Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis

AbstractUnraveling of microbial genome data has indicated that two distantly related truncated hemoglobins (trHbs), HbN and HbO, might occur in many species of slow-growing pathogenic mycobacteria. Involvement of HbN in bacterial defense against NO toxicity and nitrosative stress has been proposed. A gene, encoding a putative HbN homolog with conserved features of typical trHbs, has been identified within the genome sequence of fast-growing mycobacterium, Mycobacterium smegmatis. Sequence analysis of M. smegmatis HbN indicated that it is relatively smaller in size and lacks N-terminal pre-A region, carrying 12-residue polar sequence motif that is present in HbN of M. tuberculosis. HbN encoding gene of M. smegmatis was expressed in E. coli as a 12.8kD homodimeric heme protein that binds oxygen reversibly with high affinity (P50∼0.081mm Hg) and autooxidizes faster than M. tuberculosis HbN. The circular dichroism spectra indicate that HbN of M. smegmatis and M. tuberculosis are structurally similar. Interestingly, an hmp mutant of E. coli, unable to metabolize nitric oxide, exhibited very low NO uptake activity in the presence of M. smegmatis HbN as compared to HbN of M. tuberculosis. On the basis of cellular heme content, specific nitric oxide dioxygenase (NOD) activity of M. smegmatis HbN was nearly one-third of that from M. tuberculosis. Additionally, the hmp mutant of E. coli, carrying M. smegmatis HbN, exhibited nearly 10-fold lower cell survival under nitrosative stress and nitrite derived reactive nitrogen species as compared to the isogenic strain harboring HbN of M. tuberculosis. Taken together, these results suggest that NO metabolizing activity and protection provided by M. smegmatis HbN against toxicity of NO and reactive nitrogen is significantly lower than HbN of M. tuberculosis. The lower efficiency of M. smegmatis HbN for NO detoxification as compared to M. tuberculosis HbN might be related to different level of NO exposure and nitrosative stress faced by these mycobacteria during their cellular metabolism

Cutting Edge: The Heat Shock Protein gp96 Activates Inflammasome-Signaling Platforms in APCs

Several heat shock proteins (HSPs) prime immune responses, which are, in part, a result of activation of APCs. APCs respond to these immunogenic HSPs by upregulating costimulatory molecules and secreting cytokines, including IL-1beta. These HSP-mediated responses are central mediators in pathological conditions ranging from cancer, sterile inflammation associated with trauma, and rheumatoid arthritis. We tested in this study the requirement of inflammasomes in the release of IL-1beta by one immunogenic HSP, gp96. Our results show that murine APCs activate NLRP3 inflammasomes in response to gp96 by K(+) efflux. This is shown to initiate inflammatory conditions in vivo in the absence of additional known inflammasome activators or infection. These results document a novel mechanism by which proteins of endogenous origin, the HSPs, can modulate an inflammatory response following their release from aberrant cells

CD91-Dependent Modulation of Immune Responses by Heat Shock Proteins: A Role in Autoimmunity

Heat shock proteins (HSPs) have been known for decades for their ability to protect cells under stressful conditions. In the 1980s a new role was ascribed for several HSPs given their ability to elicit specific immune responses in the setting of cancer and infectious disease. These immune responses have primarily been harnessed for the immunotherapy of cancer in the clinical setting. However, because of the ability of HSPs to prime diverse immune responses, they have also been used for modulation of immune responses during autoimmunity. The apparent dichotomy of immune responses elicited by HSPs is discussed here on a molecular and cellular level. The potential clinical application of HSP-mediated immune responses for therapy of autoimmune diseases is reviewed

Immunotherapy of tumors with α2-macroglobulin-antigen complexes pre-formed in vivo.

The cell surface receptor CD91/LRP-1 binds to immunogenic heat shock proteins (HSP) and α(2)M ligands to elicit T cell immune responses. In order to generate specific immune responses, the peptides chaperoned by HSPs or α(2)M are cross-presented on MHC molecules to T cells. While the immunogenic HSPs naturally chaperone peptides within cells and can be purified as an intact HSP-peptide complex, the peptides have had to be complexed artificially to α(2)M in previous studies. Here, we show that immunogenic α(2)M-peptide complexes can be isolated from the blood of tumor-bearing mice without further experimental manipulation in vitro demonstrating the natural association of tumor antigens with α(2)M. The naturally formed immunogenic α(2)M-peptide complexes are effective in prophylaxis and therapy of cancer in mouse models. We investigate the mechanisms of cross-presentation of associated peptides and co-stimulation by APCs that interact with α(2)M. These data have implications for vaccine design in immunotherapy of cancer and infectious disease

Responses of Mycobacterium tuberculosis Hemoglobin Promoters to In Vitro and In Vivo Growth Conditions▿

The success of Mycobacterium tuberculosis as one of the dreaded human pathogens lies in its ability to utilize different defense mechanisms in response to the varied environmental challenges during the course of its intracellular infection, latency, and reactivation cycle. Truncated hemoglobins trHbN and trHbO are thought to play pivotal roles in the cellular metabolism of this organism during stress and hypoxia. To delineate the genetic regulation of the M. tuberculosis hemoglobins, transcriptional fusions of the promoters of the glbN and glbO genes with green fluorescent protein were constructed, and their responses were monitored in Mycobacterium smegmatis and M. tuberculosis H37Ra exposed to environmental stresses in vitro and in M. tuberculosis H37Ra after in vivo growth inside macrophages. The glbN promoter activity increased substantially during stationary phase and was nearly 3- to 3.5-fold higher than the activity of the glbO promoter, which remained more or less constant during different growth phases in M. smegmatis, as well as in M. tuberculosis H37Ra. In both mycobacterial hosts, the glbN promoter activity was induced 1.5- to 2-fold by the general nitrosative stress inducer, nitrite, as well as the NO releaser, sodium nitroprusside (SNP). The glbO promoter was more responsive to nitrite than to SNP, although the overall increase in its activity was much less than that of the glbN promoter. Additionally, the glbN promoter remained insensitive to the oxidative stress generated by H2O2, but the glbO promoter activity increased nearly 1.5-fold under similar conditions, suggesting that the trHb gene promoters are regulated differently under nitrosative and oxidative stress conditions. In contrast, transition metal-induced hypoxia enhanced the activity of both the glbN and glbO promoters at all growth phases; the glbO promoter was induced ∼2.3-fold, which was found to be the highest value for this promoter under all the conditions evaluated. Addition of iron along with nickel reversed the induction in both cases. Interestingly, a concentration-dependent decrease in the activity of both trHb gene promoters was observed when the levels of iron in the growth media were depleted by addition of an iron chelator. These results suggested that an iron/heme-containing oxygen sensor is involved in the modulation of the trHb gene promoter activities directly or indirectly in conjunction with other cellular factors. The modes of promoter regulation under different physiological conditions were found to be similar for the trHbs in both M. smegmatis and M. tuberculosis H37Ra, indicating that the promoters might be regulated by components that are common to the two systems. Confocal microscopy of THP-1 macrophages infected with M. tuberculosis carrying the trHb gene promoter fusions showed that there was a significant level of promoter activity during intracellular growth in macrophages. Time course evaluation of the promoter activity after various times up to 48 h by fluorescence-activated cell sorting analysis of the intracellular M. tuberculosis cells indicated that the glbN promoter was active at all time points assessed, whereas the activity of the glbO promoter remained at a steady-state level up to 24 h postinfection and increased ∼2-fold after 48 h of infection. Thus, the overall regulation pattern of the M. tuberculosis trHb gene promoters correlates not only with the stresses that the tubercle bacillus is likely to encounter once it is in the macrophage environment but also with our current knowledge of their functions. The in vivo studies that demonstrated for the first time expression of trHbs during macrophage infection of M. tuberculosis strongly indicate that the hemoglobins are required, and thus important, during the intracellular phase of the bacterial cycle. The present study of transcriptional regulation of M. tuberculosis hemoglobins in vitro under various stress conditions and in vivo after macrophage infection supports the hypothesis that biosynthesis of both trHbs (trHbN and trHbO) in the native host is regulated via the environmental signals that the tubercle bacillus receives during macrophage infection and growth in its human host

α₂M from tumor-bearing mice is associated with antigen.

<p>a&b) α₂M was purified from mice bearing solid tumors, tumors as ascites or from naïve mice. The purified α₂M was monitored for purity by SDS-PAGE (a,i and b,i). a) Mice bearing established CMS5 tumors were treated with α₂M that was derived from tumor bearing or naïve mice as indicated. Tumor growth was monitored. b) BALB/c mice were immunized with α₂M that was derived from mice bearing solid tumors, tumors as ascites or from naïve mice (no tumor). One week later mice were challenged with MethA tumor cells. Tumor growth was monitored. p<0.05 between the two groups (a) or p<0.001 for each group compared to PBS (b). c) α₂M purified from the indicated sources (lanes 1,4,5) were analyzed on 5% native polyacrylamide gels. α₂M was additionally analyzed following complexing to peptides with mild thermal manipulation (lane 2). A “fast” form of α₂M was made by treatment with methylamine (lane 3).</p

Cross-presentation of α₂M-chaperoned peptides is dependent on proteasomes and vesicular trafficking.

<p>a) α₂M complexed to the ova 19-mer peptide (P) was incubated with bone marrow-derived dendritic cells in the presence or absence of the proteasome inhibitor lactacystin. Controls include cells incubated with peptide alone, ova8 or ovalbumin protein (ova) or PBS (none). B3Z activation was monitored at A₅₉₅ after addition of substrate. b) α₂M or gp96 complexed to the ova 19-mer peptide (P) were incubated with bone marrow-derived dendritic cells in the presence or absence of BFA. Controls include cells incubated with ova 19-mer peptide alone (P), ova8 or ovalbumin protein (ova) or PBS (none). B3Z activation was monitored at A₅₉₅ after addition of substrate. Error bars in (a,b) represent standard deviation of duplicates.</p

α₂M-tumor peptides complexes are effective in immunotherapy.

<p>a&b) cohorts of BALB/c mice were challenged with the CMS5 tumor as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050365#s2" target="_blank">Methods</a>. Mice bearing 3×3 mm diameter tumors were treated with α₂M complexed to tumor-derived peptides (a) or with gp96 complexed to peptides, peptides alone or PBS (b). Tumor growth was monitored. * is p<0.01 and ** is p<0.005 compared to α₂M-irrelevant peptide group. Purifications of α₂M were apparently homogenous as monitored by SDS-PAGE and immunoblotting with an α₂M-specific antibody (inset).</p

α₂M provides signals necessary for T cell priming.

<p>a) RAW264.7 cells were pulsed with α₂M, LPS or PBS. The β-chain of CD91/LRP-1 was immunoprecipitated and probed with α-pTyr antibodies. b) Splenic dendritic cells were pulsed with α₂M (left panel) or PBS (right panel) for 20 h. Supernatants from either culture were incubated with cytokine profiler arrays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050365#s2" target="_blank">Methods</a> and developed. Signals higher (blue boxes) or lower (red boxes) than background (PBS pulsed cells) are highlighted. Standardization signals on each blot (with+signs in Black boxes) are used to normalize signals to each other. c) Splenic dendritic cells were pulsed with α₂M or LPS in the presence or absence of the NF-κB inhibitor cardamonin for 20 h. Supernatants were analyzed by ELISA for TNF-α. Error bars represent standard deviation of duplicates.</p