Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum

Reviewer #1 (Public Review):

This is a straightforward paper that uses TraDIS (high-density TnSeq) with Klebsiella pneumoniae to infer essential genes, and genes required for survival under various infection-relevant conditions. The gene sets identified, together with the raw sequence data, will be valuable resources for the Klebsiella research community. The evidence to support the lists of essential and conditionally-important genes is solid, although a few additional follow-up experiments would strengthen some of the claims made based on the TraDIS data.

1. The data strongly suggest that iron depletion in urine leads to conditional essentiality of some genes. It would be informative to test the single gene deletions (Figure 3G) for growth in urine supplemented with iron, to determine how many of those genes support growth in urine due to iron limitation.
2. Line 641. The authors raise the intriguing possibility that some mutants can "cheat" by benefitting from the surrounding cells that are phenotypically wild-type. Growing a fepA deletion strain in urine, either alone or mixed with wild-type cells, would address this question. Given that other mutants may be similarly "masked", it is important to know whether this phenomenon occurs.
3. In cases where there are disparities between studies, e.g., for genes inferred to be essential for serum resistance, it would be informative to test individual deletions for genes described as essential in only one study.

Reviewer #2 (Public Review):

This study presents a useful inventory of essential genes from an antibiotic-resistant K. pneumoniae strain to grow in a rich medium. The study also includes a catalogue of genes required to grow/survive in urine and in serum. The former is particularly interesting. The data is analyzed using adequate tools.

The authors leveraged TraDIS to identify essential genes of K. pneumoniae in LB, and those required to survive in urine, and serum. TraDIS is a well-established approach to investigate these aspects, and in fact, has also been already exploited in the case of K. pneumoniae to identify essential genes and those required for serum resistance. The strain used by the team is not probed by many other laboratories, making it difficult to assess the relevance in the context of K. pneumoniae population biology. Nonetheless, the authors have tried to compare their results against other published studies.

The descriptions of the method and analysis of the data are quite detailed; however considering that this work is mostly a bioinformatics one, it would have been interesting to go beyond the Ecl8 strain and make a detailed comparison against the other published data sets as well as consider the genes identified in the wider population structure of K. pneumonaie and other Enterobactericease (particularly E. coli and Salmonella).

The catalogue of genes may spark additional research to provide mechanistic insights into the contribution of the loci to the phenotypes (either urine and/or serum survival). These experiments are not included in the manuscript beyond the validation level achieved by constructing additional mutants using the Red system.

Reviewer #3 (Public Review):

In this study, Gray and coworkers use a transposon mutant library in order to define: (i) essential genes for K. pneumoniae growth in LB medium, (ii) genes required for growth in urine, (iii) genes required for resistance to serum, and complement-mediated killing. Although there are previous studies, using a similar strategy, to describe essential genes for K. pneumoniae growth and genes required for serum resistance, this is the first work to perform such a study in urine. This is important because these types of pathogens can cause urinary tract infections. Moreover, the authors performed the work using a highly saturated library of mutants, which makes the results more robust, and use a clinically relevant strain from a pathotype for which similar studies have not been performed yet. Besides applying the transposon mutant library coupled with high-throughput sequencing, the authors validate some of the most relevant genes required for each condition using targeted mutagenesis. This is clearly an important step to confirm that the results obtained from the library are reliable. Moreover, in vitro experiments involving complementation of urine with iron provide additional support to the results obtained with the mutants suggesting the importance of genes required for iron acquisition in a limiting-iron environment such as urine. Overall, the study is well-designed and written, and the methodology and analysis performed are adequate. The study would have benefited from in vivo experiments, including a mouse model of bacterial sepsis or urinary tract infections which could have demonstrated the role of the identified genes in the infection process. Nevertheless, the results obtained are informative for the scientific community in order to understand which genes are potentially more relevant in infections caused by K. pneumoniae. The identified genes could represent future targets for developing new therapies against a type of pathogen that is acquiring resistance to all available antibiotics. Below I include several comments regarding potential weaknesses in the methodology used:

- The study was done with biological duplicates. In vitro studies usually require 3 samples for performing statistical robust analysis. Thus, are two duplicates enough to reach reproducible results? This is important because many genes are analyzed which could lead to false positives. That said, I acknowledge that genes that were confirmed through targeted mutagenesis led to similar phenotypic results. However, what about all those genes with higher p and q values that were not confirmed? Will those differences be real or represent false positives? Could this explain the differences obtained between this and other studies?

- Two approaches are performed to investigate genes required for K.pneumoniae resistance to serum. In the first approach, the resistance to complement in serum is investigated. And here a total of 356 genes were identified to be relevant. In contrast, when genes required for overall resistance to serum are studied, only 52 genes seem to be involved. In principle, one would expect to see more genes required for overall resistance to serum and within them identify the genes required for resistance to complement. So this result is unexpected. In addition, it seems unlikely that 356 genes are involved in resistance to complement. Thus, is it possible false positives account for some of the results obtained?