NusA to save the day during heat shock: commentary and review of Li, et al 2013

A review of Li, et al Escherichia coli transcription termination factor NusA: heat-induced oligomerization and chaperone activity (2013) Scientific Reports, vol 3 (2347) PMCID: PMC3731644

In previous blog posts, I have taken a look at two slightly dated articles concerning drug development against mycobacterium tuberculosis. Here I will continue my series of reviews but with a new direction. My last review provided a new perspective on a study that had already received press from others. For this review, I have chosen a much more recent paper, which has not been analyzed (to my knowledge) by another other blogger, science journalist, or microbiology enthusiast.

Why have I chosen a paper on NusA molecular biology in E. coli? Other than the fact that the article is fresh off the press (from the Nature sub-journal Scientific Reports), the claims (which are fairly well supported) the authors make are another example of how bacterial proteins often function as swiss army knifes: they have multiple functions, sometimes not revealed until environmental conditions are changed or cellular stresses are introduced. 

I will begin this review with a short summary, known as a Capsule. This style of synopsis / abstract is being pioneered by the Journal for Biological Chemistry (JBC), and I think it is a great idea for making summaries primary research literature more accessible to a general audience. One way to think of it is a shorter abstract, written not for experts but for the public (and policy makers, I suppose!). Authors of manuscripts submitted to JBC must provide a capsule statement; here, the capsule below is my own, not written by the authors of the NusA study in Scientific Reports (and not conforming to JBC's strict 60 word limit).

Capsule

Background:  NusA is a protein factor known to be involved in transcription termination and anti-termination (transcription is part of the process of turning genetic information in DNA into proteins and enzymes).

Results: Upon heat shock, NusA forms oligomers (multiple copies of the same protein factor bound together) which help prevent other proteins from aggregating.  

Conclusion: NusA contributes to the heat-shock resistance in E. coli by acting as a buffer to protein aggregation.

Significance: Describes a new role for NusA and expands the knowledge of how bacteria cope with stress; these abilities (in general) are important for many bacteria, including pathogenic bacteria that must resist stress from our immune system and medicines.

I invite readers to form their own capsule of this article, especially if you disagree with my choice of areas to emphasis.

Manuscript Highlights

1. NusA is the latest example of a multi-functional protein with latent chaperone 'buffer' activity during heat-shock. GreA, another transcription related factor, is also recent example.

2. NusA oligomerization (distinct from aggregation), mediated by the C-terminal repeat domains, is thought to be responsible for the chaperone buffer activity.

3. NusA's role in heat-shock conditions is not demonstrated under physiological conditions; experiments are done in vitro or with over-expressed and tagged NusA. This may reflect technical limitations.

Select 'Read More' to see the rest of the review

Waging Warfarin against Tuberculosis: A Review of Dutton et al, 2010

A review of Dutton et al. Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin. PNAS (2010) vol. 107 (1) pp. 297-301 PMCID: PMC2806739

This PNAS study from a few years ago looks at the potential antibacterial use of a popular anticoagulant, warfarin. Although my attention was drawn to this article at first through a journal club, I think that this is an interesting paper to review for several reasons. First, it is a paper published in a relatively high profile journal, and it also note worthy because it is research that has a more direct connection to clinical applications and drug development. 

This work has also been reviewed by other blogs and science writers (in a more timely fashion than I have done). For example, see a guest post in Small Things Considered blog written by two graduate students entitled "All is fair in love and warfarin". These students do a good job of summarizing the article, especially for a wider audience. A more in-depth review (and slightly more critical) entitled "Warfarin: Not Just Rat Poison" is also worth a read.

Here, I would like to add my commentary to this work, going further than the above two reviews / summaries in pointing out some flaws. The very rationale of this work is questionable, and might provide a glimpse into the misalignment of public interest and the motivation of the scientists. This review also happens to continue an unwitting trend of reviews on anti-tuberculosis drug papers, following my review of Shi et al, 2011, entitled "Grainy Westerns and Fuzzy Logic".

Manuscript Highlights

1. High doses of warfarin (and lower doses of the anticoagulant phenindione) are capable of inhibiting the growth of Mycobacterium smegmatis and tuberculosis (only warfain data for M. tb)

2. Mycobacterium VKOR, the homolog of the human target of Warfarin, is active when expressed in E. coli. The activity of this enzyme in these experiments is sensitive to Warfarin.

3. Surprisingly, inhibition of Mycobacterium VKOR by warfarin does not appear to be the reason why this drug inhibits M. tuberculosis growth. Should either VKOR or Warfarin really be the focus of drug development?

Synopsis:

In this study, Dutton and colleagues explore the use of the anticoagulant warfarin to inhibit the Mycobacterium tuberculosis VKOR enzyme (MtbVKOR). This work could lead to the development of novel, VKOR-targeting anti-tuberculosis therapeutics. This bacterial enzyme, MtbVKOR, is the ortholog of the human target of warfarin, Vitamin K epOxide Reductase (VKOR). While human VKOR is involved in the clotting pathway and bacterial VKOR is thought to be responsible for intracellular disulfide bond formation, the underlying enzymatic activity for these orthologs are very similar. In a previous study, the authors demonstrated that MtbVKOR is active in disulfide bond formation when expressed in E. coli, thereby providing a convenient system for studying the properties of this enzyme in vivo.

Utilizing this heterologous E. coli / MtbVKOR expression system, the authors demonstrate that high doses of warfarin can inhibit the activity of the Mycobacterium enzyme in this setting. This demonstrates that the bacterial and human VKOR enzymes share more than just some sequence similarity. Selection and analysis of warfarin-resistant variants of MtbVKOR supports this conclusion. Finally, the authors demonstrate that warfarin has antibacterial properties against Mycobacterium species, although they cannot identify VKOR as the target in this case.

Select 'Read More' to see the rest of the review.

Grainy Westerns and Fuzzy Logic: A Review of Shi, et al 2011

A review of Shi et al. Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis. Science (2011) vol. 333 (6049) pp. 1630-2

PMCID: PMC3502614

This study was recently published in the prestigious journal Science attempted to explain the mode of action for an important antibiotic, Pyrazinamide, used in treating tuberculosis. Due both to the high profile of this work, and the conclusion they draw regarding trans-translation, I will provide below a review of this article.

The interested reader should be aware of one important fact when approaching this article: there are different versions of it, depending on how you access it. The open access version at Pubmed is outdated; a more recent version (with updated Figure 3) is available from the journal Science itself. 

Manuscript Highlights:

1. Demonstrates binding of ribosomal protein S1 to POA (active form of Pyrazinamide drug)
2. Suggests mechanism of action for POA: binding to S1 inhibits trans-translation.
3. Quality of trans-translation related data (western blots) make the conclusion drawn by the authors questionable.
4. Alternative explanations to trans-translation rescue of stalled ribosomes in their in vitro system are not ruled out, undermining the conclusions drawn.

Synopsis:

In this study, Shi and colleagues sought to identify the target and antibiotic mechanism of the anti-tuberculosis drug Pyrazinamide. This drug is important in treatment of tuberculosis, particularly in clearing persister cells through combination with other compounds. In order to accomplish this task, the authors used affinity chromatography to capture M. tuberculosis proteins that are capable of interacting with the drug. Through this approach, ribosomal protein S1 is identified as the primary target.

Using ribosomal protein S1 (RpsA) as a lead, the authors attempt to explain the mode of action for PZA. Binding studies using isothermal titration demonstrate more conclusively that PZA is capable of binding RpsA Furthermore, a PZA resistant strain with mutant RpsA genes that cannot bind the drug is identified. Finally, the authors use an in vitro translation system to assess the effect of PZA on translation and trans-translation. The conclusion drawn from these studies is that PZA specifically inhibits trans-translation, but only in the context of M. tuberculosis ribosomes.