Segmented Filamentous Bacteria – Metabolism Meets Immunity

Segmented filamentous bacteria (SFB) are a group of host-adapted, commensal organisms that attach to the ileal epithelium of vertebrate and invertebrate hosts. A genetic relative of the genus Clostridium, these morphologically unique bacteria display a replication and differentiation lifecycle initiated by epithelial tissue binding and filamentation. SFB intimately bind to the surface of absorptive intestinal epithelium without inducing an inflammatory response. Rather, their presence impacts the generation of innate and differentiation of acquired immunity, which impact the clearance of extracellular bacterial or fungal pathogens in the gastrointestinal and respiratory tracts. SFB have recently garnered attention due to their role in promoting adaptive and innate immunity in mice and rats through the differentiation and maturation of Th17 cells in the intestinal tract and production of immunoglobulin A (IgA). SFB are the first commensal bacteria identified that impact the maturation and development of Th17 cells in mice. Recently, microbiome studies have revealed the presence of Candidatus Arthromitus (occasionally designated as Candidatus Savagella), a proposed candidate species of SFB, in higher proportions in higher-performing flocks as compared to matched lower-performing flocks, suggesting that SFB may serve to establish a healthy gut and protect commercial turkeys from pathogens resulting in morbidity and decreased performance. In this review we seek to describe the life cycle, host specificity, and genetic capabilities of SFB, such as bacterial metabolism, and how these factors influence the host immunity and microbiome. Although the role of SFB to induce antigen-specific Th17 cells in poultry is unknown, they may play an important role in modulating the immune response in the intestinal tract to promote resistance against some infectious diseases and promote food-safety. This review demonstrates the importance of studying and further characterizing commensal, host-specific bacteria in food-producing animals and their importance to animal health

Acid stress damage of DNA is prevented by Dps binding in Escherichia coli O157:H7

<p>Abstract</p> <p>Background</p> <p>Acid tolerance in <it>Escherichia coli </it>O157:H7 contributes to persistence in its bovine host and is thought to promote passage through the gastric barrier of humans. Dps (DNA-binding protein in starved cells) mutants of <it>E. coli </it>have reduced acid tolerance when compared to the parent strain although the role of Dps in acid tolerance is unclear. This study investigated the mechanism by which Dps contributes to acid tolerance in <it>E. coli </it>O157:H7.</p> <p>Results</p> <p>The results from this study showed that acid stress lead to damage of chromosomal DNA, which was accentuated in <it>dps </it>and <it>recA </it>mutants. The use of <it>Bal</it>31, which cleaves DNA at nicks and single-stranded regions, to analyze chromosomal DNA extracted from cells challenged at pH 2.0 provided <it>in vivo </it>evidence of acid damage to DNA. The DNA damage in a <it>recA </it>mutant further corroborated the hypothesis that acid stress leads to DNA strand breaks. Under <it>in vitro </it>assay conditions, Dps was shown to bind plasmid DNA directly and protect it from acid-induced strand breaks. Furthermore, the extraction of DNA from Dps-DNA complexes required a denaturing agent at low pH (2.2 and 3.6) but not at higher pH (>pH4.6). Low pH also restored the DNA-binding activity of heat-denatured Dps. Circular dichroism spectra revealed that at pH 3.6 and pH 2.2 Dps maintains or forms α-helices that are important for Dps-DNA complex formation.</p> <p>Conclusion</p> <p>Results from the present work showed that acid stress results in DNA damage that is more pronounced in <it>dps </it>and <it>recA </it>mutants. The contribution of RecA to acid tolerance indicated that DNA repair was important even when Dps was present. Dps protected DNA from acid damage by binding to DNA. Low pH appeared to strengthen the Dps-DNA association and the secondary structure of Dps retained or formed α-helices at low pH. Further investigation into the precise interplay between DNA protection and damage repair pathways during acid stress are underway to gain additional insight.</p

The evolution of metabolic networks of E. coli

<p>Abstract</p> <p>Background</p> <p>Despite the availability of numerous complete genome sequences from <it>E. coli </it>strains, published genome-scale metabolic models exist only for two commensal <it>E. coli </it>strains. These models have proven useful for many applications, such as engineering strains for desired product formation, and we sought to explore how constructing and evaluating additional metabolic models for <it>E. coli </it>strains could enhance these efforts.</p> <p>Results</p> <p>We used the genomic information from 16 <it>E. coli </it>strains to generate an <it>E. coli </it>pangenome metabolic network by evaluating their collective 76,990 ORFs. Each of these ORFs was assigned to one of 17,647 ortholog groups including ORFs associated with reactions in the most recent metabolic model for <it>E. coli </it>K-12. For orthologous groups that contain an ORF already represented in the MG1655 model, the gene to protein to reaction associations represented in this model could then be easily propagated to other <it>E. coli </it>strain models. All remaining orthologous groups were evaluated to see if new metabolic reactions could be added to generate a pangenome-scale metabolic model (iEco1712_pan). The pangenome model included reactions from a metabolic model update for <it>E. coli </it>K-12 MG1655 (iEco1339_MG1655) and enabled development of five additional strain-specific genome-scale metabolic models. These additional models include a second K-12 strain (iEco1335_W3110) and four pathogenic strains (two enterohemorrhagic <it>E. coli </it>O157:H7 and two uropathogens). When compared to the <it>E. coli </it>K-12 models, the metabolic models for the enterohemorrhagic (iEco1344_EDL933 and iEco1345_Sakai) and uropathogenic strains (iEco1288_CFT073 and iEco1301_UTI89) contained numerous lineage-specific gene and reaction differences. All six <it>E. coli </it>models were evaluated by comparing model predictions to carbon source utilization measurements under aerobic and anaerobic conditions, and to batch growth profiles in minimal media with 0.2% (w/v) glucose. An ancestral genome-scale metabolic model based on conserved ortholog groups in all 16 <it>E. coli </it>genomes was also constructed, reflecting the conserved ancestral core of <it>E. coli </it>metabolism (iEco1053_core). Comparative analysis of all six strain-specific <it>E. coli </it>models revealed that some of the pathogenic <it>E. coli </it>strains possess reactions in their metabolic networks enabling higher biomass yields on glucose. Finally the lineage-specific metabolic traits were compared to the ancestral core model predictions to derive new insight into the evolution of metabolism within this species.</p> <p>Conclusion</p> <p>Our findings demonstrate that a pangenome-scale metabolic model can be used to rapidly construct additional <it>E. coli </it>strain-specific models, and that quantitative models of different strains of <it>E. coli </it>can accurately predict strain-specific phenotypes. Such pangenome and strain-specific models can be further used to engineer metabolic phenotypes of interest, such as designing new industrial <it>E. coli </it>strains.</p

H-NS controls metabolism and stress tolerance in Escherichia coli O157:H7 that influence mouse passage

BACKGROUND: H-NS is a DNA-binding protein with central roles in gene regulation and nucleoid structuring in Escherichia coli. There are over 60 genes that are influenced by H-NS many of which are involved in metabolism. To determine the significance of H-NS-regulated genes in metabolism and stress tolerance, an hns mutant of E. coli O157:H7 was generated (hns::nptI, FRIK47001P) and its growth, metabolism, and gastrointestinal passage compared to the parent strain (43895) and strain FRIK47001P harboring pSC0061 which contains a functional hns and 90-bp upstream of the open-reading frame. RESULTS: The hns mutant grew slower and was non-motile in comparison to the parent strain. Carbon and nitrogen metabolism was significantly altered in the hns mutant, which was incapable of utilizing 42 carbon, and 19 nitrogen sources that the parent strain metabolized. Among the non-metabolized substrates were several amino acids, organic acids, and key metabolic intermediates (i.e., pyruvate) that limit carbon acquisition and energy generation. Growth studies determined that the parent strain grew in LB containing 14 to 15% bile or bile salts, while the hns mutant grew in 6.5 and 9% of these compounds, respectively. Conversely, log-phase cells of the hns mutant were significantly (p < 0.05) more acid tolerant than the parent strain and hns mutant complemented with pSC0061. In mouse passage studies, the parent strain was recovered at a higher frequency (p < 0.01) than the hns mutant regardless of whether log- or stationary-phase phase cells were orally administered. CONCLUSION: These results demonstrate that H-NS is a powerful regulator of carbon and nitrogen metabolism as well as tolerance to bile salts. It is likely that the metabolic impairments and/or the reduced bile tolerance of the E. coli O157:H7 hns mutant decreased its ability to survive passage through mice. Collectively, these results expand the influence of H-NS on carbon and nitrogen metabolism and highlight its role in the ability of O157:H7 strains to respond to changing nutrients and conditions encountered in the environment and its hosts

Using Comparative Genomics for Inquiry-Based Learning to Dissect Virulence of Escherichia coli O157:H7 and Yersinia pestis

Genomics and bioinformatics are topics of increasing interest in undergraduate biological science curricula. Many existing exercises focus on gene annotation and analysis of a single genome. In this paper, we present two educational modules designed to enable students to learn and apply fundamental concepts in comparative genomics using examples related to bacterial pathogenesis. Students first examine alignments of genomes of Escherichia coli O157:H7 strains isolated from three food-poisoning outbreaks using the multiple-genome alignment tool Mauve. Students investigate conservation of virulence factors using the Mauve viewer and by browsing annotations available at the A Systematic Annotation Package for Community Analysis of Genomes database. In the second module, students use an alignment of five Yersinia pestis genomes to analyze single-nucleotide polymorphisms of three genes to classify strains into biovar groups. Students are then given sequences of bacterial DNA amplified from the teeth of corpses from the first and second pandemics of the bubonic plague and asked to classify these new samples. Learning-assessment results reveal student improvement in self-efficacy and content knowledge, as well as students’ ability to use BLAST to identify genomic islands and conduct analyses of virulence factors from E. coli O157:H7 or Y. pestis. Each of these educational modules offers educators new ready-to-implement resources for integrating comparative genomic topics into their curricula

Bordetella pertussis Infection or Vaccination Substantially Protects Mice against B. bronchiseptica Infection

Although B. bronchiseptica efficiently infects a wide range of mammalian hosts and efficiently spreads among them, it is rarely observed in humans. In contrast to the many other hosts of B. bronchiseptica, humans are host to the apparently specialized pathogen B. pertussis, the great majority having immunity due to vaccination, infection or both. Here we explore whether immunity to B. pertussis protects against B. bronchiseptica infection. In a murine model, either infection or vaccination with B. pertussis induced antibodies that recognized antigens of B. bronchiseptica and protected the lower respiratory tract of mice against three phylogenetically disparate strains of B. bronchiseptica that efficiently infect naïve animals. Furthermore, vaccination with purified B. pertussis-derived pertactin, filamentous hemagglutinin or the human acellular vaccine, Adacel, conferred similar protection against B. bronchiseptica challenge. These data indicate that individual immunity to B. pertussis affects B. bronchiseptica infection, and suggest that the high levels of herd immunity against B. pertussis in humans could explain the lack of observed B. bronchiseptica transmission. This could also explain the apparent association of B. bronchiseptica infections with an immunocompromised state

Using genome-scale metabolic models to compare serovars of the foodborne pathogen <i>Listeria monocytogenes</i>

<div><p><i>Listeria monocytogenes</i> is a microorganism of great concern for the food industry and the cause of human foodborne disease. Therefore, novel methods of control are needed, and systems biology is one such approach to identify them. Using a combination of computational techniques and laboratory methods, genome-scale metabolic models (GEMs) can be created, validated, and used to simulate growth environments and discern metabolic capabilities of microbes of interest, including <i>L</i>. <i>monocytogenes</i>. The objective of the work presented here was to generate GEMs for six different strains of <i>L</i>. <i>monocytogenes</i>, and to both qualitatively and quantitatively validate these GEMs with experimental data to examine the diversity of metabolic capabilities of numerous strains from the three different serovar groups most associated with foodborne outbreaks and human disease. Following qualitative validation, 57 of the 95 carbon sources tested experimentally were present in the GEMs, and; therefore, these were the compounds from which comparisons could be drawn. Of these 57 compounds, agreement between <i>in silico</i> predictions and <i>in vitro</i> results for carbon source utilization ranged from 80.7% to 91.2% between strains. Nutrient utilization agreement between <i>in silico</i> predictions and <i>in vitro</i> results were also conducted for numerous nitrogen, phosphorous, and sulfur sources. Additionally, quantitative validation showed that the <i>L</i>. <i>monocytogenes</i> GEMs were able to generate <i>in silico</i> predictions for growth rate and growth yield that were strongly and significantly (p < 0.0013 and p < 0.0015, respectively) correlated with experimental results. These findings are significant because they show that these GEMs for <i>L</i>. <i>monocytogenes</i> are comparable to published GEMs of other organisms for agreement between <i>in silico</i> predictions and <i>in vitro</i> results. Therefore, as with the other GEMs, namely those for <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, <i>Vibrio vulnificus</i>, and <i>Salmonella</i> spp., they can be used to determine new methods of growth control and disease treatment.</p></div

Comparison of <i>in silico</i> predictions to experimental results for 39 individual carbon sources.

<p>The 39 carbon sources displayed are those with at least one disagreement.</p