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The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii

Dana Aghabi, Megan Sloan, Zhicheng Dou, Alfredo J. Guerra, Clare R. Harding

Preprint posted on September 09, 2021 https://www.biorxiv.org/content/10.1101/2021.09.08.458725v1

Ironing-out iron storage in T. gondii

Selected by Jennifer Ann Black

Categories: cell biology

Background

Iron is a very reactive metal, making it useful for biological reactions such as oxygen transport, but it also means iron reacts with other cellular metabolites leading to cell damage. For instance, iron reacting with hydrogen peroxide (H2O2) can form dangerous molecules called radicals which injure DNA and cellular structures like membranes. To avoid this, organisms store free iron. In mammals, iron is stored within the protein ferritin, whereas others (like yeast and plants) can pump free iron into membrane-bound structures (vacuoles) using transporters (such as the vacuolar iron transporter [VIT] in plants) (1). This difference in how iron is managed between mammalian cells and other organisms, including pathogens (disease-causing organisms), could be exploited to design better drugs targeting their ability to store iron. Toxoplasma gondii is a parasite which causes the disease Toxoplasmosis, a serious infection in immunocompromised individuals and for unborn babies. In the acute phase of disease, the parasites, known as tachyzoites, replicate and quickly lyse the host cells. In some cells, tachyzoites can differentiate into hard-to-treat cyst forms (bradyzoites), where they live for a long time and can transmit, for example, if the infected cell is eaten (i.e. undercooked or raw meat) (2).

Little is known about how T. gondii regulates and stores iron; a gap in the field the authors address within this study. By characterising the T. gondii homolog of the VIT (3), they show that VIT is involved in regulating iron storage in T. gondii, revealing how T. gondii respond to excess or scarce iron levels, and furthermore how iron management can contribute to the ability of T. gondii to cause disease in mammals.

 

Key Findings

In this study, the authors use CRISPR/Cas9 genome engineering to generate tachyzoite cells lacking VIT. This cell line forms the basis of their study.

 

A) T. gondii can use the VIT to store iron

VIT is non-essential for parasite survival in culture, but VIT-lacking cells do not grow as well as controls, i.e. the parasite replicates more slowly within host cells. The authors show that VIT loss renders parasites up to 25,000-fold more sensitive to excessive concentrations of iron.

To then ask if the VIT acts in storing iron, the authors depleted host cell iron using the iron-chelating compound deferoxamine (DFO), let the parasites invade then looked at how the parasites replicated. They show that the loss of the VIT compromises parasite replication. Furthermore, the parasites themselves contained less iron. Together, these data support a role for the VIT in iron storage and suggest that iron level regulation is important to support normal parasite growth.

A selection of data from Aghabi et al. Fig, 2C shows that parasites lacking VIT are more sensitive to excessive levels of FAC relative to control cells. Fig. 3D shows the localisation of VIT across the lytic cycle of the parasite, starting extracellularly (singular foci) and ending at 24 hrs post host cell invasion ( fragmented). Scale bar = 5 μm. Fig. 5A shows that VIT cells have higher levels of CellROX Deep Red dye suggesting they have higher levels of ROS relative to controls. Fig. 6B and C show that though parasites lacking VIT can infect mouse macrophages, their survival is reduced relative to control cells. Figures adapted from Aghabi et al and reproduced under a CC-BY-NC-ND 4.0 International license.

 

B) VIT has a dynamic localisation

To locate the VIT in the parasites, the authors fused protein epitopes (an HA or myc tag) to the C-terminus of the endogenous protein then examined the location by high resolution microscopy. They found that in extracellular parasites (outside of a host cell), a single focus of signal appeared but once the parasites invaded host cells, several foci were seen. They also provide evidence that the VIT may localise to the Vacuolar Associated Compartment (VAC), which is an understudied organelle with similarities to a lysosome (i.e. a digestive organelle). The VAC contains proteases and now may also act as a putative site of iron storage in T. gondii.

C) External iron levels effects VIT expression

The authors then reasoned that the behaviour of the VIT may alter in response to changes in iron levels. They tested for possible changes by qRT-PCR (transcript levels), western blotting (protein levels) and the localisation by immunofluorescence (location). Under excess iron, both the VIT transcript and the protein levels become reduced, correlating with less VIT foci. Whereas, when iron was scarce, no changes in the transcript levels were seen, but paradoxically, less VIT protein was expressed, correlating with less VIT foci in the parasites. These data suggest that both excess and scarce iron levels alter the behaviour of the T. gondii VIT.

D) T. gondii can prioritise iron usage

Next, VIT-lacking cells and control cells, were subject to RNA-sequencing (RNAseq) to examine their transcriptomes. Interestingly, when looking at factors and pathways requiring iron, the authors found some differential expression of iron-associated pathways whereas for others, no significant changes were found in the absence of improper iron storage. Thus, some iron-associated pathways (and factors) can alter expression in response to changes in iron levels whereas others do not. As proposed by the authors, it is possible that T. gondii prioritises certain pathways over others when iron levels are dysregulated. In addition, up-regulation of a putative transporter involved in the removal of drugs from cells was observed, leading the authors to suggest T. gondii may remove excess iron from the cell if it is unable to store it correctly.

E) Dysregulated iron storage leads to oxidative stress

One reason as to why parasites lacking VIT grow poorly and are more sensitive to excess iron could be due to high levels of oxidative stress related to increased free iron reacting to produce reactive oxygen species (ROS). To test this, the authors exposed control and VIT lacking cells to excessive iron then measure the fluorescence of a dye (CellROX Deep Red) added to the cells. The signal of CellROX Deep Red corresponds to ROS in the cytosol. In support, they show that excess iron does induce more ROS in VIT-lacking parasites, but only in the cytosol and not under routine growth conditions. Thus, excessive, and incorrectly stored iron in T. gondii can cause more ROS to form which could reduce parasite survival, and though the authors found no changes at the transcript level for factors involved in resolving ROS damage, they did note that the activity of catalase (an enzyme which removes hydrogen peroxide) became enhanced in VIT lacking parasites under normal culture conditions.

In all, despite the parasites containing less iron, these data suggest the iron they do contain may be incorrectly stored and perhaps more freely available to react with other metabolites. As suggested by the authors, the increased catalase levels (and possibly the up-regulation of transporters) could help to explain why there is no significant increase in ROS levels (and a modest replication defect) in VIT-lacking cells in the absence of excess iron if the cells are able to remove iron-reactive metabolites or the iron itself and limit potential negative consequences.

F) VIT is needed for T. gondii virulence

Lastly, the authors asked if the VIT was required during a host infection. By infecting mice with control and VIT-lacking parasites, they were able to show that mice infected with VIT-lacking parasites survived better, presenting evidence that this loss of fitness in the host might be due to parasites failing to thrive in host macrophages.

 

What I Liked about this Preprint

On the one hand iron plays essential roles in most cells, whereas if left to its own devices, iron cause cell damage and even death. Understanding this fine balance between the good and the bad of iron is already an exciting field of research. What attracted me to this study was how surprisingly little we knew about iron related processes in this parasite and the curiosity to find out more. Something the authors also highlight as an interesting reason for studying iron storage in this parasite. Here, the authors have used interesting and informative experiments to ask key questions about iron storage in this parasite, now shedding light on this largely uncharacterised process in T. gondii. Though further work is needed to fully understand the intricacies of this process, their results will certainly create numerous new avenues of investigation into this process and open discussions about the roles of iron in this parasite. The more insight we gain into how pathogenic organisms operate, the higher the chance we can pinpoint key weaknesses which we can exploit for new therapies.

 

References

  1. Kaplan and D.M. Ward. The essential nature of iron usage and regulation. Current Biology (2013).
  2. Attias, D.E. Teixeira, M. Benchimol, R.C. Vommaro, P. Henrique Crepaldi and W. De Souza. The life-cycle of Toxoplasma gondii reviewed using animations. Parasites&Vectors (2020).
  3. Slavic, S. Krishna, A. Lahree, G. Bouver, K.K. Hanson, I. Vera, J.K. Pittman, H.M. Staines, M.M. Mota. A Vacuolar iron-transporter homologue acts as a detoxifier in Plasmodium. Nat. Comms (2016).

 

Tags: #toxoplasma, iron, iron storage, ros, t. gondii

Posted on: 19th October 2021

doi: https://doi.org/10.1242/prelights.30835

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Author's response

The author team shared

Questions for the Authors

Q1: Why do you think the HA-tagged VIT does not co-localise with the VAC but your myc-tagged VIT does? And why do you think extracellular parasites likely just have one singular focus of the VIT compared to those inside a host cell?

A1: We are not sure! Our current idea is that some of HA tag is being degraded, possibly by the proteases or by the low pH in the vacuole, we are doing a few more experiments to look at this. As for the change in localisation through the cell cycle, a few people have seen that the VAC is very dynamic through the replication cycle. We don’t know why, but it appears to fragment when the parasites start to divide. This is something we would love to look into further.

 

Q2: Why do you think the parasites decrease the protein levels of the VIT under iron-limited conditions?

A2: This is a great question, it’s possible that when iron is limiting, storage is no longer required and so the parasites down-regulate VIT to allow iron to be used for metabolic processes. What we found even more surprising is that VIT levels also go down when iron is high. We didn’t expect this, and you would expect them to go up. What we did see, was a redistribution of VIT upon changing iron levels, it’s possible that its function is closely linked to its localisation, but we will have to investigate this in future work.

 

Q3: You see a down-regulation of the enzyme RNR, which produces nucleotides required for DNA synthesis. Do you think that some of the defective replication of your parasites could also be related to reduced DNA synthesis? Do your parasites exhibit signs of replication stress?

A3: We have not looked at replication stress specifically, but it may well be a factor. Given the up-regulation of catalase activity under normal conditions, our belief is that mislocalized iron leading to increased ROS production is the major driver of the reduced growth. However, we are currently further investigating how the cells respond to changing iron levels and hopefully we will be able to investigate this question in more detail.

 

Q4: Your RNA-seq shows that a putative drug transporter becomes up-regulated when you remove VIT from your cells. If this transporter performed a similar function as reported in Plasmodium and mammalian cells, do you think that the up regulation of this type of transporter could prove challenging to design drugs targeting iron storage factors in T. gondii?

A4: We do see this up-regulation, and this transporter has proved to be an important factor in drug resistance in Plasmodium. However, clearly even under normal conditions this is not enough to enable normal growth of the parasite in vitro or in vivo. The mechanisms of iron storage and how the parasites react to altered iron levels are not yet understood enough to know whether this would be a good drug target, hopefully we can investigate this further in the future.

 

Q5: Ordinarily mitochondria are major sites of ROS production, and excess iron can cause increased ROS production in these organelles, but you do not see an increase in mitochondrial ROS levels by flow cytometry. Could this relate to the sensitivity of the flow cytometry assay or why do you think the mitochondria is not the main location of ROS when iron storage is dysregulated in T. gondii?

A5: We currently believe that the main area of ROS formation is the cytosol, simply from our results in this study. However, it is very possible that excess ROS formation occurs in the mitochondria which we were not able to detect under our conditions. We don’t currently know how iron enters the mitochondrion, or how iron entry is regulated and so these are questions that we are examining now.

 

Q6: Bradyzoites can form under conditions of stress (i.e reduced nutrients). Are your cells able to form bradyzoites in response to low iron?

A6: This is something we have not yet looked at in detail but we hope to in the future. Toxoplasma appears able to induce the bradyzoite programme under a number of different stress conditions, so we believe that it is very possible low iron induces bradyzoite formation. This may even have a role in vivo, as some areas where bradyzoites form (e.g. the eye) have low levels of available iron. We would be very interested in how this occurs, and how the parasite is sensing and responding to low iron levels.

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