The nuclear factor-κB (NF-κB) family of transcription factors plays a central part in the host response to infection by microbial pathogens, by orchestrating the innate and acquired host immune responses. The NF-κB proteins are activated by diverse signalling pathways that originate from many different cellular receptors and sensors. Many successful pathogens have acquired sophisticated mechanisms to regulate the NF-κB signalling pathways by deploying subversive proteins or hijacking the host signalling molecules. Here, we describe the mechanisms by which viruses and bacteria micromanage the host NF-κB signalling circuitry to favour the continued survival of the pathogen.

Modulation of NF-κB signalling by microbial pathogens

Toxoplasma gondii and Plasmodium falciparum are important human pathogens. These parasites and many of their apicomplexan relatives undergo a complex developmental process in the cells of their hosts, which includes genome replication, cell division and the assembly of new invasive stages. Apicomplexan cell cycle progression is both globally and locally regulated. Global regulation is carried out throughout the cytoplasm by diffusible factors that include cell cycle-specific kinases, cyclins and transcription factors. Local regulation acts on individual nuclei and daughter cells that are developing inside the mother cell. We propose that the centrosome is a master regulator that physically tethers cellular components and that provides spatial and temporal control of apicomplexan cell division.

Cell division in apicomplexan parasites

The major heat shock protein, Hsp70, can protect against cell death by directly interfering with mitochondrial apoptosis pathways. However, Hsp70 also sensitizes cells to certain apoptotic stimuli like TNF. Little is known about how Hsp70 enhances apoptosis. We demonstrate here that Hsp70 promotes TNF killing by specifically binding the coiled-coil domain of IκB kinase γ (IKKγ) to inhibit IKK activity and consequently inhibit NF-κB-dependent antiapoptotic gene induction. An IKKγ mutant, which interacts with Hsp70, competitively inhibits the Hsp70–IKKγ interaction and relieves heat-mediated NF-κB suppression. Depletion of Hsp70 expression with RNA interference rescues TNF-mediated cell death. Although TNF may or may not be sufficient to trigger apoptosis on its own, TNF-triggered apoptosis was initiated or made worse when Hsp70 expression increased to high levels to disrupt NF-κB signaling. These results provide significant novel insights into the molecular mechanism for the pro-apoptotic behavior of Hsp70 in death-receptor-mediated cell death.

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Hsp70 promotes TNF-mediated apoptosis by binding IKKγ and impairing NF-κB survival signaling

Theileria are economically important, intra-cellular protozoa, transmitted by ixodid ticks, which infect wild and domestic ruminants. In the mammalian host, parasites infect leukocytes and erythrocytes. In the arthropod vector they develop in gut epithelial cells and salivary glands. All four intra-cellular stages of Theileria survive free in the cytoplasm. The schizont stages of certain Theileria species induce a unique, cancer-like, phenotype in infected host leukocytes. Theileria undergoes an obligate sexual cycle, involving fusion of gametes in the tick gut, to produce a transiently diploid zygote. The existence of sexual recombination in T. parva has been confirmed in the laboratory, and is presumed to contribute to the extensive polymorphism observed in field isolates. Key parameters in T. parva population dynamics are the relative importance of asymptomatic carrier cattle and animals undergoing severe disease, in transmission of the parasite to ticks, and the extent of transmission by nymphs as compared to adult ticks. Tick populations differ in vector competence for specific T. parva stocks. Recombinant forms of T. parva and T. annulata sporozoite surface antigens induce protection against parasite challenge in cattle. In future, vaccines might be improved by inclusion of tick peptides in multivalent vaccines.

Theileria: Intracellular protozoan parasites of wild and domestic ruminants transmitted by ixodid ticks

Upregulation of the matrix metalloproteinase (MMP)-9 plays a central role in tumor progression and metastasis by stimulating cell migration, tumor invasion, and angiogenesis. To gain insights into MMP-9 expression, we investigated its epigenetic control in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. Gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. Notably, we found that the H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. SMYD3 is overexpressed in many types of cancer cells, but its contributions to malignant pathophysiology are unclear. We found that overexpression of SMYD3 was sufficient to induce MMP-9 expression in transformed leukocytes and fibrosarcoma cells and that proinflammatory phorbol esters further enhanced this effect. Furthermore, SMYD3 was sufficient to increase cell migration associated with MMP-9 expression. In contrast, RNA interference-mediated knockdown of SMYD3 decreased H3K4me3 modification of the MMP-9 promoter, reduced MMP-9 expression, and reduced tumor cell proliferation. Furthermore, SMYD3 knockdown also reduced cellular invasion in a zebrafish xenograft model of cancer. Together, our results define SMYD3 as an important new regulator of MMP-9 transcription, and they provide a molecular link between SMYD3 overexpression and metastatic cancer progression.

https://cancerres.aacrjournals.org/content/canres/72/3/810.full.pdf

SMYD3 Promotes Cancer Invasion by Epigenetic Upregulation of the Metalloproteinase MMP-9

Parasites have evolved a plethora of mechanisms to ensure their propagation and evade antagonistic host responses. The intracellular protozoan parasite Theileria is the only eukaryote known to induce uncontrolled host cell proliferation. Survival of Theileria-transformed leukocytes depends strictly on constitutive nuclear factor kappa B (NF-kappaB) activity. We found that this was mediated by recruitment of the multisubunit IkappaB kinase (IKK) into large, activated foci on the parasite surface. IKK signalosome assembly was specific for the transforming schizont stage of the parasite and was down-regulated upon differentiation into the nontransforming merozoite stage. Our findings provide insights into IKK activation and how pathogens subvert host-cell signaling pathways.

https://science.sciencemag.org/content/sci/298/5595/1033.full.pdf

Hijacking of host cell IKK signalosomes by the transforming parasite Theileria

The intracellular protozoan parasites Theileria parva and Theileria annulata transform leucocytes by interfering with host cell signal transduction pathways. They differ from tumour cells, however, in that the transformation process can be entirely reversed by elimination of the parasite from the host cell cytoplasm using a specific parasiticidal drug. We investigated the state of activation of Akt/PKB, a downstream target of PI3-K-generated phosphoinositides, in Theileria-transformed leucocytes. Akt/PKB is constitutively activated in a PI3-K- and parasite-dependent manner, as judged by the specific phosphorylation of key residues, in vitro kinase assays and its cellular distribution. In previous work, we demonstrated that the parasite induces constitutive activation of the transcription factor NF-kappaB, providing protection against spontaneous apoptosis that accompanies transformation. In a number of other systems, a link has been established between the PI3-K-Akt/PKB pathway and NF-kappaB activation, resulting in protection against apoptosis. In Theileria-transformed leucocytes, activation of the NF-kappaB and the PI3-K-Akt/PKB pathways are not directly linked. The PI3-K-Akt/PKB pathway does not contribute to the persistent induction of IkappaBalpha phosphorylation, NF-kappaB DNA-binding or transcriptional activity. We show that the two pathways are downregulated with different kinetics when the parasite is eliminated from the host cell cytoplasm and that NF-kappaB-dependent protection against apoptosis is not dependent on a functional PI3-K-Akt/PKB pathway. We also demonstrate that Akt/PKB contributes, at least in part, to the proliferation of Theileria-transformed T cells.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1462-5822.2001.00134.x

The Akt/PKB pathway is constitutively activated in Theileria-transformed leucocytes, but does not directly control constitutive NF-kappaB activation.

The IkappaB kinase (IKK) complex controls processes such as inflammation, immune responses, cell survival and the proliferation of both normal and tumor cells. By activating NFkappaB, the IKK complex contributes to G1/S transition and first evidence has been presented that IKKalpha also regulates entry into mitosis. At what stage IKK is required and whether IKK also contributes to progression through mitosis and cytokinesis, however, has not yet been determined. In this study, we use BMS-345541, a potent allosteric small molecule inhibitor of IKK, to inhibit IKK specifically during G2 and during mitosis. We show that BMS-345541 affects several mitotic cell cycle transitions, including mitotic entry, prometaphase to anaphase progression and cytokinesis. Adding BMS-345541 to the cells released from arrest in S-phase blocked the activation of Aurora A, B and C, Cdk1 activation and histone H3 phosphorylation. Additionally, treatment of the mitotic cells with BMS-345541 resulted in precocious cyclin B1 and securin degradation, defective chromosome separation and improper cytokinesis. BMS-345541 was also found to override the spindle checkpoint in nocodazole-arrested cells. In vitro kinase assays using BMS-345541 indicate that these effects are not primarily due to a direct inhibitory effect of BMS-345541 on mitotic kinases such as Cdk1, Aurora A or B, Plk1 or NEK2. This study points towards a new potential role of IKK in cell cycle progression. Since deregulation of the cell cycle is one of the hallmarks of tumor formation and progression, the newly discovered level of BMS-345541 function could be useful for cell cycle control studies and may provide valuable clues for the design of future therapeutics.

Rebekka Schwab

Papers

Hijacking of host cell IKK signalosomes by the transforming parasite Theileria

The Akt/PKB pathway is constitutively activated in Theileria-transformed leucocytes, but does not directly control constitutive NF-kappaB activation.

The IKK inhibitor BMS-345541 affects multiple mitotic cell cycle transitions.