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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.

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…


Next week’s #microtwjc paper comes from the September 2012 issue of the SGM’s ‘Microbiology’ and is titled:

Molecular characterization of a novel mosaic tet(S/M) gene encoding tetracycline resistance in foodborne strains of Streptococcus bovis

Simona Barile, Chiara Devirgiliis, and Giuditta Perozzi

(Microbiology September 2012 158:2353-2362; published ahead of print June 21, 2012, doi:10.1099/mic.0.058206-0 )

The abstract:

The presence of antibiotic-resistance (AR) genes in foodborne bacteria of enteric origin represents a relevant threat to human health in the case of opportunistic pathogens, which can reach the human gut through the food chain. Streptococcus bovis is a human opportunistic pathogen often associated with infections in immune-compromised or cancer patients, and it can also be detected in the environment, including fermented foods. We have focused on the molecular characterization of a tetracycline (Tet)-resistance gene present in 39 foodborne isolates of S. bovis phenotypically resistant to this drug. The gene was identified as a novel tet(S/M) fusion, encoding a mosaic protein composed of the N-terminal 33 amino acids of Tet(S), in-frame with the Tet(M) coding sequence. Heterologous expression of the mosaic gene was found to confer Tet resistance upon Escherichia coli recipients. Moreover, the tet(S/M) gene was found to be transcriptionally inducible by Tet under the endogenous tet(S) promoter in both S. bovis and E. coli. Nucleotide sequencing of the surrounding genomic region of 16.2 kb revealed large blocks of homology with the genomes of Streptococcus infantarius and Lactococcus lactis. A subregion of about 4 kb containing mosaic tet(S/M) was flanked by two copies of the IS1216 mobile element. PCR amplification with primers directed outwards from the tet(S/M) gene identified the presence of a 4.3 kb circular form corresponding to the intervening chromosomal region between the two IS1216 elements, but lacking a replication origin. The circular element shared extensive overall homology with a region of the multidrug-resistance plasmid pK214 from Lc. lactis, containing tet(S), as well as the IS1216 transposase-containing element and intervening non-coding sequences. Linear reconstruction of the insertion events likely to have occurred within this genomic region, inferred from sequence homology, provides further evidence of the chromosomal rearrangements that drive genomic evolution in complex bacterial communities such as the gut and food microbiota.


A few discussion points to consider (if you have any other points please comment below)

  1. Was the paper well written, clearly laid out and easy to follow?
  2. Were the experiments well designed?  Were there any experiments you would have liked to see there that weren’t?
  3. How does this work contribute to our knowledge of antibiotic resistance and are there any practical suggestions/outcomes/considerations that stem from this work?
  4. If you were the researchers what future work would you want to do?


We hope you can join us next week, Tues 11th September, 8pm UK time for #microtwjc 🙂


With thanks to CRV Arnhem for making this available via a Creative Commons licence

This week the paper we will be discussing is:

The authors examined the effect of probiotics and antibiotics on intestinal homeostasis using a  a computer controlled model of the human large intestine. Antibiotic associated diarrhea (AAD) and Clostridium difficile infection (CDI) are complications in broad spectrum antibiotic therapy, probiotics have been suggested to be promising agents in the prevent of AAD and CDI. The authors suggest that mechanistic studies in vivo are difficult to perform due to sampling and ethical difficulties and the model simulates the colon to a high degree. The model is a unique tool as its able to study the stability, release, dissolution, absorption of nutrients, chemicals, bioactive compounds and pharmaceuticals in the gastrointestinal tract. They found that short chain fatty acids, ammonia, branched chain fatty acids and lactate were increased when using probiotic therapy compared to the controls and combination therapy (Clindamycin and probiotics).  Probiotics following antibiotic therpay did not help to recover the intestinal microbiota lost during the therapy. The main finding is that the simultaneous application of antibiotics and probiotics stablises the intestinal microbiota.  The authors conclude that probiotics could be a resonable strategy to prevent antibiotic associated disturbances of the gastrointestinal tract.

Discussion points

* How do you feel about the application of an in vitro model to study this?

* What  other experiments could be performed ?

* Do you think probiotics are useful in this application?

* What other data would be needed to convince you that probiotics work?

* Should we be using the probiotic mixture they described? What could the long term consequences be?

As ever please feel free to leave any other discussion points below.

Zoonotica here posting on behalf of @flashton2003 the summary and discussion points for the next #microtwjc on Tues 19th June at 8pm BST.

The paper we’ll be discussing is:

Hfq virulence regulation in enterohemorrhagic Escherichia coli O157:h7 strain 86-24, Kendall et al., J. Bac., v193, p6843, 2011

The authors examined the role of the small RNA chaperone Hfq in the regulation of virulence in Enterohemorrhagic Escherichia coli O157:H7 (EHEC). They found that the deletion of hfq down regulated the transcriptional activator of a pathogenicity associated locus (locus of enterocyte effacement, LEE) as well as a two component system (qseBC) which is involved in interkingdom signalling and virulence gene regulation (it upregulates shiga toxin production) in EHEC. They also examined the production of shiga toxin protein and the virulence of the mutant in a cell model and found both to be decreased. The author’s findings differ from work done on another EHEC strain in which deletion of hfq led to the upregulation of virulence genes. They ascribe this difference to the different genetic background of the two strains which have each acquired the virulence genes by horizontal gene transfer.

Discussion points

* How do you think that the thoroughness and technical aspects of this work compared with the previous work on the related strain which showed that deletion of hfq increase the production of various virulence associated proteins?

* What experiments could be performed to elucidate the different roles of Hfq in the different strains?

* Interesting that strains which exhibit such similar phenotypes (i.e. EHEC) have such different ways of regulating the genes which result in this phenotype. What evolutionary mechanism could explain this?

* What other interesting phenotypes are associated with post-transcriptional regulation by e.g. Hfq?

As ever please feel free to leave any other discussion points below, plus if any of you write/have written a blog post on the paper please feel free to plug that below too. 🙂

The paper for 22nd May journal club presents the genome of a Yersinia pestis strain isolated from a victim of the Black Death from the 14th century.

The authors developed a novel capture array to fish out bacterial DNA from the dental pulp of a plague victim buried in London’s East Smithfield cemetery.  The captured sequences were then sequenced and mapped to a contemporary Y. pestis strain. Differences in gene content and synteny, as well as polymorphic sites, were investigated through a combination of BLAST searches and reference-guided assembly of sequence reads.  The study concludes that the 14th century strain does not contain any unique polymorphisms, or significant genetic changes, that would make it more virulent than modern strains.

The authors go on to place the 14th century strain in the phylogenetic context of other Yersinia strains, and show that it sits “close to the ancestral node of all extant human pathogenic Y. pestis strains”.  Using a Bayesian coalescent method, they estimate a date for the emergence of human-associated Y. pestis to between 1282–1343 AD, calling into question the commonly accepted hypothesis that the earlier Justinian Plague was caused by the same pathogen.

The authors feel that the study of ancient pathogens can inform the study of mechanisms of host adaptation and pandemic spread of modern pathogens.  The authors close by stating “At our current resolution, we posit that molecular changes in pathogens are but one component of a constellation of factors contributing to changing infectious disease prevalence and severity, where genetics of the host population, climate, vector dynamics, social conditions and synergistic interactions with concurrent diseases should be foremost in discussions of population susceptibility to infectious disease and host–pathogen relationships with reference to Y. pestis infections.”

Discussion points

  • What are the drawbacks of the capture array/sequencing method used?
  • Is the method used sufficient to support the conclusions the authors have drawn?
  • Do you feel that the study of ancient pathogens is useful for informing the study of contemporary strains?
  • Is the conclusion that the causative agent of the Black Death is distinct from earlier supposed plagues convincing?

Links to other relevant discussions

The paper received quite a bit of coverage when it was published, thought I’d link to the NY Times article and the TWiM podcast that discussed a bit about the previous work done.