In situ profiling of bacterial growth activity during the early stages of Pseudomonas aeruginosa infection in airway epithelia

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen of major clinical importance, which frequently gives rise to persistent antibiotic resilient infections. To investigate the connection between the bacterial physiological activity and the host environment during the early stages of bacterial infections, we have established a dual-fluorescent reporter system to monitor growth activity of P. aeruginosa during infection of a human airway epithelial model. This approach enables quantitative and spatially resolved analysis of bacterial growth within distinct infection micro-niches. Using this infection model, we compared the infection routes for the reference strain PAO1 as well as for two patho-adaptive mutants, PAO1{Delta}pscC ({Delta}pscC) and PAO1{Delta}mexZ ({Delta}mexZ). All three strains colonized apical-, intracellular-, interepithelial-, and epithelial barrier breach sites, but with strain-specific patterns of localisation. PAO1 was rarely observed intracellularly,{Delta} pscC was detected in few cases at epithelial-barrier breach- and interepithelial sites, and{Delta} mexZ colonized apical and intracellular niches in only few cases. Measurements of bacterial growth activities in these niches further revealed distinct hierarchies of bacterial growth activity among the tissue sites: PAO1 bacteria were most active at epithelial-barrier breach sites,{Delta} pscC at interepithelial sites, and{Delta} mexZ in intracellular niches. Taken together, these data support a model in which P. aeruginosa follows a progressive infection continuum from apical colonization to barrier breach, involving interepithelial spread, whereas the mutant strains represented truncated or altered versions of this infection program. More broadly, this study demonstrates the utility of unstable fluorescent reporters for capturing dynamic, niche-specific growth activity patterns during host-pathogen interactions, with implications for both basic pathogenesis research and therapeutic development. Author SummaryIn this study, we set out to better understand how bacterial infections develop and progress on human tissues. To do so, we created fluorescent "reporter" strains of Pseudomonas aeruginosa, a bacterium that commonly infects the lungs of people with cystic fibrosis and other chronic lung diseases. These genetically modified bacteria allow us to directly observe their growth activity in living human cell cultures using advanced microscopy. Here, we can see how the bacteria grow and spread at different locations within the tissue, and if there are any sites they prefer relative to others. We can also see if those patterns change, depending on any mutations the bacteria might have. In the future, the same genetic engineering can be applied to other bacteria, and the fluorescence can inform us of a range of important bacterial functions; for instance, at precisely what point of the infection process - and where in the tissues - the bacteria produce toxins, are stressed, produce antibiotic-resistance proteins or divide, for example. Our experiments revealed that some genetic mutations, that are often found in bacterial isolates from hospital infections, change the preference of colonization to distinct tissue sites and confer specialized and unique patterns - and peaks - of growth activity. Some bacterial strains tend to grow on the surface of the tissue, while others are more likely to move between or inside human cells. These behaviours reflect how bacteria evolve during long-term infections and adapt to different environments within the body tissues. The approach we present here provides a new tool for studying how bacterial infections unfold and may ultimately help identify more effective ways to treat or control them.