https://orcid.org/0009-0002-8469-1701
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Figures
Abstract
Toxoplasma gondii (T. gondii) is an intracellular parasite that infects nearly all warm-blooded animals, including humans. The susceptibility to T. gondii infection varies among hosts. In this study, we found that Microtus fortis (M. fortis) exhibited a naturally high level of resistance to the T. gondii RH strain, as evidenced by survival assays. We further observed that M. fortis activated the complement system via the lectin pathway to lyse T. gondii tachyzoites, whereas serum from Kunming (KM) mice showed no such effect. Furthermore, the ability of M. fortis to clear T. gondii tachyzoites was significantly impaired when the complement system was inhibited by cobra venom factor (CVF). These findings indicate that M. fortis exhibits a naturally high resistance to T. gondii. This resistance is mediated, in part, by the complement system, which is activated through the lectin pathway and directly lyses extracellular tachyzoites. Thus, the complement system plays an essential role in controlling T. gondii infection in this species.
Author summary
T. gondii can infect nearly all warm-blooded animals. Mice infected with T. gondii type I strains typically develop acute toxoplasmosis symptoms and often succumb to the infection. However, we observed that M. fortis infected with the T. gondii type I strain did not develop acute toxoplasmosis and effectively cleared the T. gondii tachyzoites. Our study revealed that T. gondii tachyzoites activate the complement system in M. fortis via the lectin pathway, leading to their destruction by lysis. In contrast, the complement system in mice failed to eliminate the tachyzoites. Furthermore, complement inhibition in M. fortis significantly impaired its ability to clear the infection. These findings suggest that the complement system plays a critical role in enabling M. fortis to control T. gondii infection. The complement-mediated killing of T. gondii offers new insights for the development of anti-T. gondii drugs and vaccines.
Citation: Xie J, Wu M, Zhou Z, Liu Y, Liu X, Wu K, et al. (2026) The resistance to Toxoplasma gondii in Microtus fortis is associated with the activation of the complement lectin pathway. PLoS Negl Trop Dis 20(3): e0014052. https://doi.org/10.1371/journal.pntd.0014052
Editor: Steven M. Singer, Georgetown University, UNITED STATES OF AMERICA
Received: February 20, 2025; Accepted: February 18, 2026; Published: March 2, 2026
Copyright: © 2026 Xie et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The dataset generated and/or analyzed during the current study is stored in the Figshare respository. DOI: https://doi.org/10.6084/m9.figshare.28448429. The data underlying the results presented in this manuscript can be accessed at: https://www.doi.org/10.6084/m9.figshare.28448429.
Funding: The work was supported by the National Natural Science Foundation of China (grant number 32170510 to L.J.), the National Key Research and Development Program of Hunan Province (grant number 2024DK2001 to Z.Z.), Natural Science Foundation of Hunan Province General Program (grant number 2024JJ5422 to Z.Z.), and the Changsha Major Special Project of Science and Technology (grant number kh2301027 to Z.Z.). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Toxoplasma gondii (T. gondii) is a globally widespread zoonotic apicomplexan parasite that can infect humans and other warm-blooded animals [1]. While toxoplasmosis is typically mild and asymptomatic in immunocompetent individuals, it can lead to severe disease and even death in immunocompromised patients, such as those with AIDS or organ transplant recipients [2,3]. Additionally, acute infection during pregnancy is a major cause of spontaneous abortion, intrauterine fetal death, preterm delivery, or serious fetal malformations, including encephalitis, ocular and cerebral syndromes [4,5]. The current first-line treatment for toxoplasmosis involves a combination of pyrimethamine (PYR) and sulfadiazine (SDZ). However, the drugs used clinically against tachyzoites have adverse side effects, including hypersensitivity and toxicity in humans. The development of new medicines and vaccines remains a primary focus of research on toxoplasmosis [6,7].
T. gondii has three major clonal lineages: type I, type II, and type III. These genotypes are associated with distinct clinical manifestations in humans and animals. Type I is more pathogenic and typically causes acute disease, while type II is more likely to lead to chronic infections [8–10]. T. gondii is regarded as one of the most successful parasites, partly due to its broad host range and its ability to infect nearly all warm-blooded animals, including humans. However, there are significant differences in host susceptibility. Mice, for example, are highly susceptible to T. gondii and develop symptoms of acute toxoplasmosis when infected with type I strains. In contrast, rat species such as Lewis (LEW) and Sprague-Dawley (SD) rats show reduced sensitivity to T. gondii infection. Specifically, in Lewis rats, the NLRP1 inflammasome can detect Toxoplasma infection, which triggers pyroptosis in infected macrophages and eliminates the parasite’s replication niche [11–15]. Interferon-γ (IFN-γ) is the major cytokine that mediates parasite resistance in human and murine cells, inducing the transcription of two major GTPase families: immune-associated GTPases (IRGs) and guanylate-binding proteins (GBPs) [16]. Oligomerization of IRG and GBP proteins on the parasitophorous vacuole membrane (PVM) is essential for membrane disintegration, with subsequent death of the parasite and host cells [17]. However, there are 23 genes coding for IRG proteins in mice, and the expression of these genes is dependent on the mouse strain. In humans, there are only 3 such genes, of which only one encodes a functional protein [18]. Additionally, T. gondii type I strains produce virulence factors such as rhoptry protein 5 (ROP5) and rhoptry protein 18 (ROP18), which help the parasite evade this intracellular innate immune response [19].
Microtus fortis (M. fortis), a rodent characterized by its short ears, legs, and tail, belongs to the class Mammalia, order Rodentia, suborder Myomorpha, family Cricetidae, subfamily Microtinae, and genus Microtus, and is mainly distributed in China and North Korea. M. fortis is widely used in research on Schistosoma japonicum (S. japonicum) due to its unique role as the only known mammalian host exhibiting strong natural resistance to S. japonicum infection [20]. This resistance can be inherited and is not affected by geographical distribution, environment (wild or laboratory-bred), or generation [21]. In contrast to the extreme susceptibility of mice to T. gondii type I strains, our experiments demonstrated that M. fortis exhibited remarkable resistance to the same strain. The underlying mechanisms responsible for this naturally high resistance to T. gondii type I strains in M. fortis remain to be further investigated.
The complement system is a central component of innate immunity, serving as a crucial line of defense against the invasion of foreign pathogens [22]. It consists of plasma proteins produced mainly by the liver or cell surface membrane proteins. The complement system can be initiated by three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. All three pathways lead to a common terminal pathway [23–25]. This cascade of protein reactions ultimately leads to the formation of a membrane attack complex on the pathogen’s membrane, resulting in the pathogen’s destruction [26,27].
In this study, we demonstrate that M. fortis exhibits strong resistance to T. gondii infection and further establish that the complement system is one of the key mediators that contributes significantly to this resistance.
Materials and methods
Ethics statement
Ethical approval was obtained from the Center for Medical Ethics of Central South University (protocol code: 2020KT-11).
Animals
The specific-pathogen-free (SPF) eight-week-old Kunming (KM) mice and the M. fortis were procured from the Department of Laboratory Animals at Central South University (Changsha, China). All procedures were conducted in accordance with the institutional guidelines for animal ethics.
Serum preparation
Blood samples from M. fortis and KM mice were collected via the eye-detachment technique, with blood from each mouse stored separately. After standing at 4 °C for two hours, the blood was centrifuged at 3,000 rpm for 10 minutes at 4 °C. The serum was then separated and filtered to remove red blood cells and other cellular debris. All sera were subsequently stored at -80 °C for further analysis.
Parasites and cell culture
Human foreskin fibroblasts (HFFs) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 1% antibiotics (10 mg/mL streptomycin solution, 25 μg/mL amphotericin B and 10,000 U/mL penicillin) (Sangon Biotech, China) and 10% fetal bovine serum (FBS, Invitrogen, USA) at 37 °C in 5% CO2. The RH-GFP-TgAtg8 and RH wild-type T. gondii strains were maintained by serial passage in confluent monolayers of HFFs [28]. Intracellular parasites were released by rapidly passing them through a 30-gauge needle 6–8 times. The parasite suspension was filtered through a 5 μm pore size filter to remove cellular debris, then centrifuged at 3000 rpm for 8 minutes to enrich the tachyzoites. The pellet was resuspended in DMEM and counted using a hemocytometer.
Survival assay
To evaluate the susceptibility of M. fortis to T. gondii RH strain tachyzoites, M. fortis served as the experimental group and KM mice served as the control group, with six mice in each group. Each animal was intraperitoneally injected with 2 × 103 tachyzoites resuspended in 0.5 ml phosphate-buffered saline (PBS). The animals were observed daily for 30 days. M. fortis injected with T. gondii survived and were sacrificed after 30 days. Their organs (brain, heart, liver, spleen, lung, and kidney) as well as ascitic fluid were collected. To further confirm the presence of T. gondii in live M. fortis, 0.5 mL of homogenate from each organ or ascitic fluid was intraperitoneally injected into KM mice, with six mice assigned to each group.
To assess the contribution of the complement system to T. gondii resistance in M. fortis, the experimental group of M. fortis was injected with cobra venom factor (CVF) (50 μg/kg) via the tail vein to deplete the complement in vivo, while the control group received an equivalent volume of PBS via the tail vein, with five mice in each group. CVF is the complement-activating protein in cobra venom. It forms a stable, fluid-phase enzyme that resists inactivation by factors H and I, thereby effectively depleting serum complement components such as complement component 3 (C3) and complement component 5 (C5) and blocking the terminal complement pathway [29–32]. Twelve hours after CVF administration, each M. fortis was intraperitoneally injected with 1 × 10⁴ tachyzoites. The experimental group received CVF (50 μg/kg every 2 days) to maintain the complement system-depleted state, while the control group was injected with an equal volume of PBS. Observations were recorded over a 30-day period.
DNA extraction and PCR
For PCR detection, total DNA was isolated from fresh organs using a genomic DNA purification kit. A 529 bp fragment was amplified from the DNA template using the following primers:(5’-CGCTGCAGGGAGGAAGACGAAAGTTG-3’ and 5’-CGCTGCAGACACAGTGCATCTGGATT-3’), which were selected from the literature [33]. PCR was performed in a 20 μl reaction volume containing 10 μl Taq PCR Master Mix, 0.2 μM primer mix, and 2 μl DNA template. The PCR cycling protocol consisted of an initial denaturation at 95 °C for 2.5 minutes, followed by 30 cycles of denaturation at 95 °C for 15 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 60 seconds, with a final extension at 72 °C for 10 minutes. PCR products were electrophoresed in Tris-acetic acid-EDTA buffer with 1% agarose, stained with Safe Green DNA stain, and visualized under ultraviolet light. Positive samples exhibited a band at 529 bp.
Viability of parasites incubated with KM mice or M. fortis serum
Trypan blue can penetrate the cell membrane of dead cells and stain the cytoplasm blue. The tachyzoites that are stained blue can be considered dead under the optical microscope. To further distinguish between dead and surviving tachyzoites, a fluorescent strain of T. gondii (RH-GFP-TgAtg8) was used, which was fluorescently labelled with a cytoplasmic protein [34]. It was observed that the fluorescence disappeared upon death of tachyzoites. The mortality of tachyzoites was assessed using a combination of fluorescent strains and trypan blue staining. Tachyzoites were prepared as described previously, and the concentration was adjusted to 2 × 107/ml with PBS. The tachyzoite suspension was then incubated for 2 hours with M. fortis serum (30%) or KM serum (30%) at 4 °C in 200 μl volumes. After centrifugation at 3000 rpm for 8 min to remove the reaction buffer, the parasites were resuspended in 50 μl PBS and stained with trypan blue (0.5%, Sigma Chemical Co, USA). The viability of the tachyzoites was immediately assessed using an upright fluorescence microscope (Leica, DM 3000 LED, China). Parasites were randomly counted in 5 microscopic fields, and the mortality rate of tachyzoites was calculated using the following formula: mortality (%) = (number of dead parasites (tachyzoites stained blue)/number of total parasites (tachyzoites stained blue plus fluorescent tachyzoites)) × 100%. These assays were performed in triplicate, with serum derived from three distinct animals for each of the three replicates within each group.
In some experiments, M. fortis serum was pretreated before being mixed with the tachyzoites suspension. The treatments included heat inactivation (56 °C for 30 minutes), 10 mM ethylenediaminetetraacetic acid (EDTA), 10 μg/ml CVF, 5 mM ethylene glycol tetraacetic acid (EGTA) with 10 mM MgCl₂, and 100 nM Narsoplimab (Aladdin, China) [35,36]. All pre-treatments, except for heat inactivation of complement, were mixed and incubated at 4 °C for 10 minutes.
Electron microscopy analysis
The ultrastructural characterization of tachyzoites treated with serum was captured by scanning electron microscopy (SEM). T. gondii tachyzoites (1 × 107) were incubated with PBS, serum from Kunming mice, and serum from M. fortis at 4°C for 2 hours. The tachyzoites treated with PBS were employed as the control group. The tachyzoites were collected by centrifugation at 3000 rpm for 8 minutes and then were washed three times with PBS and fixed with 0.01 mol/L PBS (pH 7.4) containing 4% glutaraldehyde at 4 °C overnight. All samples were dehydrated in ethanol dilutions (70%, 80%, 90%, and 100%). The tachyzoites were then subjected to critical point drying, mounted on stubs, coated with a metal layer, and observed under a Hitachi S-3400N scanning electron microscope [37].
Indirect immunofluorescence staining
Tachyzoites co-incubated with M. fortis serum or PBS were centrifuged to collect the precipitate, which was then resuspended in 50 μL of PBS. A 10 μL aliquot of the tachyzoite suspension was used to prepare smears. After drying, the smears were fixed with methanol for 10 minutes, washed three times with PBS for 5 minutes, incubated with 2% (w/v) BSA at 37 °C for 30 minutes, and washed three times with PBS for 5 minutes. They were then stained with mannan-binding lectin serine protease 2 (MASP2) recombinant rabbit polyclonal antibody (Affinity Biosciences, China) or membrane attack complex (MAC) recombinant rabbit polyclonal antibody (Bioss, China) at 37 °C for 1 hour, washed three times with PBS for 5 minutes. Stained with AF488 fluorescein conjugated sheep anti-rabbit antibody (Servicebio, China) at 37 °C for 1 hour under light-protected conditions and washed three times with PBS for 5 minutes. The slides were sealed with an anti-fluorescence quencher containing the fluorescent dye DAPI, and subsequently photographed under an upright fluorescence microscope (Leica, DM 3000 LED, China).
Statistical analysis
Data were analyzed and graphs were constructed using Photoshop version 2020 (Adobe Systems Inc., SAN Jose, CA, USA) and GraphPad Prism version 8.4 (GraphPad Software, San Diego, CA, USA). Various statistical analyses were calculated using the two-tailed Student’s t-test and p < 0.05 was considered statistically significant.
Results
M. fortis is not susceptible to the RH strain of T. gondii
In this study, we observed that M. fortis infected with T. gondii tachyzoites via intraperitoneal injection did not exhibit the severe acute toxoplasmosis symptoms in KM mice. The morphology of the M. fortis is shown in Fig 1A. To determine the susceptibility of M. fortis to T. gondii tachyzoites, M. fortis and KM mice were intraperitoneally injected with 2 × 103 tachyzoites and monitored daily for 30 days. KM mice, which are sensitive to T. gondii, died within 7 days. However, M. fortis survived for 30 days (Fig 1B), and no T. gondii tachyzoites were detected by microscopy in the brain, heart, liver, spleen, lung, kidney, or ascitic fluid of the surviving M. fortis. To further confirm whether T. gondii was still present in the surviving M. fortis, we performed PCR to detect T. gondii DNA in the brain, heart, liver, spleen, lung, kidney, and ascitic fluid of infected M. fortis. Additionally, we inoculated the peritoneal cavity of KM mice with organ homogenates and ascitic fluid from surviving M. fortis to detect viable tachyzoites. PCR did not detect T. gondii DNA in the brain, heart, liver, lung, or kidney of infected M. fortis (Fig 1C). Furthermore, all KM mice intraperitoneally injected with organ homogenates or ascitic fluid survived for 30 days (Fig 1D). These results show the absence of T. gondii in M. fortis that survived 30 days after intraperitoneal infection with T. gondii.
(A) Morphology of M. fortis. (B) Survival curve of M. fortis and KM mice infected with 2 × 103 tachyzoites of T. gondii in 0.5 ml PBS. The survival of mice was monitored for 30 days (n = 6 for each group). (C) PCR detection of the T. gondii DNA in six surviving M. fortis. Lanes 1–8 in order: Positive control (DNA extracted from tachyzoites), brain, heart, liver, spleen, lung, kidney, and negative control, lane M: DNA ladder. (D) The survival curve of KM mice inoculated with the homogenates of brain, heart, liver, spleen, lung, kidney, and ascitic fluid derived from infected M. fortis. The survival of mice was recorded for 30 days (n = 6 for each group).
M. fortis serum can kill the T. gondii tachyzoites
M. fortis is frequently used to study host resistance to Schistosoma japonicum because it is naturally resistant to the parasites [38,39]. Previous studies have shown that M. fortis serum can directly exert cytotoxic effects on schistosomula in vitro [40]. Our study found that M. fortis serum also exhibits significant killing activity against T. gondii tachyzoites in vitro. After exposure to 50% M. fortis serum or 50% KM mouse serum for 2 hours, more than 80% of parasites survived in PBS or KM mouse serum. However, more than 90% of M. fortis serum-treated parasites were stained with trypan blue and lost green fluorescence, indicating loss of viability (Fig 2A, 2B). We found that the mortality rate of tachyzoites increased with the concentration of M. fortis serum. Incubation of tachyzoites with 30% M. fortis serum for 2 hours resulted in the death of over 90% of them (Fig 2C). The ultrastructure of T. gondii tachyzoites treated with PBS, KM mouse serum, or M. fortis serum was observed by scanning electron microscopy (SEM). In the PBS-treated group (Fig 3A-3C), tachyzoites displayed a smooth, regular, and intact surface with typical crescent shapes. A similar morphology was observed in the KM serum-treated group (Fig 3D-3F). In contrast, parasites treated with M. fortis serum exhibited significant morphological changes. Most tachyzoites lost their characteristic crescent shape, with numerous deep ridges, pits, and wrinkles on the surface (Fig 3G-3I). These alterations suggested that M. fortis serum treatment disrupted the tachyzoite membrane structure.
(A) Tachyzoites were treated with PBS, 50% M. fortis serum (MF), or 50% KM serum (KM) for 2 hours, and then stained with trypan blue and photographed under bright-field and fluorescence microscopy. Dead and surviving tachyzoites were identified based on the merged images. (B) Statistical data on tachyzoite mortality after treatment with PBS, 50% M. fortis serum (MF), or 50% KM serum (KM) for 2 hours (ns: p ＞ 0.05, ****p < 0.0001). (C) Statistical data on tachyzoite mortality after treatment with PBS, 10%, 20%, 30%, 40%, or 50% M. fortis serum for 2 hours (ns: p ＞ 0.05, ****p < 0.0001).
Scale bars: 10 μm (A, D, G); 5 μm (B, E, H); 2 μm (C, F, I).
M. fortis serum kills T. gondii by activating the complement system
The complement system plays an important role in the recognition and elimination of invading pathogens. To ascertain whether the complement system is responsible for killing T. gondii tachyzoites in M. fortis serum, we employed several approaches: heat inactivation of serum, inhibition of complement activation with 10 mM EDTA, and complement depletion through pretreatment of M. fortis serum with CVF. The killing effect of tachyzoites was completely inhibited by heat inactivation of the M. fortis serum (Fig 4A). Pre-treatment of M. fortis serum with EDTA to chelate calcium and magnesium ions, which are required for complement activation [41,42], completely inhibited the serum’s ability to kill tachyzoites (Fig 4B). In addition, the ability of M. fortis serum to kill tachyzoites was significantly inhibited by pretreatment with CVF, which depletes serum complement components and blocks complement system activation (Fig 4C). These results suggested that the mechanism underlying the killing of T. gondii tachyzoites in M. fortis serum was associated with the activation of the complement system.
Statistical data on tachyzoite mortality after heat inactivation at 56 °C (A), EDTA pretreatment (B), and CVF pretreatment (C) of 30% M. fortis serum co-incubated with T. gondii tachyzoites for 2 hours. M. fortis serum (MF), KM serum (KM), M. fortis heat-inactivated serum (MFH), KM heat-inactivated serum (KMH) (ns: p > 0.05, ***p < 0.001, ****p < 0.0001).
T. gondii tachyzoites activate the complement system via the lectin pathway in the serum of M. fortis
To determine the complement activation pathway activated by tachyzoites in M. fortis serum, we pretreated the serum with 5 mM EGTA to chelate calcium ions. Calcium ions are required for the initiation of the classical and lectin pathways, but not for the alternative pathway [22,43]. As a result, we observed that the tachyzoite-killing activity of M. fortis serum was markedly diminished (Fig 5A). The classical pathway is often referred to as antibody-dependent because of its strong initiation by IgM/IgG clusters [22]. However, no anti-T. gondii antibodies were detected in M. fortis serum by indirect haemagglutination assay (S1 Table). MASP2 is an essential protease in the initiation of the lectin pathway [44,45]. Indirect immunofluorescence assay with an anti-MASP2 antibody revealed fluorescence on the tachyzoite membrane after incubation with M. fortis serum (Fig 5B). This observation indicated that MASP2 was deposited on the surface of T. gondii membranes. To further confirm the involvement of MASP2 in complement activation, M. fortis serum was pretreated with Narsoplimab (a MASP2 inhibitor) and then co-incubated with tachyzoites. This treatment resulted in a notable inhibition of the serum’s killing effect on tachyzoites (Fig 5C), indicating that MASP2 was essential for complement system activation by T. gondii. To verify whether complement system activation directly results in the formation of the membrane attack complex (C5b-9, MAC) on the surface of tachyzoites and subsequently mediates their lysis, indirect immunofluorescence was performed to assess MAC formation. The results revealed distinct MAC deposition on the tachyzoite surface following incubation with non-immune serum from M. fortis. In contrast, no MAC formation was detected on tachyzoites incubated with non-immune serum from KM mice or humans (Fig 5D).
(A) Statistical data on tachyzoite mortality after EGTA-MgCl2 pretreatment of 30% M. fortis serum co-incubated with T. gondii tachyzoites for 2 hours (ns: p > 0.05, ****p< 0.0001). (B), (D) Bright-field microscopy was used to locate the tachyzoites. DAPI (blue) staining reveals the localization of the tachyzoite nuclei, and AF488 fluorescein (green) shows the localization of MASP2 or MAC. The merged images show the co-localization of DAPI and AF488 fluorescein signals. Scale bars: 10 μm. (C) Statistical data on tachyzoite mortality after Narsoplimab pretreatment of 30% M. fortis serum co-incubated with T. gondii tachyzoites for 2 hours (ns: p > 0.05, ****p < 0.0001).
The capacity of M. fortis to eliminate T. gondii is impaired when the complement system is inhibited
To determine whether the complement system in M. fortis plays a key role in its high resistance to T. gondii, we depleted complement components C3 and C5 in M. fortis by tail vein administration of CVF. Twelve hours post-CVF administration, the lethal effect of M. fortis serum on T. gondii was significantly inhibited, confirming the inhibitory effect of CVF on the complement system in M. fortis (Fig 6A). Although M. fortis with complement depletion survived T. gondii infection for 30 days (Fig 6B), the T. gondii DNA remained detectable in the organs and ascitic fluid of M. fortis (Fig 6C). Ascitic fluid from surviving M. fortis in the experimental group was injected into the peritoneal cavity of KM mice, the KM mice died within 11 days, and a significant number of fluorescent T. gondii tachyzoites were observed in the ascitic fluid (Fig 6D). In contrast, no T. gondii DNA were detected in the control M. fortis, and all KM mice that received ascitic fluid from these controls survived for 30 days. These findings suggested that T. gondii remained viable in the experimental group of M. fortis.
(A) Serum was collected from M. fortis 12 hours after CVF injection. Statistical data on tachyzoite mortality after co-incubation of 30% M. fortis serum with T. gondii tachyzoites for 2 hours. (ns: p ＞ 0.05, ****p < 0.0001) (B) M. fortis was injected with CVF in the experimental group and an equal amount of PBS in the control group. Survival curve of the experimental and control groups M. fortis infected with 1 × 104/0.5 ml tachyzoites of T. gondii. The survival time of M. fortis was recorded for 30 days (n = 5 per group). (C) PCR detection of the T. gondii DNA in the experimental and control groups of M. fortis at day 30. Lanes 1-8 in order: Heart, liver, spleen, lung, kidney, brain, ascitic fluid, positive control (DNA extracted from tachyzoites), lane M: DNA ladder. (D) Fluorescence microscopy of tachyzoites in ascitic fluid from infected KM mice. Scale bars: 20 μm.
Discussion
In this study, M. fortis was observed to exhibit natural high resistance to T. gondii RH strain infection, whereas KM mice showed extreme susceptibility. We demonstrated that T. gondii tachyzoites triggered the complement system via the lectin pathway in M. fortis serum, resulting in tachyzoite lysis. In contrast, the complement system of KM mice was unable to effectively lyse T. gondii tachyzoites. And it was demonstrated that the complement system in M. fortis played a crucial role in resisting the infection of T. gondii as no M. fortis died acutely, unlike KM mice.
T. gondii is recognized as one of the most successful parasites, primarily because of its extensive host range, which includes nearly all warm-blooded animals. To investigate the disparity in infection outcomes between KM mice and M. fortis after infection with a virulent T. gondii RH strain, we conducted subsequent studies. These experiments revealed that serum from M. fortis effectively killed T. gondii tachyzoites in vitro, whereas KM mouse serum was ineffective. Further experiments established a critical role for the complement system in the in vitro tachyzoite-killing activity of M. fortis serum. The complement system, a vital component of the innate immune system, plays a pivotal role in the protection against the invasion of foreign pathogens. In M. fortis injected with CVF to inhibit complement activity, viable tachyzoites remained detectable 30 days post-infection. In contrast, no tachyzoites were found in the control group. These findings demonstrate a critical role for the complement system controlling T. gondii infection in M. fortis. However, in contrast to the KM mice, M. fortis did not develop uncontrolled symptoms of acute toxoplasmosis. This suggested that factors other than the complement system contribute to infection control, or that complement activity was not completely abolished in M. fortis.
In order to establish a persistent infection, parasites circulating in the host bloodstream or requiring circulatory passage must evade complement-mediated destruction. In vitro experiments have shown that the T. b. gambiense invariant surface glycoprotein 65 (ISG65) specifically inhibits the activity of the alternative pathway C5 converting enzyme, thereby preventing the formation of membrane attack complexes and subsequent cell lysis [46]. The Plasmodium falciparum proteins VAR2CSA, TM284VAR1, DBLMSP, and DBLMSP2 bind IgM in different ways, interfering with IgM-mediated complement activation and promoting Plasmodium immune evasion [47]. Amoebae can fuse host proteins to their cell membranes to evade complement-mediated lysis after ingestion of host cells [48,49]. T. gondii is an obligate intracellular parasite, and the role of the complement system in the clearance of T. gondii appears to be limited. Our findings indicate that the complement system plays a crucial role in the killing of extracellular tachyzoites. The NLRP1 inflammasome in the Lewis rats can detect T. gondii infection, which triggers pyroptosis in infected macrophages and eliminates the parasite’s replication niche [14,15,50]. Our findings in the similarly resistant M. fortis demonstrate that the complement system mediates the direct killing of extracellular tachyzoites. Thus, we speculate that a collaborative defense strategy may exist in naturally resistant rodent hosts, whereby intracellular pyroptosis and extracellular complement-mediated lysis cooperate to achieve comprehensive parasite clearance.
Despite the resistance of T. gondii tachyzoites to complement-mediated lysis in mice, the complement component C3 remains a key factor in regulating parasite proliferation and transmission, as well as promoting chronic infection [51]. Moreover, the production of anti-T. gondii antibodies in humoral immunity is imperative for resistance to acute T. gondii infection [52,53]. In this study, some M. fortis inoculated with 1 × 10⁴ T. gondii tachyzoites for 30 days developed low titers of T. gondii-specific antibodies, regardless of CVF treatment, indicating the establishment of a specific humoral immune response (S2 Table). Theoretically, antibody production can activate the complement system via the classical pathway, contributing to the clearance of tachyzoites [51,54]. However, in M. fortis, the complement system is already effectively activated through the lectin pathway to lyse tachyzoites; thus, antibody-mediated complement activation may represent a functionally redundant mechanism in this context. Nevertheless, antibody production may still enhance the overall resistance of M. fortis to T. gondii infection by facilitating the phagocytic clearance of tachyzoites by macrophages [55,56]. In human serum, T. gondii has been observed to recruit Factors H and C4b-binding proteins, which mediate resistance to serum killing [51]. This mechanism may underpin the ineffectiveness of the complement system against T. gondii in mice.
M. fortis is a non-susceptible rodent host for Schistosoma japonicum. The clearance of Schistosoma by M. fortis is achieved through a mechanism involving contact-dependent trogocytosis by M. fortis macrophages, which is dependent on complement C3 and its receptor CR3 [57]. T. gondii is a unicellular eukaryotic protozoan, whereas Schistosoma is a multicellular worm. The complement system’s capacity to lyse cells is sufficient to cause the death of T. gondii, whereas the killing of Schistosoma is dependent on trogocytosis by macrophages. Further investigation is required to ascertain whether complement activation via the lectin pathway enhances macrophage clearance of T. gondii in M. fortis. We also conducted a study to evaluate the killing effect of M. fortis serum on ME49 tachyzoites and demonstrated that M. fortis serum effectively killed ME49 tachyzoites in vitro (S1 Fig). However, further investigation is required to determine whether M. fortis can inhibit cyst formation during infection with the T. gondii type II strain.
Conclusions
This study reveals that M. fortis is naturally resistant to infection by T. gondii type I strain RH. Furthermore, the complement system in M. fortis is activated through the lectin pathway, leading to direct lysis of extracellular tachyzoites and contributing to infection control. These findings highlight the significant role of the complement system in controlling T. gondii infection and provide new insights into host-parasite interactions.
Supporting information
S1 Fig. In vitro killing effect of M. fortis serum on ME49 tachyzoites.
Statistical data on ME49 tachyzoites mortality after treatment with PBS or 30% M. fortis serum (MF) for 2 hours (***p < 0.001).
https://doi.org/10.1371/journal.pntd.0014052.s001
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S1 Table. Results of indirect immunohemagglutination assay for T. gondii antibody detection.
The antibody titers of experimental samples were determined based on the highest dilution showing visible agglutination (≥1+). MF: M. fortis, KM: Kunming mice. The numbering corresponds to serum from different individuals.
https://doi.org/10.1371/journal.pntd.0014052.s002
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S2 Table. Detection of anti-T. gondii antibodies in M. fortis serum 30 days post-infection.
The antibody titers of experimental samples were determined based on the highest dilution showing visible agglutination (≥1+). PBS-MF: M. fortis control group receiving tail vein injection of PBS after challenge with 10⁴ tachyzoites, CVF-KM: M. fortis experimental group receiving tail vein injection of CVF after challenge with 10⁴ tachyzoites. The numbering corresponds to serum from different individuals.
https://doi.org/10.1371/journal.pntd.0014052.s003
(DOCX)
Acknowledgments
The authors would like to thank Professor Hongjuan Peng of Southern Medical University (Guangzhou, China) for providing the T. gondii RH strain and Professor Qianyun Sun of the Key Laboratory of Natural Products Chemistry, Chinese Academy of Sciences (Guizhou, China) for providing cobra venom factor (CVF). The authors also gratefully acknowledge the instrumentation support provided by the Top-Notch Innovation Base of Basic Medicine, Central South University.
Consent for publication: All authors have provided their consent for publication.
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