Fungal persister cells: The basis for recalcitrant infections?

Abstract

Persister cells are a small subpopulation within fungal biofilms that are highly resistant to high concentrations of antifungals and therefore most likely contribute to the resistance and recalcitrance of biofilm infections. Moreover, this subpopulation is defined as a nongrowing, phenotypic variant of wild-type cells that can survive high doses of antifungals. There are high degrees of heterogeneity and plasticity associated with biofilm formation, resulting in a strong variation in the amount of persister cells. The fraction of these cells in fungal biofilms also appear to be dependent on the type of substrate. The cells can be observed immediately after their adhesion to that substrate, which makes up the initial step of biofilm formation. Thus far, persister cells have primarily been studied in Candida spp. These fungi are the fourth most common cause of nosocomial systemic infections in the United States, with C. albicans being the most prevalent species. Remarkably, persisters exhibit characteristics of a dormant state similar to what is observed in cells deprived of glucose. This dormant state, together with attachment to a substrate, appears to provide the cells with characteristics that help them overcome the challenges with fungicidal drugs such as amphotericin B (AmB). AmB is known to induce apoptosis, and persister cells are able to cope with the increase in reactive oxygen species (ROS) by activating stress response pathways and the accumulation of high amounts of glycogen and trehalose-two known stress-protecting molecules. In this review, we discuss the molecular pathways that are involved in persister cell formation in fungal species and highlight that the eradication of persister cells could lead to a strong reduction of treatment failure in a clinical setting.

Fig 1. Persister cells are phenotypic variants of wild-type cells.

An overnight culture of C. albicans SC5314 (wild-type) was diluted to OD₆₀₀ 0.1 and seeded to a flat bottomed 96-well plate (CELLSTAR Greiner) containing RPMI-MOPS medium to allow biofilm formation. Biofilms were grown at 37°C for 24 hours, washed with 1× PBS and challenged with 100 μg/mL AmB dissolved in fresh RPMI-MOPS medium or left to mature in fresh RPMI-MOPS medium. After 24 hours, the medium was removed, and the remaining biofilm was again washed with 1× PBS and stained with 100 μg/mL fluorescein diacetate (Sigma Aldrich) and 500 μg/mL Texas Red conjugated to concavalin A (Molecular Probes) for 60 minutes. Fluorescein diacetate stains living cells green, while Texas Red conjugated to concavalin A was used to stain the fungal cell wall red. Pictures were taken using the scanning confocal microscope at a magnification of 600× (white scale bar represents 20 μm). (A) A C. albicans biofilm treated with AmB lacks green fluorescent cells (indicating that most of the cells are dead). However, a bright fluorescent cell is also present (indicated by the black arrow). This cell meets the definition of a persister cell. We were able to identify several persister cells in the treated biofilm but none of them were of the hyphal form. (B) A nontreated biofilm is thicker in appearance and therefore has a higher background of green fluorescence. Most of the cells in focus are green and thus appear to be viable (indicated by the black arrow). AmB, amphothericin B.

Fig 2. Persister cells of C. albicans are committed to energy storage.

Several enzymes of energy storage pathways are up-regulated in persister cells (indicated in green), whereas glycolysis is down-regulated (indicated in red), suggesting that C. albicans persister cells are adapted to the absence of glucose. Icl1 and Mls1 are up-regulated in persister cells. This results in an increase in oxaloacetate that enters gluconeogenesis that is converted to phophoenolpyruvate by Pck1. Pck1, together with Fbp1, are also up-regulated in persister cells and are both key enzymes in the gluconeogenesis pathway. The up-regulation of these enzymes will likely result in an increased flux towards the production of energy storage molecules such as trehalose and glycogen. Additionally, up-regulation of Fbp1 also results in a decreased amount of fructose-1,6-biphosphate, which is the activator of Ras1. This suggests that persister cells have a lower proportion of active Ras1. Finally, Hsp90, which is up-regulated in persister cells may also inhibit the activation of Ras1 in persister cells. Ras1 is up-regulated in persister cells, and because persister cells appear to have a lower proportion of active Ras1, this may prepare persister cells for a rapid metabolic switch to restart proliferation. Dashed bold lines: likely to occur in persister cells; bold lines: proven in persister cells; dashed lines: likely to occur in cells growing in the presence of glucose. All other lines: proven to occur in cells growing in the presence of glucose. Fbp1, fructose-1,6-biphosphatase; GDP, guanosine-5'-diphosphate; GTP, guanosine-5'-triphosphate; Hsp90, heat shock protein 90;Icl1, Isocitrate lysase; Mls1, malate synthase; Pck1, phosphoenolpyruvate carboxykinase; Ras1, rat sarcoma.

Fig 3. Persister cells are able to survive ROS accumulation induced by fungicidal drugs.

Fungicidal drugs induce ROS accumulation in the mitochondria, and persister cells appear to use several mechanisms to cope with the high amount of ROS. (A) Miconazole-induced ROS in the mitochondria is detoxified by the overexpression of several SODs. (B) AmB treatment results in ROS accumulation. This leads to calcium accumulation and activation of the calcineurin pathway, thereby increasing expression of CaCMA1 via Crz1. CaCMA1 expression results in decreased expression of TPS1 and TPS2 and concomitantly results in apoptosis. In order to survive the apoptotic response induced by ROS, persister cells need to break the apoptotic feedback loop. They do so by up-regulation of stress response proteins (indicated in green). First, up-regulation of Hsp21 in persister cells may result in an increased glycogen content that protects the cells from the oxidative stress. Second, Hsp90 is also up-regulated in persister cells. Paradoxically, Hsp90 also activates the apoptotic pathway by activation of the calcineurin pathway, and concomitantly inhibits Ras1-cAMP-PKA signaling, decreasing the apoptotic response. Therefore, depending on the conditions, Hsp90 may determine the cellular fate of biofilm cells under stringent oxidative stress conditions and may direct the cells to apoptosis or the persister cell state. Dashed bold lines: likely to occur in persister cells; bold lines: proven in persister cells; all other lines: proven to occur in cells undergoing apoptosis. AmB, Amphotericin B; Crz1, calcineurin responsive zinc finger 1; Hsp21, heat shock protein 21; PKA, protein kinase A; Ras1, rat sarcoma; ROS, reactive oxygen species; SOD, superoxide dismutase; Tps1, trehalose-6-phosphate synthase.