Rodent malaria parasites (RMPs) serve as an essential experimental model, playing a crucial role in parasitology research and being widely applied in various aspects of malaria studies. These applications include parasite development within the host, drug resistance mechanisms, pathogenesis, and vaccine efficacy evaluation. Plasmodium berghei is a rodent malaria parasite widely used in malaria research, incapable of infecting humans, thereby exhibiting minimal biosafety risks during laboratory manipulations, simplified experimental procedures, highly reproducible results, and particular suitability for laboratories lacking infrastructure for high-risk pathogen handling. Although P. falciparum directly correlates with human malaria, its cultivation requires humanized mice or ethically complex human-mosquito cycle systems, alongside prolonged experimental timelines and high costs. The P. berghei model serves as an alternative system for rapid screening of candidate intervention strategies, followed by targeted validation in P. falciparum systems. P. berghei demonstrates high developmental efficiency and synchronization across all life cycle stages within mosquito vectors and murine hosts, enabling precise control of infection timepoints and suitability for investigating parasite–host-vector interaction mechanisms (Dehghan et al., 2018). During transmission of P. berghei via the bite of a female Anopheles mosquito, sporozoites are deposited into the murine host through the insect’s salivary glands, thereby initiating a new infection cycle. Genetic diversity exists between different strains of P. berghei, with the ANKA strain being a highly virulent Plasmodium capable of causing experimental cerebral malaria (ECM) and widely used to study how host factors affect ECM. The genome of P. berghei contains 14 chromosomes and has extensive genetic homology with human Plasmodium (Pattaradilokrat et al., 2022).

Plasmodium yoelii, a RMP widely utilized in murine infection models, serves as a cornerstone experimental system for malaria research. A landmark study by Swardson-Olver et al. (2002) demonstrated that P. yoelii employs the murine Duffy antigen receptor for chemokines (DARC) as a primary receptor for invading mature erythrocytes (normocytes). Using DARC-knockout mice, the authors revealed that normocyte invasion was drastically reduced in the absence of DARC, whereas reticulocytes remained susceptible, indicating a DARC-independent invasion pathway for immature erythrocytes. This study highlighted the species’ ability to exploit distinct receptors on different erythrocyte subsets, underscoring its utility in dissecting host–parasite interactions. The findings also revealed conservation of DARC-dependent invasion mechanisms between rodent and human malaria parasites, solidifying P. yoelii as a pivotal model for studying invasion biology and immune evasion. This species comprises multiple subspecies, which exhibit divergent virulence and host tropism, thereby providing a valuable resource for studying Plasmodium evolution.

Plasmodium chabaudi, a key representative of RMPs, with a life cycle similar to that of human Plasmodium. A comprehensive review by Stephens et al. (2012) highlights that P. chabaudi infection in mice recapitulates critical aspects of human malaria pathogenesis, including synchronized erythrocytic cycles, sequestration of infected red blood cells (iRBCs) in hepatic microvasculature, and induction of severe anemia with dyserythropoiesis. Infection with P. chabaudi in mice recapitulates the four major mechanisms underlying human malarial anemia, thereby establishing a severe anemia model. Compared to the P. berghei model—which often induces acute lethal cerebral malaria and shows significant pathological discrepancies from human cerebral malaria (CM)—P. chabaudi’s chronic disease progression is more suitable for investigating non-lethal pathologies (e.g., anemia). In contrast, the P. yoelii model, which primarily infects immature erythrocytes and lacks chronic infection characteristics, fails to simulate the long-term immune responses observed in human malaria.

While the three established RMPs, P. berghei, P. yoelii, and P. chabaudi have been developed as experimental models, Plasmodium vinckei has remained understudied due to limited genetic manipulation tools and phenotypic data. Carlton (2021) highlighted recent advances in understanding this fourth RMP species, which exhibits the broadest geographic distribution among the four RMPs. The authors emphasize that P. vinckei, containing four subspecies with marked genetic diversity, fills critical gaps of existing RMP models, such as the restricted genetic background of P. berghei, and emerges as an ideal system for investigating Plasmodium evolution, host adaptation, and multifunctional gene family dynamics.

Plasmodium, the causative agent of malaria, is a protozoan parasite with a complex life cycle that involves both vertebrate hosts and Anopheles mosquitoes. The life cycle of Plasmodium begins when an infected mosquito injects sporozoites into the host’s bloodstream during a blood meal. These sporozoites quickly travel to the liver, where they invade hepatocytes and undergo asexual replication exoerythrocytic schizogony, resulting in the formation of thousands of merozoites. The infected hepatocytes eventually rupture, releasing merozoites into the bloodstream, where they invade red blood cells (RBCs) and initiate the erythrocytic cycle. During the erythrocytic cycle, merozoites replicate within RBCs, leading to the formation of schizonts. The RBCs eventually rupture, releasing new merozoites that can infect additional RBCs. This cyclical destruction of RBCs is responsible for the clinical symptoms of malaria, including fever, anemia, and organ damage. Some merozoites differentiate into male and female gametocytes, which can be taken up by a mosquito during a blood meal. Within midgut of mosquito parasite undergoes sexual cycle and differentiate into ookiente, oocyst and finally sporozoite, thereby completing the cycle.

The pathogenesis of malaria is multifaceted and involves both parasite and host factors. The liver stage of Plasmodium development involves hepatocyte invasion, which may indirectly influence gut-liver axis signaling and systemic immune responses (Denny et al., 2019). The destruction of RBCs during the erythrocytic cycle leads to anemia and the release of toxic heme, which can cause oxidative stress and tissue damage, potentially disrupting gut barrier integrity and microbiota composition (Sriboonvorakul et al., 2023). The parasite’s interaction with the endothelium can result in the sequestration of infected RBCs in vital organs, such as the brain, leading to severe complications like cerebral malaria. The host’s immune response, while essential for controlling the infection, can also contribute to pathology. TNF-α and IL-1β directly amplify the inflammatory cascade by activating neutrophils, promoting the expression of endothelial cell adhesion molecules, and inducing the release of inflammatory mediators (Mehdi et al., 2025). The host’s immune response exemplifies a dual role: while pro-inflammatory mediators may induce immunopathology, they remain essential for controlling infection. This paradox is mechanistically validated by Anand et al. (2015b), who demonstrated that oral administration of alginate-enclosed, chitosan-conjugated lactoferrin nanocapsules in P. berghei infected mice significantly elevated pro-inflammatory cytokines TNF-α and IFN-γ, alongside increased reactive oxygen species (ROS) and nitric oxide (NO) production. This heightened inflammatory response was associated with reduced parasite load in spleen and liver tissues, highlighting the dual role of immune activation in both pathogen clearance and potential tissue damage. The study also showed that nanoformulated lactoferrin modulated gut microbiota-mediated immune signaling, balancing protective immunity against excessive inflammation. Excessive production of pro-inflammatory cytokines, such as TNF-α and IFN-γ, can cause systemic inflammation and tissue damage.

Understanding the biology and pathogenesis of Plasmodium is crucial for developing effective interventions against malaria. Through these investigations, which integrate epidemiological surveillance and molecular pathogenesis studies, a deeper mechanistic understanding of malaria transmission dynamics can be achieved, thus informing the development of next-generation antimalarial drugs and vaccine. Such advances are critical for disrupting parasite dissemination and mitigating the global burden of this disease. Concurrently, it is imperative to prioritize multidisciplinary collaborations and sustained funding initiatives to accelerate translational research in malaria therapeutics. By addressing these priorities, the international community can strengthen health systems and safeguard global public health security, particularly in endemic regions where socioeconomic disparities exacerbate vulnerability.

The gut microbiota is composed of bacteria, viruses, fungi, archaea, and other microorganisms. Bacteria dominate this ecosystem, which is taxonomically classified into six major phyla: Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia. Among these, Firmicutes and Bacteroidetes collectively account for over 90% of the total bacterial population, emerging as the predominant lineages (Hou et al., 2022).

Guo et al. (2022) conducted 16S rRNA sequencing on fecal samples from BALB/c and C57BL/6 J mice, which were categorized into disease-associated (DRA), immunodeficient (IDA), gene-edited (GED), and normal control groups. Their analysis revealed that host genetic background exerted the greatest influence on gut microbiota composition, with C57BL/6 J mice exhibiting greater microbial stability compared with BALB/c mice. Notably, Bacteroidetes and Firmicutes, two dominant phyla, displayed the most significant variations in microbial richness across the normal, IDA, GED, and DRA groups. Hildebrand et al. (2013) compared the microbiome composition of mice of different genotypes of mice and found that the more similar the genotypes between different strains of mice, the more similar the composition of their intestinal microbiome, so it is important to select strains of mice with identical genotypes for microbiome studies.

Xiao et al. (2015) found that mice of identical strains maintained distinct microbial signatures despite identical feed protocols across different laboratories. High-fat diets consistently elevated alpha diversity while reducing beta diversity in gut microbiota, driving microbial structural convergence and enriching genera such as Clostridium and Butyrivibrio. In contrast, low-fat diets correlated with higher abundances of Prevotella and Bacteroides. Laboratory environmental variables (e.g., bedding materials, operational protocols) further modified microbial metabolic functions. Additionally, strain-specific effects governed the colonization of particular taxa (e.g., Akkermansia and Lactobacillus). These multifactorial interactions underscore the necessity to strictly control housing variables in experimental designs to ensure comparability across experimental outcomes.

It is necessary to evaluate the structure of the gut microbiota of mice and to consider the influence of the gut microbiota of the experimental animals on the results of the experiments in future basic and clinical studies. Understanding the status of murine intestinal microbiome in mice and using high quality laboratory animals can significantly enhance the accuracy of experimental results. Recent investigations of intestinal microbiome have focused on their roles in diverse pathological conditions. In contrast, few investigations have been conducted on the bacterial community structure of representative mouse strains from the same environmental context excluding manufacturers and environmental facilities, and these studies are critical for addressing this knowledge gap and refining mechanistic interpretations of microbiome-host interactions.

The functional diversity of gut microbiota is directly influenced by its compositional structure (Hildebrand et al., 2013; Guo et al., 2022). Variations in microbiota composition and diversity induced by genetic backgrounds and rearing conditions lead to significant alterations in key functional modules, including SCFAs biosynthesis pathways and bile acid metabolism-associated gene abundance. These findings collectively indicate that structural characteristics of microbial communities constitute the foundation of their functional phenotypes. Increased microbiota α-diversity promotes Clostridium genus enrichment (Xiao et al., 2015), which enhances intestinal mucosal barrier function through IgA secretion stimulation and IL-12 signaling pathway activation, while simultaneously remodeling microbial ecology via induction of antimicrobial peptide REG3β expression (Stefka et al., 2014). Conversely, abnormal proliferation of Proteobacteria phylum exacerbates systemic inflammation through lipopolysaccharide (LPS) synthesis (Panzer and Lynch, 2015; Agus et al., 2016). This evidence demonstrates that microbial community composition serves as the biological basis driving host immune response and metabolic homeostasis remodeling. The following sections will systematically dissect how dynamic microbiota composition influences host physiology through immune activation and metabolic regulatory networks.

The gut microbiota plays an indispensable role in the development and regulation of host immune systems. Members of the phylum Firmicutes and Bifidobacterium spp. generate short-chain fatty acids (SCFAs) through dietary fiber fermentation, which are critical for maintaining intestinal homeostasis and systemic metabolic balance by enhancing mucosal barrier integrity, modulating immune responses, and suppressing inflammatory pathways. SCFAs exert anti-inflammatory effects through histone deacetylase (HDAC) inhibition, a process that promotes transcriptional activation of immunoregulatory genes and facilitates regulatory Treg cell differentiation while suppressing pro-inflammatory Th17 cell polarization. Furthermore, SCFAs activate G protein-coupled receptors (GPCRs), triggering signaling cascades that reduce pro-inflammatory cytokines and elevate anti-inflammatory mediators, thereby preserving epithelial barrier function. Intestinal epithelial cells and immune cells recognize microbial molecules through pattern recognition receptors (PRRs) and other mechanisms, initiating immune responses. Gut-associated lymphoid tissue (GALT) generates IgA that neutralizes pathogens and constrains the excessive proliferation of commensal bacteria, thereby establishing immune tolerance (Yoo et al., 2020).

Distinct microbial taxa directly orchestrate immune cell differentiation (Ivanov et al., 2022), as exemplified by segmented filamentous bacteria (SFB), which elicit Th17 cell polarization through epithelial adherence-triggered antigen presentation, thereby fortifying intestinal barrier integrity. Conversely, Clostridium spp. promote regulatory T cell (Treg) differentiation, while Akkermansia muciniphila drives T follicular helper cell (Tfh) commitment, collectively shaping immunological equilibrium. These mechanisms, operating at the interface of microbial ecology and host immunometabolism, establish the gut microbiota’s central role in maintaining mucosal homeostasis, combating enteric pathogens, and coordinating systemic immune responses.

Furthermore, the gut microbiota modulates host metabolism. Takeuchi et al. (2023) elucidated in murine models that gut microbiota associated with insulin sensitivity ameliorates host insulin resistance through multifactorial mechanisms. By generating SCFAs, these microbial communities activate intestinal L-cell secretion of glucagon-like peptide-1 (GLP-1), which enhances insulin signaling and glucose homeostasis. Concurrently, microbiota-mediated modulation of bile acid metabolism suppresses hepatic gluconeogenesis. Reduced circulating endotoxin levels further attenuate adipose tissue inflammation and macrophage M1 polarization, while strengthened expression of tight junction proteins in the intestinal epithelium preserves barrier integrity, mitigating systemic low-grade inflammation. Notably, specific commensals such as A. muciniphila upregulate peroxisome proliferator-activated receptor gamma (PPARγ) and mitochondrial biogenesis-related genes, a process that promotes white adipose tissue browning and energy expenditure, synergistically resolving glucolipid metabolic dysregulation.

Overall, the composition and function of the gut microbiota have a profound impact on the health and well-being of the host. Maintaining a healthy and diverse gut microbial community is essential for optimal physiological function.

Studies demonstrated that genetically similar mice from distinct commercial suppliers exhibited divergent gut microbiota profiles, which subsequently led to differential parasite burdens and mortality rates following P. yoelii infection (Villarino et al., 2016). Resistant mice, characterized by specific microbial compositions, displayed lower parasitic loads, reduced clinical severity, and elevated survival post-infection. In contrast, susceptible mice manifested heightened parasite burdens, severe clinical manifestations, and diminished survival. Mandal et al. (2020) revealed that spatiotemporal variations in gut microbiota of mice obtained from the same supplier at different timepoints significantly modulated host immune responses to Plasmodium infection. Early-stage microbiota facilitated rapid clearance of P. yoelii, whereas late-stage microbiota resulted in elevated peak parasitemia and delayed pathogen elimination.

Stough et al. (2016) observed significant differences in resistance patterns among C57BL/6 mice sourced from Taconic Biosciences versus Charles River Laboratories. Mice originating from Taconic exhibited markedly lower parasite burdens and accelerated recovery trajectories, while those acquired from Charles River Laboratories manifested higher parasitemia levels and delayed resolution kinetics. Taniguchi et al. (2015) revealed that P. berghei ANKA infection induces pathophysiological alterations in the intestinal mucosa, manifesting as epithelial apoptosis and compromised barrier function in C57BL/6 mice. These structural disruptions correlate with significant compositional changes in the gut microbiome, including a marked reduction in Firmicutes abundance and concomitant expansion of Proteobacteria taxa, hallmarks of microbial dysbiosis. Notably, this pathological phenotype was significantly attenuated in BALB/c mice.

While the genetic and microbial characteristics of the host have been shown to play a pivotal role in malaria—related microbiota changes, the unique attributes of Plasmodium species also exert a profound impact on the composition and dynamics of the gut microbiota. Taniguchi et al. (2015) found that C57BL/6 mice infected with P. berghei ANKA exhibited intestinal pathological alterations concurrent with pronounced gut microbiota dysbiosis. This study represents the first experimental demonstration that Plasmodium infection directly induces microbiota dysbiosis, with the degree of microbial disruption closely linked to the severity of intestinal and cerebral pathological damage.

Even within the same mouse strain, intestinal microbiota exhibit marked divergence following infection with distinct Plasmodium species. Guan et al. (2022) reported that BALB/c mice infected with P. yoelii 17XL developed severe malaria accompanied by significant shifts in gut microbiota richness and diversity, with Lactobacillaceae emerging as the dominant family across all samples. Guan et al. (2023) further investigated the relationship between mild malaria and microbiota by analyzing gut microbial changes in BALB/c mice infected with the non-lethal strain Py 17XNL and evaluating parasitemia and survival rates at multiple post-infection timepoints. Significant alterations in microbiota richness and diversity were observed at days 9 and 15 post-infection, with near-complete restoration by day 28. These gut microbiota dynamics were closely associated with the progression of malaria pathology. Parasite presence activates host immune responses, disrupts microbial homeostasis, or engages in metabolic competition with commensal microbes for nutrients and ecological niches (Drew et al., 2021; Table 1. Differential Impacts of Plasmodium Species on Gut Microbiota in Murine Models).

The compositional shifts in gut microbiota represent adaptive restructuring rather than simple dysbiosis, through integrated multi-omics analyses, Fan et al. (2019) identified specific operational taxonomic units (OTUs) associated with malaria pathogenesis and convalescence. Their findings highlighted substantial microbiome remodeling during infection, with persistent alterations that failed to revert to baseline composition post-recovery. This dynamic reorganization emerged from tripartite interactions involving direct parasitic manipulation, immunological modulation and environmental perturbations including antibiotic exposure.

Intestinal cells including enterocytes, goblet cells, Paneth cells, and tuft cells establish a physical barrier via tight junctions, preventing pathogen invasion (Neurath et al., 2025). Goblet cells produce mucus to trap pathogens (McCauley and Guasch, 2015), while Paneth cells release antimicrobial molecules including lysozymes and defensins while supporting intestinal stem cell maintenance (Clevers and Bevins, 2013). Tuft cells initiate anti-parasite immunity through IL-25 secretion, a process dependent on the transcription factor Pou2f3 (Gerbe et al., 2016; Howitt et al., 2016). During parasitic infection, gut cells detect pathogen-derived signals through pattern recognition receptors such as TLRs, triggering alarmins like IL-25 to activate Th2-type immune responses. This stimulates enhanced mucus secretion, intestinal fluid leakage, and amplified smooth muscle contractions, collectively forming a secretory-sweeping mechanism for parasite expulsion. Mast cell-derived proteases simultaneously degrade tight junctions to promote luminal fluid flow and synergize with muscular contractions to eliminate parasites (Maizels et al., 2012).

The resolution of malaria infection and the development of adaptive immunity depend critically on host T follicular helper (Tfh) cell and germinal center (GC) B cell differentiation and function (Figueiredo et al., 2017; Pérez-Mazliah et al., 2017), serving as pivotal determinants of disease severity and long-term immunological memory. These immunological mechanisms collectively provide a theoretical framework for microbiota-based interventions to enhance malaria control strategies (Figure 1).

The gut microbiota plays a critical role in nutritional metabolism and nutrient availability, with its compositional changes during malaria infection potentially altering nutrient accessibility to influence parasite growth and survival. Probiotics such as Lactobacillus spp. enhance serum nitric oxide (NO) levels to suppress intraerythrocytic parasite proliferation, thereby reducing parasitemia. Hepatic-derived bile acids suppress pathogenic bacterial growth by inducing antimicrobial peptide expression, thereby maintaining gut microbiota homeostasis, while intestinal microbes modulate hepatic bile acid and lipid synthesis, establishing a hepatic-intestinal metabolic crosstalk network (Tripathi et al., 2018). Disruption of this axis directly or indirectly impacts hepatic-stage Plasmodium infection (Mancio-Silva et al., 2017; Zuzarte-Luís et al., 2017). Liver damage is particularly pronounced in severe malaria, accompanied by alterations in bile acid metabolism that further destabilize gut microbial homeostasis (Denny et al., 2019).

While SCFAs exert pleiotropic effects on host immunity, Chakravarty et al. (2019) revealed that the regulatory role of gut microbiota in malaria severity exhibits no direct correlation with fecal SCFA levels. Gut microbiota may metabolize antimalarial drugs, altering their bioavailability and efficacy. The presence or absence of specific microbial strains could either enhance or suppress drug metabolic rates, thereby affecting therapeutic outcomes against malaria. Studies have shown that different types of antibiotics exhibit significant variations in their effects on gut microbiota. Although the specific patterns of species depletion depend on microbial community composition and antibiotic types, antibiotic treatment is consistently associated with marked reductions in the relative abundance of core taxa, including Bacteroidetes, Firmicutes, and Actinobacteria. This loss of diversity creates an ecological niche vacancy that facilitates colonization or overgrowth of opportunistic pathogens from the Proteobacteria phylum, contributing to various gastrointestinal disorders. These opportunistic pathogens often harbor transferable antibiotic resistance genes or possess intrinsic resilience traits such as sporulation, enabling their survival under antibiotic pressure (Fishbein et al., 2023).

In mosquito malaria models, Vinayagam et al. (2023) systematically reviewed that gut microbiota in mosquitoes directly suppress Plasmodium development by activating the immune deficiency IMD pathway and peptidoglycan recognition protein PGRP-mediated immune responses, thereby inducing thioester-containing protein TEP1 expression and antimicrobial peptide secretion. Concurrently, microbiota stimulate host-derived reactive oxygen species production to eliminate parasites while modulating microbial colonization, synthesize secondary metabolites targeting Plasmodium developmental stages, and regulate antioxidant enzymes such as catalase to mitigate oxidative stress, thereby preventing host tissue damage and mortality. This immune-metabolic synergy highlights microbiota-mediated dual suppression of Plasmodium infection via immunological and metabolic axes. These regulatory strategies exhibit cross-species conservation in arthropod vectors and mammalian hosts, though specific metabolites may vary due to microbiome compositional divergence.

Current evidence (Wilson and Nicholson, 2017) indicates that structural variations in gut microbial communities regulate drug bioavailability and metabolic pathways via enzymatic transformations such as nitro-reduction and hydrolytic deconjugation, a phenomenon hypothesized to extend to antimalarial compound metabolism. Gut microbiota may metabolize antimalarial drugs such as artemisinin and chloroquine, altering their bioavailability and therapeutic efficacy. The presence or absence of specific bacterial strains may enhance or attenuate drug metabolic rates, thereby modulating treatment outcomes in Plasmodium infection.

Notably, certain antimalarial agents exhibit broad-spectrum antimicrobial properties. Zhang et al. (2022) first identified that bacteriophage Gp46 protein inhibits bacterial and Plasmodium growth by binding to the highly conserved HU protein, a critical DNA-binding protein in both organisms, through competitive occupation of its DNA-binding sites, thereby blocking HU-DNA interactions and inducing filamentous morphology due to impaired chromosome segregation. This dual antimicrobial-antimalarial activity suggests that some antimalarial drugs may disrupt gut commensal microbiota by suppressing specific strains or promoting resistance gene dissemination. Additionally, Dada et al. (2018) demonstrated that antibiotic treatments alter microbiota diversity in insecticide-resistant mosquitoes: vancomycin reduced resistance, whereas streptomycin and gentamicin enhanced resistance phenotypes. Resistance-exhibiting mosquitoes exhibited lower microbial diversity compared to susceptible counterparts, implying that antibiotic supplementation could modulate microbial load and diversity. Specific microbiota may contribute to resistance mechanisms via insecticide degradation or induction of detoxifying metabolic enzymes. This microbiota-resistance interplay highlights the potential of microbial-targeted strategies to reverse insecticide resistance.

Understanding the intricate interplay between gut microbiota, host defense mechanisms, and malaria pathogenesis is essential for identifying novel therapeutic targets and intervention strategies. Notably, this field remains under active investigation, necessitating further research to delineate the molecular pathways governing these complex interactions.

To address the potential risks of probiotics, future studies should prioritize establishing precision-guided and personalized probiotic application strategies, integrating multi-dimensional host factors (genetic background, gut microbiota profiles, dietary metabolism) to develop AI-driven colonization prediction models and screen next-generation probiotic strains with well-defined molecular mechanisms. Multi-center, large-scale randomized controlled trials (RCTs) must be advanced, harmonizing efficacy evaluation criteria and incorporating long-term safety monitoring (particularly in children, immunocompromised individuals, and critically ill patients) to elucidate probiotics long-term impacts on microbiota reconstitution, metabolic programming, and immune maturation. Simultaneously, leveraging single-cell sequencing, metabolomics, and organoid models can dissect the probiotic-host-microbiota tripartite interaction network, dismantling the “black-box” application paradigm to establish direct associations between strain functionality and clinical endpoints. Finally, regulatory frameworks require enhancement to standardize strain identity, dosage, and indication specifications, fostering interdisciplinary collaborations to accelerate evidence-based clinical translation (Waide and Schmidt, 2020).

Although the human and mouse gut microbiota share many taxonomic similarities, translational accuracy is hampered by notable variations in microbial quantity and composition. A possible method for more accurately simulating human gut ecology in experimental models is the use of humanized gnotobiotic mice that have been colonized with microbiota obtained from humans. To refine murine models, developing “humanized microbiota mice” by colonizing germ-free mice with fecal microbiota from healthy donors or malaria patients, combined with humanized immune systems, can better mimic human host–microbe interaction dynamics. Multi-center harmonized protocols should standardize diet formulations, housing conditions, and antibiotic pretreatment regimens, enabling cross-laboratory comparability of murine microbiota backgrounds through shared metagenomic databases. Introducing synthetic microbial consortia to precisely control colonization abundance of specific taxa (e.g., Bacteroidetes/Firmicutes ratios), alongside CRISPR-Cas9-mediated strain gene editing, will elucidate functional microbial mechanisms underlying Plasmodium inhibition.

Future directions in translational research should prioritize bridging insights from murine models with human clinical data through a two-pronged approach. First, validated pathways identified in humanized mouse models, such as those regulating liver-stage infection (e.g., EphA2-mediated hepatocyte invasion) or immune evasion mechanisms, should be corroborated using transcriptomic and proteomic datasets from malaria patients, particularly those with severe disease or treatment resistance (Vaughan et al., 2015; Minkah et al., 2018). Second, next-generation humanized mouse models must be developed to recapitulate human physiology more faithfully, integrating functional human hepatocytes (e.g., FRG huHep mice), erythropoiesis, and immune systems (HIS mice) to enable study of the complete P. falciparum life cycle, including hypnozoite persistence in P. vivax and sequestration-mediated pathology (Mikolajczak et al., 2015; Soulard et al., 2015). Such models should also incorporate human endothelial receptors (e.g., EPCR, ICAM1) to investigate tissue-specific sequestration and cerebral malaria pathogenesis, while dual-chimeric systems (HIS huHep) could resolve the role of human T cell subsets and antibody responses in vaccine-mediated protection (Huang et al., 2015; Li et al., 2016). Long-term, integrating these models with clinical omics data will accelerate translation of preclinical findings into novel therapeutics and vaccines.

For human studies, interventional clinical trials should evaluate the adjunctive efficacy of probiotics (e.g., Lactobacillus, Bifidobacterium) or microbiota-derived metabolites (SCFAs, bile acid derivatives) in enhancing antimalarial therapy, particularly focusing on improved treatment responses against artemisinin-resistant strains. Establishing malaria patient gut/blood microbiome cohorts with longitudinal sampling across infection phases (acute, chronic, post-treatment) will capture microbiota dynamics. Integrating host immune phenotypes and metabolomic profiles can identify diagnostic or prognostic microbial biomarkers.

Despite these challenges, the therapeutic potential of gut microbiota manipulation in malaria prevention continues to attract considerable interest. Future research will deepen our understanding of the host-microbiome-malaria axis through integrated multi-omics analyses, ultimately advancing the development of safe, efficacious microbiome-based interventions that complement existing malaria control frameworks.