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Tuesday’s #microtwjc (8pm GMT) will be looking at this paper:
Active Transport of Phosphorylated Carbohydrates Promotes Intestinal Colonization and Transmission of a Bacterial Pathogen
Brandon Sit , Shauna M. Crowley , Kirandeep Bhullar, Christine Chieh-Lin Lai, Calvin Tang, Yogesh Hooda, Charles Calmettes, Husain Khambati, Caixia Ma, John H. Brumell, Anthony B. Schryvers, Bruce A. Vallance , Trevor F. Moraes
Efficient acquisition of extracellular nutrients is essential for bacterial pathogenesis, however the identities and mechanisms for transport of many of these substrates remain unclear. Here, we investigate the predicted iron-binding transporter AfuABC and its role in bacterial pathogenesis in vivo. By crystallographic, biophysical and in vivo approaches, we show that AfuABC is in fact a cyclic hexose/heptose-phosphate transporter with high selectivity and specificity for a set of ubiquitous metabolites (glucose-6-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate). AfuABC is conserved across a wide range of bacterial genera, including the enteric pathogens EHEC O157:H7 and its murine-specific relative Citrobacter rodentium, where it lies adjacent to genes implicated in sugar sensing and acquisition. C. rodentium ΔafuA was significantly impaired in an in vivo murine competitive assay as well as its ability to transmit infection from an afflicted to a naïve murine host. Sugar-phosphates were present in normal and infected intestinal mucus and stool samples, indicating that these metabolites are available within the intestinal lumen for enteric bacteria to import during infection. Our study shows that AfuABC-dependent uptake of sugar-phosphates plays a critical role during enteric bacterial infection and uncovers previously unrecognized roles for these metabolites as important contributors to successful pathogenesis.
Essentially all Gram-negative pathogens are reliant on specific transport machineries termed binding protein-dependent transporters (BPDTs) to transport solutes such as amino acids, sugars and metal ions across their membranes. In this study we investigated AfuABC, a predicted iron-transporting BPDT found in many bacterial pathogens. We show by structural and functional approaches that AfuABC is not an iron transporter. Instead, AfuABC is a trio of proteins that bind and transport sugar-phosphates such as glucose-6-phosphate (G6P). In doing so, we present the first structural solution of a G6P-specific transport protein and add to the few known unique machineries for sugar-phosphate uptake by bacteria. Furthermore, we show that AfuABC is required by the intestinal pathogen C. rodentium to effectively transmit between mice and re-establish infection, leading us to propose that the transport of sugar-phosphates is an important part of general bacterial pathogenesis.
Discussion points to follow…
It’s a pretty straight forward paper which details the preparation of an ancient antimicrobial recipe and some studies on it’s antimicrobial efficacy. Some of the authors are on twitter (@friendlymicrobe, @sbi5ar and @stevediggle) which means we may have the opportunity to ask them questions directly.
Please keep the following discussion points/questions in mind when reading the paper and feel free to tweet to @microtwjc or comment below with any points/questions you might have during reading as well as any question you might have for the authors directly.
- How does the introduction support the research and (in addition to the discussion) help contextualize the findings in the field?
- What are the advantages and limitations of the experimental models chosen to study the antimicrobial effect of Bald’s eyesalve?
- Are the conclusions supported by the data (methods and results) in the paper?
- What have we learnt after reading the paper?
- Given what we have learnt, what future work would be of interest to conduct?
Here’s the abstract and hope to see you online!
Plant-derived compounds and other natural substances are a rich potential source of compounds that kill or attenuate pathogens that are resistant to current antibiotics. Medieval societies used a range of these natural substances to treat conditions clearly recognizable to the modern eye as microbial infections, and there has been much debate over the likely efficacy of these treatments. Our interdisciplinary team, comprising researchers from both sciences and humanities, identified and reconstructed a potential remedy for Staphylococcus aureus infection from a 10th century Anglo-Saxon leechbook. The remedy repeatedly killed established S. aureus biofilms in an in vitro model of soft tissue infection and killed methicillin-resistant S. aureus (MRSA) in a mouse chronic wound model. While the remedy contained several ingredients that are individually known to have some antibacterial activity, full efficacy required the combined action of several ingredients, highlighting the scholarship of premodern doctors and the potential of ancient texts as a source of new antimicrobial agents.
This week, Tues 25th November, we will be looking at this paper
Shed GP of Ebola Virus Triggers Immune Activation and Increased Vascular Permeability
- Is the paper well written?
- Do the results support the conclusions?
- What work would you like to see done in the future?
Hope to see you there on Tuesday
The next #microtwjc will take place on Tues 12th August at 8pm BST (and won’t clash with the Great British Bake Off this series!)
We will be looking at another Salmonella paper but this paper focuses on population dynamics: both a mechanism to study the dynamics and how vaccination interacts with them. The paper can be found here
Independent Bottlenecks Characterize Colonization of Systemic Compartments and Gut Lymphoid Tissue by Salmonella
Sabrina Voedisch, Benjamin Wahl, Syed Fazle Rouf, Robert Geffers, Mikael Rhen, Oliver Pabst
Vaccination represents an important instrument to control typhoid fever in humans and protects mice from lethal infection with mouse pathogenic serovars of Salmonella species. Mixed infections with tagged Salmonella can be used in combination with probabilistic models to describe the dynamics of the infection process. Here we used mixed oral infections with taggedSalmonella strains to identify bottlenecks in the infection process in naïve and vaccinated mice. We established a next generation sequencing based method to characterize the composition of tagged Salmonella strains which offers a fast and reliable method to characterise the composition of genome-tagged Salmonella strains. We show that initial colonization ofSalmonella was distinguished by a non-Darwinian selection of few bacteria setting up the infection independently in gut associated lymphoid tissue and systemic compartments. Colonization of Peyer’s patches fuels the sustained spread of bacteria into mesenteric lymph nodes via dendritic cells. In contrast, infection of liver and spleen originated from an independent pool of bacteria. Vaccination only moderately reduced invasion of Peyer’s patches but potently uncoupled bacterial populations present in different systemic compartments. Our data indicate that vaccination differentially skews the capacity of Salmonella to colonize systemic and gut immune compartments and provide a framework for the further dissection of infection dynamics.
Pathogens have evolved strategies to invade, replicate and spread within their hosts. On the contrary, vertebrates have developed sophisticated immune defence mechanisms that limit, and ideally clear, the infection. This dynamic interplay between host and pathogens determines the course of the infection and the development of clinical disease. Knowledge on particularly vulnerable steps in the infection process, i.e. the “Achilles heel” of a pathogen, may guide the development of anti-infective therapies and vaccines. However, for most pathogens we lack detailed information on the dynamics of the infection process. Here we determined bottlenecks, i.e. critical steps during pathogen invasion and spread, after oral Salmonella infection in non-manipulated and vaccinated mice. We infected mice with mixtures of tagged Salmonella strains and analysed the strain composition in different compartments by high throughput sequencing. This information allowed us to estimate the number of Salmonella invading a given tissue and to describe routes of pathogen dissemination. We show that vaccination only modestly reduces invasion of intestinal lymphoid tissue but had a profound effect on the spread of Salmonella to systemic compartments.
- Was the paper clearly written, figures clear etc?
- Was the method sound? How novel was it? Does it have other applications?
- Are the conclusions supported by the results
- What further work would you do? Would you use the method to study something else? What questions does it raise about vaccination?
Our next #microtwjc session will be on Tuesday 8th July 8pm (BST).
We will be discussing the following paper Fructose-Asparagine Is a Primary Nutrient during Growth of Salmonella in the Inflamed Intestine by Ali et al published in PLoS pathogens in June 2014. The link to the paper is here http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1004209
Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that Salmonella encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn), which is essential for Salmonella fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10−/− mice). The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1− SPI2− or ttrA mutants, respectively). The severe fitness defect of a Salmonella fra mutant suggests that F-Asn is the primary nutrient utilized by Salmonella in the inflamed intestine and that this system provides a valuable target for novel therapies.
It has long been thought that the nutrient utilization systems of Salmonella would not make effective drug targets because there are simply too many nutrients available to Salmonella in the intestine. Surprisingly, we have discovered that Salmonella relies heavily on a single nutrient during growth in the inflamed intestine, fructose-asparagine (F-Asn). A mutant of Salmonella that cannot obtain F-Asn is severely attenuated, suggesting that F-Asn is the primary nutrient utilized by Salmonella during inflammation. No other organism has been reported to synthesize or utilize this novel biological compound. The novelty of this nutrient and the apparent lack of utilization systems in mammals and most other bacteria suggest that the F-Asn utilization system represents a specific and potent therapeutic target for Salmonella.
1. Is the paper well written and easy to follow and understand?
2. Are the methods adequate?
3. Do the results further our knowledge?
4. Any other experiments you would do?