Abstract
Nitric oxide (NO) and its derivatives are important effectors of host innate immunity, disrupting cellular function of infecting pathogens. Transcriptome analysis of Vibrio vulnificus, an opportunistic human pathogen, identified a set of genes induced upon exposure to NO. Among them, VvhmpA (V. vulnificus hmpA), encoding a multidomain NO dioxygenase, was the most greatly induced upon exposure to NO and was thus further characterized. Absorption spectra demonstrated that VvHmpA is a heme protein in which the heme iron can exist in either reduced, NO-bound, or oxidized state. Biochemical studies revealed that VvHmpA is a flavohemoglobin containing equimolar amounts of heme and FAD as cofactors. The KM and kcat values of VvHmpA for NO at 37°C, the temperature encountered by V. vulnificus in the host, were greater than those at 30°C, indicating that VvHmpA detoxifies high levels of NO effectively during infection. Compared with the wild type, the VvhmpA mutant exhibited a lower NO-decomposition activity and impaired growth in the presence of NO in vitro. Also, the cytotoxicity and survival of the VvhmpA mutant infecting the NO-producing murine macrophage cells were lower than those of the wild type. Furthermore, the mouse lethality of the VvhmpA mutant was reduced compared to that of the parental wild type. The combined results revealed that VvHmpA is a potent virulence factor that is induced upon exposure to NO and important for the survival and pathogenesis of V. vulnificus during infection.
Highlights
Nitric oxide (NO) and its derivatives, collectively called reactive nitrogen species (RNS), are among the most important components of the host innate immune system, the first line of defense against infecting pathogens (Fang, 2004)
Among the genes upregulated upon exposure to NO, 8 genes potentially involved in nitrosative stress defense were selected (Figure 1)
Expression of the 8 genes in V. vulnificus exposed to NO/PPNPs was reevaluated by using quantitative real-time PCR analyses, further confirming that NO exposure induced transcription of the genes (Figure 1)
Summary
Nitric oxide (NO) and its derivatives, collectively called reactive nitrogen species (RNS), are among the most important components of the host innate immune system, the first line of defense against infecting pathogens (Fang, 2004). NO produced by iNOS can subsequently be converted into derivatives such as nitrogen dioxide (NO2), peroxynitrite (ONOO−), and dinitrogen trioxide (N2O3) (Fang, 2004; Stern and Zhu, 2014). RNS can lead to the damage of cellular components, including metal centers of proteins, membrane lipids and nucleotide bases, and thereby inhibit respiration and interfere with DNA replication of pathogens (Fang, 1999). Pathogens have evolved sophisticated mechanisms to overcome nitrosative stress caused by the increased level of RNS, and the mechanisms are closely linked to their virulence (Bang et al, 2006; Richardson et al, 2006; Stern et al, 2012)
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