Endotoxin-driven systemic immune activation is a common hallmark across various clinical conditions. During acute critical illness, elevated plasma lipopolysaccharide triggers non-specific systemic immune activation. In addition, a compositional shift in the gut microbiota, including an increase in gut-luminal opportunistic pathogens, is observed. Whether a causal link exists between acute endotoxemia and abundance of gut-luminal opportunistic pathogens is incompletely understood. Here, we model acute, pathophysiological lipopolysaccharide concentrations in mice and show that systemic exposure promotes a 100-10'000-fold expansion of Klebsiella pneumoniae, Escherichia coli, Enterococcus faecium and Salmonella Typhimurium in the gut within one day, without overt enteropathy. Mechanistically, this is driven by a Toll-like receptor 4-dependent increase in gut-luminal oxygen species levels, which transiently halts microbiota fermentation and fuels growth of gut-luminal facultative anaerobic pathogens through oxidative respiration. Thus, systemic immune activation transiently perturbs microbiota homeostasis and favours opportunistic pathogens, potentially increasing the risk of infection in critically ill patients.

Antagonistic interactions play a key role in determining microbial community dynamics. Here, we report that one of the most widespread contact-dependent effectors in human gut microbiomes, Bte1, directly targets the PpiD-YfgM periplasmic chaperone complex in related microbes. Structural, biochemical, and genetic characterization of this interaction reveals that Bte1 reverses the activity of the chaperone complex, promoting substrate aggregation and toxicity. Using Bacteroides, we show that Bte1 is active in the mammalian gut, conferring a fitness advantage to expressing strains. Recipient cells targeted by Bte1 exhibit sensitivity to membrane-compromising conditions, and human gut microbes can use this effector to exploit pathogen-induced inflammation in the gut. Further, Bte1 allelic variation in gut metagenomes provides evidence for an arms race between Bte1-encoding and immunity-encoding strains in humans. Together, these studies demonstrate that human gut microbes alter the protein-folding capacity of neighboring cells and suggest strategies for manipulating community dynamics.

Endogenous antimicrobial peptides and proteins are essential for shaping and maintaining a healthy gut microbiota, contributing to anti-inflammatory responses and resistance to pathogen colonization. Salmonella enterica subsp. enterica serovar Typhimurium (ST) infection is one of the most frequently reported bacterial diseases worldwide. Manipulation of the gut microbiota through exogenous antimicrobial peptides may protect against ST colonization and improve clinical outcomes.

This study demonstrated that oral administration of the antimicrobial peptide AP2 (2 µg /mouse), an optimized version of native apidaecin IB (AP IB), provided protective effects against ST infection in mice. These effects were evidenced by reduced ST-induced body weight loss and lower levels of serum inflammatory cytokines. A 16 S rRNA-based analysis of the cecal microbiota revealed that AP2 significantly modulated the gut microbiota, increasing the relative abundance of Bifidobacterium while decreasing that of Akkermansia at the genus level. Furthermore, the transplantation of fecal microbiota from AP2-treated donor mice, rather than from Control mice, significantly reduced cecal damage caused by ST and decreased the concentration of ST by one order of magnitude after infection.

These findings reveal a novel mechanism by which exogenous antimicrobial peptides mitigate Salmonella Typhimurium infection through the modulation of gut microbiota.

Antimicrobial peptides (AMPs) are recognized as one of the most ancient components of innate immunity, playing a pivotal role as the first line of host defense systems. These evolutionarily conserved molecules have been identified in various organisms, from prokaryotes to humans. AMPs establish a delicate balanced relationship between host and microbes, by simultaneously regulating the biological activities of pathogens and commensal microbes. Given the escalating global concern over antibiotic resistance, there is an urgent need to explore alternative strategies to combat challenging infectious diseases. AMPs have emerged as promising candidates employed in clinical practice due to their sustainable bactericidal properties. Witnessed by deep understanding of AMPs actions toward host and bacteria, the potential applications of AMPs extend far beyond infection control. Emerging developments harnessed natural capabilities of AMPs to optimize their roles in modulating host signaling, treating diverse diseases, advancing biosensing and bioimaging technologies. In this Concept paper, we provide a comprehensive overview of the diversity and properties of AMPs. Additionally, we elaborate on the mechanisms underlying AMP activity and bacterial responses counteracting AMPs' functions. Most importantly, we discuss potential biomedical applications of AMPs and offer perspectives on their future development.

The gut microbiota plays a pivotal role in influencing host health, through the production of metabolites and other key signalling molecules. While the impact of specific metabolites or taxa on host cells is well-documented, the broader impact of a disrupted microbiota on immune homeostasis is less understood, which is particularly important in the context of the increasing overuse of antibiotics.

Female C57BL/6 mice were gavaged twice daily for four weeks with Vancomycin, Polymyxin B, or PBS (control). Caecal microbiota composition was assessed via 16S rRNA sequencing and caecal metabolites were quantified with NMR spectroscopy. Immune profiles of spleen and mesenteric lymph nodes (MLNs) were assessed by flow cytometry, and splenocytes assessed for ex vivo cytokine production. A generalised additive model approach was used to examine the relationship between global antibiotic consumption and IBD incidence.

Antibiotics significantly altered gut microbiota composition, reducing alpha-diversity. Acetate and butyrate were significantly reduced in antibiotic groups, while propionate and succinate increased in Vancomycin and PmB-treated mice, respectively. The MLNs and spleen showed changes only to DC numbers. Splenocytes from antibiotic-treated mice stimulated ex vivo exhibited increased production of TNF. Epidemiological analysis revealed a positive correlation between global antibiotic consumption and IBD incidence.

Our findings demonstrate that antibiotic-mediated dysbiosis results in significantly altered short-chain fatty acid levels but immune homeostasis in spleen and MLNs at steady state is mostly preserved. Non-specific activation of splenocytes ex vivo, however, revealed mice with perturbed microbiota had significantly elevated production of TNF. Thus, this highlights antibiotic-mediated disruption of the gut microbiota may program the host towards dysregulated immune responses, predisposing to the development of TNF-associated autoimmune or chronic inflammatory disease.

Encrypted peptides (EPs) have been recently described as a new class of antimicrobial molecules. They have been found in numerous organisms and have been proposed to have a role in host immunity and as alternatives to conventional antibiotics. Intriguingly, many of these EPs are found embedded in proteins unrelated to the immune system, suggesting that immunological responses extend beyond traditional host immunity proteins. To test this idea, we synthesized and analyzed representative peptides derived from non-immune human proteins for their ability to exert antimicrobial and immunomodulatory properties. Most of the tested peptides from non-immune proteins, derived from structural proteins as well as proteins from the nervous and visual systems, displayed potent in vitro antimicrobial activity. These molecules killed bacterial pathogens by targeting their membrane, and those originating from the same region of the body exhibited synergistic effects when combined. Beyond their antimicrobial properties, nearly 90% of the peptides tested exhibited immunomodulatory effects, modulating inflammatory mediators, such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein-1 (MCP-1). Moreover, eight of the peptides identified, collagenin-3 and 4, zipperin-1 and 2, and immunosin-2, 3, 12, and 13, displayed anti-infective efficacy in two different preclinical mouse models, reducing bacterial infections by up to four orders of magnitude. Altogether, our results support the hypothesis that peptides from non-immune proteins may have a role in host immunity. These results potentially expand our notion of the immune system to include previously unrecognized proteins and peptides that may be activated upon infection to confer protection to the host.

The mechanism(s) underlying gut microbial metabolite (GMM) contribution towards alcohol-mediated cardiovascular disease (CVD) is unknown. Herein we observe elevation in circulating phenylacetylglutamine (PAGln), a known CVD-associated GMM, in individuals living with alcohol use disorder. In a male murine binge-on-chronic alcohol model, we confirm gut microbial reorganization, elevation in PAGln levels, and the presence of cardiovascular pathophysiology. Fecal microbiota transplantation from pair-/alcohol-fed mice into naïve male mice demonstrates the transmissibility of PAGln production and the CVD phenotype. Independent of alcohol exposure, pharmacological-mediated increases in PAGln elicits direct cardiac and vascular dysfunction. PAGln induced hypercontractility and altered calcium cycling in isolated cardiomyocytes providing evidence of improper relaxation which corresponds to elevated filling pressures observed in vivo. Furthermore, PAGln directly induces vascular endothelial cell activation through induction of oxidative stress leading to endothelial cell dysfunction. We thus reveal that the alcohol-induced microbial reorganization and resultant GMM elevation, specifically PAGln, directly contributes to CVD.

The overuse of antibiotics has led to lethal multi-antibiotic-resistant microorganisms around the globe, with restricted availability of novel antibiotics. Compared to conventional antibiotics, evolutionarily originated antimicrobial peptides (AMPs) are promising alternatives to address these issues. The gut microbiome of Blattella germanica represents a previously untapped resource of naturally evolving AMPs for developing antimicrobial agents.

Using the in-house designed tool "AMPidentifier," AMP candidates were mined from the gut microbiome of B. germanica, and their activities were validated both in vitro and in vivo. Among filtered candidates, AMP1, derived from the symbiotic microorganism Blattabacterium cuenoti, demonstrated broad-spectrum antibacterial activity, low cytotoxicity towards mammalian cells, and a lack of hemolytic effects. Mechanistic studies revealed that AMP1 rapidly permeates the bacterial cell and accumulates intracellularly, resulting in a gradual and mild depolarization of the cell membrane during the initial incubation period, suggesting minimal direct impact on membrane integrity. Furthermore, observations from fluorescence microscopy and scanning electron microscopy indicated abnormalities in bacterial binary fission and compromised cell structure. These findings led to the hypothesis that AMP1 may inhibit bacterial cell wall synthesis. Furthermore, AMP1 showed potent antibacterial and wound healing effects in mice, with comparable performances of vancomycin.

This study exemplifies an interdisciplinary approach to screening safe and effective AMPs from natural biological tissues, and our identified AMP 1 holds promising potential for clinical application.

The inflammation-inducing properties of lipopolysaccharides (LPS) of Gram-negative bacteria reside in their lipid A moiety. Bacillus fragilis, which is a commensal Gram-negative bacterium, biosynthesises lipid A that is structurally distinct from that of E. coli and other enteric bacteria. It is composed of a β1,6-linked glucosamine (GlcN) disaccharide that is only phosphorylated at the anomeric center. The major species of B. fragilis has five fatty acids and the amine of the distal GlcN moiety carries the unusual (R)-3-(13-methyltetradecanoyloxy)-1.5-methylhexadecanoic acid. A recent study indicates that the LPS of B. fragilis has anti-viral activity by selective induction of interferon (IFN)-β and is protective in mouse models of vesicular stomatitis virus (VSV) and influenza A. Heterogeneity in the structures of LPS and lipid A and possible contamination with other inflammatory components make it difficult to unambiguously define the immune-modulatory properties of LPS or lipid A. Therefore, we developed a synthetic approach for the preparation of the unusual major lipid A species derived from B. fragilis, which includes a synthetic approach for (R)-3-(13-methyltetradecanoyloxy)-1.5-methylhexadecanoic acid by the Wittig olefination to install the terminal isopropyl moiety. The proinflammatory and antiviral responses of synthetic B. fragilis lipid A were investigated in several cell lines and primary human monocytes by examining the production of interleukin (IL)-6 and IFN-β. It was found that B. fragilis does not induce the production of IL-6 and IFN-β but can partially antagonize the production of pro-inflammatory cytokines induced by E. coli LPS and lipid A.

Understanding factors influencing the composition and maintenance of beneficial host-associated microbial communities is central to understanding their ecological, evolutionary, and health consequences for hosts. Host immunity is often implicated as a regulator of these microbiota, but immunity may also play a disruptive role, with responses to infection perturbing beneficial communities. Such effects may be more prominent from innate immune responses, with more rapid-acting and often non-specific components, compared to adaptive responses. We investigated how upregulation of antibacterial immunity in the bumble bee Bombus impatiens affects its core gut microbiota, testing the hypothesis that immunity-induced perturbation impacts the microbiota structure. Freshly emerged adult bees were fed a microbiota inoculum before receiving a non-pathogenic immune stimulation injection. We quantified microbial communities using 16S rRNA amplicon sequencing and targeted quantitative PCR. Coarse community membership shows apparent robustness, but we find that immune stimulation alters the abundance of two core community members, Gilliamella and Snodgrassella. Moreover, a positive association in communities between these bacteria is perturbed following a Gram-negative challenge. The observed changes in the gut microbial community are suggestive of immune response-induced dysbiosis, linking ecological interactions across levels between hosts, their pathogens, and their beneficial gut microbiota. The potential for collateral perturbation of the natural gut microbiota following an innate immune response may contribute to immune costs, shaping the evolutionary optimization of immune investment depending on the ecological context.

Our work demonstrates how innate immunity may influence the host-associated microbiota. While previous work has demonstrated the role of adaptive immunity in regulating the microbiota, we show that stimulation of an innate immune response in bumble bees may disrupt the native gut microbial community by shifting individual abundances of some members and pairwise associations. This work builds upon previous work in bumble bees demonstrating factors determining microbe colonization of hosts and microbiota membership, implicating immune response-induced changes as a factor shaping these important gut communities. While some microbiota members appear unaffected, changes in others and the community overall suggests that collateral perturbation of the native gut microbiota upon an innate immune response may serve as an additional selective pressure that shapes the evolution of host innate immunity.

Drug-resistant bacteria are outpacing traditional antibiotic discovery efforts. Here, we computationally screened 444,054 previously reported putative small protein families from 1,773 human metagenomes for antimicrobial properties, identifying 323 candidates encoded in small open reading frames (smORFs). To test our computational predictions, 78 peptides were synthesized and screened for antimicrobial activity in vitro, with 70.5% displaying antimicrobial activity. As these compounds were different compared with previously reported antimicrobial peptides, we termed them smORF-encoded peptides (SEPs). SEPs killed bacteria by targeting their membrane, synergizing with each other, and modulating gut commensals, indicating a potential role in reconfiguring microbiome communities in addition to counteracting pathogens. The lead candidates were anti-infective in both murine skin abscess and deep thigh infection models. Notably, prevotellin-2 from Prevotella copri presented activity comparable to the commonly used antibiotic polymyxin B. Our report supports the existence of hundreds of antimicrobials in the human microbiome amenable to clinical translation.

We previously reported that a linear cationic 12-amino acid cell-penetrating peptide (CPP) was bactericidal for Neisseria gonorrhoeae. In this study, our objectives were to determine the effect of cyclization of the linear CPP on its antibacterial activity for N. gonorrhoeae and cytotoxicity for human cells. We compared the bactericidal effect of 4-hour treatment with the linear CPP to that of CPPs cyclized by a thioether or a disulfide bond on human challenge and multi-drug resistant (MDR) strains of N. gonorrhoeae grown in cell culture media with 10% fetal bovine serum (FBS). The effect of lipooligosaccharide (LOS) sialylation on bactericidal activity was analyzed. We determined the ability of the CPPs to treat human cells infected in vitro with N. gonorrhoeae, to reduce the inflammatory response of human monocytic cells to gonococci, to kill strains of three commensal Neisseria species, and to inhibit gonococcal biofilms. The cyclized CPPs killed 100% of gonococci from all strains at 100 µM and >90% at 20 µM and were more potent than the linear form. The thioether-linked but not the disulfide-linked CPP was less cytotoxic for human cervical cells compared to the linear CPP. LOS sialylation had minimal effect on bactericidal activity. In treating infected human cells, the thioether-linked CPP at 20 µM killed >60% of extra- and intracellular bacteria and reduced TNF-α expression by THP-1 cells. The potency of the CPPs for the pathogenic and the commensal Neisseria was similar. The thioether-linked CPP partially eradicated gonococcal biofilms. Future studies will focus on determining efficacy in the female mouse model of gonorrhea.IMPORTANCENeisseria gonorrhoeae remains a major cause of sexually transmitted infections with 82 million cases worldwide in 2020, and 710,151 confirmed cases in the US in 2021, up 25% from 2017. N. gonorrhoeae can infect multiple tissues including the urethra, cervix, rectum, pharynx, and conjunctiva. The most serious sequelae are suffered by infected women as gonococci ascend to the upper reproductive tract and cause pelvic inflammatory disease, chronic pelvic pain, and infertility in 10%-20% of women. Control of gonococcal infection is widely recognized as increasingly challenging due to the lack of any vaccine. N. gonorrhoeae has quickly developed resistance to all but one class of antibiotics and the emergence of multidrug-resistant strains could result in untreatable infections. As such, gonorrhea is classified by the Center for Disease Control (CDC) as an urgent public health threat. The research presented herein on new therapeutics for gonorrhea has identified a cyclic cell-penetrating peptide (CPP) as a potent molecule targeting N. gonorrhoeae.

Multiple peptide resistance factor (MprF) confers resistance to cationic antimicrobial peptides (AMPs) in several pathogens, thereby enabling evasion of the host immune response. The role of MprF in commensals remains, however, uncharacterized. To close this knowledge gap, we used a common gut commensal of animals, Lactiplantibacillus plantarum, and its natural host, the fruit fly Drosophila melanogaster, as an experimental model to investigate the role of MprF in commensal-host interactions. The L. plantarum ΔmprF mutant that we generated exhibited deficiency in the synthesis of lysyl-phosphatidylglycerol (Lys-PG), resulting in increased negative cell surface charge and increased susceptibility to AMPs. Susceptibility to AMPs had no effect on ΔmprF mutant's ability to colonize guts of uninfected flies. However, we observed significantly reduced abundance of the ΔmprF mutant after infection-induced inflammation in the guts of wild-type flies but not of flies lacking AMPs. Additionally, we found that the ΔmprF mutant compared to wild-type L. plantarum induces a stronger intestinal immune response in flies due to the increased release of immunostimulatory peptidoglycan fragments, indicating an important role of MprF in promoting host tolerance to commensals. Our further analysis suggests that MprF-mediated lipoteichoic acid modifications are involved in host immunomodulation. Overall, our results demonstrate that MprF, besides its well-characterized role in pathogen immune evasion and virulence, is also an important commensal resilience factor.

Novel antibiotics are urgently needed to combat the antibiotic-resistance crisis. We present a machine-learning-based approach to predict antimicrobial peptides (AMPs) within the global microbiome and leverage a vast dataset of 63,410 metagenomes and 87,920 prokaryotic genomes from environmental and host-associated habitats to create the AMPSphere, a comprehensive catalog comprising 863,498 non-redundant peptides, few of which match existing databases. AMPSphere provides insights into the evolutionary origins of peptides, including by duplication or gene truncation of longer sequences, and we observed that AMP production varies by habitat. To validate our predictions, we synthesized and tested 100 AMPs against clinically relevant drug-resistant pathogens and human gut commensals both in vitro and in vivo. A total of 79 peptides were active, with 63 targeting pathogens. These active AMPs exhibited antibacterial activity by disrupting bacterial membranes. In conclusion, our approach identified nearly one million prokaryotic AMP sequences, an open-access resource for antibiotic discovery.

Rheumatoid arthritis (RA) is a chronic, autoimmune rheumatic disease characterized by synovial joint inflammation with subsequent destruction as well as systemic manifestation, leading to impaired mobility and impaired quality of life. The etiopathogenesis of RA is still unknown, with genetic, epigenetic and environmental factors (incl. tobacco smoking) contributing to disease susceptibility. The link between genetic factors like "shared epitope alleles" and the development of RA is well known. However, why only some carriers have a break in self-tolerance and develop autoimmunity still needs to be clarified. The presence of autoantibodies in patients' serum months to years prior to the onset of clinical manifestations of RA has moved the focus to possible epigenetic factors, including environmental triggers that could contribute to the initiation and perpetuation of the inflammatory reaction in RA. Over the past several years, the role of microorganisms at mucosal sites (i.e., microbiome) has emerged as an essential mediator of inflammation in RA. An increasing number of studies have revealed the microbial role in the immunopathogenesis of autoimmune rheumatic diseases. Interaction between the host immune system and microbiota initiates loss of immunological tolerance and autoimmunity. The alteration in microbiome composition, the so-called dysbiosis, is associated with an increasing number of diseases. Immune dysfunction caused by dysbiosis triggers and sustains chronic inflammation. This review aims to provide a critical summary of the literature findings related to the hypothesis of a reciprocal relation between the microbiome and the immune system. Available data from studies reveal the pivotal role of the microbiome in RA pathogenesis.

Molecular de-extinction aims at resurrecting molecules to solve antibiotic resistance and other present-day biological and biomedical problems. Here we show that deep learning can be used to mine the proteomes of all available extinct organisms for the discovery of antibiotic peptides. We trained ensembles of deep-learning models consisting of a peptide-sequence encoder coupled with neural networks for the prediction of antimicrobial activity and used it to mine 10,311,899 peptides. The models predicted 37,176 sequences with broad-spectrum antimicrobial activity, 11,035 of which were not found in extant organisms. We synthesized 69 peptides and experimentally confirmed their activity against bacterial pathogens. Most peptides killed bacteria by depolarizing their cytoplasmic membrane, contrary to known antimicrobial peptides, which tend to target the outer membrane. Notably, lead compounds (including mammuthusin-2 from the woolly mammoth, elephasin-2 from the straight-tusked elephant, hydrodamin-1 from the ancient sea cow, mylodonin-2 from the giant sloth and megalocerin-1 from the extinct giant elk) showed anti-infective activity in mice with skin abscess or thigh infections. Molecular de-extinction aided by deep learning may accelerate the discovery of therapeutic molecules.

The gastrointestinal (GI) tract and the brain form bidirectional nervous, immune, and endocrine communications known as the gut-brain axis. Several factors can affect this axis; among them, various studies have focused on the microbiota and imply that alterations in microbiota combinations can influence both the brain and GI. Also, many studies have shown that the immune system has a vital role in varying gut microbiota combinations. In the current paper, we will review the multidirectional effects of gut microbiota, immune system, and nervous system on each other. Specifically, this review mainly focuses on the impact of Peyer's patches as a critical component of the gut immune system on the gut-brain axis through affecting the gut's microbial composition. In this way, some factors were discussed as proposed elements of missing gaps in this field.

The spatial organization of gut microbiota is crucial for the functioning of the gut ecosystem, although the mechanisms that organize gut bacterial communities in microhabitats are only partially understood. The gut of the insect Riptortus pedestris has a characteristic microbiota biogeography with a multispecies community in the anterior midgut and a monospecific bacterial population in the posterior midgut. We show that the posterior midgut region produces massively hundreds of specific antimicrobial peptides (AMPs), the Crypt-specific Cysteine-Rich peptides (CCRs) that have membrane-damaging antimicrobial activity against diverse bacteria but posterior midgut symbionts have elevated resistance. We determined by transposon-sequencing the genetic repertoire in the symbiont Caballeronia insecticola to manage CCR stress, identifying different independent pathways, including AMP-resistance pathways unrelated to known membrane homeostasis functions as well as cell envelope functions. Mutants in the corresponding genes have reduced capacity to colonize the posterior midgut, demonstrating that CCRs create a selective barrier and resistance is crucial in gut symbionts. Moreover, once established in the gut, the bacteria differentiate into a CCR-sensitive state, suggesting a second function of the CCR peptide arsenal in protecting the gut epithelia or mediating metabolic exchanges between the host and the gut symbionts. Our study highlights the evolution of an extreme diverse AMP family that likely contributes to establish and control the gut microbiota.

Respiratory syncytial virus is the major cause of acute lower respiratory tract infections in young children, causing extensive mortality and morbidity globally, with limited therapeutic or preventative options. Cathelicidins are innate immune antimicrobial host defence peptides and have antiviral activity against RSV. However, upper respiratory tract cathelicidin expression and the relationship with host and environment factors in early life, are unknown. Infant cohorts were analysed to characterise early life nasal cathelicidin levels, revealing low expression levels in the first week of life, with increased levels at 9 months which are comparable to 2-year-olds and healthy adults. No impact of prematurity on nasal cathelicidin expression was observed, nor were there effects of sex or birth mode, however, nasal cathelicidin expression was lower in the first week-of-life in winter births. Nasal cathelicidin levels were positively associated with specific inflammatory markers and demonstrated to be associated with microbial community composition. Importantly, levels of nasal cathelicidin expression were elevated in infants with mild RSV infection, but, in contrast, were not upregulated in infants hospitalised with severe RSV infection. These data suggest important relationships between nasal cathelicidin, upper airway microbiota, inflammation, and immunity against RSV infection, with interventional potential.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are located in the outer membrane of Gram-negative bacteria and are comprised of three distinctive parts: lipid A, core oligosaccharide (OS), and O-antigen. The structure of each region influences bacterial stability, toxicity, and pathogenesis. Here, we highlight the use of targeted activated-electron photodetachment (a-EPD) tandem mass spectrometry to characterize LPS and LOS from two crucial players in the human gut microbiota, Escherichia coli Nissle and Bacteroides fragilis. a-EPD is a hybrid activation method that uses ultraviolet photoirradiation to generate charge-reduced radical ions followed by collisional activation to produce informative fragmentation patterns. We benchmark the a-EPD method for top-down characterization of triacyl LOS from E. coli R2, then focus on characterization of LPS from E. coli Nissle and B. fragilis. Notably, a-EPD affords extensive fragmentation throughout the backbone of the core OS and O-antigen regions of LPS from E. coli Nissle. This hybrid approach facilitated the elucidation of structural details for LPS from B. fragilis, revealing a putative hexuronic acid (HexA) conjugated to lipid A.

Mammalian α-defensins are a family of abundant effector peptides of the mucosal innate immune system. Although primarily considered to be antimicrobial, α-defensins can increase rather than block infection by certain prominent bacterial and viral pathogens in cell culture and in vivo. We have shown previously that exposure of mouse and human adenoviruses (HAdVs) to α-defensins is able to overcome competitive inhibitors that block cell binding, leading us to hypothesize a defensin-mediated binding mechanism that is independent of known viral receptors. To test this hypothesis, we used genetic approaches to demonstrate that none of several primary receptors nor integrin co-receptors are needed for human α-defensin-mediated binding of HAdV to cells; however, infection remains integrin dependent. Thus, our studies have revealed a novel pathway for HAdV binding to cells that bypasses viral primary receptors. We speculate that this pathway functions in parallel with receptor-mediated entry and contributes to α-defensin-enhanced infection of susceptible cells. Remarkably, we also found that in the presence of α-defensins, HAdV tropism is expanded to non-susceptible cells, even when viruses are exposed to a mixture of both susceptible and non-susceptible cells. Therefore, we propose that in the presence of sufficient concentrations of α-defensins, such as in the lung or gut, integrin expression rather than primary receptor expression will dictate HAdV tropism in vivo. In summary, α-defensins may contribute to tissue tropism not only through the neutralization of susceptible viruses but also by allowing certain defensin-resistant viruses to bind to cells independently of previously described mechanisms.

Mammalian α-defensins are a family of abundant effector peptides of the mucosal innate immune system. Although primarily considered to be antimicrobial, α-defensins can increase rather than block infection by certain prominent bacterial and viral pathogens in cell culture and in vivo . We have shown previously that exposure of mouse and human adenoviruses (HAdVs) to α-defensins is able to overcome competitive inhibitors that block cell binding, leading us to hypothesize a defensin-mediated binding mechanism that is independent of known viral receptors. To test this hypothesis, we used genetic approaches to demonstrate that none of several primary receptors nor integrin co-receptors are needed for human α-defensin-mediated binding of HAdV to cells; however, infection remains integrin dependent. Thus, our studies have revealed a novel pathway for HAdV binding to cells that bypasses viral primary receptors. We speculate that this pathway functions in parallel with receptor-mediated entry and contributes to α-defensin-enhanced infection of susceptible cells. Remarkably, we also found that in the presence of α-defensins, HAdV tropism is expanded to non-susceptible cells, even when viruses are exposed to a mixture of both susceptible and non-susceptible cells. Therefore, we propose that in the presence of sufficient concentrations of α-defensins, such as in the lung or gut, integrin expression rather than primary receptor expression will dictate HAdV tropism in vivo . In summary, α-defensins may contribute to tissue tropism not only through the neutralization of susceptible viruses but also by allowing certain defensin-resistant viruses to bind to cells independently of previously described mechanisms.

In this study, we demonstrate a novel mechanism for binding of human adenoviruses (HAdVs) to cells that is dependent upon interactions with α-defensin host defense peptides but is independent of known viral receptors and co-receptors. To block normal receptor-mediated HAdV infection, we made genetic changes to both host cells and HAdVs. Under these conditions, α-defensins restored cell binding; however, infection still required the function of HAdV integrin co-receptors. This was true for multiple types of HAdVs that use different primary receptors and for cells that are either naturally devoid of HAdV receptors or were engineered to be receptor deficient. These observations suggest that in the presence of concentrations of α-defensins that would be found naturally in the lung or intestine, there are two parallel pathways for HAdV binding to cells that converge on integrins for productive infection. Moreover, these binding pathways function independently, and both operate in mixed culture. Thus, we have found that viruses can co-opt host defense molecules to expand their tropism.

Encrypted peptides have been recently described as a new class of antimicrobial molecules. They have been proposed to play a role in host immunity and as alternatives to conventional antibiotics. Intriguingly, many of these peptides are found embedded in proteins unrelated to the immune system, suggesting that immunological responses may extend beyond traditional host immunity proteins. To test this idea, here we synthesized and tested representative peptides derived from non-immune proteins for their ability to exert antimicrobial and immunomodulatory properties. Our experiments revealed that most of the tested peptides from non-immune proteins, derived from structural proteins as well as proteins from the nervous and visual systems, displayed potent in vitro antimicrobial activity. These molecules killed bacterial pathogens by targeting their membrane, and those originating from the same region of the body exhibited synergistic effects when combined. Beyond their antimicrobial properties, nearly 90% of the peptides tested exhibited immunomodulatory effects, modulating inflammatory mediators such as IL-6, TNF-α, and MCP-1. Moreover, eight of the peptides identified, collagenin 3 and 4, zipperin-1 and 2, and immunosin-2, 3, 12, and 13, displayed anti-infective efficacy in two different preclinical mouse models, reducing bacterial infections by up to four orders of magnitude. Altogether, our results support the hypothesis that peptides from non-immune proteins may play a role in host immunity. These results potentially expand our notion of the immune system to include previously unrecognized proteins and peptides that may be activated upon infection to confer protection to the host.

The limited existing knowledge regarding resistance to antimicrobial peptides (AMPs) is hindering their broad utilization. The aim of this study is to enhance the understanding of AMP resistance, a pivotal factor in the exploration of alternative drug development in response to the escalating challenge of antibiotic resistance.

We utilized metagenomic functional selection to analyze genes resistant to AMPs, with a specific focus on the microbiota in soil and the human gut. Through a combination of experimental methods and bioinformatics analyses, our investigation delved into the possibilities of the evolution of resistance to AMPs, as well as the transfer or interchange of resistance genes among the environment, the human body, and pathogens. Additionally, we examined the cross-resistance between AMPs and evaluated interactions among AMPs and conventional antibiotics.

The presence of AMP resistance, including various resistance mechanisms, was observed in both soil and the human gut microbiota, as indicated by our findings. Significantly, the study underscored the facile evolution of AMP resistance and the potential for gene sharing or exchange among different environments. Notably, cross-resistance among AMPs was identified as a phenomenon, while cross-resistance between AMPs and antibiotics was found to be relatively infrequent.

The results of our study highlight the significance of taking a cautious stance when considering the extensive application of AMPs. It is imperative to thoroughly assess potential resistance risks, with a particular focus on the development of resistance to AMPs across diverse domains. A comprehensive grasp of these aspects is essential for making well-informed decisions and ensuring the responsible utilization of AMPs in the ongoing fight against antibiotic resistance.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract that results from dysfunction in innate and/or adaptive immune responses. Impaired innate immunity, which leads to lack of control of an altered intestinal microbiota and to activation of the adaptive immune system, promotes a secondary inflammatory response that is responsible for tissue damage. Neutrophils are key players in innate immunity in IBD, but their roles have been neglected compared with those of other immune cells. The latest studies on neutrophils in IBD have revealed unexpected complexities, with heterogeneous populations and dual functions, both deleterious and protective, for the host. In parallel, interconnections between disease development, intestinal microbiota and neutrophils have been highlighted. Numerous IBD susceptibility genes (such as NOD2, NCF4, LRRK2, CARD9) are involved in neutrophil functions related to defence against microorganisms. Moreover, severe monogenic diseases involving dysfunctional neutrophils, including chronic granulomatous disease, are characterized by intestinal inflammation that mimics IBD and by alterations in the intestinal microbiota. This observation demonstrates the dialogue between neutrophils, gut inflammation and the microbiota. Neutrophils affect microbiota composition and function in several ways. In return, microbial factors, including metabolites, regulate neutrophil production and function directly and indirectly. It is crucial to further investigate the diverse roles played by neutrophils in host-microbiota interactions, both at steady state and in inflammatory conditions, to develop new IBD therapies. In this Review, we discuss the roles of neutrophils in IBD, in light of emerging evidence proving strong interconnections between neutrophils and the gut microbiota, especially in an inflammatory context.

The genus Bacteroides, a predominant group in the human gut microbiome, presents significant potential for microbiome engineering and the development of live biotherapeutics aimed at treating gut diseases. Despite its promising capabilities, tools for effectively engineering Bacteroides species have been limited. In our study, we have made a breakthrough by identifying novel signal peptides in Bacteroides thetaiotaomicron and Akkermansia muciniphila. These peptides facilitate efficient protein transport across cellular membranes in Bacteroides, a critical step for therapeutic applications. Additionally, we have developed an advanced episomal plasmid system. This system demonstrates superior protein secretion capabilities compared to traditional chromosomal integration plasmids, making it a vital tool for enhancing the delivery of therapeutic proteins in Bacteroides species. Initially, the stability of this episomal plasmid posed a challenge; however, we have overcome this by incorporating an essential gene-based selection system. This novel strategy not only ensures plasmid stability but also aligns with the growing need for antibiotic-free selection methods in clinical settings. Our work, therefore, not only provides a more robust secretion system for Bacteroides but also sets a new standard for the development of live biotherapeutics.

The critical role of the intestinal microbiota in human health and disease is well recognized. Nevertheless, there are still large gaps in our understanding of the functions and mechanisms encoded in the genomes of most members of the gut microbiota. Genome-scale libraries of transposon mutants are a powerful tool to help us address this gap. Recent advances in barcoded transposon mutagenesis have dramatically lowered the cost of mutant fitness determination in hundreds of in vitro and in vivo experimental conditions. In an accompanying review, we discuss recent advances and caveats for the construction of pooled and arrayed barcoded transposon mutant libraries in human gut commensals. In this review, we discuss how these libraries can be used across a wide range of applications, the technical aspects involved, and expectations for such screens.

Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes. We discuss the advantages and limitations of each approach, as well as factors to consider when selecting a strategy. In addition, we highlight experimental and computational advances in arraying condensed libraries from mutant pools. We focus on examples of successful library construction in gut bacteria and their application to gene function studies and drug discovery. Given the need for understanding gene function and organization in gut bacteria, we provide a comprehensive guide for researchers to construct randomly barcoded transposon mutant libraries.

The infant gut microbiome is impacted by early-life feeding, as human milk oligosaccharides (HMOs) found in breastmilk cannot be digested by infants and serve as nutrients for their gut bacteria. While the vast majority of HMO-utilization research has focused on Bifidobacterium species, recent studies have suggested additional HMO-utilizers, mostly Bacteroides, yet their utilization mechanism is poorly characterized. Here, we investigate Bacteroides dorei isolates from breastfed-infants and identify that polysaccharide utilization locus (PUL) 33 enables B. dorei to utilize sialylated HMOs. We perform transcriptional profiling and identity upregulated genes when growing on sialylated HMOs. Using CRISPR-Cas12 to knock-out four PUL33 genes, combined with complementation assays, we identify GH33 as the critical gene in PUL33 for sialylated HMO-utilization. This demonstration of an HMO-utilization system by Bacteroides species isolated from infants opens the way to further characterization of additional such systems, to better understand HMO-utilization in the infant gut.

Antimicrobial peptides/proteins (AMPs) constitute a critical component of gut immunity in animals, protecting the gut from pathogenic bacteria. However, the interactions between AMPs and gut microbiota remain elusive. In this study, we show that leukocyte-derived chemotaxin-2 (LECT2)-b, a recently discovered AMP, helps maintain gut homeostasis in grass carp (Ctenopharyngodon idella), one of the major farmed fish species globally, by directly regulating the gut microbiota. Knockdown of LECT2-b resulted in dysregulation of the gut microbiota. Specifically, LECT2-b deficiency led to the dominance of Proteobacteria, consisting of proinflammatory bacterial species, over Firmicutes, which includes anti-inflammatory bacteria. In addition, the opportunistic pathogenic bacteria genus Aeromonas became the dominant genus replacing the probiotic bacteria Lactobacillus and Bacillus. Further analysis revealed that this effect was due to the direct and selective inhibition of certain pathogenic bacterial species by LECT2-b. Moreover, LECT2-b knockdown promoted biofilm formation by gut microbiota, resulting in tissue damage and inflammation. Importantly, LECT2-b treatment alleviated the negative effects induced by LECT2-b knockdown. These findings highlight the crucial role of LECT2-b in maintaining the gut microbiota homeostasis and mucosal health. Overall, our study provides important data for understanding the roles of AMPs in the regulation of gut homeostasis in animals.

Mammals do not eat continuously, instead concentrating their feeding to a restricted portion of the day. This behavior presents the mammalian gut microbiota with a fluctuating environment with consequences for host-microbiome interaction, infection risk, immune response, drug metabolism, and other aspects of health. We demonstrate that in mice, gut microbes elevate levels of an intracellular signaling molecule, (p)ppGpp, during the fasting phase of a time-restricted feeding regimen. Disabling this response in a representative human gut commensal species significantly reduces colonization during this host-fasting phase. This response appears to be general across species and conserved across mammalian gut communities, highlighting a pathway that allows healthy gut microbiomes to maintain stability in an unstable environment.

Due to huge diversity and dynamic competition, the human gut microbiome produces a diverse array of antimicrobial peptides (AMPs) that play an important role in human health. The gut microbiome has an important role in maintaining gut homeostasis by the AMPs and by interacting with other human organs via established connections such as the gut-lung, and gut-brain axis. Additionally, gut AMPs play a synergistic role with other gut microbiota and antimicrobials to maintain gut homeostasis by fighting against multi-antibiotic resistance (MAR) bacteria. Further, conventional antibiotics intake creates a synergistic evolutionary pressure for gut AMPs, where antibiotics and gut AMPs fight synergistically against MAR. Overall, gut AMPs are evolving under a complex and highly synergistic co-evolutionary pressure created by the various interactions between gut microbiota, gut AMPs, and antibiotics; however, the complete mechanism is not well understood. The current review explores the synergistic action of gut AMPs and antibiotics along with possibilities to fight against MAR bacteria.

Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.

The past decade has seen growing interest in bacterial engineering for therapeutically relevant applications. While early efforts focused on repurposing genetically tractable model strains, such as Escherichia coli, engineering gut commensals is gaining traction owing to their innate capacity to survive and stably propagate in the intestine for an extended duration. Although limited genetic tractability has been a major roadblock, recent advances in systems and synthetic biology have unlocked our ability to effectively harness native gut commensals for therapeutic and diagnostic purposes, ranging from the rational design of synthetic microbial consortia to the construction of synthetic cells that execute "sense-and-respond" logic operations that allow real-time detection and therapeutic payload delivery in response to specific signals in the intestine. In this review, we outline the current progress and latest updates on microbial therapeutics, with particular emphasis on gut commensal engineering driven by synthetic biology and systems understanding of their molecular phenotypes. Finally, the challenges and prospects of engineering gut commensals for therapeutic applications are discussed.

During intestinal inflammation, host nutritional immunity starves microbes of essential micronutrients, such as iron. Pathogens scavenge iron using siderophores, including enterobactin; however, this strategy is counteracted by host protein lipocalin-2, which sequesters iron-laden enterobactin. Although this iron competition occurs in the presence of gut bacteria, the roles of commensals in nutritional immunity involving iron remain unexplored. Here, we report that the gut commensal Bacteroides thetaiotaomicron acquires iron and sustains its resilience in the inflamed gut by utilizing siderophores produced by other bacteria, including Salmonella, via a secreted siderophore-binding lipoprotein XusB. Notably, XusB-bound enterobactin is less accessible to host sequestration by lipocalin-2 but can be "re-acquired" by Salmonella, allowing the pathogen to evade nutritional immunity. Because the host and pathogen have been the focus of studies of nutritional immunity, this work adds commensal iron metabolism as a previously unrecognized mechanism modulating the host-pathogen interactions and nutritional immunity.