De novo mutations mediate phenotypic switching in an opportunistic human lung pathogen.

Bacteria evolving within human hosts encounter selective tradeoffs that render mutations adaptive in one context and deleterious in another. Here, we report that the cystic fibrosis-associated pathogen Burkholderia dolosa overcomes in-human selective tradeoffs by acquiring successive point mutations that alternate phenotypes. We sequenced the whole genomes of 931 respiratory isolates from two recently infected patients and an epidemiologically-linked, chronically-infected patient. These isolates are contextualized using 112 historical genomes from the same outbreak strain. Within both newly infected patients, diverse parallel mutations that disrupt O-antigen expression quickly arose, comprising 29% and 63% of their B. dolosa communities by 3 years. The selection for loss of O-antigen starkly contrasts with our previous observation of parallel O-antigen-restoring mutations after many years of chronic infection in the historical outbreak. Experimental characterization revealed that O-antigen loss increases uptake in immune cells while decreasing competitiveness in the mouse lung. We propose that the balance of these pressures, and thus whether O-antigen expression is advantageous, depends on tissue localization and infection duration. These results suggest that mutation-driven alternation during infection may be more frequent than appreciated and is underestimated without dense temporal sampling.

Rapid expansion and extinction of antibiotic resistance mutations during treatment of acute bacterial respiratory infections.

Acute bacterial infections are often treated empirically, with the choice of antibiotic therapy updated during treatment. The effects of such rapid antibiotic switching on the evolution of antibiotic resistance in individual patients are poorly understood. Here we find that low-frequency antibiotic resistance mutations emerge, contract, and even go to extinction within days of changes in therapy. We analyzed Pseudomonas aeruginosa populations in sputum samples collected serially from 7 mechanically ventilated patients at the onset of respiratory infection. Combining short- and long-read sequencing and resistance phenotyping of 420 isolates revealed that while new infections are near-clonal, reflecting a recent colonization bottleneck, resistance mutations could emerge at low frequencies within days of therapy. We then measured the in vivo frequencies of select resistance mutations in intact sputum samples with resistance-targeted deep amplicon sequencing (RETRA-Seq), which revealed that rare resistance mutations not detected by clinically used culture-based methods can increase by nearly 40-fold over 5-12 days in response to antibiotic changes. Conversely, mutations conferring resistance to antibiotics not administered diminish and even go to extinction. Our results underscore how therapy choice shapes the dynamics of low-frequency resistance mutations at short time scales, and the findings provide a possibility for driving resistance mutations to extinction during early stages of infection by designing patient-specific antibiotic cycling strategies informed by deep genomic surveillance.

Overproduction of the AlgT Sigma Factor Is Lethal to Mucoid Pseudomonas aeruginosa.

Pseudomonas aeruginosa isolates from chronic lung infections often overproduce alginate, giving rise to the mucoid phenotype. Isolation of mucoid strains from chronic lung infections correlates with a poor patient outcome. The most common mutation that causes the mucoid phenotype is called mucA22 and results in a truncated form of the anti-sigma factor MucA that is continuously subjected to proteolysis. When a functional MucA is absent, the cognate sigma factor, AlgT, is no longer sequestered and continuously transcribes the alginate biosynthesis operon, leading to alginate overproduction. In this work, we report that in the absence of wild-type MucA, providing exogenous AlgT is toxic. This is intriguing, since mucoid strains endogenously possess high levels of AlgT. Furthermore, we show that suppressors of toxic AlgT production have mutations in mucP, a protease involved in MucA degradation, and provide the first atomistic model of MucP. Based on our findings, we speculate that mutations in mucP stabilize the truncated form of MucA22, rendering it functional and therefore able to reduce toxicity by properly sequestering AlgT.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen capable of causing chronic lung infections. Phenotypes important for the long-term persistence and adaption to this unique lung ecosystem are largely regulated by the AlgT sigma factor. Chronic infection isolates often contain mutations in the anti-sigma factor mucA, resulting in uncontrolled AlgT and continuous production of alginate in addition to the expression of ∼300 additional genes. Here, we report that in the absence of wild-type MucA, AlgT overproduction is lethal and that suppressors of toxic AlgT production have mutations in the MucA protease, MucP. Since AlgT contributes to the establishment of chronic infections, understanding how AlgT is regulated will provide vital information on how P. aeruginosa is capable of causing long-term infections.

The histone-like protein AlgP regulon is distinct in mucoid and nonmucoid Pseudomonas aeruginosa and does not include alginate biosynthesis genes.

The opportunistic bacterial pathogen Pseudomonas aeruginosa causes acute and chronic infections that are notoriously difficult to treat. In people with cystic fibrosis, P. aeruginosa can cause lifelong lung infections, and isolation of mucoid P. aeruginosa, resulting from the overproduction of alginate, is associated with chronic infection. The histone-like protein AlgP has previously been implicated in the control of alginate gene expression in mucoid strains, but this regulation is unclear. To explore AlgP in further detail, we deleted algP in mucoid strains and demonstrated that the deletion of algP did not result in a nonmucoid phenotype or a decrease in alginate production. We showed that the algP promoter is expressed by both the nonmucoid strain PAO1 and the isogenic mucoid strain PDO300, suggesting that there may be genes that are differentially regulated between these strains. In support of this, using RNA sequencing, we identified a small AlgP regulon that has no significant overlap between PAO1 and PDO300 and established that alginate genes were not differentially regulated by the deletion of algP. Of note, we found that deleting algP in PAO1 increased expression of the nitric oxide operon norCBD and the nitrous oxide reductase genes nosRZ and subsequently promoted growth of PAO1 under anaerobic conditions. Altogether, we have defined a narrow regulon of genes controlled by AlgP and provided evidence that alginate production is not greatly affected by AlgP, countering the long-standing premise in the field.

The Natural Product Elegaphenone Potentiates Antibiotic Effects against Pseudomonas aeruginosa.

Natural products represent a rich source of antibiotics that address versatile cellular targets. The deconvolution of their targets via chemical proteomics is often challenged by the introduction of large photocrosslinkers. Here we applied elegaphenone, a largely uncharacterized natural product antibiotic bearing a native benzophenone core scaffold, for affinity-based protein profiling (AfBPP) in Gram-positive and Gram-negative bacteria. This study utilizes the alkynylated natural product scaffold as a probe to uncover intriguing biological interactions with the transcriptional regulator AlgP. Furthermore, proteome profiling of a Pseudomonas aeruginosa AlgP transposon mutant provided unique insights into the mode of action. Elegaphenone enhanced the elimination of intracellular P. aeruginosa in macrophages exposed to sub-inhibitory concentrations of the fluoroquinolone antibiotic norfloxacin.

Remodeling of O Antigen in Mucoid Pseudomonas aeruginosa via Transcriptional Repression of wzz2.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in people with cystic fibrosis (CF). Chronic P. aeruginosa isolates generally do not express O antigen and often have a mucoid phenotype, which is characterized by the overproduction of the exopolysaccharide alginate. Therefore, O antigen expression and the mucoid phenotype may be coordinately regulated upon chronic adaption to the CF lung. Here we demonstrate that PDO300, a mucoid strain derived from the nonmucoid laboratory isolate PAO1, does not produce very long O antigen due to decreased expression of Wzz2, the very long O antigen chain length control protein, and that mucoid clinical isolates express reduced levels of Wzz2 compared to nonmucoid isolates. Further, we show that forcing the expression of very long O antigen by PDO300, by providing wzz2 in trans, does not alter alginate production, suggesting that sugar precursors are not limited between the two biosynthesis pathways. Moreover, we confirm that AmrZ, a transcription factor highly expressed in mucoid strains, is a negative regulator of wzz2 promoter activity and very long O antigen expression. These experiments identify the first transcriptional regulator of O antigen chain length in P. aeruginosa and support a model where transition to a chronic mucoid phenotype is correlated with downregulation of very long O antigen through decreased Wzz2 production.IMPORTANCE Detection of mucoid Pseudomonas aeruginosa, characterized by the overproduction of alginate, is correlated with the establishment of a chronic pulmonary infection and disease progression in people with cystic fibrosis (CF). In addition to the overproduction of alginate, loss of O antigen lipopolysaccharide production is also selected for in chronic infection isolates. In this study, we have identified the regulatory network that inversely regulates O antigen and alginate production. Understanding the regulation of these chronic phenotypes will elucidate mechanisms that are important for the establishment of a long-term P. aeruginosa lung infection and ultimately provide an opportunity for intervention. Preventing P. aeruginosa from chronically adapting to the CF lung environment could provide a better outcome for people who are infected.