Haemoglobin C, which carries a glutamate-to-lysine mutation in the beta-globin chain, protects West African children against Plasmodium falciparum malaria. Mechanisms of protection are not established for the heterozygous (haemoglobin AC) or homozygous (haemoglobin CC) states. Here we report a marked effect of haemoglobin C on the cell-surface properties of P. falciparum-infected erythrocytes involved in pathogenesis. Relative to parasite-infected normal erythrocytes (haemoglobin AA), parasitized AC and CC erythrocytes show reduced adhesion to endothelial monolayers expressing CD36 and intercellular adhesion molecule-1 (ICAM-1). They also show impaired rosetting interactions with non-parasitized erythrocytes, and reduced agglutination in the presence of pooled sera from malaria-immune adults. Abnormal cell-surface display of the main variable cytoadherence ligand, PfEMP-1 (P. falciparum erythrocyte membrane protein-1), correlates with these findings. The abnormalities in PfEMP-1 display are associated with markers of erythrocyte senescence, and are greater in CC than in AC erythrocytes. Haemoglobin C might protect against malaria by reducing PfEMP-1-mediated adherence of parasitized erythrocytes, thereby mitigating the effects of their sequestration in the microvasculature.

It is thought that both helper and effector functions of CD4+ T cells contribute to protective immunity to blood stage malaria infection. However, malaria infection does not induce long-term immunity and its mechanisms are not defined. In this study, we show that protective parasite-specific CD4+ T cells were depleted after infection with both lethal and nonlethal species of rodent Plasmodium. It is further shown that the depletion is confined to parasite-specific T cells because (a) ovalbumin (OVA)-specific CD4+ T cells are not depleted after either malaria infection or direct OVA antigen challenge, and (b) the depletion of parasite-specific T cells during infection does not kill bystander OVA-specific T cells. A significant consequence of the depletion of malaria parasite–specific CD4+ T cells is impaired immunity, demonstrated in mice that were less able to control parasitemia after depletion of transferred parasite-specific T cells. Using tumor necrosis factor (TNF)-RI knockout– and Fas-deficient mice, we demonstrate that the depletion of parasite-specific CD4+ T cells is not via TNF or Fas pathways. However, in vivo administration of anti–interferon (IFN)-γ antibody blocks depletion, suggesting that IFN-γ is involved in the process. Taken together, these data suggest that long-term immunity to malaria infection may be affected by an IFN-γ–mediated depletion of parasite-specific CD4+ T cells during infection. This study provides further insight into the nature of immunity to malaria and may have a significant impact on approaches taken to develop a malaria vaccine.

Plasmodium falciparum malaria parasites, living in red blood cells, express proteins of the erythrocyte membrane protein-1 (PfEMP1) family on the red blood cell surface. The binding of PfEMP1 molecules to human cell surface receptors mediates the adherence of infected red blood cells to human tissues. The sequences of the 60 PfEMP1 genes in each parasite genome vary greatly from parasite to parasite, yet the variant PfEMP1 proteins maintain receptor binding. Almost all parasites isolated directly from patients bind the human CD36 receptor. Of the several kinds of highly polymorphic cysteine-rich interdomain region (CIDR) domains classified by sequence, only the CIDR1α domains bind CD36. Here we describe the CD36-binding portion of a CIDR1α domain, MC179, as a bundle of three α-helices that are connected by a loop and three additional helices. The MC179 structure, containing seven conserved cysteines and 10 conserved hydrophobic residues, predicts similar structures for the hundreds of CIDR sequences from the many genome sequences now known. Comparison of MC179 with the CIDR domains in the genome of the P. falciparum 3D7 strain provides insights into CIDR domain structure. The CIDR1α three-helix bundle exhibits less than 20% sequence identity with the three-helix bundles of Duffy-binding like (DBL) domains, but the two kinds of bundles are almost identical. Despite the enormous diversity of PfEMP1 sequences, the CIDR1α and DBL protein structures, taken together, predict that a PfEMP1 molecule is a polymer of three-helix bundles elaborated by a variety of connecting helices and loops. From the structures also comes the insight that DBL1α domains are approximately 100 residues larger and that CIDR1α domains are approximately 100 residues smaller than sequence alignments predict. This new understanding of PfEMP1 structure will allow the use of better-defined PfEMP1 domains for functional studies, for the design of candidate vaccines, and for understanding the molecular basis of cytoadherence.

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4 ؉ T cells, have never been defined. We generated CD4 ؉ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-␥, and tumor necrosis factor-␣, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. Nterminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.A lthough it is known that immunity to malaria can be mediated by different immune mechanisms (1-4), to date only target antigens for antibodies have been defined, and these targets are either variant or demonstrate allelic polymorphism (5). CD4 ϩ T cells can adoptively transfer resistance to malaria (3, 6, 7) in rodent models, but target antigens have not been defined. Of interest, CD4 ϩ T cells displaying a Th1 cytokine profile (IL-2-and IFN-␥-secreting) and specific for the major merozoite surface protein 1 (MSP1 19 ) of Plasmodium yoelii (a leading vaccine candidate homologue) are unable to transfer resistance, and immunization of mice with defined T cell epitopes from MSP1 19 did not render the mice resistant (8). Identification of a target antigen(s) would provide an additional vaccine strategy for vaccine design. The goal of this study was to define such target antigens in the rodent model, P. yoelii. Materials and MethodsMice. Four-to 6-week-old normal BALB͞c, athymic BALB͞c nude, and BALB͞c severe combined immunodeficient (SCID) mice were used when 6-8 weeks old.Parasites and Parasitemia. P. yoelii 17XNL was maintained by passaging between infected and uninfected mice via the i.p. route. A thin blood smear was air-dried and stained using Diff-Quick (Lab Aids, Narrabeen, Australia).Preparation of Parasite Antigens. Preparation of whole parasite antigen (pRBC). Parasitized red blood cells (pRBC) in PBS were lysed by incubation in erythrocyte lysis buffer (0.17 M Tris-hydroxymethyl aminomethane͞0.16 M ammonium chloride; pH 7.2) at 37°C for 10 min. The parasites were then freeze-thawed at least three times, sonicated at 4°C to break up the parasites, aliquoted, and stored at Ϫ70°C to be used for in vitro cell cultures. Preparation of soluble parasite antigen (sAg).RBCs were lysed by incubation of the blood in 0.01% saponin͞PBS at 37°C for 20 min in the presence of protease inhibitors (Sigma). The blood was then given another wash in saponin͞PBS buffer before the pRBCs were sonicated in cold PBS at 4°C.After sonication, the lysate was ...

The most evident challenge to treatment of Helicobacter pylori, a bacterium responsible for gastritis, peptic ulcers and gastric cancer, is the increasing rate of resistance to all currently used therapeutic antibiotics. Thus, the development of novel therapies is urgently required. N-geranyl-N'-(2-adamantyl) ethane-1, 2-diamine (SQ109) is an ethylene diamine-based antitubercular drug that is currently in clinical trials for the treatment of tuberculosis (TB). Previous pharmacokinetic studies of SQ109 revealed that persistently high concentrations of SQ109 remain in the stomach 4 hours post oral administration in rats. This finding, combined with the need for new anti- Helicobacter therapies, prompted us to define the in vitro efficacy of SQ109 against H. pylori. Liquid broth micro-dilution was used for susceptibility studies to determine the antimicrobial activity of SQ109 against a total of 6 laboratory strains and 20 clinical isolates of H. pylori; the clinical isolates included a multi-drug resistant strain. All strains tested were susceptible to SQ109 with MIC and MBC ranges of 6-10 µM and 50-60 µM, respectively. SQ109 killing kinetics were concentration- and time-dependent. SQ109 killed H. pylori in 8-10 h at 140 µM (2MBCs) or 4-6 h at 200 µM (~3MBCs). Importantly, though the kinetics of killing were altered, SQ109 retained potent bactericidal activity against H. pylori at low pH. Additionally, SQ109 demonstrated robust thermal stability and was effective at killing slow growing or static bacteria. In fact, pretreatment of cultures with a bacteriostatic concentration of chloramphenicol (Cm) synergized the effects of typically bacteriostatic concentrations of SQ109 to the level of five-logs of bacterial killing. A molar-to-molar comparison of the efficacy of SQ109 as compared to metronidazole (MTZ), amoxicillin (AMX), rifampicin (RIF) and clarithromycin (CLR), revealed that SQ109 was superior to MTZ, AMX and RIF but not to CLR. Finally, the frequency of resistance to SQ109 was low and electron microscopy studies revealed that SQ109 interacted with bacterial inner membrane and cytoplasmic content(s). Collectively, our in vitro data demonstrate that SQ109 is an effective monotherapy against susceptible and multi-drug resistant strains of H. pylori and may be useful alone or in combination with other antibiotics for development as a new class of anti- Helicobacter drugs.

The gastric pathogen Helicobacter pylori has developed resistance to virtually all current antibiotics; thus, there is a pressing need to develop new anti-H. pylori therapies. The goal of this work was to evaluate the antibacterial effect of oligo-acyl-lysyl (OAK) antimicrobial peptidomimetics to determine if they might represent alternatives to conventional antibiotic treatment of H. pylori infection. A total of five OAK sequences were screened for growthinhibitory and/or bactericidal effects against H. pylori strain G27; four of these sequences had growth-inhibitory and bactericidal effects. The peptide with the highest efficacy against strain G27, C 12 K-2␤ 12 , was selected for further characterization against five additional H. pylori strains (26695, J99, 7.13, SS1, and HPAG1). C 12 K-2␤ 12 displayed MIC and minimum bactericidal concentration (MBC) ranges of 6.5 to 26 M and 14.5 to 90 M, respectively, across the six strains after 24 h of exposure. G27 was the most sensitive H. pylori strain (MIC ‫؍‬ 6.5 to 7 M; MBC ‫؍‬ 15 to 20 M), whereas 26695 was the least susceptible strain (MIC ‫؍‬ 25 to 26 M; MBC ‫؍‬ 70 to 90 M). H. pylori was completely killed after 6 to 8 h of incubation in liquid cultures containing two times the MBC of C 12 K-2␤ 12 . The OAK demonstrated strong in vitro stability, since efficacy was maintained after incubation at extreme temperatures (4°C, 37°C, 42°C, 50°C, 55°C, 60°C, and 95°C) and at low pH, although reduced killing kinetics were observed at pH 4.5. Additionally, upon transient exposure to the bacteria, C 12 K-2␤ 12 showed irreversible and significant antibacterial effects and was also nonhemolytic. Our results show a significant in vitro effect of C 12 K-2␤ 12 against H. pylori and suggest that OAKs may be a valuable resource for the treatment of H. pylori infection.Helicobacter pylori is a microaerophilic gram-negative bacterium that colonizes the gastric mucosa. It is known to be a principal gastric pathogen of humans and is associated with the development of gastritis, gastric ulcers, duodenal ulcers, and gastric cancer (46,55,56,60). Approximately half of the world's population is infected with H. pylori (79). Thus, the bacterium poses a significant public health problem, which is further compounded by the fact that H. pylori has developed antimicrobial resistance to virtually all current antibiotics, a phenomenon that is hampering efforts to treat the infection (40, 51).Since the original isolation of H. pylori in the early 1980s, treatment of the bacterial infection has undergone a significant evolutionary development from initial monotherapy to dual, triple, and in more recent trials quadruple therapy (8, 18). Current treatment strategies employ combination therapy, since single-antibiotic therapy often results in failure to eradicate the infection (21). The highest H. pylori eradication rates have been reported with triple therapy, which involves the utilization of two antibiotics in combination with bismuth or a proton pump inhibitor, PPI (34,44). Amoxicillin (amoxicil...

Helicobacter pylori has developed antimicrobial resistance to virtually all current antibiotics. Thus, there is a pressing need to develop new anti-H. pylori therapies. We recently described a novel oligo-acyl-lysyl (OAK) antimicrobial peptidomimetic, C 12 K-2␤ 12 , that shows potent in vitro bactericidal activity against H. pylori. Herein, we define the mechanism of action and evaluate the in vivo efficacy of C 12 K-2␤ 12 against H. pylori after experimental infection of Mongolian gerbils. We demonstrate using a 1-N-phenylnaphthylamine (fluorescent probe) uptake assay and electron microscopy that C 12 K-2␤ 12 rapidly permeabilizes the bacterial membrane and creates pores that cause bacterial cell lysis. Furthermore, using nucleic acid binding assays, Western blots, and confocal microscopy, we show that C 12 K-2␤ 12 can cross the bacterial membranes into the cytoplasm and tightly bind to bacterial DNA, RNA, and proteins, a property that may result in inhibition of enzymatic activities and macromolecule synthesis. To define the in vivo efficacy of C 12 K-2␤ 12 , H. pylori-infected gerbils were orogastrically treated with increasing doses and concentrations of C 12 K-2␤ 12 1 day or 1 week postinfection. The efficacy of C 12 K-2␤ 12 was strongest in animals that received the largest number of doses at the highest concentration, indicating dose-dependent activity of the peptide (P < 0.001 by analysis of variance [ANOVA]) regardless of the timing of the treatment with C 12 K-2␤ 12 . Overall, our results demonstrate a dual mode of action of C 12 K-2␤ 12 against the H. pylori membrane and cytoplasmic components. Moreover, and consistent with the previously reported in vitro efficacy, C 12 K-2␤ 12 shows significant in vivo efficacy against H. pylori when used as monotherapy. Therefore, OAK peptides may be a valuable resource for therapeutic treatment of H. pylori infection.

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