The OxyR regulon in nontypeable Haemophilus influenzae

Abstract

Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium and a common commensal organism of the upper respiratory tract in humans. NTHi causes a number of diseases, including otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During the course of colonization and infection, NTHi must withstand oxidative stress generated by insult due to multiple reactive oxygen species produced endogenously by other copathogens and by host cells. Using an NTHi-specific microarray containing oligonucleotides representing the 1821 open reading frames of the recently sequenced NTHi isolate 86-028NP, we have identified 40 genes in strain 86-028NP that are upregulated after induction of oxidative stress due to hydrogen peroxide. Further comparisons between the parent and an isogenic oxyR mutant identified a subset of 11 genes that were transcriptionally regulated by OxyR, a global regulator of oxidative stress. Interestingly, hydrogen peroxide induced the OxyR-independent upregulation of expression of the genes encoding components of multiple iron utilization systems. This finding suggested that careful balancing of levels of intracellular iron was important for minimizing the effects of oxidative stress during NTHi colonization and infection and that there are additional regulatory pathways involved in iron utilization.

FIG. 1.

The growth of NTHi 86-028NP strain (A) and the NTHi 86-028NP oxyR mutant strain (B) in increasing concentrations of H₂O₂. Both parent and mutant strains were grown to exponential phase, and then the designated concentration of H₂O₂ was added (arrowhead). Growth was then allowed to continue in the presence of H₂O₂.

FIG. 2.

Recovery of the NTHi 86-028NP strain and the NTHi 86-028NP oxyR mutant after a 10-min treatment with 250 μM H₂O₂. Both parent and mutant strains were grown to exponential phase and then treated with 250 μM H₂O₂ for 10 min (arrowhead indicates the point of H₂O₂ addition). The medium was then removed, and the cells were washed and resuspended in fresh medium. Growth was then allowed to continue.