T. gondii is a food-borne parasite found worldwide that can infect almost all warm-blooded animals, including humans [24]. Chronic T. gondii infection often results in few clinical symptoms in immunocompetent individuals. However, some studies have suggested that it can result in changes in immunity, brain transcriptomic signatures, microbiota, and even behavior and personality in the host [6, 25, 26]. Some studies have revealed that a healthy intestinal microbiota is a complex ecological community that can carry out critical physiological functions with the intestinal mucosa of the host [27]. Moreover, changes in the gut flora composition can prevent or alleviate intestinal protozoan infection and affect the ultimate outcomes of parasitic diseases. There is also a close connection among the host, microbiota and pathogens. An interesting example is that a certain gut flora composition in the host can impede gut colonization by Helminths or reduce the colonization time [28]. To date, there is no study that associates the host microbiota with the treatment of T. gondii infection. Shao et al only discussed the changes in the intestinal flora 13 days and 21 days after infection with T. gondii [25]. In this study, we investigated not only the effect of koumiss treatment on the intestinal flora of mice chronically infected with T. gondii but also the long-term changes in the gut flora caused by T. gondii infection, including the changes that occur in the acute infection stage, chronic infection stage and cyst formation stage.
In this study, we determined the 16S rRNA gene sequences of the gut microbiota of the C, G, S and X groups at 7 dpi, 14 dpi, 21 dpi, 28 dpi, 35 dpi and 42 dpi. The intestinal flora community structure at the phylum level was mainly analyzed. Bacteroidota was the most abundant phylum in the C, G, S and X groups, and the relative abundance of Bacteroidota decreased after infection with T. gondii. There was an obvious increase in the relative abundance of Firmicutes in all the experimental groups, which was different from a previous study [25]. In a previous study, mice were infected with 100 purified sporulated oocysts of the ME49 strain, which may have caused inflammation in the intestine and was associated with a low F/B (Firmicutes/Bacteroidota) index [29]. In this study, mice were inoculated with 3 cysts to establish chronic infection, and no intestinal inflammation was observed (data not shown), but this treatment resulted in a high F/B index. The gut-brain axis plays an important and complex bidirectional role in communication between the intestine and the central nervous system (CNS) and is involved in different pathways and systems, including the autonomic and enteric nervous system, the endocrine system, the hypothalamic-pituitary-adrenal axis (HPA), the immune system, and the regulation of the microbiota and its metabolites [30-32]. Therefore, we considered that one of the reasons that explains our results is that parasites might influence the intestinal microbiota through the gut-brain axis. Moreover, this parasite is distributed all around the host during chronic infection, which makes the mechanism more difficult to understand; this is an interesting phenomenon worthy of further study.
In addition, we found that T. gondii infection dramatically changed the community structure of the microbiota. The C group and other treatment groups showed an obvious separation in the PCoA plot at 21 dpi, indicating that T. gondii infection would alter the intestinal flora composition, which was consistent with previous studies [25]. Nevertheless, the C group was not separately clustered from the other treatment groups at 14 dpi. This result may have occurred because a large number of parasites had appeared in the brain. The strains of T. gondii and the inoculation dose and time may affect the distribution of T. gondii. Zenner et al confirmed that T. gondii first reached the mesenteric lymph nodes (MLNs) through the intestinal wall, then reached spleen, and finally reached other organs [33]. Saeij et al thought that a virulent strain of T. gondii first appeared in the head and neck, whereas an attenuated strain appeared preferentially in the abdomen [34]. The changes in the microbiota composition caused by T. gondii in the brain were also associated with the gut-brain axis. Interestingly, there was also an apparent separation of 35 and 42 dpi from 7, 14, 21 and 28 dpi in the G, S and X groups, which indicated that the community structure of the gut flora could change over time during T. gondii infection. The results are shown in the PCA plot, PCoA plot, NMDS plot and Venn diagrams. The Ace and Chao1 indices of the G, S and X groups were obviously increased at 35 and 42 dpi. The results demonstrated that there was an obvious increase in the gut flora diversity during T. gondii cyst formation and that this increased diversity persisted for a long time. Traditionally, pathogen infection is thought to decrease the microbiota richness. However, the results of this study were completely opposite of that previous theory. This discovery has important implications for understanding the connection between pathogens and the intestinal flora in the host.
The effect of koumiss treatment on intestinal flora was also discussed in this study. It should be noted that the infected mice treated with koumiss were not significantly different from the untreated mice in terms of OTUs, the Shannon index or the Simpson index. However, we still found that koumiss treatment altered the relative abundance of certain bacteria, such as Actinobacteriota, Lactobacillaceae and Acetobacter. The most important alteration was that the relative abundance of Lachnospiraceae continuously increased after the mice infected with T. gondii were orally gavaged with koumiss. There were significant differences in the relative abundance of Lachnospiraceae between the S and C group, the S and X group at 42 dpi. Lachnospiraceae are a family of obligate anaerobic bacteria that are abundant in healthy humans. Lachnospiraceae can facilitate colonization resistance against intestinal pathogens by producing the short-chain fatty acids (SCFAs) acetate and butyrate [35], converting primary to secondary bile acids [36] or producing lantibiotics [37]. Therefore, it was thought that the reduction in the numbers of cysts in the brain in the S group, a key metric indicating decreased parasitic burden during chronic T. gondii infection, may be associated with a high level of Lachnospiraceae. Moreover, the results published in Science showed that Lachnospiraceae can allow mice to survive after receiving a lethal dose of total body radiation by promoting hematopoiesis restoration and gastrointestinal repair [38]. More importantly, Lachnospiraceae abundances can be altered by changes in diet [39]. This will be further explored for the treatment of T. gondii infection. Furthermore, we expectedly found that the relative abundance of A. muciniphila in the S group at 7 dpi dramatically differed from that in the C, G and X groups (P < 0.001). A. muciniphila is an emerging beneficial bacterium that controls the essential regulatory system of glucose and energy metabolism [40]. In addition, A. muciniphila can also decrease blood lipid levels and affect metabolic modulation, immune response regulation, and gut health maintenance [41-44]. Interestingly, it was also found that A. muciniphila could facilitate the expulsion of intestinal Trichinella spiralis through the development of goblet cell hyperplasia and secretion of mucus [45, 46]. Although A. muciniphila was isolated from human feces in 2004, the relationship between A. muciniphila and intracellular pathogens is still unknown. This study provides a novel focus for establishing connections between these species through the intestinal flora.
Koumiss treatment also influenced the function of the flora in the intestine. The results showed that at 14 dpi, the mice in the S group had more bacteria than those in the C, G and X groups that functioned in metabolism, such as carbohydrate metabolism, lipid metabolism and amino acid metabolism. Host metabolites can not only promote the growth of T. gondii but also directly govern its development and differentiation [47]. The glycolysis pathway is very important for the T. gondii tachyzoite stage, and this pathway not only provides a carbon source for fatty acid synthesis but also drives T. gondii invasion of host cells [48]. After hosts are infected by T. gondii, dendritic cells and macrophages drive an immune response but also improve the dependence of metabolism on glycolysis, which supports T. gondii growth [49]. A. muciniphila may inhibit T. gondii growth by regulating glucose and energy metabolism. Furthermore, research has suggested that T. gondii infection increases the relative abundance of Escherichia coli in the intestine [50]. This may be the reason why the relative abundance of Escherichia coli was higher in the S group than in the C, G and X groups at 14 dpi. Lipids also participate in innate immunity and may play an important role in controlling T. gondii infection [51]. Lachnospiraceae may facilitate colonization resistance against intestinal pathogens through the lipid metabolism pathway. T. gondii infection also interferes with arginine uptake, synthesis and metabolization in the host cell. A study demonstrated that T. gondii induced the integrated stress response (ISR) by depleting arginine from host cells, which caused increased expression of the host cationic amino acid transporter CAT1 to ensure a constant supply of arginine [52]. Compared with that in uninfected cells, the expression of arginine succinate synthase 1 (ASS) in T. gondii-infected cells was upregulated by 5-fold [53]. A study also indicated that T. gondii differentiated toward bradyzoites when essential nutrient supplies, such as arginine and cysteine, were limited [54]. Moreover, we strikingly found that the S group had fewer bacteria that function in membrane transport at 14 dpi. T. gondii secretes rhoptries (ROPs) and dense granules (GRAs) that interfere with biological processes in host cells, resulting in slight evasion of host cell autonomous defense mechanisms [55]. Secreted proteins are also regarded as nonspecific small molecule channels that facilitate the flux of metabolites between the parasitophorous vacuole (PV) space and the host cytoplasm [56]. Therefore, koumiss may inhibit T. gondii energy supplementation by reducing metabolite transport. In addition, it was found that at 14 dpi, the S group had more bacteria in the organismal system, including the immune system, which were beneficial for preventing T. gondii infection.
In conclusion, we investigated brain cyst counts and explored long-term changes in the microbiota and the effect of koumiss treatment on the microbiota in mice infected with T. gondii. Strikingly, we found that koumiss treatment significantly reduced brain cyst counts in mice infected with T. gondii. Moreover, T. gondii infection altered the intestinal flora community structure, whereas koumiss treatment could enhance the relative abundance of Lachnospiraceae and A. muciniphila; this effect of ameliorating T. gondii infection occurred via special metabolic pathways. The study established a close interaction among the host, intracellular pathogens and intestinal flora and provided a novel direction for preventing and eradicating T. gondii infection.