Role of an inducible single-domain hemoglobin in mediating resistance to nitric oxide and nitrosative stress in Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from Vitreoscilla stercoraria and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of C. jejuni was hypersensitive to nitrosating agents (S-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress, cgb expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the cgb mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the cgb mutant. Based on similar studies in Campylobacter coli, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.     

Structural and functional properties of a truncated hemoglobin from a food-borne pathogen Campylobacter jejuni

Campylobacter jejuni contains two hemoglobins, Cgb and Ctb. Cgb has been suggested to perform an NO detoxification reaction to protect the bacterium against NO attack. On the other hand, the physiological function of Ctb, a class III truncated hemoglobin, remains unclear. By using CO as a structural probe, resonance Raman data show that the distal heme pocket of Ctb exhibits a positive electrostatic potential. In addition, two ligand-related vibrational modes, nu(Fe-O(2)) and nu(O-O), were identified in the oxy derivative, with frequencies at 542 and 1132 cm(-1), respectively, suggesting the presence of an intertwined H-bonding network surrounding the heme-bound ligand, which accounts for its unusually high oxygen affinity (222 microm(-1)). Mutagenesis studies of various distal mutants suggest that the heme-bound dioxygen is stabilized by H-bonds donated from the Tyr(B10) and Trp(G8) residues, which are highly conserved in the class III truncated hemoglobins; furthermore, an additional H-bond donated from the His(E7) to the Tyr(B10) further regulates these H-bonding interactions by restricting the conformational freedom of the phenolic side chain of the Tyr(B10). Taken together, the data suggest that it is the intricate balance of the H-bonding interactions that determines the unique ligand binding properties of Ctb. The extremely high oxygen affinity of Ctb makes it unlikely to function as an oxygen transporter; on the other hand, the distal heme environment of Ctb is surprisingly similar to that of cytochrome c peroxidase, suggesting a role of Ctb in performing a peroxidase or P450-type of oxygen chemistry.     

Purification and spectroscopic characterization of Ctb, a group III truncated hemoglobin implicated in oxygen metabolism in the food-borne pathogen Campylobacter jejuni

Campylobacter jejuni is a food-borne bacterial pathogen that possesses two distinct hemoglobins, encoded by the ctb and cgb genes. The former codes for a truncated hemoglobin (Ctb) in group III, an assemblage of uncharacterized globins in diverse clinically and technologically significant bacteria. Here, we show that Ctb purifies as a monomeric, predominantly oxygenated species. Optical spectra of ferric, ferrous, O(2)- and CO-bound forms resemble those of other hemoglobins. However, resonance Raman analysis shows Ctb to have an atypical nu(Fe)(-)(CO) stretching mode at 514 cm(-)(1), compared to those of the other truncated hemoglobins that have been characterized so far. This implies unique roles in ligand stabilization for TyrB10, HisE7, and TrpG8, residues highly conserved within group III truncated hemoglobins. Because C. jejuni is a microaerophile, and a ctb mutant exhibits O(2)-dependent growth defects, one of the hypothesized roles of Ctb is in the detoxification, sequestration, or transfer of O(2). The midpoint potential (E(h)) of Ctb was found to be -33 mV, but no evidence was obtained in vitro to support the hypothesis that Ctb is reducible by NADH or NADPH. This truncated hemoglobin may function in the facilitation of O(2) transfer to one of the terminal oxidases of C. jejuni or, instead, facilitate O(2) transfer to Cgb for NO detoxification.     

Growth of Campylobacter jejuni on nitrate and nitrite: electron transport to NapA and NrfA via NrfH and distinct roles for NrfA and the globin Cgb in protection against nitrosative stress

Pathways of electron transport to periplasmic nitrate (NapA) and nitrite (NrfA) reductases have been investigated in Campylobacter jejuni, a microaerophilic food-borne pathogen. The nap operon is unusual in lacking napC (encoding a tetra-haem c-type cytochrome) and napF, but contains a novel gene of unknown function, napL. The iron-sulphur protein NapG has a major role in electron transfer to the NapAB complex, but we show that slow nitrate-dependent growth of a napG mutant can be sustained by electron transfer from NrfH, the electron donor to the nitrite reductase NrfA. A napL mutant possessed approximately 50% lower NapA activity than the wild type but showed normal growth with nitrate as the electron acceptor. NrfA was constitutive and was shown to play a role in protection against nitrosative stress in addition to the previously identified NO-inducible single domain globin, Cgb. However, nitrite also induced cgb expression in an NssR-dependent manner, suggesting that growth of C. jejuni with nitrite causes nitrosative stress. This was confirmed by lack of growth of cgb and nssR mutants, and slow growth of the nrfA mutant, in media containing nitrite. Thus, NrfA and Cgb together provide C. jejuni with constitutive and inducible components of a robust defence against nitrosative stress.     

The response of Paracoccidioides spp. to nitrosative stress

Paracoccidioidomycosis (PCM) is an endemic disease in Latin America caused by species belonging to the genus Paracoccidioides. During infection, immune cells present a variety of defense mechanisms against pathogens. One of these defensive strategies is the production and release of nitric oxide (NO) and S-nitroso thiols (e.g., S-nitrosoglutathione, GSNO), which produce reactive nitrogen species (RNS). This results in damage to DNA and membranes, inhibition of respiration and inactivation of cellular enzymes. In response to nitrosative stress, human pathogenic fungi possess defense mechanisms to prevent the adverse effects of NO, which helps them survive during initial contact with the host immune system. To understand how Paracoccidioides spp. respond to nitrosative stress, we conducted this study to identify genes and proteins that might contribute to this response. The results of proteomic analysis demonstrated that nitrosative stress induced a reduction in the expression of proteins related to the mitochondrial electron transport chain. This hypothesis was supported by the reduced mitochondrial activity observed in the presence of GSNO. Additionally, lipids and branched chain amino acid metabolism enzymes were altered. The role played by enzymes acting in oxidative stress in the RNS response was remarkable. This interface among enzymes acting in both stress responses was confirmed by using a RNA approach to silence the ccp gene in Paracoccidioides. It was observed that mutants with low expression of the ccp gene were more sensitive to nitrosative stress.     

Helicobacter pylori proteins response to nitric oxide stress

Helicobacter pylori is a highly pathogenic microorganism with various strategies to evade human immune responses. Nitric oxide (NO) and reactive nitrogen species (RNS) generated via nitric oxide synthase pathway are important effectors during the innate immune response. However, the mechanisms of H. pylori to survive the nitrosative stress are not clear. Here the proteomic approach has been used to define the adaptive response of H. pylori to nitrosative stress. Proteomic analysis showed that 38 protein spots were regulated by NO donor, sodium nitroprusside (SNP). These proteins were involved in protein processing, anti-oxidation, general stress response, and virulence, as well as some unknown functions. Particularly, some of them were participated in iron metabolism, potentially under the control of ferric uptake regulator (Fur). Real time PCR revealed that fur was induced under nitrosative stress, consistent with our deduction. One stress-related protein up-regulated under nitrosative conditions was thioredoxin reductase (TrxR). Inactiva-tion of fur or trxR can lead to increased susceptivity to nitrosative stress respectively. These studies described the adaptive response of H. pylori to nitric oxide stress, and analyzed the relevant role of Fur regulon and TrxR in nitrosative stress management.     

Mechanisms of nitric oxide crosstalk with reactive oxygen species scavenging enzymes during abiotic stress tolerance in plants

Nitric oxide (NO) acts in a concentration and redox-dependent manner to counteract oxidative stress either by directly acting as an antioxidant through scavenging reactive oxygen species (ROS), such as superoxide anions (O₂^?*), to form peroxynitrite (ONOO^?) or by acting as a signaling molecule, thereby altering gene expression. NO can interact with different metal centres in proteins, such as heme-iron, zinc–sulfur clusters, iron–sulfur clusters, and copper, resulting in the formation of a stable metal–nitrosyl complex or production of varied biochemical signals, which ultimately leads to modification of protein structure/function. The thiols (ferrous iron–thiol complex and nitrosothiols) are also involved in the metabolism and mobilization of NO. Thiols bind to NO and transport it to the site of action whereas nitrosothiols release NO after intercellular diffusion and uptake into the target cells. S-nitrosoglutathione (GSNO) also has the ability to transnitrosylate proteins. It is an NO˙ reservoir and a long-distance signaling molecule. Tyrosine nitration of proteins has been suggested as a biomarker of nitrosative stress as it can lead to either activation or inhibition of target proteins. The exact molecular mechanism(s) by which exogenous and endogenously generated NO (or reactive nitrogen species) modulate the induction of various genes affecting redox homeostasis, are being extensively investigated currently by various research groups. Present review provides an in-depth analysis of the mechanisms by which NO interacts with and modulates the activity of various ROS scavenging enzymes, particularly accompanying ROS generation in plants in response to varied abiotic stress.     

Structural and functional properties of a single domain hemoglobin from the food-borne pathogen Campylobactor jejuni

Campylobacter jejuni contains two globins, a truncated hemoglobin, Ctb, and a single domain hemoglobin, Cgb. The physiological function of Ctb remains unclear, whereas Cgb has been linked to NO detoxification. With resonance Raman scattering, the iron-histidine stretching mode of Cgb was identified at 251 cm(-1). This frequency is unusually high, suggesting an imidazolate character of the proximal histidine as a result of the H-bonding network linking the catalytic triad involving the F8His, H23Glu, and G5Tyr residues. In the CO-complex, two conformers were identified with the nuC-O/nuFe-CO at 529/1914 cm(-1) and 492/1963 cm(-1). The former is assigned to a "closed" conformation, in which the heme-bound CO is stabilized by the H-bond(s) donated from the B10Tyr-E7Gln residues, whereas the latter is assigned to an "open" conformer, in which the H-bonding interaction is absent. The presence of the two alternative conformations demonstrates the plasticity of the protein matrix. In the O2-complex, the iron-O2 stretching frequency was identified at 554 cm(-1), which is unusually low, indicating that the heme-bound O2 is stabilized by strong H-bond(s) donated by the B10Tyr-E7Gln residues. This scenario is consistent with its low O2 off-rate (0.87 s(-1)). Taken together the data suggest that the NO-detoxifying activity of Cgb is facilitated by the imidazolate character of the proximal F8His and the distal positive polar environment provided by the B10Tyr-E7Gln. They may offer electronic "push" and "pull," respectively, for the O-O bond cleavage reaction required for the isomerization of the presumed peroxynitrite intermediate to the product, nitrate.     

Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni

CosR (Campylobacter oxidative stress regulator; Cj0355c) is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a leading foodborne pathogen causing human gastroenteritis worldwide. Despite importance, the function of CosR remains completely unknown mainly because of cell death caused by its knockout mutation. To overcome this technical limitation, in this study, antisense technology was used to investigate the regulatory function of CosR by modulating the level of CosR expression. Two-dimensional gel electrophoresis (2DGE) was performed to identify the CosR regulon either by suppressing CosR expression with antisense peptide nucleic acid (PNA) or by overexpressing CosR in C. jejuni. According to the results of 2DGE, CosR regulated 32 proteins involved in various cellular processes. Notably, CosR negatively regulated a few key proteins of the oxidative stress response of C. jejuni, such as SodB, Dps, Rrc and LuxS, whereas CosR positively controlled AhpC. Electrophoretic mobility shift assay showed that CosR directly bound to the promoter region of the oxidative stress genes. DNase I footprinting assays identified 21-bp CosR binding sequences in the sodB and ahpC promoters, suggesting CosR specifically recognizes and binds to the regulated genes. Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide. Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type. Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.     

Ferrous Campylobacter jejuni truncated hemoglobin P displays an extremely high reactivity for cyanide - a comparative study

Campylobacter jejuni hosts two hemoglobins (Hbs). The Camplylobacter jejuni single-domain Hb (called Cgb) is homologous to the globin domain of flavohemoglobin, and it has been proposed to protect the bacterium against nitrosative stress. The second Hb is called Ctb (hereafter Cj-trHbP), belongs to truncated Hb group III, and has been hypothesized to be involved in O(2) chemistry. Here, the kinetics and thermodynamics of cyanide binding to ferric and ferrous Cj-trHbP [Cj-trHbP(III) and Cj-trHbP(II), respectively] are reported and analyzed in parallel with those of related heme proteins, with particular reference to those from Mycobacterium tuberculosis. The affinity of cyanide for Cj-trHbP(II) is higher than that reported for any known (in)vertebrate globin by more than three orders of magnitude (K = 1.2 x 10(-6) m). This can be fully attributed to the highest (ever observed for a ferrous Hb) cyanide-binding association rate constant (k(on) = 3.3 x 10(3) m(-1).s(-1)), even though the binding process displays a rate-limiting step (k(max) = 9.1 s(-1)). Cj-trHbP(III) shows a very high affinity for cyanide (L = 5.8 x 10(-9) m); however, cyanide association kinetics are independent of cyanide concentration, displaying a rate-limiting step (l(max) = 2.0 x 10(-3) s(-1)). Values of the first-order rate constant for cyanide dissociation from Cj-trHbP(II)-cyanide and Cj-trHbP(III)-cyanide (k(off) =5.0 x 10(-3) s(-1) and l(off) > or = 1 x 10(-4) s(-1), respectively) are similar to those reported for (in)vertebrate globins. The very high affinity of cyanide for Cj-trHbP(II), reminiscent of that of horseradish peroxidase(II), suggests that this globin may participate in cyanide detoxification.