Our next Journal club will be on the 28th of October 2014 at 8 pm on this paper here:

Regulation of the AbrA1/A2 Two-Component System in Streptomyces coelicolor and the Potential of Its Deletion Strain as a Heterologous Host for Antibiotic Production by Sergio Rico, Ana Yepes, Héctor Rodríguez, Jorge Santamaría, Sergio Antoraz, Eva M. Krause, Margarita Díaz, Ramón I. Santamaría, published on October 10, 2014 DOI:10.1371/journal.pone.0109844

Link: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0109844

Background
Streptomyces is a genus of actinobacteria, a group of Gram-positive soil bacteria with an unusual life cycle, involving substrate mycelium, formation of aerial hyphae and spore formation. They are prolific producers of bioactive metabolites, such as clavulinic acid or FK-506 and thus are of great interest for industry. The onset for the production of these metabolites usually happens when nutrients are limited and come along with the developmental stages. The regulation of antibiotic production has been extensively studied mainly in S.coelicolor which is one of the model organisms and many genetic tools are available. For more details see this review here: http://mmbr.asm.org/content/77/1/112.short
Regulation of the AbrA1/A2 Two-Component System in Streptomyces coelicolor and the Potential of Its Deletion Strain as a Heterologous Host for Antibiotic Production
By Sergio Rico, Ana Yepes, Héctor Rodríguez, Jorge Santamaría, Sergio Antoraz, Eva M. Krause, Margarita Díaz, Ramón I. Santamaría
Published on October 10, 2014DOI: 10.1371/journal.pone.0109844
Abstract
The Two-Component System (TCS) AbrA1/A2 from Streptomyces coelicolor M145 is a negative regulator of antibiotic production and morphological differentiation. In this work we show that it is able to auto-regulate its expression, exerting a positive induction of its own operon promoter, and that its activation is dependent on the presence of iron. The overexpression of the abrA2 response regulator (RR) gene in the mutant ΔabrA1/A2 results in a toxic phenotype. The reason is an excess of phosphorylated AbrA2, as shown by phosphoablative and phosphomimetic AbrA2 mutants. Therefore, non-cognate histidine kinases (HKs) or small phospho-donors may be responsible for AbrA2 phosphorylation in vivo. The results suggest that in the parent strain S. coelicolor M145 the correct amount of phosphorylated AbrA2 is adjusted through the phosphorylation-dephosphorylation activity rate of the HK AbrA1. Furthermore, the ABC transporter system, which is part of the four-gene operon comprising AbrA1/A2, is necessary to de-repress antibiotic production in the TCS null mutant. Finally, in order to test the possible biotechnological applications of the ΔabrA1/A2strain, we demonstrate that the production of the antitumoral antibiotic oviedomycin is duplicated in this strain as compared with the production obtained in the wild type, showing that this strain is a good host for heterologous antibiotic production. Thus, this genetically modified strain could be interesting for the biotechnology industry.

Discussion points:
1. Is the paper well written?
2. Were the methods appropriate?
3. Do the results reflect the conclusion?
4. What else could be done?

Hi everyone!

Welcome to #MicroTwJC 55, hosted on 14/10/2014 by @_LisaKWilliams_ and @Stewart_Barker.

This week we will be discussing the fascinating world of virophages – that is viruses which attack other viruses. The paper can be found here: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0094923

Following a previous #MicroTwJC discussion on the largest discovered viruses, this fairly recent paper describes a satellite virus (or virophage) named Zamilon that is associated with the giant virus family Mimiviridae. Zamilon is similar to other described virophages which have negative effects on their host – Sputnik virophages, yet does not appear to inhibit it’s host in any way…

 

“The host-specificity of the Zamilon virophage supports the distinction between satellite viruses (opportunistic entities associated with a virus) and virophages, which target specific hosts.”

 

Aside from what we think is an interesting paper and concept, the above quote is a crucial point of the paper. We leave you with the abstract and questions to consider below, and look forward to a lively debate!

Abstract

Virophages, which are potentially important ecological regulators, have been discovered in association with members of the order Megavirales. Sputnik virophages target the Mimiviridae, Mavirus was identified with the Cafeteria roenbergensis virus, and virophage genomes reconstructed by metagenomic analyses may be associated with the Phycodnaviridae. Despite the fact that the Sputnik virophages were isolated with viruses belonging to group A of theMimiviridae, they can grow in amoebae infected by Mimiviridae from groups A, B or C. In this study we describe Zamilon, the first virophage isolated with a member of group C of theMimiviridae family. By co-culturing amoebae with purified Zamilon, we found that the virophage is able to multiply with members of groups B and C of the Mimiviridae family but not with viruses from group A. Zamilon has a 17,276 bp DNA genome that potentially encodes 20 genes. Most of these genes are closely related to genes from the Sputnik virophage, yet two are more related to Megavirus chiliensis genes, a group B Mimiviridae, and one to Moumouvirus monve transpoviron.

 

Points to consider

  1. Is the paper well written?
  2. Do the methods fully investigate whether the Zamilon virophage has a disruptive effect on its host?
  3. Are the results and discussion appropriate?
  4. Is Zamilon a virophage, or a satellite virus? Is there actually a difference between the two terms?

 

Streptococcus_pyogenesThis Tuesday’s paper will be on the consequences of viral/bacterial co-infection in a mouse model, focusing on bacterial transmission and its genetic determinants. As you can imagine, pathogen transmission is a hot-topic right now, especially when viruses such as influenza are involved. So have a read, critique the paper and join us on Tuesday night at 8 pm BST on the 30th of September.

The paper was published recently in PloS pathogens, see link.

For discussion, I think we should focus on these points:

  • what is the model telling us? and is it a good model for this important question?
  • does this paper providing convinving evidence for the role or TLR2-driven inflammation? and what is the role of influenza virus in this?
  • what are the real-world consequences of viral/bacterial co-infection?

TLR2 Signaling Decreases Transmission of Streptococcus pneumoniae by Limiting Bacterial Shedding in an Infant Mouse Influenza A Co-infection Model.

  • Aimee L. Richard,
  • Steven J. Siegel,
  • Jan Erikson,
  • Jeffrey N. Weiser mail

Abstract

While the importance of transmission of pathogens is widely accepted, there is currently little mechanistic understanding of this process. Nasal carriage of Streptococcus pneumoniae (the pneumococcus) is common in humans, especially in early childhood, and is a prerequisite for the development of disease and transmission among hosts. In this study, we adapted an infant mouse model to elucidate host determinants of transmission of S. pneumoniae from inoculated index mice to uninfected contact mice. In the context of co-infection with influenza A virus, the pneumococcus was transmitted among wildtype littermates, with approximately half of the contact mice acquiring colonization. Mice deficient for TLR2 were colonized to a similar density but transmitted S. pneumoniae more efficiently (100% transmission) than wildtype animals and showed decreased expression of interferon α and higher viral titers. The greater viral burden intlr2−/− mice correlated with heightened inflammation, and was responsible for an increase in bacterial shedding from the mouse nose. The role of TLR2 signaling was confirmed by intranasal treatment of wildtype mice with the agonist Pam3Cys, which decreased inflammation and reduced bacterial shedding and transmission. Taken together, these results suggest that the innate immune response to influenza virus promotes bacterial shedding, allowing the bacteria to transit from host to host. These findings provide insight into the role of host factors in the increased pneumococcal carriage rates seen during flu season and contribute to our overall understanding of pathogen transmission.

Author Summary

In this study, we sought to identify factors contributing to the transmission of the bacterial pathogen Streptococcus pneumoniae (the pneumococcus), a major cause of otitis media, pneumonia, and septicemia. Often found as a co-infection with other bacterial and viral pathogens, the pneumococcus is commonly carried by young children and is spread by close human contact, most likely through large droplet respiratory secretions. The specific determinants of bacterial transmission, however, have not been identified. This report details our use of an infant mouse model of transmission, which includes influenza A co-infection, to elucidate the mechanism of host-to-host transmission. We found that the inflammatory response to influenza, which is aggravated in the context of weakened host defense, promotes transmission by inducing bacterial shedding from the mouse nose. These results show how a bacterial pathogen exploits the host immune response to spread from one host to the next.

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?

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