Pathogenic and virulence characterization of colonial mutants of Nocardia asteroides GUH-2

The pathogenicities in mice (comparing LD50 determinations) of two mutant strains and one wild-type strain of Nocardia asteroides GUH-2, each possessing a colonial morphology distinct from the other, were compared at respective stages of growth. Despite the three strains' colinear growth curves and similar physiological properties, unique patterns of pathogenicity emerged for each strain upon analysis. Ultrastructural and fatty acid profiles of cultures at the various growth stages were monitored. The mutant strain SCII-A1 was consistently less virulent than the other strains of N. asteroides GUH-2 (SCII-P and SCII-C). Further, its fatty acid profiles as well as the shape and consistency of its colonies differed greatly from those of the wild-type strain. The fatty acid composition and the colonial morphology of strain SCII-C more closely resembled those of the parent, although its virulence was both greater than (before 28 h of growth) and less than the parent's depending upon the specific stage of growth. The comparative degrees of cellular fragmentation and complexity, as determined by scanning and transmission electron microscopy, were found to coincide with changes in relative degrees of pathogenicity.     

Effect of growth stage on mycolic acid structure in cell walls of Nocardia asteroides GUH-2

Mycolic acids were extracted from the cell walls of Nocardia asteroides GUH-2 during different phases of growth at 37 degrees C. These were subjected to structural analysis by combining thin-layer chromatography and gas-liquid chromatography with UV and infrared spectrophotometry and mass spectroscopy of both methyl esters and trimethyl silyl derivatives. By analyzing the fragmentation patterns of these derivatives by three different methods of mass spectroscopy combined with gas-liquid chromatographic separation, the different structural subclasses of mycolic acids were quantitated. Significant qualitative and quantitative modifications of specific mycolic acid subclasses occurred in the cell walls of N. asteroides GUH-2 that were growth stage dependent. The mycolic acids that were predominant in the log phase were polyunsaturated (greater than 2 double bonds per molecule), with long chain lengths and even carbon atom numbers (i.e., C54, C56). In contrast, those that were prominent in the stationary phase were more saturated (few or no double bonds) and of shorter overall carbon chain length (less than or equal to C52). Furthermore, stationary-phase cells had significantly increased amounts of mycolic acids with odd-numbered carbon chain lengths (i.e., C49, C51, C53).     

Comparison of PCR and culture for detection of Nocardia asteroides in brain specimens from experimentally infected BALB/c mice

Systemic infection of BALB/c mice with Nocardia asteroides strain GUH-2 results in widespread replication of the organism in the brain, followed by its immune-mediated clearance. The present study compared the sensitivity of polymerase chain reaction (PCR) to bacterial culture for detection of cerebral nocardial infection in this experimental system. Mice (n=4/time point) were administered N. asteroides by intravenous injection, and brain specimens were evaluated for Nocardia by PCR and culture at post-infection days 2, 7, 14 and 21. Nocardia was detected by PCR in all infected animals on post-infection days 2, 7, and 14, and in one of four mice on post-infection day 21; in contrast, the organism was detected by culture only on post-infection days 2 and 7. These findings suggest that PCR may be more sensitive than culture for the detection of low numbers of Nocardia in the brain.     

Siderophores Produced by Nitrogen-Fixing Azotobacter vinelandii OP in Iron-Limited Continuous Culture

Azotobacter vinelandii requires a high complement of iron and an efficient iron acquisition system to support nitrogen fixation. To circumvent problems inherent in batch culture trace metal studies, continuous cultures were used to measure the response of A. vinelandii to iron stress. Iron was found to be growth limiting for nitrogen-fixing A. vinelandii at a concentration as high as 12.5 muM; iron was growth sufficient at 25 muM. Iron-stressed A. vinelandii in continuous culture formed 2,3-hydroxybenzoic acid (DHB), 2-N,6-N-di-(2,3-dihydroxybenzoyl)-l-lysine (DHBL), and a chromophoric yellow-green fluorescent peptide (YGFP). At a fixed dilution rate of 0.1 h, steady-state growth occurred at growth-limiting iron concentrations. DHB and DHBL were quantitatively measured during iron-limited steady states and iron-sufficient states by Arnow colorimetric assays. YGFP was determined by absorbance measurements taken at 380 nm, and the concentration was calculated from the reported specific absorption coefficient. Biomass increased and DHBL, DHB, and YGFP concentrations decreased as the concentration of growth-limiting iron was increased in the culture vessel and medium reservoirs. DHBL was the major siderophore and YGFP was the minor siderophore species produced during iron-limited equilibrium growth. A low level of DHB and YGFP, but no DHBL, was formed under iron-sufficient conditions. These results provide further physiological evidence that DHB, YGFP, and especially DHBL may function as siderophores in nitrogen-fixing A. vinelandii.     

Nocardia as a pathogen of the brain: mechanisms of interactions in the murine brain--a review.

Nocardia asteroides strain GUH-2 invades the brain without inducing an early inflammatory response in normal mice. This strain can grow within the neurons as well as adjacent areas causing neurological damage without abscess formation. At low inoculum doses, this organism is gradually cleared from the brain after the initial burst of growth. Therefore, at two weeks, the brains of these mice appear to become sterile, but the animals begin to develop a variety of neurological signs including an L-dopa responsive headshake. It was found that mice that recovered from sublethal infection with N. asteroides strain GUH-2 had progressive and permanent neuron damage in regions of the brain, the extent of which appeared to correlate with specific neurological signs. The mechanisms of this response are not known, but current data indicate that the level of nonspecific phagocyte activation (microglia?) within the brain represents an important first line of defense. Next, T-lymphocytes are important in the secondary response in the brain to nocardial invasion. Finally, B-lymphocytes and a humoral immune response may be involved in these complex interactions; however, it is not clear how these responses relate to the permanent and progressive brain damage induced by Nocardia. It is possible that these latter responses may exacerbate neurodegeneration.     

Purification and properties of a unique superoxide dismutase from Nocardia asteroides

A unique form of superoxide dismutase was isolated and characterized from Nocardia asteroides GUH-2. This enzyme contains 1 to 2 g atoms each of Fe, Mn, and Zn per mol and exhibits spectral properties suggestive of Fe- or Mn-containing superoxide dismutases. Its Mr = 100,000, and it is composed of four subunits of equal size which are not covalently joined. The amino acid composition of the enzyme was more closely related to the Mn- or Fe-containing enzymes of Mycobacterium species and was least related to the Cu-Zn enzyme of eukaryotes. Azide at 1 and 20 mM inhibits the activity 10 and 41%, respectively, and 5 mM H2O2 inhibits 40%, but 1 or 5 mM cyanide caused trivial effect. The immunofluorescent staining, which was specific for superoxide dismutase of N. asteroides, indicated the association of this enzyme to the outer cell wall of the organism. Further, the enzyme was shown to be selectively secreted into the medium.     

Immunodiagnosis of nocardiosis.

A specific immunodominant 54-kDa antigen was purified from a culture filtrate of Nocardia asteroides by immunoaffinity chromatography. The chromatography column was prepared with immunoglobulin G obtained from sera from patients with lepromatous leprosy. Unbound solutes consisted of specific, partially purified N. asteroides antigens, primarily a 54-kDa band, accompanied by two others of 31 and 62 kDa. The Western blot (immunoblot) technique was applied to detecting the immunologic response to nocardiae in the serum of nocardiosis patients. Each of the serum samples from immunosuppressed or immunocompetent patients infected with N. asteroides reacted with the 54-kDa band, and two reacted with the 31- and 62-kDa bands. There was no reaction to either the 54- or the 31-kDa antigen with all serum samples obtained from patients with tuberculosis, except for one, with all serum samples obtained from patients with leprosy, or with all sera obtained from healthy controls. The partially purified 54-kDa antigen, specific for N. asteroides, was used as the immunogen to generate monoclonal antibodies (mAbs) and two mAbs were selected. As determined by Western blot, both mAbs reacted with the 54-kDa band. Using indirect immunofluorescence or enzyme immunoassay with whole N. asteroides micro-organisms, the mAbs did not react with N. asteroides cells. No cross-reactivity with mycobacterial antigens, either culture-filtrate antigens or tuberculin, was exhibited with any of the two mAbs. These mAbs are candidates to be used for the development of a sensitive and specific diagnostic test for nocardiosis.     

Tinctorial Properties of Spherical Bodies in Broth Cultures of Nocardia asteroides GUH-2

In yeast extract-supplemented brain heart infusion (BHI) broth cultures of Nocardia asteroides GUH-2, many spherical bodies (SBs) were frequently seen nearby filamentous cells. They showed no Gram-positivity when Gram stain was applied. When acridine orange stain was applied, many of them showed different green fluorescence from bright orange fluorescence of the filamentous nocardiae under ultraviolet light. Their acid-fastness appeared to depend on the presence of paraffin. Using the polymerase chain reaction (PCR) method, 16S rRNA genes were detected in SB-containing broth cultures inoculated with culture filtrates from broth cultures of the strain and identical to that of N. asteroides. These results suggest that SBs are cell wall-defective (CWD) forms which result from the spontaneous mutation of N. asteroides GUH-2.     

An unusual murine behavior following infection with log-phase Nocardia asteroides type 6 strain GUH-2 (Nocardia cyriacigeorgica GUH-2)

Nocardia asteroides (Nocardia cyriacigeorgica) strain GUH-2 infects the brains of mice following intravenous injection. Non-lethal infections resulted in a transitory increase of bacterial numbers in the brain followed by the development of permanent impaired movements at a time when bacteria appeared to be eliminated from the brain. These signs included headshake, rigidity, stooped posture, dyskinesia, retropulsion, and abnormal tail positioning in approximately 20% of infected animals. The attached video presents a typical mouse following infection with this organism, as compared to an age- and gender-matched uninfected control mouse.     

Characterization of Lactobacillus bulgaricus Bacteriophage ch2

Bacteriophage ch2, a virulent bacteriophage of Lactobacillus bulgaricus CH2, was characterized according to its morphology, genome size, structural proteins, and growth kinetics. Electron micrographs revealed that bacteriophage ch2 has an icosahedral head of 50-nm diameter and a long tail of 170 nm. Its genome is linear and 35 kilobases in length, and its structural proteins consist of two major and eight minor proteins. One-step growth kinetics of bacteriophage ch2 under optimal conditions (45°C in MRS medium [Oxoid Ltd.]) showed that the latent time was 40 min, the rise period was 15 min, and the burst size was 130 bacteriophages per cell. To monitor the effects of bacteriophage infection on host growth and β-galactosidase production, the absorbance of the culture and the β-galactosidase activity were followed during the infection cycle. Before lysis the infected culture continued to grow and produce β-galactosidase at the same rate as the uninfected culture.     

Itoic Acid Synthesis in Bacillus subtilis

Under conditions of iron deficiency, strains of Bacillus subtilis produced 2,3-dihydroxybenzoic acid (DHB), 2,3-dihydroxybenzolyglycine (DHBG), or both of these compounds. DHB(G) production [production of DHB(G) refers to the production of DHB, or DHBG, or both] was proportional to the amount of iron present and occurred logarithmically, paralleling growth. Supplementation of media with more than 150 mug of iron per liter at zero-time inhibited DHB accumulation completely. In the presence of DHB, lower levels of iron inhibited DHB(G) production, so that the actual inhibitor of synthesis may involve the Fe(3+):[DHB(G)](3) complex. The strains producing DHBG also produced coproporphyrin III during iron-deficient growth, whereas a strain producing DHB did not produce coproporphyrin III under these conditions. Accumulation of DHB(G) was influenced by the levels of aromatic amino acids and anthranilic acid in the medium. In vivo experiments with strain B-1471 demonstrated that DHB was coupled to added glycine to form DHBG. Metabolism of DHB(G) was observed in two of the strains studied.     

Morphological characteristics of Nocardia asteroides]

Morphology--by growth phases-- was studied in the N. asteroides strain isolated from cows suffering from mastitis in Cuba. During the growth N. asteroides culture underwent a number of morphological cyclic changes; definite morphology was typical for each growth phase. There was a similarity between the structure of the surface N. asteroides and mycobacteria; cyclic changes in the structure of the membranes in nocardia were also shown.     

Experimental pathogenicity of isolates of Nocardia asteroides, Nocardia brasiliensis and Nocardia caviae from different sources

The experimental pathogenicity of 14 isolates of Nocardia brasiliensis, 15 of N. asteroides, and 5 of N. caviae was investigated for the white Swiss mice inoculated intraperitoneally and in the foot pad, and for the guinea-pig and the hamster (Mesocricetus auratus) both inoculated intratesticularly. The guinea-pig was remarkably sensitive to N. asteroides, with an apparent relationship between pathogenicity and thermotolerance, confirming previous observations. Mice were in general less susceptible to this species. In both guinea-pigs and hamsters it was possible to observe typical granules with or without clubs. N. caviae was highly pathogenic for the guinea-pig and the hamster but no mycetomas were produced in the mice inoculated in the foot pad. Isolates of N. brasiliensis from natural sources were scarcely virulent for the different animals. Those of human origin produced significant lesions in the mice inoculated intraperitoneally with granules. Foot pad inoculation of mice with N. brasiliensis caused mycetomas in several animals.     

Mycolic acid analysis in Nocardia species. The mycolic acid compositions of Nocardia asteroides, N. farcinica, and N. nova.

Mycolic acids from twelve Nocardia species were analyzed for structure using capillary gas chromatography and mass spectrometry. This high-resolution procedure permitted good separation of the trimethylsilyl (TMS) ether derivatives of mycolic acid methyl ester according to the total number of carbon and double bonds. The profiles of the mycolic acid molecular species were used as models to illustrate the difference in the structures of each species, even in the case of N. asteroides complex; N. asteroides, N. farcinica and N. nova. Although N. asteroides and N. farcinica had similar lengths of carbon skeleton, i.e., 51.9-53.7 was the average carbon number (Av.Nc.), they had different compositions of unsaturated acids. Mycolic acids from N. asteroides were composed of abundant saturated acids and less than 1% tetraenoic acids; mycolic acids from N. farcinica were composed of unsaturated acids, which were composed of abundant dienoic acids, 2-12% of tetraenoic acids and a trace of pentaenoic acids. In contrast, Av.Nc. of mycolic acids from N. nova were 55.7-56.3, which were relatively longer than those from N. asteroides or N. farcinica. Regarding the characteristics of the structure of alpha-branch, major components were C16:0 and C18:0 for N. asteroides 23206T, and C16:0 and C14:0 for N. farcinica 23157T, respectively. The presence of monounsaturated alpha-branch (C18:1 and C16:1) was characteristic of N. nova.     

Nocardia asteroides culture filtrates cause dopamine depletion and cytotoxicity in PC12 cells

Experimental infection of BALB/c mice with the gram-positive bacterium Nocardia asteroides produces marked loss of nigrostriatal dopamine neurons, resulting in striatal dopamine depletion. To investigate the mechanism(s) responsible for this neuronal loss, we evaluated the influence of N. asteroides cell-free culture filtrates on rat pheochromocytoma PC12 cells, an in vitro model for dopamine neurons. Changes in cell viability and cell numbers were minimal after 24 h, but increased with longer incubation. In contrast, dopamine depletion occurred after 30 min incubation, and was greater with GUH-2 filtrate than with filtrate from the less virulent strain 10905. Incubation with the culture filtrate decreased viability in neuroblastoma and glioma cell lines, indicating that cytotoxic effects were not limited to dopaminergic cells. These findings suggest that the loss of nigrostriatal dopamine neurons and concomitant striatal dopamine depletion in Nocardia-infected mice may be due, at least in part, to the neurotoxicity of nocardial secretory products.     

Ethyl-3,4-dihydroxybenzoate inhibits myoblast differentiation: evidence for an essential role of collagen

To study the role of (pro)collagen synthesis in the differentiation of rat L6 skeletal myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB), was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium before the cell alignment stage. The inhibition is alleviated partially by ascorbate, which along with alpha-ketoglutarate serves as cofactor for the enzyme, prolyl hydroxylase. DHB drastically decreases the secretion of procollagen despite an increase in the levels of the mRNA for pro alpha 1(I) and pro alpha 2(I) chains. Probably, the procollagen chains produced in the presence of DHB, being underhydroxylated, are unable to fold into triple helices and are consequently degraded in situ. Along with the inhibition of procollagen synthesis, DHB also decreases markedly the production of a collagen-binding glycoprotein (gp46) present in the ER. The results suggest that procollagen production and/or processing is needed as an early event in the differentiation pathway of myoblasts.     

Immunochemical characterization of Nocardia asteroides antigens: support for a single species concept

Nocardia asteroides strains are highly heterogeneous. They show morphological, physiological, and immunological differences. In a previous study, we delineated seven immunotypes of N. asteroides. In the present study, we compared the culture filtrate antigens of these immunotypes by antigen-antibody crossed-immunoelectrophoresis and by rocket electrophoresis. We have also compared the antigen preparations by two-dimensional electrophoresis. While unique components constitute the major portion of the components, the results indicate that similar components are present in the culture filtrates of all strains. This finding supports the view of retaining all the immunotypes in the species Nocardia asteroides rather than designating different species such as N. farcinica and N. sebivorans.     

Production and Purification of Remazol Brilliant Blue R Decolorizing Peroxidase from the Culture Filtrate of Pleurotus ostreatus

An extracellular H(inf2)O(inf2)-requiring Remazol brilliant blue R (RBBR) decolorizing enzymatic activity was found in the culture medium of Pleurotus ostreatus. The enzymatic activity was maximally obtained in idiophase, and the optimum C/N ratio was 24. High C/N ratios repressed the enzymatic activity, and addition of veratryl alcohol had no effect on the production of enzyme. The enzyme was purified by ammonium sulfate fractionation, Sephacryl S-200 HR chromatography, DEAE Sepharose CL-6B chromatography, and Mono Q chromatography. The purification of RBBR decolorizing peroxidase, as judged by the final specific activity of 6.00 U/mg, was 54.5-fold, with a yield of 9.9%. The molecular mass of the native enzyme determined by gel permeation chromatography was found to be about 73 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was a monomer with a molecular mass of 71 kDa. The enzyme was optimally active at pH 3.0 to 3.5 and at 25(deg)C. Under standard assay conditions, the apparent K(infm) values of the enzyme toward RBBR and H(inf2)O(inf2) were 10.99 and 32.97 (mu)M, respectively. The enzyme had affinity toward various phenolic compounds and artificial dyes, and it was inhibited by Na(inf2)S(inf2)O(inf5), potassium cyanide, NaN(inf3), and cysteine. The absorption spectrum of the enzyme exhibited maxima at 407, 510, and 640 nm. The addition of H(inf2)O(inf2) to the enzyme resulted in an absorbance decrease at 407 and 510 nm.     

Pathogenesis of Cryptococcus neoformans is Associated with Quantitative Differences in Multiple Virulence Factors

Two isolates of Cryptococcus neoformans were previously described as being highly divergent in their level of capsule synthesis in vivo and in their virulence for mice. The highly virulent isolate (NU-2) produced more capsule than a weakly virulent isolate (184A) in vitro under tissue culture conditions and in vivo. This investigation was done to determine if there were differences between the two isolates in other factors that might also contribute to virulence. Growth rate was not a factor as NU-2 grew more slowly than 184A. Based on PCR fingerprinting the two isolates were genetically different providing an opportunity to examine differences in multiple virulence traits. Quantitative analysis revealed that NU-2 expressed significantly more melanin and mannitol than did 184A. Although the isolates expressed the same capsular chemotype, NU-2 produced an additional structure reporter group (SRG)under tissue culture conditions that was not present when grown in glucose salts/urea/basal medium (GSU).Capsular polysaccharide SRGs of 184A were unaffected by shifting the growth conditions from GSU to tissue culture conditions. Our results suggest that pathogenesis of a C. neoformans strain is dictated by the quantitative expression of the strain's combined virulence traits. Regulators of the expression of these genes may be playing key roles in virulence.This revised version was published online in October 2005 with corrections to the Cover Date.     

In vitro spheroplast and L-form induction within the pathogenic nocardiae.

Six strains of Nocardia asteroides, two strains of N. caviae, and two strains of N. braziliensis were grown in medium supplementted with glycine, lysozyme, D-cycloserine, glycine plus lysozyme, and glycine plus D-cycloserine. It was shown that three strains of N. asteroides, and two strains of N. caviae, readily formed spheroplasts and/or protoplasts when grown in the presence of glycine plus either lysozyme or D-cycloserine. This process was studied by both phase contrast microscopy and electron microscopy. The induced cultures were then plated on hypertonic medium for the isolation of L-forms. It was shown that the organisms differed greatly in their ability to produce spheroplasts and subsequently grew as L-forms or transitional-phase variants.