Robert E. Molestina

Activation of NF-κB by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated IκB to the parasitophorous vacuole membrane

Mammalian cells infected with Toxoplasma gondii are resistant to apoptosis induced by a variety of stimuli. We have demonstrated that the host transcription factor NF-κB plays a pivotal role in the T.-gondii-mediated blockade of apoptosis because inhibition is lost in cells lacking the p65 (RelA) subunit of NF-κB (p65–/–). In the present study, we examined the effects of T. gondii infection on NF-κB activation and the expression of genes involved in the apoptotic cascade. Infection of wild-type mouse embryonic fibroblasts (MEFs) with T.-gondii-induced nuclear translocation of the p50 and p65 subunits of NF-κB as examined by immunoblotting of nuclear extracts, immunofluorescence and electrophoretic mobility shift assays. A comparison of apoptotic gene expression profiles from wild-type and p65–/– MEFs revealed distinct patterns of induction in response to T. gondii infection. In particular, the differences seen in the Bcl-2 and IAP families are consistent with the antiapoptotic responses observed in the resistant wild-type cells compared with the sensitive p65–/– fibroblasts. Consistent with NF-κB activation, T. gondii infection promoted phosphorylation of the inhibitor IκB. Interestingly, phosphorylated IκB was concentrated on the parasitophorous vacuole membrane (PVM), suggesting a parasite-directed event. Results from this study suggest that activation of NF-κB plays an important role in stimulation of antiapoptotic gene expression by T. gondii. Furthermore, recruitment of phosphorylated IκB to the PVM implies the presence of intrinsic factor(s) in T. gondii that might be used to manipulate the NF-κB signaling pathway in the host to elicit a survival response during infection.

Inhibition of caspase activation and a requirement for NF-κB function in the Toxoplasma gondii-mediated blockade of host apoptosis

Mammalian cells infected with the protozoan parasite Toxoplasma gondii are resistant to many apoptotic stimuli transmitted along both the mitochondrial and death receptor pathways. Apoptosis, and its inhibition in infected cells, was examined using multiple morphological, molecular and biochemical approaches. The data strongly indicate manipulation of the host apoptotic machinery at multiple levels, focusing on the inhibition of host caspases. Activation of the pro-apoptotic caspase family of proteases is a biochemical hallmark of apoptosis. Caspase activation occurs in a highly ordered cascade triggered by the initiator caspases 8 and 9, which activate the executioner caspase, caspase 3. Our findings indicate a profound blockade of caspase activation and activity as the molecular basis for the inhibition of apoptosis in T.-gondii-infected cells. Caspase inhibition was demonstrated using multiple intrinsic and synthetic substrates. Although the specific inhibitory molecule remains to be identified, data indicate an absolute requirement for the host transcription factor NF-κB and, by extension, genes regulated by it. We propose that T. gondii activates the host survival response, thereby increasing the overall resistance of infected cells to apoptotic stimuli.

Proteomic Analysis of Differentially Expressed Chlamydia pneumoniae Genes during Persistent Infection of HEp-2 Cells

ABSTRACT Recent data have shown that the respiratory pathogen Chlamydia pneumoniae expresses an altered gene transcription profile during gamma interferon (IFN-γ)-induced persistent infection in vitro. In the present study, we examined, by proteomics, expression of C. pneumoniae proteins labeled intracellularly with [35S]methionine/cysteine under normal conditions or IFN-γ-mediated persistence. The identity of differentially expressed proteins during persistent infection was determined by matching spots to those of proteins identified in C. pneumoniae elementary bodies by matrix-assisted laser desorption ionization mass spectrometry. Upon treatment with 50 U of IFN-γ per ml, a marked upregulation of major outer membrane protein (MOMP), heat shock protein 60 (Hsp-60/GroEL), and proteins with functions in DNA replication (GyrA), transcription (RpoA, PnP), translation (Rrf), glycolysis (PgK, GlgP), and type III secretion (SctN) was observed at 24 h of infection. In contrast, no significant decreases in bacterial protein expression were found in C. pneumoniae-infected cells due to IFN-γ treatment. Upregulation of C. pneumoniae proteins involved in diverse functions during persistent infection may allow the organism to resist the inhibitory effects of IFN-γ while retaining basic functions. Future studies should examine the differential expression of chlamydial proteins during the developmental cycle under IFN-γ pressure to obtain a finer representation of the gene products involved in establishing persistence.

Characterization of Chlamydia pneumoniae persistence in HEp-2 cells treated with gamma interferon.

ABSTRACT Infection with Chlamydia pneumoniae has been implicated as a potential risk factor for atherosclerosis. This study demonstrated the effects of gamma interferon (IFN-γ)-mediated indoleamine 2,3-dioxygenase activity on C. pneumoniaepersistence in HEp-2 cells, inclusion morphology, and ultrastructure.C. pneumoniae replication showed a dose-dependent decrease when treated with increasing concentrations of IFN-γ and a phenotypic switch resulting in a decrease in typical inclusions with an increase in smaller, less-dense atypical inclusions. Ultrastructural analysis of IFN-γ-treated C. pneumoniae revealed atypical inclusions containing large reticulatate-like aberrant bodies with no evidence of redifferentiation into elementary bodies.

Inhibition of Chlamydia pneumoniae replication in human aortic smooth muscle cells by gamma interferon-induced indoleamine 2, 3-dioxygenase activity

ABSTRACT Infection with Chlamydia pneumoniae, a human respiratory pathogen, has been implicated as a potential risk factor in atherosclerosis, possibly because the pathogen can exist in a persistent form similar to that described for Chlamydia trachomatis. The present study investigated whether gamma interferon (IFN-γ) can induce indoleamine 2,3-dioxygenase (IDO) activity in aortic smooth muscle cells, leading to a marked inhibition of C. pneumoniae growth. Our data indicate a stimulation of IDO mRNA expression and dose-dependent enzymatic activity following IFN-γ treatment. IDO-mediated increase in tryptophan catabolism resulted in a dose-dependent marked inhibition of C. pneumoniae replication.

Detection of a novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkappaBalpha.

Toxoplasma gondii activates the NF‐κB pathway in the infected host cell resulting in upregulation of pro‐survival genes and prevention of apoptosis. Manipulation of the NF‐κB cascade by T. gondii correlates with the localization of phosphorylated IκB at the parasitophorous vacuole membrane (PVM). This suggests a parasite‐mediated event, involving the recruitment and activation of the host IκB kinase (IKK) complex, as has been observed with the related protozoan Theileria parva. In contrast to Theileria, confocal microscopy studies showed no apparent hijacking of IKKα, IKKβ, or their activated phosphorylated forms at the T. gondii PVM. Remarkably, phosphorylation of IκBα at Ser 32/36 was observed at the PVM of T. gondii‐infected IKKα–/–, IKKβ–/– and IKKα/β double‐knockout (IKKα/β–/–) fibroblasts, suggesting the involvement of a parasite kinase activity independent of host IKK. The presence of a putative T. gondii IκB kinase was examined by in vitro kinase assays using GST–IκBα constructs and protein extracts from both extracellular parasites and PVM fractions. Interestingly, an activity capable of phosphorylating IκBα at the critical Ser 32/36 sites was identified in parasite extracts, a property restricted to the IKK signalosome. Taken together, our data support the role for a T. gondii kinase involved in phosphorylation of host cell IκBα and suggest an unusual mechanism utilized by an intracellular pathogen capable of manipulating the NF‐κB pathway.

Host and parasite-derived IKK activities direct distinct temporal phases of NF-κB activation and target gene expression following Toxoplasma gondii infection

Activation of NF-κB by the intracellular pathogen Toxoplasma gondii is associated with the localization of phosphorylated IκBα to the parasitophorous vacuole membrane (PVM). This is mediated by a parasite-derived IκB kinase (TgIKK) activity and is independent of host IKK function. In the present study, we examined the roles of host IKK and parasite-derived TgIKK on the temporal modulation of NF-κB activation. Despite the presence of TgIKK activity at the PVM, nuclear translocation of NF-κB and subsequent gene expression exhibited a requirement for the host IKK complex. A detailed kinetic analysis of NF-κB activation revealed a biphasic, hierarchical and temporally regulated response. We propose a novel paradigm for the modulation of NF-κB-dependent gene expression by T. gondii that involves both the host IKK complex and TgIKK activity at different phases of infection. Thus, T. gondii effectively alters gene expression in a temporal dimension by exploiting the NF-κB signaling machinery and subsequently rewiring the activation circuits of the infected host cell.

Infection with Toxoplasma gondii results in dysregulation of the host cell cycle

Mammalian cells infected with Toxoplasma gondii are characterized by a profound reprogramming of gene expression. We examined whether such transcriptional responses were linked to changes in the cell cycle of the host. Human foreskin fibroblasts (HFFs) in the G0/G1 phase of the cell cycle were infected with T. gondii and FACS analysis of DNA content was performed. Cell cycle profiles revealed a promotion into the S phase followed by an arrest towards the G2/M boundary with infection. This response was markedly different from that of growth factor stimulation which caused cell cycle entry and completion. Transcriptional profiles of T. gondii‐infected HFF showed sustained increases in transcripts associated with a G1/S transition and DNA synthesis coupled to an abrogation of cell cycle regulators critical in G2/M transition relative to growth factor stimulation. These divergent responses correlated with a distinct temporal modulation of the critical cell cycle regulator kinase ERK by infection. While the kinetics of ERK phosphorylation by EGF showed rapid and sustained activation, infected cells displayed an oscillatory pattern of activation. Our results suggest that T. gondii infection induces and maintains a ‘proliferation response’ in the infected cell which may fulfill critical growth requirements of the parasite during intracellular residence.

Effects of Fluoroquinolones on the Migration of Human Phagocytes through Chlamydia pneumoniae-Infected and Tumor Necrosis Factor Alpha-Stimulated Endothelial Cells

ABSTRACT The anti-inflammatory activities of three quinolones, levofloxacin, moxifloxacin, and gatifloxacin, were investigated with an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVEC) were seeded in Transwell inserts, treated with serial dilutions of antibiotics, infected with Chlamydia pneumoniae, or stimulated with tumor necrosis factor alpha (TNF-α). Neutrophils or monocytes were also preincubated with serial dilutions of each antibiotic. TEM was assessed by light microscopic examination of the underside of the polycarbonate membrane, and levels of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. In HUVEC infected with C. pneumoniae or stimulated with TNF-α, all fluoroquinolones significantly decreased neutrophil and monocyte TEM, compared to antibiotic-free controls. Moxifloxacin and gatifloxacin produced a significant decrease in IL-8 in C. pneumoniae-infected and TNF-α-stimulated HUVEC; however, moxifloxacin was the only fluoroquinolone that produced a significant decrease in MCP-1 levels under both conditions. Results from this study indicate similarities in the anti-inflammatory activities of these fluoroquinolones, although no statistically significant decrease in chemokine secretion was observed when levofloxacin was used. Mechanisms of neutrophil and monocyte TEM inhibition by fluoroquinolone antibiotics are unknown but may be partially due to inhibition of IL-8 and MCP-1 production, respectively.

The apicomplexan pathogen Neospora caninum inhibits host cell apoptosis in the absence of discernible NF-kappa B activation.

ABSTRACT Neospora caninum, a causative agent of bovine abortions, is an apicomplexan parasite that is closely related to the human pathogen Toxoplasma gondii. Since a number of intracellular parasites, including T. gondii, have been shown to modulate host cell apoptosis, the present study was conducted to establish whether N. caninum is similarly capable of subverting apoptotic pathways in its host cells. Our results indicated that death receptor-mediated apoptosis is repressed during N. caninum infection, and the data further showed that the executioner caspase, caspase 3, does not become activated in the infected cells. Surprisingly, nuclear translocation of the NF-κB subunit p65 was not detected in N. caninum-infected cells, although this host transcription factor has been shown to upregulate prosurvival genes in cells infected with T. gondii. Consistent with these findings, the distinct accumulation of phosphorylated IκB that is seen at the parasitophorous vacuole membrane (PVM) of T. gondii was not apparent on the N. caninum PVM. Although a putative IκB kinase activity was detected in N. caninum extracts, thereby implying that this parasite is capable of modulating NF-κB translocation into the host cell nucleus, the data collectively suggest that a profound and sustained activation of the NF-κB pathway is not central to the ability of N. caninum to prevent apoptosis of their host cells.