ABSTRACT
The increase in emerging drug resistant Gram-negative bacterial infections is a global concern. In addition, there is growing recognition that compromising the microbiota through the use of broad-spectrum antibiotics can impact long term patient outcomes. Therefore, there is the need to develop new bactericidal strategies to combat Gram-negative infections that would address these specific issues. In this study, we report and characterize one such approach, an antibody-drug conjugate (ADC) that combines (i) targeting the surface of a specific pathogenic organism through a monoclonal antibody with (ii) the high killing activity of an antimicrobial peptide. We focused on a major pathogenic Gram-negative bacterium associated with antibacterial resistance: Pseudomonas aeruginosa. To target this organism, we designed an ADC by fusing an antimicrobial peptide to the C-terminal end of the VH and/or VL-chain of a monoclonal antibody, VSX, that targets the core of P. aeruginosa lipopolysaccharide. This ADC demonstrates appropriately minimal levels of toxicity against mammalian cells, rapidly kills P. aeruginosa strains, and protects mice from P. aeruginosa lung infection when administered therapeutically. Furthermore, we found that the ADC was synergistic with several classes of antibiotics. This approach described in this study might result in a broadly useful strategy for targeting specific pathogenic microorganisms without further augmenting antibiotic resistance.
Subject(s)
Bacterial Infections , Immunoconjugates , Animals , Mice , Pseudomonas aeruginosa , Antibodies, Monoclonal/pharmacology , Anti-Bacterial Agents/pharmacology , Antimicrobial Peptides , MammalsABSTRACT
OBJECTIVES: Alterations in tryptophan (Trp) metabolism have been reported in inflammatory diseases, including rheumatoid arthritis (RA). However, understanding whether these alterations participate in RA development and can be considered putative therapeutic targets remains undetermined.In this study, we combined quantitative Trp metabolomics in the serum from patients with RA and corrective administration of a recombinant enzyme in experimental arthritis to address this question. METHODS: Targeted quantitative Trp metabolomics was performed on the serum from 574 previously untreated patients with RA from the ESPOIR (Etude et Suivi des POlyarthrites Indifférenciées Récentes) cohort and 98 healthy subjects. A validation cohort involved 69 established patients with RA. Dosages were also done on the serum of collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) mice and controls. A proof-of-concept study evaluating the therapeutic potency of targeting the kynurenine pathway was performed in the CAIA model. RESULTS: Differential analysis revealed dramatic changes in Trp metabolite levels in patients with RA compared with healthy controls. Decreased levels of kynurenic (KYNA) and xanthurenic (XANA) acids and indole derivatives, as well as an increased level of quinolinic acid (QUIN), were found in the serum of patients with RA. They correlated positively with disease severity (assessed by both circulating biomarkers and disease activity scores) and negatively with quality-of-life scores. Similar profiles of kynurenine pathway metabolites were observed in the CAIA and CIA models. From a mechanistic perspective, we demonstrated that QUIN favours human fibroblast-like synoviocyte proliferation and affected their cellular metabolism, through inducing both mitochondrial respiration and glycolysis. Finally, systemic administration of the recombinant enzyme aminoadipate aminotransferase, responsible for the generation of XANA and KYNA, was protective in the CAIA model. CONCLUSIONS: Altogether, our preclinical and clinical data indicate that alterations in the Trp metabolism play an active role in the pathogenesis of RA and could be considered as a new therapeutic avenue.
Subject(s)
Arthritis, Experimental , Arthritis, Rheumatoid , Humans , Animals , Mice , Tryptophan/therapeutic use , Kynurenine/therapeutic use , Biomarkers , Arthritis, Experimental/pathologyABSTRACT
Newborns, whether born prematurely or at term, have a fully formed but naive immune system that must adapt to the extra-uterine environment to prevent infections. Maternal immunity, transmitted through the placenta and breast milk, protects newborns against infections, primarily via immunoglobulins (IgG and IgA) and certain maternal immune cells also known as microchimeric cells. Recently, it also appeared that the maternal gut microbiota played a vital role in neonatal immune maturation via microbial compounds impacting immune development and the establishment of immune tolerance. In this context, maternal vaccination is a powerful tool to enhance even more maternal and neonatal health. It involves the transfer of vaccine-induced antibodies to protect both mother and child from infectious diseases. In this work we review the state of the art on maternal immune factors involved in the prevention of neonatal bacterial infections, with particular emphasis on the role of maternal vaccination in protecting neonates against bacterial disease.
Subject(s)
Bacterial Infections , Communicable Diseases , Pregnancy , Female , Child , Infant, Newborn , Humans , Milk, Human , Immunologic Factors , Bacterial Infections/prevention & control , Antibodies, ViralABSTRACT
Several bacteria in the gut microbiota have been shown to be associated with inflammatory bowel disease (IBD), and dozens of IBD genetic variants have been identified in genome-wide association studies. However, the role of the microbiota in the etiology of IBD in terms of host genetic susceptibility remains unclear. Here, we studied the association between four major genetic variants associated with an increased risk of IBD and bacterial taxa in up to 633 IBD cases. We performed systematic screening for associations, identifying and replicating associations between NOD2 variants and two taxa: the Roseburia genus and the Faecalibacterium prausnitzii species. By exploring the overall association patterns between genes and bacteria, we found that IBD risk alleles were significantly enriched for associations concordant with bacteria-IBD associations. To understand the significance of this pattern in terms of the study design and known effects from the literature, we used counterfactual principles to assess the fitness of a few parsimonious gene-bacteria-IBD causal models. Our analyses showed evidence that the disease risk of these genetic variants were likely to be partially mediated by the microbiome. We confirmed these results in extensive simulation studies and sensitivity analyses using the association between NOD2 and F. prausnitzii as a case study.
Subject(s)
Gastrointestinal Microbiome/genetics , Host Microbial Interactions/genetics , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/microbiology , Adult , CARD Signaling Adaptor Proteins/genetics , Clostridiales/genetics , Clostridiales/isolation & purification , Clostridiales/pathogenicity , Faecalibacterium prausnitzii/genetics , Faecalibacterium prausnitzii/isolation & purification , Faecalibacterium prausnitzii/pathogenicity , Female , Genetic Association Studies , Genetic Predisposition to Disease , Genetic Variation , Humans , Inflammatory Bowel Diseases/etiology , Male , Middle Aged , Models, Genetic , Nod2 Signaling Adaptor Protein/genetics , Polymorphism, Single NucleotideABSTRACT
A time series analysis of 871 543 pediatric emergency visits revealed that the coronavirus disease 2019 (COVID-19) lockdown and school closures were associated with a significant decrease in infectious diseases disseminated through airborne or fecal-oral transmission: common cold, gastroenteritis, bronchiolitis, and acute otitis. No change was found for urinary tract infections.
Subject(s)
COVID-19 , Pandemics , Child , Communicable Disease Control , Humans , SARS-CoV-2 , SchoolsABSTRACT
Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is presenting as a systemic disease associated with vascular inflammation and endothelial injury. Severe forms of SARS-CoV-2 infection induce acute respiratory distress syndrome (ARDS) and there is still an ongoing debate on whether COVID-19 ARDS and its perfusion defect differs from ARDS induced by other causes. Beside pro-inflammatory cytokines (such as interleukin-1 ß [IL-1ß] or IL-6), several main pathological phenomena have been seen because of endothelial cell (EC) dysfunction: hypercoagulation reflected by fibrin degradation products called D-dimers, micro- and macrothrombosis and pathological angiogenesis. Direct endothelial infection by SARS-CoV-2 is not likely to occur and ACE-2 expression by EC is a matter of debate. Indeed, endothelial damage reported in severely ill patients with COVID-19 could be more likely secondary to infection of neighboring cells and/or a consequence of inflammation. Endotheliopathy could give rise to hypercoagulation by alteration in the levels of different factors such as von Willebrand factor. Other than thrombotic events, pathological angiogenesis is among the recent findings. Overexpression of different proangiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) or placental growth factors (PlGF) have been found in plasma or lung biopsies of COVID-19 patients. Finally, SARS-CoV-2 infection induces an emergency myelopoiesis associated to deregulated immunity and mobilization of endothelial progenitor cells, leading to features of acquired hematological malignancies or cardiovascular disease, which are discussed in this review. Altogether, this review will try to elucidate the pathophysiology of thrombotic complications, pathological angiogenesis and EC dysfunction, allowing better insight in new targets and antithrombotic protocols to better address vascular system dysfunction. Since treating SARS-CoV-2 infection and its potential long-term effects involves targeting the vascular compartment and/or mobilization of immature immune cells, we propose to define COVID-19 and its complications as a systemic vascular acquired hemopathy.
Subject(s)
COVID-19/metabolism , Myelopoiesis , Neovascularization, Pathologic/metabolism , Respiratory Distress Syndrome/metabolism , SARS-CoV-2/metabolism , Thrombosis/metabolism , COVID-19/pathology , COVID-19/therapy , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelial Cells/virology , Fibrin Fibrinogen Degradation Products/metabolism , Fibroblast Growth Factor 2/metabolism , Humans , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Membrane Proteins/metabolism , Neovascularization, Pathologic/pathology , Neovascularization, Pathologic/therapy , Neovascularization, Pathologic/virology , Respiratory Distress Syndrome/pathology , Respiratory Distress Syndrome/therapy , Respiratory Distress Syndrome/virology , Thrombosis/pathology , Thrombosis/therapy , Thrombosis/virology , Vascular Endothelial Growth Factor A/metabolism , von Willebrand Factor/metabolismABSTRACT
"Antibiotic resistance is usually associated with a fitness cost" is frequently accepted as common knowledge in the field of infectious diseases. However, with the advances in high-throughput DNA sequencing that allows for a comprehensive analysis of bacterial pathogenesis at the genome scale, including antibiotic resistance genes, it appears that this paradigm might not be as solid as previously thought. Recent studies indicate that antibiotic resistance is able to enhance bacterial fitness in vivo with a concomitant increase in virulence during infections. As a consequence, strategies to minimize antibiotic resistance turn out to be not as simple as initially believed. Indeed, decreased antibiotic use may not be sufficient to let susceptible strains outcompete the resistant ones. Here, we put in perspective these findings and review alternative approaches, such as preventive and therapeutic anti-bacterial immunotherapies that have the potential to by-pass the classic antibiotics.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/pathogenicity , Bacterial Infections/drug therapy , Drug Resistance, Bacterial , Genome, Bacterial , Animals , Bacteria/drug effects , Bacteria/genetics , Bacterial Infections/microbiology , DNA Transposable Elements , High-Throughput Nucleotide Sequencing , Humans , VirulenceABSTRACT
OBJECTIVE: The bacterial intestinal microbiota plays major roles in human physiology and IBDs. Although some data suggest a role of the fungal microbiota in IBD pathogenesis, the available data are scarce. The aim of our study was to characterise the faecal fungal microbiota in patients with IBD. DESIGN: Bacterial and fungal composition of the faecal microbiota of 235 patients with IBD and 38 healthy subjects (HS) was determined using 16S and ITS2 sequencing, respectively. The obtained sequences were analysed using the Qiime pipeline to assess composition and diversity. Bacterial and fungal taxa associated with clinical parameters were identified using multivariate association with linear models. Correlation between bacterial and fungal microbiota was investigated using Spearman's test and distance correlation. RESULTS: We observed that fungal microbiota is skewed in IBD, with an increased Basidiomycota/Ascomycota ratio, a decreased proportion of Saccharomyces cerevisiae and an increased proportion of Candida albicans compared with HS. We also identified disease-specific alterations in diversity, indicating that a Crohn's disease-specific gut environment may favour fungi at the expense of bacteria. The concomitant analysis of bacterial and fungal microbiota showed a dense and homogenous correlation network in HS but a dramatically unbalanced network in IBD, suggesting the existence of disease-specific inter-kingdom alterations. CONCLUSIONS: Besides bacterial dysbiosis, our study identifies a distinct fungal microbiota dysbiosis in IBD characterised by alterations in biodiversity and composition. Moreover, we unravel here disease-specific inter-kingdom network alterations in IBD, suggesting that, beyond bacteria, fungi might also play a role in IBD pathogenesis.
Subject(s)
Ascomycota/isolation & purification , Basidiomycota/isolation & purification , Candida albicans/isolation & purification , Colitis, Ulcerative/microbiology , Crohn Disease/microbiology , Dysbiosis/microbiology , RNA, Ribosomal, 16S/analysis , Bacteria/isolation & purification , Case-Control Studies , Colitis, Ulcerative/genetics , Crohn Disease/genetics , Feces/microbiology , Gastrointestinal Microbiome , Humans , Polymorphism, Single Nucleotide , Saccharomyces cerevisiae/isolation & purificationABSTRACT
Burkholderia dolosa caused an outbreak in the cystic fibrosis (CF) clinic at Boston Children's Hospital from 1998 to 2005 and led to the infection of over 40 patients, many of whom died due to complications from infection by this organism. To assess whether B. dolosa significantly contributes to disease or is recognized by the host immune response, mice were infected with a sequenced outbreak B. dolosa strain, AU0158, and responses were compared to those to the well-studied CF pathogen Pseudomonas aeruginosa In parallel, mice were also infected with a polar flagellin mutant of B. dolosa to examine the role of flagella in B. dolosa lung colonization. The results showed a higher persistence in the host by B. dolosa strains, and yet, neutrophil recruitment and cytokine production were lower than those with P. aeruginosa The ability of host immune cells to recognize B. dolosa was then assessed, B. dolosa induced a robust cytokine response in cultured cells, and this effect was dependent on the flagella only when bacteria were dead. Together, these results suggest that B. dolosa can be recognized by host cells in vitro but may avoid or suppress the host immune response in vivo through unknown mechanisms. B. dolosa was then compared to other Burkholderia species and found to induce similar levels of cytokine production despite being internalized by macrophages more than Burkholderia cenocepacia strains. These data suggest that B. dolosa AU0158 may act differently with host cells and is recognized differently by immune systems than are other Burkholderia strains or species.
Subject(s)
Burkholderia Infections/immunology , Cystic Fibrosis/complications , Cytokines/immunology , Flagella/immunology , Flagellin/genetics , Animals , Bronchoalveolar Lavage , Burkholderia/genetics , Burkholderia/immunology , Burkholderia Infections/microbiology , Cell Line , Cystic Fibrosis/microbiology , Disease Models, Animal , Epidemics , Female , Flagella/genetics , Humans , Lung/microbiology , Mice , Mice, Inbred C57BL , Mutation , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/immunologyABSTRACT
BACKGROUND: Many antibiotics are prescribed inappropriately in pediatric emergency departments (PEDs), but little data are available in these settings about effective interventions based on guidelines that follow the antimicrobial stewardship principle. Our aim was to assess the impact of implementing the 2011 national guidelines on antibiotic prescriptions for acute respiratory tract infection (ARTI) in PEDs. METHOD: We conducted a multicentric, quasiexperimental, interrupted time series analysis of prospectively collected electronic data from 7 French PEDs. We included all pediatric patients who visited a participating PED during the study period from November 2009 to October 2014 and were diagnosed with an ARTI. The intervention consisted of local protocol implementation, education sessions, and feedback. The main outcome was the antibiotic prescription rate of discharge prescriptions for ARTI per 1000 PED visits before and after implementation, analyzed using the segmented regression model. RESULTS: We included 242534 patients with an ARTI. The intervention was associated with a significant change in slope for the antibiotic prescription rate per 1000 PED visits (-0.4% per 15-day period, P = .04), and the cumulative effect at the end of the study was estimated to be -30.9%, (95% CI [-45.2 to -20.1]), representing 13136 avoided antibiotic prescriptions. The broad-spectrum antibiotic prescription relative percentage decreased dramatically (-62.7%, 95% CI [-92.8; -32.7]) and was replaced by amoxicillin. CONCLUSION: Implementation of the 2011 national French guidelines led to a significant decrease in the antibiotic prescription rate for ARTI and a dramatic drop in broad-spectrum antibiotic prescriptions, in favor of amoxicillin.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Antimicrobial Stewardship , Drug Prescriptions/statistics & numerical data , Respiratory Tract Infections/epidemiology , Adolescent , Child , Child, Preschool , Emergency Service, Hospital , Humans , Infant , Infant, Newborn , Interrupted Time Series Analysis , Practice Guidelines as Topic , Prospective Studies , Respiratory Tract Infections/drug therapy , Treatment OutcomeABSTRACT
In the context of the great concern about the impact of human activities on the environment, we studied 403 commensal Escherichia coli/Escherichia clade strains isolated from several animal and human populations that have variable contacts to one another. Multilocus sequence typing (MLST) showed a decrease of diversity 1) in strains isolated from animals that had an increasing contact with humans and 2) in all strains that had increased antimicrobial resistance. A specific B1 phylogroup clonal complex (CC87, Institut Pasteur schema nomenclature) of animal origin was identified and characterized as being responsible for the increased antimicrobial resistance prevalence observed in strains from the environments with a high human-mediated antimicrobial pressure. CC87 strains have a high capacity of acquiring and disseminating resistance genes with specific metabolic and genetic determinants as demonstrated by high-throughput sequencing and phenotyping. They are good mouse gut colonizers but are not virulent. Our data confirm the predominant role of human activities in the emergence of antimicrobial resistance in the environmental bacterial strains and unveil a particular E. coli clonal complex of animal origin capable of spreading antimicrobial resistance to other members of microbial communities.
Subject(s)
Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli/genetics , Genetic Variation , Animals , Anti-Infective Agents/adverse effects , Drug Resistance, Multiple, Bacterial/drug effects , Escherichia coli/drug effects , Escherichia coli Proteins/genetics , Genotype , Humans , Mice , Multilocus Sequence Typing , PhylogenyABSTRACT
Bacillus subtilis is intensively studied as a model organism for the development of bacterial biofilms or pellicles. A key component is currently undefined exopolysaccharides produced from proteins encoded by genes within the eps locus. Within this locus are four genes, epsHIJK, known to be essential for pellicle formation. We show they encode proteins synthesizing the broadly expressed microbial carbohydrate poly-N-acetylglucosamine (PNAG). PNAG was present in both pellicle and planktonic wild-type B. subtilis cells and in strains with deletions in the epsA-G and -L-O genes but not in strains deleted for epsH-K. Cloning of the B. subtilis epsH-K genes into Escherichia coli with in-frame deletions in the PNAG biosynthetic genes pgaA-D, respectively, restored PNAG production in E. coli. Cloning the entire B. subtilis epsHIJK locus into pga-deleted E. coli, Klebsiella pneumoniae, or alginate-negative Pseudomonas aeruginosa restored or conferred PNAG production. Bioinformatic and structural predictions of the EpsHIJK proteins suggest EpsH and EpsJ are glycosyltransferases (GT) with a GT-A fold; EpsI is a GT with a GT-B fold, and EpsK is an α-helical membrane transporter. B. subtilis, E. coli, and pga-deleted E. coli carrying the epsHIJK genes on a plasmid were all susceptible to opsonic killing by antibodies to PNAG. The immunochemical and genetic data identify the genes and proteins used by B. subtilis to produce PNAG as a significant carbohydrate factor essential for pellicle formation.
Subject(s)
Acetylglucosamine/physiology , Bacillus subtilis/physiology , Biofilms , Acetylglucosamine/chemistry , Antibodies, Bacterial/physiology , Bacterial Proteins/chemistry , Bacterial Proteins/physiology , Biosynthetic Pathways , Escherichia coli , HL-60 Cells , Humans , Models, Molecular , Opsonin Proteins/physiology , Phagocytosis , Polysaccharides, Bacterial , Protein Structure, TertiarySubject(s)
COVID-19 , Child , Communicable Disease Control , France/epidemiology , Humans , SARS-CoV-2 , SchoolsABSTRACT
BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) are responsible for worldwide outbreaks and antibiotic treatments are problematic. The polysaccharide poly-(ß-1,6)-N-acetyl glucosamine (PNAG) is a vaccine target detected on the surface of numerous pathogenic bacteria, including Escherichia coli. Genes encoding PNAG biosynthetic proteins have been identified in two other main pathogenic Enterobacteriaceae, Enterobacter cloacae and Klebsiella pneumoniae. We hypothesized that antibodies to PNAG might be a new therapeutic option for the different pan-resistant pathogenic species of CRE. METHODS: PNAG production was detected by confocal microscopy and its role in the formation of the biofilm (for E. cloacae) and as a virulence factor (for K. pneumoniae) was analysed. The in vitro (opsonophagocytosis killing assay) and in vivo (mouse models of peritonitis) activity of antibodies to PNAG were studied using antibiotic-susceptible and -resistant E. coli, E. cloacae and K. pneumoniae. A PNAG-producing strain of Pseudomonas aeruginosa, an organism that does not naturally produce this antigen, was constructed by adding the pga locus to a strain with inactive alg genes responsible for the production of P. aeruginosa alginate. Antibodies to PNAG were tested in vitro and in vivo as above. RESULTS: PNAG is a major component of the E. cloacae biofilm and a virulence factor for K. pneumoniae. Antibodies to PNAG mediated in vitro killing (>50%) and significantly protected mice against the New Delhi metallo-ß-lactamase-producing E. coli (Pâ=â0.02), E. cloacae (Pâ=â0.0196) and K. pneumoniae (Pâ=â0.006), against K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae (Pâ=â0.02) and against PNAG-producing P. aeruginosa (Pâ=â0.0013). Thus, regardless of the Gram-negative bacterial species, PNAG expression is the sole determinant of the protective efficacy of antibodies to this antigen. CONCLUSIONS: Our findings suggest antibodies to PNAG may provide extended-spectrum antibacterial protective activity.
Subject(s)
Antibodies, Bacterial/pharmacology , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Bacterial Vaccines/immunology , Enterobacteriaceae Infections/microbiology , Enterobacteriaceae Infections/prevention & control , Enterobacteriaceae/genetics , beta-Glucans/immunology , beta-Lactamases/biosynthesis , beta-Lactamases/genetics , Animals , Biofilms , Drug Resistance, Bacterial/drug effects , Enterobacteriaceae/enzymology , Mice , Virulence Factors/immunologyABSTRACT
An important question regarding the biologic implications of antibiotic-resistant microbes is how resistance impacts the organism's overall fitness and virulence. Currently it is generally thought that antibiotic resistance carries a fitness cost and reduces virulence. For the human pathogen Pseudomonas aeruginosa, treatment with carbapenem antibiotics is a mainstay of therapy that can lead to the emergence of resistance, often through the loss of the carbapenem entry channel OprD. Transposon insertion-site sequencing was used to analyze the fitness of 300,000 mutants of P. aeruginosa strain PA14 in a mouse model for gut colonization and systemic dissemination after induction of neutropenia. Transposon insertions in the oprD gene led not only to carbapenem resistance but also to a dramatic increase in mucosal colonization and dissemination to the spleen. These findings were confirmed in vivo with different oprD mutants of PA14 as well as with related pairs of carbapenem-susceptible and -resistant clinical isolates. Compared with OprD(+) strains, those lacking OprD were more resistant to killing by acidic pH or normal human serum and had increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that an oprD mutant showed dramatic changes in the transcription of genes that may contribute to the various phenotypic changes observed. The association between carbapenem resistance and enhanced survival of P. aeruginosa in infected murine hosts suggests that either drug resistance or host colonization can cause the emergence of more pathogenic, drug-resistant P. aeruginosa clones in a single genetic event.
Subject(s)
Carbapenems/pharmacology , Drug Resistance, Bacterial/genetics , Mutation , Porins , Pseudomonas aeruginosa , Animals , Disease Models, Animal , Drug Resistance, Bacterial/drug effects , Female , High-Throughput Nucleotide Sequencing , Humans , Hydrogen-Ion Concentration , Macrophages/metabolism , Macrophages/microbiology , Macrophages/pathology , Male , Mice , Porins/biosynthesis , Porins/genetics , Pseudomonas Infections/drug therapy , Pseudomonas Infections/genetics , Pseudomonas Infections/metabolism , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicityABSTRACT
Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A ß-(1â6)-linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule is synthesized by four proteins encoded in genetic loci designated intercellular adhesion in Staphylococcus aureus or polyglucosamine in selected Gram-negative bacterial pathogens. We report that many microbial pathogens lacking an identifiable intercellular adhesion or polyglucosamine locus produce PNAG, including Gram-positive, Gram-negative, and fungal pathogens, as well as protozoa, e.g., Trichomonas vaginalis, Plasmodium berghei, and sporozoites and blood-stage forms of Plasmodium falciparum. Natural antibody to PNAG is common in humans and animals and binds primarily to the highly acetylated glycoform of PNAG but is not protective against infection due to lack of deposition of complement opsonins. Polyclonal animal antibody raised to deacetylated glycoforms of PNAG and a fully human IgG1 monoclonal antibody that both bind to native and deacetylated glycoforms of PNAG mediated complement-dependent opsonic or bactericidal killing and protected mice against local and/or systemic infections by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, Candida albicans, and P. berghei ANKA, and against colonic pathology in a model of infectious colitis. PNAG is also a capsular polysaccharide for Neisseria gonorrhoeae and nontypable Hemophilus influenzae, and protects cells from environmental stress. Vaccination targeting PNAG could contribute to immunity against serious and diverse prokaryotic and eukaryotic pathogens, and the conserved production of PNAG suggests that it is a critical factor in microbial biology.
Subject(s)
Acetylglucosamine/immunology , Antibodies, Bacterial/immunology , Bacterial Infections/immunology , Malaria/immunology , Mycoses/immunology , Staphylococcus aureus/immunology , Animals , Antibodies, Bacterial/pharmacology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Bacterial Capsules/immunology , Bacterial Capsules/metabolism , Bacterial Infections/microbiology , Bacterial Infections/prevention & control , Fungi/immunology , Fungi/physiology , Gram-Negative Bacteria/immunology , Gram-Negative Bacteria/physiology , Gram-Positive Bacteria/immunology , Gram-Positive Bacteria/physiology , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/immunology , Humans , Immunoglobulin G/immunology , Immunoglobulin G/pharmacology , Malaria/parasitology , Malaria/prevention & control , Mice , Mice, Inbred C57BL , Mycoses/microbiology , Mycoses/prevention & control , Opsonin Proteins/immunology , Plasmodium berghei/immunology , Plasmodium berghei/physiology , Protein Binding/immunology , Staphylococcus aureus/metabolism , Survival Analysis , Time FactorsABSTRACT
High-throughput sequencing of transposon (Tn) libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in different environments. We used insertion-sequencing (INSeq) to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of â¼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200-1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian host.
Subject(s)
Bacterial Proteins/genetics , DNA Transposable Elements/genetics , Gene Library , High-Throughput Nucleotide Sequencing , Pseudomonas aeruginosa , Virulence Factors/genetics , Animals , DNA, Bacterial/genetics , Genes, Bacterial/physiology , Humans , Mice , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/pathogenicityABSTRACT
Sepsis, a leading cause of death worldwide, involves proinflammatory responses and inefficient bacterial clearance. Phagocytic cells play a crucial part in the prevention of sepsis by clearing bacteria through host innate receptors. Here we show that the FcRgamma adaptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing signal transduction subunit of the Fc receptor family, has a deleterious effect on sepsis. FcRgamma(-/-) mice show increased survival during peritonitis, owing to markedly increased E. coli phagocytosis and killing and to lower production of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. The FcRgamma-associated receptor that inhibits E. coli phagocytosis is FcgammaRIII (also called CD16), and its absence protects mice from sepsis. FcgammaRIII binds E. coli, and this interaction induces FcRgamma phosphorylation, recruitment of the tyrosine phosphatase SHP-1 and phosphatidylinositide-3 kinase (PI3K) dephosphorylation. Decreased PI3K activity inhibits E. coli phagocytosis and increases TNF-alpha production through Toll-like receptor 4. We identified the phagocytic receptor negatively regulated by FcRgamma on macrophages as the class A scavenger receptor MARCO. E. coli-FcgammaRIII interaction induces the recruitment of SHP-1 to MARCO, thereby inhibiting E. coli phagocytosis. Thus, by binding FcgammaRIII, E. coli triggers an inhibitory FcRgamma pathway that both impairs MARCO-mediated bacterial clearance and activates TNF-alpha secretion.
Subject(s)
Escherichia coli Infections/immunology , Inflammation Mediators/physiology , Phagocytosis/immunology , Receptors, IgG/physiology , Sepsis/immunology , Signal Transduction/immunology , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Cell Line , Cells, Cultured , Escherichia coli Infections/metabolism , Escherichia coli Infections/microbiology , Escherichia coli K12/immunology , Inflammation Mediators/metabolism , Macrophages/immunology , Macrophages/metabolism , Macrophages/pathology , Mice , Mice, Inbred BALB C , Mice, Knockout , Protein Tyrosine Phosphatase, Non-Receptor Type 6/physiology , Receptors, IgG/deficiency , Receptors, IgG/genetics , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/physiology , Sepsis/metabolism , Sepsis/microbiology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
ESKAPE pathogens are responsible for complicated nosocomial infections worldwide and are often resistant to commonly used antibiotics in clinical settings. Among ESKAPE, vancomycin-resistant Enterococcus faecium (VREfm) and methicillin-resistant Staphylococcus aureus (MRSA) are two important Gram-positive pathogens for which non-antibiotic alternatives are urgently needed. We previously showed that the lipoprotein AdcA of E. faecium elicits opsonic and protective antibodies against E. faecium and E. faecalis. Prompted by our observation, reported here, that AdcA also elicits opsonic antibodies against MRSA and other clinically relevant Gram-positive pathogens, we identified the dominant epitope responsible for AdcA cross-reactive activity and designed a hyper-thermostable and multi-presenting antigen, Sc(EH)3. We demonstrate that antibodies raised against Sc(EH)3 mediate opsonic killing of a wide-spectrum of Gram-positive pathogens, including VREfm and MRSA, and confer protection both in passive and active immunisation models. Our data indicate that Sc(EH)3 is a promising antigen for the development of vaccines against different Gram-positive pathogens.