium for degradation. IcsB counteracts this process by competitively binding to VirG, thereby concealing it from ATG5 and enabling the pathogen to escape autophagic clearance [63]. Additionally, S. flexneri effectors IcsB and VirA act synergistically to facilitate bacterial escape from autophagic recognition. IcsB directly masks the bacterial surface protein IcsA to prevent its detection by the autophagy component ATG5, while VirA inactivates the small GTPase Rab1. Collectively, these actions inhibit LC3 recruitment to the vacuolar membrane, enabling the pathogen to evade xenophagic targeting [103].

Similarly, L. monocytogenes evades autophagic recognition through its surface protein ActA, which recruits the host Arp2/3 complex and Ena/VASP to disguise the bacterial surface [104, 105]. In contrast, Brucella subverts autophagy by selectively co-opting the host autophagy initiation machinery. The pathogen transforms its replicative vacuole into an autophagic-like compartment (aBCV) by hijacking components including ULK1, Beclin1, ATG14L, and PI3‑kinase activity. Formation of the aBCV is required for completion of the intracellular cycle and promotes bacterial cell-to-cell spread, illustrating how pathogens can adapt autophagy-related pathways to facilitate infection [106].

In conclusion, these findings illustrate a convergent evolutionary strategy among diverse bacterial pathogens to sabotage multiple stages of the autophagic pathway, thereby ensuring their persistence within the host. Understanding these mechanisms provides valuable insights into host-pathogen interactions and reveals potential therapeutic targets for enhancing host defenses against intracellular infections.

Outstanding research questions in bacterial xenophagy

Bacterial xenophagy is an important cell-autonomous defense pathway by which host cells recognize and eliminate intracellular bacteria. Despite substantial progress, several key questions remain unresolved. Here, we outline major directions for future research, focusing on the molecular mechanisms of bacterial recognition and autophagic targeting, the interplay between xenophagy and immune signaling, its potential relevance to antibiotic resistance and cell death, and its therapeutic implications (Fig. 4).

Fig. 4.

Outstanding research questions in bacterial xenophagy: the membrane source for xenophagy, the interplay between xenophagy and immune response, the role of xenophagy in vivo, and the potential implications for antibiotic resistance

Molecular mechanisms of autophagosome formation at the bacterial surface

The precise origin of the xenophagic membrane and the sequence of ATG protein recruitment remain incompletely understood. Key questions include whether the membrane is derived primarily from the endoplasmic reticulum (ER), Golgi apparatus, plasma membrane, or other membrane sources, and whether this origin varies depending on the pathogen or host cell type. Advanced imaging techniques, such as correlative light-electron microscopy (CLEM) and high-temporal-resolution live-cell imaging, will be important for visualizing nascent sequestration event in real time and to define the molecular triggers that specify the site of autophagosome nucleation.

Another unresolved issue is how bacterial pathogens manipulate host autophagic responses through secretion systems and effector proteins. For example, Salmonella deploys type III secretion systems to inject effector proteins into host cells, which can subvert autophagic responses [107]. Determining the precise interactions between these bacterial effectors and host autophagy components will help delineate the intricate molecular mechanisms of xenophagy.

Interplay between xenophagy and the immune system

The interplay between bacterial xenophagy and the immune system is complex and multifaceted. Although xenophagy functions as an antimicrobial mechanism, how it is integrated with broader immune responses remains incompletely understood. For instance, studies suggest that xenophagy may influence cytokine production, which could modulate inflammation and other immune responses such as antigen presentation [108–110]. Elucidating the extent of xenophagy’s role in modulating immune signaling, particularly in coordinating responses between autophagy and adaptive immunity, represents an emerging field of investigation.

Xenophagy does not operate in isolation. Its crosstalk with other clearance pathways, including the ubiquitin-proteasome system, LC3-associated phagocytosis (LAP), and programmed cell death (apoptosis, pyroptosis) remains poorly understood. Under what conditions does xenophagy trigger inflammation or apoptosis? Future research should aim to define the molecular switches that determine these cell fate decisions following pathogen recognition.

Notably, many studies have relied on immortalized cell lines, leaving the role of xenophagy in primary cells, specialized phagocytes, and in vivo infection models less well defined. Future work should clarify how xenophagy contributes to tissue homeostasis, chronic infection, antimicrobial immunity, and non-canonical functions such as antigen presentation.

Impact of xenophagy on bacterial antibiotic resistance

Antibiotic resistance is a major global health threat, highlighting the need to understand how xenophagy influences bacterial persistence and antibiotic efficacy. Some studies suggest that autophagy could contribute to bacterial persistence by providing a niche for intracellular pathogens, allowing them to evade antibiotics that cannot effectively penetrate host cells [111]. The relationship between autophagy and antibiotic efficacy is complex, as certain antibiotics can modulate autophagic responses, which in turn affects bacterial survival and the host’s ability to clear infections.

Xenophagy represents a promising target for the development of novel therapeutic interventions against intracellular bacterial infections. Although some drugs targeting autophagy pathways have been developed, their clinical efficacy remains limited, and xenophagy-specific therapies have not yet been established. A major challenge is the development of drugs that can specifically modulate xenophagy without affecting general autophagy, as disrupting normal autophagic processes can lead to unintended side effects. Moreover, combinatorial approaches that pair xenophagy-enhancing agents with conventional antibiotics may prove effective, particularly in combating antibiotic-resistant pathogens. The design of such strategies requires a deeper understanding of xenophagy-specific molecular pathways and the identification of specific biomarkers that can guide therapeutic intervention.

In conclusion, bacterial xenophagy represents a dynamic interface between cell biology, microbiology, and immunology. Addressing the outstanding questions in this field will require interdisciplinary approaches that combine mechanistic biochemistry, advanced imaging, microbial genetics, and in vivo physiology. These efforts will not only deepen our understanding of this cell-autonomous defense pathway but may also open new avenues for therapeutic intervention in infectious diseases and other conditions involving proteostasis and inflammatory regulation.

Summary

Xenophagy, as a specialized form of autophagy, represents an important cell-autonomous defense mechanism against intracellular bacterial pathogens. This review summarizes the molecular mechanisms by which host cells recognize, sequester, and degrade invading bacteria through the autophagy pathway. At the same time, intracellular bacteria are not passive targets. Many pathogens have evolved strategies to evade, inhibit, or exploit xenophagy to promote their intracellular survival. These bacterial countermeasures range from interference with autophagosome formation to disruption of autophagosome–lysosome fusion, highlighting the complex interplay between host defense mechanisms and bacterial survival strategies within infected cells.

Despite substantial progress, several fundamental questions remain unresolved. What are the precise molecular signaling cascades that differentiate xenophagy from general autophagy? How do different pathogens exploit unique molecular adaptations to circumvent xenophagy across various cellular environments? And, how can insights into bacterial evasion mechanisms be used to develop therapeutic strategies that enhance xenophagic clearance of persistent intracellular pathogens? Addressing these questions will require integrated approaches combining molecular biology, microbiology, immunology, imaging, and pharmacology. A deeper understanding of bacterial xenophagy may not only inform new host-directed therapies for infectious diseases but also provide broader insights into immune regulation and cellular homeostasis.

Authors contributions

Yinjuan Song: Conceptualization, Literature search, Data curation, Writing-original draft, Figure preparation, Funding acquisition; Li Tang: Data curation, Figure preparation; Na Li: Literature search, Visualization; Xiangmei Zhou: Conceptualization, Supervision, Writing-review & editing; Yuefeng Chu: Supervision, Writing-review & editing, Funding acquisition.

Funding

This work was supported by the grants from National Key Research and Development Program of China (2023YFD1800703); Open Fund Project of the State Key Laboratory of Veterinary Public Health and Safety (2026SKLVPHS01); Natural Science Foundation of Gansu Province (25JRRA438); the Key Program of Gansu Joint Research Foundation (24JRRA809); The Major Project of Joint Research Fund of Gansu Province (25JRRA1085); Key Research and Development Program of Xinjiang (2025B02022-3); the Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2021-LVRI).

Data availability

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Declarations

Conflict of interest

No potential conflict of interest was reported by the authors.