Essential genome of <em>Pseudomonas aeruginosa</em> in cystic fibrosis sputum
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Author contributions: K.H.T., A.K.W., and M.W. designed research; K.H.T., A.K.W., and J.L.M. performed research; K.H.T., G.C.P., and J.L.M. contributed new reagents/analytic tools; K.H.T., A.K.W., and M.W. analyzed data; and K.H.T., A.K.W., J.L.M., and M.W. wrote the paper.
Significance
The opportunistic pathogen Pseudomonas aeruginosa thrives in cystic fibrosis (CF) lung sputum. Here, we define the essential genome of two P. aeruginosa strains in laboratory media and in CF sputum. We also use genomic methods to profile P. aeruginosa genetic requirements for fitness in both natural and synthetic CF sputum. Finally, we show that the essential genomes of different strains of P. aeruginosa are distinct, suggesting that the architecture of genetic networks is a primary determinant of a gene’s role in fitness. This has implications for the development of strain-independent therapeutics and underscores the importance of functional studies in pathogenic strains of interest.
Abstract
Defining the essential genome of bacterial pathogens is central to developing an understanding of the biological processes controlling disease. This has proven elusive for Pseudomonas aeruginosa during chronic infection of the cystic fibrosis (CF) lung. In this paper, using a Monte Carlo simulation-based method to analyze high-throughput transposon sequencing data, we establish the P. aeruginosa essential genome with statistical precision in laboratory media and CF sputum. Reconstruction of the global requirements for growth in CF sputum compared with defined growth conditions shows that the latter requires several cofactors including biotin, riboflavin, and pantothenate. Comparison of P. aeruginosa strains PAO1 and PA14 demonstrates that essential genes are primarily restricted to the core genome; however, some orthologous genes in these strains exhibit differential essentiality. These results indicate that genes with similar molecular functions may have distinct genetic roles in different P. aeruginosa strains during growth in CF sputum. We also show that growth in a defined growth medium developed to mimic CF sputum yielded virtually identical fitness requirements to CF sputum, providing support for this medium as a relevant in vitro model for CF microbiology studies.
The opportunistic pathogen Pseudomonas aeruginosa is a common cause of chronic cystic fibrosis (CF) lung infection. In the CF lung, P. aeruginosa grows to high densities (10–10 cfu/mL) within airway sputum, which likely serves as the nutritional source during infection (1, 2). Sputum is a complex mixture of airway mucus, inflammatory substances, and bacterial products. The inflammatory components include large numbers of polymorphonuclear leukocytes, dead host cells, and serum components that enter the airway due to vascular leakage and pulmonary hemorrhage (1). The generation time of P. aeruginosa in CF sputum is variable but can be as short as 40 min, suggesting that sputum provides a robust growth environment for P. aeruginosa (3, 4). Long-term colonization of the CF lung leads to the evolution of numerous presumed adaptive phenotypes including mucoidy, amino acid auxotrophy, loss of acute virulence factors, and antibiotic resistance (5–7). Despite the intense interest in P. aeruginosa CF lung infections, very little is currently known regarding the genetic requirements for P. aeruginosa survival and proliferation in sputum. The goal of this study was to address this knowledge gap using high-throughput genomics.
High-throughput genomic approaches such as transposon sequencing (Tn-seq) have been used to identify genetic elements required for in vitro and in vivo fitness (8). Tn-seq allows for simultaneous assessment of the abundance of tens or hundreds of thousands of individual transposon mutants after growth in a selective condition (e.g., in vivo infection model) (9–11). Comparing the abundance of mutants before and after growth in the selective condition allows for rapid identification of mutants with reduced fitness in that condition. Our laboratory recently used Tn-seq to reveal fitness requirements for P. aeruginosa during acute and chronic murine wound infection (12). A major finding of this study was that transcriptome-based approaches such as RNA sequencing cannot be used to predict fitness requirements (12, 13), thus calling into question the utility of previous P. aeruginosa CF sputum transcriptome results (3, 14) for elucidating fitness requirements in the CF lung.
Analysis of bacterial mutant fitness in an experimental condition can reveal many key features of bacterial physiology compared with appropriate controls. For example, the relative lack of mutants in a particular genetic element in a high-density transposon library can indicate the essentiality of that element. Previous studies have used several criteria to determine the immutable regions of a bacterial genome from Tn-seq data in the absence of control conditions, including the prevalence of transposon insertions detected per genetic element or the probability of encountering a DNA segment with no insertions given that segment’s length (15–17). However, these methods do not always account for two types of information available in Tn-seq data: (i) the abundance of each transposon mutant and (ii) the variability observed in Tn-seq biological replicates. These are important criteria to consider when making statistically sound declarations about the absence of transposon insertions in a particular gene. For example, if a few insertions interrupting a gene are tolerated, yet drastically impair fitness, that gene may inappropriately fail to be identified as essential without considering the expected abundance of mutants in that gene. Recently, more sophisticated methods using hidden Markov models have incorporated mutant abundance data successfully but do not consider biological variability (18, 19).
In this work, Tn-seq and a Monte Carlo simulation-based approach was used to determine the essential genome of two P. aeruginosa strains in laboratory medium and CF sputum with statistical precision. The results show that although essential genes are contained in both the core and accessory genomes they are enriched in the core genome. However, the essentiality of these core genes can differ between strains, suggesting that the mere presence or absence of a gene does not necessarily predict how its function integrates into the networks that define fitness in CF sputum. Finally, we show that growth in a defined growth medium developed to mimic CF sputum yielded fitness requirements virtually identical to CF sputum, providing evidence that this medium is a valid in vitro model to study P. aeruginosa CF colonization and persistence.
Acknowledgments
We thank M.W. laboratory members for critical discussions of the manuscript. This work was supported by a grant from the Cystic Fibrosis Foundation (to M.W.) K.H.T. is a Cystic Fibrosis Foundation Postdoctoral Research Fellow. M.W. is a Burroughs Wellcome Investigator in the Pathogenesis of Infectious Disease.
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequence reported in this paper has been deposited in the National Center for Biotechnology Information Sequence Read Archive (accession no. PRJNA265367).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1419677112/-/DCSupplemental.
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