Detection and purification of a catalase-peroxidase from Mycobacterium sp. Pyr-1

A catalase-peroxidase from Mycobacterium sp. Pyr-1, a strain capable of growth on pyrene, was purified to homogeneity by anion exchange and hydroxyapatite column chromatography. The enzyme, like the M. tuberculosis T-catalase, reduced nitroblue tetrazolium in the presence of isoniazid (INH) and H2O2. It also oxidized 3,3',5,5'-tetramethylbenzidine and other substrates of the catalase-peroxidase of M. tuberculosis in the presence of either tert-butyl hydroperoxide or H2O2. It had a UV/ visible absorption spectrum (Soret peak at 406 nm) similar to that of the catalase-peroxidase of M. tuberculosis (Soret peak at 408 nm) and identical to that of the catalase-peroxidase of M. smegmatis. After electrophoresis on non-denaturing gels the enzyme showed one single protein band with both catalase and peroxidase activity, which were lost after electrophoresis on SDS-PAGE. The enzyme was inhibited by sodium azide, glutathione, 2-mercaptoethanol, and isoniazid, but not by isonicotinic acid. The optimum enzyme activity was obtained at pH 4.5 and at 25 degrees C.     

Bacteriostatic and bactericidal activity of antituberculosis drugs against Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium intracellulare complex and Mycobacterium kansasii in different growth phases.

Bacteriostatic and bactericidal activities of rifampicin, isoniazid, streptomycin, enviomycin and ethambutol against Mycobacterium tuberculosis, Mycobacterium avium--M. intracellulare complex and Mycobacterium kansasii were studied in different growth phases. Bacteriostatic activities of the drugs were similar in different growth phases, except isoniazid. M. tuberculosis was much less susceptible to isoniazid in the lag phase than in the log and the stationary phases. In contrast, bactericidal activity was influenced by the growth phase. M. tuberculosis was killed by isoniazid, streptomycin and rifampicin. The bactericidal activity of isoniazid was strongest. The bactericidal activity of isoniazid and streptomycin was most marked in the log phase. M. avium complex and M. kansasii resisted the bactericidal activity, but some strains of M. avium complex were killed by streptomycin and enviomycin, and the activities of these two drugs were most marked in the lag phase.     

Antimicrobial Protegrin-1 Forms Ion Channels: Molecular Dynamic Simulation, Atomic Force Microscopy, and Electrical Conductance Studies

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among β-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a β-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the β-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.     

Tissue-specific human beta-defensins (HBD)-1, HBD-2 and HBD-3 secretion profile from human amniochorionic membranes stimulated with candida albicans in a two-compartment tissue culture system

ABSTRACT: BACKGROUND: During intrauterine infection, amniochorionic membranes represent a mechanical and immunological barrier against dissemination of infection. Human beta defensins (HBD)-1, HBD-2, and HBD-3 are key elements of innate immunity that represent the first line of defense against different pathogen microorganisms associated with preterm labor. The aim of this work was to characterize the individual contribution of the amnion (AMN) and choriodecidua (CHD) regions to the secretion of HBD-1, HBD-2 and HBD-3, after stimulation with Candida albicans. METHODS: Full-thickness human amniochorionic membranes were obtained after delivery by elective cesarean section from women at 37-40 wk of gestation with no evidence of active labor. The membranes were cultured in a two-compartment experimental model in which the upper compartment is delimited by the amnion and the lower chamber by the choriodecidual membrane. One million of Candida albicans were added to either the AMN or the CHD face or to both and compartmentalized secretion profiles of HBD-1, HBD-2, and HBD-3 were quantified by ELISA. Tissue immunolocalization was performed to detect the presence of HBD-1, -2, -3 in tissue sections stimulated with Candida albicans. RESULTS: HBD-1 secretion level by the CHD compartment increased 2.6 times (27.30 [20.9-38.25] pg/micrograms protein) when the stimulus with Candida albicans was applied only on this side of the membrane and 2.4 times (26.55 [19.4-42.5] pg/micrograms protein) when applied to both compartments simultaneously. HBD-1 in the amniotic compartment remained without significant changes.HBD-2 secretion level increased significantly in the CHD when the stimulus was applied only to this region (2.49 [1.49-2.95] pg/micrograms protein) and simultaneously to both compartments (2.14 [1.67- 2.91] pg/micrograms protein). When the stimulus was done in the amniotic compartment HBD-2 remained without significant changes in both compartments.HBD-3 remained without significant changes in both compartments regardless of the stimulation modality. Localization of immune-reactive forms of HBD-1, HBD-2, and HBD-3 was carried out by immunohistochemistry confirming the cellular origin of these peptides. CONCLUSION: Selective stimulation of amniochorionic membranes with Candida albicans resulted in tissue-specific secretion of HBD-1 and HBD-2, mainly in the CHD, which is the first region to become infected during an ascending infection.     

IL-4 and IL-13 negatively regulate TNF-alpha- and IFN-gamma-induced beta-defensin expression through STAT-6, suppressor of cytokine signaling (SOCS)-1, and SOCS-3

Human beta-defensins (HBDs) are a major class of antimicrobial peptides that play an important role in the innate immune response, however, the induction and regulation of these antimicrobial peptides is not well understood. We demonstrate here that stimulation of keratinocytes with TNF-alpha/IFN-gamma induces HBD-2 and HBD-3 by activating STAT-1 and NF-kappaB signaling. We further demonstrate that IL-4 and IL-13 activate STAT-6 and induce the suppressors of cytokine signaling (SOCS)-1 and -3. This interferes with STAT-1 and NF-kappaB signaling, thereby inhibiting TNF-alpha/IFN-gamma-mediated induction of HBD-2 and HBD-3. These data suggest that targeting the STAT-1-signaling pathway or suppressor of cytokine signaling expression enhances beta-defensin expression and represents a new therapeutic strategy for reduction of infection in human diseases associated with beta-defensin deficiency.     

Antimicrobial protegrin-1 forms amyloid-like fibrils with rapid kinetics suggesting a functional link

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich β-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-β (Aβ_1–42) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Aβ_1–42 fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Aβ_1–42 fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing β-rich AMPs and amyloids such as Aβ, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and β-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function.     

Bovine aortic chondroitin sulphate- and dermatan sulphate-containing proteoglycans. Isolation, fractionation and chemical characterization.

1. Guanidinium chloride (4M) in the presence of proteinase inhibitors extracted 90% of bovine aorta galactosaminoglycans as proteoglycans that were subsequently purified by ion-exchange and gel chromatography. 2. Fractionation of the calcium salts of the purified proteoglycans with increasing concentration of ethanol yielded fractions PG-25 (28%), PG-35 (45%) and PG-50 (37%). 3. Fraction PG-50 contained proteochondroitin 6-sulphate, whereas fractions PG-25 and PG-35 were proteodermatan sulphates of greatly different carbohydrate composition; the molar proportions of L-iduronate-N-acetylgalactosamine 4-sulphate, D-glucuronate-N-acetyl-galactosamine 4-sulphate and D-glucuronate-N-acetylgalactosamine 6-sulphate were 75: 18 :7 in fraction PG-25 and 14 :46 :40 in fraction PG-35. 4. The presence of alternating or mixed sequences with L-iduronate- and D-glucuronate-containing repeating disaccharides was indicated by the formation of tetrasaccharides after chondroitinase AC digestion (single L-iduronate residues) and by the release of fragments containing four or five consecutive D-glucuronate-N-acetylgalactosamine repeats after periodate oxidation and alkaline elimination. 5. The amino acid compositions of fractions PG-25 and PG-35 were similar and markedly different from that of fraction PG-50, which also contained more side chains.     

Characteristics of Mycobacterium tuberculosis clinical strains genotype A1

The Mycobacterium tuberculosis strains of genotype A1 (LAM family, VNTR profile 222222) are often resulted from people with pulmonary tuberculosis, who live in Central Russia. Among strains of this family, drug-resistant strains, including those with simultaneous resistance to isoniazid and rifampicin (MDR), are common. The strains of the genotype A1 tend to spread as clones.     

Antitubercular activity of trifluoperazine, a calmodulin antagonist

Trifluoperazine, a calmodulin antagonist, completely inhibited the growth of mycobacteria. The minimum inhibitory concentrations in shake cultures in a synthetic medium containing 0.2% Tween 80 were 5 and 8 micrograms/ml, respectively, for the human pathogenic strain Mycobacterium tuberculosis H37Rv and M. tuberculosis resistant to isoniazid. When added to a growing culture of M. tuberculosis H37Rv on the 10th day (mid exponential phase), trifluoperazine 50 micrograms/ml further arrested growth of this organism. It is suggested that trifluoperazine or similar calmodulin antagonists might be useful as antitubercular drugs.     

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: Effect of head group electrostatics and tail group packing

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a β-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.     

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: effect of head group electrostatics and tail group packing

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a beta-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.     

Modulation of human β-defensin-2 expression by 17β-estradiol and progesterone in vaginal epithelial cells

We investigated the expression of HBD-1 and -2 in vaginal epithelial cells treated with lipopolysaccharide (LPS) and the effects on HBD-2 expressions by 17β-estradiol and progesterone. Primary vaginal epithelial cells were isolated from a segment of normal anterior vaginal wall obtained during vaginoplasty and were cultured in keratinocyte growth medium and were allowed to undergo their 3rd passage. Expression of HBD-1 and -2 by different stimuli using LPS 0.5 μg/ml, 17β-estradiol 2 nM and progesterone 1 μM was measured by RT-PCR, ELISA and real-time RT-PCR, respectively. HBD-1 was produced constitutively in vaginal epithelial cells and the production of HBD-1 was not influenced by LPS, 17β-estradiol and progesterone, but the production of HBD-2 was increased inducibly by LPS. 17β-Estradiol and progesterone did not change the production of HBD-2 in normal state, but 17β-estradiol increased the production of HBD-2 and progesterone suppressed the production of HBD-2 under the circumstances with infection. The HBD-2 plays an important role at innate host defense on genitourinary tract. The lacks of estrogen during menopause or uses of a progesterone-based oral contraceptive in sexually active women may influence production of HBD-2 in vaginal epithelium and may increase susceptibility to bacterial vaginitis or recurrent UTI.     

AhpC, oxidative stress and drug resistance in Mycobacterium tuberculosis

The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidant proteins with alkylhydroperoxidase (Ahp) and thioredoxin-dependent peroxidase (TPx) activities. AhpC expression is associated with resistance to the front-line antitubercular drug isoniazid in the naturally resistant organisms E. coli and M. smegmatis. We identified several isoniazid-resistant M. tuberculosis isolates with ahpC promoter mutations resulting in AhpC overexpression. These strains were more resistant to cumene hydroperoxide than were wild-type strains. However, these strains were unchanged in their sensitivity to isoniazid, refuting a role for AhpC in detoxification of this drug. All the isoniazid-resistant, AhpC-overexpressing strains were also deficient in activity of the mycobacterial catalase-peroxidase KatG. KatG, the only known catalase in M. tuberculosis, is required for activation of isoniazid. We propose that compensatory ahpC promoter mutations are selected from KatG-deficient, isoniazid-resistant M. tuberculosis during infections, to mitigate the added burden imposed by organic peroxides on these strains.     

Photochromogenic and scotochromogenic mycobacteria: their clinical significance

In the period 1973--1977, Mycobacterium tuberculosis was isolated by cultivation in 4408 cases from the clinical specimens of patients with positive X-ray findings. On the basis of atypical colony morphology or pigment formation, 263 other mycobacterial strains were identified: of these 23 were photochromogenic and belonged to Mycobacterium kansasii. The strains were cultured on several occasions from the specimens of 4 patients with broncho-pulmonary mycobacteriosis. The strains were resistant to isoniazid and streptomycin, sensitive to ethambutol and rifampicin. A total of 18 scotochromogenic isolates cultured from 14 patients with positive X-ray findings were identified as Mycobacterium aquae (M. gordonae) and its variants: strains showing slow Tween hydrolysis and 1 strain of rapid growth. In 5 cases M. tuberculosis was also obtained, indicating the presence of a mixed mycobacterial population. All scotochromogens were resistant to isoniazid and sensitive to ethambutol, with the exception of two strains sensitive to rifampicin.     

Induction of human beta-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines

Defensins are cationic peptides with broad-spectrum antimicrobial activity. They are members of a supergene family consisting of alpha and beta subtypes and each subtype is comprised of a number of different isoforms. For example, human alpha-defensin (HAD) has six isoforms, which are expressed by polymorphonuclear leukocytes and Paneth cells. In contrast, human beta-defensin (HBD) has two isoforms that are expressed by epithelial cells of the skin, gut, respiratory and urogenital tracts. Recently, HBD-1 was detected in human brain biopsy tissue. However, little is known about the expression of HBD-1 or HBD-2 in the CNS and whether neural cells can secrete these peptides. For the present study, human astrocyte, microglial, meningeal fibroblast and neuronal cultures were probed for the expression of HBD-1 and HBD-2 mRNA and protein. Each cell type was either maintained in tissue culture medium alone or in medium containing lipopolysaccharide (LPS) at concentrations ranging from 0.1 to 1 microgram/mL, interleukin-1 beta (IL-1beta) at 1-50 ng/mL, or tumor necrosis factor alpha (TNF-alpha) at the same concentrations. The expression of HBD-1 and HBD-2 mRNAs was monitored by RT-PCR. The cDNA products were sequenced to characterize the gene product. HBD-2 protein was detected by immunoblot, immunoprecipitation and immunocytochemistry. Results of these studies showed that HBD-1 mRNA was detected in all cell cultures except in those enriched for neurons. In contrast, HBD-2 mRNA was detected only in astrocyte cultures that were treated with LPS, IL-1beta or TNF-alpha. The detection of the respective proteins correlated positively with the mRNA results. As such, these data represent the first demonstration of HBD-2 expression by astrocytes and suggest that this peptide may play a role in host defense against bacterial CNS pathogenesis.     

Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid

Isoniazid is a key drug used in the treatment of tuberculosis. Isoniazid is a pro-drug, which, after activation by the katG-encoded catalase peroxidase, reacts nonenzymatically with NAD(+) and NADP(+) to generate several isonicotinoyl adducts of these pyridine nucleotides. One of these, the acyclic 4S isomer of isoniazid-NAD, targets the inhA-encoded enoyl-ACP reductase, an enzyme essential for mycolic acid biosynthesis in Mycobacterium tuberculosis. Here we show that the acyclic 4R isomer of isoniazid-NADP inhibits the M. tuberculosis dihydrofolate reductase (DHFR), an enzyme essential for nucleic acid synthesis. This biologically relevant form of the isoniazid adduct is a subnanomolar bisubstrate inhibitor of M. tuberculosis DHFR. Expression of M. tuberculosis DHFR in Mycobacterium smegmatis mc(2)155 protects cells against growth inhibition by isoniazid by sequestering the drug. Thus, M. tuberculosis DHFR is the first new target for isoniazid identified in the last decade.     

Characterization of Mycobacterium smegmatis mutants defective in 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside and mycothiol biosynthesis

Mycothiol (MSH) is the major low molecular weight thiol in mycobacteria. Two chemical mutants with low MSH and one with no MSH (strain 49) were produced in Mycobacterium smegmatis mc2155 to assess the role of MSH in mycobacteria. Strain 49 was shown to not produce 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside (GlcN-Ins), an intermediate in MSH biosynthesis. Relative to the parent strain, mutant 49 formed colonies more slowly on solid media and was more sensitive to H2O2 and rifampin, but less sensitive to isoniazid. Complementation of mutant 49 with DNA from M. tuberculosis H37Rv partially restored production of GlcN-Ins and MSH, and resistance to H2O2, but largely restored colony growth rate and sensitivity to rifampin and isoniazid. The results indicate that MSH and GlcN-Ins are not essential for in vitro survival of mycobacteria but may play significant roles in determining the sensitivity of mycobacteria to environmental toxins.