Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells.

Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin-mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.

Recent advances in Chlamydia subversion of host cytoskeletal and membrane trafficking pathways.

Chlamydia species are obligate intracellular bacteria that cause sexually transmitted disease, ocular infections and atypical pneumonia. This review highlights recent advances describing the mechanisms by which Chlamydia subvert host cytoskeleton and membrane trafficking pathways to create a replication competent niche.

The Anaplasma phagocytophilum-occupied vacuole selectively recruits Rab-GTPases that are predominantly associated with recycling endosomes.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell-derived vacuole. The A. phagocytophilum-occupied vacuole (ApV) fails to mature along the endocytic pathway and is non-fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)-tagged Rab GTPases in A. phagocytophilum-infected HL-60 cells. GFP-Rab4A, GFP-Rab10, GFP-Rab11A, GFP-Rab14, RFP-Rab22A and GFP-Rab35, which regulate endocytic recycling, and GFP-Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome-associated GFP-Rabs and acquisition of GFP-Rab5, GFP-Rab7, and the lysosomal marker, LAMP-1. Wild-type and GTPase- deficient versions, but not GDP-restricted versions of GFP-Rab1, GFP-Rab4A and GFP-Rab11A, localize to the ApV. Strikingly, GFP-Rab10 recruitment to the ApV is guanine nucleotide-independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.

Multiple host proteins that function in phosphatidylinositol-4-phosphate metabolism are recruited to the chlamydial inclusion.

Chlamydiae replicate within a nonacidified vacuole, termed an inclusion. As obligate intracellular bacteria, chlamydiae actively modify their vacuole to exploit host signaling and trafficking pathways. Recently, we demonstrated that several Rab GTPases are actively targeted to the inclusion. To define the biological roles of inclusion localized Rab GTPases, we have begun to identify inclusion-localized Rab effectors. Here we demonstrate that oculocerebrorenal syndrome of Lowe protein 1 (OCRL1), a Golgi complex-localized phosphatidylinositol (PI)-5-phosphatase that binds to multiple Rab GTPases, localizes to chlamydial inclusions. By examining the intracellular localization of green fluorescent protein (GFP) fusion proteins that bind to unique phosphoinositide species, we also demonstrate that phosphatidylinositol-4-phosphate (PI4P), the product of OCRL1, is present at the inclusion membrane. Furthermore, two additional host proteins, Arf1, which together with PI4P mediates the recruitment of PI4P-binding proteins to the Golgi complex, and PI4KII alpha, a major producer of Golgi complex-localized PI4P, also localize to chlamydial inclusions. Depletion of OCRL1, Arf1, or PI4KII alpha by small interfering RNA (siRNA) decreases inclusion formation and the production of infectious progeny. Infectivity is further decreased in cells simultaneously depleted for all three host proteins, suggesting partially overlapping functions in infected cells. Collectively, these data demonstrate that Chlamydia species create a unique replication-competent vacuolar environment by modulating both the Rab GTPase and the PI composition of the chlamydial inclusion.

Manipulation of rab GTPase function by intracellular bacterial pathogens.

Intracellular bacterial pathogens have evolved highly specialized mechanisms to enter and survive within their eukaryotic hosts. In order to do this, bacterial pathogens need to avoid host cell degradation and obtain nutrients and biosynthetic precursors, as well as evade detection by the host immune system. To create an intracellular niche that is favorable for replication, some intracellular pathogens inhibit the maturation of the phagosome or exit the endocytic pathway by modifying the identity of their phagosome through the exploitation of host cell trafficking pathways. In eukaryotic cells, organelle identity is determined, in part, by the composition of active Rab GTPases on the membranes of each organelle. This review describes our current understanding of how selected bacterial pathogens regulate host trafficking pathways by the selective inclusion or retention of Rab GTPases on membranes of the vacuoles that they occupy in host cells during infection.

Chlamydia pneumoniae inclusion membrane protein Cpn0585 interacts with multiple Rab GTPases.

Chlamydiae are intracellular bacteria that develop within a membrane-bound vacuole called an inclusion. To ensure that the inclusion is a safe niche for chlamydial replication, chlamydiae exploit a number of host cell processes, including membrane-trafficking pathways. Recently, several Rab GTPases were found to associate with the inclusions of various chlamydial species. Here we report that Cpn0585, a Chlamydia pneumoniae inclusion membrane protein (Inc), interacts with multiple Rab GTPases. The results from yeast two-hybrid experiments revealed that an amino-terminally truncated form of Cpn0585 (Cpn0585(102-651)) interacts with Rab1, Rab10, and Rab11 but not with Rab4 or Rab6. Cpn0585-Rab GTPase interactions are direct and GTP dependent as shown in glutathione S-transferase pull-down assays using native and recombinant Cpn0585. In C. pneumoniae-infected HEp-2 cells transfected with enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, the colocalization with Cpn0585 at the inclusion membrane was partial for EGFP-Rab1 and EGFP-Rab10, but extensive for wild-type EGFP-Rab11A and the constitutively active GTPase-deficient EGFP-Rab11AQ70L. Moreover, Cpn0585 colocalized with EGFP-Rab11AQ70L as early as 2 h postinfection. Upon delivery into live C. pneumoniae-infected cells, Cpn0585(628-651)-specific antibodies bound to the inclusion membrane, demonstrating that the Rab GTPase-interacting domain of Cpn0585 faces the host cell cytosol. Finally, ectopic expression of Cpn0585(102-651) partially inhibited the development of C. pneumoniae inclusions in EGFP. but not in EGFP-Rab11AQ70L-expressing HEp-2 cells. Collectively, these data suggest that Cpn0585 is involved in the recruitment of Rab GTPases to the inclusion membrane and that interfering with this function may adversely impact the fitness of the C. pneumoniae inclusion for chlamydial replication.

A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium.

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome-lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

Interaction of the Salmonella-containing vacuole with the endocytic recycling system.

Upon entry of the pathogen Salmonella enterica serovar Typhimurium into host cells, the majority of bacteria reside in a membrane-bound compartment called the Salmonella-containing vacuole (SCV). Previous studies have established that the SCV transiently interacts with early endosomes but only acquires a subset of late endosomal/lysosomal proteins. However, the complete set of interactions between the SCV and the endocytic machinery has yet to be characterized. In this study, we have shown that four characterized regulators of endocytic recycling were present on the SCV after invasion. Interaction kinetics were different for each of the regulators; ARF6 and Rab4 associated immediately, but their presence was diminished 60 min post-infection, whereas syntaxin13 and Rab11 association peaked at 60 min. Using a dominant negative approach, we determined that Rab11 regulates the recycling of CD44 from the vacuole but had no effect on major histocompatibility complex (MHC) class I recycling. In contrast, syntaxin13 regulated the recycling of MHC class I but not of CD44. We also determined that maturation of the SCV, measured by the acquisition of lysosomal associated membrane protein-1, slowed when recycling was impaired. These findings suggest that protein movement through the endocytic recycling system is regulated through at least two concurrent pathways and that efficient interaction with these pathways is necessary for maturation of the Salmonella-containing vacuole. We also demonstrate the utility of using Salmonella invasion as a model of endosomal recycling events.

Cultivation and Laboratory Maintenance of Chlamydia trachomatis.

Chlamydia species are obligate intracellular bacteria that require growth inside mammalian cells for propagation and survival. As a result, Chlamydia cannot be grown on conventional bacteriological medium. This property makes Chlamydia difficult organisms to grow and maintain in the laboratory. Up until 1965, passage in the yolk sac of the embryonated hen egg was the only way to isolate and propagate the organism. Since then, a tissue culture system has been available that allows easier laboratory culture of the Chlamydia species. However, with the exception of the LGV serovars, most C. trachomatis strains do not readily infect tissue culture cells. Chemical or mechanical assistance is used to increase their infectivity. Today, large numbers of infectious organisms can be purified through Renografin density gradient centrifugation of infected cell lysates. The ability to propagate C. trachomatis in the laboratory has greatly increased the understanding of the pathogenesis of C. trachomatis organisms.

Rab GTPases are recruited to chlamydial inclusions in both a species-dependent and species-independent manner.

Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the peri-Golgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFP-Rab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to Chlamydia trachomatis, Chlamydia muridarum, and Chlamydia pneumoniae inclusions, whereas GFP-Rab5, GFP-Rab7, and GFP-Rab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with C. trachomatis inclusions but not with C. pneumoniae or C. muridarum inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11's association with the inclusion is not mediated solely through Rab11's association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion.

Restricted fusion of Chlamydia trachomatis vesicles with endocytic compartments during the initial stages of infection.

The chlamydial inclusion occupies a unique niche within the eukaryotic cell that does not interact with endocytic compartments but instead is fusogenic with a subset of sphingomyelin-containing exocytic vesicles. The Chlamydia trachomatis inclusion acquires these distinctive properties by as early as 2 h postinfection as demonstrated by the ability to acquire sphingomyelin, endogenously synthesized from 6[N-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproylsphingosine] (C(6)-NBD-ceramide). The molecular mechanisms involved in transformation of the properties and cellular interactions of the inclusion are unknown except that they require early chlamydial transcription and translation. Although the properties of the inclusion are established by 2 h postinfection, the degree of interaction with endocytic pathways during the brief interval before fusogenicity with an exocytic pathway is established is unknown. Using a combination of confocal and electron microscopy to localize endocytic and lysosomal markers in C. trachomatis infected cells during the early stages of infection, we demonstrate a lack of these markers within the inclusion membrane or lumen of the inclusion to conclude that the nascent chlamydial inclusion is minimally interactive with endosomal compartments during this interval early in infection. Even when prevented from modifying the properties of the inclusion by incubation in the presence of protein synthesis inhibitors, vesicles containing elementary bodies are very slow to acquire lysosomal characteristics. These results imply a two-stage mechanism for chlamydial avoidance of lysosomal fusion: (i) an initial phase of delayed maturation to lysosomes due to an intrinsic property of elementary bodies and (ii) an active modification of the vesicular interactions of the inclusion requiring chlamydial protein synthesis.

Proteins in the chlamydial inclusion membrane.

The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole (the inclusion) during their entire developmental cycle. Several proteins have recently been identified that are localized to the inclusion membrane. The following is a discussion of how inclusion membrane proteins might participate in the chlamydial developmental process.