How does the microbiota control systemic innate immunity?

The intestinal microbiota has a pervasive influence on mammalian innate immunity fortifying defenses to infection in tissues throughout the host. How intestinal microbes control innate defenses in systemic tissues is, however, poorly defined. In our opinion, there are three core challenges that need addressing to advance our understanding of how the intestinal microbiota controls innate immunity systemically: first, deciphering how signals from intestinal microbes are transmitted to distal tissues; second, unraveling how intestinal microbes prime systemic innate immunity without inducing widespread immunopathology; and third, identifying which intestinal microbes control systemic immunity. Here, we propose answers to these problems which provide a framework for understanding how microbes in the intestine can regulate innate immunity systemically.

Symbiotic Firmicutes establish mutualism with the host via innate tolerance and resistance to control systemic immunity.

The intestinal microbiota regulates immunity across organ systems. Which symbionts control systemic immunity, the mechanisms they use, and how they avoid widespread inflammatory damage are unclear. We uncover host tolerance and resistance mechanisms that allow Firmicutes from the human microbiota to control systemic immunity without inducing immunopathology. Intestinal processing releases Firmicute glycoconjugates that disseminate, resulting in release of cytokine IL-34 that stimulates macrophages and enhances defenses against pneumonia, sepsis, and meningitis. Despite systemic penetration of Firmicutes, immune homeostasis is maintained through feedback control whereby IL-34-mediated mTORC1 activation in macrophages clears polymeric glycoconjugates from peripheral tissues. Smaller glycoconjugates evading this clearance mechanism are tolerated through sequestration by albumin, which acts as an inflammatory buffer constraining their immunological impact. Without these resistance and tolerance mechanisms, Firmicutes drive catastrophic organ damage and cachexia via IL-1ß. This reveals how Firmicutes are safely assimilated into systemic immunity to protect against infection without threatening host viability.

Antibiotics promote intestinal growth of carbapenem-resistant Enterobacteriaceae by enriching nutrients and depleting microbial metabolites.

The intestine is the primary colonisation site for carbapenem-resistant Enterobacteriaceae (CRE) and serves as a reservoir of CRE that cause invasive infections (e.g. bloodstream infections). Broad-spectrum antibiotics disrupt colonisation resistance mediated by the gut microbiota, promoting the expansion of CRE within the intestine. Here, we show that antibiotic-induced reduction of gut microbial populations leads to an enrichment of nutrients and depletion of inhibitory metabolites, which enhances CRE growth. Antibiotics decrease the abundance of gut commensals (including Bifidobacteriaceae and Bacteroidales) in ex vivo cultures of human faecal microbiota; this is accompanied by depletion of microbial metabolites and enrichment of nutrients. We measure the nutrient utilisation abilities, nutrient preferences, and metabolite inhibition susceptibilities of several CRE strains. We find that CRE can use the nutrients (enriched after antibiotic treatment) as carbon and nitrogen sources for growth. These nutrients also increase in faeces from antibiotic-treated mice and decrease following intestinal colonisation with carbapenem-resistant Escherichia coli. Furthermore, certain microbial metabolites (depleted upon antibiotic treatment) inhibit CRE growth. Our results show that killing gut commensals with antibiotics facilitates CRE colonisation by enriching nutrients and depleting inhibitory microbial metabolites.

Correction: Abiotic microcompartments form when neighbouring droplets fuse: an electrochemiluminescence investigation.

[This corrects the article DOI: 10.1039/D2SC06553C.].

Abiotic microcompartments form when neighbouring droplets fuse: an electrochemiluminescence investigation.

Many studies have shown chemistry proceeds differently in small volumes compared to bulk phases. However, few studies exist elucidating spontaneous means by which small volumes can form in Nature. Such studies are critical in understanding the formation of life in microcompartments. In this study, we track in real-time the coalescence of two or more water microdroplets adsorbed on an electrified surface in a 1,2-dichloroethane continuous phase by electrogenerated chemiluminescence (ECL) imaging, uncovering the spontaneous generation of multiple emulsions inside the resulting water droplets. During the fusion of adsorbed water droplets with each other on the electrode surface, volumes of organic and water phases are entrapped in between and detected respectively as ECL not-emitting and emitting regions. The diameter of those confined environments inside the water droplets can be less than a micrometer, as described by scanning electron microscopy data. This study adds a new mechanism for the generation of micro- and nano-emulsions and provides insight into confinement techniques under abiotic conditions as well as new potential strategies in microfluidic devices.

Interfacial solute flux promotes emulsification at the water|oil interface.

Emulsions are critical across a broad spectrum of industries. Unfortunately, emulsification requires a significant driving force for droplet dispersion. Here, we demonstrate a mechanism of spontaneous droplet formation (emulsification), where the interfacial solute flux promotes droplet formation at the liquid-liquid interface when a phase transfer agent is present. We have termed this phenomenon fluxification. For example, when HAuCl4 is dissolved in an aqueous phase and [NBu4][ClO4] is dissolved in an oil phase, emulsion droplets (both water-in-oil and oil-in-water) can be observed at the interface for various oil phases (1,2-dichloroethane, dichloromethane, chloroform, and nitrobenzene). Emulsification occurs when AuCl4- interacts with NBu4+, a well-known phase-transfer agent, and transfers into the oil phase while ClO4- transfers into the aqueous phase to maintain electroneutrality. The phase transfer of SCN- and Fe(CN)63- also produce droplets. We propose a microscopic mechanism of droplet formation and discuss design principles by tuning experimental parameters.

Preferential Electroreduction at the Oil|Water|Conductor Interface.

Chemistry in confined volumes, such as aqueous droplets, is different from bulk, continuous water. However, few techniques are available to probe interfacial reactivity in complex, multiphase environments. Here, we demonstrate preferential electroreduction at the oil|water|conductor (three-phase) interface. Electrodeposition of cobalt and nickel results in ringlike structures that can be characterized with tens of nanometers precision in scanning electron microscopy and energy dispersive X-ray spectroscopy. To demonstrate the generalizability of these observations, we show that electroreduction of resazurin to fluorescent resorufin occurs preferentially at the three-phase boundary. The preferential electroreduction does not depend on droplet geometry. These results, grounded in three-phase boundary reactivity, are highly important across all fields of chemistry and biology because they highlight how the interface can change chemistry in unexpected ways.

Airway mucins promote immunopathology in virus-exacerbated chronic obstructive pulmonary disease.

The respiratory tract surface is protected from inhaled pathogens by a secreted layer of mucus rich in mucin glycoproteins. Abnormal mucus accumulation is a cardinal feature of chronic respiratory diseases, but the relationship between mucus and pathogens during exacerbations is poorly understood. We identified elevations in airway mucin 5AC (MUC5AC) and MUC5B concentrations during spontaneous and experimentally induced chronic obstructive pulmonary disease (COPD) exacerbations. MUC5AC was more sensitive to changes in expression during exacerbation and was therefore more predictably associated with viral load, inflammation, symptom severity, decrements in lung function, and secondary bacterial infections. MUC5AC was functionally related to inflammation, as Muc5ac-deficient (Muc5ac-/-) mice had attenuated RV-induced (RV-induced) airway inflammation, and exogenous MUC5AC glycoprotein administration augmented inflammatory responses and increased the release of extracellular adenosine triphosphate (ATP) in mice and human airway epithelial cell cultures. Hydrolysis of ATP suppressed MUC5AC augmentation of RV-induced inflammation in mice. Therapeutic suppression of mucin production using an EGFR antagonist ameliorated immunopathology in a mouse COPD exacerbation model. The coordinated virus induction of MUC5AC and MUC5B expression suggests that non-Th2 mechanisms trigger mucin hypersecretion during exacerbations. Our data identified a proinflammatory role for MUC5AC during viral infection and suggest that MUC5AC inhibition may ameliorate COPD exacerbations.

Microbiota-mediated protection against antibiotic-resistant pathogens.

Colonization by the microbiota provides one of our most effective barriers against infection by pathogenic microbes. The microbiota protects against infection by priming immune defenses, by metabolic exclusion of pathogens from their preferred niches, and through direct antimicrobial antagonism. Disruption of the microbiota, especially by antibiotics, is a major risk factor for bacterial pathogen colonization. Restoration of the microbiota through microbiota transplantation has been shown to be an effective way to reduce pathogen burden in the intestine but comes with a number of drawbacks, including the possibility of transferring other pathogens into the host, lack of standardization, and potential disruption to host metabolism. More refined methods to exploit the power of the microbiota would allow us to utilize its protective power without the drawbacks of fecal microbiota transplantation. To achieve this requires detailed understanding of which members of the microbiota protect against specific pathogens and the mechanistic basis for their effects. In this review, we will discuss the clinical and experimental evidence that has begun to reveal which members of the microbiota protect against some of the most troublesome antibiotic-resistant pathogens: Klebsiella pneumoniae, vancomycin-resistant enterococci, and Clostridioides difficile.

Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane.

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

Antibiotics are life-saving medicines, but many bacteria now have the ability to resist their effects. For some infections, all frontline antibiotics are now ineffective. To treat infections caused by these highly resistant bacteria, clinicians must use so-called 'antibiotics of last resort'. These antibiotics include a drug called colistin, which is moderately effective, but often fails to eradicate the infection. One of the challenges to making colistin more effective is that its mechanism is poorly understood. Bacteria have two layers of protection against the outside world: an outer cell membrane and an inner cell membrane. To kill them, colistin must punch holes in both. First, it disrupts the outer membrane by interacting with molecules called lipopolysaccharides. But how it disrupts the inner membrane was unclear. Bacteria have evolved several different mechanisms that make them resistant to the effects of colistin. Sabnis et al. reasoned that understanding how these mechanisms protected bacteria could reveal how the antibiotic works to damage the inner cell membrane. Sabnis et al. examined the effects of colistin on Escherichia coli bacteria with and without resistance to the antibiotic. Exposing these bacteria to colistin revealed that the antibiotic damages both layers of the cell surface in the same way, targeting lipopolysaccharide in the inner membrane as well as the outer membrane. Next, Sabnis et al. used this new information to make colistin work better. They found that the effects of colistin were magnified when it was combined with the experimental antibiotic murepavadin, which caused lipopolysaccharide to build up at the inner membrane. This allowed colistin to punch more holes through the inner membrane, making colistin more effective at killing bacteria. To find out whether this combination of colistin and murepavadin could work as a clinical treatment, Sabnis et al. tested it on mice with Pseudomonas aeruginosa infections in their lungs. Colistin was much better at killing Pseudomonas aeruginosa and treating infections when combined with murepavadin than it was on its own. Pseudomonas aeruginosa bacteria can cause infections in the lungs of people with cystic fibrosis. At the moment, patients receive colistin in an inhaled form to treat these infections, but it is not always successful. The second drug used in this study, murepavadin, is about to enter clinical trials as an inhaled treatment for lung infections too. If the trial is successful, it may be possible to use both drugs in combination to treat lung infections in people with cystic fibrosis.

Immunological design of commensal communities to treat intestinal infection and inflammation.

The immunological impact of individual commensal species within the microbiota is poorly understood limiting the use of commensals to treat disease. Here, we systematically profile the immunological fingerprint of commensals from the major phyla in the human intestine (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria) to reveal taxonomic patterns in immune activation and use this information to rationally design commensal communities to enhance antibacterial defenses and combat intestinal inflammation. We reveal that Bacteroidetes and Firmicutes have distinct effects on intestinal immunity by differentially inducing primary and secondary response genes. Within these phyla, the immunostimulatory capacity of commensals from the Bacteroidia class (Bacteroidetes phyla) reflects their robustness of TLR4 activation and Bacteroidia communities rely solely on this receptor for their effects on intestinal immunity. By contrast, within the Clostridia class (Firmicutes phyla) it reflects the degree of TLR2 and TLR4 activation, and communities of Clostridia signal via both of these receptors to exert their effects on intestinal immunity. By analyzing the receptors, intracellular signaling components and transcription factors that are engaged by different commensal species, we identify canonical NF-κB signaling as a critical rheostat which grades the degree of immune stimulation commensals elicit. Guided by this immunological analysis, we constructed a cross-phylum consortium of commensals (Bacteroides uniformis, Bacteroides ovatus, Peptostreptococcus anaerobius and Clostridium histolyticum) which enhances innate TLR, IL6 and macrophages-dependent defenses against intestinal colonization by vancomycin resistant Enterococci, and fortifies mucosal barrier function during pathological intestinal inflammation through the same pathway. Critically, the setpoint of intestinal immunity established by this consortium is calibrated by canonical NF-κB signaling. Thus, by profiling the immunological impact of major human commensal species our work paves the way for rational microbiota reengineering to protect against antibiotic resistant infections and to treat intestinal inflammation.

Staphylococcal DNA Repair Is Required for Infection.

To cause infection, Staphylococcus aureus must withstand damage caused by host immune defenses. However, the mechanisms by which staphylococcal DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as being important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double-strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double-strand breaks through reactive oxygen species (ROS) generated by the respiratory burst, which are repaired by RexAB, leading to the induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted the survival of these pathogens in human blood, suggesting that DNA double-strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that the repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.IMPORTANCE To cause infection, bacteria must survive attack by the host immune system. For many bacteria, including the major human pathogen Staphylococcus aureus, the greatest threat is posed by neutrophils. These immune cells ingest the invading organisms and try to kill them with a cocktail of chemicals that includes reactive oxygen species (ROS). The ability of S. aureus to survive this attack is crucial for the progression of infection. However, it was not clear how the ROS damaged S. aureus and how the bacterium repaired this damage. In this work, we show that ROS cause breaks in the staphylococcal DNA, which must be repaired by a two-protein complex known as RexAB; otherwise, the bacterium is killed, and it cannot sustain infection. This provides information on the type of damage that neutrophils cause S. aureus and the mechanism by which this damage is repaired, enabling infection.

Commensal Bacteroidetes protect against Klebsiella pneumoniae colonization and transmission through IL-36 signalling.

The microbiota primes immune defences but the identity of specific commensal microorganisms that protect against infection is unclear. Conversely, how pathogens compete with the microbiota to establish their host niche is also poorly understood. In the present study, we investigate the antagonism between the microbiota and Klebsiella pneumoniae during colonization and transmission. We discover that maturation of the microbiota drives the development of distinct immune defence programmes in the upper airways and intestine to limit K. pneumoniae colonization within these niches. Immune protection in the intestine depends on the development of Bacteroidetes, interleukin (IL)-36 signalling and macrophages. This effect of Bacteroidetes requires the polysaccharide utilization locus of their conserved commensal colonization factor. Conversely, in the upper airways, Proteobacteria prime immunity through IL-17A, but K. pneumoniae overcomes these defences through encapsulation to effectively colonize this site. Ultimately, we find that host-to-host spread of K. pneumoniae occurs principally from its intestinal reservoir, and that commensal-colonization-factor-producing Bacteroidetes are sufficient to prevent transmission between hosts through IL-36. Thus, our study provides mechanistic insight into when, where and how commensal Bacteroidetes protect against K. pneumoniae colonization and contagion, providing insight into how these protective microorganisms could be harnessed to confer population-level protection against K. pneumoniae infection.

Shigella sonnei infection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence.

Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.

Inhaled corticosteroid suppression of cathelicidin drives dysbiosis and bacterial infection in chronic obstructive pulmonary disease.

Bacterial infection commonly complicates inflammatory airway diseases such as chronic obstructive pulmonary disease (COPD). The mechanisms of increased infection susceptibility and how use of the commonly prescribed therapy inhaled corticosteroids (ICS) accentuates pneumonia risk in COPD are poorly understood. Here, using analysis of samples from patients with COPD, we show that ICS use is associated with lung microbiota disruption leading to proliferation of streptococcal genera, an effect that could be recapitulated in ICS-treated mice. To study mechanisms underlying this effect, we used cellular and mouse models of streptococcal expansion with Streptococcus pneumoniae, an important pathogen in COPD, to demonstrate that ICS impairs pulmonary clearance of bacteria through suppression of the antimicrobial peptide cathelicidin. ICS impairment of pulmonary immunity was dependent on suppression of cathelicidin because ICS had no effect on bacterial loads in mice lacking cathelicidin (Camp -/-) and exogenous cathelicidin prevented ICS-mediated expansion of streptococci within the microbiota and improved bacterial clearance. Suppression of pulmonary immunity by ICS was mediated by augmentation of the protease cathepsin D. Collectively, these data suggest a central role for cathepsin D/cathelicidin in the suppression of antibacterial host defense by ICS in COPD. Therapeutic restoration of cathelicidin to boost antibacterial immunity and beneficially modulate the lung microbiota might be an effective strategy in COPD.

Exploitation of Antibiotic Resistance as a Novel Drug Target: Development of a ß-Lactamase-Activated Antibacterial Prodrug.

Expression of ß-lactamase is the single most prevalent determinant of antibiotic resistance, rendering bacteria resistant to ß-lactam antibiotics. In this article, we describe the development of an antibiotic prodrug that combines ciprofloxacin with a ß-lactamase-cleavable motif. The prodrug is only bactericidal after activation by ß-lactamase. Bactericidal activity comparable to ciprofloxacin is demonstrated against clinically relevant E. coli isolates expressing diverse ß-lactamases; bactericidal activity was not observed in strains without ß-lactamase. These findings demonstrate that it is possible to exploit antibiotic resistance to selectively target ß-lactamase-producing bacteria using our prodrug approach, without adversely affecting bacteria that do not produce ß-lactamase. This paves the way for selective targeting of drug-resistant pathogens without disrupting or selecting for resistance within the microbiota, reducing the rate of secondary infections and subsequent antibiotic use.

Microbial bile salt hydrolases mediate the efficacy of faecal microbiota transplant in the treatment of recurrent Clostridioides difficile infection.

OBJECTIVE: Faecal microbiota transplant (FMT) effectively treats recurrent Clostridioides difficile infection (rCDI), but its mechanisms of action remain poorly defined. Certain bile acids affect C. difficile germination or vegetative growth. We hypothesised that loss of gut microbiota-derived bile salt hydrolases (BSHs) predisposes to CDI by perturbing gut bile metabolism, and that BSH restitution is a key mediator of FMT's efficacy in treating the condition. DESIGN: Using stool collected from patients and donors pre-FMT/post-FMT for rCDI, we performed 16S rRNA gene sequencing, ultra performance liquid chromatography mass spectrometry (UPLC-MS) bile acid profiling, BSH activity measurement, and qPCR of bsh/baiCD genes involved in bile metabolism. Human data were validated in C. difficile batch cultures and a C57BL/6 mouse model of rCDI. RESULTS: From metataxonomics, pre-FMT stool demonstrated a reduced proportion of BSH-producing bacterial species compared with donors/post-FMT. Pre-FMT stool was enriched in taurocholic acid (TCA, a potent C. difficile germinant); TCA levels negatively correlated with key bacterial genera containing BSH-producing organisms. Post-FMT samples demonstrated recovered BSH activity and bsh/baiCD gene copy number compared with pretreatment (p<0.05). In batch cultures, supernatant from engineered bsh-expressing E. coli and naturally BSH-producing organisms (Bacteroides ovatus, Collinsella aerofaciens, Bacteroides vulgatus and Blautia obeum) reduced TCA-mediated C. difficile germination relative to culture supernatant of wild-type (BSH-negative) E. coli. C. difficile total viable counts were ~70% reduced in an rCDI mouse model after administration of E. coli expressing highly active BSH relative to mice administered BSH-negative E. coli (p<0.05). CONCLUSION: Restoration of gut BSH functionality contributes to the efficacy of FMT in treating rCDI.

Inhibiting Growth of Clostridioides difficile by Restoring Valerate, Produced by the Intestinal Microbiota.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is effective for treating recurrent Clostridioides difficile infection (CDI), but there are concerns about its long-term safety. Understanding the mechanisms of the effects of FMT could help us design safer, targeted therapies. We aimed to identify microbial metabolites that are important for C difficile growth. METHODS: We used a CDI chemostat model as a tool to study the effects of FMT in vitro. The following analyses were performed: C difficile plate counts, 16S rRNA gene sequencing, proton nuclear magnetic resonance spectroscopy, and ultra-performance liquid chromatography and mass spectrometry bile acid profiling. FMT mixtures were prepared using fresh fecal samples provided by donors enrolled in an FMT program in the United Kingdom. Results from chemostat experiments were validated using human stool samples, C difficile batch cultures, and C57BL/6 mice with CDI. Human stool samples were collected from 16 patients with recurrent CDI and healthy donors (n = 5) participating in an FMT trial in Canada. RESULTS: In the CDI chemostat model, clindamycin decreased valerate and deoxycholic acid concentrations and increased C difficile total viable counts and valerate precursors, taurocholic acid, and succinate concentrations. After we stopped adding clindamycin, levels of bile acids and succinate recovered, whereas levels of valerate and valerate precursors did not. In the CDI chemostat model, FMT increased valerate concentrations and decreased C difficile total viable counts (94% decrease), spore counts (86% decrease), and valerate precursor concentrations; concentrations of bile acids were unchanged. In stool samples from patients with CDI, valerate was depleted before FMT but restored after FMT. Clostridioides difficile batch cultures confirmed that valerate decreased vegetative growth, and that taurocholic acid was required for germination but had no effect on vegetative growth. Clostridioides difficile total viable counts were decreased by 95% in mice with CDI given glycerol trivalerate compared with phosphate buffered saline. CONCLUSIONS: We identified valerate as a metabolite that is depleted with clindamycin and only recovered with FMT. Valerate is a target for a rationally designed recurrent CDI therapy.

RitR is an archetype for a novel family of redox sensors in the streptococci that has evolved from two-component response regulators and is required for pneumococcal colonization.

To survive diverse host environments, the human pathogen Streptococcus pneumoniae must prevent its self-produced, extremely high levels of peroxide from reacting with intracellular iron. However, the regulatory mechanism(s) by which the pneumococcus accomplishes this balance remains largely enigmatic, as this pathogen and other related streptococci lack all known redox-sensing transcription factors. Here we describe a two-component-derived response regulator, RitR, as the archetype for a novel family of redox sensors in a subset of streptococcal species. We show that RitR works to both repress iron transport and enable nasopharyngeal colonization through a mechanism that exploits a single cysteine (Cys128) redox switch located within its linker domain. Biochemical experiments and phylogenetics reveal that RitR has diverged from the canonical two-component virulence regulator CovR to instead dimerize and bind DNA only upon Cys128 oxidation in air-rich environments. Atomic structures show that Cys128 oxidation initiates a "helical unravelling" of the RitR linker region, suggesting a mechanism by which the DNA-binding domain is then released to interact with its cognate regulatory DNA. Expanded computational studies indicate this mechanism could be shared by many microbial species outside the streptococcus genus.

The microbiota protects against respiratory infection via GM-CSF signaling.

The microbiota promotes resistance to respiratory infection, but the mechanistic basis for this is poorly defined. Here, we identify members of the microbiota that protect against respiratory infection by the major human pathogens Streptococcus pneumoniae and Klebsiella pneumoniae. We show that the microbiota enhances respiratory defenses via granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, which stimulates pathogen killing and clearance by alveolar macrophages through extracellular signal-regulated kinase signaling. Increased pulmonary GM-CSF production in response to infection is primed by the microbiota through interleukin-17A. By combining models of commensal colonization in antibiotic-treated and germ-free mice, using cultured commensals from the Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria phyla, we found that potent Nod-like receptor-stimulating bacteria in the upper airway (Staphylococcus aureus and Staphylococcus epidermidis) and intestinal microbiota (Lactobacillus reuteri, Enterococcus faecalis, Lactobacillus crispatus and Clostridium orbiscindens) promote resistance to lung infection through Nod2 and GM-CSF. Our data reveal the identity, location, and properties of bacteria within the microbiota that regulate lung immunity, and delineate the host signaling axis they activate to protect against respiratory infection.