Extracellular H2O2, peroxiredoxin, and glutathione reductase alter Neospora caninum invasion and proliferation in Vero cells

Neosporosis is a relevant cause of abortion in cattle and neurodegeneration in dogs (Dubey et al., 2006; Dubey and Schares, 2011). This disease draws attention by the significant worldwide financial impact, around 1.3 billion dollars in the milk and meat industries (Reichel et al., 2013). Up to date, the control and prevention of neosporosis have not been effective through drugs or vaccines (Horcajo et al., 2016). Neospora caninum, neosporosis etiological agent, is an Apicomplexa protozoan that has the tachyzoite form capable of actively invading and proliferating in host cells. In addition, the parasite has a resistance form, bradyzoite, fundamental for evasion and resistance from the immune system mechanisms (Dubey et al., 2006; Fereig and Nishikawa, 2020). Knowledge about N. caninum biology has been increasing since its discovery in 1988 by Dubey, evidencing a plethora of similarities and differences compared to Toxoplasma gondii (Dubey et al., 1988).

Apicomplexa parasites must deal not only with radical oxygen species (ROS) produced by their metabolism but also by host mitochondrial aerobic respiration and further by host defense mechanisms or drugs (Müller, 2004; Müller et al., 2003). To avoid the death induced by oxidative stress, parasites exhibit plenty of antioxidants and redox enzymes, such as superoxide dismutase, peroxiredoxin, glutathione reductase, and catalase that assure the appropriated ROS scavenge. In addition, as described in Plasmodium, parasites can be benefited from the host redox apparatus to resist oxidative stress (Brizuela et al., 2014; Koncarevic et al., 2009). Superoxide dismutase catalyzes the decomposition of superoxide anion into hydrogen peroxide (H2O2) and molecular oxygen (Wang et al., 2018). High levels of H2O2 can damage cellular structures inducing apoptosis (Calabrese et al., 2019). Therefore, H2O2 is scavenged by peroxiredoxins or catalase, protecting the cells from its harmful effects. Glutathione reductase catalyzes the conversion of oxidized glutathione into reduced glutathione, an essential antioxidant agent that regulates cellular redox homeostasis.

Thioredoxin-dependent peroxide reductase (NcPrx) (Venancio-Brochi et al., 2022), superoxide dismutase (Cho et al., 2004), glutaredoxins (Song et al., 2021), and glutathione reductase (NcGR) (Venancio-Brochi et al., 2021) have been described in N. caninum, supporting the presence of a conserved redox mechanism in this parasite.

The thioredoxin-dependent peroxide reductase, or peroxiredoxins, are enzymes that detoxify hydrogen peroxide, organic peroxide, and peroxynitrite (ONOO). Peroxiredoxin also regulates H2O2 levels in cell compartments, acts as a chaperone in cellular signaling, and plays a role in immune regulation (Rhee and Woo, 2020; Rhee, 2016; Robinson et al., 2010). NcPrx has been experimentally described as a 2-Cys-Prx peroxidase cytoplasmic enzyme in N. caninum (Venancio-Brochi et al., 2022), as well as performing a mechanism of H2O2 sensing in this parasite (Venancio-Brochi et al., 2022). Additionally, the recombinant NcPrx has antioxidant properties to protect plasmid DNA in oxidative environments and peroxidase activity in vitro (Venancio-Brochi et al., 2022).

Glutathione is a tripeptide antioxidant that protects cells from oxidative damage by removing radical oxygen species and xenobiotics (Grant and Dawes, 1996). Glutathione reductase is a key enzyme that maintains high levels of glutathione in cells, converting oxidized glutathione (GSSG) into reduced glutathione (GSH). In this mechanism, the nicotinamide adenine dinucleotide phosphate (NADPH) is used as an electron donor(Deponte, 2013; Rietveld et al., 1994) NcGR of N. caninum was described as a cytosolic enzyme and the recombinant form of NcGR successfully recycles reduced glutathione in the presence of NADPH. Additionally, our group demonstrated rNcGR inhibition by phenothiazinium dyes (Venancio-Brochi et al., 2021).

ROS produced by macrophages restrain T. gondii proliferation in the early stages of infection; however, this parasite can evade the ROS challenge through cellular respiratory burst suppression (Shrestha et al., 2006). Similarly, N. caninum early infection also increases oxidative stress (Glombowsky et al., 2017; Tonin et al., 2014), inducing inflammasome activation and controlling parasite proliferation (Mota et al., 2020).

Although redox regulation is crucial to host defense against Neospora caninum (Mota et al., 2020), its impact on the N. caninum in vivo and in vitro invasion and proliferation remains unclear. Therefore, this study investigated the proliferation and invasion facing different levels of H2O2 and recombinant redox enzymes (rNcPrx and rNcGR). Our results contribute to understanding the redox dynamics in N. caninum, elucidating some features of the antioxidant enzymes activities. Furthermore, it is the first time that apicomplexan invasion and proliferation in the host cell are evaluated in the presence of recombinant antioxidant enzymes.

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