Reprogramming in Candida albicans Gene Expression Network under Butanol Stress Abrogates Hyphal Development.

Candida albicans is the causative agent of invasive fungal infections. Its hyphae-forming ability is regarded as one of the important virulence factors. To unravel the impact of butanol on Candida albicans, it was placed in O+ve complete human serum with butanol (1% v/v). The Candida transcriptome under butanol stress was then identified by mRNA sequencing. Studies including electron microscopy demonstrated the inhibition of hyphae formation in Candida under the influence of butanol, without any significant alteration in growth rate. The numbers of genes upregulated in the butanol in comparison to the serum alone were 1061 (20 min), 804 (45 min), and 537 (120 min). Candida cells exhibited the downregulation of six hypha-specific transcription factors and the induction of four repressor/regulator genes. Many of the hypha-specific genes exhibited repression in the medium with butanol. The genes related to adhesion also exhibited repression, whereas, among the heat-shock genes, three showed inductions in the presence of butanol. The fungal-specific genes exhibited induction as well as repression in the butanol-treated Candida cells. Furthermore, ten upregulated genes formed the core stress gene set in the presence of butanol. In the gene ontology analysis, enrichment of the processes related to non-coding RNA, ribosome biosynthesis, and metabolism was observed in the induced gene set. On the other side, a few GO biological process terms, including biofilm formation and filamentous growth, were enriched in the repressed gene set. Taken together, under butanol stress, Candida albicans is unable to extend hyphae and shows growth by budding. Many of the genes with perturbed expression may have fitness or virulence attributes and may provide prospective sites of antifungal targets against C. albicans.

The Multistage Antimalarial Compound Calxinin Perturbates P. falciparum Ca2+ Homeostasis by Targeting a Unique Ion Channel.

Malaria elimination urgently needs novel antimalarial therapies that transcend resistance, toxicity, and high costs. Our multicentric international collaborative team focuses on developing multistage antimalarials that exhibit novel mechanisms of action. Here, we describe the design, synthesis, and evaluation of a novel multistage antimalarial compound, 'Calxinin'. A compound that consists of hydroxyethylamine (HEA) and trifluoromethyl-benzyl-piperazine. Calxinin exhibits potent inhibitory activity in the nanomolar range against the asexual blood stages of drug-sensitive (3D7), multidrug-resistant (Dd2), artemisinin-resistant (IPC4912), and fresh Kenyan field isolated Plasmodium falciparum strains. Calxinin treatment resulted in diminished maturation of parasite sexual precursor cells (gametocytes) accompanied by distorted parasite morphology. Further, in vitro liver-stage testing with a mouse model showed reduced parasite load at an IC50 of 79 nM. A single dose (10 mg/kg) of Calxinin resulted in a 30% reduction in parasitemia in mice infected with a chloroquine-resistant strain of the rodent parasite P. berghei. The ex vivo ookinete inhibitory concentration within mosquito gut IC50 was 150 nM. Cellular in vitro toxicity assays in the primary and immortalized human cell lines did not show cytotoxicity. A computational protein target identification pipeline identified a putative P. falciparum membrane protein (Pf3D7_1313500) involved in parasite calcium (Ca2+) homeostasis as a potential Calxinin target. This highly conserved protein is related to the family of transient receptor potential cation channels (TRP-ML). Target validation experiments showed that exposure of parasitized RBCs (pRBCs) to Calxinin induces a rapid release of intracellular Ca2+ from pRBCs; leaving de-calcinated parasites trapped in RBCs. Overall, we demonstrated that Calxinin is a promising antimalarial lead compound with a novel mechanism of action and with potential therapeutic, prophylactic, and transmission-blocking properties against parasites resistant to current antimalarials.

Dissecting The role of Plasmodium metacaspase-2 in malaria gametogenesis and sporogony.

The family of apicomplexan specific proteins contains caspases-like proteins called "metacaspases". These enzymes are present in the malaria parasite but absent in human; therefore, these can be explored as potential drug targets. We deleted the MCA-2 gene from Plasmodium berghei genome using a gene knockout strategy to decipher its precise function. This study has identified that MCA-2 plays an important role in parasite transmission since it is critical for the formation of gametocytes and for maintaining an appropriate number of infectious sporozoites required for sporogony. It is noticeable that a significant reduction in gametocyte, oocysts, ookinete and sporozoites load along with a delay in hepatocytes invasion were observed in the MCA-2 knockout parasite. Furthermore, a study found the two MCA-2 inhibitory molecules known as C-532 and C-533, which remarkably inhibited the MCA-2 activity, abolished the in vitro parasite growth, and also impaired the transmission cycle of P. falciparum and P. berghei in An. stephensi. Our findings indicate that the deletion of MCA-2 hampers the Plasmodium development during erythrocytic and exo-erythrocytic stages, and its inhibition by C-532 and C-533 critically affects the malaria transmission biology.

Novel Antiplasmodial Compounds Leveraged with Multistage Potency against the Parasite Plasmodium falciparum: In Vitro and In Vivo Evaluations and Pharmacokinetic Studies.

Hydroxyethylamine (HEA)-based novel compounds were synthesized and their activity against Plasmodium falciparum 3D7 was assessed, identifying a few hits without any apparent toxicity. Hits 5c and 5d also exhibited activity against resistant field strains, PfRKL-9 and PfC580Y. A single dose, 50 mg/Kg, of hits administered to the rodent parasite Plasmodium berghei ANKA exhibited up to 70% reduction in the parasite load. Compound 5d tested in combination with artesunate produced an additional antiparasitic effect with a prolonged survival period. Additionally, compound 5d showed 50% inhibition against hepatic P. berghei infection at 1.56 ± 0.56 µM concentration. This compound also considerably delayed the progression of transmission stages, ookinete and oocyst. Furthermore, the toxicity of 5d assessed in mice supported the normal liver and kidney functions. Altogether, HEA analogues (5a-m), particularly 5d, are nontoxic multistage antiplasmodial agents with therapeutic and transmission-blocking efficacy, along with favorable preliminary pharmacokinetic properties.

Conserved Plasmodium Protein (PF3D7_0406000) of Unknown Function: In-silico Analysis and Cellular Localization.

In spite of a decrease in malaria cases, the threat of malaria due to Plasmodium falciparum still prevails. The sequencing of Plasmodium falciparum reveals that approximately 60% of the Plasmodium genes code for hypothetical/putative proteins. Here we report an in silico characterization and localization of one such protein. This was encoded by one of the hub genes, in a weighted gene co-expression based systems network, from in-vivo samples of patients suffering from uncomplicated malaria or complicated malaria disease like jaundice and jaundice with renal failure. Interestingly, the protein PF3D7_0406000 (PFD0300w) is classified as a conserved protein of unknown function and shows no identity with any protein from the human host. The transcriptomic data shows up-regulation of transcripts in cases of malaria induced disease complications. PFD0300w peptide antibody based immunolocalization studies using a, gametocyte producing P. falciparum strain RKL-9, shows presence of the protein in the cytoplasm of both asexual and sexual stage parasites.

Pathogenic Pore Forming Proteins of Plasmodium Triggers the Necrosis of Endothelial Cells Attributed to Malaria Severity.

Severe malaria caused by Plasmodium falciparum poses a major global health problem with high morbidity and mortality. P. falciparum harbors a family of pore-forming proteins (PFPs), known as perforin like proteins (PLPs), which are structurally equivalent to prokaryotic PFPs. These PLPs are secreted from the parasites and, they contribute to disease pathogenesis by interacting with host cells. The severe malaria pathogenesis is associated with the dysfunction of various barrier cells, including endothelial cells (EC). Several factors, including PLPs secreted by parasites, contribute to the host cell dysfunction. Herein, we have tested the hypothesis that PLPs mediate dysfunction of barrier cells and might have a role in disease pathogenesis. We analyzed various dysfunctions in barrier cells following rPLP2 exposure and demonstrate that it causes an increase in intracellular Ca2+ levels. Additionally, rPLP2 exposed barrier cells displayed features of cell death, including Annexin/PI positivity, depolarized the mitochondrial membrane potential, and ROS generation. We have further performed the time-lapse video microscopy of barrier cells and found that the treatment of rPLP2 triggers their membrane blebbing. The cytoplasmic localization of HMGB1, a marker of necrosis, further confirmed the necrotic type of cell death. This study highlights the role of parasite factor PLP in endothelial dysfunction and provides a rationale for the design of adjunct therapies against severe malaria.

Plasmodium falciparum metacaspase-2 capture its natural substrate in a non-canonical way.

Programmed cell death (PCD) is a multi-step process initiated by a set of proteases, which interacts and cleaves diverse proteins, thus modulating their biochemical and cellular functions. In metazoans, PCD is mediated by proteolytic enzymes called caspases, which triggered cell death by proteolysis of human Tudor staphylococcus nuclease (TSN). Non-metazoans lack a close homologue of caspases but possess an ancestral family of cysteine proteases termed 'metacaspases'. Studies supported that metacaspases are involved in PCD, but their natural substrates remain unknown. In this study, we performed the Plasmodium falciparum TSN (PfTSN) cleavage assay using wild and selected mutants of P. falciparum metacaspases-2 (PfMCA-2) in vitro and in vivo. Interestingly, PfMCA-2, cleaved a phylogenetically conserved protein, PfTSN at multiple sites. Deletion or substitution mutation in key interacting residues at the active site, Cys157 and His205 of PfMCA-2, impaired its enzymatic activity with the artificial substrate, z-GRR-AMC. However, the mutant Tyr224A did not affect the activity with z-GRR-AMC but abolished the cleavage of PfTSN. These results indicated that the catalytic dyad, Cys157 and His205 of PfMCA-2 was essential for its enzymatic activity with an artificial substrate, whereas Tyr224 and Cys157 residues were responsible for its interaction with the natural substrate and subsequent degradation of PfTSN. Our results suggested that MCA-2 interacts with TSN substrate in a non-canonical way using non-conserved or conformationally available residues for its binding and cleavage. In future, it would be interesting to explore how this interaction leads to the execution of PCD in the Plasmodium.

Plasmodium Perforin-Like Protein Pores on the Host Cell Membrane Contribute in Its Multistage Growth and Erythrocyte Senescence.

The pore forming Plasmodium Perforin Like Proteins (PPLP), expressed in all stages of the parasite life cycle are critical for completion of the parasite life cycle. The high sequence similarity in the central Membrane Attack Complex/ Perforin (MACPF) domain among PLPs and their distinct functional overlaps define them as lucrative target for developing multi-stage antimalarial therapeutics. Herein, we evaluated the mechanism of Pan-active MACPF Domain (PMD), a centrally located and highly conserved region of PPLPs, and deciphered the inhibitory potential of specifically designed PMD inhibitors. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to hemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence which can be hypothesized to account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing malaria anemia. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress processes and protected erythrocytes against rPMD induced senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage, transmission-blocking potential of these inhibitors. Concievably, our study has introduced a novel set of anti-PMD inhibitors with pan-inhibitory activity against all the PPLPs members which can be developed into potent cross-stage antimalarial therapeutics along with erythrocyte senescence protective potential to occlude PPLPs mediated anemia in severe malaria.

A nonpeptidyl molecule modulates apoptosis-like cell death by inhibiting P. falciparum metacaspase-2.

Metacaspases are novel cysteine proteases found in apicomplexan whose function is poorly understood. Our earlier studies on Plasmodium falciparum metacaspase-2 (PfMCA-2) revealed that the caspase inhibitor, Z-FA-FMK efficiently inhibited PfMCA-2 activity and, expression, and significantly blocked in vitro progression of the parasite developmental cycle via apoptosis-like parasite death. Building on these findings, we synthesized a set of novel inhibitors based on structural modification of Z-FA-FMK with the amides of piperic acid and investigated their effect on PfMCA-2. One of these analogs, SS-5, specifically inhibited the activity and expression of PfMCA-2. The activities of some other known malarial proteases (falcipains, plasmepsins and vivapain), and human cathepsins-B, D and L, and caspase-3 and -7 were not inhibited by SS-5. SS-5 blocked the development of P. falciparum in vitro (IC50 1 µM) and caused prominent morphological distortions. Incubation with SS-5 led to persistent parasite oxidative stress accompanied by depolarization of mitochondrial potential and accumulation of intracellular Ca2+. SS-5 also inhibited the development of P. berghei in a murine model. Our results suggest that the inhibition of PfMCA-2 results in oxidative stress, leading to apoptosis-like parasite death. Thus, SS-5 offers a starting point for the optimization of new antimalarials, and PfMCA-2 could be a novel target for antimalarial drug discovery.

Identification and characterization of protective CD8+ T-epitopes in a malaria vaccine candidate SLTRiP.

INTRODUCTION: Efforts are required at developing an effective vaccine that can inhibit malaria prevalence and transmission. Identifying the critical immunogenic antigens and understanding their interactions with host proteins forms a major focus of subunit vaccine development. Previously, our laboratory showed that SLTRiP conferred protection to the liver stage of Plasmodium growth in rodents. In the follow-up of earlier research, we demonstrate that SLTRiP-mediated protection is majorly concentrated in specific regions of protein. METHOD: To identify particular protective regions of protein, we synthesized multiple nonoverlapping fragments from SLTRiP protein. From this, we designed a panel of 8-20mer synthetic peptides, which were predicted using T-epitope-based prediction algorithm. We utilized the IFN-γ enzyme-linked immunosorbent spot assay to identify immunodominant peptides. The latter were used to immunize mice, and these mice were challenged to assess protection. RESULTS: The protective polypeptide fragment SLTRiP C3 and SLTRiP C4 were identified, by expressing and testing multiple fragments of PbSLTRiP protein. The immune responses generated by these fragments were compared to identify the immunodominant fragment. The T-epitopes were predicted from SLTRiP protein using computer-based algorithms. The in vitro immune responses generated by these peptides were compared with each other to identify the immunodominant T-epitope. Immunization using these peptides showed significant reduction in parasite numbers during liver stage. CONCLUSION: Our findings show that the protective efficacy shown by SLTRiP is localized in particular protein fragments. The peptides designed from such regions showed protective efficacy equivalent to whole protein. The sequence conservation analysis with human Plasmodium species also showed that these peptides were conserved. In conclusion, these peptides or their equivalent from other Plasmodium species could impart protection against malaria in their respective hosts too. Our studies provide a basis for the inclusion of these peptides in clinical vaccine constructs against malaria.

Metacaspase-3 of Plasmodium falciparum: An atypical trypsin-like serine protease.

Metacaspases are clan CD cysteine peptidases found in plants, fungi and protozoa that possess a conserved Peptidase_C14 domain, homologous to the human caspases and a catalytic His/Cys dyad. Earlier reports have indicated the role of metacaspases in cell death; however, metacaspases of human malaria parasite remains poorly understood. In this study, we aimed to functionally characterize a novel malarial protease, P. falciparum metacaspase-3 (PfMCA3). Unlike other clan CD peptidases, PfMCA3 has an atypical active site serine (Ser1865) residue in place of canonical cysteine and it phylogenetically forms a distinct branch across the species. To investigate whether this domain retains catalytic activity, we expressed, purified and refolded the Peptidase_C14 domain of PfMCA3 which was found to express in all asexual stages. PfMCA3 exhibited trypsin-like serine protease activity with ser1865 acting as catalytic residue to cleave trypsin oligopeptide substrate. PfMCA3 is inhibited by trypsin-like serine protease inhibitors. Our study found that PfMCA3 enzymatic activity was abrogated when catalytic serine1865 (S1865A) was mutated. Moreover, PfMCA3 was found to be inactive against caspase substrate. Overall, our study characterizes a novel metacaspase of P. falciparum, different from human caspases and not responsible for the caspase-like activity, therefore, could be considered as a potential chemotherapeutic target.

Molecular characterization and expression profile of an alternate proliferating cell nuclear antigen homolog PbPCNA2 in Plasmodium berghei.

Proliferative cell nuclear antigen (PCNA) is the processivity factor for various DNA polymerases and it functions in response to DNA damage in eukaryotic system. Plasmodium falciparum contains two PCNAs, while PCNA1 has been attributed to DNA replication, the role of PCNA2 has been assigned to DNA damage response in erythrocytic developmental stages. Although a recent transposon mediated knockout strategy qualified pcna2 as a nonessential gene in Plasmodium berghei, a conventional homologous recombination-based knockout strategy has not been employed for this gene yet. Moreover, the cellular dynamics of PCNA2 in extraerythrocytic stages still remain elusive in Plasmodium. We attempted multiple times to knock out PbPCNA2 from the parasite genome using homologous recombination strategy without much success. However, we were able to generate PbPCNA2-GFP tagged transgenic parasites confirming that the pcna2 locus is amenable to genetic manipulation. The GFP-tagged parasites showed similar growth phenotype, compared to wild-type parasites, in both erythrocytic and sporogonic cycle, suggesting that tagging had no effect on parasite physiology. PbPCNA2 expression was also observed during the sporogonic cycle in midgut oocyst and salivary gland sporozoites. The PbPCNA2 expression was upregulated in the presence of DNA damaging agents like hydroxyurea and methyl methanesulphonate. Our inability to knock out PCNA2 suggested its essentiality in the parasite development and elevated expression during DNA damaging condition hint at a critical role of the protein in parasite physiology. © 2019 IUBMB Life, 71(9):1293-1301, 2019.

Tryptophan-kynurenine pathway attenuates ß-catenin-dependent pro-parasitic role of STING-TICAM2-IRF3-IDO1 signalosome in Toxoplasma gondii infection.

Recent studies have documented the diverse role of host immunity in infection by the protozoan parasite, Toxoplasma gondii. However, the contribution of the ß-catenin pathway in this process has not been explored. Here, we show that AKT-mediated phosphorylated ß-catenin supports T. gondii multiplication which is arrested in the deficiency of its phosphorylation domain at S552 position. The ß-catenin-TCF4 protein complex binds to the promoter region of IRF3 gene and initiates its transcription, which was also abrogated in ß-catenin knockout cells. TBK-independent phosphorylation of STING(S366) and its adaptor molecule TICAM2 by phospho-AKT(T308S473) augmented downstream IRF3-dependent IDO1 transcription, which was also dependent on ß-catenin. But, proteasomal degradation of IDO1 by its tyrosine phosphorylation (at Y115 and Y253) favoured parasite replication. In absence of IDO1, tryptophan was catabolized into melatonin, which supressed cellular reactive oxygen species (ROS) and boosted parasite growth. Conversely, when tyrosine phosphorylation was abolished by phosphosite mutations, IDO1 escaped its ubiquitin-mediated proteasomal degradation system (UPS) and the stable IDO1 prevented parasite replication by kynurenine synthesis. We propose that T. gondii selectively utilizes tryptophan to produce the antioxidant, melatonin, thus prolonging the survival of infected cells through functional AKT and ß-catenin activity for better parasite replication. Stable IDO1 in the presence of IFN-γ catabolized tryptophan into kynurenine, promoting cell death by suppressing phospho-AKT and phospho-ß-catenin levels, and circumvented parasite replication. Treatment of infected cells with kynurenine or its analogue, teriflunomide suppressed kinase activity of AKT, and phosphorylation of ß-catenin triggering caspase-3 dependent apoptosis of infected cells to inhibit parasite growth. Our results demonstrate that ß-catenin regulate phosphorylated STING-TICAM2-IRF3-IDO1 signalosome for a cell-intrinsic pro-parasitic role. We propose that the downstream IRF3-IDO1-reliant tryptophan catabolites and their analogues can act as effective immunotherapeutic molecules to control T. gondii replication by impairing the AKT and ß-catenin axis.

Fast-Acting Small Molecules Targeting Malarial Aspartyl Proteases, Plasmepsins, Inhibit Malaria Infection at Multiple Life Stages.

The eradication of malaria remains challenging due to the complex life cycle of Plasmodium and the rapid emergence of drug-resistant forms of Plasmodium falciparum and Plasmodium vivax. New, effective, and inexpensive antimalarials against multiple life stages of the parasite are urgently needed to combat the spread of malaria. Here, we synthesized a set of novel hydroxyethylamines and investigated their activities in vitro and in vivo. All of the compounds tested had an inhibitory effect on the blood stage of P. falciparum at submicromolar concentrations, with the best showing 50% inhibitory concentrations (IC50) of around 500 nM against drug-resistant P. falciparum parasites. These compounds showed inhibitory actions against plasmepsins, a family of malarial aspartyl proteases, and exhibited a marked killing effect on blood stage Plasmodium. In chloroquine-resistant Plasmodium berghei and P. berghei ANKA infected mouse models, treating mice with both compounds led to a significant decrease in blood parasite load. Importantly, two of the compounds displayed an inhibitory effect on the gametocyte stages (III-V) of P. falciparum in culture and the liver-stage infection of P. berghei both in in vitro and in vivo. Altogether, our findings suggest that fast-acting hydroxyethylamine-phthalimide analogs targeting multiple life stages of the parasite could be a valuable chemical lead for the development of novel antimalarial drugs.

Biochemical characterization of unusual cysteine protease of P. falciparum, metacaspase-2 (MCA-2).

Earlier studies on Plasmodium apoptosis revealed the presence of proteases with caspases like- activity, which are known as "metacaspases". Although this family of cysteine proteases is structurally similar to caspases with Cys-His dyad but their evolutionary significance and functional relevance remains largely unknown. These proteases are considered to be an important target against malaria due to their absence in humans. In this report, we have biochemically characterized metacaspase-2 (PfMCA-2) of P.falciparum. Enzymatic assay showed that PfMCA-2 efficiently cleaved arginine/lysine specific peptide, but not caspase-specific substrate. Consistently, PfMCA-2 activity was sensitive to effector caspases inhibitor, Z-FA-FMK, and mildly inhibited by aprotinin and E-64. However, general caspase inhibitors such as Z-VAD-FMK and Z-DEVD-FMK had no effect on PfMCA-2 activity. Z-FA-FMK inhibits parasite growth with an IC50 value of 2.7 µM along with the notable morphological changes. PfMCA-2 specifically expressed in schizonts and gametocyte stages and there was a notable depletion of PfMCA-2 expression in Z-FA-FMK treated schizonts and gametocytes stages of parasite. Notably, PfMCA-2 cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease) and the proteolysis of PfTSN did not occur after treatment with the Z-FA-FMK. The production of large amount of reactive oxygen species in presence of Z-FA-FMK caused oxidative stress which in turn leads to loss of cell viability. The oxidative stress further generates positive feedback for the occurrence of cell death in term of phosphatidylserine externalization and DNA fragmentation in vitro.

Frequent inoculations with radiation attenuated sporozoite is essential for inducing sterile protection that correlates with a threshold level of Plasmodia liver-stage specific CD8+ T cells.

Whole sporozoite vaccine (WSV) is shown to induce sterile protection that targets Plasmodium liver-stage infection. There are many underlying issues associated with induction of effective sterile protracted protection. In this study, we have addressed how the alterations in successive vaccine regimen could possibly affect the induction of sterile protection. We have demonstrated that the pattern of vaccination with RAS (radiation attenuated sporozoites) induces varying degrees of protection among B6 mice. Animals receiving four successive doses generated 100% sterile protection. However, three successive doses, though with the same parasite inoculum as four doses, could induce sterile protection in ∼50% mice. Interestingly, mice immunized with the same 3 doses, but with longer gap, could not survive the challenge. We demonstrate that degree of protection correlates with the frequencies of IFN-γ+ and multifunctional (IFN-γ+ CD107a+) CD8+ TEM cells present in liver. The failure to achieve protective threshold frequency of these cells in liver might make the host more vulnerable to parasite infection during infectious sporozoite challenge.

Cytokines induce effector T-helper cells during invasive aspergillosis; what we have learned about T-helper cells?

Invasive aspergillosis caused by Aspergillus species (Aspergillus fumigatus, A. flavus, and A. terreus) is life-threatening infections in immunocompromised patients. Understanding the innate and adaptive immune response particularly T-helper cells (TH-cells) against these Aspergillus species and how the different sub-set of TH-cells are regulated by differentiating cytokines at primary target organ site like lung, kidney and brain is of great significance to human health. This review focuses on presentation of Aspergillus through Antigen presenting cells (APCs) to the naive CD4(+) T-cells in the host. The production of differentiating/effector cytokines that activate following TH-cells, e.g., TH1, TH2, TH9, and TH17 has been reported in association or alone in allergic or invasive aspergillosis. Chemokines (CXCL1, CXCL2, CCL1, and CCL20) and their receptors associated to these TH-cells have also been observed in invasive aspergillosis. Thus, further study of these TH-cells in invasive aspergillosis and other elements of adaptive immune response with Aspergillus species are required in order to have a better understanding of host response for safer and effective therapeutic outcome.

Cytokine milieu in renal cavities of immunocompetent mice in response to intravenous challenge of Aspergillus flavus leading to aspergillosis.

Investigations in mice have demonstrated that Aspergillus flavus is more virulent than all other Aspergillus species except A. tamari. However, there is a complete lack of information on the immune responses elicited by A. flavus in systemic model. This communication reports the progression of infection and cytokine profile in BALB/c mice in response to intravenous challenge of A. flavus. The pathogenesis of infection was evaluated morphologically and by the analysis of Colony Forming Units (CFUs) in kidney homogenates. The kinetics of regulated cytokines was determined in kidneys by cytokine-specific murine ELISA. During the initial phase of infection the rate of clearance of A. flavus was high, most likely through recruited neutrophils and the resident renal macrophages with concurrent significant release of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-12/IL-23p40, IL-6) indicating antifungal innate immune response to be active at the site. However, at 24h PI there was a significant rise of IL-17 and IL-23 suggesting the activation of IL-17/IL-23 axis of inflammation resulting in rise of CFU. The lack of significant induction in the anti-inflammatory cytokines like IL-4 and IL-10 confirmed the absence of Th2 type of response. In the late phase, after 3days post-infection, there was a rise in the number of pathogen in the kidneys as determined by histopathology and CFU counts. The A. flavus hyphae were evident in the renal pelvis and ureter and we propose the production of blastoconidia by metamorphosed hyphae.

Targeted deletion of Plasmodium knowlesi Duffy binding protein confirms its role in junction formation during invasion.

Red cell invasion by Plasmodium merozoites involves multiple steps such as attachment, apical reorientation, junction formation and entry into a parasitophorous vacuole. These steps are mediated by specific molecular interactions. P. vivax and the simian parasite P. knowlesi require interaction with the Duffy blood group antigen to invade human erythrocytes. P. vivax and P. knowlesi Duffy binding proteins (PvDBP and PkDBP), which bind the Duffy antigen during invasion, share regions of sequence homology and belong to a family of erythrocyte binding proteins (EBPs). By deletion of the gene that encodes PkDBP, we demonstrate that interaction of PkDBP with the Duffy antigen is absolutely necessary for invasion of human erythrocytes by P. knowlesi. Electron microscopy studies reveal that PkDBP knockout parasites are unable to form a junction with human erythrocytes. The interaction of PkDBP with the Duffy antigen is thus necessary for the critical step of junction formation during invasion. These studies provide support for development of intervention strategies that target EBPs to inhibit junction formation and block erythrocyte invasion by malaria parasites.

Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC).

Plasmodium vivax is one of four Plasmodium species that cause human malaria. P. vivax and a related simian malaria parasite, Plasmodium knowlesi, invade erythrocytes by binding the Duffy antigen/receptor for chemokines (DARC) through their respective Duffy binding proteins. Here we show that tyrosines 30 and 41 of DARC are modified by addition of sulphate groups, and that the sulphated tyrosine 41 is essential for association of the Duffy binding proteins of P. vivax (PvDBP) and P. knowlesi (PkDaBP) with DARC-expressing cells. These sulphated tyrosines also participate in the association of DARC with each of its four known chemokine ligands. Alteration of tyrosine 41 to phenylalanine interferes with MCP-1, RANTES and MGSA association with DARC, but not with that of IL8. In contrast, alteration of tyrosine 30 to phenylalanine interferes with the association of IL8 with DARC. A soluble sulphated amino-terminal domain of DARC, but not one modified to phenylalanine at residue 41, can be used to block the association of PvDBP and PkDaBP with red blood cells, with an IC50 of approximately 5 nM. These data are consistent with a role for tyrosine sulphation in the association of many or most chemokines with their receptors, and identify a key molecular determinant of erythrocyte invasion by P. vivax.