Mechanisms of probiotic Bacillus against enteric bacterial infections

Gastrointestinal infection is a leading cause of gut diseases attracting global health concerns. The emerging antimicrobial resistance in enteric pathogens drives the search of viable and renewable alternatives to antibiotics for the health of both human beings and animals. Spore-forming probiotic Bacillus have received extensively interests for their multiple health benefits, including the restoration of microbiota dysbiosis and the reduction of drug-resistant pathogens. These promising benefits are mainly attributed to the activity of structurally diverse Bacillus-derived metabolites, such as antibacterial compounds, short-chain fatty acids, and other small molecules. Such metabolites show the capacity to directly target either the individual or community of bacterial pathogens, and to potentiate both host cells and gut microbiota. The better understanding of the mechanisms by which probiotic Bacillus and the metabolites modulate the metabolism of hosts and microbiota will advance the screening and development of probiotic Bacillus. In this review, we discuss the interaction among probiotic Bacillus, microbiota and host, and summarize the Bacillus-derived metabolites that act as key players in such interactions, shedding light on the mechanistic understanding of probiotic Bacillus against enteric bacterial infections.


Introduction
Gut microbiota is a huge community of microbes engaged in multiple interaction that is vital for ensuring gastrointestinal (GI) system health.It is estimated that the number of microbial cells within gut lumen, containing a density of up to 10 11 -10 12 bacteria per gram, is ten times more than somatic and germ cells in mammals [1].Most of gut microbes enrich in the caecum and proximal colon and build up as microbial barrier adjacent to physical (epithelial cells) and chemical (mucus, etc.) barriers.The maintenance of gut microbiota is well-known associated with the health of host, not only affect physiological processes such as appetite and digestion but also shape psychological state [2].Many factors drive the change to composition and function of microbiota, including host genetics, dietary and lifestyle habits, and microbial infections.Notably, pathogenic invasion has been regarded as the critical factor that contributes to the alteration of microbiota [3].Diverse pathogenic microbes are competitively competed with resident bacteria and decrease a plethora of 'good' bacteria, which compromise the gut barrier leading to metabolic disorder.Antibiotics are usually used as an effective approach to reduce the load of pathogenic microbes in intestinal infection.However, the therapy with antibiotic frequently leads to gut microbial dysbiosis and polymicrobial infection [4].In some cases, antibiotics can cause the development of MDR mutants.In particular, sublethal levels of antibiotics improve the production of virulence factors to enhance the persistence of bacteria in epithelial cells [5].Although the emergence of antimicrobial resistance (AMR) is a natural phenomenon no matter of antibiotic use, it can be promoted by the wasteful and uncritical use of antibiotics without adequate consideration.To conquer the AMR emerges and spreads globally, several novel antibacterial approaches are in development, including anti-virulence agents, engineered phages, and probiotics.
Probiotics have been used for long time historically and are generally recognized as safe (GRAS) and effective that can confer a range of benefits to its host.Additionally, probiotics have received increasing interests both in human healthcare and animal husbandry because they rarely induce the AMR and even reverse it [6].Among numerous microorganisms, spore-forming Bacillus strains, with the ability of sporulation to survive in harsh environment of gut lumen, exhibit a wide range of activities in manipulating host immunity and eliminating invasive pathogens.Normally, probiotic Bacillus via oral administration can temporarily remain in intestinal tract, reaching from 10 5 to 10 8 CFUs/g in different intestinal section [7].This colonization allows Bacillus to continuously employ multiple mechanisms to provide protection against infections [8].Thus, probiotic Bacillus are increasingly selected and used as dietary supplements or live biotherapeutic products (LBPs) for the probiotic potential [9].Nowadays, more than 40 species of probiotic Bacillus have been used in treating enteric diseases and other diseases for their antibacterial bioactivity and relatively strong stability [10].It's noticeable that diverse Bacillus-derived metabolites can be diffused into the gut lumen and modify the collective community, resulting in elimination of enteric pathogens such as pathogenic E. coli, Salmonella, or other drug-resistant bacteria [11].Some unique proteins exposed on the surface of spore show colonization resistance and host immunomodulatory effect in gut [12].Metabolites produced by Bacillus is the key mediator in interaction with gut microbiota or host, such as antimicrobial compounds that directly inhibit the growth of pathogen and secondary metabolites like vitamins promote the health of host.
Reviews about the metabolites derived from the Bacillus genus and their structure classes and activities have been published elsewhere [13][14][15].In this review, we focus on elucidating the mechanisms underlying the interaction between probiotic Bacillus and both the microbial community and host system.By shedding light on the prominent classes of Bacillus-derived metabolites with probiotic potential properties, we aim to gain deeper insights into the ecological role of probiotic Bacillus mediated through metabolites, which advance our understanding of the beneficial mechanism on the host.

Bacteria to bacteria interaction
Numerous of intestinal microbes colonized in nutrient limited intestinal lumen, namely gut microbiota, tend to find a suitable niche for survive and replication.These microbes densely colonize the mucous surface and are in close proximity to each other engaging in multiple interactions.Microbial interactions are associated with the homeostasis of gut microenvironment especially when foreign species introduce.Colonization of probiotic Bacillus has been reported to reduce pathogen adaptability in gut [16] and confer a range of benefits on the host, such as increased production of short chain fatty acids (SCFAs) [17].So far, probiotic Bacillus mediate colonization resistance against enteropathogenic bacteria through bacterial interactions can be summarized as niche occupation, nutrient and oxygen competition, and metabolites mediated exploitation (Fig. 1).

Niche occupation
Dynamic ecological interactions are dominated by two opposite relationships: competition and cooperation [18].In these relationships, positive cooperation is common in gut microbiota [19].Commensal microbes employ a myriad of mechanisms to keep a certain elastic fluctuation in community and exclude alien species.As for probiotic Bacillus, these species can transit unimpeded and occupy a niche in the nutrient-limited gastrointestinal tract (GIT) mostly attributed to the particularity of cell structure (spore).Spores are the dormant form of life in Bacillus, characterized by thick proteinaceous coat, peptidoglycan cortex, and a dehydrated core abundant in dipicolinic acid (DPA), divalent metal ions, and acid-soluble proteins (SASPs).These components collectively contribute to the exceptional resistance against heat, radiation, reactive chemicals, and extreme physical processing [20].Besides, the outer sporular layer is responsible for environmental sensing [21], adhesion [22], host protection [23], host cell uptake [24] and immune inhibition [25].Mutation in exosporium layer, an outer layer of spore, showed less hydrophobic than the wild-type strains [26], while hydrophobicity of the bacterial surface is correlated with the adhesion [27] suggested that the adhesion of spore depend on exosporium proteins.In vegetative form, specific components in cell membrane, such as surface layer (S-layer) proteins, pilus, and mucusbinding protein, exhibit a strong affinity for intestinal epithelial cells [28].In addition, flagellins are relevant to strengthen the adhesion between bacteria and epithelial cells [29].The presence of certain carbohydrate like sucrose, enhanced the length of flagellum so as to promote the colonization of Bacillus [30].Collectively, diverse surface protein in dormant and vegetative cells promote the colonization of Bacillus in gut.Recent research demonstrated that B. subtilis employ an interesting strategy to compete with phylogenetically distinct pathogens by the increased production of antibiotics when encountering the peptidoglycan from pathogens in the same niche [31].Therefore, Bacillus utilize multiple mechanism for outcompeting other gut bacteria in space competition.

Nutrient and oxygen competition
Microbial communities are commonly shaped by biotic and abiotic factors under the nutrient scarcity [32].In normal, microbes with multiple metabolic pathways have a competitive advantage in auxotrophic environments.Probiotic Bacillus have the ability to utilize a wide range (1) Bacillus adapt itself in suitable niche against niche-occupying competitors.It approaches the intestinal mucous layer and competitively binds to intestinal epithelial cells and mucous layer components via surface proteins.Thus, the effect of niche occupation by Bacillus expels harmful bacteria from the host intestinal epithelial barrier and reduces pathogen invasion.(2) Competitive utilization of nutrients for Bacillus growth.Bacillus secrete various enzymes to rapidly exploit both the macronutrients and micronutrients in gut environment, resulting in limited availability of nutrition to pathogenic bacteria.(3) Bacillus produce an arsenal of antibacterial metabolites that directly inhibit the growth of pathogens.The metabolites included lipopeptides, bacteriocins, polyketides, and SCFAs are effective against the expansion and invasion of pathogens.(4) Bacillus can consume excess oxygen from gut lumen and host circulation for maintaining the intestinal environment in a state of hypoxia, which drive a dominance of bacteria such as lactate acid bacteria that use fermentation for energy production of sugars, organic acids and other organic compounds as sources of cell structure and energy regeneration [33].An intriguing phenomenon is Bacillus has secondary growth phase during the entire life cycle.This growth capability contributes to the survive under nutrient scarcity through selective nutrients utilization.In the context of limited nutrient condition such as gut lumen, Bacillus are prone to use glucose and enable themselves to rapidly capture the niche [34,35].Although the major source of energy generation is derived from carbohydrate, nitrates and nitrites can also be directly used as electron acceptors to maintain the energy balance in Bacillus [36].In addition to the competition of carbohydrate and nitrogenous compounds, the sequestration of the essential nutrient metal is a powerful mean in combating the invasion of bacterial pathogens [37].For instances, Bacillus product high affinity siderophore bacillibactin to create iron limited environment and restrain the expansion of pathogen due to iron starvation [38].Additionally, Bacillus probiotics produce a range of nutrients, including extracellular polysaccharides, vitamins, and exoenzymes, that promote the growth of beneficial microbiota.For instance, the extracellular polysaccharides produced by Bacillus can serve as a carbon source for lactobacilli and enhance their capacity of adhesion and acetate production [39]; the organic acids produced by Bacillus and the hyporedox state mediated by Bacillus acidify the gut environment, thereby promoting an enrichment of beneficial SCFA-producing bacteria.Therefore, there are several metabolic features for Bacillus to outcompete enteropathogens and maintain the growth of beneficial microorganisms in the limited nutrients environment.
Oxygen availability is extreme limited (below 1 mmHg) in a healthy intestine [40].Once the gut barriers disruption, the level of intestinal oxygen will be increased and contribute to the growth of invasive bacteria [41].The maintenance of hypoxic environment in gut lumen is attributed by intestinal epithelium metabolism mainly directing toward oxidative phosphorylation [42] and oxygen consumption by microbiota such as the phyla of Firmicutes and Bacteroidetes [43].Bacillus as facultative anaerobicity bacteria can ferment in hypoxic environment and consume excessive oxygen to maintain low oxidation state in the lumen.Although there is no direct research supporting the modulation of gut microbiota is due to the oxygen-capturing capability of Bacillus, the metabolites from Bacillus may contribute to the biological oxygen capturing capability resulting in microbiota regulation.The fermentation product surfactin enhance oxygen diffusion in the growth of early stationary phase and maintain viability during oxygen depletion by shift in metabolic profile and membrane depolarization [44], which acquire the advantage in interspecies bacterial competition under hypoxia.When oxygen complete depletion, Bacillus turn to dormant and form resistant spore to keep viability for germination in nutrient rich condition.Thus, probiotic Bacillus exhibit the extraordinary capability in depleting gut pathogens by competitively consuming limited nutrients and oxygens in distinct section of intestinal.

Metabolites mediated exploitation
Antagonistic microorganisms often have an advantage in a limited microbial environment due to their arsenal of antimicrobial compounds.The driving force and intricate pattern of microbial competition mainly attribute to the activities of antimicrobials.Bacillus are generally considered with strong capability in producing structurally diverse antimicrobial peptide (AMPs) for competitor inhibition [45,46].Although there are no evidences verified that antimicrobials from Bacillus directly mediate the exclusion of pathogenic bacteria in vivo, the production of antimicrobial metabolites, such as AMPs and bacteriocins, possibly dominate the bacterial interference between Bacillus and enteropathogenic bacteria in GI tract [47,48].Notably, Bacillus can regulate antibiotic production in response to the component from competitors [31].We do know that Bacillus employ multiple distinct compounds that have been proven with antimicrobial activity to maintain viability while interact with other microbes, but no literature is available for summarizing the structural classification and corresponding biosynthetic gene clusters (BGCs) of these compounds in probiotic Bacillus.Thus, we summarized the information about antimicrobial metabolites produced by probiotic Bacillus, including the biosynthesis mechanisms, molecular targets within bacterial cells or communities, and antimicrobial spectrum.

Distribution of biosynthetic gene clusters in probiotic Bacillus
Bacterial metabonomic profile can be pre-identified by gene function prediction [49].Regardless of the bacterial gene expressions being silent or unknown, this strategy expedites the discovery of active compounds with the ability to control the microbiota [50,51].BGCs are responsible for the synthesis of secondary metabolites involved in microbial exploitation.Previous research has revealed the positive association between BGCs and antagonistic activity in Bacillus [52].This relevance provides a simple and efficacious way to find the important antibacterial compounds that dominate the interaction between Bacillus and other bacteria.Thus, to search most abundant BGCs in Bacillus, we first search the NCBI database for establishing genome assemblage of Bacillus with probiotic potential.A total of 452 isolates are selected and used for further bioinformatics analysis.Prediction using antiSMASH database and in-house database showed that three major classes of biosynthetic gene cluster (BGCs) were predominated in probiotic Bacillus as: ribosomally synthesized and post-translationally modified peptides (RiPPs), polyketide synthases (PKS), and non-ribosomal peptide synthetases (NRPS), which have similar distribution as previous study [53].Among these, probiotic Bacillus accommodates a high abundance of NRPS and RiPPs reaching up to 100% in B. subtilis group (the group including B. subtilis, B. licheniformis, B. velezensis, etc.).The peptide antibiotic such as lipopeptide synthesized by NRPS showed broad spectrum antibacterial activity, while ribosomally synthesized peptides selectively inhibit certain pathogens.Bacillusderived antibiotics can contribute to enhance niche adaptation and spatially outcompete between different microbes [32].
In the analysis, the diversity and concrete distribution of the BGCs and functional genes were relevant to the phylogenetic relatedness.Probiotic B. cereus strains were all in the presence of the genes or BGCs responsible for synthesizing bacteriocins, quorum sensing molecules, terpenes, and vitamins, but in absence of PKS gene cluster and phosphonates synthesized genes (Table 1).In B. subtilis group, high abundance of PKS, NRPS, and NRPS/ PKS hybrid BGCs were detected, many of which were involved in growth interference.Additionally, RiPPs were distributed in wide range of Bacillus species.The RiPPs products are documented that play a role in bacterial physiology and niche competition [54].The antibacterial metabolites related to above BGCs were predicted by antiSMASH and listed in Table 2.As probiotic Bacillus can product diverse metabolites with antimicrobial activities that mediate the beneficial interaction between host and microbiota, we further highlight the notable example of bacteriocin, lipopeptide, and polyketide antibiotics for their modes of action and antimicrobial spectrum (Table 3 and Fig. 2).

Bacteriocins and lipopetides
Bacterial cell membrane, cell wall synthesis, DNA synthesis, transcription, as well as folate synthesis are traditional targets by most of available antibiotic [120].Bacteriocins or antimicrobial peptides employ multiple mechanisms to directly show antibacterial function by targeting cell membrane or cell wall leading to collapse of bacterial metabolism.Bacteriocins are ribosomally synthesized (poly)peptides produced by almost prokaryotic lineages [121], and non-ribosomal peptides is an indispensable part for bacterial adaptation [44].Due to the flexible biosynthetic mechanism of NRPS, these compounds are structural diversity and exhibit relatively wide range of activity [17,122].In this section, we introduced the mode of action of bacteriocin and lipopeptide antibiotic and their function in microsystem regulation.As for post-translationally modified peptides, the characteristic feature is containing intramolecular ring that form by thioether bonds between amino acids.Most of these lantibiotics targets cell membrane and disrupt the balance of energy metabolism, such as subtilin, subtilosin A, sublichenin, sublancin 168, lichenicidin, and cerecidins.Subtilin [74], entianin [60], and sublichenin [73] have strong structural similarities to each other with identical organization of lanthionine-bridging structure.Subtilinlike lantibiotics show potent MIC as low as 0.25 μg/mL against extended spectrum of foodborne Gram-positive (G + ) pathogens via cell wall biosynthesis interference and pores formation to cause leakage of the cytoplasmic    small molecules.Subtilosin A, a cyclic lantibiotic protein, can interact with the lipid head group region of bilayer membranes in a concentration dependent manner [76] and act as a autoinducer-2 inhibitor to inhibit quorum sensing [77].Sublancin 168 is a glycosylated bacteriocin with unique antibacterial mechanism to against G + bacteria.This compound affects the bacterial glucose uptake system rather than the integrity of cell wall or membrane to exert its activity.The deletion of the ptsGHI, the major glucose transporter components, results in resistance of sublancin [72].Subtilomycin was identified from marine sponge associated B. subtilis.It exhibits a wide range of antibacterial activity towards important enteric pathogens including L. monocytogenes, MRSA and P. aeruginosa and resistance to certain extent of heat, acidic, enzymatic treatments [75].Amylolysin, a type-B lantibiotic produced by B. amyloliquefaciens, also has the similar stability and antimicrobial activity as subtilomycin with pore-forming ability by depolarizing the cell membrane leads to cell leaking [55].Lichenicidin is the first lanthipeptide showed antimicrobial activity on G + bacteria, that also targets bacterial membrane [64,65].

Bacteriocins As presented in
Cerecidins is novel lanthipeptide from B. cereus against G + bacteria, its variants cerecidins A7 showed inhibition activity on MDRSA and VRE [56], which may also target the cell membrane.Formicin is a novel member in twopeptide lantibiotic with reduced hydrophobic α peptide and unusual negative charge β peptide, displaying a broad spectrum of foodborne G + pathogens inhibition such as C. difficile and S. aureus [61].Mersacidin, an efficacious bactericidal lantibiotic specifically targeting G + bacteria, serves as a potent inducer of the cell wall stress response and a peptidoglycan synthesis inhibitor [68].Furthermore, it exhibits superior activity compared to vancomycin in a mouse infection model [69].Clausin displays high antimicrobial activity against G + bacteria by binding to lipid precursors of the bacterial cell wall to inhibit bacterial cell integrity [57,58].Haloduracin [62,63] and pseudomycoicidin [70] are effective anti-G + lantibiotics originally found in B. halodurans and B. pseudomycoides, respectively.Thiocillins are members of the thiazolyl peptide class of natural product antibiotics not only known act as target G + bacteria [81], but also as a biofilm matrix inducer to modulate bacterial cellular physiology [123].However, not all of bacteriocins secreted from Bacillus are effective to suppress multiple pathogenic bacteria.Plantazolicin is the highly post-translationally modified lanthipeptide with narrow-spectrum antibacterial activity toward the causative agent of anthrax.This antimicrobial compound exerts its action by penetrating the outer layer of bacteria and subsequently disrupting the integrity of the plasma membrane through the formation of pores, leading to complete depolarization of the membrane [71].
As for nonmodified peptides, thuricin and thurincin are the representative nonmodified bacteriocins produced by B. thuringiensis exhibit inhibitory activity against Grampositive pathogens.However, there are different mode of action among them.Thuricin binds to the membrane of target cell membrane leading to membrane permeabilization while thurincin causes loss of cell integrity without affecting membrane permeability and the detailed mechanisms is still unclear [78][79][80].Coagulin is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus with the specific characteristics of genetic environment that its structural gene harbor in plasmid I 4 [59].It exhibits both bactericidal and bacteriolytic activity against multiple pathogenic bacteria, including Listeria, Pediococcus and Enterococcus [124].Other BLIS such as megacin [66,67] is a single polypeptides with approximately 2000 amino acids displaying antibacterial activity in close related species by inhibition of protein synthesis.
Antimicrobial lipopeptides Bacterial lipopeptides are non-ribosomal natural product biosynthesized by NRPSs or PKS-NRPS [125], with the majority of these compounds originating from species belonging to the B. subtilis group.Bacillus-derived lipopeptides can be can be categorized into three groups based on their chemical structures: cyclic cationic lipopeptides, non-cyclic cationic lipopeptides, and linear lipopeptides.

1) Cyclic cationic lipopeptides
Cyclic cationic lipopeptides are composed of a cyclic oligopeptide interlinked with feasible fatty acid chain, such as the antimicrobial compounds like circulin [83], polymyxins [85], polypeptins [84], and octapeptins [86].Cationic peptides generally involve in formation of the channels through ions passing the channels and disrupting bacterial cytoplasmic membranes.Circulin group, polymyxin analogues and octapeptin analogues show potent activity against Gram-negative (G − ) bacteria by permeabilizing cell membrane.Circulin group cover broader spectrum antibacterial activity than Bacitracin and the other two group analogues.Octapeptin exhibits selective antibacterial activity by binding to lipid A and inducing membrane depolarization [87].The cationic sug-ars, when combined with lipid A, reduce its efficacy; however, this occurs through distinct mechanisms compared to polymyxins [126].Bacitracin from Bacillus strains inhibits G + bacteria via interference with the dephosphorylation of C55-undecaprenyl pyrophosphate (bactoprenol) resulting in block of cell wall synthesis [82].
2) Cyclic noncationic lipopeptides Non-cationic peptides may bind to bacterial surface bilayer and change the linkage of negatively charged lipid tissue resulting in lipid bilayer restructure.The cyclic noncationic lipopeptides are iturin group, surfactin analogues, fengycin analogues, and fusaricidin analogues.Iturin group contain a β-hydroxy fatty acid with a 14-carbon chain, including iturins (variants A, C, D, and E), bacillomycins [90][91][92] (variants D, F, L, and Lc), and mycosubtilin [92,94].Iturns shows antibacterial activity by targeting cytoplasmic membrane resulting in formation of ion-conducting pores and increased K + permeability [93], but recent studies found that fungal DNA and biofilm matrix are also the target of some iturins.Surfactin is one of the most powerful known biosurfactants secreted by Bacillus.It is reported that surfactin exert multiple activities to impact the colonization and adherence of pathogens and acts not only as an antibiotic but also a competition factor to pathogen and contributor to its fitness in bacterial community [100,127].Lichenysin produced by B. licheniformis show similar structural and physiochemical properties with surfactin.This compound can not only cause permeabilization of phospholipid membrane but also decrease the load of pathogens through reduction of bacterial biofilm [96,97,128].A novel lipopeptide, bacaucin, identified from B. subtilis, shows broad antibacterial activity against MDR G + pathogens by membranedisruptive mechanism without induction of bacterial resistance [95].Fengycin and Plipastatin have a strong antifungal activity and a restricted antibacterial activity against certain species.Their targets are the specific membrane component such as glycerol-3-phosphate transporter to affect membrane homeostasis [102].Notably, fengycin can block S. aureus quorum sensing for colonization resistance acted as the analogue of autoinducer AIP [16].Differ from the antimicrobial spectrum of fengycin group, fusaricidin analogues are more effective to against G + bacteria and included the activity to disrupt the balance of cellular metabolism [104,105].The natural product Bogorol A, derived from Bacillus sp., was first discovered in 2001.It exhibits inhibitory activity against MRSA and VRE, but the precise target of its action remains unknown [106].Bacilotetrins A and B [88] are two new cyclic-lipotetrapeptides produced by B. subtilis exhibit anti-MRSA activity with minimum inhibitory concentration (MIC) values of 8-32 µg/mL and show no cytotoxicity.Similarly, locillomycin is a novel family of cyclic lipopeptides produced by B. subtilis with low cytotoxicity, characterized by a unique nonapeptide sequence and macrocyclization.It has inhibitory activity against both bacteria and virus [89].

3) Linear lipopeptides
Although cyclic lipopeptide tend to be more stable than linear lipopeptide for its circular structure, linear lipopeptide has several advantages as follow: (1) reduced toxicity; (2) easier to synthesize; (3) multiple target within the target cells and microbiota modulation [129].
Linear noncationic lipopeptide such as bacillin [113] and bacilysin are both produced from B. subtilis possessing antimicrobial activity toward G + and negative bacteria, among which bacilysin act as an important factor in microcosm to shape interaction between species [130].Gageopeptides [108], gageostatins [109], gageotetrins [110] were Leu-rich linear lipopeptide discovered from B. subtilis share the similar physicochemical and bioactive properties such as a broad spectrum antimicrobial activity on both bacteria and fungus, among which gageopeptides displays noncytotoxic character and extraordinary antimicrobial activity with MIC values of 0.02-0.09µM.Tridecaptins are a re-emerging class of non-ribosomal antibacterial peptides (NRAPs) with potent activity against G − bacteria [111].Zwittermicin was initially discovered for its role in the competitive interactions between different bacterial species.It acts as a potent inhibitor against the growth of other microorganisms, giving the producing strain a competitive advantage in its ecological niche [131].The compound is effective against a wide range of bacteria, including both G + and G − species [112].

Polyketides and PKs/NRPs hybrids
Polyketides (PKs) are structurally diverse compounds with numerous biological activities particularly as antibacterial activity in Bacillus.The PKs machinery are linear assembly and minimally comprises three core domains: ketosynthase (KS), an acyltransferase (AT), and an acyl-carrier protein (ACP) domain, to orderly synthesis variable compounds.Unlike lipopeptide antibiotics often target cell membrane or cell wall to exert their activities, polyketide commonly interfere with the process of protein synthesis.Three main polyketides are found in Bacillus, including bacillaene, difficidin, and macrolactin, which play a crucial role in microbiota modulation.Bacillaene is an instable polyene antibiotic that inhibit bacteria by hindering prokaryotic protein synthesis [116].It is reported that the competition between B. subtilis and Salmonella typhimurium in vitro is mediated by bacillaene through interrupt the growth of S. typhimurium under nutrient-rich condition [117].Likewise, macrocyclic polyene difficidin regulate rhizosphere microbiota by suppressing the metabolism and virulence of phytopathogenic bacteria, which might exhibit same mechanistically action in gut microbiota regulation [114].
Macrolactin with both antifungal activity and broad antibacterial spectrum exerts the antagonistic activity by means of disturbance of bacterial cell wall synthesis.This compound could effectively suppress the colonization of multi-drug resistance bacteria in intestine [118], and in some cases, reduced the diversity of bacterial community and changed the collective metabolic pathways [119].Amicoumacin is a ribosome-targeting antibiotic and vital for the negative interaction with anti-Helicobacter pylori and anti-vibrio activity [48,115].Along with the repeated discoveries of the genomic biosynthetic clusters and natural derivatives of amicoumacins in Bacillus species, the ecological role of amicoumacin was found to function as major antibacterial metabolite driving the reduce of competitor population [132].

Bacteria-host interactions
Metabolic crosstalk among commensals, host, and invaders contributes to a state of dynamic balance.Administration of probiotic Bacillus has been associated with a range of benefits to host.These include enhancement of pathogenic resistance [133], alteration of inflammation response [134], activation of innate immunity [135], and amelioration of intestinal damage [136,137].
The benefits are likely mediated by the Bacillus-derived metabolites such as lactate secreted by B. coagulans that helps maintain an acidic environment in the gut and exoenzymes produced by B. subtilis that promote host digestibility.Although metabolomics studies reveal the diverse array of Bacillus-derived metabolites [138,139], many of their functional role in host remain unclear.In this section, we summarize the reported metabolites that exert effects on the host, primarily through two mechanisms: (1) Involvement in intestinal cell metabolism to enhance the intestinal physical barrier; (2) Activation of innate immune responses to drive host's clearance of pathogens (Fig. 3).

Organic acids
Bacteria have the capability of biosynthesis in organic acids, such as SCFAs, secondary bile acids (BAs), amino acids, and their derivatives [140], that deeply affect host metabolism [141].Bacillus spp.employ these abundant secondary metabolites to participate in host circulation.We summarized the organic acids produced by Bacillus that have reportedly metabolic function in host (Table 4), many of which serve as important factors to regulate host homeostasis by immunity modulation.SCFAs are most studied metabolites that derive from the fermentation of dietary fibre.In the context of normal GI environment in mice and humans, acetate, propionate, and butyrate with a molar ratio of 60: 20: 20 comprise the majority of SCFAs pool in gut [142].These compounds not only have the role in regulation of immunity system but also exert their antibacterial activities by directly inhibiting the growth of pathogenic bacteria [143], or act as adjuvants by enhancing the potency of antibiotic [144].Mechanically, SCFAs mediate intracellular acidification that disrupt the respiration [145] Fig. 3 Probiotic Bacillus produces various metabolites to activate intestinal immune.Metabolites trigger B cells and T cells through M cell (1) and dendritic cell (2), as well as improve the phagocytosis ability of macrophages (3) resulting in enhanced clearance of pathogens; and stimulate the intestinal associated lymphoid tissue to produce CD8 + and T cell (4), alleviate some kinds of inflammation.In another aspect, the metabolites improve the diversity of commensal microbiota (5), induce paneth cell producing AMPs (6) as well as enhance the expression of tight junction protein in epithelial cell (7), to strengthen the local barriers configuration and perturb the accumulation of anion [146].The antibacterial activity or synergistic effect of SCFAs promote the recovery of gut microbiota through upregulation of lactic acid bacteria and other commensal flora [147].However, the function of SCFAs mostly exhibit in intracellular processes involving in cell proliferation, differentiation and gene expression.For example, SCFAs can target G-protein coupled receptors (GPCRs) to activate host immune signaling cascades against IBD [148,149], as well as regulate T cells to increase anti-inflammatory factors [150] and reduce pro-inflammatory factors [151].Lactate and pyruvate can enhance immune responses by inducing GPCRs-mediated dendrite protrusion of intestinal C-X3-C motif chemokine receptor 1 + cells [152].The other secondary bile acid metabolized by Bacillus, can improve the permeability of the intestines and avoid the unnecessary increase of BAs production [153].

Exoenzymes
Various enzymes excreted by probiotic Bacillus have multiple functions, including inhibition of pathogenic microbes, decrease of virulence in enteric pathogens, and rebalance of intestinal homeostasis by regulating host immunity.Antimicrobial enzymes produced by probiotic Vitamin B12 B. megaterium Affecting DNA synthesis and regulation, fatty acid synthesis and energy production [186,187] Vitamin K B. subtilis Prevention of osteoporosis and cardiovascular disease [188][189][190] Bacillus significantly against pathogenic growth [191].For example, two kinds of chitinases (ChiS and ChiL) [192] degrade butyrin and the peptidoglycan component of the fungal cell wall [193] or catalase and serine protease that reduce the pH and decrease the oxygen concentration of the intestinal tract [194].Similarly, 1-3-glucanase is also reported with directly antimicrobial activity [195].Others enzymes, like amylases, cellulases, lipase phytase or protease, are closely related to degradation of foods.Since quorum-sensing is important in regulating bacterial population, Bacillus is reported to use quorum-sensing molecules (QSMs)-pentapeptidescompetence inducing cytoprotective heat shock proteins to protect intestinal epithelial cell from oxidative stress and loss of barrier function [196].Additionally, they collectively regulate production of surfactin in B. subtilis [197] and enhances digestive enzyme activity to promotes host growth performance [198].Pheromone produced by B. subtilis involved in bacterial quorum sensing that regulate bacterial competence and surfactant production [199].Interestingly, a kind of serine protease secreted by B. clausii could inhibit hemolytic and cytotoxic effects of Clostridium difficile and toxic B. cereus [174].Another intriguing function attributed to probiotic Bacillus is its potential in treating allergies.This effect is mediated by a specific sporular protein, which hinders the development of eosinophilia and goblet cell hyperplasia that are typically associated with allergic responses [200].

Other metabolites
In addition to the organic acids and exoenzymes, other metabolites such as vitamins also enhance the survival or growth fitness of commensal bacteria and serve as public goods in maintenance of host homeostasis.Given that about 45-60% of gut bacteria are genomic active in producing certain or all of the B-vitamins [201], some Bacillus strains show potential to biosynthesize vitamin B 1 , B 2 , B 3 , B 5 , B 6 , B 7 , B 9 and B 12 [202], many of which contribute the absorption of food proteins in host [203], regulation of fatty acid synthesis, regeneration of energy production, as well as promoting the elimination of pathogens to maintain the microbiota homeostasis [204].K-vitamin is emerged as fitness determinant for host to fight against cancer owing to that vitamin K 2 is both vital to cell respiration both in host and bacteria as well as participating in bone formation and absorption [188,189].Recently, researchers found that Bacillus could excrete vitamin B 3 to nourish the surrounding colonies [181,205].Vitamin B 3 is a vitamin family containing nicotinamide adenine dinucleotide (NAD) and the related precursors.These substances are vital for both host cell and bacterial growth that involved in many enzymatic processes [206].Bacillus spp. is reported to produce these factors for boosting host energy metabolism as effective antiaging intervention [182].Besides, most of Bacillus are biofilm-forming strains, which implies that they secrete numerous extracellular products such as exopolysaccharides (EPS) during their growth.EPS can not only act as prebiotics to provide nourishment for beneficial bacteria, but also inhibit enterotoxigenic Escherichia adherence via binding to colonization factor fimbriae on the cell surface [39].Mucosal integrity and inflammatory responses are also regulated by Bacillus EPS.These extracellular components have been shown to enhance the expression of tight junction-related proteins (claudin-1, claudin-2 and occluding) and inhibit the secretion of the pro-inflammatory cytokine IL-6 and the activation of nuclear factor-κB pathway in macrophage, thereby alleviate gut inflammation [179,207].

Concluding remarks and future perspectives
Collectively, probiotic Bacillus can perform probiotic benefit through directly interact with pathogenic bacteria or mediate by structurally diverse metabolites, which contribute to the stability and homeostasis of intestinal flora.However, the probiotic properties of Bacillus are strain-specific and activity-dependent [47].This suggested that the qualification of Bacillus required to be determine before application.The probiotic activities are not only associated with their inherent species properties, but more importantly, are determined by the metabolites they secrete.Given that the intricate nature of microbial communities and host environment, the advantages of Bacillus in niche and resource competition against enteropathogens are conditional upon specific metabolites.Thus, determining the profiles of metabolites secreted from Bacillus under different culture conditions is a prerequisite for probiotic selection.The transition between dormant spore and vegetative cell in Bacillus contribute to more resistance than other gut bacteria, which not only protects cell from harsh environment but also promotes self-colonization and growth in gut.Compared to most of bacteria (like some strictly anaerobic or aerobic), Bacillus species exhibit the ability to thrive in extreme environment by regulating cellular physiology through the overlapping regulatory systems of key metabolites to maintain viability [35].Notably, the flexible metabolic regulatory network of Bacillus would be advantageous for causing nutrients stress to competitors, because nutrient intervention is new target for treating pathogens infections [208].As the intestinal microbiota establishes itself within the resource-limited niches, the properties of Bacillus make it more suitable for self-survival and predisposed to outcompete invasive species.
Probiotic Bacillus harbors remarkable ability to directly mediate colonization resistance of pathogens by various antibacterial compounds [51,209].Some of the compounds are promising agents, such as amicoumacin and surfactin, with potent antimicrobial activity and exert multiple function in host-microbiota system awaiting for further application.Besides, it is noteworthy that antimicrobial substances in Bacillus are predominantly synthesized by BGCs.Thus, we can selectively screen the Bacillus strains that possess abundant secondary metabolite gene clusters, particularly NRPS, for the development of probiotics [210].In addition to exploring traditional antimicrobial compound, SCFAs have also garnered attention from antibacterial developers due to their synergistic effects when combined with antibiotics.Small molecular organic acid and vitamin derived from probiotic Bacillus can be developed as prebiotics and postbiotics for enhancing the host resistance to environment changes and pathogenic infections.Therefore, the ability to produce metabolites that mediate the interaction between Bacillus and host system are attracting direction to develop a novel probiotic Bacillus preparation.Furthermore, understanding the mechanisms that mediated by Bacillus metabolites in the intestine, could advance the development of stratagem of enteric infections.However, most studies demonstrated the positive result when probiotic Bacillus applied in disease model, but partial of them elaborated the underlying mechanisms of probiotic Bacillus, leaving ample scope for further mechanistic research.Currently, some studies unraveled the underlying beneficial mechanism between probiotic and host [211][212][213], but the metabolites that dominated the interaction remained unclear.
The use of Bacillus for probiotic and feed additive have been last for at least 50 years since the well-known Italian product (Enterogermina ® ) used for OTC medicinal supplement in 1958.Currently, probiotic Bacillus are widely used as a nutrient supplement and for the treatment enteric infection, such as the product NutriCommit ® , Lactopure ® , and Biosubtyl ® .As the fast expansion of Bacillus probiotic in multiple field, increasing researches have demonstrated that the safety assessment of Bacillus probiotic is insufficient and should be laid more emphasis [214,215].In some specific species like B. cereus, they were found to secrete the enterotoxin Nhe, hemolysin Hly, and emetic cereulide causing severe foodborne disease.Around the year 2000, two commercial B. cereuscontaining product Paciflor ® and Esporafeed Plus ® have been withdrawn by SCAN in Europe for the toxigenic potential and antimicrobial resistance found in probiotic B. cereus.Thus, to avoid the side effect and maximum the benefit bringing by probiotic Bacillus, selected strains should be comprehensively evaluated for their safety through in vitro and in vivo experiments.Conclusively, our work provides advanced insight into the host interaction mechanism of probiotic Bacillus, particularly in relation to metabolites and strain properties.

Fig. 1
Fig. 1 Probiotic Bacillus employ multifactorial competition mechanism to restrict the expansion of pathogens through four pathways.(1) Bacillus adapt itself in suitable niche against niche-occupying competitors.It approaches the intestinal mucous layer and competitively binds to intestinal epithelial cells and mucous layer components via surface proteins.Thus, the effect of niche occupation by Bacillus expels harmful bacteria from the host intestinal epithelial barrier and reduces pathogen invasion.(2) Competitive utilization of nutrients for Bacillus growth.Bacillus secrete various enzymes to rapidly exploit both the macronutrients and micronutrients in gut environment, resulting in limited availability of nutrition to pathogenic bacteria.(3) Bacillus produce an arsenal of antibacterial metabolites that directly inhibit the growth of pathogens.The metabolites included lipopeptides, bacteriocins, polyketides, and SCFAs are effective against the expansion and invasion of pathogens.(4) Bacillus can consume excess oxygen from gut lumen and host circulation for maintaining the intestinal environment in a state of hypoxia, which drive a dominance of bacteria such as lactate acid bacteria that use fermentation for energy production

Fig. 2
Fig. 2 The mode of action of AMPs and SCFAs derived from probiotic Bacillus.The antibacterial activity exhibited by Bacillus is mainly attributed to the production of AMPs and SCFAs.AMPs and SCFAs produced by probiotic Bacillus can directly (a) kill/inhibit pathogenic bacteria or antagonize the colonization of pathogenic bacteria by destroying bacterial cell membrane, genetic material and (b) interfere with bacterial quorum sensing system.Additionally, (c) the produced SCFAs can easily penetrate into the lipid membrane of the bacterial cell and cause the acidification of cytoplasm or require excess energy consumption to export the dissociated protons from SCFAs.These effects result in inhibition of pathogen's growth

Table 3 ,
members of the B. subtilis group stands out for its abundance of BGCs responsible for antimicrobial compound production.With this group, notable species such as B. amyloliquefaciens, B. subtilis, B. licheniformis, and B. velezensis synthesize diverse lantibiotics.Strains belonging to the B. cereus group also produce diverse bacteriocin, such as B. cereus and B. thuringiensis.Based on the biosynthesis mechanism and chemical structure, probiotic Bacillusderived bacteriocins can be classified into post-translationally modified peptides, nonmodified peptides, and other linear bacteriocin-like inhibitory substances (BLIS).

Table 1
Distribution of BGCs and functional genes in probiotic Bacillus a The abbreviations were listed after the main

Table 2
Antimicrobial secondary metabolites in probiotic Bacillus predicted by antiSMASH a The percentage represent the average identity with the reference sequence.And numbers in the parentheses indicate numbers of the BGCs-carrying Bacillus isolates Species Predicted

Table 3
Antimicrobial metabolites in probiotic Bacillus

Polyketide compounds synthesized by polyketide synthetase or polyketide synthetase/non-ribosomal peptide synthetase
Antimicrobial metabolites listed in the table are commonly produced by producer species but not limited in these speciesbThe type of BGCs is categorized according to the published researches and MIBiG database c Their activities are shown in the way of whether exhibit the inhibition of the growth of pathogenic bacteria (Gram-positive G + and Gram-negative G − ), fungus or virus a d It represents unknown antibacterial targets.The abbreviations in the table can be found in the abbreviation list

Table 4
Bacillus-derived organic acids, exoenzymes and other metabolites that involved in bacteria to host interaction a The producer species listed in the table are representative of the producers of the corresponding metabolites; however, it should be noted that these strains are not the exclusive producers of these metabolites within the genus of Bacillus AbbreviationsAMPsAntimicrobial peptide.AMPs are oligopeptides with a varying number (from five to over a hundred) of amino acids, showing potent biological activity to inhibit diverse pathogenic bacteria AMR Antimicrobial resistance.Certain antibiotic that cannot inhibit the growth of the bacteria Bas Secondary bile acids.The secondary bile acids are derived from primary bile acids produced by the liver and are more hydrophobic than primary bile acids.The major secondary bile acids are deoxycholic acid (DCA) and lithocholic acid (LCA) BGCs Biosynthetic gene clusters.The BGCs are a locally clustered group of two or more genes that together encode a biosynthetic pathway for the production of a secondary metabolite BLIS Bacteriocin-like inhibitory substances.The BLIS has similar chemical struture with bacteriocin and not entirely characterized as bacteriocin CSF Competence and sporulation factor.A regulatory protein involved in the regulation of bacterial competence and sporulation processes plays a critical role in the regulation of competence development DPA Dipicolinic acid.DPA is a major component of Bacillus spore and functions as protective molecules to increase the stability of DNA EPS Exopolysaccharide.EPS are complex carbohydrate molecules produced and secreted by bacteria.These polysaccharides are synthesized and released into the surrounding environment, forming a protective matrix or biofilm GI Gastrointestinal.The part of the digestive system that consists of the stomach and intestines GPCRs G-protein coupled receptors.G protein-coupled receptor (GPCR), also called seven-transmembrane receptor or heptahelical receptor, locate in the cell membrane that binds extracellular substances and transmits signals from these substances to an intracellular molecule LBPs Live biotherapeutic products.LBPs are defined as live organisms designed and developed to treat, cure, or prevent a disease or condition, excluding vaccines, filterable viruses and so on MDR Multidrug resistance.The bacteria show resistance to a wide range of structurally unrelated antibiotics MIC Minimum inhibitory concentration.MIC defines in vitro levels of susceptibility or resistance of specific bacterial strains to applied antibiotic NAD Nicotinamide adenine dinucleotide.NAD is a molecule that participate in multiple cellular processes such as redox reactions and energy generations, to maintain the homestasis of metabolism NRPS Non-ribosomal peptide synthetases.The NRPS are large multienzyme machineries that assemble numerous peptides with large structural and functional diversity NRPS-PKS NRPS-PKS hybrids.They are responsible for the production of complex natural products that possess both peptide and polyketide components PKS Polyketide synthases.The PKS are multifunctional enzyme that use primary metabolites (acetyl-CoA and malonyl-CoA) to biosynthesize numorous natural product, many of which are antibiotics PTS Phosphoenolpyruvate: sugar phosphotransferase system.A bacterial transport system that facilitates the uptake and phosphorylation of sugars QSMs Quorum-sensing molecules.It also called autoinducer that acts as a cell-to-cell communication intermediator in response to fluctuations in cell-population density RiPPs Ribosomally synthesized and post-translationally modified peptides.The synthesis of RiPPs is ribosomal-dependent that produce polypeptide with modification by various dedicated enzymes SASPs Acid-soluble proteins.SASPs stabilise the DNA in an A-helix configuration and confer a protection benefit from cleavage by enzymes or UV light SCFAs Short chain fatty acids.A type of fatty acid with less than six carbon atoms mainly comprises acetate, propionate, and butyrate