The Extracytoplasmic Sigma Factor, ς E , Is Required for Intracellular Survival of Nontypeable Haemophilus influenzae in J774 Macrophages

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Abstract
Nontypeable Haemophilus influenzae (NTHi) causes a wide variety of respiratory tract infections in humans. It is capable of invading and surviving in epithelial cells and has also been shown to persist in macrophage-like cell line J774A.1. To determine the molecular mechanisms which enable NTHi to survive in an intracellular environment, differential display reverse transcriptase PCR was used to identify genes which were either induced or upregulated by NTHi residing in macrophages. Using this approach, we identified one transcript which was consistently amplified from intracellular NTHi cDNA. Nucleotide sequence analysis of this product revealed that it spanned the 3′ and 5′ ends of rpoE and rseB , respectively, which form part of the extracytoplasmic stress operon that encodes and regulates expression of alternate sigma factor sigma E (ς E ). To confirm that expression of rpoE was upregulated following uptake of NTHi by macrophages, an rpoE-lacZ transcriptional fusion was constructed, and expression of β-galactosidase activity in broth-grown NTHi was compared with expression of β-galactosidase activity in intracellular NTHi. The level of β-galactosidase activity in NTHi 4 h after phagocytosis by macrophages was found to be 100-fold higher than that of broth-grown organisms, suggesting that genes of the ς E regulon may be important for persistence of NTHi in mammalian cells. The hypothesis that ς E plays a role in the intracellular survival of NTHi was subsequently confirmed by the decreased ability of an rpoE insertion mutant to survive in macrophages.