Rottlerin-Mediated Inhibition of Chlamydia trachomatis Growth and Uptake of Sphingolipids Is Independent of p38-Regulated/Activated Protein Kinase (PRAK)

We previously found that rottlerin, a plant-derived small molecule compound, profoundly inhibited Chlamydia trachomatis growth and blocked sphingolipid trafficking from host cell Golgi into chlamydial inclusions. Since the p38-regulated/activated protein kinase (PRAK) is a known target of rottlerin and is activated in Chlamydia trachomatis-infected cells, we investigated the potential role of this kinase in rottlerin-mediated anti-chlamydial activity. However, we found that a PRAK-specific inhibitor failed to inhibit chlamydial growth, suggesting that the kinase activity of PRAK may not be required for chlamydial intracellular replication. This conclusion was supported by the observation that chlamydial organisms replicated equally well in mouse embryonic fibroblast cells with or without PRAK. Moreover, neither the PRAK inhibitor nor PRAK deficiency altered host sphingolipid trafficking into chlamydial inclusions. Finally, rottlerin maintained its anti-chlamydial activity in PRAK-deficient cells. Together, these observations have demonstrated that PRAK is not required for either the rottlerin-mediated anti-chlamydial activity or rottlerin inhibition of sphingolipid trafficking, suggesting that rottlerin may achieve its inhibitory role by targeting other host factors.     

A Mathematical Model of Chlamydial Infection Incorporating Movement of Chlamydial Particles

We present a spatiotemporal mathematical model of chlamydial infection, host immune response, and movement of infectious particles. The resulting partial differential equations model both the dynamics of the infection and changes in infection profile observed spatially along the length of the host genital tract. This model advances previous Chlamydia modelling by incorporating spatial change. Numerical solutions and model analysis are carried out, and we present a hypothesis regarding the potential for treatment and prevention of infection by increasing chlamydial particle motility.     

CPAF: A Chlamydial Protease in Search of an Authentic Substrate

Bacteria in the genus Chlamydia are major human pathogens that cause an intracellular infection. A chlamydial protease, CPAF, has been proposed as an important virulence factor that cleaves or degrades at least 16 host proteins, thereby altering multiple cellular processes. We examined 11 published CPAF substrates and found that there was no detectable proteolysis when CPAF activity was inhibited during cell processing. We show that the reported proteolysis of these putative CPAF substrates was due to enzymatic activity in cell lysates rather than in intact cells. Nevertheless, Chlamydia-infected cells displayed Chlamydia-host interactions, such as Golgi reorganization, apoptosis resistance, and host cytoskeletal remodeling, that have been attributed to CPAF-dependent proteolysis of host proteins. Our findings suggest that other mechanisms may be responsible for these Chlamydia-host interactions, and raise concerns about all published CPAF substrates and the proposed roles of CPAF in chlamydial pathogenesis.     

Golgi Fragmentation and Sphingomyelin Transport to Chlamydia trachomatis during Penicillin-Induced Persistence Do Not Depend on the Cytosolic Presence of the Chlamydial Protease CPAF

Chlamydia grows inside a cytosolic vacuole (the inclusion) that is supplied with nutrients by the host through vesicular and non-vesicular transport. It is unclear in many respects how Chlamydia organizes this transport. One model posits that the Chlamydia-induced fragmentation of the Golgi-apparatus is required for normal transport processes to the inclusion and for chlamydial development, and the chlamydial protease CPAF has been controversially implicated in Golgi-fragmentation. We here use a model of penicillin-induced persistence of infection with Chlamydia trachomatis to test this link. Under penicillin-treatment the inclusion grew in size for the first 24 h but after that growth was severely reduced. Penicillin did not reduce the number of infected cells with fragmented Golgi-apparatus, and normal Golgi-fragmentation was found in a CPAF-deficient mutant. Surprisingly, sphingomyelin transport into the inclusion and into the bacteria, as measured by fluorescence accumulation upon addition of labelled ceramide, was not reduced during penicillin-treatment. Thus, both Golgi-fragmentation and transport of sphingomyelin to C. trachomatis still occurred in this model of persistence. The portion of cells in which CPAF was detected in the cytosol, either by immunofluorescence or by immune-electron microscopy, was drastically reduced in cells cultured in the presence of penicillin. These data argue against an essential role of cytosolic CPAF for Golgi-fragmentation or for sphingomyelin transport in chlamydial infection.     

Imbalanced Oxidative Stress Causes Chlamydial Persistence during Non-Productive Human Herpes Virus Co-Infection

Both human herpes viruses and Chlamydia are highly prevalent in the human population and are detected together in different human disorders. Here, we demonstrate that co-infection with human herpes virus 6 (HHV6) interferes with the developmental cycle of C. trachomatis and induces persistence. Induction of chlamydial persistence by HHV6 is independent of productive virus infection, but requires the interaction and uptake of the virus by the host cell. On the other hand, viral uptake is strongly promoted under co-infection conditions. Host cell glutathione reductase activity was suppressed by HHV6 causing NADPH accumulation, decreased formation of reduced glutathione and increased oxidative stress. Prevention of oxidative stress restored infectivity of Chlamydia after HHV6-induced persistence. We show that co-infection with Herpes simplex virus 1 or human Cytomegalovirus also induces chlamydial persistence by a similar mechanism suggesting that Chlamydia -human herpes virus co-infections are evolutionary shaped interactions with a thus far unrecognized broad significance.     

Interleukin-22 Promotes T Helper 1 (Th1)/Th17 Immunity in Chlamydial Lung Infection

The role of interleukin-22 (IL-22) in intracellular bacterial infections is a controversial issue, although the contribution of this cytokine to host defense against extracellular bacterial pathogens has been well established. In this study, we focused on an intra-cellular bacterium, Chlamydia, and evaluated the production and function of IL-22 in host defense against chlamydial lung infection using a mouse model. We found that Chlamydia muridarum infection elicited quick IL-22 responses in the lung, which increased during infection and were reduced when bacterial loads decreased. More importantly, blockade of endogenous IL-22 using neutralizing anti-IL-22 monoclonal antibodies (mAb) resulted in more severe disease in the mice, leading to significantly higher weight loss and bacterial growth and much more severe pathological changes than treatment with isotype control antibody. Immunological analyses identified significantly lower T helper 1 (Th1) and Th17 responses in the IL-22–neutralized mice. In contrast, intranasal administration of exogenous IL-22 significantly enhanced protection following chlamydial lung infection, which was associated with a significant increase of Th17 response. The data demonstrate that IL-22 is a critical cytokine, mediating host defense against chlamydial lung infection and coordinating the function of distinct Th-cell subsets, particularly Th1 and Th17, in the process.     

EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.     

Benzylidene Acylhydrazides Inhibit Chlamydial Growth in a Type III Secretion- and Iron Chelation-Independent Manner

Chlamydiae are widespread Gram-negative pathogens of humans and animals. Salicylidene acylhydrazides, developed as inhibitors of type III secretion system (T3SS) in Yersinia spp., have an inhibitory effect on chlamydial infection. However, these inhibitors also have the capacity to chelate iron, and it is possible that their antichlamydial effects are caused by iron starvation. Therefore, we have explored the modification of salicylidene acylhydrazides with the goal to uncouple the antichlamydial effect from iron starvation. We discovered that benzylidene acylhydrazides, which cannot chelate iron, inhibit chlamydial growth. Biochemical and genetic analyses suggest that the derivative compounds inhibit chlamydiae through a T3SS-independent mechanism. Four single nucleotide polymorphisms were identified in a Chlamydia muridarum variant resistant to benzylidene acylhydrazides, but it may be necessary to segregate the mutations to differentiate their roles in the resistance phenotype. Benzylidene acylhydrazides are well tolerated by host cells and probiotic vaginal Lactobacillus species and are therefore of potential therapeutic value.     

Water-Filtered Infrared A Irradiation in Combination with Visible Light Inhibits Acute Chlamydial Infection

New therapeutic strategies are needed to overcome drawbacks in treatment of infections with intracellular bacteria. Chlamydiaceae are Gram-negative bacteria implicated in acute and chronic diseases such as abortion in animals and trachoma in humans. Water-filtered infrared A (wIRA) is short wavelength infrared radiation with a spectrum ranging from 780 to 1400 nm. In clinical settings, wIRA alone and in combination with visible light (VIS) has proven its efficacy in acute and chronic wound healing processes. This is the first study to demonstrate that wIRA irradiation combined with VIS (wIRA/VIS) diminishes recovery of infectious elementary bodies (EBs) of both intra- and extracellular Chlamydia (C.) in two different cell lines (Vero, HeLa) regardless of the chlamydial strain (C. pecorum, C. trachomatis serovar E) as shown by indirect immunofluorescence and titration by subpassage. Moreover, a single exposure to wIRA/VIS at 40 hours post infection (hpi) led to a significant reduction of C. pecorum inclusion frequency in Vero cells and C. trachomatis in HeLa cells, respectively. A triple dose of irradiation (24, 36, 40 hpi) during the course of C. trachomatis infection further reduced chlamydial inclusion frequency in HeLa cells without inducing the chlamydial persistence/stress response, as ascertained by electron microscopy. Irradiation of host cells (HeLa, Vero) neither affected cell viability nor induced any molecular markers of cytotoxicity as investigated by Alamar blue assay and Western blot analysis. Chlamydial infection, irradiation, and the combination of both showed a similar release pattern of a subset of pro-inflammatory cytokines (MIF/GIF, Serpin E1, RANTES, IL-6, IL-8) and chemokines (IL-16, IP-10, ENA-78, MIG, MIP-1α/β) from host cells. Initial investigation into the mechanism indicated possible thermal effects on Chlamydia due to irradiation. In summary, we demonstrate a non-chemical reduction of chlamydial infection using the combination of water-filtered infrared A and visible light.     

The novel chlamydial adhesin CPn0473 mediates the lipid raft‐dependent uptake of Chlamydia pneumoniae

Chlamydiae are Gram‐negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down‐stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473‐coupled fluorescent latex beads adhere to human epithelial HEp‐2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp‐2 cells with rCPn0473 does not attenuate adhesion but promotes dose‐dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473‐dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae.     

Inclusion Biogenesis and Reactivation of Persistent Chlamydia trachomatis Requires Host Cell Sphingolipid Biosynthesis

Chlamydiae are obligate intracellular pathogens that must coordinate the acquisition of host cell-derived biosynthetic constituents essential for bacterial survival. Purified chlamydiae contain several lipids that are typically found in eukaryotes, implying the translocation of host cell lipids to the chlamydial vacuole. Acquisition and incorporation of sphingomyelin occurs subsequent to transport from Golgi-derived exocytic vesicles, with possible intermediate transport through endosomal multivesicular bodies. Eukaryotic host cell-derived sphingomyelin is essential for intracellular growth of Chlamydia trachomatis, but the precise role of this lipid in development has not been delineated. The present study identifies specific phenotypic effects on inclusion membrane biogenesis and stability consequent to conditions of sphingomyelin deficiency. Culturing infected cells in the presence of inhibitors of serine palmitoyltransferase, the first enzyme in the biosynthetic pathway of host cell sphingomyelin, resulted in loss of inclusion membrane integrity with subsequent disruption in normal chlamydial inclusion development. Surprisingly, this was accompanied by premature redifferentiation to and release of infectious elementary bodies. Homotypic fusion of inclusions was also disrupted under conditions of sphingolipid deficiency. In addition, host cell sphingomyelin synthesis was essential for inclusion membrane stability and expansion that is vital to reactivation of persistent chlamydial infection. The present study implicates both the Golgi apparatus and multivesicular bodies as key sources of host-derived lipids, with multivesicular bodies being essential for normal inclusion development and reactivation of persistent C. trachomatis infection.     

Chlamydia-secreted protease CPAF degrades host antimicrobial peptides

Chlamydia trachomatis infection in the lower genital tract, if untreated, can ascend to the upper genital tract, potentially leading to complications such as tubal factor infertility. The ascension involves cell-to-cell spreading, which may require C. trachomatis organisms to overcome mucosal extracellular effectors such as antimicrobial peptides. We found that among the 8 antimicrobial peptides tested, the cathelicidin LL-37 that is produced by both urogenital epithelial cells and the recruited neutrophils possessed a most potent antichlamydial activity. Interestingly, this antichlamydial activity was completely inhibited by CPAF, a C. trachomatis-secreted serine protease. The inhibition was dependent on CPAF's proteolytic activity. CPAF selectively degraded LL-37 and other antimicrobial peptides with an antichlamydial activity. CPAF is known to secrete into and accumulate in the infected host cell cytoplasm at the late stage of chlamydial intracellular growth and may be released to confront the extracellular antimicrobial peptides before the intra-inclusion organisms are exposed to extracellular environments during host cell lysis and chlamydial spreading. Thus, the finding that CPAF selectively targets host antimicrobial peptides that possess antichlamydial activities for proteolysis suggests that CPAF may contribute to C. trachomatis pathogenicity by aiding in ascending infection.     

Effect of 6-thioguanine on Chlamydia trachomatis growth in wild-type and hypoxanthine-guanine phosphoribosyltransferase-deficient cells.

Chlamydiae have evolved a biphasic life cycle to facilitate their survival in two discontinuous habitats. The unique growth cycle is represented by two alternating forms of the organism, the elementary body and the reticulate body. Chlamydiae have an absolute nutritional dependency on the host cell to provide ribonucleoside triphosphates and other essential intermediates of metabolism. This report describes the pleiotropic effects of the purine antimetabolite 6-thioguanine on chlamydial replication. In order to display cytotoxicity, 6-thioguanine must first be converted to the nucleotide level by the host cell enzyme hypoxanthine-guanine phosphoribosyltransferase. Our results show that 6-thioguanine is an effective inhibitor of chlamydial growth with either wild-type or hypoxanthine-guanine phosphoribosyltransferase-deficient cell lines as the host. Interestingly, the mechanism of 6-thioguanine-induced inhibition of chlamydial growth is different depending on which cell line is used. With wild-type cells as the host, the cytotoxic effects of 6-thioguanine on chlamydial growth are relatively fast and irreversible. Under these circumstances, cytotoxicity likely results from the combined effect of starving chlamydiae for purine ribonucleotides and incorporation of host-derived 6-thioguanine-containing nucleotides into chlamydial nucleic acids. With hypoxanthine-guanine phosphoribosyltransferase-deficient cells as the host, 6-thioguanine must be present at the start of the chlamydial infection cycle to be effective and the growth inhibition is reversible upon removal of the antimetabolite. These findings suggest that in hypoxanthine-guanine phosphoribosyltransferase-deficient cells, the free base 6-thioguanine may inhibit the differentiation of elementary bodies to reticulate bodies. With hypoxanthine-guanine phosphoribosyltransferase-deficient cells as the host, 6-thioguanine was used as a selective agent in culture to isolate a Chlamydia trachomatis isolate resistant to the effects of the drug. This drug resistant C. trachomatis isolate was completely resistant to 6-thioguanine in hypoxanthine-guanine phosphoribosyltransferase-deficient cells; however, it displayed wildtype sensitivity to 6-thioguanine when cultured in wild-type host cells.     

Chlamydial infection induces host cytokinesis failure at abscission

Chlamydia trachomatis is an obligate intracellular bacteria and the infectious agent responsible for the sexually transmitted disease Chlamydia. Infection with Chlamydia can lead to serious health sequelae such as pelvic inflammatory disease and reproductive tract scarring contributing to infertility and ectopic pregnancies. Additionally, chlamydial infections have been epidemiologically linked to cervical cancer in patients with a prior human papilomavirus (HPV) infection. Chlamydial infection of cultured cells causes multinucleation, a potential pathway for chromosomal instability. Two mechanisms that are known to initiate multinucleation are cell fusion and cytokinesis failure. This study demonstrates that multinucleation of the host cell by Chlamydia is entirely due to cytokinesis failure. Moreover, cytokinesis failure is due in part to the chlamydial effector CPAF acting as an anaphase promoting complex mimic causing cells to exit mitosis with unaligned and unattached chromosomes. These lagging and missegregated chromosomes inhibit cytokinesis by blocking abscission, the final stage of cytokinesis.     

Immune Control of Chlamydial Growth in the Human Epithelial Cell Line RT4 Involves Multiple Mechanisms That Include Nitric Oxide Induction,Tryptophan Catabolism and Iron Deprivation

The antimicrobial activity of T cell-derived cytokines, especially interferon (IFN)-γ, against intracellular pathogens, such as Chlamydia trachomatis, involves the induction of 3 major biochemical processes: tryptophan catabolism, nitric oxide (NO) induction and intracellular iron (Fe) deprivation. Since the epithelial cell is the natural target of chlamydial infection, the presence of these antimicrobial systems in the cell would suggest that they may be involved in T cell control of intracellular multiplication of Chlamydia. However, the controversy over whether these 3 antimicrobial processes are present in both mice and humans has precluded the assessment of the relative contribution of each of the 3 mechanisms to chlamydial inhibition in the same epithelial cell from either mice or humans. In the present study, we identified a Chlamydia-susceptible human epithelial cell line, RT4, that possesses the 3 antimicrobial systems, and we examined the role of nitric oxide (NO) induction, and deprivation of tryptophan or Fe in cytokine-induced inhibition of chlamydiae. It was found that the 3 antimicrobial systems contributed to cytokine-mediated inhibition of the intracellular growth of Chlamydia. NO induction accounted for ~20% of the growth inhibition; tryptophan catabolism contributed approximately 30%; iron deprivation was least effective; but the combination of the 3 systems accounted for greater than 60% of the inhibition observed. These results indicate that immune control of chlamydial growth in human epithelial cells may involve multiple mechanisms that include NO induction, tryptophan catabolism and Fe deprivation.     

Immunobiological Outcomes of Repeated Chlamydial Infection from Two Models of Within-Host Population Dynamics

Background

Chlamydia trachomatis is a common human pathogen that mediates disease processes capable of inflicting serious complications on reproduction. Aggressive inflammatory immune responses are thought to not only direct a person''s level of immunity but also the potential for immunopathology. With human immunobiology being debated as a cause of prevailing epidemiological trends, we examined some fundamental issues regarding susceptibility to multiple chlamydial infections that could have implications for infection spread. We argue that, compared to less-frequent exposure, frequent exposure to chlamydia may well produce unique immunobiological characteristics that likely to have important clinical and epidemiological implications.

Methods and Results

As a novel tool for studying chlamydia, we applied principles of modeling within-host pathogen dynamics to enable an understanding of some fundamental characteristics of an individual''s immunobiology during multiple chlamydial infections. While the models were able to reproduce shorter-term infection kinetics of primary and secondary infections previously observed in animal models, it was also observed that longer periods between initial and second infection may increase an individual''s chlamydial load and lengthen their duration of infectiousness. The cessation of short-term repeated exposure did not allow for the formation of long-lasting immunity. However, frequent re-exposure non-intuitively linked the formation of protective immunity, persistent infection, and the potential for immunopathology.

Conclusions

Overall, these results provide interesting insights that should be verified with continued study. Nevertheless, these results appear to raise challenges for current evidence of the development of long-lasting immunity against chlamydia, and suggest the existence of a previously unidentified mechanism for the formation of persistent infection. The obvious next goal is to investigate the qualitative impact of these results on the spread of chlamydia.     

Chlamydial envelope components and pathogen-host cell interactions

Few bacterial pathogens are as widespread in nature or as capable of eliciting such a diversity of disease syndromes as are the chlamydiae. As obligate intra-cellular organisms, they pose a special research challenge in defining the molecular components and mechanisms for productive growth within host cells and the overall progress of infection throughout host tissue. Although a comprehensive view of chlamydial envelope composition and respective functions in pathogenesis is far from complete, ongoing investigations continue to expose new and intriguing avenues for exploration.     

Chlamydial Lung Infection Induces Transient IL-9 Production Which Is Redundant for Host Defense against Primary Infection

IL-9/Th9 responses are recently found to be important for innate and adaptive immunity particularly in parasitic infections. To date, the study on the role of IL-9 in bacterial infections is limited and the reported data are contradictory. One reported function of IL-9/Th9 is to modulate Th1/Th17 responses. Since our and others’ previous work has shown a critical role of Th1 and Th17 cells in host defense against chlamydial lung infection, we here examined the role of IL-9 responses in Chlamydia muridarum (Cm) lung infection, particularly its effect on Th1 and Th17 responses and outcome infection. Our data showed quick but transient IL-9 production in the lung following infection, peaking at day 3 and back to baseline around day 7. CD4+ T cell was the major source of IL-9 production in the lung infection. Blockade of endogenous IL-9 using neutralizing antibody failed to change Interferon-γ (IFN-γ) and IL-17 production by cultured spleen mononuclear cells isolated from Cm infected mice. Similarly, in vivo neutralization of IL-9 failed to show significant effect on T cell (Th1 and Th17) and antibody responses (IgA, IgG1 and IgG2a). Consistently, the neutralization of IL-9 had no significant effect on disease process, including body weight change, bacterial burden and histopathological score. The data suggest that IL-9 production following chlamydial lung infection is redundant for host defense against the intracellular bacteria.