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Chlamydial infections in humans cause severe health problems, including blinding trachoma and sexually transmitted diseases. Although the involved pathogenic mechanisms remain unclear, the ability to replicate and maintain long-term residence in the infected cells seems to significantly contribute to chlamydial pathogenicity. These obligate intracellular parasites maintain a delicate balance between exploiting and protecting their host: they occupy intracellular space and acquire nutrients from the infected cells, but at the same time they have to maintain the integrity of the host cells for the completion of their intracellular growth. For this purpose, chlamydiae hijack certain signaling pathways that prevent the host cells from undergoing apoptosis induced by intracellular stress, and protect the infected cells from recognition and attack by host defenses. Interestingly, one of the strategies that chlamydiae use for these purposes is the induction of limited proteolysis of host proteins, which is the main focus of this article.
There exist multiple chlamydial species, which have a diverse range of tissue tropisms and are involved in various disease processes 1. Chlamydia trachomatis 2 and C. pneumoniae 3 are human pathogens causing ocular and urogenital tract infections, and respiratory infection, respectively, whereas C. muridarum 3 and C. caviae 4 infect mucosal tissues of mice and guinea pigs; C. psittaci mainly affects birds but can also be transmitted to humans 5. Despite profound differences in host range, all these parasites display a remarkable similarity in their genome sequences 2-4, 6 and possess a conserved intracellular growth cycle with distinct biphasic stages (Figure 1). The infectious particle (also called elementary body or EB) can invade non-phagocytic epithelial cells via induced phagocytosis 7, 8. The EB-laden cytoplasmic vacuole (also called inclusion) migrates to the peri-Golgi region as the EB starts to differentiate into a noninfectious but metabolically active reticulate body (RB) that can undergo rapid replication. The progeny RBs eventually differentiate back to EBs for spreading to other cells. The entire intracellular growth cycle occurs within the initial inclusion that expands to occupy a large portion of the host cytoplasmic space as the parasites replicate. It takes several days for most human chlamydiae to complete a productive infection cycle in cell culture. However, infection in humans can become persistent, during which the RBs, instead of undergoing rapid replication and differentiating into infectious EBs, are limited in numbers and each becomes the so called aberrant or persistent body with enlarged size and multiple nucleoids. The aberrant body-laden inclusions may persist in the infected hosts for long periods of time 9. Intracellular survival and growth are considered major contributors to chlamydial pathogenesis 10. Some infected women, if untreated, can develop inflammatory pathologies, including pelvic inflammatory diseases, ectopic pregnancy and infertility 9, 11.
Because of their obligate intracellular lifestyle, chlamydiae have evolved strategies for manipulating host cell signaling pathways. After an EB induces its own entry into a non-phagocytic epithelial cell 7, the EB-containing vacuole diverges from the default endocytic pathway (which would lead to destruction of the microbes) and intercepts with the ER/Golgi secretory pathway 12. The availability, from the mammalian cell cytoplasm, of numerous metabolites not normally found in the external environment has provided selective pressure for chlamydiae to evolve a unique capacity to utilize host metabolic intermediates 13. Genome sequence analysis 2-4 has revealed that the microbes have not only shortened many of their biosynthetic pathways 13 but also possess numerous novel genes that might be involved in the manipulation of the host cells 14-17. Chlamydiae can take up host lipid molecules via various strategies including vesicle fusion 18, enzymatic conversion 19 and lipid droplet recruitment 20, 21. The expansion of the inclusion in the cytoplasm and the alteration of host signaling pathways during parasite acquisition of nutrients are inevitably destructive to the infected cells. Indeed, infected cells often display altered metabolic 22, immunological 23, 24 and cell biological 25, 26 characteristics. However, at the same time, the microbes have to maintain the integrity and viability of host cells before completing their own intracellular replication. To achieve this goal, chlamydiae have evolved the ability to both prevent the infected cells from undergoing apoptosis induced by intracellular stress and to protect these cells from recognition and attack by lymphocytes 14, 23, 24. Remarkably, the parasites can utilize the degradation of host proteins as a strategy to ensure both intracellular growth and evasion of host defense mechanisms, which is the main focus of this article.
In order to invade mucosal epithelial cells and to establish long-term infection in mammalian hosts, chlamydiae must overcome various host defense mechanisms belonging to both the innate and the adaptive immunity systems.
Before entering mucosal epithelial cells for the first time or spreading to neighboring cells, an infectious EB must avoid attack by various anti-microbial factors present in the mucosa, including defensins, lysozymes, complements and natural antibodies, as well as C-type lectins such as surfactant protein D 27, 28. These preexisting extracellular defense molecules might be able to inactivate most EBs during a given infection.
Once inside the host cell, chlamydiae face a different set of innate defense mechanisms. They must inhibit the fusion between the EB-containing vacuole (or phagosome) and the lysosomes, which would result in the destruction of the invader 29, and they have to acquire nutrients from the host cells 12. However, the precise mechanisms for some of these processes remain unknown.
During chlamydial invasion and intracellular growth, the host innate immunity sensors (or pattern recognition receptors, PRR) can detect the infection by recognizing microbial components (or pathogen-associated molecular patterns, PAMPs). Chlamydial PAMPs such as HSP60 and Mip are recognized by host PRR TLR4 30 and TLR2 31 respectively. The intracellular PRR NOD1 can also detect the infection, although the corresponding chlamydial ligands have not been identified 32. These PRRs, upon ligand binding, can lead to activation of various inflammatory signaling pathways including NF-κB, NF-IL-6 and MAP kinases.
In chlamydiae-infected cells, the MAP kinase ERK1/2 pathway is activated along the entire infection cycle (Figure 2). Consistent with this, it has been shown that the inhibition of this pathway can suppress chlamydial growth and acquisition of triglycerophospholipids, host cPLA2 activity, and parasite-induced cytokine production 19,33, 34. It is worth noting that many viruses can also activate host ERK1/2-mediated signaling pathways 35, 36.
Activation of the ERK1/2/cPLA2 signaling pathway can contribute to inflammation in two ways. First, as a result of cPLA2 activation, arachidonic acid (AA) release is greatly increased in infected cells 33. AA can be converted into prostaglandins, including prostaglandin E2 (PGE2). Remarkably, cyclooxygenase 2 (COX2), which catalyzes the rate-limiting step of AA conversion to prostaglandins, is up-regulated in infected cells33. PGE2 can both amplify inflammation by inducing the production of cytokines 33 and enhance chlamydial survival by suppressing IFNγ production by NK cells 37. Second, the MAP kinase ERK1/2 pathway can also participate in cytokine gene activation. In chlamydiae-infected cells, IL-8 production is dependent on ERK1/2 but not on other MAP kinases 34. The IL-8 gene activation also correlates with chlamydiae-induced nuclear localization of intracellular IL-1α 38. Nuclear IL-1α is known to promote transcription of inflammatory cytokine genes via an ancient intracellular pathway 39. The question is whether the activated ERK1/2 pathway also participates in the promotion of IL-1α nuclear localization.
Inflammatory responses are meant to strengthen host defense against infection. However, because chlamydiae hide inside the host cells, the induced inflammatory responses can not only fail to effectively clear the infection but also contribute to inflammatory pathologies 10. For example, mice deficient in caspase-1 (which is required for the maturation of IL-1β, IL-18 and IL-33) were as susceptible to chlamydial infection as wild type mice but developed significantly less severe pathologies in the upper genital tract 40.
B lymphocytes can secrete IgA antibodies directed to chlamydial antigens for neutralizing extracellular EBs 41, while T lymphocytes can detect the infected cells 42. CD4+ T cells can enhance the intracellular defense mechanisms to restrict chlamydial replication and to make the uninfected cells more resistant to infection 43. CD8+ T cells should be able to induce apoptosis of infected cells so that these cells along with the intracellular chlamydiae can be cleared 44. Various studies using animal models have shown that both the IgA secreting B cells and IFNγ-producing CD4+ TH1 T cells are the most important adaptive immunity mechanisms in controlling chlamydial infection 43 although other immune components also play some roles 45. It is worth noting that among all the T cell cytokines, IFNγ is the only one that can directly inhibit chlamydial growth 46-49.
Despite these powerful host defense mechanisms, acute chlamydial infection (if not treated) can persist in some infected hosts, leading to inflammatory pathologies including infertility and ectopic pregnancy 9, 11, 50. Thus, understanding the mechanisms of chlamydial immune evasion might provide important information for developing new therapies to attenuate these pathologies.
A bioinformatics analysis of the C. trachomatis genome identified more than two dozens of ORFs encoding proteins with potential proteolytic activity 2. Additionally, a substrate-oriented functional assay unveiled a novel serine protease, designated as chlamydial proteasome/protease-like activity factor (CPAF) 14. Besides their roles in chlamydial biology, some of these proteases might also be used to target host cell proteins since some proteins are cleaved and/or degraded in infected cells. The attacked host proteins include the transcriptional factors USF-1 23, RFX5 24, NF-κB 51, 52 and HIF-1 53, the BH3-only proteins (proapoptotic members of the Bcl-2 family) 54-56, the DNA repairing enzyme PARP (Poly-(ADP-ribose) polymerase) 57, cyclin B1 58, various cytoskeleton proteins such as keratin 8, keratin 18 and vimentin 59-61, golgin-84 62 and even cell surface proteins CD1d 63 and nectin-1 64. Degradation of these and other yet to be identified host proteins might allow chlamydiae to effectively manipulate host cell signaling pathways for both evading host defense and supporting their own intracellular growth (Table 1).
Extracellular pathogens can be easily attacked by both humoral and cellular immune effector mechanisms such as defensins or complement-mediated lysis, antibody-mediated neutralization and phagocyte-mediated digestion. However, chlamydiae as well as other successful intracellular pathogens can hide in privileged intracellular compartments to make themselves invisible to these immune mechanisms. In particular, chlamydiae restrict themselves within a cytoplasmic inclusion that neither become acidic nor fuse with host cell lysosomes 29, which makes it difficult for host MHC class II molecules to capture peptides derived from live EBs. However, the inhibition of lysosomal fusion is only restricted to vacuoles containing live EBs 29, and infected cells are still able to carry out lysosomal processing of other antigens, including dead chlamydiae. Indeed, there is a high probability for a given host cell to be infected with live EBs and simultaneously internalize chlamydial antigens when a neighboring infected cell is burst to release both live EBs and a large amount of bacterial noninfectious materials. Epitopes processed from the endocytosed noninfectious antigens can be presented to CD4+ T cells by the infected cells. In addition, chlamydial proteins secreted into the inclusion membranes and host cell cytosol by live EBs can be presented to CD8+ T cells.
Protecting the infected cells from detection by lymphocytes is essential for the parasite to safely complete intracellular replication. This is because immune recognition of the infected cells can lead to total destruction of both the host cells and the intracellular microbes. As a strategy for evading host immune recognition, chlamydiae can inhibit IFNγ-inducible MHC class II expression 23, which might affect antigen presentation to CD4+ T cells. Chlamydiae also suppress both constitutive and IFNγ-inducible MHC class I expression 24, which might reduce the recognition of the infected cells by CD8+ T cells. The parasite achieves these goals by secreting CPAF into the host cell cytoplasm, leading to degradation of the host transcription factors RFX5 and USF-1, which are required for MHC gene activation 14. Other mechanisms can also be used by the microbe to regulate the MHC antigen expression on the infected cells. For example, the infected cells were found to secrete IFNβ that is known to block IFNγ-inducible MHC class II expression 65.
Besides evading immune recognition, many intracellular pathogens have also evolved strategies for escaping immune effector mechanisms to double ensure that they can survive in their infected hosts for long periods of time. One efficient host defense mechanism is apoptosis, or programmed cell death, which plays a critical role in both innate and adaptive immunity against intracellular pathogens. It is noticeable that many viruses have evolved strategies to counteract host cell apoptosis 66.
A profound chlamydial antiapoptotic activity was first reported in 1998 67 and subsequently confirmed in many laboratories under different experimental conditions 25, 56, 68-71. Although there were also reports describing a chlamydial proapoptotic activity 72, it seems that most of the apoptotic cells detected in infected cultures were not successfully infected 73. It is worth noting that chlamydiae have been known to induce an immediate cytotoxicity when added in extremely high numbers (MOI of 50 or higher) to cell cultures 74, 75, but this is a different process from the pro- or anti-apoptotic activities detected in cultures that have been productively or persistently infected at lower MOIs. More importantly, the chlamydial antiapoptotic activity was correlated with inhibition of caspase 3 activation67, blockade of mitochondrial cytochrome c release 67, inhibition of Bax/Bak activation 76 and lack of NF-κB activation 52, 77.
Finally, recent studies have shown that the intracellular stress sensor molecules such as Puma and Bim are degraded in cells infected by chlamydiae 54, 56. These BH3-only proteins are normally sequestered in various intracellular organelles in the cytoplasm. Upon detecting overwhelming intracellular stress signals, these BH3-only proteins can migrate to mitochondria and activate the multi-domain proapoptotic Bax and Bak to overcome the inhibition of anti-apoptotic BcL-2 proteins to launch apoptosis. Interestingly, CPAF is both necessary and sufficient for degrading the BH3-only domain proteins in chlamydiae-infected cells 55. Thus, CPAF-mediated degradation of host proteins contributes to evasion of both immune recognition and effector mechanisms, making this protease a bona-fide virulence factor. However, one must realize that the CPAF-mediated mechanism cannot account for all the chlamydial antiapoptotic activity. For example, CPAF is only made and secreted at about 16 hours after infection 78, and it is not known how the parasite prevents host cell apoptosis before CPAF is secreted. It is equally unclear how the microbe inhibits the apoptosis induced by death receptors 67, since BH3-only proteins are not required for apoptosis induced by death receptors such as Fas in some cell types.
As described in a previous section, chlamydial infection can activate inflammation and induce the production of a wide variety of inflammatory cytokines, including IL-1, IL-6, IL-8 and TNFα 38, 79. Since NF-κB can activate many of these cytokine genes and often plays a dominant role in the induction of inflammation, it was expected that chlamydial infection would induce NF-κB activation. However, no significant NF-κB activation is detected in infected cells 52, 77. Instead, the microbe appears to use the MAP kinase pathway to activate inflammatory responses, probably because this is also useful for nutrient acquisition. Therefore, the question is how the parasite manages to silence the NF-κB inflammatory pathway. Examination of infected cells revealed that NF-κB p65 was cleaved into two major fragments (p40 and p20), and the chlamydial tail-specific protease (Tsp) was found to be responsible for the cleavage 52. Since the N-terminal fragment p40 maintains the ability to interact with I-κBα (a cytoplasmic inhibitor of NF-κB) and to bind to DNA but lacks transactivation ability, overexpression of the p40 might block the residual full length p65-mediated response via a dominant negative effect 52. The biological relevance of this finding was further demonstrated in an experiment showing that expressing chlamydial Tsp in mammalian cells alone can block NF-κB pathways 52.
Expansion of the chlamydial inclusion in the host cell cytoplasm is necessary to accommodate the rapid replication of the parasite. Epithelial cells, the natural target of chlamydiae, are enriched with cytoskeletal structures including the rigid intermediate filaments (IF) for maintaining cell shape. Therefore, the microbe has to overcome the host cell cytoskeletal restriction for expanding its inclusion, while at the same time maintain the integrity of the apparently fragile inclusion (note that these inclusions can quickly loss its integrity upon microinjection 80, 81). How is this accomplished? The discovery of chlamydial cleavage of cytokeratin 8 60 and other cytoskeletal proteins 59, 61 might provide partial answers to the question. The parasite can use CPAF to cleave off both head and tail domains of cytoskeletal proteins, and the residual rod domain might have a dominant negative effect on the polymerization of the residual cytoskeletal proteins; this might lead to loosing up portions of the cytoskeleton surrounding the inclusions. The residual levels of IF along with other cytoskeletal structures such as filamentous actin or stress fibers might be required for maintaining the integrity of both the infected cells and the rapidly expanding inclusions 59.
Chlamydiae can acquire sphingomyelin 82 and cholesterol 83 from the Golgi exocytic pathways, and host kinases sensitive to rottlerin inhibition appear to be required for this process 84. However, the precise mechanism on how Golgi-derived lipids are transported to the inclusion remains unclear. A recent study from Meyer's group shows that chlamydial proteases can target the Golgi apparatus to facilitate the uptake of Golgi-derived lipids 62. This finding has provided a mechanistic link between the acquisition of Golgi-derived lipids and a long standing observation that Golgi apparatus closely associates with the inclusions and forms ministacks around them 38, 82. In infected cells, golgin-84 is cleaved, which triggers Golgi fragmentation and the formation of ministacks surrounding the inclusions. The recruitment of the fragmented Golgi around inclusions seems to be necessary for both acquisition of nutrients and maturation of the microbe 62. Although it is still unknown how the fragmented Golgi is recruited, and what protease is responsible for the cleavage of golgin-84, the fact that the parasite can hijack the entire organelle via its proteolytic force re-highlights the importance of proteolysis as an strategy to manipulate host signaling pathways.
The degradation of such a broad spectrum of host proteins is likely accomplished via multiple pathways. First, it is possible that chlamydial infection could induce modification of host proteins and make the modified proteins more susceptible to host proteosomal degradation. Although this is a common viral strategy 85, 86, there is still lack of biochemical evidence for chlamydiae to use such a pathway. Second, infection could activate host proteolytic systems that are normally silenced in uninfected cells. This is definitely a testable hypothesis in need to be pursued. Third, the chlamydial genome appears to encode several dozens of predicted proteases 2, many of which might participate in the degradation of host proteins. However, so far only Tsp and CPAF have been demonstrated to be able to cleave host proteins. Finally, only CPAF has been shown to target multiple host proteins, suggesting that this protease might play a dominant role in the process. The recently unveiled crystal structure of CPAF suggests that it might have a broad substrate specificity, since the contacts between a putative substrate peptide and the active site are mainly via hydrophobic interactions 87, which might enable CPAF to cleave many different types of proteins. However, not all accessible host proteins are degraded by CPAF in infected cells 23. How does this protease recognize and attack certain proteins and not others? To address this question we need to understand how CPAF is activated and secreted in the infected cells.
Since degradation of host proteins, due to either direct attack by chlamydial proteases or by activation of host proteolytic systems, can occur both inside cells during infection and outside cells during sample processing (after cell lysis), it is extremely important to distinguish between intracellular and extracellular proteolysis. Obviously, only the degradation occurring inside infected cells would be biologically relevant to the study of the mechanisms of host protein degradation by chlamydiae. Thus, caution should be taken to block all proteolytic activities when harvesting and analyzing cellular samples.
The extensive cleavage of host proteins in infected cells has inevitably alerted us to question the fate of the degradation products. Host protein degradation might be intended to (i) enrich the nutritional supply in the infected cell cytosol for chlamydial uptake or (ii) alter host cell signaling pathways for protecting the intracellular parasite. The question boils down to which is more important for chlamydial survival: food or war. Without a lengthy debate on the rather philosophic question, one can hypothesize that the degradation products might effectively serve as nutrients. This hypothesis is supported by the fact that the chlamydial genome appears to encode multiple amino acid and oligopeptide transporters 2, which might equip the microbe with the tools for taking in nutrients and building blocks from host cells. Although chlamydial proteases might not directly generate oligo-peptides and single amino acids from host proteins, the host proteosomal systems might be able to do the follow-up job by chewing up the damaged proteins. Furthermore, most chlamydiae-targeted proteins appear to be constitutively expressed in the host cells. It makes more sense to target preexisting and abundant proteins for the purpose of generating food. It would be much more efficient to target less abundant and transiently expressed proteins for the purpose of inactivating the targets' function. Interestingly, Protochlamydia amoebophila UWE25, a member of the phylum Chlamydiae and obligate intracellular symbiont of a free-living amoeba, also encodes a CPAF homolog 88. Since there is no need to protect the infected amoeba from lymphocyte detection and attack, one can speculate that the protochlamydial CPAF might help UWE25 to fight off the amoeba's digestive power and to generate nutrients for UWE25. Finally, the extent of host protein degradation seems to correlate with the speed of chlamydial growth. In most cases, host protein degradation reaches the highest level at middle cycle when parasite biosynthesis and replication peak. Whether host protein-derived amino acids can indeed end up in chlamydial proteins requires experimental demonstration. The degradation of host proteins might even allow the endoparasite to literally eat its way out since chlamydiae-mediated lysis of host cells is also dependant on a proteolytic activity 89. It will be interesting to test whether CPAF plays any role in chlamydial exiting and spreading.
In summary, chlamydiae appear to use a basic strategy (degradation of host proteins) for ensuring its own intracellular replication while maintaining the integrity of the infected host cells for long periods of time. However, emphasizing the role of host protein degradation does not in any way mean that this is the only major chlamydial strategy for immune evasion. On the contrary, the parasite has probably evolved other strategies, most of which we still do not know. Further research on the biology of these microorganisms will likely unveil new mechanisms and, hopefully, will result in novel therapeutic approaches to treat chlamydial pathologies.
This work was supported in part by grants from the US National Institutes of Health.
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