Protozoan persister-like cells and drug treatment failure

Abstract

Antimicrobial treatment failure threatens our ability to control infections. In addition to antimicrobial resistance, treatment failures are increasingly understood to derive from cells that survive drug treatment without selection of genetically heritable mutations. Parasitic protozoa, such as Plasmodium species that cause malaria, Toxoplasma gondii and kinetoplastid protozoa, including Trypanosoma cruzi and Leishmania spp., cause millions of deaths globally. These organisms can evolve drug resistance and they also exhibit phenotypic diversity, including the formation of quiescent or dormant forms that contribute to the establishment of long-term infections that are refractory to drug treatment, which we refer to as 'persister-like cells'. In this Review, we discuss protozoan persister-like cells that have been linked to persistent infections and discuss their impact on therapeutic outcomes following drug treatment.

Figure 1. Persister-like cell types of Plasmodium spp.

A.The Plasmodium vivax hypnozoite. P. vivax sporozoites injected into the host by infected Anopheles mosquitoes rapidly infect hepatocytes. In the liver cells, the parasites undergo a proliferative stage, including substantial nuclear division without cell division to produce a schizont (schizogony). The schizont differentiates into merozoite forms that burst from hepatocytes and enter the bloodstream where they invade erythrocytes. Alternatively, some sporozoites will form hypnozoites that can persist in a growth-arrested state for weeks to many months before entering schizogony and completing the developmental cycle in the liver. B. Artemisinin refractory dormant ring stages in P. falciparum Exposure of synchronous ring stage P. falciparum parasites to dihydroartemisinin (DHA) induces cell-cycle arrest. These dormant ring stage forms are characterized morphologically by condensed chromatin and reduced cytoplasm. By contrast, pre-exposed ring stage forms have a relatively diffuse nucleus and large cytoplasmic area. Dormant ring stage parasites persist for several days before resuming normal growth. DHA-induced ring stage dormancy is observed in artemisinin-susceptible and artemisinin–resistant P. falciparum^, . The top panel shows Giemsa stained parasites (purple) inside erythrocytes. The lower panel provides a representation of the upper panel in which key structures visible in light microscopy are visible. The diameter of the erythrocyte is 6 micrometers. Figure 1aadapted from Ref. . Fig 1b courtesy of Dennis E. Kyle (University of Georgia, Athens, USA).

Figure 2:. Toxoplasma gondii tachyzoites and bradyzoites.

T. gondii, the causative agent of toxoplasmosis, undergoes asexual replication in nucleated host cells of intermediate hosts (such as rodents and humans). During initial infection tachyzoites disseminate throughout the host and cause acute disease. In response to immune pressure and environmental stress, the parasite differentiates into bradyzoites, which form tissue cysts within host cells and give rise to persistent infection (chronic stage). Shown are microscopy images of the tachyzoite (visualized using polyclonal rabbit anti- surface antigen 1 antibody with Alexa Fluor 488) and a tissue cyst containing bradyzoites (brightfield image). Current therapies are successful only against the acute phase, but fail to eliminate the chronic stage. See reference for further detail on bradyzoite development. Image on the right courtesy of, L. David Sibley (Washington University School of Medicine, USA). Image on the left reproduced from Ref .

Figure 3.. Dormant amastigote forms of Trypanosoma cruzi.

Following invasion of host cells, T. cruzi trypomastigotes (shown in blue; indicating no or minimal proliferation) differentiate into non-replicating amastigotes. The presence or absence of dormant parasites determines treatment outcomes. Amastigotes may not replicate (bottom panel; blue amastigotes, dormant parasites), undergo minimal replication (top and middle panels, red amastigotes) before becoming dormant, or actively replicate (top, second round of invasion, red amastigotes). Replicating parasites are eliminated by drug treatment and under appropriate conditions (for example, long-treatment period), possibly resulting in sterile cure (top panel). However, parasites that are dormant during the time of drug exposure (middle and bottom, blue amastigotes) resist drug clearance and can revive the infection by resuming replication after drug treatment (bottom panel).

Figure 4.. Leishmania life cycle showing impact of persisters.

Leishmania spp. alternate between motile promastigotes in the sand fly vector and intracellular amastigotes in the mammalian host. Following transmission of infective, non-dividing metacyclic promastigotes via the bite of a sandfly, promastigotes enter host phagocytic cells and differentiate within phagolysosomes into amastigote forms (with truncated flagellum) and undergo replication. Subsequently, host cells burst and the released amastigotes enter further host cells. Alternatively amastigotes may be taken up by a sandfly during a blood meal in which they differentiate into proliferating promastigotes (procyclic promastogotes) in the midgut Studies have identified, discrete replicating and non-dividing intracellular amastigote populations and it is likely that growth-arrested leishmanial amastigote forms contribute to drug treatment failure and persistent infections (top right panel). Promastigotes have been shown to enter a non-proliferative but viable state in purine-depleted medium (bottom left panel). Figure reproduced with adaptation from Reference .

Figure 5.. Metabolic changes in protozoan persisters.

Currently, our knowledge is limited regarding metabolic changes across protozoan persister-like cells. Studies have shown that in Plasmodium spp. and Toxoplasma gondii DNA replication, general transcription and protein synthesis are decreased in persister-like cells. However, some biochemical pathways are sustained, such as protein export in Plasmodium cynomolgi hypnozoites forms and also oxidative and other stress responses in Plasmodium and T. gondii bradyzoites. In addition, in purine-depleted culture conditions, Leishmania donovani non-proliferative promastigotes exhibit decreased DNA replication and repair, and protein synthesis, whereas oxidative stress response pathways were increased. Finally, in the apicomplexan protozoa phosphorylation of parasite eukaryotic initiation factor-2α (eIF2α) has been implicated in the establishment of dormancy (possibly by inhibiting global protein synthesis), although the role of role eIF2α phosphorylation in the formation of persister-like cells in the kinetoplastid protozoa has yet to be investigated.