Acquisition of nutrients by Chlamydiae: unique challenges of living in an intracellular compartment

Summary

The Chlamydiae are obligate intracellular pathogen that replicate within a membrane-bound vacuole, termed the “inclusion”. From this compartment, bacteria acquire essential nutrients by selectively redirecting transport vesicles and hijacking intracellular organelles. Re-routing is achieved by several mechanisms including proteolysis-mediated fragmentation of the Golgi apparatus, recruitment of Rab GTPases and SNAREs, and translocation of cytoplasmic organelles into the inclusion lumen. Given Chlamydiae’s extended co-evolution with eukaryotic cells, it is likely that co-option of multiple cellular pathways is a strategy to provide redundancy in the acquisition of essential nutrients from the host and has contributed to the success of these highly adapted pathogens.

Introduction

The Chlamydiaceae comprise a distinct family of closely related, obligate intracellular bacteria that diverged early in evolution and parasitize a wide range of hosts [1]. The human pathogens are widely distributed and include Chlamydia trachomatis (leading cause of genital infections and infectious blindness), C. pneumoniae (common cause of respiratory tract infections and community-acquired pneumonia), and C. psittaci (a zoonotic pathogen that can cause severe pneumonia in humans) [2–6].

All Chlamydiaceae undergo a biphasic developmental cycle involving the infectious and environmentally resistant form or elementary body (EB), and the replicative, non-infectious form or reticulate body (RB). After entering their target eukaryotic cells, EBs differentiate into RBs, and replicate within a membrane-bound parasitophorous vacuole, termed the “inclusion”. Finally, RBs differentiate back into EBs, which are released to the extracellular medium to infect neighboring cells [7].

Chlamydia species have undergone massive genome condensation and lack in several biosynthetic pathways [8–12], suggesting that they have acquired compensatory mechanisms to allow the import and incorporation of nucleotides, amino acids, lipids and other nutrients from the host cell [13–18]. Though it is a crucial step in their pathogenesis, the molecular mechanisms used by Chlamydia species to acquire nutrients are poorly characterized largely due to the lack of a system for mutational analysis and cell-free cultivation methods.

In this review, we discuss our understanding of Chlamydia nutrient acquisition pathways, based on information from genome sequencing and new cell biological observations detailing the extensive interactions between the inclusion and host organelles.

Nutrient uptake systems

The current estimation of Chlamydia’s metabolic capacity is founded on hints from genomic sequencing. As for any living organism, Chlamydia needs pyrimidine and purine nucleotides for energy transduction and nucleic acid biosynthesis [19,20], but is unable to synthesize them de novo. Chlamydia encodes enzymes that can generate ATP via substrate level phosphorylation, and CTP from UTP through a CTP synthetase; however, these organisms still require host cell–derived ATP, GTP and UTP [10,12,14,15,21,22]. The bacteria import these nucleotides by an unusual transport system that is found only in a small number of obligate intracellular bacteria and plant plastids [23–25]. C. trachomatis has at least two nucleotide transport proteins (Npt): Npt1 and Npt2 [26]. Npt1 mediates the import of ATP from the host cell into the bacteria coupled with the export of ADP. Npt2 catalyzes the uptake of GTP, UTP, CTP and ATP, in a proton-dependent manner [26]. These transport systems are found in several Chlamydia species, Chlamydia-related amoeba symbionts, and Rickettsia [8,12,26–29]. Chlamydia species differ in their ability to metabolize nucleotides. For instance, C. muridarum harbors guaAB-add and upp genes whose predicted products enable ATP to GTP conversion and the uracil phosphoribosyl tranferase-mediated biosynthesis of UTP from uracil, respectively. C. pneumoniae encodes udk, which may mediate uridine kinase-dependent synthesis of UTP from uracil, a UMP synthetase (PyrE), and a non-functional guaAB-add operon [11,12]. C. caviae encodes an intact guaAB-add operon and pyrE [11]. Chlamydia also lacks the components for NAD⁺ synthesis, indicating that this essential molecule must be scavenged from the host as has been demonstrated in the amoeba symbiont Parachlamydia UWE25 [23].

Genomic comparisons reveal that Chlamydia has several incomplete amino acid biosynthesis pathways [8,10–12,30]. Not surprisingly, Chlamydia also contains several amino acid transporters including aat (neutral amino acid transporter), xasA (amino acid antiporter), brnQ-like (branched amino acid transporter), and a substantial number of ABC transporters (at least 13 in C. trachomatis) that are likely associated with amino acid and oligopeptide transport [8,10–12,31]. The regulation of tryptophan biosynthesis is of particular interest for Chlamydia infections. Interferon-gamma (IFN-γ) secreted by immune cells activates indoleamine 2,3-dioxygenase, which inhibits chlamydial replication by depleting intracellular pools of tryptophan [32]. The ability of different Chlamydia species to synthesize or to acquire tryptophan precursors correlates with their susceptibility to IFN-γ-mediated killing and is linked to tissue tropism [33–35]. IFN-γ inhibits the replication of C. pneumoniae, C. muridarum, and ocular strains of C. trachomatis, but has limited effect on genital strains that contain genes for converting scavenged indole tryptophan [33]. Indeed, the anti-chlamydial activity of IFN-γ in human cells can be abrogated in vitro by addition of tryptophan to the culture media [35]. Although Chlamydia has several efficient and specialized uptake systems, these systems are confined to bacterial membranes. How nutrients cross from the host cytoplasm through the inclusion membrane, which is not permissive to the diffusion of molecules > 520 Da [36], is largely unknown.

Co-option of host organelles and trafficking pathways promote delivery of nutrients to the inclusion

One potential mechanism for nutrient acquisition may involve the interception of vesicular transport intermediates. Even though the chlamydial inclusion is predominantly segregated from classical endo/lysosomal transport pathways, it can exploit membrane trafficking events [37–44] and host lipases [45] to acquire lipids. The molecular basis for these events is unclear, but a subset of Rab GTPases and SNARE proteins, which regulate membrane trafficking, are recruited to the inclusion membrane [46–50]. Indeed, recent findings indicate that interaction with multivesicular bodies, the Golgi-apparatus, mitochondria, and lipid droplets, are required for optimal chlamydial replication [51–57] and suggests that membrane fusion events between host-derived vesicles and the inclusion may deliver nutrients to Chlamydia.

Interception of exocytic vesicles

Chlamydia acquires eukaryote-specific lipids [58] from the host cell. Sphingomyelin, glucosylceramide and cholesterol are delivered to the inclusion, and subsequently incorporated into chlamydial membranes [43–45,59,60]. Cholesterol and sphingolipid transport to C. trachomatis are brefeldin A-sensitive and microtubule-dependent. Since brefeldin A inhibits anterograde vesicular traffic from the Golgi, Golgi-derived exocytic vesicles are likely involved in the delivery of these lipids to the bacteria. Furthermore, chlamydial protein synthesis is required for sphingomyelin uptake, suggesting that co-option of these vesicles is a bacteria-driven process [59]. The utilization of exocytic pathways is more pronounced when analyzing Chlamydia infection of polarized epithelial cells [40]. In these cells sphingomyelin, but not glucosylceramide, is retained by the Chlamydia inclusion and EBs due to preferential interception of basolaterally trafficked exocytic vesicles. However, the bulk protein cargo normally present within Golgi-derived secretory vesicles does not appear to accumulate in the inclusion, suggesting that a subset of vesicles may be preferentially targeted. The requirement of Golgi-derived vesicles for chlamydial replication is controversial because brefeldin A does not inhibit chlamydial replication [43]. However, RNAi screens identified COPI coat proteins as important for chlamydial growth [57,61]. The exact role of COPI in in bacterial survival, remains to be determined. It is possible that a subpopulation of brefeldin A-insensitive COPI vesicles deliver nutrients to the inclusion, or that other COPI-dependent membrane trafficking events (endosomal sorting, lipid droplet biogenesis) are required by Chlamydia. Recently, the GPI-anchored plasma membrane protein CD59 was found to traffic to the inclusion in a brefeldin A-insensitive manner, which suggests that Chlamydia also exploits Golgi-independent pathways to acquire nutrients [62].

Chlamydia-mediated reprogramming of the Golgi apparatus

The Golgi-apparatus processes and sorts newly synthesized proteins and lipids to their subcellular destinations [63]. Recent findings by Heuer and coworkers detail the importance of Golgi architecture in C. trachomatis replication [53]. At early stages of infection, Golgi ribbon-like structures of normal morphology are found closely associated with the chlamydial inclusion. These structures progressively fragment into ministacks during the course of infection and fragmentation correlates with the cleavage of the Golgi protein golgin-84. The processing of golgin-84 is sensitive to caspase and calpain inhibitors, but it is unclear if secreted bacterial proteases also contribute to this process. Nonetheless, disruption of golgin-84 processing with the caspase inhibitor,Z-WEHD-FMK impaired both bacterial replication and sphingolipid transport into the inclusion. Moreover, when Golgi-fragmentation was induced by knockdown of different Golgi matrix proteins, bacterial replication was increased. These results demonstrate that Golgi-fragmentation enhances chlamydial replication and is required for the efficient transport of sphingolipids into the inclusion.

Recruitment of Rab GTPases and SNARE proteins: subversion of vesicular trafficking

Rab GTPases are pivotal regulators of membrane trafficking processes and organelle identity [64] and SNARE proteins are key components of the intracellular membrane fusion machinery [65]. Scidmore and colleagues were the first to show that a subset Rab GTPases are recruited to the inclusion [46]. Rab1, 4 and 11 are recruited to inclusions of all chlamydial species tested, while recruitment of Rab6 is species-specific for C. trachomatis and Rab10 associates with both C. pneumoniae and C. muridarum. In addiiton, in RNAi screens several Rabs have been identified as important factors required for Chlamydia replication and development [57,61]. Recent observation indicate that this is partially due to the role of Rab GTPases in remodeling the Golgi-apparatus [54]. Depletion of Rab6 or Rab11 by RNAi inhibited Chlamydia-induced Golgi-fragmentation despite processing of golgin-84, suggesting that these Rabs act downstream of golgin-84 cleavage. Interestingly, the impairment of chlamydial replication in Rab6 or Rab11 silenced cells is reversed when Golgi-fragmentation is induced through disruption of the Golgi-tethering protein p115. These findings provide additional evidence for a functional link between Golgi-fragmentation and chlamydial replication.

How Chlamydia mediates recruitment of Rab proteins is not fully understood. A family of integral inclusion membrane proteins (Incs) are attractive candidates for bacterial factors that may facilitate interaction between Rabs and the inclusion membrane [66]. The C. trachomatis Inc CT229 interacts with Rab4 [47], and the C. pneumoniae inclusion membrane protein Cpn0585 interacts with Rabs 1, 10, and 11 [48]. These interactions are important for bacterial development as ectopic overexpression of Cpn0585 in C. pneumoniae-infected cells [48] or the microinjection of anti-CT229 in C. trachomatis-infected cells [47] negatively impact bacterial replication.

Like Rab GTPases, a group of SNARE proteins is targeted to the inclusion. Subtil and coworkers determined that the R-SNAREs Vamp3, Vamp7, and Vamp8 preferentially localize around the inclusion [49]. Through bioinformatics and structural modeling, SNARE-like motifs were identified in at least three C. trachomatis Incs (IncA, CT813 and CT223) suggesting the potential for interaction with host SNAREs. IncA interacts with Vamp3, Vamp7 and Vamp8, and SNARE recruitment to the inclusion is reduced in C. trachomatis isolates lacking IncA. Furthermore, ectopically expressed CT813 interacts with Vamp-7 and Vamp-8 in co-immunoprecipitation assays. Importantly, the SNARE-like motif of these proteins was essential for these interactions. More recently, recombinant IncA was shown to block SNARE-mediated membrane fusion in an in vitro liposome fusion assay [50]. These findings lead to the speculation that Chlamydia subverts host SNARE-mediated fusion events by expressing inhibitory SNARE-like proteins [50].

Modulation of signaling pathways

Chlamydial membranes contain lipid species that are normally associated with eukaryotic membranes [67]. Using CHO cell lines deficient in various phospholipid components, the lipid composition of chlamydial membranes was found to closely mimic that of the host [67]. The bacteria modify host-derived glycerophospholipids by replacing the straight chain fatty acid at the sn2 position with a bacteria-derived branched chain fatty acid [17]. The sn2 deacylation of phospholipids requires phospholipase A2 (PLA2) activity. Because chlamydial genomes do not encode any obvious PLA2 homologues, it is speculated that Chlamydia relies on a host PLA2 [45]. In support of this, pharmacological inhibition of the Ca²⁺-dependent cytosolic PLA2 (cPLA2) prevents Chlamydia uptake of host-derived phospholipids and severely limits replication [45]. Furthermore, cPLA2 is activated by the extracellular signal-regulated map kinase (ERK1/2) which is fully activated during chlamydial infections [45]. These results suggest that Chlamydia manipulates ERK/cPLA2 signaling pathways to facilitate the acquisition of glycerophospholipids. However, this story is likely more complex than originally proposed, as components of these signaling pathways have not been recognized as necessary for infection by RNAi-based screens. Indeed, we have found through genetic ablation experiments that cPLA2 has an innate immune signaling function in Chlamydia-infected cells, and that these processes vary between host cell species (unpublished observation).

Targeting of endosomal compartments

Multivesicular bodies (MVB) are a specialized subset of late endosomes involved in the sorting of cargo proteins and lipids to lysosomes for degradation [68,69]. By immunofluorescence and immunoelectron microscopy, two different MVB markers, CD63 and MLN64, and the lipid LBPA (lysobisphosphatidic acid, highly enriched in intra-luminal vesicles of MVBs) were found in the inclusion lumen. Inhibition of MVB biogenesis with pharmacological inhibitors or by exogenous addition of anti-CD63 antibodies impairs chlamydial replication and disrupts the traffic of sphingomyelin and cholesterol into the inclusion [51,52]. However, RNAi-mediated silencing of CD63 synthesis does not prevent MVB interactions with the inclusion [52], and MVB components have not been identified in the host genome RNAi screens [57,61]. It is likely that Golgi and MVBs partially overlap in their ability to deliver essential nutrients to replicating Chlamydia. This would explain why disruption of individual components of either pathway is not sufficient to inhibit chlamydial replication.

Co-option of lipid droplets

Lipid droplets (LD) are endoplasmic reticulum-derived organelles composed of a neutral lipid core surrounded by a phospholipid monolayer [70]. These lipid storage compartments are being increasingly recognized as dynamic organelles involved in a multiple biological processes [71]. These organelles proliferate and are recruited to the periphery of the inclusion during Chlamydia infection [55,56]. Three chlamydial proteins (Lda1 to Lda3) were identified as potentially involved in targeting LDs based on their ability to localize to LDs when expressed exogenously in mammalian cells [55]. Strikingly, LDs enter into the inclusion lumen [56]. These observations are significant because they demonstrate that intact organelles can be translocated into the inclusion and that vesicle fusion is not the only means to deliver potential nutrients into the inclusion lumen. What role these organelles play in Chlamydia biology is less clear, although pharmacological inhibition of neutral lipid biosynthesis, and thus LD biogenesis, impairs chlamydial development, suggesting a function for these organelles in Chlamydia pathogenesis [55].

Conclusions

In contrast with the numerous trafficking pathways intercepted by Chlamydiae during infection contrasts, little is known about the bacterial factors which mediate these processes. The molecular underpinnings of these complex host-pathogen interactions may be difficult to decipher without the development of a robust system for mutational analysis in Chlamydiae. Here we reviewed how some of the most recent cell biological findings present new potential mechanisms for Chlamydia acquisition of nutrients while sequestered in an intracellular vacuole (Figure 1). Like many highly adapted and successful pathogens we predict that Chlamydia has evolved to tap several redundant mechanisms to obtain nutrients. However, it should also be noted that the host cell is not a passive partner in this interaction and actively seeks to destroy the microbial invader. Therefore, the rerouting of membrane and protein transport events cannot be solely viewed as a “feeding” mechanism but also as bacterial countermeasure against innate immune functions.

Figure 1.

Schematic representation of host cell pathways exploited by Chlamydiae to acquire nutrients. (A) Golgin-84 is proteolytically processed during infection through activation of host caspases, calpains and potentially by bacterial protease(s). This leads to Golgi fragmentation into ribbon-like structures in a process mediated by Rab-6 and Rab-11. These Golgi ministacks are recruited around the inclusion. Formation of Golgi-ministacks is necessary for chlamydial uptake of host sphingolipids and replication. Golgi-derived exocytic vesicles containing sphingolipids and cholesterol fuse with the inclusion. (B) GPI-anchored plasma membrane protein CD59 is trafficked to the inner face of the inclusion in a Golgi-independent manner. (C) Infection leads to increased phosphorylation of extracellular signal-regulated map kinase (ERK) which, in turn, activates calcium-dependent cytosolic phospholipase A2 (cPLA2). cPLA2 removes straight chain fatty acids from the sn2 position of host glycerophospholipids to generate lyso-phospholipids (Lyso-PL). Chlamydia-derived branched fatty acids (b-FA) are incorporated into Lyso-PL to generate Chlamydia-modified phospholipids (Chl-PL). (D) Chlamydia targets multivesicular bodies (MVB) to sequester required nutrients like sphingolipids and cholesterol. (E) Lipid droplets, the main store of neutral lipids, are targeted by Chlamydia lipid droplet-associated proteins (Lda) and translocated into the inclusion. (F) Pan-species recruitment of Rab-1, Rab-4, and Rab-11, whereas Rab-6 and Rab-10 are recruited to C. trachomatis and C. pneumoniae inclusions, respectively. Candidate bacterial recruitment factors have been identified (Cpn0585 and CT229). (G) SNARE proteins Vamp-3, 7 and 8 are recruited to the inclusion, possibly by chlamydial proteins containing SNARE-like motifs (like incA and CT813) in the inclusion membrane.