# Ureaplasma-driven inhibition of the epithelial Na+ transport in fetal alveolar cells: A novel mechanism of Ureaplasma-mediated preterm lung disease

## TL;DR

This study shows how Ureaplasma infection impairs lung function in preterm infants by reducing sodium transport in fetal lung cells, potentially leading to breathing difficulties.

## Contribution

The study identifies ammonia as a Ureaplasma virulence factor and shows that urease inhibitors can restore sodium transport in infected cells.

## Key findings

- Ureaplasma infection reduces epithelial Na+ transport by 30–90% in fetal lung cells.
- Ammonia from Ureaplasma, not pH shifts, mimics the infection's effects on Na+ transport.
- Urease inhibitor flurofamide restores Na+ transport in infected cells.

## Abstract

Respiratory tract colonization with Ureaplasma species has been repeatedly associated with the development of acute and long-term pulmonary morbidity in preterm infants. However, despite strong evidence from observational studies and animal models, apart from inflammation, underlying mechanisms of Ureaplasma-driven lung disease, such as potential functional impairments, are mainly unknown. Knowledge of Ureaplasma-lung interaction and Ureaplasma virulence factors is scarce. The present investigation is the first to examine the influence of perinatal Ureaplasma infection on critical mechanisms of alveolar fluid clearance (AFC) in immature lung cells, which drive perinatal transition from fluid-filled lungs before birth to alveolar fluid absorption, enabling lung breathing. Disruption or impairment of these mechanisms could worsen respiratory distress in preterm infants and contribute to acute lung injury. Moreover, the present study addressed Ureaplasma-derived ammonia and the accompanying pH shift as potential virulence factors driving Ureaplasma-host interactions. Both have long been discussed as virulence factors, but remain unexamined, so far. We report that viable Ureaplasma isolates induced a 30–90% decrease in epithelial Na+ transport of primary rat fetal distal lung epithelial (FDLE) cells upon 24 hours of infection. Moreover, the decrease was linked to a significant inhibition of the epithelial Na+ channel (ENaC) and Na,K-ATPase activities, both mediating the essential AFC. It was observed that acute Ureaplasma infection induced phosphorylation of Erk1/2 – a well-known inhibitor of ENaC activity. Notably, exposure of FDLE cells to Ureaplasma-driven NH3 – in contrast to the hydrolysis-driven pH shift – fully mimicked Ureaplasma-driven effects and inhibited the epithelial Na+ transport. Co-incubation with the urease inhibitor flurofamide entirely restored Na+ transport in Ureaplasma-infected FDLE cells. Ureaplasma infection differentially modulated ENaC subunit and surfactant protein mRNA expression. In summary, the present study revealed a functional impairment of fetal pulmonary epithelial cells upon acute Ureaplasma infection and identified NH3 as a Ureaplasma virulence factor in this context. Co-incubation with flurofamide restored Na+ transport. This study describes a novel mechanism of Ureaplasma-driven early preterm lung disease, which might be of great significance for a deeper understanding of Ureaplasma-host interactions. Notably, the present findings offer a potential therapeutic role for urease inhibitors in Ureaplasma-colonized preterm infants.

Long dismissed as pathogens in preterm infants, Ureaplasma species represent a potentially actionable factor causing acute and chronic preterm lung disease, including bronchopulmonary dysplasia (BPD), especially in the most immature infants. Yet, data on Ureaplasma-host interactions and Ureaplasma virulence factors are scarce, and underlying mechanisms of Ureaplasma-associated preterm lung disease remain largely unaddressed. However, against the background of perinatal respiratory tract colonization in 30–60% of very preterm infants, a comprehensive understanding of Ureaplasma-driven preterm lung disease remains paramount to identifying addressable pathways and establishing focused therapeutic approaches. Our study is the first to examine the interaction of Ureaplasma with fetal distal lung epithelial cells as an established model of otherwise unavailable preterm alveoli and to investigate the influence of perinatal Ureaplasma infection on key mechanisms of alveolar fluid clearance (AFC) in the developing lung – an essential event enabling postnatal lung breathing. Our data shed light on a direct crosstalk between Ureaplasma and alveolar epithelial cells with potential impairment of the key mechanisms driving AFC. The present study reveals a Ureaplasma-mediated affection of alveolar cell function that might significantly impact postnatal transition to air breathing. We report a significant decrease in the epithelial Na+ transport in Ureaplasma-infected cells resulting from inhibition of epithelial Na+ channel (ENaC) and Na,K-ATPase activities, and we describe Ureaplasma-mediated NH3 as the potential causal virulence factor, offering a potential therapeutic role for urease inhibitors in Ureaplasma-colonized preterm infants.