Transcriptomic Identification and Biochemical Characterization of HmpA, a Nitric Oxide Dioxygenase, Essential for Pathogenesis of Vibrio vulnificus

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 K _M and k _cat 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.

Keywords: Vibrio vulnificus; flavohemoglobins; gene expression profiling; microbiology; nitric oxide dioxygenase; virulence factors.

FIGURE 1

Genes upregulated by NO exposure and possibly involved in nitrosative stress defense. Among the NO-upregulated genes (fold change ≥2; p value of <0.05) identified by transcriptome analysis, 8 genes potentially involved in nitrosative stress defense were selected and their upregulation was confirmed by qRT-PCR. Each column represents the mRNA expression level of V. vulnificus MO6-24/O exposed to NO/PPNPs relative to that exposed to PPNPs (negative control). Error bars represent the S.D. Locus tags are based on the V. vulnificus MO6-24/O genome sequence (GenBank^TM accession numbers: CP002469 and CP002470) and the products of the genes are presented on the right. NO/PPNPs, NO-releasing poly(lactic-co-glycolic acid)-polyethylenimine nanoparticles; PPNPs, poly(lactic-co-glycolic acid)-polyethylenimine nanoparticles.

FIGURE 2

Expression of VvhmpA under nitrosative stress. RNAs and proteins were extracted from V. vulnificus MO6-24/O exposed either to Spermine NONOate or M9G (negative control). (A) The VvhmpA mRNA levels were determined by qRT-PCR analyses, and expressed using the VvhmpA mRNA level of the culture unexposed to NO as 1. Error bars represent the S.D. ^∗p < 0.05 relative to the culture unexposed to NO. (B) Total proteins of the cultures were resolved on reducing SDS-PAGE, and VvHmpA and VvDnaK were immunoblotted using the rabbit anti-VvHmpA antibody and the mouse anti-DnaK antibody, respectively. The protein size markers (Bio-Rad) on the left are in kilodaltons.

FIGURE 3

Sequence analysis of V. vulnificus HmpA (VvHmpA), E. coli Hmp (EcHmp), S. Typhimurium Hmp (StHmp), and V. cholerae HmpA (VcHmpA). The amino acid sequences retrieved from the NCBI protein database (accession numbers: WP_013570994.1 for VvHmpA, NP_417047.1 for EcHmp, WP_000883146.1 for StHmp, and WP_000957477.1 for VcHmpA) were aligned using the Clustal Omega program. Identical (black boxes), conserved (gray boxes), and missing (dashes) sequences are indicated. The conserved amino acid residues potentially involved in the binding of heme and FAD are indicated above the amino acid sequences by asterisks and dots, respectively. The putative NAD-binding domain is boxed by a black line.

FIGURE 4

Absorption spectra and cofactors of VvHmpA. (A) The absorption spectra of the purified VvHmpA in the reduced (solid line), NO-bound (dashed line), and oxidized (spotted line) states were measured using a UV–vis spectrophotometer. The numbers above the peaks indicate the wavelengths of the observed Soret peaks. (B) The ratios of heme and FAD per one molecule of purified and reconstituted VvHmpA.

FIGURE 5

Kinetic analysis of VvHmpA. (A) The VvHmpA protein and various concentrations of NO were delivered into the reaction buffer. The initial rates of NO decomposition were determined by measuring the residual NO in the reaction mixture and plotted against the corresponding initial concentrations of NO. Error bars represent the S.D. (B) V_max, K_M, and k_cat values for NO were determined by fitting the curve (A) to a classical Michaelis–Menten enzyme kinetic equation. The turnover rate (k_cat) is expressed relative to heme.

FIGURE 6

NO decomposition and survival of the V. vulnificus strains under nitrosative stress. (A) The V. vulnificus strains were pre-exposed to Spermine NONOate to induce VvHmpA, and then 2 μM of NO was administered to the strains at the time designated by an arrow. The residual NO in the mixtures was measured to determine the NO decomposition. (B) Survival of the V. vulnificus strains exposed to excess NO was monitored by counting viable cells at time intervals. Error bars represent the S.D. PBS, control; WT (pJH0311), wild type; DY171 (pJH0311), VvhmpA mutant; DY171 (pDY1701), VvhmpA-complemented strain.

FIGURE 7

Survival and cytotoxicity of the V. vulnificus strains infecting the RAW 264.7 cells. The NO-producing RAW 264.7 cells were infected with the V. vulnificus strains at the MOI of 1 for various incubation times in the absence (A,C) or presence (B,D) of the NO synthase inhibitor, L-NMMA. (A,B) The V. vulnificus cells adherent to the RAW 264.7 cells were enumerated in cfu per macrophage at each time point after infection. (C,D) The cytotoxicity was expressed using the total LDH activity of the RAW 264.7 cells completely lysed by 1.5% Triton X-100 as 100%. Error bars represent the S.D. ^∗p < 0.05 and ^∗∗p < 0.005 relative to groups infected with the wild type at each incubation time. WT (pJH0311), wild type; DY171 (pJH0311), VvhmpA mutant; DY171 (pDY1701), VvhmpA-complemented strain.

FIGURE 8

Mouse lethality of the V. vulnificus strains. Groups (n = 15) of 7-week-old specific pathogen-free female ICR mice were intraperitoneally infected with either the wild type or the VvhmpA mutant of V. vulnificus at doses of 1.0 × 10⁶ cfu. Mouse survival percentage was monitored for 24 h. ^∗p < 0.05; WT, wild type; DY171, VvhmpA mutant.