Divergent adaptation of hepatitis C virus genotypes 1 and 3 to human leukocyte antigen-restricted immune pressure

Many hepatitis C virus (HCV) infections worldwide are with the genotype 1 and 3 strains of the virus. Cellular immune responses are known to be important in the containment of HCV genotype 1 infection, and many genotype 1 T cell targets (epitopes) that are presented by host human leukocyte antigens (HLAs) have been identified. In contrast, there is almost no information known about the equivalent responses to genotype 3. Immune escape mechanisms used by HCV include the evolution of viral polymorphisms (adaptations) that abrogate this host-viral interaction. Evidence of HCV adaptation to HLA-restricted immune pressure on HCV can be observed at the population level as viral polymorphisms associated with specific HLA types. To evaluate the escape patterns of HCV genotypes 1 and 3, we assessed the associations between viral polymorphisms and specific HLA types from 187 individuals with genotype 1a and 136 individuals with genotype 3a infection. We identified 51 HLA-associated viral polymorphisms (32 for genotype 1a and 19 for genotype 3a). Of these putative viral adaptation sites, six fell within previously published epitopes. Only two HLA-associated viral polymorphisms were common to both genotypes. In the remaining sites with HLA-associated polymorphisms, there was either complete conservation or no significant HLA association with viral polymorphism in the alternative genotype. This study also highlights the diverse mechanisms by which viral evasion of immune responses may be achieved and the role of genotype variation in these processes. CONCLUSION: There is little overlap in HLA-associated polymorphisms in the nonstructural proteins of HCV for the two genotypes, implying differences in the cellular immune pressures acting on these viruses and different escape profiles. These findings have implications for future therapeutic strategies to combat HCV infection, including vaccine design.

Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

Background The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. Methodology/Principal Findings We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. Conclusions Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins.

A new approach to generate a safe double-attenuated Plasmodium liver stage vaccine

Recently it has been shown in rodent malaria models that immunisation with genetically attenuated Plasmodium parasites can confer sterile protection against challenge with virulent parasites. For the mass production of live attenuated Plasmodium parasites for vaccination, safety is a prerequisite. Knockout of a single gene is not sufficient for such a strategy since the parasite can likely compensate for such a genetic modification and a single surviving parasite is sufficient to kill an immunised individual. Parasites must therefore be at least double-attenuated when generating a safe vaccine strain. Genetic double-attenuation can be achieved by knocking out two essential genes or by combining a single gene knockout with the expression of a protein toxic for the parasite. We generated a double-attenuated Plasmodium berghei strain that is deficient in fatty acid synthesis by the knockout of the pdh-e1α gene, introducing a second attenuation by the liver stage-specific expression of the pore-forming bacterial toxin perfringolysin O. With this double genetically attenuated parasite strain, a superior attenuation was indeed achieved compared with single-attenuated strains that were either deficient in pyruvate dehydrogenase (PDH)-E1 or expressed perfringolysin O. In vivo, both single-attenuated strains resulted in breakthrough infections even if low to moderate doses of sporozoites (2,000-5,000) were administered. In contrast, the double genetically attenuated parasite strain, given at moderate doses of 5,000 sporozoites, did not result in blood stage infection and even when administered at 5- to 20-fold higher doses, only single and delayed breakthrough infections were observed. Prime booster immunisation with the double genetically attenuated parasite strain completely protected a susceptible mouse strain from malaria and even a single immunisation conferred protection in some cases and lead to a markedly delayed onset of blood stage infection in others. Importantly, premature rupture of the parasitophorous vacuole membrane by liver stage-specific perfringolysin O expression did not induce host cell death and soluble parasite proteins, which are released into the host cell cytoplasm, have the potential to be processed and presented via MHC class I molecules. This, in turn, might support immunological responses against Plasmodium-infected hepatocytes.

Analysis of liver stage development in and merozoite release from hepatocytes

Exoerythrocytic Plasmodium parasites infect hepatocytes and develop to huge multinucleated schizonts inside a parasitophorous vacuole. Finally, thousands of merozoites are formed and released into the host cell cytoplasm by complete disintegration of the parasitophorous vacuole membrane. This, in turn, results in death and detachment of the infected hepatocyte, followed by the formation of merosomes. The fast growth of the parasite and host cell detachment are hallmarks of liver stage development and can easily be monitored. Here, we describe how to translate these observations into assays for characterizing parasite development. Additionally, other recently introduced techniques and tools to analyze and manipulate liver stage parasites are also discussed.

Immune recognition of self nucleic acids driven by endogenous antimicrobial peptides: role in autoimmunity

Innate immune recognition of extracellular host-derived self-DNA and self-RNA is prevented by endosomal seclusion of the Toll-like receptors (TLRs) in the dendritic cells (DCs). However, in psoriasis plasmacytoid dendritic cells have been found to be able to sense self-DNA molecules in complex with the endogenous cationic antimicrobial peptide LL37, which are internalized into the endosomal compartments and thus can access TLR9. We investigated whether this endogenous peptide can also interact with extracellular self-RNA and lead to DC activation. We found that LL37 binds self-RNA as well as self-DNA going into an electrostatic interaction; forms micro-aggregates of nano-scale particles protected from enzymatic degradation and transport it into the endosomal compartments of both plasmacytoid and myeloid dendritic cells. In the plasmacytoid DCs, the self-RNA-LL37 complexes activate TLR7 and like the self-DNA-LL37 complexes, trigger the production of IFN-α in the absence of induction of maturation or production of IL-6 and TNF-α. In contrast to the self-DNA-LL37 complexes, the self-RNA-LL37 complexes are also internalized into the endosomal compartments of myeloid dendritic cells and trigger activation through TLR8, leading to the production of TNF-α and IL-6, and the maturation of the myeloid DCs. Furthermore, we found that these self nucleic acid-LL37 complexes can be found in vivo in the skin lesions of the cutaneous autoimmune disease psoriasis, where they are associated with mature mDCs in situ. On the other hand, in the systemic autoimmune disease systemic lupus erythematosus, self-DNA-LL37 complexes were found to be a constituent of the circulating immune complexes isolated from patient sera. This interaction between the endogenous peptide with the self nucleic acid molecules present in the immune complexes was found to be electrostatic and it confers resistance to enzymatic degradation of the nucleic acid molecules in the immune complexes. Moreover, autoantibodies to these endogenous peptides were found to trigger neutrophil activation and release of neutrophil extracellular traps composed of DNA, which are potential sources of the self nucleic acid-LL37 complexes present in SLE immune complexes. Our results demonstrate that the cationic antimicrobial peptide LL37 drives the innate immune recognition of self nucleic acid molecules through toll-like receptors in human dendritic cells, thus elucidating a pathway for innate sensing of host cell death. This pathway of autoreactivity was found to be pathologically relevant in human autoimmune diseases psoriasis and SLE, and thus this study provides new insights into the mechanisms autoimmune diseases.

Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence.

A Plasmodium phospholipase is involved in disruption of the liver stage parasitophorous vacuole membrane

The coordinated exit of intracellular pathogens from host cells is a process critical to the success and spread of an infection. While phospholipases have been shown to play important roles in bacteria host cell egress and virulence, their role in the release of intracellular eukaryotic parasites is largely unknown. We examined a malaria parasite protein with phospholipase activity and found it to be involved in hepatocyte egress. In hepatocytes, Plasmodium parasites are surrounded by a parasitophorous vacuole membrane (PVM), which must be disrupted before parasites are released into the blood. However, on a molecular basis, little is known about how the PVM is ruptured. We show that Plasmodium berghei phospholipase, PbPL, localizes to the PVM in infected hepatocytes. We provide evidence that parasites lacking PbPL undergo completely normal liver stage development until merozoites are produced but have a defect in egress from host hepatocytes. To investigate this further, we established a live-cell imaging-based assay, which enabled us to study the temporal dynamics of PVM rupture on a quantitative basis. Using this assay we could show that PbPL-deficient parasites exhibit impaired PVM rupture, resulting in delayed parasite egress. A wild-type phenotype could be re-established by gene complementation, demonstrating the specificity of the PbPL deletion phenotype. In conclusion, we have identified for the first time a Plasmodium phospholipase that is important for PVM rupture and in turn for parasite exit from the infected hepatocyte and therefore established a key role of a parasite phospholipase in egress.

Inhibition of apoptosis by intracellular protozoan parasites

Protozoan parasites which reside inside a host cell avoid direct destruction by the immune system of the host. The infected cell, however, still has the capacity to counteract the invasive pathogen by initiating its own death, a process which is called programmed cell death or apoptosis. Apoptotic cells are recognised and phagocytosed by macrophages and the parasite is potentially eliminated together with the infected cell. This potent defence mechanism of the host cell puts strong selective pressure on the parasites which have, in turn, evolved strategies to modulate the apoptotic program of the host cell to their favour. Within the last decade, the existence of cellular signalling pathways which inhibit the apoptotic machinery has been demonstrated. It is not surprising that intracellular pathogens subvert these pathways to ensure their own survival in the infected cell. Molecular mechanisms which interfere with apoptotic pathways have been studied extensively for viruses and parasitic bacteria, but protozoan parasites have come into focus only recently. Intracellular protozoan parasites which have been reported to inhibit the apoptotic program of the host cell, are Toxoplasma gondii, Trypanosoma cruzi, Leishmania sp., Theileria sp., Cryptosporidium parvum, and the microsporidian Nosema algerae. Although these parasites differ in their mechanism of host cell entry and in their final intracellular localisation, they might activate similar pathways in their host cells to inhibit apoptosis. In this respect, two families of molecules, which are known for their capacity to interrupt the apoptotic program, are currently discussed in the literature. First, the expression of heat shock proteins is often induced upon parasite infection and can directly interfere with molecules of the cellular death machinery. Secondly, a more indirect effect is attributed to the parasite-dependent activation of NF-kappaB, a transcription factor that regulates the transcription of anti-apoptotic molecules.

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

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.

Transformation of leukocytes by Theileria parva and T. annulata.

Theileria parva and T. annulata provide intriguing models for the study of parasite-host interactions. Both parasites possess the unique property of being able to transform the cells they infect; T. parva transforms T and B cells, whereas T. annulata affects B cells and monocytes/macrophages. Parasitized cells do not require antigenic stimulation or exogenous growth factors and acquire the ability to proliferate continuously. In vivo, parasitized cells undergo clonal expansion and infiltrate both lymphoid and non-lymphoid tissues of the infected host. Theileria-induced transformation is entirely reversible and is accompanied by the expression of a wide range of different lymphokines and cytokines, some of which may contribute to proliferation or may enhance spread and survival of the parasitized cell in the host. The presence of the parasite in the host-cell cytoplasm modulates the state of activation of a number of signal transduction pathways. This, in turn, leads to the activation of transcription factors, including nuclear factor-kappa B, which appear to be essential for the survival of Theileria-transformed T cells.

AN APPROACH TO THE CHARACTERIZATION OF FIBRONECTIN-INTERACTION WITH MAMMALIAN CELLS

Cell adhesion is a fundamentally important process which has been implicated in morphogenesis, metastasis and wound healing. Fibronectin (Fn), a large glycoprotein present in body fluids, the extracellular matrix, and on the cell surface, mediates adhesion of fibroblastic cells. To study the interaction of Fn with Chinese Hamster Cell (CHO) cell membranes, latex beads coated with H('3)-Fn (Fn-beads) were used as surface probes. Binding of Fn-beads was independent of temperature, divalent cations, and metabolic activity. Identification of fibronectin-receptors has been problematical. To study Fn binding components, Fn-beads were pre-incubated with purified glycosaminoglycans (GAGs) and glycolipids. Among the GAGs tested, heparin and heparan sulfate blocked bead binding. Only sialylated glycolipids, GT(,1) and GD(,1) were inhibitory; however, neuraminidase treatment of cells had no effect. It was further shown that Fn-bead binding could be blocked by pre-treating cells with papain. Furthermore, papain digestion releases cellular material which blocks Fn-bead-cell binding. Beads coated with a fragment of Fn which binds to cells but not heparin (F105) were also blocked by soluble papain digests. It was observed that the ability of F105-beads to bind to CHO cells was dependent on surface charge as F105 on uncharged beads did not bind to cells; whereas, F105 on positive or negative beads displayed cell binding activity. The active component in the papain digests was apparently macromolecular (i.e. non-dialysable) and heat stable (i.e. 100(DEGREES)C for 15 min.). This suggested the inhibitory factor is more likely a glycopeptide, rather than a GAG or glycolipid. The findings of this research can be summarized as follows: (1) the expression of cell binding of Fn and Fn fragments can be modulated by the chemical nature of the surface used for adsorption; (2) factors can be released by proteolytic digestion which block Fn and Fn-fragment bead binding; and (3) since bead binding can be done under conditions which reflect initial Fn-cell interaction, it seems likely that the component(s) identified in this way may play a direct role in the recognition phases of cell adhesion to Fn. ^

Influence of mycobacterial trehalose 6,6'-dimycolate (TDM) on macrophage responses

Mycobacterium tuberculosis, the causative agent of tuberculosis, survives within macrophages by altering host cell activation and by manipulating phagosomal trafficking and acidification. Part of the success of M. tuberculosis as a major human pathogen has been attributed to its cell wall, a unique structure largely comprised of mycolic acids. Trehalose 6,6′-dimycolate (TDM) is the major glycolipid component on the surface of the mycobacterial cell wall. This study examines the contribution of TDM during mycobacterial infection of murine macrophages. Virulent M. tuberculosis was chemically depleted of surface-exposed TDM using petroleum ether extraction. Compared to their native counterparts, delipidated M. tuberculosis showed similar growth in broth culture. Bone marrow-derived macrophages (BMM) or the murine macrophage-like cell line J774A.1 were infected with delipidated M. tuberculosis, and responses were compared to cells infected with native M. tuberculosis. Delipidated M. tuberculosis demonstrated significantly decreased viability in macrophages by seven days after infection. Reconstitution of delipidated organisms with pure TDM restored viability. Infection with native M. tuberculosis led to high cellular production of cytokines (IL-1β, IL-6, IL-12, and TNF-α) and chemokines (MCP-1 and MIP-1α); infection with delipidated M. tuberculosis significantly abrogated responses. Cytokine and chemokine production were restored when delipidated organisms were reconstituted with TDM. Responses were specifically induced by TDM; all measured cytokines were elicited from macrophages incubated with TDM-coated beads, while control beads coated with bovine serum albumin (BSA) did not induce cytokine production. Visualization of mycobacterial localization in J774A.1 cells using fluorescence microscopy revealed that delipidated M. tuberculosis were significantly more likely to traffic to acidic vesicles (lysosomes) than native organisms. Reconstitution with TDM restored trafficking to non-acidic vesicles. Similarly, TDM-coated beads demonstrated significantly delayed localization to acidic vesicles compared to BSA-coated beads. In summary, the interaction of TDM with macrophages may regulate the outcome of M. tuberculosis infection by influencing cellular cytokine production and intracellular localization of organisms. This research has elucidated a novel and necessary role for TDM in survival of virulent M. tuberculosis in host macrophages during in vitro infection. ^

Yersinia signals macrophages to undergo apoptosis and YopJ is necessary for this cell death

Pathogenic Yersinia spp. carry a large common plasmid that encodes a number of essential virulence determinants. Included in these factors are the Yersinia-secreted proteins called Yops. We analyzed the consequences of wild-type and mutant strains of Yersinia pseudotuberculosis interactions with the macrophage cell line RAW264.7 and murine bone marrow-derived macrophages. Wild-type Y. pseudotuberculosis kills ≈70% of infected RAW264.7 macrophages and marrow-derived macrophages after an 8-h infection. We show that the cell death mediated by Y. pseudotuberculosis is apoptosis. Mutant Y. pseudotuberculosis that do not make any Yop proteins no longer cause host cell death. Attachment to host cells via invasin or YadA is necessary for the cell death phenotype. Several Yop mutant strains that fail to express one or more Yop proteins were engineered and then characterized for their ability to cause host cell death. A mutant with a polar insertion in YpkA Ser/Thr kinase that does not express YpkA or YopJ is no longer able to cause apoptosis. In contrast, a mutant no longer making YopE or YopH (a tyrosine phosphatase) induces apoptosis in macrophages similar to wild type. When yopJ is added in trans to the ypkAyopJ mutant, the ability of this strain to signal programmed cell death in macrophages is restored. Thus, YopJ is necessary for inducing apoptosis. The ability of Y. pseudotuberculosis to promote apoptosis of macrophages in cell culture suggests that this process is important for the establishment of infection in the host and for evasion of the host immune response.

Induction of host signal transduction pathways by Helicobacter pylori

Adherence of Helicobacter pylori to cultured gastric epithelial cells is associated with several cellular events, including the tyrosine phosphorylation of a 145-kDa host protein; the reorganization of the host cell actin and associated cellular proteins, like vasodilator-stimulated phosphoprotein, adjacent to the attached bacterial cell; and the subsequent release of the cytokine, interleukin 8 (IL-8). H. pylori isolated from patients with ulcer disease and gastric cancer contain a DNA insertion, the cag pathogenicity island (PAI), that is not present in bacteria isolated from individuals with asymptomatic infection. Mutations in a number of PAI genes abolish tyrosine phosphorylation and IL-8 synthesis but not the cytoskeletal rearrangements. Kinase inhibition studies suggest there are two distinct pathways operative in stimulating IL-8 release from host cells and one of these H. pylori pathways is independent of the tyrosine phosphorylation step.

Growth of Toxoplasma gondii is inhibited by aryloxyphenoxypropionate herbicides targeting acetyl-CoA carboxylase

Aryloxyphenoxypropionates, inhibitors of the plastid acetyl-CoA carboxylase (ACC) of grasses, also inhibit Toxoplasma gondii ACC. Clodinafop, the most effective of the herbicides tested, inhibits growth of T. gondii in human fibroblasts by 70% at 10 μM in 2 days and effectively eliminates the parasite in 2–4 days at 10–100 μM. Clodinafop is not toxic to the host cell even at much higher concentrations. Parasite growth inhibition by different herbicides is correlated with their ability to inhibit ACC enzyme activity, suggesting that ACC is a target for these agents. Fragments of genes encoding the biotin carboxylase domain of multidomain ACCs of T. gondii, Plasmodium falciparum, Plasmodium knowlesi, and Cryptosporidium parvum were sequenced. One T. gondii ACC (ACC1) amino acid sequence clusters with P. falciparum ACC, P. knowlesi ACC, and the putative Cyclotella cryptica chloroplast ACC. Another sequence (ACC2) clusters with that of C. parvum ACC, probably the cytosolic form.