Production of Ghosts with Gs Signaling and Malarial Infection that Closely Mimic Normal Erythrocytes
As summarized in Methods
and in , ghosts were prepared by dialysis of erythrocytes in a low MWCO membrane, simultaneously equilibrated with cargo and then resealed in a potassium-rich buffer in the presence of ATP (1 mM) and GTP (1 mM), and finally washed successively in RPMI and cRPMI and used as described below. Non-outdated blood was used to make erythrocyte ghosts; freshly drawn erythrocytes were used for cAMP and malarial infection assays. These resealed ghosts morphologically resembled normal biconcave erythrocytes (A). Early reports suggested that “white” ghosts depleted of hemoglobin remain permeable to small molecules [9
]. However, ghosts prepared by our procedure contained 40%–50% of starting hemoglobin and maintained the millimolar levels of ATP in which they were prepared, indicating that, at a minimum, they retained critical energy pools. These levels were half of those measured in intact erythrocytes (B), but within the range of ATP levels reported for erythrocytes [20
]. Clinical hematogram analysis of a ghost preparation suggested that they contained less hemoglobin (lower MCH), were slightly microcytic (lower MCV), and were more heterogeneous (higher RDW) than normal, intact erythrocytes (B). The ghosts could be efficiently and homogenously loaded with a variety of experimental cargoes, such as high molecular weight rhodamine-labeled antibody (C), as well as with smaller cargoes (not shown) such as low molecular weight LY (457 g/mol), 11-residue FITC-labeled Gs
peptides, 10-kDa LY-D, and 70-kDa FITC-dextran. Analyses of ghosts loaded with 70-kDa FITC-dextran by flow cytometry indicated that >99% of all cells were homogenously loaded with cargo (D). Further tests of loading and retention of purified GST suggested that proteinaceous cargoes at 10–20 μM could be reliably encapsulated within ghosts and retained under culture conditions; this GST was resistant to degradation by exogenous protease (E). These results indicated that ghosts stably maintain nucleotide pools and loaded cargoes.
Hematological and Signaling Characteristics and Cargo Loading of Erythrocyte Ghosts
One of our major interests in developing reconstituted erythrocyte ghosts was to study β-adrenergic signaling and its regulation by Gs
. This investigation required the presence of active β-adrenergic signaling in ghosts. As shown in F and G, cAMP production stimulated in ghosts by 10 μM isoproterenol (an agonist of β-ARs) was comparable to that seen in normal erythrocytes. This stimulation was quenched by the addition of equimolar racemic propranolol (the antagonist) in both cell types. Propranolol alone did not reduce basal cAMP levels in either cell type. The basal and isoproterenol-stimulated cAMP levels were similar to those previously measured in erythrocytes [22
]. These data show that the β-adrenergic signaling pathway is active in these ghosts.
As we were also interested in testing the effects of signaling on malarial infection in ghosts, we next characterized ghosts for their ability to support malarial infection. The intraerythrocytic lifecycle of malaria begins when extracellular merozoites rapidly (<2 min) invade mature erythrocytes. Intracellular ring-stage parasites result and subsequently enlarge into trophozoites. Schizogony follows when an average of 16 daughter parasites assemble and finally lyse out of infected cells to invade new erythrocytes. Therefore, ring formation provides a measurement of invasion and early development, while the appearance of trophozoites and schizonts indicates parasite maturation within the erythrocyte. In P. falciparum, this intracellular cycle lasts 48 h.
As shown, the resealed ghosts were infected by P. falciparum (A). Remarkably, infection occurred with the same efficiency as in normal erythrocytes, and ghosts supported normal parasite maturation at both low and high parasitemias (B). In cultures containing equal numbers of ghosts and normal erythrocytes, no preference was observed for invasion or growth in either cell type (C). Furthermore, parasites in ghost cultures appeared morphologically similar to their counterparts in normal erythrocytes at the three distinct ring, trophozoite, and schizont stages (D). Schizonts that mature in ghosts are capable of rupturing and reinvading ghosts (E). Furthermore, parasites grown in either ghosts or normal erythrocytes when sub-cultured into normal erythrocytes showed no difference in growth over the next infective cycle (E). Over three life cycles, completion of maturation of parasites in ghosts can extend from 48 to 54 h per cycle (unpublished data). We do not know why this occurs, but it is within an acceptable range of the asexual life cycle in normal P. falciparum cultures.
Erythrocyte Ghosts Support Malarial Invasion and Growth
Collectively, these data show that erythrocyte ghosts, active in supporting β-adrenergic signaling and malarial invasion and growth, can be produced and stably loaded with exogenous cargo. Remarkably, the ghosts are essentially equivalent to normal erythrocytes in a number of critical respects, suggesting they may be useful for investigating multiple functions of the human erythrocyte and its infection by malaria parasites.
Targeting Erythrocyte Gs
Our previous studies had shown that a peptide designed to inhibit Gs
protein interaction with its receptors blocked malarial entry into erythrocytes [7
]. However, since the peptide does not diffuse into normal erythrocytes, there was no evidence that it blocked endogenous Gs
function in the red cell. In addition, erythrocytes are terminally differentiated cells, their signaling mechanisms are not well understood, and direct evidence that erythrocyte G proteins are active is not available. We therefore introduced the Gs
peptides into ghosts to establish whether it acted on its designated target Gs
. As shown in A, Gs
peptide blocked cAMP production in response to the β-AR agonist, isoproterenol. The extent of this inhibition was comparable to that seen with propranolol, a β-adrenergic antagonist that blocks Gs
signaling. This inhibitory effect was achieved at 40 μM Gs
peptide, which is known to be effective at 10–50 μM in other cell types [24
]. In contrast, the control scrambled peptide (Gscr
) had no effect on cAMP production stimulated by isoproterenol. These data establish that β-adrenergic signaling in erythrocytes is mediated through Gs
and that the Gs
peptide directly targets and blocks activation of erythrocyte Gs
Targeting Erythrocyte Gs Down-Regulates β-Adrenergic Signaling and Inhibits Malarial Infection
To directly link this inhibition of host Gs
to malarial infection, we assayed the effect of the peptide on parasite invasion and intracellular maturation in ghosts. As shown in B, Gs
peptide blocked malarial invasion at low micromolar concentrations. Importantly, there was a marked reduction in infection at the same peptide concentration (40 μM) that blocked Gs
activation. In contrast, control Gscr
peptide had no effect on parasite invasion into ghosts, even at a higher concentration (up to 400 μM, B). In addition, peptides designed to block Gi
proteins also had no effect on invasion (Figure S1
). These data establish that the Gs
peptide can block malarial entry at the same concentrations that quench receptor-mediated Gs
signaling. However, invasion is not completely abolished, suggesting that there may be additional mechanisms that regulate entry. Thus, if Gs
only regulates invasion, it may not provide a satisfactory target for development of antimalarials.
Our results presented in suggest that ghosts support normal invasion and intracellular development of parasites, and thus allow us to examine erythrocyte functions needed in malarial entry as well as intracellular growth. To determine the requirement of Gs signaling for intraerythrocytic parasite growth, we followed the development of rings formed in the presence of 40 μM peptide to schizogony. As shown in C, at this concentration of peptide, there was no measurable effect on trophozoites formed, but the number of schizonts detected was reduced. Since maturing trophozoites are known to actively ingest as much as 80% of the erythrocyte cytoplasm, the net intraerythrocytic peptide concentration at schizont stages of growth may be lower. Nonetheless, the data presented in B and C provide a pharmacological link between the inhibition of host erythrocyte signaling, malaria parasite invasion, and intraerythrocytic development.
A second, independent way of down-regulating Gs
signaling in erythrocytes is by using β-antagonists. The β-blocker propranolol was previously known to inhibit isoproterenol-mediated stimulation of invasion at ~10 μM [7
], but effects on P. falciparum
intracellular maturation and growth were not studied. In standard growth assays, we found that propranolol inhibited [3
H]-labeled hypoxanthine uptake by half (IC50
) at 1.2 μM and by 90% inhibitory concentration (IC90
) at 7.1 μM (A). Since hypoxanthine incorporation is a standard measure of parasite proliferation, the data confirm that, in addition to invasion, propranolol can block one or more processes needed for intracellular parasite survival. The inactive isomer of propranolol was not inhibitory, suggesting that the antimalarial effect of racemic propranolol is due to specific down-regulation of erythrocyte Gs
signaling. Examination of Giemsa-stained slides from treated cultures (B) showed that 2 μM propranolol blocked intracellular maturation of parasites to the schizont stage. At 2 μM, propranolol had no significant effect on invasion, which is consistent with our earlier data indicating that significant inhibition of invasion required ~10 μM drug [7
]. Hence, the observed IC50
of 1.2 μM suggests that most of the inhibitory activity of propranolol is due to blockage of intracellular parasite maturation. Other β2
-antagonists such as ICI118,551 and alprenolol also blocked parasite growth, as measured by hypoxanthine incorporation (C). One β1
–antagonist, nadolol, was ineffective at 10 μM. All three β1
-specific antagonists had no effect, and this may reflect a lack of β1
-adrenergic receptors on the erythrocyte. The three most active β-antagonists (including propranolol) appear to show IC50
values of between 1 and 10 μM. Testing additional β-antagonists (particularly β2
antagonists) may yield additional active inhibitors.
β-Blockers Inhibit Maturation of P. falciparum in In Vitro Cultures
To examine intraerythrocytic process(es) that may be inhibited by propranolol treatment, we tested several general functions of malaria parasites. First, we tested the sensitivity of control and propranolol-treated parasites to lysis in the presence of 5% sorbitol, which is known to be actively imported into trophozoite- and schizont-stage parasites—likely via the plasmodial nutrient transport channel PESAC [25
]. No difference was found between control and drug-treated parasites (A), suggesting that maturation to the trophozoite stage occurred in both 2 and 10 μM propranolol-treated infected cells. Second, export of a parasite-encoded protein PfHRPII fused to GFP to the erythrocyte [13
] was not blocked by either 2 or 10 μM propranolol (B), suggesting that the drug did not act by altering protein export to host cells. Nonetheless, examination of the morphology of 2 μM propranolol-treated parasites at 44 h post-invasion (C) revealed a lack of the segmented, mature schizonts characteristically found in control cultures at 44 h. Control cultures also showed the presence of new rings (marked by an asterisk in B) indicating their acceleration past segmented schizonts to new rings. (This variation of the 48-h intraerythrocytic cycle is frequently observed in culture.) The effect of propranolol on parasite morphology was greater at 10 μM (compared to 2 μM), but the precise nature of one or more steps regulated by Gs
signaling and needed for maturation to the schizont stage remains unknown.
Analysis of the Maturation Defect in Propranolol-Treated Parasites
Inhibitors of Erythrocyte Gs Signaling May Be Useful as Novel Antimalarial Therapeutics
The emergence of drug-resistant parasites has led to the need for combinations of two or more antimalarials to improve microbial killing and reduce further resistance. Propranolol was therefore combined and tested with two widely used antimalarials, chloroquine and artemisinin. IC50
values were first independently determined for each individual drug in P. falciparum
3D7 and/or FCB strains (Figure S2
). Using those values, fixed ratios of propranolol:chloroquine (or propranolol:artemisinin) were tested by in vitro hypoxanthine assays [16
]. When used in combination against P. falciparum
strain 3D7, propranolol reduced the amount of chloroquine required to achieve an IC90
dose by >1 log (A and Table S1
; the 3:1 and 1:1 propranolol:chloroquine combinations had 11- and 4-fold effects, respectively). Isobologram analysis (Figure S3
) indicated that there was an additive antimalarial effect between propranolol and chloroquine. Similar results were found for this combination against the more resistant P. falciparum
FCB (B and S3
). Propranolol did not reverse chloroquine resistance, but did reduce the effective dose in the resistant (FCB) and sensitive (3D7) strains. This result was different than the well-known chloroquine reversal seen in previous fixed-ratio drug combinations using chloroquine and the calcium-channel blocker verapamil [16
]. Importantly, this β-blocker did not antagonize chloroquine against either strain, suggesting that blocking Gs
function may enable utilization of chloroquine at lower concentrations and thus may facilitate its use against resistant parasites.
Propranolol Inhibits Malarial Growth in Combination with Existing Antimalarial Drugs
Artemisinin is a newer, schizonticidal antimalarial that is gaining acceptance for treatment of severe and uncomplicated malaria because of its rapid action and lower prevalence of resistance [26
]. Its mode of action is thought to be distinct from that of chloroquine [28
]. When used in combination, propranolol also reduced the amount of artemisinin required to achieve an effective dose 5-fold (C) and acted in a potent, additive fashion (Figure S3
; Table S1
). Since propranolol was additive with two distinct drugs, it may be effective when combined with a range of existing antimalarials in order to also reduce their required doses.
Our previous results suggested that the requirement for host signaling via β-ARs and Gs
was conserved across parasite species. We therefore further tested propranolol-containing drug combinations in the P. berghei
ANKA mouse model of malarial infection. When tested alone in mice, the IC50
of racemic propranolol was 7.5 mg/kg/d [7
]. As shown in D and S3
, a combination of 2:1 propranolol:chloroquine reduced the IC50
of chloroquine from 1.64 to 0.66 mg/kg/d. The 1:1 and 1:2 propranolol:chloroquine combinations had less effect. This finding is in contrast to our results in vitro where 1:1 propranolol:chloroquine combinations had a 4-fold effect. This discrepancy may be due to the fact that the half-life of propranolol is 3–4 h [29
], and therefore multiple treatments and/or more stable compounds may prove more effective. Furthermore, since mice metabolize drugs at higher levels than humans [31
], mouse-derived data likely overestimate the amount of drug required in humans. Importantly, as in in vitro studies, the combinations tested were not antagonistic (Figure S3
Thus, both our in vitro and in vivo data suggest that targeting erythrocyte Gs
in combination with existing antimalarial drugs may have therapeutic potential and may reduce the amount of existing drug needed to effectively treat patients.
In this paper, we describe the development of erythrocyte “ghosts” active in β-adrenergic signaling that support robust P. falciparum growth in culture. With this system, we show that a Gs peptide inhibits activation of erythrocyte β-AR signaling by β-agonists, providing definitive evidence that the peptide targets erythrocyte Gs signaling. At the same pharmacological concentrations, the peptide significantly inhibits malarial invasion of ghosts as well as intracellular maturation of parasites. Furthermore, we find that inhibiting Gs signaling with β-blockers also inhibits intracellular parasite growth. Finally, we show that a β-blocker such as propranolol can be used to reduce doses of existing antimalarials in both in vitro and in vivo infections. We therefore establish that Gs offers a host target to fight infection and to develop new antimalarial chemotherapies.
Erythrocyte ghost preparations have been described previously for studying the erythrocyte membrane and cytoskeleton and malarial infection [33
]. However, malarial infection rates of earlier ghosts were either untested or lower than in normal erythrocytes. In contrast, the ghosts described here retained mature erythrocyte double-discoidal morphology, Gs
signaling responses, and support of robust malarial infection. Several differences between our procedure and procedures described in prior reports include the lysis of erythrocytes in 3.5-kDa MWCO dialysis membranes rather than in 12–14-kDa MWCO membranes used previously, and our use of a potassium-rich resealing buffer rather than isotonic saline. Dialysis pore size may be critical since a 10–13-kDa dialyzable, cytoplasmic erythrocyte protein was reported necessary for malarial invasion of ghosts [39
]. Prior studies did not include GTP, which was incorporated here to ensure the support of a GTP cycle. Ghosts prepared in this manner can be loaded with a variety of peptidic, proteinaceous, fluorescent, or other cargoes and are likely suitable for studying many aspects of erythrocyte function and malarial infection. We have used them to definitively establish that erythrocyte β-adrenergic signaling is indeed mediated by Gs
and thus to explain why inhibitors of receptor-mediated Gs
signaling (such as propranolol) block malaria parasite invasion and intracellular growth. The findings strongly support the possibility that the erythrocyte Gs
pathway might be targeted to treat malaria.
The concept of host-targeted therapies for treating infectious diseases has only emerged recently and, to the best of our knowledge, is unexplored for parasitic infections. Since malaria parasites must invade host cells to proliferate, host pathways necessary for parasite survival present opportunities for intervention. Here we have shown that targeting erythrocyte Gs
inhibits parasite growth within erythrocytes as well as at the initial entry event. One obvious possible disadvantage of host-targeted therapy is potential toxicity to the host since Gs
regulates many cellular pathways by modulating transcription factors, ion channels, metabolic enzymes, and other molecules [41
]. However, Gs
signaling alone is insufficient to drive vacuole formation (e.g., vacuoles do not form in isoproterenol-treated erythrocytes), and therefore parasite components are expected to engage this host signaling pathway during invasion [42
]. Other parasite proteins may interact with this pathway during intracellular growth. Thus, optimal inhibitors should target interactions between host Gs
and parasite proteins, and these may have significantly reduced toxicity, particularly if they can be made additionally selective for erythrocyte Gs
. The wide array of pharmacological GPCR-inhibitors currently available suggest chemical backbones that may be useful in rational design of such antimalarials.
In addition, antihypertensive drugs that down-regulate Gs
via host receptors (i.e., β-blockers such as the first-generation drug propranolol) are extremely well-tolerated, partly because the host can adapt to drug treatment by producing additional adrenergic receptors and Gs
. Since the mature erythrocyte is incapable of de novo protein synthesis, it cannot mount such an adaptive response. Thus, erythrocytes may be more vulnerable to inhibition of β-AR/Gs
signaling, while other host tissues could adapt to avoid toxicity. A well-developed, host-targeted antimicrobial therapy directed at human chemokine receptor 5 (CCR5) for the treatment of drug-resistant HIV is currently in clinical trials. Short-term treatment with CCR5 antagonists reduced viral loads in infected patients [43
], providing proof of concept for targeting a host determinant to treat a major infectious disease.
Malaria is a major threat to global health, and there is an imperative to use existing drugs to treat patients while the search for vaccines and better drugs continues. Development of a new drug is expensive and can take more than a decade [44
]. Propranolol and a wide range of β-blockers are currently approved for human use. In humans, racemic propranolol is typically prescribed at ~60–320 mg/d (~0.8–4.5 mg/kg/d in an average 70-kg person) [45
] with a maximum clinical dose of ~10 mg/kg/d. The maximum concentration of propranolol in blood approaches 1 μM [29
], and thus it is likely that treatments can be designed that achieve IC50
s in the bloodstream (0.4–1.2 μM in the work discussed in this paper; 0.4 μM, Drug Screening Program, Strategic and Discovery Research UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, personal communication) using a range of dosages comparable to those used for hypertension. Furthermore, higher concentrations of the drug can also be administered [45
Malaria patients may be hypotensive [46
], and therefore treatment with β-blockers must be considered carefully. However, at normal dosages, propranolol does not usually cause orthostatic hypotension because the α-adrenergic system maintains peripheral vasoconstriction [47
]; some patients may show mild orthostatic hypotension [49
]. Other quinoline antimalarials (e.g., quinine and mefloquine) may also exacerbate orthostatic hypotension in malaria patients [46
]. The main cause of hypotension in malaria patients is acidosis secondary to hypovolemia, which can be managed with intravenous saline. Propranolol has been found to mitigate some hyper-adrenergic effects that occur in patients with shock secondary to hypovolemia. In such cases, β-adrenergic blockade by propranolol is thought to reduce muscle lactate production and to limit metabolic acidosis [51
]. Thus, this drug may have unseen benefits for clinically ill patients.
As with any new drug, care would obviously need to be taken in any clinical malaria study using propranolol-containing combinations. Propranolol is contraindicated for use with the quinoline antimalarial quinidine [47
], which also has α-adrenergic–blocking activities [53
]. Other quinoline antimalarials (e.g., quinine and mefloquine) that exacerbate orthostatic hypotension also produce electrocardiographic abnormalities (i.e., long corrected QT interval), although not of the severity seen with halofantrine [54
]. There is one report of a patient with a previous myocardial infarction who was taking propranolol and suffered a cardiopulmonary arrest 5 h after taking a single dose of mefloquine; the patient made a full recovery [55
]. Mefloquine and propranolol are also associated independently with depression. Thus, some quinolines may be contraindicated for use with propranolol. However, only minor, asymptomatic corrected QT interval disturbances are found with other antimalarial drugs such as chloroquine (a quinoline) [56
], sulfadoxine-pyrimethamine [57
], and artemisinin [58
]; no electrocardiographic changes were associated with atovaquone-proguanil alone or in combination with artemisinin [59
]. Overall, propranolol has few serious side effects, is recommended for use in pregnant women, is widely approved by regulatory agencies, and is frequently taken for life.
In addition to its safety and efficacy, propranolol is made even more attractive by its low off-patent cost, high stability, and ease of production. In addition, second- and third-generation β-blockers have been developed, and all generations of β-blockers are routinely taken for many years to treat chronic conditions. Thus, it may be an appropriate time to evaluate combinations containing β-blockers to treat human clinical infections that fail to respond to optimized antimalarial therapy.