Gene synteny and translational coupling of sctS and sctT facilitate assembly of the unique helical T3SS export apparatus in Salmonella Typhimurium

Reviewer #1 (Public review):

Summary:

The authors set out to understand the complex regulation of the assembly of the Type 3 Secretion System of S. typhimurium. They found that the gene synteny as well as specific mRNA stem loops were important for the translational coupling of sctS and sctT. Without this regulation, SctT self-oligomerizes, which disrupts the export of effector proteins and leads to a decreased fitness of the pathogen. The work was done using a variety of convincing methods and leads to an updated picture of how T3SS assembly occurs. Since the same genetic synteny is found in a large majority of T3SS in different bacteria, it is likely that this is a general mechanism, but one that needs to be further experimentally validated.

Strengths:

The paper uses an impressive amount of experiments, with different techniques, to describe how they identified the genetic regulation of SctT production.

Weaknesses:

Only minor weaknesses are found.

(1) Regarding the use of the complex being unique. It is not well explained what makes this a unique complex.

(2) The paper would benefit from a discussion regarding how regulation might work in the minority of bacterial strains where the T3SS gene synteny is largely different. One would expect that those bacteria would have a different way of regulating T3SS assembly, but that is not discussed at all by the authors.

Reviewer #2 (Public review):

Summary:

In this manuscript, Samuel Wagner and colleagues describe an elegant mechanism to prevent promiscuous assembly of a core virulence type III secretion system protein, SctS. Starting from a bioinformatic standpoint, they demonstrate that synteny is highly conserved, and sctT occurs immediately downstream of sctS. Secretion is greatly reduced when sctT is removed or scrambled from its genomic context, and sctT expression is accordingly reduced (sctS synteny is also important, though less so). The distance between sctS and sctT is crucial. An elegant series of genetic experiments leads the authors to pinpoint a stem loop structure that occludes the Shine-Dalgarno sequence of sctT. This property is independent of the actual gene preceding sctT. In sum, this means that SctS is already expressed before SctT is expressed, preventing SctT from forming cytotoxic homooligomers.

Strengths:

The manuscript is very well-written, easy to follow, and describes a substantial amount of genetic detective work to identify the underlying mechanism. I have only a number of textual suggestions, mainly for the Introduction text, which I believe could be revised for a flagellar and broader audience.

Weaknesses:

Major concern:

While the work is rigorous and substantial, I am unsure as to whether its findings will appeal beyond a niche audience.

Minor points:

(1) Line 117: The number here seems to be very small. RefSeq has ~200,000 genomes. My guess is that at least 100,000 of these will be bacterial. Many (most?) bacteria have flagella, and some unflagellated strains have injectisomes, meaning I would have guessed that the authors would have ~50,000 genomes with SctRSTU. This estimate is error-prone, but not by too much. Can the authors explain the discrepancy between my estimate and their figure of almost two orders of magnitude? (SctRSTU/FliPQGFlhB should also be easy to pick up by sequence searches, so I don't think this is due to false negatives).

(2) Discussion: I would appreciate some discussion of how species that do not conserve the synteny of sctS and sctT prevent problems of sctT oligomerisation? It doesn't need to be evidence-based at this stage, but I'm sure the authors have thought about this, and the Discussion is an appropriate place to share their speculations.

Reviewer #3 (Public review):

At the core of the bacterial type III secretion system (T3SS), a nanomachine used to inject effector proteins into eukaryotic cells, five highly conserved proteins, SctRSTUV, form the export apparatus, which is the actual gate for effector proteins. Not only are these proteins the most strongly conserved parts of the system, but also their gene order is conserved, which is not the case for most other components of the T3SS. Interestingly, this order does not completely recapitulate the assembly order, which is SctR5-T4-S-U-V. Looking into the reasons for the conserved synteny, the authors noted a stem-loop in the mRNA of the Salmonella SPI-1 sctS gene, which is present in many other T3SS as well (and in fact had been found in Yersinia before). They then use an array of clever gene permutations and modifications to discern the benefit of this order for the bacteria. The combination of thorough sequence analysis with different, partly quantitative, protein expression and secretion assays and growth curves, both in the native Salmonella background and in heterologous systems, provides strong evidence for the interpretation of the authors: The stem-loop in sctS prevents the premature expression of SctT, which can otherwise assemble into "futile multimers" that can lead to ion loss. The presence of stem-loops in many other sctS/T genes gives weight to this finding.

This is a very nice and thorough study addressing an important point in the assembly of type III secretion systems. I only have a few suggestions.

(1) Conserved gene orders have been shown for many complexes, and the findings presented in this manuscript might be applicable to other membrane complexes.

The conservation of gene order and the presence of the stem loop give weight to the authors' findings. However, it is only mentioned quite late in the discussion that a similar stem loop was found in Yersinia upstream sctT earlier, and was interpreted differently. The authors' current discussion is somewhat evasive on this point. Why would these similar structures be used differently? Why would temperature not play a role in Salmonella SPI-1? And wouldn't the stem-loop also couple sctS and sctT expression in Yersinia? This should be addressed, if possible, by experiments (at least, the influence of temperature on the SPI-1 mRNA structure should be testable for the authors) and by a more detailed discussion (given the redundancy of RNA thermometers in the Yersinia T3SS, the interpretation in the current paper might well be the more compelling one).

(2) A point that deserves more attention is that a similar finding in Yersinia has been interpreted differently before (as a temperature sensor rather than translational coupling) - are these systems really different? Testing the different interpretations in the respective other system (at least the influence of temperature in the Salmonella SPI-1 system used in this manuscript) would have made the interpretation even more compelling.

(3) Another point that should be discussed in more detail is why this mechanism is present when replacement of the sctT ATG by weaker start codons and the simple omission of a separate SD sequence upstream sctT would achieve the same outcome. This could be tested in one of the nice heterologous systems, as used in Figure 4.