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?

 

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

Abstract

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.

Author Summary

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.

Discussion points
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?

Hi everyone, in our next session on Tuesday, the 24th of June at 8 pm (BST) we will look at the following paper:

“Circulating Avian Influenza Viruses Closely Related to the 1918 VirusHave Pandemic Potential” by Watanabe et al from Japan & the US published on the 11th of June 2014 in cell Host&Microbe available here:

http://www.cell.com/cell-host-microbe/pdf/S1931-3128(14)00163-2.pdf

SUMMARY:

Wild birds harbor a large gene pool of influenza A viruses that have the potential to cause influenza pandemics. Foreseeing and understanding this potentialis important for effective surveillance. Our phylogenetic and geographic analyses revealed the global prevalence of avian influenza virus genes whose proteins differ only a few amino acids from the 1918 pandemic influenza virus, suggesting that 1918-like pandemic viruses may emerge in the future. To assess this risk, we generated and characterized a virus composed of avian influenza viral segments with high homology to the 1918 virus. This virus exhibited pathogenicity in mice and ferrets higher than that in an authentic avian influenza virus. Further, acquisition of seven amino acid substitutions in the viral polymerases and the hemagglutinin surface glycoprotein conferred respiratory droplet transmission to the 1918-like avian virus in ferrets, demonstrating that contemporary avian influenza viruses with 1918 virus-like proteins may have pandemic potential.

The paper also got some news coverage, some examples can be found here:

http://www.sciencedaily.com/releases/2014/06/140611131551.htm

http://www.independent.co.uk/news/science/american-scientists-controversially-recreate-deadly-spanish-flu-virus-9529707.html

http://www.dailymail.co.uk/sciencetech/article-2655227/Bird-flu-viruses-unleash-pandemic-ferocious-one-killed-50-MILLION-people-1918-study-shows.html

Discussion points:

1. Was the publication written for an easy understanding?

2. Are the methods chosen appropiately?

3. Is the data well presented?

4. Does the paper achieve what the authors set it out?

5. What do you think of this research? Is it ethically to perform this high-risk research? Or would it be better to study it only theoretically? (to include the public discussion aspect)

Hope to see many of you there,

@Janitensen

Hi,

@Stewart_Barker here, very happy to be running the 50th meeting of the #MicroTwJC!

I will be tweeting from @MicroTwJC on the night (10th June, 8pm GMT), please remember to use #MicroTwJC on all tweets!

I stuck with the paper’s original title (I mean how can you re-word that?!), which can be found here: http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0098514&representation=PDF

Although some may flinch at the term ‘vaginal microbiome’, it is an important area of cross-interdisciplinary study that has implications for infections during pregnancy. This study looks at n=12 caucasian women, and compares to a ‘complementary study’ on african-american women.

The usual questions apply!:

  1. Is the paper easy to understand?
  2. Are the methods used suitable and correct?
  3. Is the data well presented and analysed?
  4. Does the paper achieve what the authors set out to do?
  5. What further work can be carried out?

 

Abstract

Objective: To assess the vaginal microbiome throughout full-term uncomplicated pregnancy.

Methods: Vaginal swabs were obtained from twelve pregnant women at 8-week intervals throughout their uncomplicated
pregnancies. Patients with symptoms of vaginal infection or with recent antibiotic use were excluded. Swabs were obtained
from the posterior fornix and cervix at 8–12, 17–21, 27–31, and 36–38 weeks of gestation. The microbial community was
profiled using hypervariable tag sequencing of the V3–V5 region of the 16S rRNA gene, producing approximately 8 million
reads on the Illumina MiSeq.

Results: Samples were dominated by a single genus, Lactobacillus, and exhibited low species diversity. For a majority of the
patients (n = 8), the vaginal microbiome was dominated by Lactobacillus crispatus throughout pregnancy. Two patients
showed Lactobacillus iners dominance during the course of pregnancy, and two showed a shift between the first and
second trimester from L. crispatus to L. iners dominance. In all of the samples only these two species were identified, and
were found at an abundance of higher than 1% in this study. Comparative analyses also showed that the vaginal
microbiome during pregnancy is characterized by a marked dominance of Lactobacillus species in both Caucasian and
African-American subjects. In addition, our Caucasian subject population clustered by trimester and progressed towards a
common attractor while African-American women clustered by subject instead and did not progress towards a common
attractor.

Conclusion: Our analyses indicate normal pregnancy is characterized by a microbiome that has low diversity and high
stability. While Lactobacillus species strongly dominate the vaginal environment during pregnancy across the two studied
ethnicities, observed differences between the longitudinal dynamics of the analyzed populations may contribute to
divergent risk for pregnancy complications. This helps establish a baseline for investigating the role of the microbiome in
complications of pregnancy such as preterm labor and preterm delivery

.Recently scientists have achieved a feat the like of which has not been seen in billions of years, they have added new letters to the genetic alphabet of a living organism.  What did they do? How did they do it ? Is it too good to be true? All these questions, and more await.

A semi-synthetic organism with an expanded genetic alphabet

Abstract

Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A–T and G–C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs)123. We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS–dNaM), which is efficiently PCR-amplified1 and transcribed45 in vitro, and whose unique mechanism of replication has been characterized67. However, expansion of an organism’s genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS–dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.

We’ll be discussing it all at 8pm BST this Tuesday (27/05/14).

Follow the #microtwjc hashtag on Twitter to join in the discussion.

Full Paper  with extended figures can be found here:

http://apinano.org.br/documentos/A-semi-synthetic-organism-with-an-expanded.pdf [edit- fixed the link- whoops!]

and here:

http://www.nature.com/nature/journal/v509/n7500/full/nature13314.html

I hope to see you there !

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