Cytoplasmic access by intracellular bacterial pathogens.

TIMI-1048; No. of Pages 10

Review

Cytoplasmic access by intracellular bacterial pathogens Jennifer Fredlund and Jost Enninga Unite´ ‘Dynamique des interactions hoˆte–pathoge`ne’, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France

Entry into host cells is a strategy widely used by bacterial pathogens, after which they either remain within membrane-bound compartments or rupture the endocytic vacuole to reach the cytoplasm. During recent years, cytoplasmic access has been documented for an increasing number of pathogens. Here we review how classical cytoplasmic bacterial pathogens rupture their endocytic vacuoles as well as the mechanisms used to accomplish this task by bacterial species for which host cytoplasmic localization has only recently been identified. We also discuss the consequences for pathogenesis resulting from this change in intracellular localization, with a particular focus on the role of the host. What emerges is that cytoplasmic access plays an important role in the pathophysiology of an increasing number of intracellular bacterial pathogens. Intracellular localization of bacterial pathogens Bacterial pathogens reside in two major niches during host infection: they either remain extracellular or are internalized via active or passive pathways [1]. When internalized, they are surrounded by host cell membranes. Subsequently, pathogens can remain vacuole-bound through the entire course of infection by blocking delivery of the lysosome or by modulating the physiological conditions of the vacuolar niche. An alternative strategy is rupture of the vacuole, or phagolysosome, giving the bacterium access to the host cell cytoplasm. The decision to remain within an endomembrane compartment or to escape into the cytoplasm has important consequences for both the invader and the infected cell. The physiological environment of each niche differs drastically with regard to nutritional access, differential pathogen recognition, and spread to neighboring cells. Consequently, induced innate and adaptive immune responses occur in different ways depending on the bacterial localization. Until recently, intracellular pathogens were neatly separated into two groups, either cytoplasmic or vacuolar. This rigid definition has been challenged by a series of scientific reports (Figure 1) suggesting that access to Corresponding author: Enninga, J. ([email protected]). Keywords: intracellular bacteria; vacuolar rupture; membrane trafficking; host cell death. 0966-842X/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.01.003

the host cytoplasm is more frequent than previously anticipated. Bacterial pathogens that reach the host cytoplasm: the classics For a number of bacteria, the mechanisms of cytosolic access have been studied in detail. We discuss known bacterial and host factors important for this process and present a brief overview of how they function during vacuolar rupture. The proteins involved that are discussed in this and in the following section are highlighted in Table 1. Shigella triggers its entry to rapidly escape from the intracellular vacuole Host cellular entry into both epithelial cells and macrophages by Shigella species has been studied for decades, mostly using Shigella flexneri [2,3]. S. flexneri invades cells via a trigger mechanism that involves injecting approximately 25 effector proteins directly into the host cytoplasm through the mxi-spa type III secretion system (T3SS). Cytoplasmic localization of Shigella within different cell types was first observed by transmission electron microscopy (Box 1, Figure 1A) [2]. Furthermore, Shigella induces ‘comet tails’ composed of host actin via the bacterial factor IcsA, and these have been commonly used as markers for bacterial cytoplasmic localization. Initially it was proposed that bacterial effectors play a major role in cytoplasmic access and vacuolar rupture by Shigella [4]. In particular, the T3SS translocator proteins IpaB and IpaC have been shown to destabilize eukaryotic cell membranes using red blood cell lysis assays at very high concentrations of the bacterial proteins [5]. Recent in vitro studies on IpaB have suggested that its assembly into multiprotein complexes [6,7] allows an influx of potassium ions through endolysosomal membranes, suggesting pore formation and an involvement in vacuolar rupture [6]. Underlining the possibility that other factors may be involved in the vacuolar rupture process, the T3SS effector IpaH7.8 has also been associated with cytoplasmic access; however, its function is not yet established [8]. Furthermore, a non-characterized region of the Shigella virulence plasmid allowed uncoupling of bacterial entry and vacuolar rupture, hinting at potential involvement of additional factors in the rupture process [9]. On vacuolar rupture, a pool of smaller vesicles formed from the vacuolar membrane remnants suggests that the autophagy machinery is recruited to the site of ruptured membranes [10,11]. Taken together, current data suggest that the rupture process represents a signaling platform with important roles for Trends in Microbiology xx (2014) 1–10

1


Review

Trends in Microbiology xxx xxxx, Vol. xxx, No. x

(A)

(C)

Shigella WT 15 min 60 min Key:

80 15 min

60

BS176 Afal M90T Afal

40 20 0 0,5

1,5 2,5 3,5 4,5 Rao 450 nm/535 nm

Number of cells

Number of cells

450 nm

535 nm

(D)

(B)

Key:

100 80 60 40 20 0

60 min

0,5

BS176 Afal M90T Afal

1,5 2,5 3,5 4,5 Rao 450 nm/535 nm TRENDS in Microbiology

Figure 1. Measuring the cytoplasmic access by bacterial pathogens. (A) Transmission electron microscopy has been extensively used as evidence of cytoplasmic access by bacteria, for example for Shigella (scale bar 500 nm; reprinted with permission from [3]). (B) The absence of markers for endosomal or lysosomal compartments hints at cytoplasmic localization. Here, dendritic cells (blue nuclei) were infected with Mycobacterium tuberculosis (green) for 4 h (upper panel) or 96 h (lower panel). At early time points, the bacteria colocalize with lysosomal associated protein 1 (LAMP1, red); later, they spread throughout the cytoplasm [38]. (C) Galectin-3 (green) flags damaged vacuolar membrane structures after their rupture by Shigella (red) [83]. (D) A fluorescence resonance energy transfer (FRET)-based approach measures access to the cytoplasm in HeLa cells infected with wild type (WT) Shigella. The cephalosporin-derived FRET probe is cleaved by b-lactamase on the surface of the invading bacteria, resulting in a signal switch from green to blue (left panel). The fluorescent ratio (450 nm/535 nm) can be plotted against the number of infected cells to highlight vacuolar rupture after 60 min of infection. Shigella BS176 AfaI cannot enter host cells and M90T AfaI is the invasive strain [84].

both bacterial and host proteins, and with effectors of the Shigella T3SS orchestrating the process. Listeria: zippering into host cells and forming pores in the vacuole Invasion by the human pathogen Listeria monocytogenes is achieved via bacterial surface proteins such as internalin A (InlA) and InlB that respectively bind to human E-cadherin and have affinity for host glycosaminoglycans, the receptor for the human globular part of the complement component gC1q-1 (gC1q-R), and the Met receptor (the main receptor for InlB) [12]. This interaction triggers a complex signaling cascade involving a battery of host factors, including kinases, GTPases, and even clathrin, leading to vacuolar formation that has been described in detail [12]. The secreted protein listeriolysin-O (LLO) is the key bacterial factor leading to vacuolar rupture15 min after Listeria internalization [12]. It is a member of the cholesterol-dependent cytolysin family, which also includes streptolysin-O and perfringolysin-O. Even though a link has been made between LLO activity and vacuolar pH that results in membrane permeation, it is still not clear whether this effect is direct or requires additional factors [13]. This is because the bacterial phospholipases PI-PLC and PC-PLC are also necessary to achieve efficient cytoplasmic access [14]. One explanation for this could be that initial membrane damage via LLO allows better cytoplasmic access for the mature phospholipases. 2

Similar to Shigella infection, it is clear that Listeria proteins function in concert with host factors to grant the bacterium cytoplasmic access. For example, host gammainterferon-inducible lysosomalthiol reductase (GILT) chemically reduces LLO, thereby increasing LLO activity [15], and the cystic fibrosis transmembrane conductance regulator (CFTCR) increases intravacuolar chloride concentrations to potentiate LLO activity [16]. In addition, subversion of the Listeria-containing vacuole from the endosomal pathway requires Ca2+ flux through the LLO pores [13]. These fluxes inhibit the normal vacuolar maturation that would eventually result in lysosomal fusion and bacterial destruction. Finally, an inducible increase in membrane resistance to pressure, termed renitence, has been identified. This mechanism may limit vacuolar membrane damage and appears to involve the host heat shock protein HSP70 [17]. Vacuolar escape within activated macrophages is reduced by the production of reactive oxygen and nitrogen intermediates (ROIs and RNIs) [18]; however, their mode of action in this process needs to be further studied. Rickettsia: concerted action of hemolysins and phospholipases Rickettsiales represent an order of important obligate human intracellular pathogens targeting mainly endothelial cells and macrophages [19]. Owing to the complex handling requirements for this pathogen, mostly genomic comparison has been used to delineate its infection


Review


Table 1. Bacterial pathogens that access the host cytoplasm Pathogen Shigella

Listeria

Francisella

Rickettsia Salmonella

Mycobacterium Legionella

Important factors a IpaB IpaC IpaH7.8 LLO PlcB PlcC GILT CFTCR HSP70 MglA FTT1103 VgrG IglL vATPase CDC27 Pld TlyC SifA SseJ PipB2 SopD2 T3SS-1 SKIP Kinesin RhoA ESAT-6 SdhA PlaA

Function (related to escape) b Membrane destabilization Membrane destabilization Needs clarification Membrane pore formation Cleaves PI Cleaves PC Chemical reduction of LLO Increases vacuolar Cl concentration Increases membrane resistance to pressure Bacterial factors that require further biochemical characterization

Refs [5–8]

Acidification of phagosome Ubiquitin ligase Hydrolyzes PC in vitro Causes hemolysis in vitro Stabilization of SCV Destabilization of SCV Destabilization of SCV Destabilization of SCV Damage to SCV membranes Kinesin regulation SCV tubule formation Tubule formation Membrane pore formation Stabilization of LCV Destabilization of LCV

[26]

[12–14]

[15] [16] [17] [25]

[20] [21] [85]

[86]

[42,43,72] [47,61]

a

Host factors are in purple.

b

Abbreviations: PI, phosphoinositide; PC, phosphadityl choline; SCV/LCV, Salmonella/Listeria-containing vacuole.

strategies. Rickettsia gains rapid access to the host cytoplasm, which was first considered dependent on the activity of its phosopholipase A2. Intriguingly, genome sequencing of Rickettsia conorii and Rickettsia prowazeki has identified another group of bacterial factors driving cytoplasmic localization, particularly a patatin-like protein, two hemolysins, and phosopholipase D (PLD) [20]. The roles of PLD and one hemolysin, TlyC, in membrane destabilization have been examined by complementation in the more tractable model Salmonella, with subsequent tracking of its intracellular localization [21]. These results were mainly obtained using genes from R. prowazeki; however, in the case of Rickettsia typhi, a gene encoding a phospholipase A2-like protein was recently identified [22].The power of comparative genomics for different Rickettsia strains and the capacity to test potential bacterial factors in more accessible pathogen models such as Salmonella will be instrumental in deciphering the mechanism of cytoplasmic access by this pathogen. Francisella: escape into the cytoplasm with a strong impact on the host The Gram-negative bacterium Francisella tularensis causes tularemia and is internalized into macrophages via a mechanism involving pseudopods and the C3 component of the host complement system, as well as other host cellular opsonins. It has been suggested that Francisella follows a similar endomembrane trafficking route as Listeria (Box 2). Subsequently, Francisella escapes from a

compartment with features of the late endosome, but devoid of lysosomal markers such as cathepsin D [23]. Temporary acidification of this compartment has also been suggested in Francisella escape [24]. On the bacterial side, factors encoded on the pathogenicity island, such as IglC, MglA, and FTT1103, are involved in cytoplasmic access. In addition, injection of bacterial effectors VgrG and IglI through a type VI secretion system are implicated in cytoplasmic localization [25]. Host factors required for vacuole rupture include an active v-ATPase and CDC27, a ubiquitin ligase identified through siRNA loss-of-function screens using Drosophila [26]. Slightly delayed compared to Shigella and Listeria, Francisella escape into the host cytoplasm occurs 30–60 min after internalization [24]. On cytoplasmic release, the pathogen replicates rapidly before breaking free to infect neighboring cells. Francisella provides an example of a bacterial species that escapes later during infection than other pathogens traditionally understood to access the cytosol. However, it also utilizes both bacterial and host factors to accomplish this task and provides a unique platform to examine protein dynamics. Pathogens localized within endomembrane compartments access the host cytosol Intriguingly, cytoplasmic access has only recently been identified as important for infection for some bacterial pathogens. These pathogens were elucidated using a variety of new and old experimental techniques (Box 1). In 3


Review Box 1. Locating intracellular microbes

Box 2. Disruption of membrane trafficking

Determination of whether a bacterium is endomembrane-bound or free in the cytosol has been a major challenge, but novel techniques are providing new insights into bacterial localization. Owing to their high resolution, electron micrographs have traditionally provided evidence about the intracellular localization of a number of bacterial pathogens. This has contributed to our understanding that Shigella, Listeria, Rickettsia, and Francisella species access the host cytoplasm upon invasion. More recently, fluorescent approaches in conjunction with differential permeabilization of host cells have distinguished bacteria within endomembrane-bound compartments from those in the cytoplasm [4]. However, some of these methods can be difficult to interpret because they measure the absence of markers of endomembrane structures around the internalized bacteria [38]. For example, lysosomal-associated protein 1 (LAMP1) has been used to infer Salmonella vacuolar localization, but the absence of a signal may not necessarily mean cytoplasmic localization, because LAMP1-negative but Salmonella-positive membrane structures have recently been identified [87]. In addition, fractionation procedures have separated cytoplasmic pathogens from those localized in other cellular compartments. To overcome the problems with these methods, reporters have been developed that monitor membrane damage or yield direct information on bacterial access to the cytoplasm (see Figure 1C,D in main text) [83,84]. First, fluorescent galectin-3 can be used to identify recently generated membrane fragments. Alternatively, another approach uses cells loaded with a FRET reporter that is cleaved by blactamase and can therefore detect contact between bacteria expressing this enzyme and the reporter-containing cytosol [84]. These new methods have confirmed the cytoplasmic localization of many bacteria classically known to reach the cytoplasm, and have also revealed many other, previously unrecognized, bacterial species that gain access to the cytoplasm.

To survive inside a vacuole, either temporarily or long term, an intracellular bacterium must interfere with normal host trafficking to avoid detection and degradation. In the case of Listeria, its vacuole (LCV) is altered via translocation of the bacterial factor Lmo2459, which inhibits activation of the small GTPase Rab5a from its GDP state into the GTP state through ADP-ribosylation [12]. It has been suggested that this modification of the endocytic pathway prevents recruitment of NADPH oxidase to the LCV, an enzyme that would likely cause the vacuole to become toxic to the invader. Other Rab GTPases have also been linked to steps of altered trafficking of the LCV during infection [88]. It is thought that Francisella follows a similar pathway to Listeria, and vacuolar membrane markers include the GTPases Rab5 and Rab7, LAMP1, and the vacuolar ATP pump [23]. To avoid the harsh environment of late endosomes/ lysosomes, the pathogen blocks activation of NADPH oxidase, although this depends on the specific bacterial strain. In Shigella, recent studies indicate that IpaB alters exocytic membrane trafficking and is involved in the dispersal of the Golgi complex during infection, which disrupts cell-surface receptor trafficking and could also disrupt maturation or trafficking of highly damaging lysosomal proteases [89]. For bacteria that reside for longer periods inside the vacuole, more extensive alteration of trafficking has been documented. For instance, Salmonella targets multiple RabGTPases throughout the course of infection and SCVs are dynamically labeled, at times containing the membrane markers Rab5, EEA1, Rab7, and Rab9, and the lysosomal markers LAMP1 and vATPase. Yet they exclude other lysosomal proteins, specifically the highly damaging mannose phosphate receptor (MPR)-associated hydrolases [90]. The SifA– SKIP complex was recently implicated in the mislocalization of MPRs to late endosomes in both macrophages and epithelial cells by acting as a Rab9 sink [91]. By disrupting the Syn10 retrograde pathway, which requires Rab9, MPRs are not recycled back to the trans-Golgi network (TGN). This allows SCVs to establish a replication niche through fusion with late endosomes yet without suffering degradation, because these compartments do not contain hydrolases. Legionella, which also spends extended time in a modified vacuole, specifically disrupts autophagy membranes, a phenomenon described in detail in Box 3.

general, less is known about the specific escape mechanisms than for those in described above. Nevertheless, the medical relevance of cytoplasmic access makes this a fastmoving field of research. Intracellular trafficking of Salmonella Similar to Shigella, Salmonella enterica serovar Typhimurium (S. Typhimurium) can invade epithelial cells via a trigger mechanism, although other modes of entry can also be employed. Unlike Shigella, Salmonella classically resides inside a modified vacuole called the Salmonellacontaining vacuole (SCV) [1]. To survive in the SCV, Salmonella must modify its progression through the normal host endocytic pathway (Box 2). SifA is important for maintenance of the SCV, because sifA mutants escape from the vacuole after a few hours but do not replicate in the macrophage cytosol [27]. SifA also controls positioning of the SCV within infected cells, mediated by its interaction with the mammalian protein SKIP, which binds the molecular motor kinesin [28]. SifA may also have guanine nucleotide exchange factor (GEF) activity that is important for membrane tubule formation [29]. Is an intravacuolar lifestyle the only one that allows Salmonella replication? It has long been understood that a small proportion of infecting Salmonella escape from the SCV, with some of these bacteria being coated in ubiquitinated proteins or associated with galectin-8 and subject to autophagy [30,31]. Knodler et al. recently reported that unlike late-escaping sifA mutants, a proportion of S. Typhimurium leave the SCV early during infection of epithelial cells and replicate very rapidly in the cytoplasm (20 min 4


per generation) [32]. Quantitative microscopic techniques demonstrated that this subpopulation occurs in a similar percentage of infected HeLa cells for both the wild type and a mutant without the T3SS-2 secretion system, which is normally important for SCV maturation. In gentamycin survival assays, which kill bacteria not taken up inside host cells, the wild type and sifA mutants show similar numbers of surviving bacteria at 8 h post infection, even though sifA mutants are unable to replicate in SCVs [33]. Malik-Kale et al. showed that T3SS-2 mutants, which should not express sifA, hyper-replicate at similar rates to wild type bacteria, thereby masking the lack of SCV replication [34]. The role of hyper-replicating bacteria during infection is unclear, but the phenotype is independent of T3SS-2, suggesting that the decision to leave the vacuole is upstream of SCV maturation. Mycobacterium tuberculosis membrane rupture Many bacterial factors important for shaping the intracellular niche within host cells have been identified by studying M. tuberculosis virulence, including the unique cell wall and several proteins [35,36]. The traditional paradigm of M. tuberculosis infection is that the bacteria take up residence within vacuoles inside professional phagocytes in the lungs, blocking fusion with lysosomes and delaying


Review maturation of the phagolysosome. Nevertheless, other mycobacteria, including the fish pathogen M. marinum, do access the host cytosol, even forming actin comet tails [37]. The intravacuolar paradigm suggested for M. tuberculosis has recently begun to shift. Van der Wel et al. were the first to convincingly challenge this view [38], although some evidence had been seen before [39,40]. They conducted a thorough study of M. tuberculosis subcellular localization in human-derived dendritic cells (DCs) using transmission electron microscopy (TEM) (Figure 1B). Surprisingly, over 50% of infected DCs contained cytoplasmic bacteria after 96 h of infection, whereas non-pathogenic vaccine strains remained in the vacuole. Cytoplasmic release of M. tuberculosis was dependent on ESX-1, a type VII secretion system (T7SS). These findings were independently confirmed in macrophages using a fluorescence resonance energy transfer (FRET) reporter (Box 1) to detect bacterial contact with the cytosol [41]. Interestingly, when the genomic RD1 region containing ESX-1, which is known to restore virulence to vaccine strains, was added to the attenuated BCG strain, these bacteria also contacted the host cytosol. Furthermore, after elimination of the secretion locus ESX-1 and, more specifically, the C terminus of ESAT-6, a T7SS substrate, bacteria remained membrane-bound [38,41]. This agrees with other evidence that M. marinum ESAT-6 alone forms small pores in macrophage vacuolar membranes [42] and that markers linked to membrane damage, such as galectin-3 and ubiquitin, colocalize with M. tuberculosis phagosomes [43]. In addition, ESAT-6 is important for recognition of M. tuberculosis by the cytoplasmic autophagic machinery (see below). Together, these results suggest not only that vacuolar rupture is an active, bacterially mediated process but also that it is essential for successful infection by M. tuberculosis and other Mycobacterium species. Legionella membranes Legionella pneumophila secretes more than 240 proteins into host cells via its type IV secretion system (T4SS), Dot/ Icm, some of which direct maturation of the Legionellacontaining vacuole (LCV) via prevention of lysosomal fusion and recruitment of endoplasmic reticulum (ER) membranes, ribosomes, and mitochondria (Box 3) [44]. Maintenance of the LCV is Dot/Icm-dependent, because non-virulent Ddot/icm mutants are found in the cytosol of human macrophages soon after infection [45]. Escape from the LCV at later time points (12–18 h post infection) is also seen for wild type strains, and although it appears to be a regulated and important part of the infection cycle, there is debate about what factors mediate vacuolar escape [46]. It was recently shown that the effector SdhA is responsible for maintaining the integrity of LCV membranes in mouse and human macrophages through antagonistic action to the effector PlaA, similar to the interaction between Salmonella SifA and SseJ [47]. However, this does not explain how Ddot/icm mutants could gain access to the cytosol. Legionella does form LCV pores during infection of its natural ameobal hosts [48] and a full-length copy of the Legionella protein IcmT is important for both pore formation and infection spread in mammalian cells


Box 3. Legionella and autophagy Legionella infection is intimately involved with the autophagy pathway, considering that LCV maturation is largely similar to autophagosome formation. Therefore, it has been used as a major model to study autophagy and bacterial infection. Many autophagy markers are found on the LCV and it is thought that Legionella effectively, or ineffectively, depending on the host, stalls autophagy to establish its niche [92]. Ubiquitinated proteins are found on the membranes soon after initial infection and multiple Legionella effectors contain motifs characteristic of E3 ubiquitin ligases. The Legionella effector AnkB increases both the accumulation of ubiquitinated proteins on the LCV and the ability of some Legionella strains to survive [93], possibly mediated by proteasomal degradation of ubiquitin-labeled host proteins, a process that may provide Legionella with amino acids that are essential for its intracellular cell growth. LubX increases survival in Drosophila through ubiquitination and subsequent degradation of other bacterial effectors [94]. However, in Dictyostelium, the autophagy pathway has no effect on Legionella growth [95], highlighting the different roles of autophagy during infection of various hosts. The RavZ effector has an antiautophagic effect through specific cleavage of Atg8–LC3 at a bond that not only unconjugates the protein from membrane phosphatidyl ethanolamine (PE) but also prevents its reattachment. However, a DravZ mutant does not display a growth defect in macrophages, suggesting that more effectors play a role in autophagy avoidance [96]. The effector LegA9 has the opposite effect, targeting the LCV to the autophagy pathway via the p62 adaptor protein. A legA9 mutant is only the second mutant found, besides flagellin mutants, that can grow in murine macrophages, suggesting that autophagic clearance is a very important host mechanism in fighting Legionella infection [97].

[49,50], although it is not required for escape from the LCV [46]. icmT mutants replicate intracellularly to similar levels as wild type bacteria, but remain trapped inside the host cells until later time points [51]. These mutant studies, in combination with cell biological techniques, support a cytosolic stage of Legionella infection as important for progression of infection, although its regulation remains to be understood. Consequences of cytoplasmic access of intracellular pathogens Cytoplasmic localization of a bacterium causes many perturbations in the host cell (Figure 2). This is because of the display and detection of bacterial surface signatures by a specific set of cytoplasmic host recognition molecules different from the receptors at the cell surface. These host factors include receptors that activate the immune system or autophagy. However, detection by host cell surveillance systems and subsequent host cell death or gene expression changes can be advantageous for the invading bacterium, and can even be an essential part of the infection process. Cell death as a result of cytoplasmic access Cell death occurs via multiple pathways in mammalian cells. Apoptosis is initiated by various signals resulting in activation of the cysteine proteases caspase-2, caspase-8, caspase-9, and caspase-10, which in turn activate effector caspases, ultimately resulting in blebbing and cell death without membrane rupture. However, pyroptosis is mediated by activation of caspase-1 and results in inflammatory cell death. In this case, the cell membrane is perforated, resulting in blebbing, spilling of cell contents, and activation of cytokines. Necrosis is a form of cell death 5


Review


(A) Bacterial effectors

RNA

DNA

Pepdoglycan

Flagellin Damaged membranes

(B)

Cell death

Necrosis-like Shigella

Non-canonical

Pyroptosis Salmonella Shigella Legionella Mycobacteria

Autophagy

Shigella Salmonella

Casp 11 Salmonella Burkholderia Legionella

Salmonella GAS Listeria Mycobacteria

Shigella

AIM2 Francisella Legionella TRENDS in Microbiology

Figure 2. The impact of cytoplasmic access by intracellular bacteria. (A) Host signaling pathways induced after vacuolar rupture are triggered by bacterial effector proteins, lipid A, flagellin, peptidoglycan, bacterial nucleic acids, the vacuolar contents or damaged vacuolar membranes. (B) Black font: different types of cell death, such as necrosis and pyroptosis, are a consequence of cytoplasmic access by bacterial pathogens. Purple font: the autophagy pathway also plays a major role in pathogen recognition and control. Left: some bacteria block autophagy. Middle: successful autophagy. Green ovals represent LC3, small circles represent ubiquitin, and squares represent NDP52. Right: very small green circles represent septins, which form cages around Shigella and show interdependence with autophagy. Abbreviations: Casp 11, caspase-11; GAS, group A Streptococcus; AIM2, absent in melanoma 2.

that also results in inflammation but is caspase-independent. It was generally thought to be uncontrolled by the affected cell, but recent evidence suggests this may not be the case [52]. Pyroptosis via caspase-1 is often induced by detection of cytosolic bacteria and can be initiated when pathogens 6

escape from a vacuole. In myeloid cells, Shigella triggers cell death through caspase-1 following endolysosomal membrane damage [6]. However, Shigella triggers cell death in non-myeloid cells via two mechanisms: (i) a caspase-1 dependent pathway triggered by ruptured vacuolar membranes, which also participate in autophagy


Review activation [10]; and (ii) a caspase-independent pathway involving loss of mitochondrial membrane potential, similar to oxidative stress. Interestingly, the bacterium also induces pro-survival signals through Nod1/NFkB/IKKb that counteract this necrotic cell death until relatively late in infection [53]. M. tuberculosis cytosolic access has only recently begun to be studied, but it appears that this change in localization results in macrophage cell death [41]. In particular, the ESX-1 and ESX-5 T7SSs are important for initiation of cell death during M. tuberculosis and M. marinum infection, likely through release of lysosomal proteases from the ruptured phagosome into the cytosol [54]. In fact, inhibition of cathepsin L [55] or cathepsin B [56] reduces macrophage cell death in response to mycobacterial infection, although these results are dependent on bacterial load. ESX-1 plays a role in activation of inflammation and cell death through nod-like receptor family, pyrin-domain-containing 3 (NLRP3) [43,57], which supports the observation that bacteria are present in the host cytosol via phagosomal escape, because NLRP3 monitors the cytoplasm and activates the inflammasome in response to cytoplasmic stimuli. Interestingly, deletion of ESX-5 effectors had a more significant impact on cell death, and it is thought that these factors only gain access to host proteins after vacuolar escape [54]. Salmonella also causes cell death as a consequence of cytoplasmic access in the subset of infected host cells harboring hyper-replicating bacteria. Transcriptional fusions of GFP to various Salmonella promoters revealed that many hyper-replicating cytosolic bacteria express both flagellar proteins and T3SS-1 genes and initiate caspase-1-mediated pyroptosis and interleukin-18 (IL-18) release [32]. This suggests a connection between cytoplasmic access and the massive inflammation that accompanies Salmonella infection. Legionella activates host cell-death pathways as well, apparently via multiple mechanisms. Cytosolic flagellin is primarily responsible for activation of caspase-1; this requires the NLRC4/Ipaf and Naip5 inflammasome components, and limits growth in restrictive cells [58,59]. Interferon b (IFNb) is increased after infection in a Dot/ Icm-dependent, but not flagellin-dependent, manner and this increase is important for Legionella restriction in the lung epithelium [60]. In a DsdhA mutant, which has an unstable LCV, flagellin is not required for induction of host immune response and cell death, nor for a large IFNb response, which instead appears to be mediated by contact of bacterial DNA with the cytosol and subsequent activation of inflammation via the AIM2 inflammasome [61]. This is consistent with bacterial degradation on LCV exit, and the growth of this mutant is severely limited within macrophages. However, the timing of cytosolic contact and the number of bacteria entering the cytosol may be important for infection progression and the ability to overwhelm host defense mechanisms, induce inflammatory responses, and achieve cellular escape. In support of recent questioning of LCV integrity, icmT mutants, which cannot form pores in the membranes of mammalian cells, do cause apoptosis via caspase-3 during early infection [51], but little necrotic morphology is seen during late infection. In addition, they do not cause red blood cell lysis or disease


in mice. Together, these data suggest that cytosolic access is required for triggering events later in infection. Dealing with autophagy Another cytoplasmic cell process is autophagy, a major catabolic pathway activated in response to nutrient depletion, misfolded proteins, and other signals. Autophagy allows for recycling of cellular contents to free up required molecules or to remove harmful protein intermediates or aggregates. The autophagic machinery recognizes ubiquitinated proteins and surrounds them with a double membrane that later fuses with lysosomes, thereby digesting the contents. In general, the proteins p62 and NDP52 are considered adaptors that bind to both ubiquitin and LC3, with LC3 then directing the autophagic complex [62]. Interesting work using Shigella links autophagy to the actin-dependent accumulation of host septins around cytosolic bacteria [63]. siRNA knockdown of certain septins or of autophagy genes resulted in loss of both septin caging and autophagy of Shigella, indicating an interdependence between the two systems for bacterial clearance. However, this was not true for other bacteria examined, including Listeria, which is also autophagocytosed but independently of actin and septins. Similar to Listeria, group A Streptococcus (GAS) utilizes a pore-forming toxin, SLO, that is important for escape from its endosomally derived uptake vacuoles. On access to the cytosol, these bacteria also induce autophagy, and are quickly marked with LC3 and limited for growth during early infection in epithelial cells [64]. Francisella also interferes with the autophagy pathway during its cytoplasmic stage and can persist within the cytoplasm for more than 10 h without detection [24,65]. The polysaccharidic O-antigen plays a main role in autophagy evasion via the Atg5 pathway; however, the surface molecules that are recognized need to be identified [66]. Interestingly, in mouse primary macrophages, a percentage of Francisella can ‘retranslocate’ in a membranebound compartment displaying lysosomal and autophagosomal features [65]. Autophagic responses to pathogens are often to cytosolic bacteria, although they can also occur in response to damaged vacuolar membranes [10,30,31]. For instance, ubiquitin coats cytosolic S. Typhimurium in both epithelial cells and macrophages. In macrophages, this leads to recruitment of proteasomes and subsequent bacterial degradation [67]. In HeLa cells, the pathway has been recently characterized. First, host sugar molecules exposed through damaged SCV membranes are labeled with galectin-8 [31] and cytosolic Salmonella are also coated with linear ubiquitin chains [68]. Then galectin-8 recruits the autophagy adapter NDP52 [31] and ubiquitin-coated cytosolic bacteria recruit the additional autophagy adapters p62, NDP52, TBK1, and optineurin [69]. Furthermore, NDP52 recognizes LC3C [70] and TBK1 phosphorylates optineurin, which leads to increased binding of the autophagy receptor LC3 and subsequent autophagy initiation [69]. However, Salmonella can also counteract autophagy through deubiquitination by the effector SseL [71]. Autophagic targeting of M. tuberculosis has a similar profile to that of Salmonella and requires the bacterial 7


Review ESX-1 system. Approximately 30% of intracellular M. tuberculosis immunostain positive for autophagy markers 4 h post-infection [72] and this co-localization is dependent primarily on ubiquitination by parkin [73]. This recruitment is dependent on ESAT-6, a bacterial effector that forms pores and is part of ESX-1, and expression of L. monocytogenes LLO restores some LC3 localization in an Desat6 strain [72]. Taken together, these data suggest that autophagy is induced by mixing of vacuolar contents with the cytoplasm due to vacuole damage. Further characterization demonstrated that cytosolic bacterial DNA is a key factor for both autophagy induction [72] and cytokine production [74]. Interestingly, Irf3 / mice, which have a limited immune response, did not die as a result of mycobacterial infection, suggesting that cytokine production and inflammation, a result of cytosolic access, are critical to the bacterium for successful infection [74]. However, Atg5 / mice, which are impaired for autophagy, were extremely sensitive to infection [72], in agreement with earlier studies in macrophages demonstrating the importance of autophagy in the control of mycobacteria. This highlights the careful balance involved in clearing infection, because both cytokine production and autophagy are activated by cytosolic bacterial DNA, but one process supports, while the other restricts, bacterial growth. Autophagy is also initiated by recognition of peptidoglycan by the intracellular surveillance proteins Nod1 and Nod2. These sensors subsequently recruit ATG16L to bacterial entry sites to allow nucleation during autophagy. Recognition of peptidoglycan also mediates autophagy initiation during L. monocytogenes infection of Drosophila, but through the pattern recognition receptor protein PGRP-LE rather than Nod [75]. One group recently found that an amino acid stress response was activated in response to membrane damage during infection by either the cytosolic bacterium Shigella or SCV-bound Salmonella [76]. In the case of Salmonella, membrane damage was SPI-1-dependent and the bacterium later relieved autophagy activation through increased amino acid uptake. For Shigella, membrane damage was a result of vacuolar escape, with the bacterial effector IcsB shielding it from degradation [77]. It is clear that autophagy is an important mechanism of bacterial clearance for the host. Because it is also a consequence of cytoplasmic access, bacteria that trigger autophagy must also have a strategy to combat or subvert it to effect successful infection. These strategies are numerous and varied and will be exciting new areas of research in pathogenesis. Non-canonical inflammasome activation Caspase-11 is an understudied, non-canonical inflammasome with an important role in limiting bacterial infection [78]. Salmonella can induce cell death via caspase-11 [79] and cytosolic sifA Salmonella mutants cause increased pyroptosis independent of the inflammasome adaptors Nlrc4 and ASC, but dependent on caspase-11 [80]. It was recently shown that Gram-negative lipid A activates caspase-11, but the compartment in which this activation occurs remains unclear, although it has been suggested that it is in the cytosol [81]. In support of this, caspase-11 / mice are sensitive to the cytosolic pathogen Burkholderia 8


[80]. In addition, caspase-11 limits growth during Legionella infection in a flagellin/Dot/Icm-dependent manner [82]. These authors reported a very different mechanism of action, suggesting that caspase-11 promotes dephosphorylation of cofilin, which leads to actin depolymerization around the LCV and fusion of the LCV with lysosomes. Concluding remarks Through the use of new approaches, it has become apparent that there are no longer two distinct groups of bacteria: those that access the cytoplasm and those that do not. Emerging evidence indicates that a much larger group of pathogens escapes from the vacuole. One consequence of this localization change is that it can resolve previously unexplained data about the induction of immune responses, for example via major histocompatibility complex class I (MHC-I). It also leads to many new questions, including the exact escape mechanisms of the newly identified pathogens and whether rupture itself, bacterial contact with the cytosol, or both is the true cause of observed host responses. Note added in proof While this review was in proof stage, an article by Knodler et al. quantifying and further characterizing hyper-replication of Salmonella was published that may be of interest to readers [98]. Acknowledgements We apologize for not being able to cite all recent work in this fast moving field because of space limitations. We would like to thank the members of J.E.’s team for helpful discussion and Beátrice de Cougny for the design of Figure 2. This work was funded by a grant from the European Research Council to J.E. (ERC StG No. 261166) and by a Pasteur Foundation fellowship to J.F.

References 1 Ray, K. et al. (2009) Life on the inside: the intracellular lifestyle of cytosolic bacteria. Nat. Rev. Microbiol. 7, 333–340 2 Hale, T.L. and Bonventre, P.F. (1979) Shigella infection of Henle intestinal epithelial cells: role of the bacterium. Infect. Immun. 24, 879–886 3 Sansonetti, P.J. et al. (1986) Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infect. Immun. 51, 461–469 4 Bobard, A. et al. (2011) Spotting the right location – imaging approaches to resolve the intracellular localization of invasive pathogens. Biochim. Biophys. Acta 1810, 297–307 5 Blocker, A. et al. (1999) The tripartite type III secretion of Shigella flexneri inserts IpaB and IpaC into host membranes. J. Cell Biol. 147, 683–693 6 Senerovic, L. et al. (2012) Spontaneous formation of IpaB ion channels in host cell membranes reveals how Shigella induces pyroptosis in macrophages. Cell Death Dis. 3, e384 7 Dickenson, N.E. et al. (2013) Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon. Protein Sci. 22, 614–627 8 Fernandez-Prada, C.M. et al. (2000) Shigella flexneri IpaH7.8 facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages. Infect. Immun. 68, 3608–3619 9 Paetzold, S. et al. (2007) Shigella flexneri phagosomal escape is independent of invasion. Infect. Immun. 75, 4826–4830 10 Dupont, N. et al. (2009) Shigella phagocytic vacuolar membrane remnants participate in the cellular response to pathogen invasion and are regulated by autophagy. Cell Host Microbe 6, 137–149 11 Ehsani, S. et al. (2012) Hierarchies of host factor dynamics at the entry site of Shigella flexneri during host cell invasion. Infect. Immun. 80, 2548–2557


Review 12 Pizarro-Cerda, J. and Cossart, P. (2009) Listeria monocytogenes membrane trafficking and lifestyle: the exception or the rule? Annu. Rev. Cell Dev. Biol. 25, 649–670 13 Shaughnessy, L.M. et al. (2006) Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles. Cell. Microbiol. 8, 781–792 14 Smith, G.A. et al. (1995) The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread. Infect. Immun. 63, 4231–4237 15 Singh, R. et al. (2008) GILT is a critical host factor for Listeria monocytogenes infection. Nature 455, 1244–1247 16 Radtke, A.L. et al. (2011) Listeria monocytogenes exploits cystic fibrosis transmembrane conductance regulator (CFTR) to escape the phagosome. Proc. Natl. Acad. Sci. U.S.A. 108, 1633–1638 17 Davis, M.J. et al. (2012) Inducible renitence limits Listeria monocytogenes escape from vacuoles in macrophages. J. Immunol. 189, 4488–4495 18 Myers, J.T. et al. (2003) Localized reactive oxygen and nitrogen intermediates inhibit escape of Listeria monocytogenes from vacuoles in activated macrophages. J. Immunol. 171, 5447–5453 19 Renvoise, A. et al. (2011) Intracellular Rickettsiales: insights into manipulators of eukaryotic cells. Trends Mol. Med. 17, 573–583 20 Renesto, P. et al. (2003) Identification and characterization of a phospholipase D-superfamily gene in Rickettsiae. J. Infect. Dis. 188, 1276–1283 21 Whitworth, T. et al. (2005) Expression of the Rickettsia prowazekii pld or tlyC gene in Salmonella enterica serovar Typhimurium mediates phagosomal escape. Infect. Immun. 73, 6668–6673 22 Rahman, M.S. et al. (2013) Rickettsia typhi possesses phospholipase A2 enzymes that are involved in infection of host cells. PLoS Pathog. 9, e1003399 23 Clemens, D.L. et al. (2009) Francisella tularensis phagosomal escape does not require acidification of the phagosome. Infect. Immun. 77, 1757–1773 24 Chong, A. et al. (2008) The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression. Infect. Immun. 76, 5488–5499 25 Barker, J.R. et al. (2009) The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence. Mol. Microbiol. 74, 1459–1470 26 Akimana, C. et al. (2010) Host factors required for modulation of phagosome biogenesis and proliferation of Francisella tularensis within the cytosol. PLoS ONE 5, e11025 27 Beuzoń, C.R. et al. (2000) Salmonella maintains the integrity of its intracellular vacuole through the action of SifA. EMBO J. 19, 3235– 3249 28 Boucrot, E. et al. (2005) The intracellular fate of Salmonella depends on the recruitment of kinesin. Science 308, 1174–1178 29 Ohlson, M.B. et al. (2008) Structure and function of Salmonella SifA indicate that its interactions with SKIP, SseJ, and RhoA family GTPases induce endosomal tubulation. Cell Host Microbe 4, 434–446 30 Birmingham, C.L. and Brumell, J.H. (2006) Autophagy recognizes intracellular Salmonella enterica serovar Typhimurium in damaged vacuoles. Autophagy 2, 156–158 31 Thurston, T.L.M. et al. (2012) Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion. Nature 482, 414– 418 32 Knodler, L.A. et al. (2010) Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. Proc. Natl. Acad. Sci. U.S.A. 107, 17733–17738 33 Beuzon, C.R. et al. (2000) Salmonella maintains the integrity of its intracellular vacuole through the action of SifA. EMBO J. 19, 3235– 3249 34 Malik-Kale, P. et al. (2012) The bimodal lifestyle of intracellular Salmonella in epithelial cells: replication in the cytosol obscures defects in vacuolar replication. PLoS ONE 7, e38732 35 Abdallah, A.M. et al. (2007) Type VII secretion – mycobacteria show the way. Nat. Rev. Microbiol. 5, 883–891 36 Philips, J.A. (2008) Mycobacterial manipulation of vacuolar sorting. Cell. Microbiol. 10, 2408–2415 37 Stamm, L.M. et al. (2003) Mycobacterium marinum escapes from phagosomes and is propelled by actin-based motility. J. Exp. Med. 198, 1361–1368


38 van der Wel, N. et al. (2007) M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 129, 1287– 1298 39 Myrvik, Q.N. et al. (1984) Disruption of phagosomal membranes of normal alveolar macrophages by the H37Rv strain of Mycobacterium tuberculosis. A correlate of virulence. Am. Rev. Respir. Dis. 129, 322– 328 40 McDonough, K.A. et al. (1993) Pathogenesis of tuberculosis: interaction of Mycobacterium tuberculosis with macrophages. Infect. Immun. 61, 2763–2773 41 Simeone, R. et al. (2012) Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death. PLoS Pathog. 8, e1002507 42 Smith, J. et al. (2008) Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole. Infect. Immun. 76, 5478–5487 43 Wong, K-W. and Jacobs, W.R., Jr (2011) Critical role for NLRP3 in necrotic death triggered by Mycobacterium tuberculosis. Cell. Microbiol. 13, 1371–1384 44 Ge, J. and Shao, F. (2011) Manipulation of host vesicular trafficking and innate immune defence by Legionella Dot/Icm effectors. Cell. Microbiol. 13, 1870–1880 45 Molmeret, M. et al. (2007) Rapid escape of the dot/icm mutants of Legionella pneumophila into the cytosol of mammalian and protozoan cells. Infect. Immun. 75, 3290–3304 46 Molmeret, M. et al. (2010) Temporal and spatial trigger of postexponential virulence-associated regulatory cascades by Legionella pneumophila after bacterial escape into the host cell cytosol. Environ. Microbiol. 12, 704–715 47 Creasey, E.A. and Isberg, R.R. (2012) The protein SdhA maintains the integrity of the Legionella-containing vacuole. Proc. Natl. Acad. Sci. U.S.A. 109, 3481–3486 48 Gao, L.Y. and Kwaik, Y.A. (2000) The mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila. Environ. Microbiol. 2, 79–90 49 Kirby, J.E. et al. (1998) Evidence for pore-forming ability by Legionella pneumophila. Mol. Microbiol. 27, 323–336 50 Bitar, D.M. et al. (2005) Structure–function analysis of the C-terminus of IcmT of Legionella pneumophila in pore formation-mediated egress from macrophages. FEMS Microbiol. Lett. 242, 177–184 51 Alli, O.A. et al. (2000) Temporal pore formation-mediated egress from macrophages and alveolar epithelial cells by Legionella pneumophila. Infect. Immun. 68, 6431–6440 52 Kroemer, G. et al. (2009) Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ. 16, 3–11 53 Carneiro, L.A. et al. (2009) Shigella induces mitochondrial dysfunction and cell death in nonmyleoid cells. Cell Host Microbe 5, 123–136 54 Abdallah, A.M. et al. (2011) Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation. J. Immunol. 187, 4744–4753 55 O’Sullivan, M.P. et al. (2007) A caspase-independent pathway mediates macrophage cell death in response to Mycobacterium tuberculosis infection. Infect. Immun. 75, 1984–1993 56 Lee, J. et al. (2006) Macrophage apoptosis in response to high intracellular burden of Mycobacterium tuberculosis is mediated by a novel caspase-independent pathway. J. Immunol. 176, 4267– 4274 57 Koo, I.C. et al. (2008) ESX-1-dependent cytolysis in lysosome secretion and inflammasome activation during mycobacterial infection. Cell. Microbiol. 10, 1866–1878 58 Molofsky, A.B. et al. (2006) Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection. J. Exp. Med. 203, 1093–1104 59 Case, C.L. et al. (2009) Asc and Ipaf Inflammasomes direct distinct pathways for caspase-1 activation in response to Legionella pneumophila. Infect. Immun. 77, 1981–1991 60 Lippmann, J. et al. (2011) Dissection of a type I interferon pathway in controlling bacterial intracellular infection in mice. Cell. Microbiol. 13, 1668–1682 61 Ge, J. et al. (2012) Preventing bacterial DNA release and absent in melanoma 2 inflammasome activation by a Legionella effector 9


Review

62 63 64 65

66

67

68

69 70

71

72

73 74

75

76

77 78 79

80

10

functioning in membrane trafficking. Proc. Natl. Acad. Sci. U.S.A. 109, 6193–6198 Boya, P. et al. (2013) Emerging regulation and functions of autophagy. Nat. Cell Biol. 15, 713–720 Mostowy, S. et al. (2010) Entrapment of intracytosolic bacteria by septin cage-like structures. Cell Host Microbe 8, 433–444 Nakagawa, I. et al. (2004) Autophagy defends cells against invading group A Streptococcus. Science 306, 1037–1040 Checroun, C. et al. (2006) Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication. Proc. Natl. Acad. Sci. U.S.A. 103, 14578–14583 Case, E.D. et al. (2013) The Francisella O-antigen mediates survival in the macrophage cytosol via autophagy avoidance. Cell. Microbiol. http://dx.doi.org/10.1111/cmi.12246 Perrin, A.J. et al. (2004) Recognition of bacteria in the cytosol of mammalian cells by the ubiquitin system. Curr. Biol. 14, 806–811 van Wijk, S.J.L. et al. (2012) Fluorescence-based sensors to monitor localization and functions of linear and K63-linked ubiquitin chains in cells. Mol. Cell 47, 797–809 Wild, P. et al. (2011) Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science 333, 228–233 von Muhlinen, N. et al. (2012) LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy. Mol. Cell 48, 329–342 Mesquita, F.S. et al. (2012) The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. PLoS Pathog. 8, e1002743 Watson, R.O. et al. (2012) Extracellular M. tuberculosis DNA targets bacteria for autophagy by activating the host DNA-sensing pathway. Cell 150, 803–815 Manzanillo, P.S. et al. (2013) The ubiquitin ligase parkin mediates resistance to intracellular pathogens. Nature 501, 512–516 Manzanillo, P.S. et al. (2012) Mycobacterium tuberculosis activates the DNA-dependent cytosolic surveillance pathway within macrophages. Cell Host Microbe 11, 469–480 Yano, T. et al. (2008) Autophagic control of Listeria through intracellular innate immune recognition in Drosophila. Nat. Immunol. 9, 908–916 Tattoli, I. et al. (2012) Amino acid starvation induced by invasive bacterial pathogens triggers an innate host defense program. Cell Host Microbe 11, 563–575 Ogawa, M. et al. (2005) Escape of intracellular Shigella from autophagy. Science 307, 727–731 Kayagaki, N. et al. (2011) Non-canonical inflammasome activation targets caspase-11. Nature 479, 117–121 Broz, P. and Monack, D.M. (2013) Noncanonical inflammasomes: caspase-11 activation and effector mechanisms. PLoS Pathog. 9, e1003144 Aachoui, Y. et al. (2013) Caspase-11 protects against bacteria that escape the vacuole. Science 339, 975–978


81 Kayagaki, N. et al. (2013) Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341, 1246–1249 82 Akhter, A. et al. (2012) Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization. Immunity 37, 35–47 83 Paz, I. et al. (2010) Galectin-3, a marker for vacuole lysis by invasive pathogens. Cell. Microbiol. 12, 530–544 84 Ray, K. et al. (2010) Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuole rupture in real time. Cell. Microbiol. 12, 545–556 85 Figueira, R. and Holden, D.W. (2012) Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors. Microbiology 158, 1147–1161 86 Leone, P. and Me´resse, S. (2011) Kinesin regulation by Salmonella. Virulence 2, 63–66 87 Mota, L.J. et al. (2009) SCAMP3 is a component of the Salmonellainduced tubular network and reveals an interaction between bacterial effectors and post-Golgi trafficking. Cell. Microbiol. 11, 1236–1253 88 Burrack, L.S. et al. (2009) Perturbation of vacuolar maturation promotes listeriolysin O-independent vacuolar escape during Listeria monocytogenes infection of human cells. Cell. Microbiol. 11, 1382–1398 89 Mounier, J. et al. (2012) Shigella effector IpaB-induced cholesterol relocation disrupts the Golgi complex and recycling network to inhibit host cell secretion. Cell Host Microbe 12, 381–389 90 Bakowski, M.A. et al. (2008) Salmonella-containing vacuoles: directing traffic and nesting to grow. Traffic 9, 2022–2031 91 McGourty, K. et al. (2012) Salmonella inhibits retrograde trafficking of mannose-6-phosphate receptors and lysosome function. Science 338, 963–967 92 Joshi, A.D. and Swanson, M.S. (2011) Secrets of a successful pathogen: Legionella resistance to progression along the autophagic pathway. Front. Microbiol. 2, 1–9 93 Al-Khodor, S. et al. (2008) A Dot/Icm-translocated ankyrin protein of Legionella pneumophila is required for intracellular proliferation within human macrophages and protozoa. Mol. Microbiol. 70, 908–923 94 Kubori, T. et al. (2010) Legionella metaeffector exploits host proteasome to temporally regulate cognate effector. PLoS Pathog. 6, e1001216 95 Otto, G.P. et al. (2004) Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum. Mol. Microbiol. 51, 63–72 96 Choy, A. et al. (2012) The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation. Science 338, 1072–1076 97 Khweek, A.A. et al. (2013) A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy. Eur. J. Immunol. 43, 1333–1344 98 Knodler, L.A. et al. (2014) Quantitative assessment of cytosolic salmonella in epithelial cells. PLoS ONE 9, e84681 http://dx.doi.org/ 10.1371/journal.pone.0084681

Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm.

Modulation of Host miRNAs by Intracellular Bacterial Pathogens.

Trogocytosis-associated cell to cell spread of intracellular bacterial pathogens.

Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

Hijacking Host Cell Highways: Manipulation of the Host Actin Cytoskeleton by Obligate Intracellular Bacterial Pathogens.

Small Non-Coding RNAs: New Insights in Modulation of Host Immune Response by Intracellular Bacterial Pathogens.

Maintenance of vacuole integrity by bacterial pathogens.

The type III secretion system apparatus determines the intracellular niche of bacterial pathogens.

Host-directed antimicrobial drugs with broad-spectrum efficacy against intracellular bacterial pathogens.

Coinfection of tick cell lines has variable effects on replication of intracellular bacterial and viral pathogens.

How old are bacterial pathogens?

Heme uptake in bacterial pathogens.

Subverting Toll-Like Receptor Signaling by Bacterial Pathogens.

Bacterial pathogens of Culicidae (mosquitos).

Host specificity of bacterial pathogens.

Nutrition-based evolution of intracellular pathogens.

Immune Response: Intracellular pathogens under attack.

Pyroptotic cell death defends against intracellular pathogens.

Intracellular delivery of biologic therapeutics by bacterial secretion systems.

Chronic Bacterial Pathogens: Mechanisms of Persistence.

Examination of faeces for bacterial pathogens.

Bacterial pathogens associated with hidradenitis suppurativa, France.

Looking in ticks for human bacterial pathogens.

Plant innate immunity against human bacterial pathogens.