Investigations into the Metabolic Requirements for Lipoic Acid and Lipid Species During the Life Cycle of the Malarial Parasite Plasmodium Berghei

Plasmodium, like many other pathogenic organisms, relies on a balance of synthesis and scavenging of lipid species for replication. How the parasite creates this balance is particularly important to successfully intervene in transmission of the disease and to generate new chemotherapies to cure infections. This study focuses on two specific aspects in the field of lipid biology of Plasmodium parasites and their hosts. Lipoic acid is a short eight-carbon chain that serves a number of different functions with the cell. By disrupting a key enzyme in the lipoic acid synthesis pathway in the rodent species of malaria, Plasmodium berghei, we sought to investigate its role during the parasite life-cycle. Deletion of the lipoyl-octanoyl transferase enzyme, LipB in P. berghei parasites demonstrate a liver-stage specific need for this metabolic pathway. In order to explore the impact of the fatty acid and triglyceride content on the pathogenesis of Plasmodium parasites, this study tests two methods to reduce lipid content in vivo and test the propagation of P. berghei parasite in these environments. Results from this study set forth new avenues of research with implications for the development of novel antimalarials and vaccine candidates

Oleic Acid Biosynthesis in Plasmodium falciparum: Characterization of the Stearoyl-CoA Desaturase and Investigation as a Potential Therapeutic Target

BACKGROUND:Plasmodium falciparum parasitization of erythrocytes causes a substantial increase in the levels of intracellular fatty acids, notably oleic acid. How parasites acquire this monounsaturated fatty acid has remained enigmatic. Here, we report on the biochemical and enzymatic characterization of stearoyl-CoA desaturase (SCD) in P. falciparum. METHODOLOGY/PRINCIPAL FINDINGS:Metabolic labeling experiments allowed us to demonstrate the production of oleic acid from stearic acid both in lysates of parasites incubated with [(14)C]-stearoyl-CoA and in parasite-infected erythrocytes labeled with [(14)C]-stearic acid. Optimal SCD activity was detected in schizonts, the stage of maximal membrane synthesis. This activity correlated with a late trophozoite stage-specific induction of PFE0555w transcripts. PFE0555w harbors a typical SCD signature. Similar to mammalian SCDs, this protein was found to be associated with the endoplasmic reticulum, as determined with PFE0555w-GFP tagged transgenic P. falciparum. Importantly, these parasites exhibited increased rates of stearic to oleic acid conversion, providing additional evidence that PFE0555w encodes the plasmodial SCD (PfSCD). These findings prompted us to assess the activity of sterculic acid analogues, known to be specific Delta9-desaturase inhibitors. Methyl sterculate inhibited the synthesis of oleic acid both with parasite lysates and infected erythrocytes, most likely by targeting PfSCD. This compound exhibited significant, rapid and irreversible antimalarial activity against asexual blood stages. This parasiticidal effect was antagonized by oleic acid. CONCLUSION/SIGNIFICANCE:Our study provides evidence that parasite-mediated fatty acid modification is important for blood-stage survival and provides a new strategy to develop a novel antimalarial therapeutic based on the inhibition of PfSCD

Parasite-induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection

Upon infection of a mammalian host, Plasmodium parasites first replicate inside hepatocytes, generating thousands of new parasites. Although Plasmodium intra-hepatic development represents a substantial metabolic challenge to the host hepatocyte, how infected cells respond to and integrate this stress remains poorly understood. Here, we present proteomic and transcriptomic analyses, revealing that the endoplasmic reticulum (ER)-resident unfolded protein response (UPR) is activated in host hepatocytes upon Plasmodium berghei infection. The expression of XBP1s-the active form of the UPR mediator XBP1-and the liver-specific UPR mediator CREBH is induced by P. berghei infection in vivo. Furthermore, this UPR induction increases parasite liver burden. Altogether, our data suggest that ER stress is a central feature of P. berghei intra-hepatic development, contributing to the success of infection

Antibody Secreting Cell Responses following Vaccination with Bivalent Oral Cholera Vaccine among Haitian Adults

Background: The bivalent whole-cell (BivWC) oral cholera vaccine (Shanchol) is effective in preventing cholera. However, evaluations of immune responses following vaccination with BivWC have been limited. To determine whether BivWC induces significant mucosal immune responses, we measured V. cholerae O1 antigen-specific antibody secreting cell (ASC) responses following vaccination. Methodology/Principal Findings We enrolled 24 Haitian adults in this study, and administered doses of oral BivWC vaccine 14 days apart (day 0 and day 14). We drew blood at baseline, and 7 days following each vaccine dose (day 7 and 21). Peripheral blood mononuclear cells (PBMCs) were isolated, and ASCs were enumerated using an ELISPOT assay. Significant increases in Ogawa (6.9 cells per million PBMCs) and Inaba (9.5 cells per million PBMCs) OSP-specific IgA ASCs were detected 7 days following the first dose (P < 0.001), but not the second dose. The magnitude of V. cholerae-specific ASC responses did not appear to be associated with recent exposure to cholera. ASC responses measured against the whole lipolysaccharide (LPS) antigen and the OSP moiety of LPS were equivalent, suggesting that all or nearly all of the LPS response targets the OSP moiety. Conclusions/Significance: Immunization with the BivWC oral cholera vaccine induced ASC responses among a cohort of healthy adults in Haiti after a single dose. The second dose of vaccine resulted in minimal ASC responses over baseline, suggesting that the current dosing schedule may not be optimal for boosting mucosal immune responses to V. cholerae antigens for adults in a cholera-endemic area

Inhibition of HIV-1 replication by eIF3f

Viruses often use host machinery in unusual ways to execute different steps during their replication. To identify host factors critical for virus replication, we screened cDNA expression libraries for genes or gene fragments that could interfere with HIV-1 vector transduction. The DNA clone that most potently inhibited HIV-1 expression encoded the N-terminal 91 aa of the eukaryotic initiation factor 3 subunit f (N91-eIF3f). Overexpression of N91-eIF3f or full-length eIF3f drastically restricted HIV-1 replication by reducing nuclear and cytoplasmic viral mRNA levels. N91-eIF3f and eIF3f specifically targeted the 3′ long terminal repeat (3′LTR) region in the viral mRNA. We show that the 3′ end cleavage of HIV-1 mRNA precursors is specifically reduced in N91-eIF3f expressing cells. Our results suggest a role of eIF3f in mRNA maturation and that it can specifically interfere with the 3′ end processing of HIV-1 mRNAs

Plasma Leptin Levels in Children Hospitalized with Cholera in Bangladesh

Vibrio cholerae, the cause of cholera, induces both innate and adaptive immune responses in infected humans. Leptin is a hormone that plays a role in both metabolism and mediating immune responses. We characterized leptin levels in 11 children with cholera in Bangladesh, assessing leptin levels on days 2, 7, 30, and 180 following cholera. We found that patients at the acute stage of cholera had significantly lower plasma leptin levels than matched controls, and compared with levels in late convalescence. We then assessed immune responses to V. cholerae antigens in 74 children with cholera, correlating these responses to plasma leptin levels on day 2 of illness. In multivariate analysis, we found an association between day 2 leptin levels and development of later anti-cholera toxin B subunit (CtxB) responses. This finding appeared to be limited to children with better nutritional status. Interestingly, we found no association between leptin levels and antibody responses to V. cholerae lipopolysaccharide, a T cell–independent antigen. Our results suggest that leptin levels may be associated with cholera, including the development of immune responses to T cell–dependent antigens

Bivalent oral cholera vaccination induces a memory B cell response to the V. cholerae O1-polysaccharide antigen in Haitian adults.

The bivalent killed whole-cell oral cholera vaccine (BivWC) is being increasingly used to prevent cholera. The presence of O-antigen-specific memory B cells (MBC) has been associated with protective immunity against cholera, yet MBC responses have not been evaluated after BivWC vaccination. To address this knowledge gap, we measured V. cholerae O1-antigen MBC responses following BivWC vaccination. Adults in St. Marc, Haiti, received 2 doses of the BivWC vaccine, Shanchol, two weeks apart. Participants were invited to return at days 7, 21, 44, 90, 180 and 360 after the initial vaccination. Serum antibody and MBC responses were assessed at each time-point before and following vaccination. We observed that vaccination with BivWC resulted in significant O-antigen specific MBC responses to both Ogawa and Inaba serotypes that were detected by day 21 and remained significantly elevated over baseline for up to 12 months following vaccination. The BivWC oral cholera vaccine induces durable MBC responses to the V. cholerae O1-antigen. This suggests that long-term protection observed following vaccination with BivWC could be mediated or maintained by MBC responses