Microbiota-Induced Radioprotection: A Novel Approach to Enhance Human Radioresistance with In-Situ Genetically Engineered Gut Bacteria

Abstract

The high sensitivity of living organic forms to space radiation remains the critical issue during spaceflight, to which they will be chronically exposed during months of interplanetary or even decades of interstellar spaceflight. In the human body, all actively dividing and poorly differentiated cells are always close to being damaged by radiological or chemical agents. The chronic exposure to ionizing radiation primarily causes changes in blood counts and intestinal damage such as fibrosis, obliterative vasculitis, changes in the gut microbiota, and atrophy or degeneration of muscle fibers. The project “MISS: Microbiome Induced Space Suit” was presented at the Giant Jamboree of the International Genetically Engineered Machine Competition 2021, with the aim to investigate the ability of the novel microbiota-mediated approach to enhance human resistance to ionizing radiation. The key innovative part of the project was the idea to create a novel radioprotector delivery mechanism based on human gut microbiota with the function of outer membrane vesicles (OMVs) secretion. The project concept proposed the feasibility of genetically modifying the human microbiota in situ through the delivery of genetic constructs to the host’s crypts using silicon nanoparticles with chemically modified surfaces. In this perspective, we discuss the advances in modifying microbiota-mediated secretory activity as a promising approach for radioprotection and as an alternative to hormone therapy and other health conditions that currently require continuous drug administration. Future clinical trials of in situ methods to genetic engineering the crypt microbiota may pave the way for indirect regulation of human cells.

1. Introduction

Under the conditions of a long space flight, humans will inevitably be exposed to high levels of chronic radiation, which are dangerous to life and health. Up-to-date defense protection engineering systems are not sufficient to fully address and provide comprehensive health care for astronauts. Pharmaceutical administration attempts to improve the human defense system through the use of radioprotectors [1,2] and natural substances derived from plants and other biological sources [3]; however, these techniques have limited effectiveness [4]. Several Food and Drug Administration (FDA)-approved drugs, including statins, nonsteroidal anti-inflammatory drugs, and angiotensin-converting enzyme inhibitors, show potential as radiation countermeasures [5]. Amifostine is currently the only FDA-approved agent for the treatment of high-dose radiation exposure, with a dose reduction factor of up to 2.4 [6]. However, its effectiveness for chronic low-dose space radiation is uncertain. For this reason, the idea of creating long-term biological protection using natural mechanisms of radioprotection, which, unlike existing low-molecular compounds, provide comprehensive protection of cells of an organism from different types of radiation, has emerged [7]. Current methods of gene therapy for radioresistance are based on the endogenous expression of the desired genes delivered to the cells via plasmid liposomes and adenoviral systems [8], but this approach and other methods associated with direct modification of human cells may not be safe. In light of the fact that the genetic modification of human beings remains questionable from both a medical and an ethical point of view [9], indirect approaches to gene editing are needed, probably based on the modification of the resident microbiota.

We take a look at the opportunities of bioengineered probiotics which could potentially enhance the radioresistance of host cells through their secretory activity or interactions with intestinal mucosal cells. Indeed, there are some cases of probiotics with similar effects, for example, Lactobacillus rhamnosus GG, which has been shown to modulate immune responses and protect intestinal cells from radiation-induced damage [10]. Bifidobacterium breve is known for its ability to enhance the mucosal barrier function and reduce inflammation [11]. Another example is Escherichia coli Nissle 1917, which has demonstrated protective effects against oxidative stress in the gut [12]. The use of gene-engineered probiotics could lead to targeted effects by enabling the precise delivery of therapeutic molecules or the modulation of specific cellular pathways. This approach holds promise for enhancing the resilience of bowel cells to radiation, potentially benefiting patients undergoing radiotherapy by reducing gastrointestinal side effects. Furthermore, the bioengineered probiotics could be tailored to produce specific enzymes or antioxidants that neutralize reactive oxygen species, thereby mitigating DNA damage and promoting cellular repair mechanisms [13]. Radiation exposure can disrupt the gastrointestinal system by damaging stem cells and altering signaling pathways crucial for intestinal homeostasis [14]. The use of radioprotectors may facilitate the stabilization of cell membranes and the reduction in permeability to harmful agents, thereby assisting in the maintenance of cellular structure under conditions of stress. For instance, probiotics have demonstrated the capacity to safeguard mucosal integrity and to diminish oxidative stress within the intestine and brain [15]. Following absorption in the intestine, radioprotectants enter the bloodstream, thereby enabling their circulation throughout the body. This enables them to reach various organs and tissues, thereby providing protection against radiation-induced damage at the systemic level. The integration of synthetic biology with probiotic therapy could revolutionize the management of radiation-induced gastrointestinal toxicity, offering a novel and personalized strategy for patient care [16].

The aim of our study was to discuss the advances in the microbiota-mediated approach to enhance human resistance to ionizing radiation.

2. Keynote Approaches to Microbiota-Mediated Human Radioresistance

There are three original ideas that could inspire space exploration bioengineers and have been applied to investigate the ability of the microbiota-mediated approach to enhance human resistance to ionizing radiation.

2.1. Modification of the Human Microbiota

Firstly, the human microbiota may be considered as a target for modification, given its integral role in maintaining health and supporting essential bodily functions. Microbial communities cover the human body and form a biological ‘suit’ that assists humans in digestion, immune response, and even mental health. In our research, we have advanced the modification of human-associated bacteria in vitro by employing silicon nanoparticles as carriers for absorbed genetic material. To facilitate the integration of the genetic construct into the bacterial genomic DNA, we utilized a sophisticated recombination editing system leveraging plasmid vectors pTmpKm employed as a template plasmid, and pRedCmOcr, functioning as a helper plasmid. Furthermore, the combination of these two plasmids facilitates double selection, as pTmpKm typically offers a resistance marker to kanamycin, while pRedCmOcr provides resistance to chloramphenicol [17]. This innovative approach is designed to encourage the engineered bacteria to produce and secrete radioprotective agents, such as radioprotective proteins, mRNA and regulation microRNAs, encapsulated within outer membrane vesicles (OMVs) [18]. These OMVs have the unique capability to fuse with human cells, potentially offering a novel method for delivering protective compounds directly to where they are needed most. Our hypothesis not only highlights the potential of microbiota modification but also opens new avenues for enhancing human resilience against environmental stressors. Previously, the idea of the in situ production of probiotics with secretory activity had not been considered.

2.2. Targeted Delivery of Plasmids to Crypt-Specific Microbiota

Secondly, targeted delivery of plasmid into the crypt-specific microbiota can be performed using nanoparticles. A crucial element of the in situ production of probiotics is the choice of an appropriate carrier, which must be capable of safeguarding the transported organisms from the deleterious effects of biological fluids and possess the capacity to be directly released into the gastrointestinal tract [3]. One promising technique involves the directed transformation of target bacteria in the human gut using silicon nanoparticles with modified surfaces, alongside the use of bacteriophages. Silicon nanoparticles are particularly advantageous due to their exceptional biocompatibility, which allows for the efficient loading of therapeutic agents. Their large surface area enhances their ability to release these agents and interact with cells in a controlled manner [19,20]. In the MISS project, the target organisms for modification are the predominantly species capable of aerobic metabolism, such as Proteobacteria [21], Akkermansia muciniphila [22], and Acinetobacter [23]. These crypt-dwelling bacteria benefit from their proximity to epithelial cells, which provide an adequate supply of oxygen by diffusion. This oxygen availability is critical for their metabolic processes, supports the successful implementation of nanoparticle-mediated interventions and enhances their secretory activity. In addition, the difficulties in identifying crypt-specific bacterial types and the mechanisms of delivery and seeding of genetically engineered microbiota into the crypt lead to appropriate in situ genetic engineering by delivery of transformation agents.

2.3. Interaction of Modified Microbiota with Intestinal Crypts and Enhancement of Overall Radioresistance

Third, secretion of outer membrane vesicles (OMVs) by crypt-specific microbiota may serve as a pathway for radioprotector transport into the cells of the host organism. The interaction of intestinal epithelial cells with bacterial cells is thought to occur through the release of outer membrane vesicles (OMVs), which are produced by Gram-negative bacteria of the intestinal crypts [24]. The intestinal crypt comprises crucial anatomical structures located within the lining of the intestines, specifically in the small and large intestines. These glandular structures are responsible for housing stem cells that continually regenerate the epithelial lining of the gut, ensuring a constant renewal process that is essential for nutrient absorption and barrier function [25]. Crypt-specific microbiota refers to the unique community of microorganisms that resides within these crypts. These microbiota species are distinct from the broader gut lumen microbiota, which occupies the central cavity of the gut and is more accessible for analysis. The crypt-specific microbiota can secrete metabolites and signaling molecules that modulate the growth and activity of other microbial communities in the gut. This interaction is crucial for maintaining a balanced microbial ecosystem, which is essential for optimal digestion, immune function, and protection against pathogenic bacteria. Moreover, alterations in the crypt-specific microbiota can have significant implications for intestinal health [26]. Crypt-specific microbiota interact with immune cells, promoting tolerance to beneficial microbes while enabling the immune system to mount effective responses against harmful pathogens [27]. This intricate relationship underscores the importance of maintaining a healthy and diverse microbiota within the crypts and throughout the intestinal tract. The phenomenon of OMVs transportation could help in understanding the mechanism of interactions with lumen microbiota and host cells. Their capacity to transport a range of biologics, including enzymes, antioxidants and transcription factors, may serve to alleviate the damage incurred as a consequence of radiation exposure. The internalization of OMVs carrying radioprotective molecules by host cells can be achieved through processes such as endocytosis, which facilitates the direct delivery of the cargo into the cellular environment, enhancing their protective effects. Therefore, increased secretion of the radioprotector’s cargo-laden OMVs could protect host cells from the effects of ionizing radiation (Figure 1).

3. Microbiome-Induced Space Suit

In the original form, our project entitled “Microbiome-Induced Space Suit” (MISS) considers the development of several types of genetic constructs under control by an arabinose-inducible promoter pBAD [1]. This means that the expression of radioprotective proteins will only be activated under the influence of arabinose, which can be administered orally if necessary, thus minimizing side effects. The effector part of the construct contained multiple sequences encoding radioprotective proteins and regulatory microRNAs. We used two approaches to identify the most prominent targets. First, we analyzed information on the transcriptome of people living in regions with a high natural radiation background. It has been shown that people who live in such areas for a long time do not have an increase in the number of diseases associated with radiation exposure compared to people who are not chronically exposed to radiation [28]. This means that humans have a natural defense mechanism against radiation that could include enhanced DNA repair processes, efficient cellular stress responses, or other protective biochemical pathways. Based on this, one of the ideas of the MISS project is to identify the specific molecular mechanisms that allow humans to tolerate high levels of radiation exposure without adverse health effects. This approach involves searching for the expression of unique genes, regulatory pathways, and protein functions that are regulated in these populations. Once the relevant molecular mechanisms are identified, genetic constructs can be designed and used to modify the human microbiome and recreate the protective effects observed in populations exposed to high levels of radiation.

The second approach is to use human proteins that are homologous to the tardigrade protein DNA damage suppressor protein (DSUP), allowing them to exist at very high levels of radiation lethal to humans [29]. As a result, we used the well-studied proteins Wnt family member 10A (Wnt10a) and Bloom’s syndrome helicase (BLM). Wnt10a plays a crucial role in wound healing and tissue regeneration by regulating collagen expression and synthesis [30]. Its absence leads to delayed wound healing and reduced fibroblast/myofibroblast numbers [31]. BLM plays an important role in DNA damage repair and genome stability by functioning as part of the BLM dissolvasome complex, which resolves bound DNA intermediates without genetic exchange [32]. Both of these proteins are involved in cellular stress resistance (Figure 2A). The proposed pathway of OMVs-mediated radioprotector delivery into intestine crypt cells through gene-engineered microbiota is presented in Figure 2B. The key challenge in the implementation of this approach is the direct transport of nanoparticles through mucin media to the bacterial cells (distance ~100–200 μm), as well as the OMV transport to host cells at a distance of 20–50 μm [33].

The proposed idea of the MISS project is that two plasmids that encode the target genes for the radioprotective molecules (BLM and Wnt10a) are conjugated with silicon nanoparticles by chemical or biological methods. Subsequently, the nanoparticles are orally administered into the intestine and may be able to transform the luminal and crypt-specific microbiota. Once the plasmids are integrated into the bacterial genome, these bacteria begin to express new genes, resulting in the formation of OMVs containing radioprotective proteins and RNA. The OMVs then fuse with the intestinal crypt cells, resulting in the release of their cargo into the cytoplasm of the host cell, with efficacy depending only on the radioprotective properties of the defense genes used.

The spectrum of radioprotective defense genes may extend far beyond the currently well-known pathways. Recently, it has been found that the induction of radiotolerance in human cells could be achieved by adopting information transfer from tardigrade cells [34]. Tardigrades, known for their remarkable resilience to extreme environmental conditions, provide a unique model for understanding the mechanisms of radiation tolerance. By studying the molecular pathways and protective proteins in tardigrades, researchers aim to develop strategies to enhance the resistance of human cells to radiation [35,36]. This issue also examines the cases of successful adaptation to radiation in humans and the challenges faced in developing comprehensive therapies to induce cellular radioresistance [37], a critical aspect for space exploration [7], and also explores using high-altitude isolated biological reservoirs as potential sources of promising radioprotectors [38]. These exotic reservoirs, often found in extreme environments, harbor organisms that have naturally evolved mechanisms to withstand high levels of radiation [39]. Tapping into these biological resources holds promise for discovering novel compounds or genes that can be used to protect human cells from radiation-induced damage.

Microbiome studies provide insight into the maintenance of gut health with probiotic consortium supplementation and will facilitate the development of probiotic-based therapeutic strategies for radiation-induced gut injury. Some types of secretory activity of gut microbiota showed the radioprotective effect on intestine cells. Espinal et al. showed the abilities of oral administration of second-generation probiotic Lactobacillus-reuteri-Interleukin-22 is an effective protector and mitigator of intestinal irradiation damage, by improving the capacity of therapeutics to stabilize the number of Lgr5+ intestinal crypt stem cells and their progeny [40]. In a mouse model, it has been shown that intestinal microflora (Lachnospiraceae and Enterococcaceae) and its production of short-chain fatty acids and specific tryptophan metabolites can tune host resistance against high doses of radiation from facilitating hematopoiesis and gastrointestinal recovery [41,42]. Fasting-induced adipose factor (angiopoietin-like 4, ANGPTL4) is a microbiota-regulated, epithelial-derived, secreted protein participating in radioresistance and suggested to be useful as a gut radioprotector [43,44]. The probiotic consortium mixture of Bifidobacterium longum BL21, Lactobacillus paracasei LC86, and Lactobacillus plantarum Lp90 attenuated radiation-induced intestinal injury by modulating the gut microbiota and metabolites, improving inflammatory symptoms, and regulating oxidative stress [45].

Advances in the genetic engineering of the gut microbiota, including transient expression systems, represent a promising frontier in the development of next-generation probiotics. These innovative approaches aim to enhance the beneficial properties of gut bacteria, enabling more precise and effective interventions to promote human health. Specific genetic modifications that enable gut bacteria to produce therapeutic compounds, enhance nutrient absorption, or modulate immune responses can be achieved using CRISPR-Cas9 gene editing systems, transposon mutagenesis, and plasmid-based systems [46]. One potential application of these modifications is the production of short-chain fatty acids, such as butyrate, which are known to have anti-inflammatory properties and play a critical role in maintaining gut health [47]. The ability of engineered bacteria to increase the production of short-chain fatty acids could be used to alleviate the symptoms of inflammatory bowel disease and other inflammatory conditions. The genetically engineered bacteria could be engineered to produce enzymes that break down complex carbohydrates, improving nutrient absorption and potentially aiding in the management of metabolic disorders such as obesity and type 2 diabetes mellitus [48].

The novel insights related to the ability of tardigrade-derived DSUP to induce radioprotection in transfected human cells [49,50,51]. Building on this basic research, our idea was to take a significant step forward by transferring radioprotective proteins, mRNA and regulatory microRNAs directly into OMVs. This innovative approach aims to harness the natural protective mechanisms of tardigrades, known for their resilience in extreme environments, and apply them in a novel context. Using OMVs as delivery vehicles, we hope to create a more efficient and targeted method for delivery of these protective agents to human intestinal cells.

4. Conclusions

Currently, mechanisms of probiotic culture are primarily based on their secretory and enzymatic activity. We believe that the microbiota-mediated mechanism of OMV secretion is a promising approach for human radioprotection and could be an alternative to hormone therapy and for other health conditions that currently require continuous drug administration. Application of in situ methods of genetic engineering of crypt microbiota could provide the way to indirect regulation of human cells.