Interaction of cord factor (alpha, alpha'-trehalose-6,6'-dimycolate) with phospholipids.

We previously reported that cord factor (alpha,alpha'-trehalose-6,6'-dimycolate) isolated from Nocardia asteroides strain GUH-2 strongly inhibits fusion between unilamellar vesicles containing acidic phospholipid. We chose to study the effects of this molecule on liposome fusion since the presence of N. asteroides GUH-2 in the phagosomes of mouse macrophages had been shown to prevent phagosomal acidification and inhibit phagosome-lysosome fusion. A virtually non-virulent strain, N. asteroides 10905, does not prevent acidification or phagosome-lysosome fusion and, further, contains only trace amounts of cord factor. In the present paper, we have investigated the effects of cord factor on phospholipid bilayers that could be responsible for the inhibition of fusion. We show that cord factor increases molecular area, measured by isothermal compression of a monolayer film, in a mixed monolayer more than would be expected based in its individual contribution to molecular area. Cord factor, as well as other glycolipids investigated, increased the overall hydration of bilayers of dipalmitoylphosphatidylcholine by 50%, as estimated from the unfrozen water fraction measured by differential scanning calorimetry. The effect of calcium on this increased molecular area and headgroup hydration was measured by fluorescence anisotropy and FTIR spectroscopy of phosphatidylserine liposomes. Both techniques showed that cord factor, incorporated at 10 mol%, increased acyl chain disorder over controls in the presence of Ca2+. However, FTIR showed that cord factor did not prevent headgroup dehydration by the Ca2+. The other glycolipids tested did not prevent either the Ca(2+)-induced chain crystallization or headgroup dehydration of phosphatidylserine bilayers. These data point to a possible role of the bulky mycolic acids of cord factor in preventing Ca(2+)-induced fusion of liposomes containing acidic phospholipids.

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.

Isolation, sequencing and expression of the superoxide dismutase-encoding gene (sod) of Nocardia asteroides strain GUH-2.

Nocardia asteroides (Na) superoxide dismutase (SOD) has been implicated as a virulence factor that allows the organism to survive intracellular killing by phagocytic cells. A full-length Na sod gene from a pathogenic strain of Na (strain GUH-2) was cloned from a recombinant phage library using the Mycobacterium tuberculosis (Mt) sod gene (Mt sod) as a probe. The promoter region and structural gene (624 bp) of Na sod was sequenced and nucleotide sequence comparisons reveal 77% homology with Mt sod. The Na sod gene also shares considerable sequence homology with sod of other mycobacterial species. In addition, conserved amino acid (aa) sequences important for metal binding indicate that Mn2+ is the preferred metal ion ligand for Na SOD. An Na sod expression plasmid, pYEX1, under transcriptional control of the Mt hsp70 promoter (pY6013), produced a 25-kDa protein product which showed SOD activity when stained in a native polyacrylamide gel and reacted with rabbit polyclonal antibody specific for Na SOD by Western blot. pYEX1, via transformation, was able to complement an Escherichia coli double sodAB mutant deficient in SOD production in the presence of paraquat (methyl viologen) which stimulates the production of superoxide radicals.

Laboratory evaluation of an outbreak of nocardiosis in immunocompromised hosts.

Seven patients in a renal unit were proved to have nocardiosis in an interval of nine months. Six of these patients had received renal transplants. Serologic investigation suggested that two additional cases of undiagnosed pulmonary disease were also nocardial, and that there were no subclinical cases in patients or staff. Clinical-serologic correlations indicate that serologic evaluation may be a useful adjunct in diagnosis of nocardiosis, if used early and repeatedly, and to follow response to therapy. Epidemiologic investigations yielded cultures of Nocardia asteroides from air and dust inside the unit and elsewhere in the hospital. Biochemical, metabolic, physical and immunologic characterization of the isolates indicated that those from patients and those from the unit environment were identical, whereas some from outside the unit could be differentiated from these. The "epidemic strain" had type III antigen, which surveys indicated is not the most common type in human nocardiosis (it occurs in association with a minority of human cases). The isolates were of subgroup B, which has been associated with virulence. The characterization methods employed could be useful in studies of nocardial epidemiology. The laboratory studies indicate epidemic spread within the unit of a single organism, and current epidemiologic guidelines, which do not recommend respiratory isolation of cases of pulmonary nocardiosis, may need reconsideration particularly when there are immunocompromised hosts in the environment.

The ultrastructure of bronchial macrophages and lymphocytes in sarcoidosis.

A physical interaction between macrophages and lymphocytes was observed more frequently in bronchial lavage fluid obtained from patients with sarcoidosis than from normal volunteers, irrespective of their history of smoking. In this spontaneous interaction more than two lymphocytes were commonly seen adhering to a macrophage without evidence of cytoplasmic bridging or membrane fusion. To a greater extent than in normal volunteers, macrophages from patients with sarcoidosis were characterized by the appearance of a more highly irregular cell surface, more membrane bound inclusions, however, was positively correlated with the smoking history of the individual, and the number of surface projections (microvilli) of macrophages from smokers appeared to be reduced. Significant differences were not apparent in the nuclear or cellular diameters of macrophages from sarcoid and normal individuals.

Development of a serologic panel for the recognition of nocardial infections in a murine model.

A murine model was used to develop a sensitive and specific serologic test for clinical and subclinical infections caused by Nocardia. The following tests were used: (a) enzyme-linked immunosorbent assays (ELISAs) with culture filtrate and cytoplasmic extract antigens from Nocardia asteroides; (b) ELISA with N. asteroides trehalose dimycolate (cord factor); (c) indirect immunofluorescent antibody assay with whole cells of N. asteroides; and (d) Western-blot analysis for the 54 to 55-kD, 36-kD, and 62-kD proteins of N. asteroides. The sera from BALB/c mice, experimentally infected with nonlethal doses of three species of Nocardia, nine species of Mycobacterium, Rhodococcus equi, two species of Actinomadura, and two species of Streptomyces were tested using this panel. The serologic tests did, indeed, identify mice infected with nocardiae and could differentiate them from mice infected with the other actinomycetes, including mycobacteria.

Occurrence of plasmids in pathogenic strains of Nocardia.

The purpose of the present study was to determine the relative distribution of plasmids in 87 clinical isolates of Nocardia, belonging to the five major pathogenic species. A correlation between plasmid content and the site of infection within the host, resistance to antibiotics and enzymic profiles was also investigated. The plasmid extraction procedure of Kado and Liu was used. Electrophoretic analysis revealed one-to-four plasmid bands, ranging in size from <8 to >50 kb, in 27 strains (31%). Based on the number of isolates tested, the incidence of plasmid-bearing strains was significantly higher among N. farcinica than N. asteroides strains. Within N. farcinica, the incidence of plasmids was higher among strains isolated in the Paris area than in strains isolated elsewhere, such as in the French provinces or outside France. A statistically significant correlation was demonstrated between the cutaneous localisation of infections and the incidence of plasmid-bearing strains. The presence of plasmids in nocardiae could not be associated with specific phenotypic traits such as resistance to antibiotics or enzymic activity. The fact that the majority of Nocardia clinical isolates (60 of 87) did not contain plasmids suggests that plasmids are not involved directly in virulence and that there is no selective pressure for plasmid acquisition.

Molecular weight determination of methyl esters of mycolic acids using thermospray mass spectrometry.

Methyl esters of normal fatty acids, corynomycolate and corynomycolenate were used as model compounds for thermospray mass spectrometric procedures for molecular weight determination of the related nocardial mycolic acids. By using ammonium acetate at the positive ion generator, in both cases, a family of ions was produced. The following members were found and corresponded to the adducts: (1) M + H; M + NH4 and M + H + NH4 for methyl esters of normal fatty acids, whereas M + H, M + 2H and M + H + NH4 were the adducts most frequently observed with methyl corynomycolates. The methyl esters of C40-C48 mycolic acids from Rhodococcus rhodochrous exhibited prominent peaks corresponding to adducts M + H + NH4 whereas those corresponding to M + 2H showed slightly lower intensities. The structure M + H had no significant representatives with this subclass of mycolic acids. A similar pattern was observed with methyl esters of C50-C54 mycolic acids from Nocardia asteroides GUH-2. Ion peaks C50-C54 representing adducts M + 2H and M + H + NH4 prevailed in the mass spectrum. In this case, the intensities of peaks corresponding to M + 2H were slightly higher than those of the M + H + NH4. Essentially three main species of nocardomycolic acids were detected: (1) monounsaturated C50:1, C52:1 and C54:1; (2) diunsaturated C50:2, C52:2 and C54:2 and (3) triunsaturated C52:3 and C54:3 mycolic acids. The most abundant mycolic acid was C52:2 followed in decreasing abundance by C52:1, C54:2, C50:2, C52:3 and C54:3 mycolic acids.

Thermospray mass spectral analyses of corynomycolic acids and their derivatives.

Thermospray mass spectral (TSP-MS) analyses were carried out on methyl corynomycolates, their 3-O-acetyl and 3-O-benzoyl derivatives, and on corynomycolic acids and their 3-O-acetyl derivatives, using an ion generating solvent system consisting of water/isopropanol (99:1, v/v) containing 0.1 M ammonium acetate. Methyl corynomycolates generated three groups of peaks corresponding to adducts M-18 + H, M + H and M + NH4, while two groups of peaks representing adducts M-60 + H and M + H + NH4 were seen in the spectra of 3-O-acetyl methyl corynomycolates. The 3-O-benzoyl methyl corynomycolates gave a series of peaks representing the adducts M-122 + H, M + 2H and M + H + NH4. In the spectra of 3-O-acetyl corynomycolic acids, a series of peaks which represented M-60 + H and M + NH4 was observed, and in turn, mass spectra of corynomycolic acids revealed peaks that represented the adducts M-18 + H and M + NH4. Therefore, methyl corynomycolates, 3-O-acyl derivatives of methyl corynomycolates,. 3-O-acetylated derivatives of corynomycolic acids and the underivatized corynomycolic acids all exhibited the formation of an adduct of the anhydro compounds. These anhydro forms were generated by a generalized process.

Composition and toxicity of diethyl ether soluble lipids from Nocardia asteroides GUH-2 and Nocardia asteroides 10905.

Virulent Nocardia asteroides GUH-2 and avirulent N. asteroides 10905 contained 8.2% and 10.6% of diethyl ether soluble lipids (DESL) per dry cell mass, respectively. Intraperitoneal (i.p.) inoculation of 250 micrograms of DESL from GUH-2 dissolved in mineral oil was toxic to mice, resulting in weight loss and death of 100% of the animals (20/20) within 20 days. In contrast, DESL from 10905 had very little toxicity, and only one mouse (1/10) died within 30 days. Three fractions resulted from DESL by silicic acid column chromatography: (1) neutral lipids and fatty acids (NFA); (2) glycolipids (GL); and (3) phospholipids (PL). Each fraction dissolved in mineral oil was inoculated into mice as described above. The NFA and PL were not toxic. GL from 10905 had low toxicity (30% of the animals died, 3/10) whereas GL from GUH-2 expressed high toxicity (100% of the animals became cachetic and died, 10/10). GL from 10905 represented a minor component (0.6% of dry cell weight) whereas GL from GUH-2 was more prominent (1.5% of dry cell wt.). Approximately 95% of GL from GUH-2 had properties of an authentic trehalose-6,6'-dimycolate standard. Infrared spectrum of the major glycolipid (GUH-2 GL) had characteristic absorptions in the fingerprint region between 800 cm-1 and 1500 cm-1. Upon alkaline hydrolysis GUH-2 GL released 76% mycolic acids in the C50 size range plus 24% trehalose.(ABSTRACT TRUNCATED AT 250 WORDS)

Recovery of microorganisms from synovial and pleural fluids of animals using hyperosmolar media.

L-phase (CWD) broth and plate media were used in parallel with conventional microbiological media during a 3-year period for culturing synovial and pleural fluids of animals. Two kinds of recoveries were obtained where parallel conventional methods were negative: (1) parent or normal bacteria, in very low numbers; and (2) Type B CWD variants in equally low numbers. Organisms in group 1 were: Streptococcus zooepidemicus from horses (2x); beta-hemolytic streptococci, Lancefield Gp. G (2x); Staphylococcus aureus; Actinobacillus, and Actinomyces viscosus. Group 2 consisted of Bacteroides sp., Propionibacterium acnes, and three "Nocardia-like" sp. Catalase + Actinomyces was not recovered equally well on CWD plates as on conventional media with fluids obtained during ampicillin treatment. This occurred in spite of the fact that the CWD media was shown to support growth and reversion of laboratory induced L-phase variants of Nocardia caviae and N. asteroides, and had facilitated recovery of a Bacteroides L-phase variant from a pleural fluid. The nature of this fault in the media is under investigation in this laboratory.

Nocardia otitidiscaviarum (GAM-5) induces parkinsonian-like alterations in mouse.

Parkinson's disease, a major neurodegenerative disorder in humans whose etiology is unknown, may be associated with some environmental factors. Nocardia otitidiscaviarum (GAM-5) isolated from a patient with an actinomycetoma produced signs similar to Parkinson's disease following iv injection into NMRI mice. NMRI mice were infected intravenously with a non-lethal dose of 5 x 10(6) colony forming units of N. otitidiscaviarum (GAM-5). Fourteen days after bacterial infection, most of the 60 mice injected exhibited parkinsonian features characterized by vertical head tremor, akinesia/bradykinesia, flexed posture and postural instability. There was a peak of nocardial growth in the brain during the first 24 h followed by a decrease, so that by 14 days nocardiae could no longer be cultured. At 24 h after infection, Gram staining showed nocardiae in neurons in the substantia nigra and occasionally in the brain parenchyma in the frontal and parietal cortex. At 21 days post-infection, tyrosine hydroxylase immunolabeling showed a 58% reduction of tyrosine hydroxylase in the substantia nigra, and a 35% reduction of tyrosine hydroxylase in the ventral tegmental region. Dopamine levels were reduced from 110 +/- 32.5 to 58 +/- 16.5 ng/mg protein (47.2% reduction) in brain from infected mice exhibiting impaired movements, whereas serotonin levels were unchanged (191 +/- 44 protein in control and 175 +/- 39 ng/mg protein in injected mice). At later times, intraneuronal inclusion bodies were observed in the substantia nigra. Our observations emphasize the need for further studies of the potential association between Parkinson's disease or parkinsonism-like disease and exposure to various nocardial species.

Subcutaneous abscesses and arthritis caused by a probable bacterial L-form in cats.

Three cats in one household developed pyogenic subcutaneous abscesses and arthritis over a period of 9.5 months. Despite vigorous surgical and antibiotic treatment, the infections in each of these cats continued to spread locally and hematogenously to involve other joints and subcutaneous sites. Although the infections did not respond to modern broad-spectrum antibiotics, they were susceptible to tetracycline. In spite of a favorable response to tetracycline, all 3 cats were euthanatized. A causative agent could not be identified by microbiologic culture of tissues obtained prior to death and at necropsy, or with special tissue stains. The infection was transmitted experimentally by sc inoculation with cell-free material from one of the naturally infected cats to a specific-pathogen-free cat. A tissue extract from the experimentally infected cat was, in turn, infectious for another specific-pathogen-free cat. The experimentally induced lesion was a rapidly enlarging necrotizing and pyogenic cellulitis and panniculitis, with no demonstrable causative agent by special tissue stains or microbiologic culture. A probable bacterial L-form was visualized in affected tissues of the experimentally infected cats and propagated in special L-form broth. Like the natural disease, infection in experimentally inoculated cats was progressive in nature, but could be treated successfully with tetracycline.

Structural and biochemical alterations of Nocardia asteroides cell walls during its growth cycle.

Analysis of Nocardia asteroides 14759 cell walls were done to determine the chemical and structural composition during the growth cycle. It was found that the ultrastructural profiles of the cell wall become altered as the cultures aged. Chemical analysis revealed corresponding shifts in cell wall components as the culture went from lag to logarithmic to stationary phases of growth. The peptidoglycan from lag-phase cells (5 h) represented 15% of the total cell wall weight, and the percentage of peptidoglycan progressively increased so that, in 1-week stationary-phase cells, it represented approximately 40% of the total wall weight. In lag-phase cells it was found that 36% of the cell wall weight was lipid in nature, whereas stationary-phase cells had only 7% lipid in their wall. The overall sugar composition of the walls remained relatively constant at about 28 to 31% however, the arabinose to galactose ratio changed from approximately 1:1 in lag-phase to 2:1 in stationary-phase cells. Gas-liquid chromatography demonstrated that the fatty acids making up the cell wall lipids changed relative to one another as the cells aged. Based upon the removal of lipids by ethanol-ether, chloroform, and alkaline methanol extraction, it was shown that the classes of loosely and firmly associated lipids changed as the cells aged. Further, it was found that a carotenoid-like pigment associated to a C22 fatty acid increased in the cell wall as the culture stopped growing. Peptidolipid or lipoprotein was found to make up a significant part of the cell wall. This component increased in amount and varied in amino acid content as the culture aged. Analysis of the totally extracted basal layer of the cell wall (peptidoglycan plus arabinogalactan) showed that it too changed as the cells grew and fragmented. The data presented established that the cell wall of N. asteroides was structurally and chemically complex and that a progression of chemical and physical processes occurred within the wall as the cells developed through their growth cycle.

Interaction of Nocardia asteroides at different phases of growth with in vitro-maintained macrophages obtained from the lungs of normal and immunized rabbits.

The interactions of cells of Nocardia asteroides GUH-2 during different stages of growth with cultured macrophages obtained from the lungs of nonimmunized and immunized rabbits were studied. The nocardial cells from all stages grew intracellularly in "normal" alveolar macrophages; however, log-phase cells increased in numbers more rapidly than did stationary-phase cells. Macrophages obtained from the lungs of specifically immunized rabbits effectively inhibited the growth of stationary-phase cells but only temporarily retarded the growth of log-phase organisms. Specific antiserum added to the nocardial cells before incubation with presensitized macrophages caused enhanced phagocytosis and inactivation of the log-phase cells but had no effect on the fate of the stationary-phase nocardia. In addition, it was fo-nd that log-phase cells were phagocytized less effectively by normal macrophages than were the stationary-phase cells, and log-phase cells were more toxic to the macrophage monolayer. From these data we conclude that secondarily induced macrophages play a major role in host resistance to pulmonary nocardial infections, and antibody may be important for effective host resistance to the filamentous form of N. asteroides. Since the nocardia were able, with time, to overcome these effects, it appears that additional host factors (such as T-lymphocytes) must be involved in an effective host response to N. asteroides.

Differential binding of Nocardia asteroides in the murine lung and brain suggests multiple ligands on the nocardial surface.

The adherence of Nocardia asteroides in the murine brain and lungs was determined. Virulent strains had increased adherence in the brain and lungs, whereas less virulent strains bound in either the brain or lungs. Nocardiae that attached apically penetrated host cells. Multiple receptors on the nocardial surface may be involved in this differential attachment and penetration.

In vitro response of rabbit alveolar macrophages to infection with Nocardia asteroides.

The interaction of Nocardia asteroides with cultured "normal" nonimmune rabbit alveolar macrophages was studied by light and electron microscopy. It was shown that the alveolar macrophage response to the more virulent strain (N. asteroides 14759) was quite different from the response to the less virulent organism (N. asteroides 10905). N. asteroides 14759 elicited a dramatic in vitro response of the macrophages toward the nocardial infection. Within a few hours postinfection, there was a migration of macrophages toward other cells actively infected with viable nocardia, so that at 6 h considerable macrophage aggregation on the cover slips had occurred. Many of the macrophages within these aggregates exhibited tight cell-to-cell contact, whereas others were observed to fuse, forming multinucleate giant cells, with many containing more than 10 nuclei. Upon continued incubation, these giant cells appeared to destroy the intracellular nocardia, so that, at 24 h postinfection, gram-positive, ultrastructurally intact bacteria could not be observed. At the same time, some of the macrophage aggregates that did not fuse appeared to be unable to stop the intracellular growth of nocardia. At 12 to 24 h large numbers of gram-positive, acid-fast filaments were observed growing out from within these macrophage aggregates. The macrophage response seemed dependent upon the strain of Nocardia infecting them, since N. asteroides 10905 did not induce a similar response within the macrophage population.

Ultrastructural analysis of growth of Nocardia asteroides during invasion of the murine brain.

BALB/c mice were infected with 10(6) CFU of log-phase cells of Nocardia asteroides GUH-2 by tail vein injection (at this lethal inoculum dose, approximately 800 to 1,000 CFU becomes deposited in the brain). At 24 h after infection, the ultrastructural interactions of the nocardiae during growth within the murine brain were investigated. The nocardiae grew perivascularly in the pons, substantia nigra, hypothalamus, and thalamus portions of the brain, where they were either within or associated with most brain cell types. There appeared to be a propensity for growth within the soma of neurons and their axonal extensions. The nocardial cells were surrounded by 1 to 30 layers of membrane, and the innermost membrane was usually tightly adherent to the cell wall. This compartmentalization of nocardiae within brain cells could contribute to their failure to induce an inflammatory response or a cytopathic effect. Nevertheless, the bacteria were able to obtain adequate nutrients from the host to grow within the brain. The nocardiae were not completely inert, since some of the brain cells showed signs of degeneration. The myelin sheaths of axons were the most strongly affected, and there was evidence of demyelinization and axonal degeneration. Nocardiae growing within brain cells were phagocytized by compact, dense cells that were probably microglia. There was no ultrastructural evidence that the nocardiae were damaged by these phagocytes 24 h after infection; nevertheless, these cells may be important for the elimination of nocardiae from the brain during a nonlethal infection.