David Skurnik

The population genetics of commensal Escherichia coli

The primary habitat of Escherichia coli is the vertebrate gut, where it is the predominant aerobic organism, living in symbiosis with its host. Despite the occurrence of recombination events, the population structure is predominantly clonal, allowing the delineation of major phylogenetic groups. The genetic structure of commensal E. coli is shaped by multiple host and environmental factors, and the determinants involved in the virulence of the bacteria may in fact reflect adaptation to commensal habitats. A better characterization of the commensal niche is necessary to understand how a useful commensal can become a harmful pathogen. In this Review we describe the population structure of commensal E. coli, the factors involved in the spread of different strains, how the bacteria can adapt to different niches and how a commensal lifestyle can evolve into a pathogenic one.

Parallel bacterial evolution within multiple patients identifies candidate pathogenicity genes

Bacterial pathogens evolve during the infection of their human host, but separating adaptive and neutral mutations remains challenging. Here we identify bacterial genes under adaptive evolution by tracking recurrent patterns of mutations in the same pathogenic strain during the infection of multiple individuals. We conducted a retrospective study of a Burkholderia dolosa outbreak among subjects with cystic fibrosis, sequencing the genomes of 112 isolates collected from 14 individuals over 16 years. We find that 17 bacterial genes acquired nonsynonymous mutations in multiple individuals, which indicates parallel adaptive evolution. Mutations in these genes affect important pathogenic phenotypes, including antibiotic resistance and bacterial membrane composition and implicate oxygen-dependent regulation as paramount in lung infections. Several genes have not previously been implicated in pathogenesis and may represent new therapeutic targets. The identification of parallel molecular evolution as a pathogen spreads among multiple individuals points to the key selection forces it experiences within human hosts.

Fungal microbiota dysbiosis in IBD

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 Spearmans 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 Crohns 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.

NADPH oxidase 1 modulates WNT and NOTCH1 signaling to control the fate of proliferative progenitor cells in the colon.

ABSTRACT The homeostatic self-renewal of the colonic epithelium requires coordinated regulation of the canonical Wnt/β-catenin and Notch signaling pathways to control proliferation and lineage commitment of multipotent stem cells. However, the molecular mechanisms by which the Wnt/β-catenin and Notch1 pathways interplay in controlling cell proliferation and fate in the colon are poorly understood. Here we show that NADPH oxidase 1 (NOX1), a reactive oxygen species (ROS)-producing oxidase that is highly expressed in colonic epithelial cells, is a pivotal determinant of cell proliferation and fate that integrates Wnt/β-catenin and Notch1 signals. NOX1-deficient mice reveal a massive conversion of progenitor cells into postmitotic goblet cells at the cost of colonocytes due to the concerted repression of phosphatidylinositol 3-kinase (PI3K)/AKT/Wnt/β-catenin and Notch1 signaling. This conversion correlates with the following: (i) the redox-dependent activation of the dual phosphatase PTEN, causing the inactivation of the Wnt pathway effector β-catenin, and (ii) the downregulation of Notch1 signaling that provokes derepression of mouse atonal homolog 1 (Math1) expression. We conclude that NOX1 controls the balance between goblet and absorptive cell types in the colon by coordinately modulating PI3K/AKT/Wnt/β-catenin and Notch1 signaling. This finding provides the molecular basis for the role of NOX1 in cell proliferation and postmitotic differentiation.

Integron-Associated Antibiotic Resistance and Phylogenetic Grouping of Escherichia coli Isolates from Healthy Subjects Free of Recent Antibiotic Exposure

ABSTRACT The study of integrons in 181 Escherichia coli isolates from three groups of healthy subjects who lived in communities and had not taken antibiotics for at least 1 month showed that the presence of integrons was associated with antibiotic resistance and phylogenetic grouping of the bacterial host and dependent on a subjects living environment.

CD16 promotes Escherichia coli sepsis through an FcR

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 FcRγ adaptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing signal transduction subunit of the Fc receptor family, has a deleterious effect on sepsis. FcRγ−/− 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)-α. The FcRγ-associated receptor that inhibits E. coli phagocytosis is FcγRIII (also called CD16), and its absence protects mice from sepsis. FcγRIII binds E. coli, and this interaction induces FcRγ phosphorylation, recruitment of the tyrosine phosphatase SHP-1 and phosphatidylinositide-3 kinase (PI3K) dephosphorylation. Decreased PI3K activity inhibits E. coli phagocytosis and increases TNF-α production through Toll-like receptor 4. We identified the phagocytic receptor negatively regulated by FcRγ on macrophages as the class A scavenger receptor MARCO. E. coli-FcγRIII interaction induces the recruitment of SHP-1 to MARCO, thereby inhibiting E. coli phagocytosis. Thus, by binding FcγRIII, E. coli triggers an inhibitory FcRγ pathway that both impairs MARCO-mediated bacterial clearance and activates TNF-α secretion.

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ABSTRACT Vaccines for pathogens usually target strain-specific surface antigens or toxins, and rarely is there broad antigenic specificity extending across multiple species. Protective antibodies for bacteria are usually specific for surface or capsular antigens. β-(1→6)-Poly-N-acetyl-d-glucosamine (PNAG) is a surface polysaccharide produced by many pathogens, including Staphylococcus aureus, Escherichia coli, Yersinia pestis, Bordetella pertussis, Acinetobacter baumannii, and others. Protective antibodies to PNAG are elicited when a deacetylated glycoform (deacetylated PNAG [dPNAG]; <30% acetate) is used in conjugate vaccines, whereas highly acetylated PNAG does not induce such antibodies. Chemical derivation of dPNAG from native PNAG is imprecise, so we synthesized both β-(1→6)-d-glucosamine (GlcNH2) and β-(1→6)-d-N-acetylglucosamine (GlcNAc) oligosaccharides with linkers on the reducing termini that could be activated to produce sulfhydryl groups for conjugation to bromoacetyl groups introduced onto carrier proteins. Synthetic 5-mer GlcNH2 (5GlcNH2) or 9GlcNH2 conjugated to tetanus toxoid (TT) elicited mouse antibodies that mediated opsonic killing of multiple S. aureus strains, while the antibodies that were produced in response to 5GlcNAc- or 9GlcNAc-TT did not mediate opsonic killing. Rabbit antibodies to 9GlcNH2-TT bound to PNAG and dPNAG antigens, mediated killing of S. aureus and E. coli, and protected against S. aureus skin abscesses and lethal E. coli peritonitis. Chemical synthesis of a series of oligoglucosamine ligands with defined differences in N acetylation allowed us to identify a conjugate vaccine formulation that generated protective immune responses to two of the most challenging bacterial pathogens. This vaccine could potentially be used to engender protective immunity to the broad range of pathogens that produce surface PNAG.

inhibitory pathway that prevents phagocytosis and facilitates inflammation

Significance Poly-N-acetylglucosamine (PNAG) has been identified as a conserved surface polysaccharide produced by major bacterial, fungal, and protozoal parasites, including malarial sporozoites and blood-stage forms, which can all be targeted for vaccination using this single antigen. Surface carbohydrates are among the most successful vaccines against human microbial pathogens but have tremendous variability that complicates vaccine development. The species of bacteria, fungi, and protozoa shown here to produce PNAG lack an identifiable genetic locus for this antigen’s biosynthetic proteins based on known loci, indicative of a possible evolutionary convergent acquisition of PNAG synthesis with potential important significance for microbial biology. Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A β-(1→6)–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.

Synthetic β-(1→6)-Linked N-Acetylated and Nonacetylated Oligoglucosamines Used To Produce Conjugate Vaccines for Bacterial Pathogens

Resistance to antibiotics is a major public health problem which might culminate in outbreaks caused by pathogenic bacteria untreatable by known antibiotics. Most of the genes conferring resistance are acquired horizontally from already resistant commensal or environmental bacteria. Food contamination by resistant bacteria might be a significant source of resistance genes for human bacteria but has never been precisely assessed, nor is it known whether organic products differ in this respect from conventionally produced products. We showed here, on a large year-long constructed sample set containing 399 products that, irrespective of their mode of production, raw fruits and vegetables are heavily contaminated by Gram-negative bacteria (GNB) resistant to multiple antibiotics. Most of these bacteria originate in the soil and environment. We focused on non-oxidative GNB resistant to third-generation cephalosporins, because of their potential impact on human health. Among them, species potentially pathogenic for immunocompetent hosts were rare. Of the products tested, 13% carried bacteria producing extended-spectrum beta-lactamases, all identified as Rahnella sp. which grouped into two phylotypes and all carrying the bla(RAHN) gene. Thus, both organic and conventional fruits and vegetables may constitute significant sources of resistant bacteria and of resistance genes.

Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens

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 ∼300,000 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.