Publications by authors named "Thomas B Clarke"

25 Publications

  • Page 1 of 1

Microbiota-mediated protection against antibiotic-resistant pathogens.

Genes Immun 2021 May 4. Epub 2021 May 4.

MRC Centre for Molecular Bacteriology and Infection, Department of Infectious Disease, Imperial College London, London, UK.

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.
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http://dx.doi.org/10.1038/s41435-021-00129-5DOI Listing
May 2021

Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane.

Elife 2021 Apr 6;10. Epub 2021 Apr 6.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom.

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 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 , 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.
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http://dx.doi.org/10.7554/eLife.65836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096433PMC
April 2021

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

PLoS Pathog 2021 01 19;17(1):e1009191. Epub 2021 Jan 19.

MRC Centre for Molecular Bacteriology and Infection, Department of Infectious Disease, Imperial College London, London, United Kingdom.

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.
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http://dx.doi.org/10.1371/journal.ppat.1009191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846104PMC
January 2021

Staphylococcal DNA Repair Is Required for Infection.

mBio 2020 11 17;11(6). Epub 2020 Nov 17.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom

To cause infection, 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 operon as being important for the survival of in whole human blood. Mutants lacking 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 and 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. To cause infection, bacteria must survive attack by the host immune system. For many bacteria, including the major human pathogen , 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 to survive this attack is crucial for the progression of infection. However, it was not clear how the ROS damaged 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 and the mechanism by which this damage is repaired, enabling infection.
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http://dx.doi.org/10.1128/mBio.02288-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7683395PMC
November 2020

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

Nat Microbiol 2020 02 6;5(2):304-313. Epub 2020 Jan 6.

MRC Centre for Molecular Bacteriology and Infection, Department of Infectious Diseases, Imperial College London, London, UK.

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.
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http://dx.doi.org/10.1038/s41564-019-0640-1DOI Listing
February 2020

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

PLoS Pathog 2019 12 12;15(12):e1008006. Epub 2019 Dec 12.

Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom.

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.
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http://dx.doi.org/10.1371/journal.ppat.1008006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980646PMC
December 2019

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

Sci Transl Med 2019 08;11(507)

National Heart and Lung Institute, St Mary's Campus, Imperial College London, London W2 1PG, UK.

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 , 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 () 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.
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http://dx.doi.org/10.1126/scitranslmed.aav3879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237237PMC
August 2019

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

J Med Chem 2019 05 1;62(9):4411-4425. Epub 2019 May 1.

MRC Centre for Molecular Bacteriology and Infection , Imperial College London , SW7 2AZ London , United Kingdom.

Expression of β-lactamase is the single most prevalent determinant of antibiotic resistance, rendering bacteria resistant to β-lactam antibiotics. In this article, we describe the development of an antibiotic prodrug that combines ciprofloxacin with a β-lactamase-cleavable motif. The prodrug is only bactericidal after activation by β-lactamase. Bactericidal activity comparable to ciprofloxacin is demonstrated against clinically relevant E. coli isolates expressing diverse β-lactamases; bactericidal activity was not observed in strains without β-lactamase. These findings demonstrate that it is possible to exploit antibiotic resistance to selectively target β-lactamase-producing bacteria using our prodrug approach, without adversely affecting bacteria that do not produce β-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.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511942PMC
May 2019

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

Gut 2019 10 11;68(10):1791-1800. Epub 2019 Feb 11.

Division of Integrative Systems Medicine and Digestive Disease, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, UK.

Objective: Faecal microbiota transplant (FMT) effectively treats recurrent infection (rCDI), but its mechanisms of action remain poorly defined. Certain bile acids affect 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 /CD genes involved in bile metabolism. Human data were validated in 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 germinant); TCA levels negatively correlated with key bacterial genera containing BSH-producing organisms. Post-FMT samples demonstrated recovered BSH activity and /CD gene copy number compared with pretreatment (p<0.05). In batch cultures, supernatant from engineered -expressing and naturally BSH-producing organisms ( and ) reduced TCA-mediated germination relative to culture supernatant of wild-type (BSH-negative) total viable counts were ~70% reduced in an rCDI mouse model after administration of expressing highly active BSH relative to mice administered BSH-negative (p<0.05).

Conclusion: Restoration of gut BSH functionality contributes to the efficacy of FMT in treating rCDI.
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http://dx.doi.org/10.1136/gutjnl-2018-317842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839797PMC
October 2019

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

Gastroenterology 2018 11 17;155(5):1495-1507.e15. Epub 2018 Jul 17.

Division of Integrative Systems Medicine and Digestive Disease, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, United Kingdom; School of Biosciences, Cardiff University, Cardiff, United Kingdom. Electronic address:

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.
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http://dx.doi.org/10.1053/j.gastro.2018.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347096PMC
November 2018

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.

PLoS Pathog 2018 05 11;14(5):e1007052. Epub 2018 May 11.

Department of Microbiology and Immunology, Loyola University Chicago; Maywood, IL, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1007052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965902PMC
May 2018

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

Nat Commun 2017 11 15;8(1):1512. Epub 2017 Nov 15.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, SW7 2AZ, UK.

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.
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http://dx.doi.org/10.1038/s41467-017-01803-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5688119PMC
November 2017

Mathematical Modeling of Colonization, Invasive Infection and Treatment.

Front Physiol 2017 2;8:115. Epub 2017 Mar 2.

Department of Bioengineering, Imperial College London London, UK.

() is a commensal bacterium that normally resides on the upper airway epithelium without causing infection. However, factors such as co-infection with influenza virus can impair the complex -host interactions and the subsequent development of many life-threatening infectious and inflammatory diseases, including pneumonia, meningitis or even sepsis. With the increased threat of infection due to the emergence of new antibiotic resistant strains, there is an urgent need for better treatment strategies that effectively prevent progression of disease triggered by infection, minimizing the use of antibiotics. The complexity of the host-pathogen interactions has left the full understanding of underlying mechanisms of -triggered pathogenesis as a challenge, despite its critical importance in the identification of effective treatments. To achieve a systems-level and quantitative understanding of the complex and dynamically-changing host- interactions, here we developed a mechanistic mathematical model describing dynamic interplays between , immune cells, and epithelial tissues, where the host-pathogen interactions initiate. The model serves as a mathematical framework that coherently explains various and studies, to which the model parameters were fitted. Our model simulations reproduced the robust homeostatic -host interaction, as well as three qualitatively different pathogenic behaviors: immunological scarring, invasive infection and their combination. Parameter sensitivity and bifurcation analyses of the model identified the processes that are responsible for qualitative transitions from healthy to such pathological behaviors. Our model also predicted that the onset of invasive infection occurs within less than 2 days from transient challenges. This prediction provides arguments in favor of the use of vaccinations, since adaptive immune responses cannot be developed in such a short time. We further designed optimal treatment strategies, with minimal strengths and minimal durations of antibiotics, for each of the three pathogenic behaviors distinguished by our model. The proposed mathematical framework will help to design better disease management strategies and new diagnostic markers that can be used to inform the most appropriate patient-specific treatment options.
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http://dx.doi.org/10.3389/fphys.2017.00115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5332394PMC
March 2017

Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids.

Nat Microbiol 2016 Oct 24;2:16194. Epub 2016 Oct 24.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK.

Daptomycin is a bactericidal antibiotic of last resort for serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA). Although resistance is rare, treatment failure can occur in more than 20% of cases and so there is a pressing need to identify and mitigate factors that contribute to poor therapeutic outcomes. Here, we show that loss of the Agr quorum-sensing system, which frequently occurs in clinical isolates, enhances S. aureus survival during daptomycin treatment. Wild-type S. aureus was killed rapidly by daptomycin, but Agr-defective mutants survived antibiotic exposure by releasing membrane phospholipids, which bound and inactivated the antibiotic. Although wild-type bacteria also released phospholipid in response to daptomycin, Agr-triggered secretion of small cytolytic toxins, known as phenol soluble modulins, prevented antibiotic inactivation. Phospholipid shedding by S. aureus occurred via an active process and was inhibited by the β-lactam antibiotic oxacillin, which slowed inactivation of daptomycin and enhanced bacterial killing. In conclusion, S. aureus possesses a transient defence mechanism that protects against daptomycin, which can be compromised by Agr-triggered toxin production or an existing therapeutic antibiotic.
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http://dx.doi.org/10.1038/nmicrobiol.2016.194DOI Listing
October 2016

The regulation of host defences to infection by the microbiota.

Immunology 2017 Jan 9;150(1):1-6. Epub 2016 Aug 9.

MRC Centre for Molecular Bacteriology and Infection, Department of Medicine, Imperial College London, London, UK.

The skin and mucosal epithelia of humans and other mammals are permanently colonized by large microbial communities (the microbiota). Due to this life-long association with the microbiota, these microbes have an extensive influence over the physiology of their host organism. It is now becoming apparent that nearly all tissues and organ systems, whether in direct contact with the microbiota or in deeper host sites, are under microbial influence. The immune system is perhaps the most profoundly affected, with the microbiota programming both its innate and adaptive arms. The regulation of immunity by the microbiota helps to protect the host against intestinal and extra-intestinal infection by many classes of pathogen. In this review, we will discuss the experimental evidence supporting a role for the microbiota in regulating host defences to extra-intestinal infection, draw together common mechanistic themes, including the central role of pattern recognition receptors, and outline outstanding questions that need to be answered.
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http://dx.doi.org/10.1111/imm.12634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5221693PMC
January 2017

Peptidoglycan from the gut microbiota governs the lifespan of circulating phagocytes at homeostasis.

Blood 2016 05 17;127(20):2460-71. Epub 2016 Mar 17.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; and Department of Microbiology, New York University School of Medicine, New York, NY.

Maintenance of myeloid cell homeostasis requires continuous turnover of phagocytes from the bloodstream, yet whether environmental signals influence phagocyte longevity in the absence of inflammation remains unknown. Here, we show that the gut microbiota regulates the steady-state cellular lifespan of neutrophils and inflammatory monocytes, the 2 most abundant circulating myeloid cells and key contributors to inflammatory responses. Treatment of mice with broad-spectrum antibiotics, or with the gut-restricted aminoglycoside neomycin alone, accelerated phagocyte turnover and increased the rates of their spontaneous apoptosis. Metagenomic analyses revealed that neomycin altered the abundance of intestinal bacteria bearing γ-d-glutamyl-meso-diaminopimelic acid, a ligand for the intracellular peptidoglycan sensor Nod1. Accordingly, signaling through Nod1 was both necessary and sufficient to mediate the stimulatory influence of the flora on myeloid cell longevity. Stimulation of Nod1 signaling increased the frequency of lymphocytes in the murine intestine producing the proinflammatory cytokine interleukin 17A (IL-17A), and liberation of IL-17A was required for transmission of Nod1-dependent signals to circulating phagocytes. Together, these results define a mechanism through which intestinal microbes govern a central component of myeloid homeostasis and suggest perturbations of commensal communities can influence steady-state regulation of cell fate.
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http://dx.doi.org/10.1182/blood-2015-10-675173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4874226PMC
May 2016

Microbial programming of systemic innate immunity and resistance to infection.

Authors:
Thomas B Clarke

PLoS Pathog 2014 Dec 4;10(12):e1004506. Epub 2014 Dec 4.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom.

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http://dx.doi.org/10.1371/journal.ppat.1004506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4256436PMC
December 2014

Early innate immunity to bacterial infection in the lung is regulated systemically by the commensal microbiota via nod-like receptor ligands.

Authors:
Thomas B Clarke

Infect Immun 2014 Nov 18;82(11):4596-606. Epub 2014 Aug 18.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom

The commensal microbiota is a major regulator of the immune system. The majority of commensal bacteria inhabit the gastrointestinal tract and are known to regulate local mucosal defenses against intestinal pathogens. There is growing appreciation that the commensal microbiota also regulates immune responses at extraintestinal sites. Currently, however, it is unclear how this influences host defenses against bacterial infection outside the intestine. Microbiota depletion caused significant defects in the early innate response to lung infection by the major human pathogen Klebsiella pneumoniae. After microbiota depletion, early clearance of K. pneumoniae was impaired, and this could be rescued by administration of bacterial Nod-like receptor (NLR) ligands (the NOD1 ligand MurNAcTri(DAP) and NOD2 ligand muramyl dipeptide [MDP]) but not bacterial Toll-like receptor (TLR) ligands. Importantly, NLR ligands from the gastrointestinal, but not upper respiratory, tract rescued host defenses in the lung. Defects in early innate immunity were found to be due to reduced reactive oxygen species-mediated killing of bacteria by alveolar macrophages. These data show that bacterial signals from the intestine have a profound influence on establishing the levels of antibacterial defenses in distal tissues.
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http://dx.doi.org/10.1128/IAI.02212-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249320PMC
November 2014

Intracellular sensors of extracellular bacteria.

Immunol Rev 2011 Sep;243(1):9-25

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6076, USA.

Initial recognition of bacteria by the innate immune system is thought to occur primarily by germline-encoded pattern recognition receptors (PRRs). These receptors are present in multiple compartments of host cells and are thus capable of surveying both the intracellular and extracellular milieu for bacteria. It has generally been presumed that the cellular location of these receptors dictates what type of bacteria they respond to: extracellular bacteria being recognized by cell surface receptors, such as certain Toll-like receptors, and bacteria that are capable of breaching the plasma membrane and entering the cytoplasm, being sensed by cytoplasmic receptors, including the Nod-like receptors (NLRs). Increasingly, it is becoming apparent that this is a false dichotomy and that extracellular bacteria can be sensed by cytoplasmic PRRs and this is crucial for controlling the levels of these bacteria. In this review, we discuss the role of two NLRs, Nod1 and Nod2, in the recognition of and response to extracellular bacteria.
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http://dx.doi.org/10.1111/j.1600-065X.2011.01039.xDOI Listing
September 2011

Invasive bacterial pathogens exploit TLR-mediated downregulation of tight junction components to facilitate translocation across the epithelium.

Cell Host Microbe 2011 May;9(5):404-14

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

Streptococcus pneumoniae and Haemophilus influenzae are members of the normal human nasal microbiota with the ability to cause invasive infections. Bacterial invasion requires translocation across the epithelium; however, mechanistic understanding of this process is limited. Examining the epithelial response to murine colonization by S. pneumoniae and H. influenzae, we observed the TLR-dependent downregulation of claudins 7 and 10, tight junction components key to the maintenance of epithelial barrier integrity. When modeled in vitro, claudin downregulation was preceded by upregulation of SNAIL1, a transcriptional repressor of tight junction components, and these phenomena required p38 MAPK and TGF-β signaling. Consequently, downregulation of SNAIL1 expression inhibited bacterial translocation across the epithelium. Furthermore, disruption of epithelial barrier integrity by claudin 7 inhibition in vitro or TLR stimulation in vivo promoted bacterial translocation. These data support a general mechanism for epithelial opening exploited by invasive pathogens to facilitate movement across the epithelium to initiate disease.
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http://dx.doi.org/10.1016/j.chom.2011.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536975PMC
May 2011

Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity.

Nat Med 2010 Feb 17;16(2):228-31. Epub 2010 Jan 17.

Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Humans are colonized by a large and diverse bacterial flora (the microbiota) essential for the development of the gut immune system. A broader role for the microbiota as a major modulator of systemic immunity has been proposed; however, evidence and a mechanism for this role have remained elusive. We show that the microbiota are a source of peptidoglycan that systemically primes the innate immune system, enhancing killing by bone marrow-derived neutrophils of two major pathogens: Streptococcus pneumoniae and Staphylococcus aureus. This requires signaling via the pattern recognition receptor nucleotide-binding, oligomerization domain-containing protein-1 (Nod1, which recognizes meso-diaminopimelic acid (mesoDAP)-containing peptidoglycan found predominantly in Gram-negative bacteria), but not Nod2 (which detects peptidoglycan found in Gram-positive and Gram-negative bacteria) or Toll-like receptor 4 (Tlr4, which recognizes lipopolysaccharide). We show translocation of peptidoglycan from the gut to neutrophils in the bone marrow and show that peptidoglycan concentrations in sera correlate with neutrophil function. In vivo administration of Nod1 ligands is sufficient to restore neutrophil function after microbiota depletion. Nod1(-/-) mice are more susceptible than wild-type mice to early pneumococcal sepsis, demonstrating a role for Nod1 in priming innate defenses facilitating a rapid response to infection. These data establish a mechanism for systemic immunomodulation by the microbiota and highlight potential adverse consequences of microbiota disruption by broad-spectrum antibiotics on innate immune defense to infection.
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http://dx.doi.org/10.1038/nm.2087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497535PMC
February 2010

Crystal structures of penicillin-binding proteins 4 and 5 from Haemophilus influenzae.

J Mol Biol 2010 Feb 1;396(3):634-45. Epub 2009 Dec 1.

Yokohama City University, Suehiro 1-7-29, Tsurumi, Yokohama 230-0045, Japan.

We have determined high-resolution apo crystal structures of two low molecular weight penicillin-binding proteins (PBPs), PBP4 and PBP5, from Haemophilus influenzae, one of the most frequently found pathogens in the upper respiratory tract of children. Novel beta-lactams with notable antimicrobial activity have been designed, and crystal structures of PBP4 complexed with ampicillin and two of the novel molecules have also been determined. Comparing the apo form with those of the complexes, we find that the drugs disturb the PBP4 structure and weaken X-ray diffraction, to very different extents. PBP4 has recently been shown to act as a sensor of the presence of penicillins in Pseudomonas aeruginosa, and our models offer a clue to the structural basis for this effect. Covalently attached penicillins press against a phenylalanine residue near the active site and disturb the deacylation step. The ready inhibition of PBP4 by beta-lactams compared to PBP5 also appears to be related to the weaker interactions holding key residues in a catalytically competent position.
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http://dx.doi.org/10.1016/j.jmb.2009.11.055DOI Listing
February 2010

Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice.

J Clin Invest 2009 Jul 8;119(7):1899-909. Epub 2009 Jun 8.

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6076, USA.

Microbial colonization of mucosal surfaces may be an initial event in the progression to disease, and it is often a transient process. For the extracellular pathogen Streptococcus pneumoniae studied in a mouse model, nasopharyngeal carriage is eliminated over a period of weeks and requires cellular rather than humoral immunity. Here, we demonstrate that primary infection led to TLR2-dependent recruitment of monocyte/macrophages into the upper airway lumen, where they engulfed pneumococci. Pharmacologic depletion of luminal monocyte/macrophages by intranasal instillation of liposomal clodronate diminished pneumococcal clearance. Efficient clearance of colonization required TLR2 signaling to generate a population of pneumococcal-specific IL-17-expressing CD4+ T cells. Depletion of either IL-17A or CD4+ T cells was sufficient to block the recruitment of monocyte/macrophages that allowed for effective late pneumococcal clearance. In contrast with naive mice, previously colonized mice showed enhanced early clearance that correlated with a more robust influx of luminal neutrophils. As for primary colonization, these cellular responses required Th17 immunity. Our findings demonstrate that monocyte/macrophages and neutrophils recruited to the mucosal surface are key effectors in clearing primary and secondary bacterial colonization, respectively.
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http://dx.doi.org/10.1172/JCI36731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2701860PMC
July 2009

Mutational analysis of the substrate specificity of Escherichia coli penicillin binding protein 4.

Biochemistry 2009 Mar;48(12):2675-83

Department of Biological Sciences, University of Warwick, Coventry, UK.

Escherichia coli PBP4 is the archetypal class C, low molecular mass penicillin binding protein (LMM-PBP) and possesses both dd-carboxypeptidase and dd-endopeptidase activity. In contrast to other classes of PBP, class C LMM-PBPs show high dd-carboxypeptidase activity and rapidly hydrolyze synthetic fragments of peptidoglycan. The recently solved X-ray crystal structures of three class C LMM-PBPs (E. coli PBP4, Bacillus subtilis PBP4a, and Actinomadura R39 dd-peptidase) have identified several residues that form a pocket in the active site unique to this class of PBP. The X-ray cocrystal structure of the Actinomadura R39 DD-peptidase with a cephalosporin bearing a peptidoglycan-mimetic side chain showed that residues of this pocket interact with the third position meso-2,6-diaminopimelic acid residue of the peptidoglycan stem peptide. Equivalent residues of E. coli PBP4 (Asp155, Phe160, Arg361, and Gln422) were mutated, and the effect on both DD-carboxypeptidase and DD-endopeptidase activities was determined. Using N-acetylmuramyl-L-alanyl-gamma-D-glutamyl-meso-2,6-diaminopimelyl-D-alanyl-D-alanine as substrate, mutation of Asp155, Phe160, Arg361, and Gln422 to alanine reduced k(cat)/K(m) by 12.7-, 1.9-, 24.5-, and 13.8-fold, respectively. None of the k(cat) values deviated significantly from wild-type PBP4. PBP4 DD-endopeptidase activity was also affected, with substitution of Asp155, Arg361, and Gln422 reducing specific activity by 22%, 56%, and 40%, respectively. This provides the first direct demonstration of the importance of residues forming a subsite to accommodate meso-2,6-diaminopimelic acid in both the DD-carboxypeptidase and DD-endopeptidase activities of a class C LMM-PBP.
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http://dx.doi.org/10.1021/bi801993xDOI Listing
March 2009

Nod1 signaling overcomes resistance of S. pneumoniae to opsonophagocytic killing.

PLoS Pathog 2007 Aug;3(8):e118

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp) leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi), provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1), which is involved in recognition of gamma-D-glutamyl-meso-diaminopimelic acid (meso-DAP) contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1(-/-) mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo.
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http://dx.doi.org/10.1371/journal.ppat.0030118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1950946PMC
August 2007