ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth in macrophages

Reviewer #1 (Public Review):

Summary:
The authors sought to establish a biochemical strategy to study ESAT-6 and CFP-10 biochemistry. They established recombinant reagents to study these protein associations in vitro revealing an unexpected relationship at low pH. They next develop much-needed reagents to study these proteins in an infection context and reveal that treatment with an ESAT-6 nanobody enhances Mtb control.

Strengths:
The biochemical conclusions are supported by multiple configurations of the experiments. They combine multiple approaches to study a complex problem.

Weaknesses:
It would be valuable to understand if the nanobody is disrupting the formation of the ESAT6-CFP10 complex. It is unclear how the nanobody is functioning to enhance control in the infection context. More detail or speculation in the discussion would have been valuable. Where is the nanobody in the cell during infection?

Reviewer #2 (Public Review):

Summary:
Bates TA. et al. studied the biochemical characteristics of ESAT-6, a major virulence factor of Mycobacterium tuberculosis (Mtb), as part of the heterodimer with CFP10, a molecular chaperon of ESAT-6, as in homodimer and in homotetramer using recombinant ESAT-6 and CFP10 expressed in E. coli by applying several biochemical assays including Biolayer Interferometry (BLI) assay. The main findings show that ESAT-6 forms a tight interaction with CFP10 as a heterodimer at neutral pH, and ESAT-6 forms homodimer and even tetramer-based larger molecular aggregates at acidic pH. Although the discussion of the potential problems associated with the contamination of ESAT-6 preparations with ASB-14 during the LPS removal step is interesting, this research does not test the potential impact of residual ASB-14 contaminant on the biochemical behavior ESAT-6-CFP10 heterodimer and ESAT-6 homodimer or tetramer and their hemolytic activity in comparison with the ones without ASB-14. The main strength of this study is the generation of ESAT-6 specific nanobodies and the demonstration of its anti-tuberculosis efficiency in THP-1 cell lines infected with Mtb strains with reporter genes.

Strengths:
Generation and demonstration of the anti-ESAT-6 nanobodies against tuberculosis infection in a cell line based Mtb infection model.

Weaknesses:
Although the biochemistry studies provide quantitative data about the interactions of ESAT-6 with its molecular chaperon CFP10 and the interaction of ESAT-6 homodimer and tetramers, the novel information from these studies is minimal.

Reviewer #3 (Public Review):

Summary:
This manuscript describes some biochemical experiments on the crucial virulence factor EsxA (ESAT-6) of Mycobacterium tuberculosis. EsxA is secreted via the ESX-1 secretion system. Although this system is recognized to be crucial for virulence the actual mechanisms employed by the ESX-1 substrates are still mostly unknown. The EsxA substrate is attracting the most attention as the central player in virulence, especially phagosomal membrane disruption. EsxA is secreted as a dimer together with EsxB. The authors show that EsxA is also able to form homodimers and even tetramers, albeit at very low pH (below 5). Furthermore, the addition of a nanobody that specifically binds EsxA blocks intracellular survival, as well as if the nanobody is produced in the cytosol of the infected macrophages.

Strengths:
-Decent biochemical characterization of EsxA and identification of a new and interesting tool to study the function of EsxA (nanobody).

-The manuscript is well-written.

Weaknesses:
The findings are not critically evaluated using extra experiments or controls.

For instance, tetrameric EsxA in itself is interesting and could reveal how EsxA works. But one would say that this is a starting point to make small point mutations that specifically affect tetramer formation and then evaluate what the effect is on phagosomal membrane lysis. Also one would like to see experiments to indicate whether these structures can be produced under in vitro conditions, especially because it seems that this mainly happens when the pH is lower than 5, which is not normally happening in phagosomes that are loaded with M. tuberculosis.

Also, the fact that the addition of the nanobody, either directly to the bacteria or produced in the cytosol of macrophages is interesting, but again it is the starting point for further experimentation. As a control, one would like to see the effect on an Esx-1 secretion mutant. Furthermore, does cytosolic production or direct addition of the nanobody affect phagosomal escape? What happens if an EsxA mutant is produced that does not bind the nanobody?

Finally, it is a bit strange that the authors use a non-native version of esxA that has not only an additional His-tag but also an additional 12 amino acids, which makes the protein in total almost 20% bigger. Of course, these additions do not have to alter the characteristics, but they might. On the other hand, they easily discard the natural acetylation of EsxA by mycobacteria itself (proven for M. marinum) as not relevant for the function because it might not happen in (the close homologue) M. tuberculosis.