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Summary:

Pathogen emergence is a complex phenomenon that is poorly understood. The paper discusses a virulence adapted polymorphism (VAP) that is associated with the OmpU gene, and outer membrane porin. In their efforts to determine the exact nature of the ompU VAP, the authors discover that ompU, an outer membrane porin has the ability to repress biofilm formation and alter the transcriptome of the bacteria, driving it towards pathogenicity. Further investigation revealed that the ability of OmpU to enact these phenotypes is associated with a small RNA (sRNA), termed OueS. The authors further show that OueS is controlled by the ompU promoter, and is a functional homologue of the toxR gene, responsible for majority of the pathogenicity associated phenotypes expressed by V. cholerae. Through bioinformatic modelling, they also show that OueS is a modular genetic element, capable of dictating whether a V. cholerae strain expresses pathogenic traits or not. Further investigation into the how these modular genetic elements recombine to produce new phenotypes will aid in deciphering how virulence factors modulate pathogenicity.

OmpU represses biofilm formation in V. cholerae and modulates its transcriptome

The authors constructed a ΔompU mutant and measured its transcriptome. As OmpU activation of pathways is regulated by RpoE, they also constructed a ΔrpoE mutant so that they can determine if there are any overlaps in the transcriptome (Fig1).

Line 140, minor comment

The authors mention sigma factor RpoE as an important factor in modulating the transcriptome of V. cholerae, but this is not immediately obvious as there is no prior mention of RpoE apart from that sentence. More background information in the introduction regarding RpoE would greatly improve the context that the ΔrpoE mutant plays in this analysis.

Line 144, Minor comment

The authors claim that a large percentage of the transcriptomic changes are independent of RpoE regulation, but it is only 144 out of 270 genes (53.3%). This means that there’s almost half of these differentially expressed genes that have overlap with the ΔRpoE. The authors may want to consider rewording this to accurately reflect the proportion of differentially expressed genes associated only with a ΔOmpU mutant, as seen in the published results. If there are any large similarities or dissimilarities between the expression patterns of the 2 mutants that led to the conclusion of “a large percentage”, these results can be included and highlighted so as to support the claim.

OueS suppresses biofilm formation by repressing iron uptake.

The authors then looked at the genes that OueS were regulating to determine the mechanism of biofilm repression and identified iron uptake repression as a key role. They proved this by either supplementing the media with extra iron, or with the use of a cell permeable iron chelator. Bacteria grown in the media with supplemented iron were able to form biofilms regardless of the genotype, thus confirming the hypothesis.

Line 246, Minor comment

The authors used 2,2- bipyridyl (2,2-BIP), an iron chelator, to deplete iron from the media, resulting in the phenotype of reduced biofilm formation associated with OueS production. However, 2,2-BIP is a cell-membrane permeable iron chelator that has the ability to bind out iron inside and outside the cell. It is thus difficult to say that the reduced biofilm formation phenotype is associated with the downregulation of iron-scavenging molecules due to OueS, and does not support the authors intention of depleting native iron in the media. The direct effect of 2,2-BIP on the growth of V. cholerae and the subsequent effect on biofilm formation is a potential confounding factor.

OueS is associated with ToxR-mediated phenotypes

As ToxR regulates the expression of OmpU, the authors sought to determine the regulatory relationships between OueS and ToxR. The authors noted that a ΔtoxR mutant produces similar biofilm repression as seen in the ΔompU mutant. This phenotype in the ΔtoxR mutant can be rescued by either ectopically rescuing with pOmpU or pOueS. As such, the authors concluded that OueS is the response element to ToxR regulation, and notes a connection between sRNA and the ToxR regulon.

Line 256, Minor comment

This sentence does not exactly line up to figure 3A. Line 256 mentions deletion of toxR being similar to ΔompU mutant, but in figure 3A, what is depicted is not that ΔtoxR is similar to ΔompU, but it is ΔtoxR +ptoxR that is similar to the ΔompU.

Line 275, Major comment

Our analyses reveal that OmpU regulates a much larger proportion of genes under AKI conditions than modulating the expression of 947 genes (560 activated and 379 repressed), compared to 270 genes (181 repressed, 89 activated) in biofilms

The intention behind this sentence is a little confusing. Based on our understanding of this, OmpU regulates more genes during biofilm formation under virulence inducing (AKI) conditions compared to after the biofilm is formed.

The text here also refers to table S4 for the genes modulated under AKI conditions, but the tables are not directly add up to 947 genes as stated.  The titles of the tables of differentially expressed gene tables in the Table S4 that reference the origins of the 947 genes, are listed below.

789 differentially-expressed genes in AKI-grown ΔompU cells, independent of ΔrpoE, compared to WT C6706

190 differentially-expressed genes in AKI-grown ΔrpoE cells, independent of ΔompU , compared to WT C6706

Despite the titles, the origin of these 947 genes is not clearly stated and leaves the reader guessing if there is a dataset that has been left out, or if there is recombination of the existing datasets to obtain this number.

OueS is expressed from the ompU promoter.

The authors wanted to explore the regulation of OueS expression. They cloned OueS with varying lengths upstream of the OueS start site into a ΔompU background to determine if there were any promoter regions within the OmpU gene. They did not find any change in the biofilm repression, indicating that the promoter region for OueS was not in the within the OmpU gene, and likely to be sharing a promoter with OmpU.

To confirm this, they deleted 300bp upstream of OmpU, and found that the biofilm formation phenotype was now similar to that of the ΔompU mutant, and suggesting lack of OueS expression. As OmpU and OueS share the same promoter, the authors also conclude that OueS expression is also likely to be controlled by ToxR.

Line 318, Minor comment

The authors indicate that there is likely a lack of OueS expression based on the phenotype of biofilm formation, and do not show quantitative evidence to confirm this claim. qPCR experiments can be conducted to rapidly confirm this claim.

Modular heterogeneity in OueS drives emergence of toxigenic strains.

As mentioned above, the specific alleles of OueS dictate the virulence associated phenotypes. The authors had previously conducted an analysis on the OmpU gene and 41 alleles of the gene. When they modelled the structures, they discovered a diverse set of conserved OueS sequences.

Based on this structural diversity, they constructed isogenic mutants to express the OueS variants from different strains and repeated the colonisation assay. They find that OueS variety acts as a preadaptation to virulence in toxigenic strains, and is also crucial for the emergence of pathogenic potential

Line 356, Major comment

Isogenic mutants were constructed based on their structural diversity, but the authors do not discuss any of the diversity, or the significance of the different mutant strains.

Further discussion on the secondary structures of the 5’ 3’ regions would also be preferred, based on the following points.

-       We note that in figure S4 there is a ‘spacer’ region from the start site to the start of the first stem loops in OueS variants possessing improved colonization ability, similar to that of C6706 WT. We would like to ask if this spacer region is relevant to the binding/ regulatory ability of the OueS gene?

-       Progressive removal of nucleotides from the 5’ end of the OueS gene may shed light onto this phenotype.

Supplementary figures

We note that 2 more strains not mentioned in the main text were included in Fig S5. The figure legend lends some explanation into this, but does not clarify the relationships between the different clades of V. cholerae

We also note an additional figure in Figure S3(as available online), documenting biofilm formation and resembles Figure S3, but is titled “Supplementary Figure 5”

Competing interests

The authors declare that they have no competing interests.