Peroxynitrite detoxification by ferryl Mycobacterium leprae truncated hemoglobin O.

During infection, Mycobacterium leprae is faced with the host macrophagic environment limiting the growth of the bacilli. However, (pseudo-)enzymatic detoxification systems, including truncated hemoglobin O (Ml-trHbO), could allow this mycobacterium to persist in vivo. Here, kinetics of peroxynitrite (ONOOH/ONOO(-)) detoxification by ferryl Ml-trHbO (Ml-trHbO-Fe(IV)=O), obtained by treatment with H(2)O(2), is reported. Values of the second-order rate constant for peroxynitrite detoxification by Ml-trHbO-Fe(IV)=O (i.e., of Ml-trHbO-Fe(III) formation; k(on)), at pH 7.2 and 22.0 degrees C, are 1.5x10(4) M(-1) s(-1), and 2.2x10(4) M(-1) s(-1), in the absence of and presence of physiological levels of CO(2) (approximately 1.2x10(-3) M), respectively. Values of k(on) increase on decreasing pH with a pK(a) value of 6.7, this suggests that ONOOH reacts preferentially with Ml-trHbO-Fe(IV)=O. In turn, peroxynitrite acts as an antioxidant of Ml-trHbO-Fe(IV)=O, which could be responsible for the oxidative damage of the mycobacterium. As a whole, Ml-trHbO can undertake within the same cycle H(2)O(2) and peroxynitrite detoxification.

H2O2 and (.)NO scavenging by Mycobacterium leprae truncated hemoglobin O.

Kinetics of ferric Mycobacterium leprae truncated hemoglobin O (trHbOFe(III)) oxidation by H2O2 and of trHbOFe(IV)O reduction by (.)NO and NO2- are reported. The value of the second-order rate constant for H2O2-mediated oxidation of trHbOFe(III) is 2.4 x 10(3) M(-1) s(-1). The value of the second-order rate constant for (.)NO-mediated reduction of trHbOFe(IV)O is 7.8 x 10(6) M(-1) s(-1). The value of the first-order rate constant for trHbOFe(III)ONO decay to the resting form trHbOFe(III) is 2.1 x 10(1) s(-1). The value of the second-order rate constant for NO2--mediated reduction of trHbOFe(IV)=O is 3.1 x 10(3) M(-1) s(-1). As a whole, trHbOFe(IV)O, generated upon reaction with H2O2, catalyzes (.)NO reduction to NO2-. In turn, (.)NO and NO2- act as antioxidants of trHbOFe(IV)O, which could be responsible for the oxidative damage of the mycobacterium. Therefore, Mycobacterium leprae trHbO could be involved in both H2O2 and (.)NO scavenging, protecting from nitrosative and oxidative stress, and sustaining mycobacterial respiration.

Scavenging of reactive nitrogen species by mycobacterial truncated hemoglobins.

Tuberculosis and leprosy are among the most challenging infectious threats to human health. The ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of effective detoxification (pseudoenzymatic) systems. Mycobacterium tuberculosis and Mycobacterium leprae truncated hemoglobins (trHbs) belonging to group I (or N; trHbN) and group II (or O; trHbO) have recently been implicated in the scavenging of nitrogen monoxide (*NO) and peroxynitrite (ONOO-/HOONO). Furthermore, M. leprae trHbO was found to act as an efficient scavenger of the strong oxidant trioxocarbonate(*1-) (CO3*-) following the reaction of peroxynitrite with carbon dioxide (CO2). Here, mechanisms for scavenging of reactive nitrogen species by mycobacterial trHbs are reviewed, and detailed protocols for assessing pseudoenzymatic kinetics are provided.

*NO dissociation represents the rate limiting step for O2-mediated oxidation of ferrous nitrosylated Mycobacterium leprae truncated hemoglobin O.

Mycobacterium leprae truncated hemoglobin O (trHbO) protects from nitrosative stress and sustains mycobacterial respiration. Here, kinetics of M. leprae trHbO(II)-NO denitrosylation and of O(2)-mediated oxidation of M. leprae trHbO(II)-NO are reported. Values of the first-order rate constant for *NO dissociation from M. leprae trHbO(II)-NO (k(off)) and of the first-order rate constant for O(2)-mediated oxidation of M. leprae trHbO(II)-NO (h) are 1.3 x 10(-4) s(-1) and 1.2 x 10(-4) s(-1), respectively. The coincidence of values of k(off) and h suggests that O(2)-mediated oxidation of M. leprae trHbO(II)-NO occurs with a reaction mechanism in which *NO, that is initially bound to heme(II), is displaced by O(2) but may stay trapped in a protein cavity(ies) close to heme(II). Next, M. leprae trHbO(II)-O(2) reacts with *NO giving the transient Fe(III)-OONO species preceding the formation of the final product M. leprae trHbO(III). *NO dissociation from heme(II)-NO represents the rate limiting step for O(2)-mediated oxidation of M. leprae trHbO(II)-NO.

Microbial community structure and dynamics of dark fire-cured tobacco fermentation.

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.

Peroxynitrite scavenging by ferrous truncated hemoglobin GlbO from Mycobacterium leprae.

Mycobacterium leprae GlbO has been proposed to represent merging of both O(2) uptake/transport and scavenging of nitrogen reactive species. Peroxynitrite reacts with M. leprae GlbO(II)-NO leading to GlbO(III) via the GlbO(III)-NO species. The value of the second order rate constant for GlbO(III)-NO formation is >1x10(8)M(-1)s(-1) in the absence and presence of CO(2) (1.2x10(-3)M). The CO(2)-independent value of the first order rate constant for GlbO(III)-NO denitrosylation is (2.5+/-0.4)x10(1)s(-1). Furthermore, peroxynitrite reacts with GlbO(II)-O(2) leading to GlbO(III) via the GlbO(IV)O species. Values of the second order rate constant for GlbO(IV)O formation are (4.8+/-0.5)x10(4) and (6.3+/-0.7)x10(5)M(-1)s(-1) in the absence and presence of CO(2) (=1.2x10(-3)M), respectively. The value of the second order rate constant for the peroxynitrite-mediated GlbO(IV)O reduction (= (1.5+/-0.2)x10(4)M(-1)s(-1)) is CO(2)-independent. These data argue for a role of GlbO in the defense of M. leprae against nitrosative stress.

Truncated hemoglobin GlbO from Mycobacterium leprae alleviates nitric oxide toxicity.

As a consequence of reductive genome evolution, the obligate intracellular pathogen Mycobacterium leprae has minimized the repertoire of genes implicated in defense against reactive oxygen and nitrogen species. Genes for multiple hemoglobin types coexist in mycobacterial genomes, but M. leprae has retained only glbO, encoding a group-II truncated hemoglobin. Mycobacterium tuberculosis GlbO has been involved in oxygen transfer and respiration during hypoxia, but a role in protection from nitric oxide (NO) has not been documented yet. Here, we report that the in vitro reaction of oxygenated recombinant M. leprae GlbO with NO results in an immediate stoichiometric formation of nitrate, concomitant with heme-protein oxidation. Overexpression of GlbO alleviates the growth inhibition of Escherichia colihmp (flavohemoglobin gene) mutants in the presence of NO-donors, partly complementing the defect in Hmp synthesis. A promoter element upstream of glbO was predicted in silico, and confirmed by using a glbO::lacZ transcriptional fusion in the heterologous Mycobacterium smegmatis system. The glbO::lacZ fusion was expressed through the whole growth cycle of M. smegmatis, and moderately induced by NO. We propose that M. leprae, by retaining the unique truncated hemoglobin GlbO, may have coupled O2 delivery to the terminal oxidase with a defensive mechanism to scavenge NO from respiratory enzymes. These activities would help to sustain the obligate aerobic metabolism required for intracellular survival of leprosy bacilli.

Nitric oxide scavenging by Mycobacterium leprae GlbO involves the formation of the ferric heme-bound peroxynitrite intermediate.

Ferrous oxygenated (Fe(II)O2) hemoglobins (Hb's) and myoglobins (Mb's) have been shown to react very rapidly with NO, yielding NO3(-) and the ferric heme-protein derivative (Fe(III)), by means of the ferric heme-bound peroxynitrite intermediate (Fe(III)OONO), according to the minimum reaction scheme: Fe(II)O2 + NO (k(on))--> Fe(III)OONO (h)--> Fe(III) + NO3(-). For most Hb's and Mb's, the first step (indicated by k(on)) is rate limiting, the overall reaction following a bimolecular behavior. By contrast, the rate of isomerization and dissociation of Fe(III)OONO (indicated by h) is rate limiting in NO scavenging by Fe(II)O2 murine neuroglobin, thus the overall reaction follows a monomolecular behavior. Here, we report the characterization of the NO scavenging reaction by Fe(II)O2 truncated Hb GlbO from Mycobacterium leprae. Values of k(on) (=2.1x10(6) M(-1) s(-1)) and h (=3.4 s(-1)) for NO scavenging by Fe(II)O2 M. leprae GlbO have been determined at pH 7.3 and 20.0 degrees C, the rate of Fe(III)OONO decay (h) is rate limiting. The Fe(III)OONO intermediate has been characterized by optical absorption spectroscopy in the Soret region. These results have been analyzed in parallel with those of monomeric and tetrameric globins as well as of flavoHb and discussed with regard to the three-dimensional structure of mycobacterial truncated Hbs and their proposed role in protection from nitrosative stress.

Nitric oxide and Mycobacterium leprae pathogenicity.

Leprosy is an old, still dreaded infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae. During the infectious process, M. leprae is faced with the host macrophagic environment, where the oxidative stress and NO release, combined with low pH, low pO2, and high pCO2, contribute to limit the growth of the bacilli. Comparative genomics has unraveled massive gene decay in M. leprae, linking the strictly parasitic lifestyle with the reductive genome evolution. Compared with Mycobacterium tuberculosis and Mycobacterium bovis, the leprosy bacillus has lost most of the genes involved in the detoxification of reactive oxygen and nitrogen species. The very low reactivity of the unique truncated hemoglobin retained by M. leprae could account for the susceptibility of this exceptionally slow-growing microbe to NO.

The truncated hemoglobin from Mycobacterium leprae.

Truncated hemoglobins (trHb's) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb's branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb's, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.

Nitric oxide and mycobacterium leprae pathogenicity

Leprosy is an old, still dreaded infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae. During the infectious process, M. leprae is faced with the host macrophagic environment, where the oxidative stress and NO release, combined with low pH, low pO2, and high pCO2, contribute to limit the growth of the bacilli. Comparative genomics has unraveled massive gene decay in M. leprae, linking the strictly parasitic lifestyle with the reductive genome evolution. Compared with Mycobacterium tuberculosis and Mycobacterium bovis, the leprosy bacillus has lost most of the genes involved in the detoxification of reactive oxygen and nitrogen species. The very low reactivity of the unique truncated hemoglobin retained by M. leprae could account for the susceptibility of this exceptionally slow-growing microbe to NO.