RHS-elements function as type II toxin-antitoxin modules that regulate intra-macrophage replication of Salmonella Typhimurium
Authors: Feifei Xu
Malin Borg
Susan Schlegel
Magnus Stårsta
Marcus Wäneskog
Sten Herschend
Disa L Hammarlöf
Sanna Koskiniemi
Arvid Heden Gynnå
Journal: PLoS genetics (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: RHS elements; toxin-delivery systems; positively selected genes; Salmonella Typhimurium; Rhs toxins
Affiliations: Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.

Abstract:  RHS elements are components of conserved toxin-delivery systems, wide-spread within the bacterial kingdom and some of the most positively selected genes known. However, very little is known about how Rhs toxins affect bacterial biology. Salmonella Typhimurium contains a full-length rhs gene and an adjacent orphan rhs gene, which lacks the conserved delivery part of the Rhs protein. Here we show that, in addition to the conventional delivery, Rhs toxin-antitoxin pairs encode for functional type-II toxin-antitoxin (TA) loci that regulate S. Typhimurium proliferation within macrophages. Mutant S. Typhimurium cells lacking both Rhs toxins proliferate 2-times better within macrophages, mainly because of an increased growth rate. Thus, in addition to providing strong positive selection for the rhs loci under conditions when there is little or no toxin delivery, internal expression of the toxin-antitoxin system regulates growth in the stressful environment found inside macrophages. 

Retracted: No

PubPeer comments:
 A reader pointed me towards some unexpected compression artifacts in the Western blots of Figure 6. In the top a-RNAP-B subunit panel in particular, there are sharp pitch-black blocks of pixels, while other areas in the same photo are compressed in a more  fuzzy way. Could the authors provide the raw blot scan, please? It is probably nothing to worry about, but I have not seen this compression artifact before, and it would be great if the authors could help me understand it. Here is a close-up example of that panel: ![file] , 
 In Figure 3 and Figure S3, there is a little speck in the background of the colony blot that is visible multiple times (shown with red rounded boxes). Other parts of the background (which I assume is the LA medium, not the table) are different. The colony strips also appear to have different resolution with the colonies on the right displaying compression artifacts and reflections not visible in the left parts of the strips, and unexpected positioning of the different colonies (with the right two ones always higher than the left colonies). Could the authors please some explanation? Just trying to understand what could explain this. ![file] ![file] , 
 #2 About the speck, my guess would be dust on the image capture device (either camera lens or glass plate of flat-bed scanner, whichever they use). About the other issues, you might need to clarify your comments: RE: * The colony strips also appear to have different resolution with the colonies on the right displaying compression artifacts and reflections not visible in the left parts of the strips... * u2022 I detect no changes in resolution or compression artifacts in these images. Are you sure you mean  resolution and  compression ? Or are you referring to differering light levels or contrast? (which could just reflect uneven illumination of the field). An illustration might be in order; I don t see any problem. RE: * ...and unexpected positioning of the different colonies (with the right two ones always higher than the left colonies). * u2022 (BTW, presumably you do not really mean to refer to  colonies , but rather to  spots , which are the circular groups of colonies.) Clarify what is  unexpected about this. I.e., what is your expectation, and what is the logical basis for it? Bear in mind that such spot tests often use a multi-channel pipettor, which naturally links the positions of multiple spots in an array. E.g., if using an 8-channel pipettor to spot 8 spots per column, and column 5 is spotted slightly higher than columns 1-4, then all 8 spots in column 5 will be higher. Similarly, if the distance between column 1 and 2 is greater than that between columns 2 and 3, this will be true for all rows. Because of this, you can actually make a pretty good deduction of which strips (rows) came from the exact same plate array. In most cases it seems that the left and right strips can from the same plate, whereas going from top to bottom are from different plates. (The Ara and Glu data necessarily come from different plates, as the medium is different.) So, unless I have misinterpreted your comment, I don t think there is really anything  unexpected about these patterns. , 
 Thank you, Leucanella, for your insights. Some very good points. ,