Whole-tissue imaging reveals intrastrain diversity shapes the spatial organization ofin a murine infection model.

Abstract

Intrastrain genetic and phenotypic heterogeneity ofis a hallmark of chronic lung infections in individuals with cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Although the coexistence of multiplelineages within a single host is well documented, the impact of this heterogeneity on infection microbiogeography remains poorly understood. We previously showed that loss of the lipopolysaccharide (LPS) O-specific antigen (OSA) altersaggregate assembly. Since OSA-deficient variants are common in chronic pulmonary infections and associated with increased pathogenesis and immune evasion, we investigated whether intrastrain OSA diversity shapes infection microbiogeography. We constructed mixed populations containing equal ratios of OSA-deficient variants and wild-type (WT) cells and examined aggregate assembly and population structures in a synthetic CF sputum model (SCFM2). To assess OSA heterogeneity, we used a murine pneumonia model combined with hybridization chain reaction (HCR) RNA-FISH and whole-tissue clearing to visualize spatial organization in the airways. In SCFM2, OSA-deficient variants increased total population size, reduced WT aggregate size, and altered spatial organization. We employed 2-plex HCR RNA-FISH to distinguish WT and OSA-deficient variants in murine lungs. Interestingly, in contrast toconditions, OSA-deficient cells led to significantly larger WT aggregates in the airways. These findings highlight the role of intrastrain genetic heterogeneity in shaping infection microbiogeography and provide a framework for understanding how population dynamics influence microbial physiology and host-pathogen interactions at the micron scale.IMPORTANCEIntrastrain genetic and phenotypic diversity withinpopulations is common in chronic pulmonary infections. While this intrastrain heterogeneity is a hallmark of chronic infection, its consequences for the spatial organization ofwithin the airways remain unclear. Here, we demonstrate that the loss of O-specific antigen in a subpopulation ofsignificantly alters the spatial architecture of, without changing the total population size or composition. Using a combination of tissue clearing and hybridization chain reaction RNA-FISH in a murine lung infection model, we mapped the localization of genetically distinctvariants in mixed populations. These findings reveal that genetic diversification within a strain can reshape the infection landscape at the micron scale, highlighting the overlooked role of intrastrain dynamics in shaping the microbiogeography of infections and influencing host-pathogen interactions.