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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

 

Abstract

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.

Author Summary

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…

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dishHi all

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

Chee Han Lim, Sabrina Voedisch, Benjamin Wahl, Syed Fazle Rouf, Robert Geffers, Mikael Rhen, Oliver Pabst

Abstract

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.

 

Author Summary

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.

 

Discussion Points

  1. Was the paper clearly written, figures clear etc?
  2. Was the method sound? How novel was it? Does it have other applications?
  3. Are the conclusions supported by the results
  4. What further work would you do? Would you use the method to study something else? What questions does it raise about vaccination?

 

The paper up for discussion on Tuesday 19th March, 8pm UK time is on Helicobacter and can be found here

http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1003189

Author summary:

Helicobacter pylori is a bacterium that colonizes the stomach of about half the world’s population, most of whom are asymptomatic. However, some strains of H. pylori express a bacterial secretion system, a sort of molecular syringe that injects a bacterial protein inside the gastric cells and causes inflammation that can lead to peptic ulcer disease or gastric cancer. One of the essential components of the H. pylori secretion system is CagY, which is unusual because it contains a series of repetitive amino acid motifs that are encoded by a very large number of direct DNA repeats. Here we have shown that DNA recombination in cagY changes the protein motif structure and alters the function of the secretion system turning it on or off. Using mouse and non-human primate models, we have demonstrated that CagY is a molecular switch that ‘‘tunes’’ the host inflammatory response, and likely contributes to persistent infection. Determining the mechanism by which CagY functions will enhance our understanding of the effects of H. pylori on human health, and could lead to novel applications for the modulation of host cell function.

Discussion points:

1. Is the paper well written, easy to understand and follow?

2. CagY variants evade the hosts immune response, any thoughts?

3. Is inflammation advantageous?

4. Anything you would do differently?

Next time (Tuesday 20th November) we will be discussing the following paper

Title: Rumen microbial and fermentation characteristics are affected differently by bacterial probiotic supplementation during induced lactic and subacute acidosis in sheep

 

available from the following link  http://www.biomedcentral.com/1471-2180/12/142

Abstract:

Background:

Ruminal disbiosis induced by feeding is the cause of ruminal acidosis, a digestive disorder prevalent in high-producing ruminants. Because probiotic microorganisms can modulate the gastrointestinal microbiota,

propionibacteria- and lactobacilli-based probiotics were tested for their effectiveness in preventing different forms of acidosis.

Results:

Lactic acidosis, butyric and propionic subacute ruminal acidosis (SARA) were induced by feed chalenges in three groups of four wethers intraruminally dosed with wheat, corn or beet pulp. In each group, wethers were either not supplemented (C) or supplemented with Propionibacterium P63 alone (P) or combined with L. plantarum (Lp + P) or L. rhamnosus (Lr + P). Compared with C, all the probiotics stimulated lactobacilli proliferation, which reached up to 25% of total bacteria during wheat-induced lactic acidosis. This induced a large increase in lactate concentration, which decreased ruminal pH. During the corn-induced butyric SARA, Lp + P decreased Prevotella spp. proportion with a concomitant decrease in microbial amylase activity and total volatile fatty acids concentration,and an increase in xylanase activity and pH. Relative to the beet pulp-induced propionic SARA, P and Lr + P improved ruminal pH without affecting the microbial or fermentation characteristics. Regardless of acidosis type, denaturing gradient gel electrophoresis revealed that probiotic supplementations modified the bacterial community structure.

Conclusion:

This work showed that the effectiveness of the bacterial probiotics tested depended on the acidosis type. Although these probiotics were ineffective in lactic acidosis because of a deeply disturbed rumen microbiota, some of the probiotics tested may be useful to minimize the occurrence of butyric and propionic SARA in sheep. However, their modes of action need to be further investigated.

Discussion points:

1. Is the paper well written and easy to follow?

2. Do the authors address the aims of the paper?

3. Was the data conclusive enough?

4. What other experiments could be done?

From the genome of ancient organisms we now move on to ancient therapies for the next #Microtwjc meeting.  This week’s paper (5th June 2012) goes “back to basics” reporting the ability of manuka honey to inhibit Streptococcus pyogenes biofilms and reduce binding to human tissue proteins.

A big thanks needs to go to The Society for General Microbiology (@SocGenMicro) and the journal Microbiology for making this week’s journal open access (until the 7th June 2012).  They have been and will continue to be very supportive of #Microtwjc.

If you have any problems accessing the paper then please let me know!

Summary:

Streptococcus pyogenes is a problematic organism of clinical significance, whereby infection of skin trauma sites can result in increased patient morbidity and mortality.  The authors first identified the antibacterial effects of manuka honey on both planktonic and biofilm cultures of Group A Streptococcus progenies by growing cells in the presence/absence of manuka honey.  The minimum inhibitory concentration (MIC) was  found to be 20% w/v and the minimum bactericidal concentration (MBC) was found to be 45% w/v with growth analysis showing a dose dependent inhibitory effect.

Having established a bactericidal mode of action, they went on to investigate the effects of manuka honey on biofilms, aggregation, and micro-colony formation.  Manuka honey was effective at permeating established biofilms, killing bacterial cells (reduction in CFU/ml), and reducing the overall biomass of the biofilm (reduction in crystal violet staining).  Microscopy looking at micro-colony formation showed the inability of S. pyogenes to form micro-colonies when grown in sub-lethal concentrations of manuka honey.  Binding of S. pyogenes to human tissue proteins was observed for fibronectin but not fibrinogen at sub-lethal concentrations which were confirmed by end point RT-PCR.

Based on the evidence provided by the authors they conclude that “manuka honey is effective at inhibiting the development of biofilms and disrupting established biofilms of S. pyogenes”.  They go on state that this most likely “mediated by the specific interruption of binding to host tissue ligands” and that manuka honey has potential as a “preventative measure against and treatment for wounds infected with S. pyogenes”.

Discussion Points:

  • Was the paper written clearly and logically with a natural progression of thoughts and ideas through the article?
  • Were the methods robust enough to draw conclusions from?
  • Would you have liked to see any other experiments, maybe some other host tissue proteins tested?
  • Do you agree with the authors closing statements?
  • Finally, is there place for this ancient remedy in modern health care practices?
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