Conventional treatment of acquired and congenital toxoplasmosis usually involves a regimen based on pyrimethamine and sulfadiazine. However, pyrimethamine is potentially teratogenic and causes reversible bone marrow suppression[9, 46, 47, 48]. In addition, various serious complications of clinical drugs for toxoplasmosis have been reported, such as agranulocytosis, Stevens-Johnson syndrome, toxic epidermal necrolysis and hepatic necrosis, ect[49, 50, 51, 52, 53]. Current treatment for toxoplasmosis is limited, with multiple side effects and long treatment durations (ranging from 4–6 weeks to more than 1 year)[9, 54]. In addition, some intrinsic factors of T. gondii, such as increased drug resistance, differential drug susceptibility among different strains, and other unknown aspects of parasite pathogenicity, also play an important role in disease progression and treatment failure[55, 56, 57]. Therefore, there is an urgent need to develop safer and more effective therapeutic alternatives for toxoplasmosis with fewer side effects, which depends on the growing knowledge of Toxoplasma pathophysiology and the discovery of promising drug targets.
In this study, the lipopeptide L-C12, L-C12-1, L-C12-2, L-C12-3, L-C12-4, L-C12-5, L-C12-6 and L-C12-7 were preliminarily evaluated for their direct anti- T. gondii activity in vitro using the trypan blue assay. It was found that L-C12-4 and L-C12-6 lipopeptides completely lost their anti- T. gondii activity in vitro. We suggested that the lysine at these two sites was the key site for Lycosin-I to exert its anti-T. gondii activity, and that lauric acid modified at these two sites altered its original physical and chemical properties. Although L-C12, L-C12-1, L-C12-2, L-C12-3, L-C12-5 and L-C12-7 all showed concentration-dependent anti- T. gondii activity in vitro, only L-C12 showed similar efficacy to Lycosin-I at 10 µM and lower concentration, the other lipopeptides were obviously weaker than Lycosin-I in anti-T. gondii activity. Therefore, we speculated that the α-amino terminus of the first lysine site of Lycosin-I (the N-terminus of Lycosin-I) was the best modification site to improve the anti- T. gondii activity, which was consistent with the previous research[32].
Since fatty acids of different lengths have different activities on peptides[45], we obtained the lipopeptide L-an by coupling another shorter fatty chain (aminocaproic acid) to the N-terminus of Lycosin-I. Trypan blue assay showed that the anti-T. gondii activity of the two lipopeptides in vitro was similar to that of Lycosin-I. However, the cytotoxicity of L-C12 was significantly higher than that of Lycosin-I. L-an showed no significant difference with Lycosin-I, indicating that the longer the fatty chain, the more toxic it was to the cells. The serum stability of Lycosin-I was increased after fatty acid modification, as shown by the mass spectrometry, which showed that Lycosin-I was degraded into smaller peptides after incubation with 10% serum for 24 h, whereas L-C12 was not degraded[32]. Interestingly, we found that Lycosin-I only slightly decreased, rather than completely lost its anti-T. gondii activity in vitro after 24 hours of serum incubation. We hypothesized that although Lycosin-I was degraded into smaller peptides, the key amino acid sequences of anti-T. gondii were still retained in the small peptide, suggesting that we could optimize the specific activity of peptides by truncating them. After incubation in serum for 24 hours, L-C12 and L-an retained substantial anti-T. gondii activity in vitro, consistent with the results of L-C12 mass spectrometry. Although L-an was not shown by mass spectrometry to be resistant to enzymatic degradation in serum, it was confirmed by trypan blue assay that L-an improved the stability of the anti-T. gondii activity of Lycosin-I in serum.
The invasion and pathogenicity of T. gondii to host is a complex process involving parasite movement and penetration, as well as interaction and attachment with host cells[58]. The proliferation of T. gondii in host cells is mainly influenced by the immune response of the host cells[59, 60]. In vitro, Lycosin-I, L-C12 and L-an showed significant anti-T. gondii activity, which was consistent with our previous results of Lycosin-I[23]. We found that two lipopeptides L-C12 and L-an showed slightly better inhibition efficiency of tachyzoites than Lycosin-I, especially at the low concentration of 5 µM. These results showed that fatty acid modification enhanced the ability of Lycosin-I to inhibit tachyzoite invasion by reducing their motility, adhesion, and locomotion. Although the parasitophorous vacuole membrane (PVM) was mainly derived from the plasma membrane of host cells, T. gondii also secreted some proteins to participate in the formation of PVM. What’s more, some proteins of T. gondii are even secreted and released into host cells through the PVM, mediating the host cell immune response and achieving the purpose of evading host immunity[61, 62, 63]. We hypothesized that Lycosin-I, L-C12 and L-an could inhibit tachyzoite proliferation by appropriately promoting the host inflammatory response and inhibiting the release of proteins secreted by tachyzoites into the host cytoplasm. In the plaque assay, Lycosin-I, L-C12, and L-an show no advantages over the positive drug SDZ at the same concentration, which may be related to the time of onset and the stability of drug efficacy. In the invasion and proliferation assays, there were 5 ⋅ 105 tachyzoites (MOI = 5, tachyzoite number/cell number) in each well, and the incubation time was relatively short (only 2 h or 24 h). Fast acting peptides therefore have an advantage over slow acting SDZ. However, only 500 tachyzoites were used in each well in the plaque assay, and the incubation time was longer than 7 days. Peptide may be degraded by serum, resulting in less good effect than positive drugs. The inhibitory effect of L-C12 and L-an was slightly better than that of Lycosin-I, which may be related to the improved serum stability of these two lipopeptides.
The experiment of animals is the most important indicator of drug efficacy. We established a mouse model acutely infected with tachyzoites, observed the survival time of the mice and deteermined the parasite load in the tissues of the mice. Lycosin-I and L-an can effectively prolong the survival time of mice. Although SDZ and L-C12 can effectively inhibit the proliferation of tachyzoites in mice, they couldn't prolong the survival time of mice at the same dose. It's a fact that the clinical dosage of SDZ is 400 mg/kg, but only 4 mg/kg was used in this work, we hypothesized that SDZ could not perform anti-T. gondii in vivo at low dosages. As for L-C12, this may be due to its excessive toxicity to mice, which resulted in early death of the mice. The cytotoxicity of L-C12 was significantly higher than that of Lycosin-I in vitro, with an IC50 of 15.62 µM compared to 46.16 µM for Lycosin-I.
Toxoplasma infection can induce a host immune response that is primarily mediated by T helper cell 1 (Th1) and requires components of both the innate and adaptive immune response[64]. In the initial stage of infection, T. gondii is recognized by innate immune response cells, stimulating dendritic cells, macrophages and neutrophils to produce interleukin-12 (IL-12) and inducing natural killer (NK) cells to produce interferon-gamma (IFN-γ). In addition, tumour necrosis factor (TNF) can interact synergistically with IL-12 to optimize IFN-γ production in these cells[65, 66]. These pro-inflammatory cytokines are produced when the adaptive immune response, mediated by CD4+ and CD8+ T lymphocytes, is activated. These lymphocytes produce and secrete several inflammatory mediators, such as nitric oxide (NO), and also cause a greater increase in IL-12 and IFN-γ levels[67, 68]. IFN-γ can activate dendritic cells, macrophages, and neutrophils to promote the reduction or elimination of T. gondii. When macrophages are activated by IFN-γ, the production of NO increases, contributing to the toxicity of T. gondii[69]. In this study, we found that IFN-γ, but not TNF-α, was significantly increased in mice acutely infected with tachyzoites compared with normal mice. SDZ, Lycosin-I, L-C12 and L-an promoted the expression of IFN-γ and TNF-α in mice. However, an excessive inflammatory response can lead to host death[65, 70]. Therefore, it is important to strike a balance between Th1 and Th2-mediated immune responses. This balance can be mediated by the production of anti-inflammatory cytokines, such as IL-4, IL-10, and transforming growth factor (TGF-β1), which play a role by reducing the production of NO in macrophages and the cytotoxic activity of NK cells[71, 72]. Besides, Toxoplasma seeks mechanisms to evade the strong immune response of the host, such as induction of anti-inflammatory cytokines. Expression of IL-10 and TGF-β1 increased the susceptibility of BeWo cells to T. gondii infection. Toxoplasma infection can induce the production of IL-4, IL-10 and TGF-β1 in host cells[73]. In this study, we found that the expression of IL-4 and IL-10 was higher in mice acutely infected with tachyzoites than in normal mice. Interestingly, SDZ, Lycosin-I, L-C12 and L-an all promoted IL-4 expression in mice compared to the negative control mice. However, SDZ, Lycosin-I and L-an inhibited the expression of IL-10, while L-C12 promoted the expression of IL-10. We hypothesized that the increase of IL-4 may regulate inflammation caused by the expression of the pro-inflammatory factors IFN-γ and TNF-α, and prevent the excessive inflammation leading to the death of the mice. The reduction in IL-10 may be related to the inhibition of tachyzoites to evade host immunity by inducing the expression of anti-inflammatory factors. As for the promotion of IL-10 expression by L-C12, this may also be the reason why L-C12 didn’t prolong the survival time of the mice in animal survival experiments, because it only focuses on promoting the expression of anti-inflammatory factors to regulate the inflammatory response, which is precisely what helps the tachyzoites to escape the host immune response.