<i>In Vivo</i> Analysis of Staphylococcus aureus-Infected Mice Reveals Differential Temporal and Spatial Expression Patterns of <i>fhuD2</i>

Abstract

<i>Staphylococcus aureus</i> is an opportunistic human pathogen and a major cause of invasive infections such as bacteremia, endocarditis, pneumonia, and wound infections. FhuD2 is a staphylococcal lipoprotein involved in the uptake of iron-hydroxymate and is under the control of the iron uptake regulator Fur. This protein is part of an investigational multicomponent vaccine formulation that has shown protective efficacy in several murine models of infection. Even though <i>fhuD2</i> expression has been shown to be upregulated in murine kidneys infected with <i>S. aureus</i>, it is not known whether the bacterium undergoes increased iron deprivation during prolonged infection. Furthermore, different <i>S. aureus</i> infection niches might provide different environments and levels of iron availability, resulting in different <i>fhuD2</i> expression patterns among organs of the same host. To address these questions, we characterized the <i>in vitro</i> expression of the <i>fhuD2</i> gene and confirmed Fur-dependent regulation of its expression. We further investigated its expression in mice infected with a bioluminescent reporter strain of <i>S. aureus</i> expressing the luciferase operon under the control of the <i>fhuD2</i> promoter. The emission of bioluminescence in different organs was followed over a 7-day time course, and quantitative real-time PCR analysis of the RNA transcribed from the endogenous <i>fhuD2</i> gene was performed. Using this approach, we were able to show that <i>fhuD2</i> expression was induced during infection in all organs analyzed and that differences in expression were observed at different time points and in different infected organs. Our data suggest that <i>S. aureus</i> undergoes increased iron deprivation during the progression of infection in diverse host organs and accordingly induces dedicated iron acquisition mechanisms. Since FhuD2 plays a central role in providing the pathogen with the required iron, further knowledge of the patterns of <i>fhuD2</i> expression <i>in vivo</i> during infection will be instrumental in better defining the role of this antigen in <i>S. aureus</i> pathogenesis and as a vaccine antigen.