I have chosen a paper based on a technique I saw being exploited at a recent conference I attended. The paper can be found using the link below and I hope it really is open access:

Long-Lived Intracellular Single-Molecule Fluorescence Using Electroporated Molecules

This is a bit different from our usual papers but there are aspects of this technique that make one think of what we can now do! However the usual rules apply:

Discussion points:

1. Is the paper well written and concise?

2. Are the experiments well designed?

3. Anything you would have done differently?

Abstract: Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; however, the scope of such studies would be increased considerably by using organic fluorophores, which are smaller and more photostable than their fluorescent protein counterparts. Here, we describe a straightforward, versatile, and high-throughput method to internalize DNA fragments and proteins labeled with organic fluorophores into live Escherichia coli by employing electroporation. We studied the copy numbers, diffusion profiles, and structure of internalized molecules at the single-molecule level in vivo, and were able to extend single-molecule observation times by two orders of magnitude compared to green fluorescent protein, allowing continuous monitoring of molecular processes occurring from seconds to minutes. We also exploited the desirable properties of organic fluorophores to perform single-molecule Förster resonance energy transfer measurements in the cytoplasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeastSaccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions addressable in microbes by single-molecule fluorescence.

Hi All

For the next #microtwjc session on Tuesday 3rd Feb 8pm GMT we will be discussing the following paper

Granulocytes Impose a Tight Bottleneck upon the Gut Luminal Pathogen Population during Salmonella Typhimurium Colitis

Published in December in PLoS Pathogens

The paper is available from the link below:

http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004557

Sorry Im posting it so close to the session I forgot it was my turn!

Abstract

Topological, chemical and immunological barriers are thought to limit infection by enteropathogenic bacteria. However, in many cases these barriers and their consequences for the infection process remain incompletely understood. Here, we employed a mouse model forSalmonella colitis and a mixed inoculum approach to identify barriers limiting the gut luminal pathogen population. Mice were infected via the oral route with wild type S. Typhimurium (S.Tm) and/or mixtures of phenotypically identical but differentially tagged S. Tm strains (“WITS”, wild-type isogenic tagged strains), which can be individually tracked by quantitative real-time PCR. WITS dilution experiments identified a substantial loss in tag/genetic diversity within the gut luminal S. Tm population by days 2–4 post infection. The diversity-loss was not attributable to overgrowth by S. Tm mutants, but required inflammation, Gr-1+ cells (mainly neutrophilic granulocytes) and most likely NADPH-oxidase-mediated defense, but not iNOS. Mathematical modelling indicated that inflammation inflicts a bottleneck transiently restricting the gut luminalS. Tm population to approximately 6000 cells and plating experiments verified a transient, inflammation- and Gr-1+ cell-dependent dip in the gut luminal S. Tm population at day 2 post infection. We conclude that granulocytes, an important clinical hallmark of S. Tm-induced inflammation, impose a drastic bottleneck upon the pathogen population. This extends the current view of inflammation-fuelled gut-luminal Salmonella growth by establishing the host response in the intestinal lumen as a double-edged sword, fostering and diminishing colonization in a dynamic equilibrium. Our work identifies a potent immune defense against gut infection and reveals a potential Achilles’ heel of the infection process which might be targeted for therapy.

Author Summary:

Salmonella Typhimurium can colonize the human intestine and cause severe diarrhea. In recent years, it has become clear that this pathogen profits from inflammatory changes in the intestinal lumen, as the inflamed gut helps Salmonella to out-compete the resident microbiota. Granulocytes transmigrating into the gut lumen were found to “foster” luminal Salmonellagrowth by providing nutrients (used by Salmonella, not the microbiota) and by releasing growth inhibitors affecting the microbiota, but not the pathogen. In this study, we extend this “fostering” concept by showing that gut luminal Salmonella Typhimurium population is itself surprisingly vulnerable to the host’s inflammatory response. Indeed, inflammation reduces the size of the gut luminal Salmonella population by as much as 105-fold at day 2 post infection. Thus, triggering of mucosal inflammation is in fact a double-edged sword by providing S. Typhimurium with a relative growth advantage against the microbiota in the gut lumen and by killing 99.999% of the gut luminal pathogen population at day 2. However, the pathogen population can recover and grow up again during the subsequent days. This changes the current view: Inflammation is not simply “beneficial” for the pathogen in the gut lumen. Instead, pathogen growth in the inflamed gut must be considered as an equilibrium between inflammation-inflicted killing and fostering growth of the surviving bacteria.

Discussion points:

1. Is the paper well written and concise?

2. Are the experiments well designed?

3. Do the results further our knowledge?

4. Anything you would have done differently?

If there is anything else you would like to discuss please use the comments box below.

See you all on Tues

Hi all

Happy New Year and welcome/ welcome back to #microtwjc. Cholera_bacteria_SEM

Next week, on Tues 20th Jan at 8pm GMT we will be looking at this paper:

Insights into Vibrio cholerae Intestinal Colonization from Monitoring Fluorescently Labeled Bacteria

Yves A. Millet, David Alvarez, Simon Ringgaard, Ulrich H. von Andrian, Brigid M. Davis, Matthew K. Waldor

PLoS Pathog 10(10): e1004405. doi: 10.1371/journal.ppat.1004405

 

Abstract

Vibrio cholerae, the agent of cholera, is a motile non-invasive pathogen that colonizes the small intestine (SI). Most of our knowledge of the processes required for V. cholerae intestinal colonization is derived from enumeration of wt and mutant V. cholerae recovered from orogastrically infected infant mice. There is limited knowledge of the distribution of V. choleraewithin the SI, particularly its localization along the villous axis, or of the bacterial and host factors that account for this distribution. Here, using confocal and intravital two-photon microscopy to monitor the localization of fluorescently tagged V. cholerae strains, we uncovered unexpected and previously unrecognized features of V. cholerae intestinal colonization. Direct visualization of the pathogen within the intestine revealed that the majority of V. choleraemicrocolonies attached to the intestinal epithelium arise from single cells, and that there are notable regiospecific aspects to V. cholerae localization and factors required for colonization. In the proximal SI, V. cholerae reside exclusively within the developing intestinal crypts, but they are not restricted to the crypts in the more distal SI. Unexpectedly, V. cholerae motility proved to be a regiospecific colonization factor that is critical for colonization of the proximal, but not the distal, SI. Furthermore, neither motility nor chemotaxis were required for proper V. choleraedistribution along the villous axis or in crypts, suggesting that yet undefined processes enable the pathogen to find its niches outside the intestinal lumen. Finally, our observations suggest that host mucins are a key factor limiting V. cholerae intestinal colonization, particularly in the proximal SI where there appears to be a more abundant mucus layer. Collectively, our findings demonstrate the potent capacity of direct pathogen visualization during infection to deepen our understanding of host pathogen interactions.

 

Author Summary

Vibrio cholerae is a highly motile bacterium that causes the diarrheal disease cholera. Despite our extensive knowledge of the genes and processes that enable this non-invasive pathogen to colonize the small intestine, there is limited knowledge of the pathogen’s fine localization within the intestine. Here, we used fluorescence microscopy-based techniques to directly monitor where and how fluorescent V. cholerae localize along intestinal villi in infected infant mice. This approach enabled us to uncover previously unappreciated features of V. cholerae intestinal colonization. We found that most V. cholerae microcolonies appear to arise from single cells attached to the epithelium. Unexpectedly, we observed considerable differences between V. cholerae fine localization in different parts of the small intestine and found that V. choleraemotility exerts a regiospecific influence on colonization. The abundance of intestinal mucins appears to be an important factor explaining at least some of the regiospecific aspects of V. cholerae intestinal localization. Overall, our findings suggest that direct observation of fluorescent pathogens during infection, coupled with genetic and/or pharmacologic manipulations of pathogen and host processes, adds a valuable depth to understanding of host-pathogen interactions.

 

Discussion Points

  1. Was the paper well written? Clear? Easy to follow?
  2. Were the methods appropriate? Were there any methods/experiments that you thought were missing? Were the stats appropriate?
  3. Were the conclusions supported by the results? What impact do you think these results will have on the wider V cholera field?
  4. What experiments would you like to see done in the future to build upon this work?
  5. Anything else?  Please comment in the box below

Looking forward to see you there (just search for #microtwjc on the night and the tweets should come up automatically).  Please spread the word to your colleagues – the more the merrier :)

This week, Tues 25th November, we will be looking at this paper 

Shed GP of Ebola Virus Triggers Immune Activation and Increased Vascular Permeability

Beatriz Escudero-Pérez, Valentina A. Volchkova, Olga Dolnik, Philip Lawrence, Viktor E. Volchkov mail

 

Discussion points

  1. Is the paper well written?
  2. Do the results support the conclusions?
  3. What work would you like to see done in the future?

 

Hope to see you there on Tuesday

 

Zoo

This week on Microbiology Twitter Journal Club, we’ll be discussing  a brand new broad spectrum antiviral Favipiravir, and it’s efficacy compared to Ribavirin against Mouse Norovirus.

Abstract

Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses.

Discussion points:

  • Is Favipiravir better than Ribavirin, and does the paper provide enough evidence for that ?
  • Was the data presented clearly ?
  • Do those stars above the graphs mean Christmas is coming, or something more foreboding ?
  • What would you have done differently?

Join the discussion at 8pm GMT tomorrow with #MicroTwJC.

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