Bacterial Pathogens Commandeer Rab GTPases to Establish Intracellular Niches

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© 2012 John Wiley & Sons A/S doi:10.1111/tra.12000

Review

Bacterial Pathogens Commandeer Rab GTPases to Establish Intracellular Niches 2 ¨ Mary-Pat Stein1,∗ , Matthias P. Muller and Angela Wandinger-Ness3 1 Department

of Biology, California State University, Northridge, Northridge, CA, USA 2 Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany 3 Department of Pathology, University of New Mexico HSC, Albuquerque, NM, 87131, USA *Corresponding author: Mary-Pat Stein, [email protected] Intracellular bacterial pathogens deploy virulence factors termed effectors to inhibit degradation by host cells and to establish intracellular niches where growth and differentiation take place. Here, we describe mechanisms by which human bacterial pathogens (including Chlamydiae; Coxiella burnetii ; Helicobacter pylori ; Legionella pneumophila; Listeria monocytogenes ; Mycobacteria; Pseudomonas aeruginosa, Salmonella enterica) modulate endocytic and exocytic Rab GTPases in order to thrive in host cells. Host cell Rab GTPases are critical for intracellular transport following pathogen phagocytosis or endocytosis. At the molecular level bacterial effectors hijack Rab protein function to: evade degradation, direct transport to particular intracellular locations and monopolize host vesicles carrying molecules that are needed for a stable niche and/or bacterial growth and differentiation. Bacterial effectors may serve as specific receptors for Rab GTPases or as enzymes that post-translationally modify Rab proteins or endosomal membrane lipids required for Rab function. Emerging data indicate that bacterial effector expression is temporally and spatially regulated and multiple virulence factors may act concertedly to usurp Rab GTPase function, alter signaling and ensure niche establishment and intracellular bacterial growth, making this field an exciting area for further study. Key words: bacterial secretion, cytoskeletal motors, membrane trafficking, pathogen containing vacuole or inclusion, phagosome, post-translational modification, regulation, replication Received 21 March 2012, revised and accepted for publication 13 August 2012, uncorrected manuscript published online 17 August 2012, published online 13 September 2012

Rab-GTPase-Regulated Trafficking to Lysosomes is a Normal Host Defense Mechanism Rab GTPases are central to the organization, maintenance and dynamics of the cellular endomembrane system

through their functions in regulating specific membrane transport pathways (Figure 1, Table 1) (1,2). In bacterial infection, Rab proteins play a pivotal role in host immunity, internalization by endocytosis or phagocytosis and directing the transport of phagocytosed pathogens to lysosomes for degradation (Table 1). The normal transport pathway to lysosomes utilizes numerous Rab proteins to efficiently deliver pathogen-containing vacuoles (PCV) from an early phagocytic compartment to a Rab5-positive early-endosomal compartment. Pathogens destined for degradation are then shuttled through a Rab7-positive lateendosome prior to reaching their final destination, the lysosomal compartment. Phagosomal maturation along this pathway has been analyzed by examination of the temporal recruitment of protein and lipid markers to phagosomes containing heat-killed pathogens, latex beads or pathogens that do not block transport to the lysosome. Targeted manipulation of Rab GTPase function through mutant protein overexpression or siRNA depletion performed in parallel has defined a large number of participating GTPases and some of their functions in phagosome maturation. Latex bead-containing phagosomes have been extensively studied over the last decade and a half to identify proteins and lipids recruited to model phagosomes due to the high degree of purity with which they can be purified from higher density cellular membranes (47,48). Proteomic studies on purified latex bead-containing phagosomes have documented the recruitment of over 40 Rab GTPases in mouse macrophages following variable uptake times (10–120 min) and monitoring kinetics of maturation after internalization for 10–180 min (49–52) (Figure 1). Some of the key principles that have emerged are that: (i) Rab GTPases associate with maturing phagosomes in a dynamic manner and change over time; (ii) heterogeneity among phagosomes makes it difficult to discern the molecular sequence of events with absolute precision; (iii) all-or-no changes in Rab compositions are rare suggesting subtle changes in concentration are biologically significant; (iv) post-translational modifications, including phosphorylation can impact Rab association with phagosomes and (v) Rab GTPases associated with maturing phagosomes derive to varying degrees from nearly all endomembranes in the cell (endosomes/lysosomes > plasma membrane > endoplasmic reticulum (ER) > Golgi > mitochondria) (52–54). Studies on the maturation of phagosomes containing heat-inactivated, mutant or non-pathogenic bacteria using immunofluorescence and western blot analyses show www.traffic.dk 1565

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Cilium Cytokinesis formation Tight junction

Rab11a (apical) Rab15

(epithelia)

23 35

Time, min

8 10 11

1

4 14

Rab11b (basolateral)

22a

35 Rab22a

23

Rab5 Rab21

Early phagosome

21 22b 39 Early 14

22 8

23

endosome

Rab7

11 35

5 14

Rab22b

3

37

7

Rab7 Rab34

9

SG 14

vATPase cathepsinD

Exocytosis

10 8

36

38

27 Rab17

Melanosome

Rab9

Rab38

7 27 32

39

14

8 6 11

22b

Lysosome

39

30-45

7 20

Intermediate

Phagolysosome

39

Rab7 Rab36

32

13

7 20 9 phagosome 22b 39 32 23 60-90 34 11 8

Late endosome MVB

SV

10-20

13

22a

5

11

Recycling endosome

23

5

Rab4

17 25

15 11

13

Phagocytosis

Endocytosis

9

23 38

120-180

34 43 37

Rab7

34

Golgi

27b 43

Mitochondrion 20 32 Rab32

Rab2

Rab1

MAM

Autophagosome

Lipid droplet 18

1

Rab24 Rab33

2

ER

Nucleus

Figure 1: Rab GTPase regulated pathways. Over 60 Rab GTPase family members regulate membrane transport on the exocytic, endocytic, phagocytic and recycling pathways. Shown are the normal functions (arrows) and localizations (black circles) of Rab GTPases that are targeted by bacterial pathogens as detailed in the text and Table 3. Phagosome and autophagosome maturation depend on the sequential fusion with early endosomes, late endosomes and lysosomes. Additional components needed for phagosome maturation are likely recruited from interactions with the secretory pathway based on the involvement of Rab GTPases with primary functions in exocytosis, organelle biogenesis, ER and Golgi dynamics, and mitochondrial function (Rab20, Rab32, Rab38, Rab43). Over 40 Rab GTPases have been identified on phagosomes at various stages of maturation; depicted are 24 Rab GTPases whose kinetic acquisition and functions have been characterized by analyses of latex bead and non-pathogenic bacterial phagosomes (see text for detail). Some Rab GTPases may be acquired in a biphasic manner (51) and others transit gradually with small changes in concentration and phosphorylation state triggering changes in activity (12,53,55). MAM, mitochondria associated membrane (thought to be of ER origin); SV, synaptic vesicle; SG, secretory granule.

significant agreement with studies on latex bead phagosomes (51,53,55). However, molecular events at early time points after internalization, as well as the functions of many of the Rab GTPases on phagosomes have remained elusive. Recently, detailed proteomic analyses of phagosomal compartments transporting live Staphylococcus aureus to lysosomes firmly established the kinetics of Rab protein recruitment to a non-pathogenic phagosome and the requirements for Rab proteins in acidification and degradative enzyme recruitment (Figure 1) (12). All 1566

Rab proteins identified on S. aureus phagosomes, with the exceptions of Rab8, Rab11 and Rab27, were also found on latex bead phagosomes, indicating that nonpathogenic bacteria are suitable models for phagosomal dynamics (52). Rab5 and Rab22 localized to S. aureuscontaining phagosomes as early as 10 min after infection with a transient recruitment of various other Rab proteins observed for up to 1 h following infection (Rab8, Rab8b, Rab11, Rab11b, Rab13, Rab14, Rab20, Rab22a, Rab32, Rab38 and Rab43) (12). Recruitment and accumulation

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Rab2

Rab26, Rab27, Rab37

Rab5a, Rab11a, Rab14

Rab4a, Rab11a, Rab15, Rab21

Rab11

Rab5a, Rab9a, Rab34, Arf6, Rac1; Rab27a, Rab33

Rab10, Rab11a

Rab3a, Rab3b, Rab3c, Rab3d

Rab4a

Rab5a,b,c

Rab6

Rab7a

Rab8a, Rab8b

GTPase partners

Rab GTPase reviews Rab1a,b

Rab GTPase

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Late endosomes and lysosomes; stage I and II melanosomes; surfactant endocytosis and signaling

Plasma membrane, clatherin coated vesicles and early endosomes Golgi

Secretory granules, synaptic vesicles Early endosomes and recycling endosomes

ER

Localization

Cell polarization

Autophagy and degradation; lysosome and lysosome-related organelle biogenesis; regulated secretion

Exocytosis

Endocytosis and recycling

Endocytosis and recycling

Exocytosis

Exocytosis

Major trafficking route

Table 1: Rab GTPases: normal functions and as targets of bacterial pathogens

Transport from early to late endosomes and late endosome to lysosome fusion; bidirectional transport of signaling endosomes, autophagosomes, multivesicular bodies, and melanosomes on microtubules in association with dynein and kinesin motor proteins; axon viability; phosphoinositide homeostasis; lipid transport; activation of mTOR signaling; lung innate and adaptive immunity Polarized transport from Golgi to basolateral plasma membrane and cilia in epithelia and photoreceptors, polarized neurite outgrowth and post-synaptic recycling

Golgi transport

Regulates sorting and endocytic recycling to the plasma membrane; trafficking of human P-glycoprotein responsible for multidrug resistance of tumors; functions with Rab14 through shared effector RUFY1/Rabip4 Endocytosis, early endosome fusion, nuclear signaling through APPL

Regulated secretion

ER to Golgi transport

Normal function

(12–18)

Early phagocytosis; excluded from L. monocytogenes phagosomes and chlamydial inclusions; Recruited to S. enterica, M. tuberculosis and C. burnetii PCV

Transient recruitment to intermediate S. aureus and M. tuberculosis containing phagosomes; recruited by L. pneumophila LidA

(8,12,28,29)

(12,21–27)

(8,19,20)

(10)

In vitro target of P. aeruginosa ExoS

Recruited to chlamydial inclusions, with effector BicD1 regulates chlamydial protein synthesis and nutrient delivery; recruited by L. pneumophila LidA Phagosome acidification and cathepsin D recruitment; phagosome maturation and fusion with lysosomal system; dissociated from M. tuberculosis phagosomes; RILP effector interaction blocked by S. enterica; facilitates H. pylori and C. burnetii niche formation

(10,11)

(3–9)

(1,2)

References review

Post-translationally modified by L. pneumophila to establish niche and gain nutrients; recruited to chlamydial inclusions In vitro target of P. aeruginosa ExoS

Role in bacterial pathogenesis

Co-opting Rab Protein Function

1567

1568

Rab20

Phagosomes, mitochondria, ER endosomes

Early endosome, Golgi

Rab4, Rab39

Golgi and recycling endosomes, early endosomes, phagosomes

Rab14

Arf4, Rab6, Rab8a, Rab10, Cdc42

Rab11a, Rab11b (neuron specific)

Base of primary cilia, Golgi

Tight junctions, Golgi, endosomes

Rab8a, Rab11a

Rab10

Late endosomes

Localization

Rab13

Rab7a

GTPase partners

Rab9a; Rab9b

Rab GTPase

Table 1: Continued

Endocytosis and recycling

Endocytosis and recycling

Cell polarization

Endocytosis and recycling; cell polarization and ciliogenesis; immune synapse formation; cytokinesis

Ciliogenesis and ciliary trafficking; immune synapse formation; cytokinesis

Endocytosis and recycling

Major trafficking route

Plasma membrane recycling; functions in concert with Rab8 and Rab11a; insulin-stimulated GLUT4 translocation; phagosome maturation; Weibel-Palade body formation and secretion of von Willebrand factor; regulated surface expression of Toll-like receptor (TLR)4 Trafficking from the trans-Golgi network to apical recycling endosomes and plasma membrane; dopamine transporter and beta2-adrenergic receptor trafficking; polarized trafficking in epithelia; phagocytosis in macrophages; functions in concert with Rab8 and Rab10 primary ciliogenesis; ciliary trafficking Associated with tight junctions and functions on trans-Golgi-endosome circuit in polarized cells Endocytic recycling of transferrin; MHC class I cross-presentation in dendritic cells; TGN to apical trafficking in epithelia; surfactant secretion in alveolar cells; insulin-dependent GLUT4 translocation; functions cooperatively with Rab4 through shared effector RUFY1/Rabip4; regulation of embryonic development through interaction with Kif16B and transport of FGF Vacuolar ATPase trafficking in kidney; target of HIF in hypoxia induced apoptosis; phagosome acidification and maturation; Gap junction biogenesis

Transport from endosome to trans-Golgi network; lipid transport; lysosome and lysosome related organelle biogenesis

Normal function

Phagosome acidification and cathepsin D recruitment through lysosome fusion; excluded from phagosomes by ESAT-6

(12)

(12,26,34,35)

(12)

Transiently on S. aureus phagosomes

Transiently associated with intermediate S. aureus phagosomes; participates in phagosome arrest of M. tuberculosis phagosomes; found on chlamydial inclusions and L. pneumophila PCV; may serve in lipid recruitment

(12,16,20,32,33)

(16,29,31,32)

(12,16,30)

References review

Temporally regulated association with chlamydial inclusions mediated by CPn0585; inhibition of Rab11-dependent recycling by H. pylori CagA inhibition of Rab11-FIP effector interactions; excluded by M. tuberculosis by ESAT-6

Recruitment to intermediate and late S. aureus and M. tuberculosis phagosomes; antagonized by S. enterica SifA and excluded from chlamydial inclusions Early phagosomes; on early M. bovis phagosomes and some chlamydial inclusions via CPn0585

Role in bacterial pathogenesis

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Traffic 2012; 13: 1565–1588 Mitochondria, autophagic vesicles Golgi and endosomes

Rab7a, Rab36

Cdc42

Rab32

Rab34

Rab35

Endosomes and plasma membrane

Golgi and vacuoles, overexpressed upon sucrose induced cell vacuolation

Rab29 (Rab7L1)

Melanosomes, lysosome related organelles

Rab27a,b

Rab7a, Rab17 Rab32, Rab38

Plasma membrane and endosomes

Rab23

Early endosome, plasma membrane

Localization

trans-Golgi

Rab5a, Rab7a

GTPase partners

Rab22b

Rab22a

Rab GTPase

Table 1: Continued

Endocytosis and recycling; ciliogenesis and ciliary Trafficking; Immune synapse formation; cytokinesis

Endocytosis and recycling

Autophagy and degradation

Lysosome and lysosome-related organelle biogenesis; regulated secretion Exocytosis

Ciliogenesis and ciliary Trafficking; immune synapse formation; cytokinesis

Endocytosis and recycling

Endocytosis and recycling

Major trafficking route

Post-Golgi trafficking of melanogenic enzymes; ER stress mediated apoptosis; mitochondrial dynamics Endosomes, macropinosome formation, phagosome maturation, lysosome morphogenesis, functions with Rab36 and Rab7 through shared effector (RILP) Fast endocytic recycling; cytokinesis; immune synapse function; MHC class I and II endocytosis and recycling; T cell receptor recycling; phosphoinositide regulation; neurite outgrowth through interfaces with Cdc42; actin remodeling through fascin effector leading to filopodia formation

Stress regulated expression, bacterial toxin trafficking

Melanosome biogenesis and trafficking; prostate marker secretion

Trafficking of sonic hedgehog signaling components; central nervous system development and ciliary trafficking

Transport of transferrin from sorting endosomes to recycling endosomes; pathogen phagocytosis; enriched in glia; shares effectors and GEFs with Rab5 (Rabex-5, EEA1) Golgi-plasma membrane recycling

Normal function

Phosphocholinated by L. pneumophila AnkX and reversed by Lem3/lpg0696

Cathepsin D recruitment to phagosomes

Export of typhoid toxin in cells infected with S. enterica serovar Typhi; cleaved by GtgE a type III secretion effector expressed in broad-host S. enterica, but not S. typhi Cathepsin D recruitment to phagosomes

Early phagocytosis; Cathepsin D recruitment to phagosomes; transiently associated with early M. tuberculosis phagosome Early phagocytosis; associated with S. aureus phagosomes throughout transit to lysosomes for degradation; transiently associated with early M. tuberculosis phagosomes S. aureus late endocytic trafficking and phagosome maturation; excluded from Mycobacteria phagosomes by ESAT-6

Transiently associated with intermediate S. aureus phagosomes; accumulates on M. tuberculosis phagosomes and participates in phagosome arrest

Role in bacterial pathogenesis

(4,42,146)

(12)

(12,41)

(39,40)

(12)

(12,29)

(12,28)

(12,36–38)

References review

Co-opting Rab Protein Function

1569

1570

Rab43

Rab14

Rab39

Golgi and early endosomes, AP1 membrane domains; lysosomes Endosomes, Golgi, phagosomes

Tyrosinase positive melanosomes; surfactant containing vesicles

Rab7a, Rab27a Rab32

Rab38

Localization Secretory granules (insulin, mast cells, macrophages)

GTPase partners

Rab37

Rab GTPase

Table 1: Continued

Endocytosis and Golgi recycling, autophagy and degradation

Lysosome and lysosome-related organelle biogenesis; regulated secretion Endocytosis and recycling

Regulated secretion

Major trafficking route

ER-Golgi transport; retrograde transport on the exocytic pathway; associates with dynein/dynactin; cathepsin D transport to phagosomes

Caspase-dependent-IL-1beta secretion; homology to Rab14; phagosomal acidification

Degranulation; regulation of wnt signaling and angiogenesis, inactivated by methionine aminopeptidase-2 (MetAP-2), TNFalpha secretion Trans-Golgi to melanosome transport; lung surfactant secretion; functions with Rab32 and Rab7 in melanosome biogenesis

Normal function

Cathepsin D recruitment to phagosomes; transiently associated with intermediate M. tuberculosis phagosomes

Phagosome acidification

(12,45,46)

(12)

(12,41)

(12,43,44)

S. aureus late endocytic trafficking; late phagosome maturation; increased expression induced by H. pylori infection Cathepsin D recruitment to phagosomes

References review

Role in bacterial pathogenesis

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Co-opting Rab Protein Function

of Rab7, Rab9, Rab34 and Rab39 after 30 min was subsequently observed and Rab27 and Rab37 were only observed on S. aureus phagosomes 1 h post-infection. Rab23 localized to S. aureus-containing phagosomes at all time points analyzed (12). Rab39 was found important for phagosome acidification while Rab22b, Rab32, Rab34 and Rab38 were crucial for Cathepsin D recruitment. Rab7 and Rab20 were central to phagosomal maturation and lysosomal fusion. These data demonstrate that the concerted actions of multiple Rab GTPases results in the acquisition of acidic pH and degradative enzymes and fusion of S. aureus-containing phagosomes with lysosomes for pathogen clearance. Furthermore, the involvement of Rab GTPases with primary functions in exocytosis, organelle biogenesis, ER and Golgi dynamics, and mitochondrial function (Rab20, Rab32, Rab38, Rab43) suggests that some of the components needed for phagosome maturation are recruited from interactions with the secretory pathway (51,53–55). One interesting player in this respect is Rab32, which modulates ER calcium handling and cargo shuttling between mitochondria associated membranes and the peripheral ER, providing a source for newly synthesized lipids and calcium (an important cofactor in regulated fusion) (56). Together, the kinetic data for Rab recruitment provides benchmarks one can use to classify when, where and how specific intracellular pathogens arrest phagosomal maturation and modulate their niche for intracellular survival.

Intracellular Pathogens and the Cell Types they Invade Intracellular pathogens often utilize phagocytic cells such as macrophages as hosts to gain intracellular access. Pathogens such as Legionella pneumophila and Mycobacterium tuberculosis are internalized by alveolar macrophage leading to establishment of an intracellular niche (Figure 2A). However, other pathogens utilize non-phagocytic cells or more than one cell type as their homes, gaining intracellular access by receptormediated endocytosis, lipid-raft mediated internalization or by manipulating host cell actin dynamics through host GTPases such as Rho, Rac and Cdc42, to achieve internalization (reviewed in 57–59). Salmonella enterica serovar Typhi, for example, may infect and replicate in intestinal epithelia or in some cases traverse the intestinal barrier by transcytosis, whereupon dendritic cells and macrophages can phagocytose bacteria and establish a vacuolar replicative niche (60,61). Systemic dissemination of Salmonella typhi causes human typhoid fever and can result in persistent infection in the bone marrow and gall bladder for life. S. enterica serovar Typhimurium is used extensively as an experimental model as it commonly causes self-resolving gastroenteritis in humans due to infection and replication in epithelia and can be studied in mice (62,63). Pathogens such as Helicobacter pylori specifically invade mucosal epithelia (Figure 2B). No

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matter what cell type serves as the host, for intracellular survival pathogens such as Chlamydiae, H. pylori, L. pneumophila, Mycobacteria, Pseudomonas aeruginosa, and S. enterica modulate the transport of their vacuoles to evade transport to and degradation in lysosomes, and to establish an environment allowing for growth and differentiation (Table 2). Two notable exceptions are Listeria monocytogenes, which escapes from the phagosome/vacuole to the cytoplasm, and Coxiella burnetii , which capitalizes on the acidic environment in lysosomes. This review focuses on mechanisms by which all of these pathogens modulate host Rab GTPase activities to establish an intracellular niche where acquisition of nutrients, lipids and other necessary factors prepare the pathogen for egress from host cells.

Pathogen Requirements for Intracellular Survival and Growth One mechanism that pathogens use to avoid destruction and promote growth and multiplication is to prohibit transport of the PCV down the endocytic pathway to lysosomes. The selective recruitment of Rab or Rab effector proteins to the PCV and the direct modulation of Rab protein activity are mechanisms utilized by pathogen virulence factors to regulate the transport of the PCV through the host cell (Table 3). In addition to evading degradation, pathogens actively direct their transport to intracellular sites where assembly of the appropriate environment for bacterial differentiation and growth may occur. Pathogens direct trafficking of the PCV to specific intracellular locales utilizing Rab-regulated host cytoskeletal motor proteins. Pathogens also modulate signaling and direct the recruitment of host vesicles laden with proteins and lipids to modify the PCV and to provide nutrients for bacterial growth. Thus, the establishment of an appropriate intracellular niche involves multiple steps where host-bacterial protein interactions modulate Rab activities. Intracellular bacterial pathogens employ complex secretion systems (summarized in Table 2) for conveying virulence factors into the host cell cytoplasm where they contact Rab proteins and modulate Rab GTPase functions. Some of the intracellular pathogens discussed in this review include Gram-negative bacteria (Chlamydiae, P. aeruginosa and S. enterica), which rely on type 3 (III) secretion systems (T3SS) comprised of flagellalike machines for protein injection (65,66). Salmonella virulence depends on two interdependent T3SS systems, T3SS1 and T3SS2 effectors that are involved in pathogen vacuole biogenesis (62,78). Other intracellular Gram-negative bacteria utilize a type 4 (IV) secretion machinery (T4SS), which resembles bacterial conjugation pili (H. pylori ) or in the case of C. burnetii and L. pneumophila use a specialized type 4B (T4BSS) assembly of Dot and Icm proteins (68,70). Mycobacteria and L. monocytogenes are Gram-positive and utilize general secretory (Sec) and twin-arginine translocation (Tat) 1571

Stein et al. A

Macrophage Host

B

Epithelial Host

Pseudomonas Chlamydia aeruginosa trachomatis, pneumoniae

Helicobacter pylori Salmonella enterica

Legionella pneumophila

Rab5

Rab7/ RILP Rab37

PI3K

e bul

le ubu

LE L

Chlamydial inclusion

Rab5 Rab7

AP

L

Golgi

Rab24

Nucleus

Rab3 Rab4 Rab5

LE

vacuole

Golgi

ER

Ct-Rab4 Rab6 Cp-Rab1 Rab11 Rab10 Rab14 Rab11

EE

Rab11 Rab13 Rab14 Rab20 Rab22 Rab27

PM blebs

EE

MTOC

rotu

Rab7/ RILP/ dynein Rab9

ZO-1, Jam-A

Mic

rot M ic Rab1 Rab6 Rab8 Rab35

Rab7/ SKIP kinesin/ Arl8b

Listeria monocytogenes TJ Mycobacterium tuberculosis AJ Coxiella burnetii Rab5

ER

Nucleus

Figure 2: Intracellular bacterial pathogens create specialized niches in macrophage and epithelial hosts by modulating Rab GTPases. Pathogens alter Rab GTPase functions to escape degradation and obtain essential nutrients for growth and survival. A) Macrophage host. Legionella pneumophila enters alveolar macrophages by coiling phagocytosis and creates a replicative niche in close apposition to the endoplasmic reticulum (ER) by modulating the activity of Rab1 and Rab35. Legionella evades fusion with lysosomes (L), although some exchange with endosomes may take place and pH is mildly acidic. S. enterica can infect enterocytes or traverse the intestinal epithelial barrier by transcytosis, and in the subluminal Peyer’s patches be phagocytosed by macrophages and dendritic cells that can promote systemic dissemination and infection. S. enterica coopts active Rab7-regulated, microtubule transport to establish a replicative niche in the peri-Golgi region and form tubules called Sifs that promote cell-to-cell spread. M. tuberculosis and C. burnetii both preferentially infect alveolar macrophages, although Mycobacteria allow only early endosome (EE) fusion and induce phagosome arrest by selective Rab GTPase recruitment to avoid fusion with late endosomes (LE) and lysosomes. Coxiella-containing phagosomes on the other hand fuse with late endosomes, lysosomes and autophagosomes (AP), therefore, C. burnetii are adapted to thrive in an acidic niche. B) Epithelial host. L. monocytogenes infects macrophages, intestinal epithelia and hepatocytes; gaining entry by specific binding to and internalization with E-cadherin or Met receptors and evading degradation by blocking Rab5 before release to the cytoplasm. H. pylori infect intestinal epithelia through the apical recruitment of tight junction proteins (ZO-1 and Jam-A) and after internalization establish a replicative niche via the vacuolating toxin VacA and Rab7-mediated fusion with endosomes. Chlamydia trachomatis (Ct ) or pneumonia (Cp) infect epithelia from the apical surface and utilize Rab-regulated, microtubule transport to establish a specialized inclusion in the peri-Golgi region that depends on Rab-regulated fusion with early endosomes, late endosomes and Golgi-derived vesicles. P. aeruginosa recruits the phosphatidylinositol 3-kinase to the apical plasma membrane (PM) where it resides within plasma membrane blebs and blocks endocytosis by ribosylation of Rab5.

pathways for translocation of unfolded and folded proteins into the extracytoplasmic-cell wall space, respectively. Mycobacteria have additional secretory systems (SecA2 and ESX) that serve in the secretion of select proteins lacking N-terminal signal sequences such as ESAT-6. Bacterial virulence factors that are delivered via specialized secretion systems, have several discrete functional domains and act in concert with other virulence factors to modulate host cell functions are collectively termed bacterial effectors, and are distinguished from toxins, which can act extracellularly (78). Here, we provide mechanistic examples of how intracellular pathogens manipulate Rab 1572

proteins through bacterial effector protein interactions to direct their intracellular lifestyle.

Normal Rab GTPase Function Rab GTPases govern vesicular trafficking through a cycle of activation (GTP-binding), inactivation (GTP-hydrolysis) and cytosolic recycling (1). Membrane-dependent activation is controlled by guanine nucleotide exchange factors (GEF), while inactivation is regulated by GTPase activating proteins (GAP) that accelerate the hydrolysis of

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Negative

Negative

Negative

Negative

Positive

C. burnetii

H. pylori

L. pneumophila

L. monocytogenes

Gram stain

Chlamydia (trachomatis and pneumoniae)

Microbe

Traffic 2012; 13: 1565–1588 Dot/Icm T4BSS (IVB) specialized type IV secretion system dependent on Dot and Icm proteins present only in Legionella and Coxiella General secretory (Sec) pathway for translocation from cytosol to extracytoplasmiccell wall space

Dot/Icm T4BSS (IVB), specialized type IV secretion system dependent on Dot and Icm proteins present only in Coxiella and Legionella. T4SS (contact- and pili-dependent)

T3SS

Secretion system

Intestinal, cervical, corneal and lung epithelia; hepatocytes, nervous tissue macrophage

Lung epithelia

Gastric and intestinal epithelia

Macrophage

Cervical and lung epithelia

Target cell type

Table 2: Intracellular bacterial pathogen caused diseases and niche requirements

Early phagosomes and cytosol

ER and Golgi associated vacuole

Late endocytic vacuoles

Parasitophorous vacuole/phagosome, Coxiella replicative vacuole, autophagic pathway

peri-Golgi associated inclusion

Intracellular niche

Accumulation of osmotically active weak bases to form large spacious vacuole; inhibition of lysosome fusion Legionella-containing vacuole with remodeled phosphoinositides and recruitment of biosynthetic vesicles and host trafficking machinery through reversible adenylylation/deadenylylation Inhibition of phagosome maturation; escape to cytosol; PI(3,4,5)P3 requirement for infection

Specialized inclusion; inhibition of lysosomal fusion; host derived lipids (sphingomyelin, cholesterol, glycerophospholipids and neutral lipids) Low pH and oxygen; cholesterol rich membrane; metabolites from autophagy; spacious cavity devoid of lysosomal enzymes; conversion to large cell

Requirements for replication

Food-borne listeriosis; meningoencephalits; fatal in 20–30% of cases

Transmission via inhalation, and water containing infected amoebae; Legionnaires’ disease, pneumonia, GI infections and diarrhea

Gastric ulcers and cancer

Transmission via inhalation; Q-fever including pneumonia, hepatitis, cardiac disease

Sexually transmitted disease; blindness; pneumonia

Human disease/ pathology

(65,73)

(68,71,72)

(69,70)

(15,67,68)

(64–66)

References

Co-opting Rab Protein Function

1573

1574

Acid-fast, Grampositive (lack outer cell membrane)

Negative

Negative

P. aeruginosa

S. enterica

Gram stain

M. tuberculosis

Microbe

Table 2: Continued

T3SS (flagella-like injectisome), Salmonella T3SS effectors are encoded by two pathogenicity islands, T3SS1 and T3SS2 that function coordinately in invasion and intracellular survival

General secretory (Sec) and twin-arginine translocation (Tat) pathways for translocation of unfolded and folded proteins, respectively, from cytosol to extracytoplasmiccell wall space; SecA2 and ESX export systems for secretion of select proteins such as ESAT-6 lacking N-terminal signal sequences T3SS

Secretion system

Intestinal epithelia, macrophages are a reservoir

Lung, skin, urinary tract and corneal epithelia

Macrophage

Target cell type

Late endocytic Salmonella containing vacuole

Plasma membrane blebs

Arrested early phagosomes

Intracellular niche

Apical plasma membrane remodeling into basolateral-like membrane via PI 3-kinase recruitment and ADP-ribosylation to inactivate Rab proteins and prevent internalization Activation of PI(3)P synthesis to recruit host trafficking machinery; manipulation of motor proteins and cytoskeletal trafficking

PI(3)P synthesis inhibited and bacterial phosphatases secreted to prevent phagosomal maturation at early stage; specialized bacterial lipids allow continuous fusion of Mycobacterium containing vacuoles with early endosomes

Requirements for replication

Human food-borne illness (serovar Typhimurium, serovar Enteritidis), human systemic disease and typhoid fever (serovar Typhi)

Opportunistic pulmonary and urinary tract infections in immune compromised patients, may cause sepsis

Tuberculosis, GI tract infections causing diarrhea and malabsorption

Human disease/ pathology

(62,70,77,78)

(66,75,76)

(65,74)

References

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Virulence protein (effector) Activity

Host cell partner

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CT119 (IncA) similar to CPn0186

CT147

CT229

CT813

C. trachomatis

C. trachomatis

C. trachomatis

C. trachomatis

Inclusion membrane protein (Inc); SNARE mimic acts in membrane fusion

Inclusion membrane protein similar to the Rab5 effector EEA1; contains Zn-finger for membrane binding but lacks Rab5 GTPase binding domain Inclusion membrane protein (Inc); Rab4 binding via cytosolic carboxy-terminal domain

Inclusion membrane protein (Inc); SNARE mimic acts in controlling membrane fusion

VAMP7 (Rab GTPases indirectly as part of SNARE complex)

Rab4

unknown

VAMP3, VAMP7, VAMP8 (Rab GTPases indirectly as part of SNARE complex)

Modulation of membrane trafficking through selective Rab recruitment and membrane fusion Brucella abortus RicA Dot/Icm Type IV secreted Rab2 protein binds Rab2 Rab1, Rab10, Rab11 C. pneumoniae CPn0585 Inclusion membrane protein (Inc); Rab GTPase binding via two overlapping cytosolic Rab binding domains with homology to host Rab binding proteins GM130, FIP3, golgin-84

Microbe

Table 3: Alteration of Rab GTPase functions by bacterial effector proteins in niche formation

GTP-dependent recruitment of Rab4 to inclusion membrane to promote dynein dependent transport on microtubules to peri-Golgi region and interaction with transferrin-containing endosomes Interaction may inhibit VAMP7 mediated fusion with lysosomes or plasma membrane

Specific recruitment of Rab2 to PCV GTP-dependent recruitment of Rab11 to bacterial inclusion for peri-nuclear transport early in infection; subsequent interaction with Rab1 and Rab10 to maintain MTOC localization and access to membrane lipids Interaction with host Rab/tethering/SNARE protein complexes on endosomes to regulate delivery of nutrients for inclusion growth, prevent recycling and lysosomal delivery Suggested to function in endosome tethering but preclude fusion possibly by blocking Rab5

Consequence of interaction

(80)

(82)

(81)

(80)

(32)

(79)

References

Co-opting Rab Protein Function

1575

1576 ?

CagA

DrrA/SidM ESAT-6

ESX-1 secreted factors

GtgE

SopB

C. burnetii

H. pylori

L. pneumophilia M. tuberculosis

M. tuberculosis

S. enterica (serovars with broad host specificity, not Typhi)

S. enterica (serovar Typhimurium)

SopE

?

C. trachomatis

S. enterica (serovar Typhimurium and dublin)

Virulence protein (effector)

Microbe

Table 3: Continued

GEF mimic, T3SS1 effector

Phosphoinositide phosphatase, Cdc42 binding and GDI activity, T3SS1 effector

Protease

specific rab receptors?

GEF mimic membrane pore formation

Lipid-raft associated cytotoxin

Activity

Rab5, Cdc42 and Rac1

Rab5, (indirectly Rab8b, Rab13, Rab23 and Rab35)

Rab29 (Rab7L1)

Rab14, Rab22a

Rab1 Rab5, Rab11, Rab11b, Rab13, Rab20, Rab27

Src

Rab5, Rab7, Rab24

Rab6, BicD1, Rab11, Rab14

Host cell partner Formation of replicative niche, Sphingolipid recruitment to inclusion via Rab14, biosynthetic and endocytic cargo via Rab6 and Rab11. Convergence with autophagic pathway suggested to block degradation in lysosomes though some degradation resistant variants reach lysosomes Src substrate, blocks Rab11-FIP association to decrease pathogen recycling Activates Rab1 on PCV Pore formation may promote dissociation of indicated Rab proteins from mycobacterial phagosomes, contributing to phagosome arrest and niche formation Rab14 may function in sphingolipid delivery as is also the case for Chlamydia, Rab22a precludes Rab7 acquisition Cleaves Rab29 in serovars with broad host specificity leading to increased replication in macrophages; ins serovar Typhi lacking GtgE Rab29 is not cleaved and mediates typhoid toxin secretion Reduction of negatively charged PI(4,5)P2 via phosphatase enables Rab5/PI 3-kinase hVps34 recruitment and also prevents electrostatic membrane interaction of other Rabs thereby modulates membrane trafficking regulators and inhibits SCV-lysosome fusion. Rab5 binding and recruitment to SCV, in vitro promotes fusion of SCV with early endosomes and nucleotide exchange on Rab5; Rac1 and Cdc42 GEF activity important for invasion

Consequence of interaction

(18,89)

(86–88)

(40)

(35,37)

(6,85) (12)

(33,84)

(15,83)

(19,34)

References

Stein et al.

Traffic 2012; 13: 1565–1588

Virulence protein (effector) Activity

Host cell partner

Traffic 2012; 13: 1565–1588 VacA

? PipB2

SifA

H. pylori

M. bovis

S. enterica (serovar Typhimurium)

S. enterica (serovar Typhimurium)

T3SS2 protein interacts with kinesin light chain of the kinesin-1 motor and cooperates with SifA T3SS2 protein with N-terminal SKIP binding domain and C-terminal Rho GEF both required for Sif formation; prenylated CAAX motif for membrane binding

Vacuole formation

RhoA-GDP, Rab7, SifA and kinesin interacting protein (SKIP)/PLEKHM2, Rab9 and RILP antagonist, Arl8b

Kinesin-1 motor (Rab7 via SifA)

Rab7-RILP

Rab7-RILP, ORP1L

Selective localization through modulation of Rab regulated cytoskeletal transport C. trachomatis ? Src, Rab11 and various FIP effectors?

Microbe

Table 3: Continued

Regulated SCV membrane tubulation through multiple host and bacterial effector protein interactions. Recruits and activates host RhoA and bacterial SseJ. The SifA-SKIP complex activates the host kinesin-1 motor (recruited in inactive state by PipB2) and promotes Sif tubule extension toward cell periphery. SifA-SKIP binding antagonizes the normal host SKIP-Rab9-GTP binding through a conserved WxxxE domain in SifA that acts as a G-protein mimic and is conserved among bacterial proteins. SifA also binds host Rab7 and blocks host RILP/dynein/dynactin association and thereby controls tubule dynamics.

Recruitment of p150(glued)dynactin/dynein complex for MTOC transport of chlamydial inclusion in conjunction with Rab11? Juxtanuclear positioning may facilitate access to nutrients from Golgi and recycling endosomes for niche formation VacA and Rab7-RILP are essential for bacterial vacuole formation though direct interaction is not demonstrated. The Rab7 effector ORP1L may participate in vacuole transport through its cholesterol sensitive regulation of dynein Evasion of lysosomal delivery by blocking Rab7 and/or RILP recruitment. Recruits autoinhibited kinesin-1 to SCV for proper SCV positioning through coordinated kinesin motor activation with SifA/SKIP and plus-end Sif tubule extension

Consequence of interaction

(22,30,89,96–105)

(62,94,95)

(25)

(23,24,92,93)

(20,90,91)

References

Co-opting Rab Protein Function

1577

1578 SopD2

Virulence protein (effector) Activity

T3SS2 protein required for intracellular growth, regulates Sif dynamics S. enterica (serovar SseF T3SS2 protein binds Typhimurium) dynein in complex with SseG S. enterica (serovar SseG T3SS2 protein required for Typhimurium) intracellular growth; forms complex with SseF to bind dynein S. enterica (serovar SseJ T3SS2 protein required for Typhimurium) intracellular growth; deacylase, phospholipase and acyl transferase activities alter SCV lipid membrane; interacts with SifA and PipB2 in regulating SCV membrane tabulation Modulation of Rab function through post-translational modification Uncharacterized proteins C. pneumoniae CPn0034; containing Macro CPn0367; domain which may CPn0369; serve in binding CPn0370; ADP-ribosylated CPn0524 proteins; Macro domain may be regulated through mono-ADP-ribosylation

S. enterica (serovar Typhimurium)

Microbe

Table 3: Continued

(62,100,108)

(13,109,110)

Deacylase activity mediates recruitment of RhoA and facilitates membrane tubulation (Sifs) may modulate GTPase function.

Parallels to L. monocytogenes ADP-ribosylating enzyme specific for Rab5?

RhoA-GTP (Rab7 via SifA and PipB2?)

Unknown, ADP-ribosylated GTPases?

Rab7/RILP via dynein or kinesin interaction?

(62,107)

(62,107)

Binds SseG peri-nuclear localization of SCV, dynein recruitment and microtubule bundling Binds SseF; required for dynein recruitment or kinesin inhibition and microtubule bundling

Rab7/RILP via dynein or kinesin interaction?

(106)

References

Connections to SifA and PipB2, may modulate Rab7 and motors?

Consequence of interaction

?

Host cell partner

Stein et al.

Traffic 2012; 13: 1565–1588

Virulence protein (effector) CT058

AnkX DrrA/SidM LepB Lem3/lpg0696 LidA SidD GAPDH from listeria Lmo2459 ExoS

Microbe

C. trachomatis

L. pneumophila

L. pneumophilia

L. pneumophila

L. pneumophila

L. pneumophila

L. pneumophila

L. monocytogenes

P. aeruginosa

Table 3: Continued

Traffic 2012; 13: 1565–1588 T3SS cytotoxin with high homology to ExoT; N-terminal GAP domain that inactivates Rho GTPases; C-terminal ADP-ribosyl transferase domain that modifies host proteins of actin cytoskeleton, Ras, Ral and Rab GTPases

Uncharacterized protein containing Macro domain which may serve in binding ADP-ribosylated proteins; Macro domain may be inactivated through mono-ADP-ribosylation Phosphocholine transferase, Type IV Dot/Icm protein Bifunctional protein with adenylyltransferase domain GAP activity, Type IV Dot/Icm protein Dephosphocholination, Type IV Dot/Icm protein Supereffector of Rab proteins, Type IV Dot/Icm protein Deadenylylation activity,Type IV Dot/Icm protein ADP-ribosylation

Activity

Rab5, Rab3, Rab4

Rab5

Rab1

Rab1, Rab6, Rab8

Rab1, Rab35

Rab1

Rab1

Rab1, Rab35

Unknown, ADP-ribosylated GTPases?

Host cell partner

ADP-ribosylation of Rab5 blocks Rab5a exchange factor Vps9 and GDI therefore blocks phagosome endosome fusion ADP-ribosylation of Rab5 and other Rab proteins controlling epithelial junctions? Rab3 and Rab4 in vitro substrates of ExoS; Co-immunoprecipitates with Rab5, Rab6 and Rab9; required for plasma membrane niche formation in epithelia

Binds multiple Rab proteins with very high affinities Reverses adenylylation

Reverses phosphocholination

Inactivation of Rab1

Phosphocholination of Rab1 and Rab35, inhibition of GDI binding Activates Rab1 on PCV

Parallels to L. monocytogenes contains an ADP-ribosylating enzyme specific for Rab5?

Consequence of interaction

(10,75,76)

(13)

(7,9)

(8)

(4,42,146)

(3,42)

(6,85)

(4,42)

(13,109,110)

References

Co-opting Rab Protein Function

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GTP to GDP. Activation in the case of Rab5, Rab7 and Rab9 may be aided by a separate guanine nucleotide dissociation inhibitor (GDI) release factor (GDF) to promote selective membrane recruitment and GTP-binding. Active GTP-bound Rab proteins are then able to interact with a carefully orchestrated sequence of downstream effectors that remodel membrane lipids or bind motor proteins and in turn recruit additional factors to facilitate vesicular translocation on the cytoskeleton, targeting and fusion (reviewed in 2, 111). Following Rab-GTP hydrolysis, a common GDI extracts GDP-bound Rab proteins and shields the membrane anchoring prenyl groups during cytosolic recycling. The hierarchical cooperation between Rab GTPases is coordinated through integrated cascades that depend on the spatial and temporal recruitment of GEF and GAP proteins that act on sequential Rab GTPases in the pathway and thus ensure seamless transitions (reviewed in 2). Bacterial pathogens have devised intricate strategies for altering various aspects of the Rab-activation and functional cycle.

Selective Rab Recruitment to Evade Lysosomal Transport Direct binding of bacterial effectors to host Rab GTPases co-opts function Chlamydia trachomatis and Chlamydia pneumoniae are significant human pathogens causing blindness, sexually transmitted disease and pneumonia (Table 2) (64). Chlamydia species enter host mucosal epithelial cells or macrophages and establish residence in a compartment termed the ‘inclusion’ that is required for Chlamydiae growth and differentiation (Figure 2). Formation of chlamydial inclusions and avoidance of transport to lysosomes both require protein synthesis by Chlamydiae, suggesting that bacterial effector proteins facilitate remodeling of the chlamydial inclusion (112). Numerous Rab proteins (Rab1, Rab4, Rab6, Rab10 and Rab11) localize to chlamydial inclusions with some displaying species specificity (16). Direct binding of C. trachomatis inclusion membrane protein (Inc), CT229 specifically to Rab4 was shown by yeast 2-hybrid and immunofluorescence studies (82). A second Inc protein, Cpn0585, sequentially binds to Rab1, Rab10 and Rab11 and thereby modulates transport (32). Structural studies suggest C. trachomatis CT147 may act as a mimic of the Rab5 effector early endosomal antigen (EEA1) (81). CT147 likely can tether endosomes together but precludes endosome fusion because it lacks the structural equivalent of a Rab5-binding domain present in EEA1, thus blocking normal protein recruitment and endosome fusion. As illustrated by these examples, direct binding of bacterial inclusion proteins to host Rab proteins and downstream effectors is emerging as an important mechanism for remodeling of bacterial inclusion membranes through regulated vesicle recruitment and fusion and is a fruitful area for further investigation (32,80–82) (Table 3). 1580

Similarly, L. pneumophila, the causative agent of a potentially lethal pneumonia called Legionnaires’ disease that afflicts primarily the elderly and the immunocompromised, translocates multiple proteins into host cells using its Dot/Icm type IV secretion apparatus. Currently, more than 250 secreted proteins are known (113,114). Several of these proteins interact with Rab1 (Table 3), which usually regulates vesicular trafficking between the ER and the Golgi apparatus. One such Rab1 interacting protein, DrrA (defect in Rab1 recruitment protein A, also called SidM) was originally described as a bifunctional protein containing GDI-displacement and GEF activities for Rab1 (6,115). Further research showed that the observed GDI-displacement activity was actually a result of the GEF activity of DrrA and that no active displacement occurs (Figure 3A) (85). Another protein secreted by L. pneumophila, the protein LepB, acts as a Rab1 GAP (3). Besides Rab1, the small GTPases Arf1, Rab7, Rab8 and Rab14 have been shown to be localized at the Legionella-containing vacuole (LCV) during infection, although mechanisms of recruitment and functions of these proteins at the LCV require further study (26,116). Notably, L. pneumophila also secretes a protein called LidA that is considered a ‘supereffector’ of Rab proteins based on its low picomolar affinity and extended protein interaction interface (8). LidA binds Rab8a and Rab6, which are important in late exocytic events from the Golgi (8). LidA binds multiple Rab proteins in the GDPand GTP-bound states with very high affinities and may thereby provide spatiotemporal regulation during infection. Thus, L. pneumophila produces a whole set of proteins for the subversion of Rab1-function and potentially other GTPases during infection in order to support intravacuolar growth.

Salmonella enterica are Gram-negative bacteria that are most often associated with food-borne illnesses resulting in diarrhea, fever and abdominal cramps. S. enterica are internalized into gastric epithelial cells or macrophages in a membrane-bound compartment termed the Salmonellacontaining vacuole (SCV). S. enterica species utilize T3SS secretion systems to deliver bacterial effector proteins into host cells. The recruitment of Rab5 to the SCV is associated with the generation of PI(3)P by a multifunctional T3SS1 protein, SopB. A phosphatase domain in SopB reduces PI(4,5)P2 levels on the SCV (87,117). In addition, SopB has a Cdc42-binding domain that acts as a Cdc42 guanine nucleotide dissociation inhibitor (GDI) (88). Thus, the activities of SopB directly regulate actin polymerization and indirectly result in the recruitment of selective Rab5 effector proteins to the SCV, including hVPS34 phosphatidyl inositol 3-kinase. SopB mutant bacteria increasingly recruit and retain Rab8b, Rab13, Rab23 or Rab35 in contrast to wild-type S. enterica suggesting that modulation of the phosphoinositides PI(4,5)P2 and PI(3)P on the SCV is also important to prohibit recruitment of specific Rab proteins and direct the maturation of the SCV (86). Traffic 2012; 13: 1565–1588

Co-opting Rab Protein Function cytoplasm

effector vesicle

adenylylation

phosphocholination

LidA?

LidA? GDI

GDI

connecdenn 1

GDP

GDI

Rab

GAPs (e.g. LepB), Mical-3

ATP

GDP

CDP-choline

Rab

GDP

Rab PC GDP

GDP

AMP AnkX

DrrA (GEF)

GTP GTP

DrrA (ATase)

Lem3 (lpg0696)

SidD

GTP

Legionella containing vacuole / other compartment

Figure 3: Schematic of bacterial effector proteins secreted by Legionella pneumophila to subvert Rab function. The figure illustrates the modification of Rab proteins by (left) phosphocholine mediated by AnkX and (right) adenosine monophosphate mediated by DrrA. Phosphocholination strongly inhibits GEF catalysis by connecdenn 1 while adenylylation strongly impairs binding of the human effector protein Mical-3 and inactivation by GAPs (e.g. LepB). In contrast, the ‘supereffector’ LidA can bind Rab1 also in the modified states and might act as a tethering factor. Interaction with GDI can only occur after removal of the modifications, indicating a possible role of the modifications in recruitment and entrapment of Rab proteins at the surface of endogenous membranes. All proteins encoded by Legionella pneumophila for subversion of Rab function are indicated in red letters.

These examples reveal interactions of bacterial secreted effector proteins with specific host cell Rab proteins on vacuolar membranes resulting in the modulation of PCV transport early after internalization. The direct binding of Chlamydiae Inc proteins to Rab proteins, the enzymatic modulation of Rab GTPases and accessory factors by Legionella, and Salmonella modulation of membrane phosphoinositides illustrate the selective recruitment and activation of Rabs and their effectors, which play an important role in early alterations necessary for PCV maturation.

Bacterial effector proteins post-translationally modify Rab protein structure In addition to the direct binding and recruitment of Rab proteins to the PCV membrane, pathogens have acquired the ability to control the activity of Rab proteins by directly altering Rab structure through post-translational modifications. L. monocytogenes, often a food-borne pathogen, infects macrophages and resides in the host phagosome for only a brief time prior to escaping into the host cell cytosol (13). Inhibition of Rab5a GEF activity was demonstrated to result in Listeria intracellular survival (118) and this inhibition is dependent on Listeria glyceraldehyde-3-phosphate dehydrogenase Traffic 2012; 13: 1565–1588

(GAPDH) protein (p40, Lmo2459). Lmo2459 binds and recruits Rab5 and ADP-ribosylates Rab5a, inhibiting exchange of GDP for GTP by inhibiting the interaction of Rab5a with Vps9 (13). Similarly, P. aeruginosa ExoS is a multidomain protein with an ADP-ribosyl transferase domain that modulates multiple Rab GTPases (Table 3) (75). Thus, ADP-ribosylation may be a common bacterial strategy for modulating trafficking and Rab GTPases that requires further study (e.g. in Chlamydiae, Table 3). Reversible adenylylation (also called AMPylation) is another post-translational modification and is used by L. pneumophila to modulate Rab GTPases. The Legionella effector DrrA, besides having GEF activity, possesses an N-terminal adenylyltransferase activity toward Rab1b Tyr77. Adenylylation of Rab1 prolongs the GTP-bound active state by preventing inactivation of Rab1 by GAPs including L. pneumophila LepB protein (Figure 3B) (5,7,9,119). The binding of the human effector protein Mical-3 is also impaired by Rab1b adenylylation, whereas the Legionella effector protein LidA still binds Rab1-AMP with high affinity (5,8). AnkX, another protein secreted by L. pneumophila catalyzes the covalent attachment of a phosphocholine moiety to the adenylylation-site-adjacent Ser76 in Rab1b and Rab35. Although phosphocholination 1581

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had only moderate effects on Rab1b binding to effector proteins Mical-3 and LidA and interactions with GAPs and GEFs, phosphocholination of Rab35 strongly impaired connecdenn 1 GEF activity (4,42). Interestingly, both adenylylation and phosphocholination drastically reduce the affinity for GDI (120). This finding indicates that L. pneumophila encode two enzymatic activities that cause the recruitment and entrapment of Rab proteins on the surface of intracellular membranes by inhibiting binding and extraction of the Rab proteins from membranes by GDI. While DrrA is localized to the LCV, the localization of AnkX during infection is not clear yet, although cell culture experiments indicate a function in vesicular trafficking to or from the Golgi apparatus (121). Adenylylation and phosphocholination of Rab1 can be reversed by the Legionella proteins SidD and Lem3 (lpg0696), respectively (7,9,42,122,146). Thus, L. pneumophila subverts Rab function in a temporally and spatially controlled manner by secreting both Rab-interacting and -modifying proteins. The emerging data identify pathogen catalyzed posttranslational modifications as a pivotal mechanism whereby pathogens modulate and usurp Rab GTPases. Taking a hint from studies on L. pneumophila, it is interesting to consider that M. tuberculosis encodes over 60 adenylylating enzymes that are considered key drug targets (123). While half of the enzyme activities are thought to function in fatty acid modification, analyses of Rab GTPase adenylylation upon M. tuberculosis infection have not yet been undertaken.

Pathogens Usurp Rab GTPases to Direct Trafficking to Specific Intracellular Locales Salmonella enterica and Chlamydiae actively manipulate cytoskeletal, motor-driven transport of their PCV to localize to specific regions of infected cells, while Mycobacteria actively block motor recruitment. Pathogendirected transport is important in both evading degradation and obtaining nutrients for survival (124,125). Microtubuledependent transport of endocytic compartments to the peri-nuclear Golgi and MTOC regions is dependent on Rab GTPase-effector complexes that in turn bind dynein/dynactin to facilitate transport (126). In contrast, transport to the cell periphery depends on Rab GTPasemediated regulation of kinesin and myosin motors (27,126,127). Salmonella enterica utilizes dynein- and kinesin-dependent transport for its peri-nuclear localization close to the MTOC early in infection and to remodel its niche through membrane tubulation late in infection (reviewed in 62, 95). The early SNX3-dependent recruitment of Rab7 and Rab7interacting lysosomal protein (RILP) to the SCV enables dynein/dynactin motor binding and peri-nuclear transport (22,128). Later in infection, the activity of the T3SS2 protein SifA in conjunction with multiple bacterial (PipB2, SseF, SseG, SseJ and SopD2) and host proteins facilitates 1582

the extension of long tubules termed Salmonella-inducedfilaments (Sifs) that are important for bacterial growth and ultimately cell-to-cell spread (99,104,105,107). SifA when present on the SCV binds Rab7 and interferes with RILP/dynein/dynactin interactions, while promoting kinesin-dependent Sif membrane tubule extension on microtubules toward the cell periphery. SifA interaction with host protein SifA-and-kinesin-interacting-protein (SKIP/PLEKHM2) antagonizes SKIP-Rab9-GTP binding, which normally directs recycling between late endosomes and the trans-Golgi (30). The SifA-SKIP complex instead activates kinesin-1 in concert with the bacterial protein PipB2 that functions upstream to recruit autoinhibited kinesin to the SCV (30,95,97). Multiple kinesin motors (Kif5B, Kif11 and Kif24), as well as interactions with additional host proteins [Arf family GTPase Arl8b and secretory vesicle membrane proteins, (SCAMP) 2 and 3] are crucial for both late endosomal dynamics and tubulation during S. enterica infection (101,103,129,130). There is a close cooperation between Arf and Rab family GTPases in the integration of membrane remodeling, e.g. in the case of Rab7 and Arl8b through the shared HOPS effector (102). Therefore, further elucidation of how S. enterica modulates its niche by co-opting GTPase function is of significant interest. The early localization of the chlamydial inclusions to a periGolgi location near the MTOC is also regulated by dyneindependent transport and requires active Src kinases (91,125). Recently, Chlamydiae inclusion membrane proteins (Inc) have been identified to bind both active Src family kinases and centrosome components (91,109). We speculate that recruitment of Src kinase together with Rab11 serves to regulate the cytoskeletal motility of chlamydial inclusions (20). Src kinase is normally activated on Rab11 and RhoB containing endosomes where it facilitates actin nucleation, and offers the potential for switching between microtubule and actin based motility of inclusions. Src could promote transport through SH2 domain interactions as has been demonstrated for lysosome clustering (131) or through phosphorylation dependent motor recruitment as demonstrated for motility of enveloped viruses (132,133). Alternatively, Src may serve to increase the pool of activated Rab11, as it normally does for Rab5 and Rab7, but which are not on inclusions (16,134). Active Rab11 would be expected to have increased interaction with effectors such as the Rab11 family interacting proteins (FIP2, FIP3 and FIP5), which in turn interact with dynein and kinesinII to control transport on microtubules (127,135) and myosin motors (MyoVb) to control actin based motility (136). Thus, the roles of Rab11 and Src in the cytoskeletal transport of chlamydial inclusions are fruitful areas for further investigation and are expected to elucidate mechanisms for maintenance and maturation of the chlamydial intracellular niche. Inhibition of Rab7-RILP interactions and reduced Rab7 recruitment are suggested to prohibit the

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Co-opting Rab Protein Function

dynein-dependent transport of PCV to peri-nuclear lysosomes and contribute to phagosomal maturation arrest as exemplified in mycobacterial infections (12,25). Grampositive Mycobacteria cause severe pulmonary and intestinal infections in humans that are highly contagious and often lethal. M. tuberculosis infects alveolar macrophages. The related Mycobacterium bovis causes pulmonary infections in cattle, but may be spread to humans through aerosols and non-pasteurized milk and is a common cause of human tuberculosis in developing countries. Nontuberculosis causing Mycobacteria include 20 species that cause human and animal disease, among them subspecies of Mycobacterium avium are implicated in chronic human lung diseases, chronic intestinal Crohn’s disease, and a primary cause of morbidity and mortality in immune compromised patients (137). All of these pathogenic Mycobacteria species reside and multiply in macrophages where they inhibit phagosome-lysosome fusion for intracellular survival. The cause of the phagosomal arrest is attributed to altered Rab7 function on Mycobacteria-containing phagosomes (12,17,138,139). In M. tuberculosis infections, reduced phagosomal Rab7 levels are suggested to account for reduced RILP-mediated transport, while in M. bovis infections RILP recruitment was blocked due to a prevalence of inactive Rab7 on Mycobacteria-phagosomes. Rab22a on the M. tuberculosis phagosome contributed to the inhibition of Rab7 recruitment to Mycobacteria-phagosomes, although the effect might be indirect (37). Rab22a normally recruits one of several Rab5 GEFs (Rabex-5) to early endosomes and is implicated in increasing early endosome fusion (38). In this regard it is interesting that M. avium depends on early endosome fusion for an adequate supply of iron and inhibition of phagosome maturation (14). However, if and how iron and recruitment of Rab22a by Mycobacteria might impact Rab5 to Rab7 conversion remains unclear. In Mycobacteria-infected cells, the expression of bacterial lipids with similarity to glycosylphosphatidylinositols antagonizes the recruitment of Rab5 and Rab7 effectors that synthesize (hVPS34) and recognize (EEA1) PI(3)P, which augment the block in phagosome maturation (140). The cumulative data suggest that mycobacterial proteins and phagosomal membrane lipids may reduce Rab5-Rab7 conversion, specifically block recruitment of RILP to Rab7 (25), or mycobacterial proteins may act as Rab7-GAPs to inactivate Rab7 (25) and inhibit interaction with RILP. Further work remains to elucidate Mycobacteria speciesspecific mechanisms that depend on their niches and the roles of other Rab GTPases associated with Mycobacteriaphagosomes.

Creation of an Intracellular Replicative Niche Active remodeling of the PCV compartment through the modulation of Rab-dependent membrane trafficking facilitates the formation of a replicative niche where bacteria differentiate into their infective forms, acquire nutrients necessary for growth and actively replicate

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in preparation for dissemination upon release from host cells. The late stage recruitment of Rab and Rab effector proteins to PCV has been described and through expression of dominant-negative or constitutively active Rab mutants or RNAi knockdown experiments, the necessity of Rab proteins derived from diverse endomembranes has been demonstrated to be crucial for pathogen growth. Here a few well-documented examples, where recruitment of host molecules is required for creation of a replicative niche, are presented. Productive formation of infectious Chlamydia (trachomatis and pneumoniae) requires a replicative niche where bacterial differentiation and maturation take place and depends on Rab6 and Rab11, GTPases involved in Golgi transport and endosomal recycling (20). Cells depleted of Rab6 and Rab11 by RNAi failed to allow C. trachomatis maturation, although the additional fragmentation of the Golgi, through loss of p115, rescued Chlamydiae maturation (20). Thus, in the absence of the redirected transport of newly synthesized host proteins and lipids by Rab6 and Rab11 to chlamydial inclusions, the complete disruption of the exocytic pathway and an intracellular accumulation of vesicular biosynthetic cargoes were able to rescue the maturation of the chlamydial inclusion. Rab14, which functions in endosomal recycling, also plays a role in chlamydial inclusion formation by providing endogenously synthesized sphingolipids to the growing inclusion body (34). A role for Rab14 in preventing lysosomal transport, as shown for Mycobacteria, has not yet been demonstrated for Chlamydia. Recruitment of both Rab6 and its effector Bicaudal D1 (BicD1) required chlamydial protein synthesis and BicD1 recruitment was independent of Rab6, suggesting that BicD1 may facilitate the recruitment of Rab6 to the inclusion in a manner distinct from normal Rab6-GTP mediated binding of BicD1 to the Golgi (19). The data demonstrate that recruitment of at least three Rab proteins to the inclusion membrane facilitates the growth and differentiation of Chlamydiae by directing the transport and fusion of nutrient-rich endosomal and Golgi derived vesicles to the inclusion. Several Rab proteins also modulate Mycobacteriaphagosome maturation and the generation of a stable niche for bacterial growth. Active mechanisms for reducing recruitment of the endosomal Rab7 GTPase are important for evading lysosomal transport as discussed above. Rab14 also plays a role in Mycobacteriaphagosome arrest (35), conceivably by modulating sphingolipid transport as occurs in chlamydial inclusion formation (34). Mycobacteria express multiple lipid hydrolases (phospholipases and ceramidases) that can efficiently catabolize sphingolipids uniquely present in the lung to fatty acids as an energy source (141). Ceramide generated from sphingolipid degradation may also impact host cell survival signaling (142). Thus, Rab14 may have a multi-functional role in precluding phagolysosomal fusion, enabling signaling, and providing nutrients and a favorable host cell environment for 1583

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bacterial growth in the replicative niche. Mycobacteriaphagosomes exhibit enhanced early endosome fusion and altered recycling, which may coordinately ensure a supply of endocytosed nutrients important for bacterial survival. Recent studies show reduced Rab10 association with Mycobacteria-phagosomes, which together with enhanced early endosome fusion promoted by bacterial lipids is speculated to be important to the replicative niche (31,140). The observed mycobacterial exclusion of Rab10 may be akin to Rab10 knockdown, which slows transferrin and glycosylphosphatidylinositol (GPI) anchored protein removal and recycling from phagosomes (31), thereby slowing recycling and ensuring access to transferrin and iron for Mycobacteria (14). GPI-anchored proteins may deliver key nutrients such as folate or provide precursors for the biosynthesis of bacterial lipids [phosphatidylinositol mannoside (PIM), and its derivatives lipomannan (LM) and lipoarabinomannan (LAM)] that promote phagosomal arrest. The known function of Rab22a in modulating communication between the biosynthetic and early endocytic pathways, may also make its recruitment to the phagosome pivotal to bacterial growth though details remain to be established (36,37). These data suggest that Rab proteins on both the endocytic and Golgi pathways play a role in Mycobacteria-phagosome maturation and generation of the replicative niche.

Helicobacter pylori is a Gram-negative bacterium that colonizes epithelial cells of the stomach and intestine resulting in massive inflammatory responses that cause gastric ulcers and frequently, gastric cancer. Although predominantly extracellular, H. pylori invade gastric epithelial cells and intracellular H. pylori persist in host cells for long periods of time in large intracellular vacuoles formed with the aid of a bacterially encoded vacuolating toxin, VacA (143). Rab7 localization on VacA induced vacuoles promotes homotypic fusion with Rab7-positive late endosomes and recruitment of RILP, promoting perinuclear localization and ensuring the continuous delivery of vesicular membranes required for VacA-dependent vacuolization (23,24,93). Selected late endosomal and lysosomal proteins such as Rab7, vacuolar ATPase, LAMP1 and Lgp110 localize to the H. pylori -vacuole, while Rab9, CI-M6PR and lysosomal enzymes such as cathepsin D are absent (23,93,144). Other Rab proteins have also been implicated in the pathogenesis of H. pylori . Proteomic analyses demonstrated an increase of Rab37 expression in H. pylori -infected cells compared to uninfected controls (43). Expression of H. pylori CagA, a Type IV secretion system effector and target of Src phosphorylation was observed to decrease the association of Rab11-FIP with detergent-resistant membranes and host membranes and may decrease pathogen recycling (33). Coxiella burnetii is a Gram-negative obligate intracellular parasite that is the causative agent of Q-fever. Infected individuals most often are infected by inhalation of aerosolized bacteria and display high fevers, headaches 1584

and general malaise that may progress to pneumonia. Q-fever has a very low mortality rate (1–2%) for acute illness, but ∼65% of individuals who develop chronic Q-fever succumb to the disease (67). Upon entry into host cells, C. burnetii initially resides in a tight parasitophorous vacuole that over hours to days matures into a compartment resembling a lysosome. The parasitophorous vacuole initially acquires markers of early and late endosomes including Rab5 and Rab7 (15,21). Data based on siRNA treatment and overexpression of dominant active and inactive GTPases indicate that both Rab5 and Rab7 play roles in the maturation of the C. burnetii parasitophorous vacuole to a replicative parasitophorous vacuole. Between 6 and 12 h after infection, the parasitophorous vacuole acquires markers of autophagy including LC3 and Rab24 (15,145), suggesting that the host autophagic pathway is necessary to create the Coxiella parasitophorous vacuole. Defects in autophagy delay the maturation of Coxiella parasitophorous vacuoles, however, further study will be necessary to understand the precise role of Rab24 and autophagy in early Coxiella infection. At 2 days postinfection, the parasitophorous vacuole is greatly enlarged (now referred to as a spacious parasitophorous vacuole) and has acquired lipid raft proteins (Flotillin1 and Flotillin2) as well as numerous lysosomal markers including 5 nucleotidase, LAMP1 and LAMP2, and an acidic pH of ∼5 (83). Rab7, Rab24 and LC3 remain associated at late time points (145). C. burnetii is currently the only identified intracellular bacteria requiring a low-pH lysosome-like intracellular niche for growth, differentiation and maturation.

Pseudomonas aeruginosa is an important pathogen in cystic fibrosis patients that creates an intracellular niche at the cell surface on respiratory epithelia through phosphatidylinositol 3-kinase recruitment and plasma membrane remodeling (75,76). The bacterial ExoS protein is pivotal in niche formation (75). ExoS is T3SS cytotoxin with an N-terminal GAP domain that inactivates Rho GTPases and a C-terminal ADP-ribosyl transferase that modifies Rab5 and thereby prevents bacterial internalization while inducing plasma membrane blebbing to create a replicative niche. Although Rab3 and Rab4 are in vitro substrates of ExoS, their roles in niche formation are untested (10).

Summary Rab GTPases, considered the master regulators of membrane trafficking, are important targets of bacterial pathogens. Soon after internalization, bacterial pathogens usurp one or more Rab GTPases to evade degradation and modulate intracellular localization. Later in infection, bacteria modulate Rab GTPase functions to obtain requisite nutrients and create an environment that is conducive to intracellular bacterial survival and growth. Intracellular localization is important for evading host

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degradation and immune detection, as well as for positioning the replicative niche in a subcellular location that facilitates bacterial access to host proteins, lipids and inorganic molecules that are essential for bacterial growth and persistence. In most cases, bacterial effector proteins, introduced by specialized secretion systems, modulate host Rab protein functions by acting as specific receptors or by blocking normal Rab protein or Rab effector function. Functional modulation of Rab GTPases occurs through changes in membrane lipid composition, Rab effector mimicry, post-translational modification of Rab GTPases, kinase signaling and alteration of Rab activation cycles. The Rab GTPases required for pathogen internalization, transport and growth are rapidly being cataloged. Yet as highlighted here, many gaps remain in our knowledge of the precise mechanisms whereby specific Rab GTPases contribute to intracellular bacterial survival and growth. Although Rab GTPases and Rabregulated pathways are important targets of disease, they are as yet underexplored therapeutic targets. Thus, further study of Rab GTPases in bacterial infection is expected to reveal insights into normal function as well as provide new ‘druggable’ targets.

Acknowledgments MPS was supported by NIH SCORE 5SC2GM086312, MPM was supported by IMPRS-CB (Dortmund, Germany) and AWN by NSF MCB0956027, NIDDK R01DK050141 and NINDS R21NS066435. We gratefully acknowledge the support of Dr. Roger Goody (Director Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany). We apologize for omissions in the citation of original work due to page and citation limitations. We gratefully acknowledge Dr. Olivia Steele-Mortimer (NIAID, Rocky Mountain Laboratories) for the image of S. enterica induced Sifs and Dr. Robert A. Heinzen (Pathogenesis Section, NIAID) for the pseudocolored scanning electron micrograph of C. burnetii contained in a lysosomelike parasitophorous vacuole. Images of L. pneumophila phagocytosis, structure of Rab1-AMP and cell graphic were prepared by the authors.

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