Tapping lipid droplets: A rich fat diet of intracellular bacterial pathogens

Lipid droplets (LDs) are dynamic and versatile organelles present in most eukaryotic cells. LDs consist of a hydrophobic core of neutral lipids, a phospholipid monolayer coat, and a variety of associated proteins. LDs are formed at the endoplasmic reticulum and have diverse roles in lipid storage, energy metabolism, membrane trafficking, and cellular signaling. In addition to their physiological cellular functions, LDs have been implicated in the pathogenesis of several diseases, including metabolic disorders, cancer, and infections. A number of intracellular bacterial pathogens modulate and/or interact with LDs during host cell infection. Members of the genera Mycobacterium, Legionella, Coxiella, Chlamydia, and Salmonella exploit LDs as a source of intracellular nutrients and membrane components to establish their distinct intracellular replicative niches. In this review, we focus on the biogenesis, interactions, and functions of LDs, as well as on their role in lipid metabolism of intracellular bacterial pathogens.

, and organelle interactions (Olzmann & Carvalho, 2019;Rakotonirina-Ricquebourg et al., 2022), as well as LD-dependent cellular signaling and innate immunity (Bosch et al., 2021;Bosch & Pol, 2022), and the role of LDs for host-pathogen interactions (Brink et al., 2021;Herker et al., 2021;Libbing et al., 2019;Welte, 2015). This review will summarize the current state of knowledge regarding the biogenesis and functions of LDs, and then focus on the modulation of lipids and LDs in the context of intracellular lipid and FA metabolism by selected pathogenic bacteria.

| LIPID DROPLE T B I OG ENE S IS AT THE ENDOPL A S MI C RE TI CULUM
LD assembly originates in the bilayer of the endoplasmic reticulum (ER) membrane, where enzymes involved in the terminal steps of neutral lipids synthesis reside ( Figure 1) (Buhman et al., 2001). These neutral lipids play important functions in energy storage and are a major source of phospholipid precursors and cholesterol for cell membranes. TAG and sterol esters (SE) are generated by diacylglycerol acyltransferases (DGAT1 and DGAT2) and acyl-CoA:cholesterol O-acyltransferases (ACAT1 and ACAT2), respectively. Cells lacking DGAT1 and DGAT2 are defective for TAG biosynthesis and LD formation (Harris et al., 2011).
As TAG and SE accumulate and diffuse laterally in the ER membrane bilayer, they eventually reach a critical concentration, where they coalesce and form an "oil lens" between the outer and the inner ER membrane leaflet (Thiam & Foret, 2016).
Cytosolic and ER-residing proteins play important roles in the formation and stabilization of nascent LDs (Figure 1). In fact, these proteins seem to favor the initial convex curvature of the cytosolic ER membrane leaflet, driving LD budding toward the cytosol and not the ER lumen. Perilipin 3 (PLIN3/TIP47), which binds to nascent LDs from the cytosol, is a good candidate for this function (Bulankina et al., 2009;Skinner et al., 2009). PLIN3 and other members of the perilipin family comprising PLIN1-5 bind to LDs through conserved amphipathic helices (Rowe et al., 2016). Seipin, a conserved ER membrane protein regulating protein and lipid trafficking into LDs (Fei et al., 2008;Salo et al., 2016;Szymanski et al., 2007), facilitates the deformation of the ER lipid bilayer by the growing lens (Henne et al., 2018) and stabilizes nascent LDs thus favoring their maturation (Wang et al., 2016). Growth of the nascent LD core requires enzymes for neutral lipids synthesis. Accordingly, enzymes involved in TAG biosynthesis, such as DGAT2 and glycerol-3-phosphate acyltransferase 4 (GPAT4), promote LD formation . F I G U R E 1 Lipid droplet biogenesis and cellular functions. LD biogenesis is based on the synthesis of neutral lipids by DGAT1/2 and ACAT1/2 in the ER membrane bilayer. The neutral lipids accumulate by lateral diffusion and form an oil lens, which gives rise to an emerging LD. Cytosolic and ER-derived proteins (PLIN3, FIT2, seipin) stabilize the nascent LD. Seipin regulates lipid and protein trafficking into the growing LD and mediates assembly of a stable ER-LD bridge allowing the transfer of, for example, DGAT2 and GPAT4 from the ER to the LD. A fraction of LDs is released via an unknown fission process to the cytosol, and the reversion of the process, that is, LD-ER reassociation depends on the small GTPase ARF1 and the COPI coat complex. Mature cytosolic and ER-bound LDs are involved in various cell processes, including cell development, motility and organelle interactions, lipid storage and metabolism, stress response, signaling, and innate immunity. ACAT1/2, acyl-CoA:cholesterol O-acyltransferase 1 and 2; COPI, coatomer complex I; DGAT2, diacylglycerol acyltransferase 2; ER, endoplasmic reticulum; FIT2, fat storage-inducing transmembrane protein 2; GPAT4, glycerol-3-phosphate acyltransferase 4; LD, lipid droplet; PLIN3, perilipin 3. For details see text.
The modulation of ER dynamics also plays a central role in LD budding. Reticulons, receptor expression-enhancing proteins (REEPs), and atlastins (ATLs), which are fundamental in maintenance of the tubular ER morphology (Hu et al., 2009;Shibata et al., 2010;Voeltz et al., 2006), have been suggested to be major players in LDs homeostasis. In fact, expression of a dominant-negative ATL mutant resulted in smaller LDs, whereas overexpression of native ATL had the opposite effect (Klemm et al., 2013). Moreover, REEP1 deletion caused lipodystrophy similar to the absence of seipin (Renvoise et al., 2016), reinforcing the idea that regulation of ER dynamics may have a strong effect on LD budding.
LDs often connect with the ER via an ER-LD lipid "bridge", which is used to transfer DGAT2, GPAT4, and possibly other enzymes and substrates, from the ER to LDs (Figure 1) (Wilfling et al., , 2014.
Most mature LDs remain in close contact with the ER, and only a subset of droplets is released through an unknown mechanism of membrane fission (Olzmann & Carvalho, 2019;Renne et al., 2020). In fact, as much as 94% of LDs in yeast and 85% of LDs in mammalian COS-7 cells seem to stay attached to the ER (Szymanski et al., 2007;Valm et al., 2017), and only a subpopulation of growing LDs is accumulating ER-derived GPAT4 . The conserved fat storage-inducing transmembrane (FIT) family of proteins is required for proper budding of LDs from the ER (Choudhary et al., 2015). Elimination or reduction of FIT proteins in yeast and higher eukaryotes causes LDs to remain in the ER membrane. The lipid bridge linking the bilayer of the ER to the LD monolayer is decorated by different enzymes and proteins including seipin Fei et al., 2008;Salo et al., 2016;Szymanski et al., 2007). Interestingly, cytosolic LDs reversibly associate with the ER and form new ER-LD lipid bridges in a process dependent on the small GTPase ARF1 and the coatomer complex I (COPI) (Thiam et al., 2013;Wilfling et al., 2013Wilfling et al., , 2014.

| LIPID DROPLE T MOTILIT Y AND ORG ANELLE INTER AC TI ON S
Active and directed motility of LDs has been described for several cell types, including cultured mammalian cells ( Figure 1) (Welte, 2009;Welte & Gould, 2017). In some cells, LDs are transported via actin-myosin filaments (Knoblach & Rachubinski, 2015;Pfisterer et al., 2017). However, in the majority of cases, LDs transport is largely directed along microtubules by the microtubule motor proteins kinesin and dynein (Orlicky et al., 2013;Welte, 2009Welte, , 2015. This transport appears to be driven by direct binding of LD-associated proteins, like PLIN3 and ARF1, to microtubule motor proteins (Gu et al., 2019;Guo et al., 2008;Rai et al., 2017). In addition to direct motion, LDs can also be transported indirectly in several ways, for example, by "hitchhiking" on other organelles such as endosomes, which are transported along microtubules (Kilwein & Welte, 2019).
LDs are not only tethered to their origin compartment, the ER, but are also observed in close proximity of the Golgi (Valm et al., 2017). Candidates for Golgi-LD linkers are the golgin family of tether proteins (Krahmer & Mann, 2019;Witkos & Lowe, 2015) and the conserved vacuolar protein sorting-associated protein 13B (VPS13B), which binds to both the trans-Golgi network (TGN) membrane and the LD monolayer, thereby promoting Golgi-LD association (Du et al., 2020). LDs have also been found inside the nucleus, though the biogenesis and biological significance of this LD population remain mostly unknown (Farese & Walther, 2016;Romanauska & Kohler, 2018). Furthermore, LDs interact with and are degraded by lysosomes in a process called lipophagy (Schulze et al., 2020;Zechner et al., 2017) (see below).
LDs and mitochondria associate in different cells and tissues, especially in those with increased capacity for storage and oxidation of FA, such as brown adipose tissue, skeletal muscle, and heart (Rakotonirina-Ricquebourg et al., 2022). LD-mitochondria interactions involve the cellular fusion proteins NSF, α-SNAP, and SNAREs (syntaxin-5; SNAP23, synaptosomal-associated protein of 23 kDa; VAMP4, vesicle-associated membrane protein 4) (Bostrom et al., 2007;Jägerström et al., 2009). Other tether proteins belong to the family of perilipins, such as PLIN1 and PLIN5, which bind to LDs and provide a direct physical and metabolic link to different mitochondrial subpopulations (Benador et al., 2018(Benador et al., , 2019Kimmel & Sztalryd, 2014. PLIN1 is phosphorylated by protein kinase A (PKA) upon adrenergic stimulation of lipolysis and interacts with mitochondrial optic atrophy 1 (OPA1) as well as with mitofusin 1 (MFN1) and MFN2 to promote the anchoring of LDs to mitochondria (Boutant et al., 2017;Pidoux et al., 2011). Moreover, LD-bound DGAT2 associates with mitochondria, thus also causing mitochondria redistribution and interaction with LDs (Stone et al., 2009).
While LD-mitochondria tethering is likely executed mainly by proteins, lipids might also facilitate this association. The contact might be mediated by fusion or hemi-fusion of the cytoplasmic leaflet of the outer mitochondrial membrane with the phospholipid monolayer of LDs, thus favoring the exchange of FA between the two organelles (Mahamid et al., 2019).
Finally, the interaction of LDs with peroxisomes has been observed in yeast (Binns et al., 2006) and mammalian cells, where tethering of LDs to peroxisomes is modulated by the peroxisomal ATPase spastin and the transporter ABCD1 (Chang et al., 2019). ABCD1 is an importer for long-chain FA, thus promoting FA trafficking between the two organelles.

| LIPID DROPLE TS AND LIPID ME TABOLIS M
LDs are an essential hub for lipid metabolism, as they provide FA for cellular processes such as the production of metabolic energy and the synthesis of membranes ( Figure 1). Depending on the cellular energy needs, lipids stored in LDs must be mobilized quickly.
FA release from TAG stored in LDs is efficiently catalyzed by cytoplasmic lipases, localizing to the LD surface, in a process called lipolysis (Zechner et al., 2017). Lipolysis is initiated by the enzymatic activity of a central triglyceride lipase, the adipose triglyceride lipase (ATGL) (Smirnova et al., 2006;Zechner et al., 2017). This is then followed by hormone-sensitive lipase (HSL), an important diacylglycerol lipase, and finally by monoacylglycerol lipase (MGL), resulting in release of fatty acids and glycerol from TAG (Zechner et al., 2017).
Intact LDs can also be metabolized through lysosome-dependent autophagy, in a process called lipophagy (Schulze et al., 2020;Zechner et al., 2017). During lipophagy, the LD is engulfed by a double-membraned autophagosome, followed by fusion and digestion by the lysosome (Singh et al., 2009). Deletion of the core autophagic protein, ATG7, impairs lipophagy and promotes the accumulation of TAG in LDs. In a process termed chaperone-mediated autophagy (Kaushik & Cuervo, 2012), cytosolic lipases and the autophagy machinery are recruited to LDs upon digestion of PLIN2 and PLIN3 at the surface of the droplets (Kaushik & Cuervo, 2016). The cellular cues required for preferential use of lipolysis or lipophagy in LD metabolism are still unclear, but likely involve metabolic signals (Cohen, 2018;Rambold et al., 2015).
Mitochondria as well as peroxisomes catalyze the β oxidation of FA and contain several enzymes involved in redox homeostasis.
Nutrient-starved cells harbor increased LD numbers and LDs associated with fused mitochondria (Rambold et al., 2015). During starvation, LDs and mitochondria relocate on detyrosinated microtubules from the cell center to adopt a dispersed distribution in the cell periphery in an AMP-activated protein kinase (AMPK)-dependent way (Herms et al., 2015). Consequently, LD-mitochondria interactions increase, and LDs are efficiently tapped as source of metabolic energy by supplying FA for mitochondrial β oxidation. PLIN1 promotes LD-mitochondria contacts, lipolysis, and FA import for mitochondrial β oxidation (Pidoux et al., 2011), and the absence of PLIN5 also causes reduced LD-mitochondria contacts and mitochondrial oxida- Analogously, the extensive interaction between LDs and peroxisomes promotes peroxisomal FA β oxidation, and thus, lipolysis is also coupled with peroxisomal FA oxidation (Binns et al., 2006). In fact, LDs, mitochondria, and peroxisomes are all implicated in a complex metabolic machinery regulating energy homeostasis in white and brown adipose tissue (Zhou et al., 2018). Therefore, it is plausible that LDs intimately interact with various different oxidative organelles to form a metabolic circuit for FA exchange and metabolism, thus avoiding FA spillover in the cytoplasm and cellular toxicity A recent study highlighted the presence of a unique peridroplet mitochondria (PDM) population that does not share components with or fuse with the rest of the cytoplasmic mitochondria (Benador et al., 2018). The distinct PDM population supports TAG synthesis and LDs expansion, instead of consuming stored TAG to fulfill the metabolic demand of the cell. In comparison to cytoplasmic mitochondria, PDM show a distinct proteome, cristae organization, elongated morphology, and reduced motility. Moreover, PDM are characterized by specific bioenergetics, as they prefer pyruvate oxidation instead of lipid oxidation. In addition to binding LDs through PLIN5, the specialization into PDM may require a multitude of other factors (Benador et al., 2018).

| LIPID DROPLE T FUN C TI ON IN VARI OUS CELLUL AR PATHWAYS
Aside from their function in lipid supply and energy metabolism, LDs participate in many other cellular pathways, such as stress response, protein homeostasis, biosynthesis of inflammatory mediators, and innate immunity ( Figure 1) (Bosch et al., 2021;Saka & Valdivia, 2012;Welte & Gould, 2017). Stress not only derives from protein misfolding in the ER due to increased protein synthesis (unfolded protein response, UPR), but also from the accumulation of saturated FA, which may perturb the composition of the ER membrane (Han & Kaufman, 2016;Volmer et al., 2013;Volmer & Ron, 2015). Upon UPR activation, a concomitant increase in LD number has been observed in many cases, suggesting a connection of LDs to ER stress. Furthermore, LDs might represent an efficient mechanism for controlling the FA concentration in the ER.
LDs are involved in the regulation of several aspects of protein homeostasis like protein maturation, storage, and turnover. Studies in yeast postulated an important role of LDs in modulation of protein folding (Moldavski et al., 2015). Protein storage on LDs has been best characterized for histones, which play a role in Drosophila development (Cermelli et al., 2006;Li et al., 2012) and innate immunity (Anand et al., 2012;Bosch et al., 2020). The antiviral protein viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) localizes to LDs as well as to the ER (Hinson & Cresswell, 2009a, 2009bRivera-Serrano et al., 2020). Moreover, LDs are involved in protein turnover  and might assist in proteasome-mediated protein degradation , or facilitate protein digestion by autophagy (Vevea et al., 2015).
The LD neutral lipid core is an appropriate storage location for hydrophobic biosynthetic precursor molecules, including ether lipids Zehmer et al., 2009) and squalene, the hydrocarbon precursor of cholesterol (Valachovic et al., 2016), as well as hydrophobic vitamins (Traber & Kayden, 1987). Intriguingly, LDs also accumulate and store hydrophobic antimicrobial drugs and toxins, thereby affecting drug bioavailability (Greenwood et al., 2019;Sandoz et al., 2014;Walther & Farese, 2019) and toxin resistance (Chang et al., 2015). The partitioning of hydrophobic compounds to LDs might either enhance their activity (as in the case of the antibiotic bedaquiline) (Greenwood et al., 2019), or quench their activity (as in the case of antifungal compounds) (Chang et al., 2015).

| THE ROLE OF LIPID DROPLE TS IN CELLUL AR S I G NALING AND INNATE IMMUNIT Y
In addition to the organelle interactions and diverse functions outlined earlier, LDs are involved in cellular signaling and innate immunity ( Figure 1). Environmental cues modulate the sequestration to and the release from LDs of transcription factors to regulate cell metabolism, and starvation regulates the partitioning of transcription factors to LDs (Bosch & Pol, 2022). FA released upon nutrient deprivation from LDs by ATGL-mediated lipolysis also activate different signaling proteins, including the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARα), PPARγ coactivator 1alpha (PGC1α), and the NAD-dependent lysine deacetylase sirtuin 1 (SIRT1) (Haemmerle et al., 2011;Khan et al., 2015;Najt et al., 2020).
Following lipolysis, the LD-mitochondria tether PLIN5 is phosphorylated by PKA, binds to LD-derived monounsaturated FA, and translocates them to the nucleus to activate SIRT1 (Najt et al., 2020).
Importantly, the hydrophobic LD core is also the storage compartment for precursors of lipid signaling molecules, such as CE, the precursors of steroid hormones involved in cell-to-cell signaling (Papackova & Cahova, 2015;Shen et al., 2016) and arachidonic acid, the precursor of eicosanoids involved in innate immunity (Bozza et al., 2011). Eicosanoids (prostaglandins and leukotrienes) are powerful mediators of inflammatory responses and are synthesized by LD-localizing cyclooxygenase 2 (COX2), which oxidizes arachidonic acid (Jarc & Petan, 2020).
While a number of intracellular pathogens exploit LDs (see the following chapters), it is becoming increasingly clear that stimulation of LD biogenesis represents a host response, which is an integral part of cell-autonomous innate immunity to counteract infections (Bosch et al., 2021;Bosch & Pol, 2022;Melo & Dvorak, 2012). In eukaryotic cells, pathogens are first detected by the recognition of common microbial constituents, known as pathogen-associated molecular patterns (PAMPs). PAMPs are bound by pattern recognition receptors (PRRs), such as the Toll-like receptors (TLRs), which localize to the host cell plasma membrane, endosomes, and the cytoplasm (Paludan et al., 2021). LD biogenesis is stimulated upon activation of PRRs in cells exposed to PAMPs such as lipopolysaccharide (LPS) (Nicolaou et al., 2012), lipoarabinomannan (D'Avila et al., 2006), or synthetic nucleic acids resembling viral DNA and RNA . Furthermore, the stimulation of different TLRs and cytokines reshape distinct macrophage lipidomes, thus enhancing inflammation (Hsieh et al., 2020). On the other hand, LPS-mediated activation markedly increases triglyceride accumulation in macrophages, which through the upregulation of the hypoxia-inducible lipid dropletassociated (HILPDA) protein downregulates ATGL, triglyceride hydrolysis, and prostaglandin E2 (PGE2) production, eventually attenuating the inflammatory response (van Dierendonck et al., 2022).
Taken together, lipid metabolism and LD biogenesis significantly contribute to cellular innate immunity.
Upon infection and stimulation by PAMPs, eukaryotic cells switch from mitochondrial oxidative metabolism (oxidative phosphorylation) to glycolysis (Kelly & O'Neill, 2015), probably as a consequence of the uncoupling between LDs and mitochondria Herms et al., 2013). Hence, LDs dissociate from mitochondria and accumulate in the cytoplasm, and the separated LDs can be instrumentalized for storage and efficient usage of antimicrobial peptides, thus avoiding mitochondrial damage and restricting "LD-avid" pathogens (Bosch & Pol, 2022).
The role of LDs in innate immunity is supported by increasing evidence revealing LD-mediated defense mechanisms during infection with viruses (Monson et al., 2018Truong et al., 2020), bacteria (Jaisinghani et al., 2018;Knight et al., 2018), and parasites (Rabhi et al., 2016). In agreement with the concept of LDs serving as innate immunity platforms, the antiviral protein viperin, histones, and the IFNγ-induced GTPase Irgm3 are immunity-related proteins that have been localized to LDs (Anand et al., 2012;Bougneres et al., 2009;Saitoh et al., 2011). The antiviral activity of viperin includes the inhibition of protein secretion, the recruitment and activation of several signaling proteins, which promote the expression of type I IFNs Saitoh et al., 2011), and the production of a ribonucleotide that blocks synthesis of viral RNA and thus inhibits viral replication (Gizzi et al., 2018). Histones adopt antimicrobial activity by disrupting the bacterial proton gradient and chromosome organization (Doolin et al., 2020).

| SUBVER S I ON OF LIPID DROPLE TS BY MICROB IAL PATHOG ENS
Viruses, bacteria, and parasites subvert LDs for their own benefit.
A variety of different viruses exploit LDs for energy provision during replication, for protecting viral particles from degradation, or for enhancing viral assembly via LD-microtubule transport (Boulant et al., 2008;Cloherty et al., 2020;Filipe & McLauchlan, 2015;Heaton & Randall, 2010). Intracellular bacterial pathogens take advantage of the host cell's vast cytoplasmic nutrient pool. To do so, they rewire host metabolic pathways, which often results in recruitment and interaction with host LDs (Figure 2) (Bosch et al., 2021).
Likewise, intracellular protozoan parasites such as Trypanosoma cruzi or Leishmania major associate with LDs to acquire host lipids Rabhi et al., 2012). Excellent reviews have been published on the role of LDs for interactions of host cells with viruses, bacteria, fungi, and parasites (Barisch & Soldati, 2017a;Brink et al., 2021;Herker et al., 2021;Libbing et al., 2019;Roingeard & Melo, 2017;Vallochi et al., 2018). In the following chapters, we will outline recent discoveries elucidating the role of LDs for selected bacteria-host cell interactions. The best studied intracellular bacteria, which are covered in detail in this review, belong to the genera Mycobacterium, Legionella, Coxiella, Chlamydia, and Salmonella.

| MODUL ATI ON OF LIPID DROPLE TS BY MYCOBAC TERIUM S PECIE S
Mycobacterium tuberculosis is the causative agent of tuberculosis, the world's most deadly infectious disease, which is still responsi- A hallmark of tuberculosis is the formation of granuloma containing "foamy", LD-filled macrophages (Peyron et al., 2008;Russell et al., 2009). Mycobacterium tuberculosis triggers the differentiation into foamy macrophages through long-chain fatty acids, namely oxygenated mycolic acids (Peyron et al., 2008), and through the manipulation of host cell metabolism (Singh et al., 2012)

F I G U R E 2
Interactions of intracellular bacterial pathogens with host lipid droplets. Mycobacterium spp. infect macrophages and form a distinct MCV dependent on T7SSs. LD formation in the host cell is induced through TLR2 and TLR6 signaling. LDs accumulate at and transverse the MCV membrane in a process that includes perilipin shedding. Mycobacterium marinum utilizes host-derived lipids to accumulate intracytosolic lipid inclusions (ILIs). Upon Coxiella burnetii infection of macrophages, an acidic CCV is formed by means of a T4SS, which upregulates Plin2 expression and LD formation. Legionella pneumophila forms a unique LCV in macrophages and amoeba. LDs intimately interact with the LCV membrane, translocate into the LCV lumen, and are implicated in FA metabolism. The large fusion GTPase Sey1 as well as the L. pneumophila T4SS and the Ran GTPase activator LegG1 promote LCV-LDs interactions. Moreover, the L. pneumophila GTPase effector LegA15 localizes to and modulates LDs. Chlamydia trachomatis employs a T3SS to form a membrane-bound inclusion in eukaryotic cells and releases the effector protein Lda3 into the cytoplasm upon infection. LDs may be tethered to the inclusion membrane by Lda3 leading to their "endocytic" translocation into the inclusion lumen and association with Chlamydia reticulate bodies. Salmonella Typhimurium forms the SCV and SIF in infected cells by means of two T3SSs. The SPI-2 T3SS translocates the effectors SseJ, SseL, and SifA, which promote cholesterol accumulation at the SCV. The accumulation of LDs is positively and negatively regulated by TLR2 and TLR4, respectively. CCV, Coxiella-containing vacuole; EE, early endosome; ILI, intracytosolic inclusions; LCV, Legionella-containing vacuole; LDs, lipid droplets; LE, late endosome; MCV, Mycobacterium-containing vacuole; Plin, perilipin; Plin2: perilipin 2; SCV, Salmonella-containing vacuole; SIF, Salmonella-induced filaments. discoideum (M. marinum) (Barisch et al., 2015). The growth of M. marinum is impaired in D. discoideum lacking the perilipin homolog PlnA (Barisch et al., 2015), but not in an amoeba mutant strain lacking Dgat1 and Dgat2 (Barisch & Soldati, 2017b). Since both mutant strains are defective for LD formation, LDs per se do not seem to be essential for intracellular growth. Moreover, hypoxia-through the IFNγ-dependent transcription factor hypoxia-inducible factor-1α (HIF-1α)-promotes LD formation, eicosanoid production, and a metabolic shift to aerobic glycolysis (Braverman et al., 2016;Knight et al., 2018).
In foamy macrophages, LDs accumulate at MCVs, but the bacteria no longer grow and adopt a dormant, nonreplicative state (Peyron et al., 2008). Mycobacterium tuberculosis uses host TAG to accumulate LDs, and inside LD-laden macrophages nearly half the bacterial population develops a dormant/persistent phenotype (Daniel et al., 2011). Furthermore, mycobacterial persistence requires the utilization of host cholesterol (Pandey & Sassetti, 2008).
The inhibition of oxygen-dependent respiration by low, nontoxic nitric oxide concentrations also induced dormancy in M. tuberculosis . The hypoxic response of M. tuberculosis is mediated by the bacterial transcription factor DosR (Park et al., 2003).
Among the 24 putative M. tuberculosis lipases, only LipY shows significant activity toward long-chain TAG, and the corresponding ∆lipY mutant strain is impaired for TAG utilization (Deb et al., 2006). Still, FA rather than carbohydrates are a major energy source of M. tuberculosis during infection and dormancy in mammalian cells (Rodriguez et al., 2014). Intriguingly, intrabacterial accumulation of LDs (intracytosolic lipid inclusions, ILI) by M. marinum in D. discoideum does not lead to dormancy (Barisch et al., 2015;Barisch & Soldati, 2017b).
In the context of Mycobacterium infection, LDs are not only a source of metabolic energy, but also accumulate mycobactins (Chao et al., 2019), a class of siderophores (low-molecular-weight Fe 3+ chelators) of Mycobacterium species including M. tuberculosis (Luo et al., 2005) and M. marinum (Knobloch et al., 2020). Water-soluble carboxy-mycobactin and lipophilic mycobactin efficiently scavenge intracellular iron in macrophages, and the iron-mycobactin complex accumulates with high selectivity in LDs, which directly contact MCVs. Finally, the antimycobacterial compound bedaquiline accumulates primarily in LDs in M. tuberculosis-infected macrophages (Greenwood et al., 2019) as well as in foamy macrophages in M.

| LIPID DROPLE T-MED IATED FAT T Y ACID ME TABOLIS M OF LEG ION ELL A PNEUMOPHIL A
Legionella pneumophila is a Gram-negative environmental bacterium, which upon inhalation of contaminated aerosols replicates in lung macrophages and causes a severe pneumonia termed Legionnaires' disease (Hilbi et al., 2011;Mondino et al., 2020;Newton et al., 2010). Legionella pneumophila is flagellated, grows strictly aerobic, and replicates in free-living protozoa (Boamah et al., 2017;Hoffmann et al., 2014). Although likely not a natural host, the genetically tractable social soil amoeba D. discoideum is a powerful and versatile model to analyze L. pneumophila-phagocyte interactions (Steinert & Heuner, 2005;Swart et al., 2018). In D. discoideum, LDs accumulate upon feeding the cells with fatty acids (in particular palmitate) or bacteria. D. discoideum LDs have a similar composition as mammalian LDs, including TAG, SE, free FA, and perilipin (Du et al., 2013), and they are coated by a polar phospholipid monolayer and distinct proteins (Du et al., 2013;Miura et al., 2002).
A recent study revealed that LCVs intimately interact with palmitate-induced LDs in D. discoideum, and the large GTPase Sey1 as well as the L. pneumophila Icm/Dot T4SS and the effector LegG1 promote LCV-LD interactions . In vitro reconstitution of LCV-LD interactions using purified LCVs and LDs from D. discoideum Ax3 or Δsey1 indicated that Sey1 and GTP promote this process. Moreover, exogenously added palmitate enhances the growth of L. pneumophila wild-type in D. discoideum Ax3 but not in Legionella pneumophila also produces an Icm/Dot-translocated effector, LegA15, which localizes to LDs (Chen et al., 2022). LegA15 is an ankyrin repeat-containing protein that shows GTPase activity, binds to the Golgi tethering factor p115 and-depending on its GTPase and p115-binding activity-modulates LD-LCV interactions in macrophages. In summary, LCVs intimately associate with LDs depending on the large fusion GTPase Sey1, and L. pneumophila produces effectors (LegG1, LegA15), which modulate LD-LCV interactions, as well as the putative FA transporter FadL, which promotes intracellular fatty acid metabolism.

| LIPID DROPLE T HOMEOS TA S IS AFFEC TS INTR ACELLUL AR G ROW TH OF COXIELL A BURNE TII
Coxiella burnetii is a Gram-negative obligate intracellular bacterium and causes the zoonosis Q fever, a severe form of culture-negative endocarditis (Delsing et al., 2011). Coxiella burnetii initially infects alveolar macrophages, but the pathogen has also been identified in LD-containing foamy macrophages in the cardiac valves of endocarditis patients. In macrophages as well as in nonphagocytic cells, C. burnetii forms a replication-permissive compartment, the Coxiella-containing vacuole (CCV) (Figure 2) (Heinzen et al., 1999;Voth & Heinzen, 2007). The nascent CCV interacts with the endosomal pathway, fuses with early and late endosomes, lysosomes, and autophagosomes, and adopts an acidic pH of ca. 4.5 (Newton et al., 2013;Voth & Heinzen, 2007). The acidic pH activates the C.
burnetii Icm/Dot T4SS and the translocation of more than 130 effector proteins into the host cytosol (Beare et al., 2011;Qiu & Luo, 2017;Segal et al., 2005). Only a few of the C. burnetii effectors have been characterized to date. The gene encoding the effector ElpA (ER-localizing protein A) is present in most C. burnetii strains, and the effector disrupts ER structure and function during infection (Graham et al., 2015). However, it has not been assessed whether ElpA also affects LD biogenesis and/or function.
Coxiella burnetii-infected macrophages upregulate the expression of PLIN2 and, dependent on the C. burnetii Icm/Dot T4SS, accumulate twice as many LDs as mock-infected macrophages (Mulye et al., 2018). Unexpectedly, blocking the LD formation with pharmacological inhibitors, or deleting ACAT1 in macrophages, increases C. burnetii growth at least twofold. Conversely, preventing LD lipolysis by blocking ATGL activity with the inhibitor atglistatin almost completely abolishes bacterial growth. Taken together, these findings suggest that LD breakdown is essential for C. burnetii, and maintenance of LD homeostasis, possibly via the C. burnetii T4SS, is critical for intracellular growth of the pathogen (Mulye et al., 2018).

| LIPID DROPLE TS ARE TR AN S P ORTED INTO THE CHL AMYDIA INCLUSION
Chlamydia pneumoniae and Chlamydia trachomatis are Gramnegative, obligate intracellular pathogens that causes a range of diseases in humans, including respiratory ailments, sexually transmitted infections, and trachoma, the leading cause of preventable blindness worldwide (Cheong et al., 2019;Wright et al., 2008). Upon infecting mucosal epithelial cells, C. trachomatis resides within a pathogen vacuole termed the inclusion (Figure 2) (Abdelrahman & Belland, 2005;Elwell et al., 2016). Chlamydia species employ a type III secretion system (T3SS) to govern their interactions with eukaryotic host cells (Ferrell & Fields, 2016;Lara-Tejero & Galan, 2019). Some T3SS-translocated effectors termed inclusion membrane (Inc) proteins are embedded in the inclusion membrane and play a role in the direct interaction of the inclusion membrane with the ER (Bugalhao & Mota, 2019;Derré, 2017;Dumoux et al., 2015). The C.
trachomatis Inc protein CT006 associates with LDs and the ER when expressed ectopically in yeast and mammalian cells, suggesting that the protein might participate in the interaction of LDs with the inclusion (Bugalhao et al., 2022).
Chlamydia trachomatis inclusions acquire host sphingomyelin (SM), a process that is essential for inclusion biogenesis as well as for bacterial replication (Elwell & Engel, 2012). Chlamydia trachomatis hijacks components of both vesicular and nonvesicular lipid trafficking pathways to acquire SM, which is then utilized by the pathogen either catabolically as an energy source for replication or anabolically for inclusion membrane growth and stability (Elwell et al., 2011). For vesicle-mediated SM acquisition and inclusion membrane growth, C. trachomatis selectively subverts the brefeldin A target, GBF1, a regulator of Arf1-dependent vesicular trafficking within the early secretory pathway. In contrast, for nonvesicular lipid trafficking and bacterial replication, C. trachomatis subverts the ceramide transfer protein (CERT), which is a key component in nonvesicular ER to TGN trafficking of ceramide (the precursor of SM) (Elwell et al., 2011). Chlamydia trachomatis recruits CERT and its ER binding partner VAP-A, as well as the SM synthases, SMS1 and SMS2, to the inclusion. Hence, a SM biosynthetic factory at or near the inclusion is likely formed (Elwell et al., 2011). Acquisition of host lipids by the C. trachomatis inclusion occurs through LDs (Cocchiaro et al., 2008). Interestingly, LDs are translocated from the host cytosol into the inclusion lumen and tightly associate with reticulate bodies, the replicative form of Chlamydia. The chlamydial effector protein Lda3 promotes the uptake of LDs into the inclusions through the formation of LDinclusion membrane contact sites, and an "endocytic" entry leading to membrane-coated, intraluminal LDs (Cocchiaro et al., 2008).
However, the metabolism of FA by C. trachomatis does not necessarily need to involve LDs, since upon LD depletion even more bacterial progeny is produced (Sharma et al., 2018).
The C. trachomatis effector IncA cofractionates with LDs and accumulates in the inclusion lumen (Cocchiaro et al., 2008).
Accordingly, the IncA-enriched inclusion subdomains might constitute the LD translocation sites. The effector IncD interacts with CERT, which in turn binds VAP on the ER (Agaisse & Derré, 2014;Derré et al., 2011). Finally, IncV directly interacts with VAP through two FFAT motifs and is enriched at ER-inclusion MCS (Stanhope et al., 2017). Depletion of CERT or VAP results in a significant decrease in inclusion size and infectious progeny production (Derré et al., 2011;Elwell et al., 2011). Taken together, IncA, the IncD-CERT-VAP complex, and the IncV-VAP complex contribute to ER-inclusion MCS and are proposed to function in the nonvesicular trafficking of host lipids to the Chlamydia inclusion, a process essential for intracellular growth (Agaisse & Derré, 2014;Derré et al., 2011;Elwell et al., 2011). So far, it has not been explored whether IncA, IncD, and IncV contribute to the formation of LD-inclusion MCS.
Dependent on the SPI-1 T3SS, S. Typhimurium induces a rapid stimulation of LD formation in macrophages (Kiarely Souza et al., 2022). LD accumulation occurs through TLR2-dependent signaling and is negatively regulated by TLR4. Pharmacological prevention of LD formation by inhibiting DGAT1 or phospholipase A2 (PLA2) reduces intracellular bacterial growth and impairs PGE2 synthesis (Kiarely Souza et al., 2022).
The SCV accumulates cholesterol as a consequence of host sterol biosynthesis perturbation due to the bacterial infection (Catron et al., 2002). The SPI-2 T3SS-translocated effectors SifA, SseJ, and SseL are necessary for cholesterol accumulation at the SCV (Greene et al., 2021;McEwan et al., 2015;Walch et al., 2021). SseJ has deacylase activity (Ohlson et al., 2005), esterifies cholesterol in vitro, in host cells and during infection, and promotes the formation of LDs (Nawabi et al., 2008). Compared to S. Typhimurium wild-type, an infection with the ΔsseJ mutant strain shows reduced levels of cholesterol ester production in HeLa cells and RAW 264.7 macrophages and a decreased number of LDs per cell. Accordingly, the ectopic production of SseJ reduces cholesterol levels in cellular membranes. On the other hand, SseJ also downregulates the production of the host cholesterol transporter ABCA1 in macrophages, leading to an increase in cellular cholesterol (Greene et al., 2021).

ACK N OWLED G M ENTS
Work in the group of H.H. was supported by the Swiss National Science Foundation (SNF; 31003A_175557, 310030_207826). Open access funding provided by Universitat Zurich.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in Pubmed at https://pubmed.ncbi.nlm.nih.gov/.

E TH I C S S TATEM ENT
This article does not contain any studies with human participants or animals.