Martha A. Clark

Host iron status and iron supplementation mediate susceptibility to erythrocytic stage plasmodium falciparum

Iron deficiency and malaria have similar global distributions, and frequently co-exist in pregnant women and young children. Where both conditions are prevalent, iron supplementation is complicated by observations that iron deficiency anaemia protects against falciparum malaria, and that iron supplements increase susceptibility to clinically significant malaria, but the mechanisms remain obscure. Here, using an in vitro parasite culture system with erythrocytes from iron-deficient and replete human donors, we demonstrate that Plasmodium falciparum infects iron-deficient erythrocytes less efficiently. In addition, owing to merozoite preference for young erythrocytes, iron supplementation of iron-deficient individuals reverses the protective effects of iron deficiency. Our results provide experimental validation of field observations reporting protective effects of iron deficiency and harmful effects of iron administration on human malaria susceptibility. Because recovery from anaemia requires transient reticulocytosis, our findings imply that in malarious regions iron supplementation should be accompanied by effective measures to prevent falciparum malaria.

Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin α-chain crosslinking.

Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size.

Influence of host iron status on Plasmodium falciparum infection

Iron deficiency affects one quarter of the worlds population and causes significant morbidity, including detrimental effects on immune function and cognitive development. Accordingly, the World Health Organization (WHO) recommends routine iron supplementation in children and adults in areas with a high prevalence of iron deficiency. However, a large body of clinical and epidemiological evidence has accumulated which clearly demonstrates that host iron deficiency is protective against falciparum malaria and that host iron supplementation may increase the risk of malaria. Although many effective antimalarial treatments and preventive measures are available, malaria remains a significant public health problem, in part because the mechanisms of malaria pathogenesis remain obscured by the complexity of the relationships that exist between parasite virulence factors, host susceptibility traits, and the immune responses that modulate disease. Here we review (i) the clinical and epidemiological data that describes the relationship between host iron status and malaria infection and (ii) the current understanding of the biological basis for these clinical and epidemiological observations.

Parasite maturation and host serum iron influence the labile iron pool of erythrocyte stage Plasmodium falciparum

Iron is a critical and tightly regulated nutrient for both the malaria parasite and its human host. The importance of the relationship between host iron and the parasite has been underscored recently by studies showing that host iron supplementation may increase the risk of falciparum malaria. It is unclear what host iron sources the parasite is able to access. We developed a flow cytometry‐based method for measuring the labile iron pool (LIP) of parasitized erythrocytes using the nucleic acid dye STYO 61 and the iron sensitive dye, calcein acetoxymethyl ester (CA‐AM). This new approach enabled us to measure the LIP of P. falciparum through the course of its erythrocytic life cycle and in response to the addition of host serum iron sources. We found that the LIP increases as the malaria parasite develops from early ring to late schizont stage, and that the addition of either transferrin or ferric citrate to culture media increases the LIP of trophozoites. Our method for detecting the LIP within malaria parasitized RBCs provides evidence that the parasite is able to access serum iron sources as part of the host vs. parasite arms race for iron.

Antimalarial Iron Chelator, FBS0701, Shows Asexual and Gametocyte Plasmodium falciparum Activity and Single Oral Dose Cure in a Murine Malaria Model

Iron chelators for the treatment of malaria have proven therapeutic activity in vitro and in vivo in both humans and mice, but their clinical use is limited by the unsuitable absorption and pharmacokinetic properties of the few available iron chelators. FBS0701, (S)3”-(HO)-desazadesferrithiocin-polyether [DADFT-PE], is an oral iron chelator currently in Phase 2 human studies for the treatment of transfusional iron overload. The drug has very favorable absorption and pharmacokinetic properties allowing for once-daily use to deplete circulating free iron with human plasma concentrations in the high µM range. Here we show that FBS0701 has inhibition concentration 50% (IC50) of 6 µM for Plasmodium falciparum in contrast to the IC50 for deferiprone and deferoxamine at 15 and 30 µM respectively. In combination, FBS0701 interfered with artemisinin parasite inhibition and was additive with chloroquine or quinine parasite inhibition. FBS0701 killed early stage P. falciparum gametocytes. In the P. berghei Thompson suppression test, a single dose of 100 mg/kg reduced day three parasitemia and prolonged survival, but did not cure mice. Treatment with a single oral dose of 100 mg/kg one day after infection with 10 million lethal P. yoelii 17XL cured all the mice. Pretreatment of mice with a single oral dose of FBS0701 seven days or one day before resulted in the cure of some mice. Plasma exposures and other pharmacokinetics parameters in mice of the 100 mg/kg dose are similar to a 3 mg/kg dose in humans. In conclusion, FBS0701 demonstrates a single oral dose cure of the lethal P. yoelii model. Significantly, this effect persists after the chelator has cleared from plasma. FBS0701 was demonstrated to remove labile iron from erythrocytes as well as enter erythrocytes to chelate iron. FBS0701 may find clinically utility as monotherapy, a malarial prophylactic or, more likely, in combination with other antimalarials.

A Quality Control Program within a Clinical Trial Consortium for PCR Protocols To Detect Plasmodium Species

ABSTRACT Malaria parasite infections that are only detectable by molecular methods are highly prevalent and represent a potential transmission reservoir. The methods used to detect these infections are not standardized, and their operating characteristics are often unknown. We designed a proficiency panel of Plasmodium spp. in order to compare the accuracy of parasite detection of molecular protocols used by labs in a clinical trial consortium. Ten dried blood spots (DBSs) were assembled that contained P. falciparum, P. vivax, P. malariae, and P. ovale; DBSs contained either a single species or a species mixed with P. falciparum. DBS panels were tested in 9 participating laboratories in a masked fashion. Of 90 tests, 68 (75.6%) were correct; there were 20 false-negative results and 2 false positives. The detection rate was 77.8% (49/63) for P. falciparum, 91.7% (11/12) for P. vivax, 83.3% (10/12) for P. malariae, and 70% (7/10) for P. ovale. Most false-negative P. falciparum results were from samples with an estimated ≤5 parasites per μl of blood. Between labs, accuracy ranged from 100% to 50%. In one lab, the inability to detect species in mixed-species infections prompted a redesign and improvement of the assay. Most PCR-based protocols were able to detect P. falciparum and P. vivax at higher densities, but these assays may not reliably detect parasites in samples with low P. falciparum densities. Accordingly, formal quality assurance for PCR should be employed whenever this method is used for diagnosis or surveillance. Such efforts will be important if PCR is to be widely employed to assist malaria elimination efforts.

The development of ciprofloxacin resistance in Pseudomonas aeruginosa involves multiple response stages and multiple proteins

ABSTRACT Microbes have developed resistance to nearly every antibiotic, yet the steps leading to drug resistance remain unclear. Here we report a multistage process by which Pseudomonas aeruginosa acquires drug resistance following exposure to ciprofloxacin at levels ranging from 0.5× to 8× the initial MIC. In stage I, susceptible cells are killed en masse by the exposure. In stage II, a small, slow to nongrowing population survives antibiotic exposure that does not exhibit significantly increased resistance according to the MIC measure. In stage III, exhibited at 0.5× to 4× the MIC, a growing population emerges to reconstitute the population, and these cells display heritable increases in drug resistance of up to 50 times the original level. We studied the stage III cells by proteomic methods to uncover differences in the regulatory pathways that are involved in this phenotype, revealing upregulation of phosphorylation on two proteins, succinate-semialdehyde dehydrogenase (SSADH) and methylmalonate-semialdehyde dehydrogenase (MMSADH), and also revealing upregulation of a highly conserved protein of unknown function. Transposon disruption in the encoding genes for each of these targets substantially dampened the ability of cells to develop the stage III phenotype. Considering these results in combination with computational models of resistance and genomic sequencing results, we postulate that stage III heritable resistance develops from a combination of both genomic mutations and modulation of one or more preexisting cellular pathways.

RBC Barcoding Allows for the Study of Erythrocyte Population Dynamics and P. falciparum Merozoite Invasion

Plasmodium falciparum invasion of host erythrocytes is essential for the propagation of the blood stage of malaria infection. Additionally, the brief extracellular merozoite stage of P. falciparum represents one of the rare windows during which the parasite is directly exposed to the host immune response. Therefore, efficient invasion of the host erythrocyte is necessary not only for productive host erythrocyte infection, but also for evasion of the immune response. Host traits, such as hemoglobinopathies and differential expression of erythrocyte invasion ligands, can protect individuals from malaria by impeding parasite erythrocyte invasion. Here we combine RBC barcoding with flow cytometry to study P. falciparum invasion. This novel high-throughput method allows for the (i) direct comparison of P. falciparum invasion into different erythrocyte populations and (ii) assessment of the impact of changing erythrocyte population dynamics on P. falciparum invasion.

Biopreservation of RBCs for in vitro Plasmodium falciparum culture

Falciparum malaria remains a devastating infectious disease, killing nearly 700 000 people annually. Further understanding of malaria pathogenesis will help identify molecular and cellular targets of next-generation therapeutics. The morbidity and mortality of malaria infection occur during the erythrocytic stage. Study of erythrocytic stage malaria is critical not only for new anti-malarial development, but also for increasing our understanding of the host pathogen relationship. For example, many host genetic polymorphisms known to impact red blood cell (RBC) physiology also alter malaria susceptibility. Methods for culturing erythrocytic stage Plasmodium falciparum were developed more than 30 years ago (Jensen & Trager, 1977). However, optimization of in vitro P. falciparum culture is still under investigation. The aspects of intraerythrocytic development that are impacted by RBC storage, as well as the effects of RBC biopreservation on the intraerythrocytic life cycle, remain unknown. Studies on RBC storage for human clinical use reveal a relationship between RBC storage and transfusion complications (Aubron et al, 2013). Current blood banking standards involve RBC storage in SAGM (a solution containing saline, adenine, mannitol and glucose) or closely related solutions for up to 42 d at 4°C (Sparrow, 2012). Many RBC storage lesions have been documented in these acidic medias, including decreased intracellular ATP, 2,3-diphosphoglycerate (2,3-DPG) and potassium; increased intracellular NaCl; oxidative damage; lipid peroxidation; membrane phospholipid changes and vesiculation; decreased deformability; reduced glycolytic capacity; decreased vasodilatory capacity; and increased cytoadhesion (Bennett-Guerrero et al, 2007; Aubron et al, 2013). Overall, these storage lesions are similar to physiological changes occurring with normal RBC ageing in the bloodstream (Franco et al, 2013) and could also impact parasite growth, as P. falciparum preferentially infects younger RBCs in circulation (Lim et al, 2013; Clark et al, 2014a). The use of biopreserved RBCs for human transfusion has been validated (Fabricant et al, 2013) and cryopreserved umbilical cord blood cells can propagate Plasmodium vivax (Borlon et al, 2012). Here we examine the impact of RBC storage and biopreservation on P. falciparum growth and development in vitro. Prolonged RBC shelf-life and biopreservation could enhance malaria research by: (i) enabling standardization of the RBC source for multiple experiments, and (ii) increasing access to RBCs from individuals with unusual blood types, nutritional deficiencies, or from remote locations. Given lingering discrepancies in standard P. falciparum culture protocols, we sought to definitively assess RBC shelflife. To begin, fresh RBCs were collected into acid citrate dextrose (ACD) and stored in aliquots as packed RBCs at 4°C for up to 6 weeks. At 2-week intervals, fresh RBCs were obtained and identically stored to allow for simultaneous comparisons of parasite growth in vitro using blood stored for 0, 2, 4 and 6 weeks. Growth in RBCs stored for 2 weeks showed no decrease in standard 96-h growth assays (Clark et al, 2014a). After 4 weeks of storage, growth rates diminished significantly (42% decline for Dd2, 65% for FCR3FMG). In RBCs stored for 6 weeks, there was very little growth (over 90% decline for both strains Dd2 and FCR3-FMG) (Fig 1A). We next compared parasite growth in RBCs stored for 0, 2 and 4 weeks at 4°C in four different storage buffers. Buffers tested were: (i) ACD; (ii) citrate-phosphate-dextrose-adenine (CPDA), commonly used for malaria culture; (iii) Alsever’s Solution, a balanced salt solution routinely used for RBC washing prior to parasite culture; and (iv) “RBC buffer” an alternative balanced salt solution. Growth rates decreased proportionally to storage length in each of the buffers, with a significant decrease after 4 weeks of storage (62% for ACD, 56% for CPDA, 54% for Alsever’s and 51% for “RBC buffer”) (Fig 1B). This confirms RBCs destined for parasite culture must be used within 2 weeks of collection and that differential storage media does not prolong their shelf-life for P. falciparum culture. We next sought to determine whether parasite replication and/or invasion were decreased in stored RBCs. To assess replication, we measured the parasite erythrocyte multiplication rate (PEMR), which reflects the number of infectious merozoites produced per schizont (Clark et al, 2014a). We found no statistically significant differences in replication between RBCs stored for 0–4 weeks (Fig 1C). Invasion rates were assayed using a RBC barcoding assay (Clark et al, 2014b) in which differentially labelled RBCs (with CellTrace membrane dyes; Life Technologies Corp., Grand Island, NY, USA) were combined in the same wells and seeded with unlabeled trophozoite stage parasitized RBCs. This assay allows direct comparison of parasite invasion into two different RBC populations. Invasion rates decreased as RBC storage time increased (Fig 1D). We hypothesize that this Correspondence

In vitro and in vivo antimalarial activity of amphiphilic naphthothiazolium salts with amine-bearing side chains.

Because of emerging resistance to existing drugs, new chemical classes of antimalarial drugs are urgently needed. We have rationally designed a library of compounds that were predicted to accumulate in the digestive vacuole and then decrystallize hemozoin by breaking the iron carboxylate bond in hemozoin. We report the synthesis of 16 naphthothiazolium salts with amine-bearing side chains and their activities against the erythrocytic stage of Plasmodium falciparum in vitro. KSWI-855, the compound with the highest efficacy against the asexual stages of P. falciparum in vitro, also had in vitro activity against P. falciparum gametocytes and in vivo activity against P. berghei in a murine malaria model.