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Infection and Immunity, February 2002, p. 708-715, Vol. 70, No. 2
0019-9567/01/$04.00+0     DOI: 10.1128/IAI.70.2.708-715.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Jane E. Craig, Angela Nobbs,,{dagger} and Nicola J. High*

School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom

Received 17 July 2001/ Returned for modification 28 August 2001/ Accepted 24 October 2001

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 ({varsigma}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 ß-galactosidase activity in broth-grown NTHi was compared with expression of ß-galactosidase activity in intracellular NTHi. The level of ß-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 {varsigma}E regulon may be important for persistence of NTHi in mammalian cells. The hypothesis that {varsigma}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.

* Corresponding author. Mailing address: School of Biological Sciences, University of Manchester, 1.800 Stopford Building, Manchester, M13 9PT, United Kingdom. Phone: 44 161 275 5749. Fax: 44 161 5656. E-mail: nicky.high{at}man.ac.uk.

Editor: D. L. Burns

{dagger} Present address: Department of Oral and Dental Science, University of Bristol Dental School, Bristol, BS1 2LY, United Kingdom.

Infection and Immunity, February 2002, p. 708-715, Vol. 70, No. 2
0019-9567/01/$04.00+0     DOI: 10.1128/IAI.70.2.708-715.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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