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.In the evolution of life, a delicate physiological balance has arisen to compensate for both an organism's need for oxygen and the attendant lethal consequences of oxygen's toxicity. For example, in the electron transport chain, electrons can be inadvertently transferred from redox-active proteins to oxygen, producing reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ). The effects of ROS on cells can then be exacerbated by the presence of free iron which, via the Fenton reaction, can react with H 2 O 2 to produce more highly reactive hydroxyl radicals (34, 40, 52). Within a host, this problem is compounded by the release of extracellular ROS by phagocytes, which again, can have deleterious effects on invading pathogens (13). Thus, bacteria have evolved an array of increasingly well-defined mechanisms to abrogate the effects of oxidative stress. Proteins involved in such processes include catalase, which decomposes H 2 O 2 , and members of both the AhpCF/TsaA family of alkylhydroperoxidases and the organic hydroperoxide resistance proteins (Ohr) that decompose organic peroxides. Also, DNA-binding ferritin-like proteins (Dps) sequester free iron and bind to DNA, thus protecting DNA from damage (27,33,45,53,59,60,67). In addition, many bacterial species possess genes encoding OxyR, a global regulator of antioxidant defenses (24, 66).Protection against oxidative stress is especially important to nontypeable Haemophilus influenzae (NTHi). NTHi is one of three bacterial commensal organisms, the others being Streptococcus pneumoniae and Moraxella catarrhalis, which can ascend a virus-compromised eustachian tube and caus...

Heme Utilization by Nontypeable Haemophilus influenzae Is Essential and Dependent on Sap Transporter Function

“…The parental NTHI strain 86-028NP is a minimally passaged clinical isolate obtained at Nationwide Children's Hospital. This strain has been extensively characterized in chinchilla models of OM and has been sequenced and annotated (13,14). The sapA mutant was constructed by insertional mutagenesis as previously described (22).…”

Activation of the CpxRA System by Deletion of cpxA Impairs the Ability of Haemophilus ducreyi To Infect Humans

“…After optimizing primers so that their efficiencies were Ͼ95%, we examined the level of transcript expression in RNA isolated from 35000HP and 35000HP⌬cpxA. Reactions utilizing bacterial RNA, the dsrA primers P16 and P17 and dnaE primers P5 and P6, and the one-step QuantiTect SYBR green reverse transcription-PCR (RT-PCR) kit (Qiagen) were performed in triplicate as described previously (19). The levels of expression were determined by using an ABI Prism 7000 sequence detector (Applied Biosystems, Carlsbad, CA).…”

Peroxide-Sensing Transcriptional Regulators in Bacteria

“…OxyR also upregulates the expression of OxyS, a small regulatory RNA that integrates peroxide stress with general stress pathways (34,122). Although there are significant differences, the OxyR regulons of other organisms tend to include similar classes of genes (39,82,96,104,116).…”