Neuroinvasive Nocardia asteroides GUH-2 induces apoptosis in the substantia nigra in vivo and dopaminergic cells in vitro.

Neurodegenerative diseases such as Parkinson's disease are increasingly prevalent in the aging population worldwide. The causes of these disorders are unknown, but many studies have suggested that the etiology is likely multifactorial and may involve exposure to something in the environment combined with the normal aging process. Nocardia asteroides are bacteria commonly found in the soil, and neuroinvasive strains of nocardiae have been described. N. asteroides strain GUH-2 invades the brains of experimentally infected animals and selectively affects dopaminergic neurons of the substantia nigra (SN), causing an L-DOPA-responsive movement disorder resembling parkinsonism. Furthermore, dopaminergic neurons undergo morphological changes characteristic of apoptosis following nocardial infection. Apoptosis has been implicated in dopaminergic neuronal dropout in Parkinson's patients as well as other parkinsonian models. Thus, in this study, in vivo and in vitro models were utilized to measure the ability of GUH-2 to induce the apoptotic death of dopaminergic cells. Following infection with GUH-2, dopaminergic apoptotic cells were identified in the SN of animals by in situ end labeling, which detects DNA fragmentation, combined with fluorescent immunolabeling of tyrosine hydroxylase-positive cells. In addition, apoptosis was observed in PC12 cell cultures incubated with GUH-2 by both in situ end labeling and the annexin V assay, which detects externalization of phosphatidylserine of the plasma membrane, indicating apoptotic death. Based on the results of these studies, it appears that experimental infection with N. asteroides provides a general model for studying apoptosis in parkinsonian disorders.

Filament tip-associated antigens involved in adherence to and invasion of murine pulmonary epithelial cells in vivo and HeLa cells in vitro by Nocardia asteroides.

The interactions of Nocardia asteroides GUH-2 with pulmonary epithelial cells of C57BL/6 mice and with HeLa cells were studied. Electron microscopy demonstrated that only the tips of log-phase cells penetrated pulmonary epithelial cells following intranasal administration, and nocardiae were recovered from the brain. Coccobacillary cells neither invaded nor disseminated. Serum from immunized mice (IMS) decreased attachment to and penetration of pulmonary epithelial cell surfaces by log-phase GUH-2 and inhibited spread to the brain. IMS was adsorbed against stationary-phase cells. Western immunoblots suggested that this adsorbed IMS was reactive primarily with 43- and 62-kDa proteins. Immunofluorescence showed that adsorbed IMS preferentially labeled the tips of log-phase GUH-2 cells. Since this IMS was reactive to culture filtrate antigens, several of these proteins were cut from gels, and mice were immunized. Sera against 62-, 55-, 43-, 36-, 31-, and 25-kDa antigens were obtained. The antisera against the 43- and 36-kDa proteins labeled the filament tips of GUH-2 cells. Only the antiserum against the 43-kDa antigen increased pulmonary clearance, inhibited apical attachment to and penetration of pulmonary epithelial cells, and prevented spread to the brain. An in vitro model with HeLa cells demonstrated that the tips of log-phase cells of GUH-2 adhered to and penetrated the surface of HeLa cells. Invasion assays with amikacin treatment demonstrated that nocardiae were internalized. Adsorbed IMS blocked attachment to and invasion of these cells. These data suggested that a filament tip-associated 43-kDa protein was involved in attachment to and invasion of pulmonary epithelial cells and HeLa cells by N. asteroides GUH-2.

Genic control of honey bee dance language dialect.

Behavioural genetic analysis of honey bee dance language shows simple Mendelian genic control over certain dance dialect differences. Worker honey bees of one parent colony (yellow) changed from round to transition dances for foraging distances of 20 m and from transition to waggle dances at 40 m. Worker bees of the other parent colony (black) made these shifts at 30 m and 90 m, respectively. F1 colonies behaved identically to their yellow parent, suggesting dominance. Progeny of backcrossing between the F1 generation and the putative recessive black parent assorted to four classes, indicating that the dialect differences studied are regulated by genes at two unlinked loci, each having two alleles. Honey bee dance communication is complex and highly integrated behaviour. Nonetheless, analysis of a small element of this behaviour, variation in response to distance, suggests that dance communication is regulated by subsets consisting of simple genic systems.

Differences in the interactions of Nocardia asteroides with macrophage, endothelial, and astrocytoma cell lines.

An in vitro model for studying host cell interactions with Nocardia asteroides was developed. Thus, macrophage cell lines J774A.1 and P388D1, pulmonary artery endothelium cell line CPAE, rat glial tumor cell line C6, and human astrocytoma cell lines CCF-STTG1 and U-373 MG were infected with either log- or stationary-phase cells of N. asteroides GUH-2, and the host cell-nocardia interactions were determined by light microscopy and electron microscopy. Polyclonal antinocardial antibody did not enhance uptake of nocardiae by any of these cell lines; however, log-phase cells of GUH-2 infected a higher percentage of J774A.1 and P388D1 than did stationary-phase organisms. When cells infected with stationary-phase GUH-2 were incubated for 6 h, filaments developed, which indicated that nocardial growth had occurred. In J774A.1 and P388D1, only 31 to 57% of the total stationary-phase coccobacillary cells that were phagocytized formed filaments within 6 h. This indicated that there was some inhibition of growth of the phagocytized nocardiae within these macrophage cell lines; however, the nocardiae grew within the endothelial (> 87% filaments) and astrocytoma (100% filaments) cell lines. Microfilament inhibitor cytochalasin B inhibited uptake of GUH-2 by macrophages and other cell lines, except that there was no effect on uptake of nocardial cells by astrocytoma cell line U-373 MG. Scanning and transmission electron microscopy showed phagocytosis of GUH-2 by the different cell lines. In cytochalasin B-treated cells, nocardiae were shown to penetrate through the cell surface and become internalized in a manner distinct from typical phagocytosis, suggesting that filamentous forms of this organism have a phagocytosis-independent invasion factor. The extent of this cytochalasin-resistant cellular penetration by the nocardiae differed in the different cell lines.

Nocardia species: host-parasite relationships.

The nocardiae are bacteria belonging to the aerobic actinomycetes. They are an important part of the normal soil microflora worldwide. The type species, Nocardia asteroides, and N. brasiliensis, N. farcinica, N. otitidiscaviarum, N. nova, and N. transvalensis cause a variety of diseases in both normal and immunocompromised humans and animals. The mechanisms of pathogenesis are complex, not fully understood, and include the capacity to evade or neutralize the myriad microbicidal activities of the host. The relative virulence of N. asteroides correlates with the ability to inhibit phagosome-lysosome fusion in phagocytes; to neutralize phagosomal acidification; to detoxify the microbicidal products of oxidative metabolism; to modify phagocyte function; to grow within phagocytic cells; and to attach to, penetrate, and grow within host cells. Both activated macrophages and immunologically specific T lymphocytes constitute the major mechanisms for host resistance to nocardial infection, whereas B lymphocytes and humoral immunity do not appear to be as important in protecting the host. Thus, the nocardiae are facultative intracellular pathogens that can persist within the host, probably in a cryptic form (L-form), for life. Silent invasion of brain cells by some Nocardia strains can induce neurodegeneration in experimental animals; however, the role of nocardiae in neurodegenerative diseases in humans needs to be investigated.

Interactions of Nocardia asteroides with murine glia cells in culture.

Astrocytes and microglia were obtained from brains of newborn mice and infected with Nocardia asteroides. Scanning electron micrographs showed nocardiae adhering to the astrocyte cell surface and entering the cytoplasm. After 6 h of incubation the intracellular nocardiae appeared as filaments, demonstrating that growth was occurring. In contrast, the microglia phagocytized nocardiae, but after 6 h the presence of coccoid cells indicated that nocardial growth was inhibited.

The timing of exposure of mononuclear phagocytes to recombinant interferon gamma and recombinant tumor necrosis factor alpha alters interactions with Nocardia asteroides.

Nocardia asteroides modulates phagocyte function and grows within macrophages. Because interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) have been shown to activate macrophages to kill a variety of microorganisms, the effects of IFN-gamma and TNF-alpha on the activation of murine macrophages and human monocytes to kill nocardiae were studied. It was found that macrophages or monocytes treated with either IFN-gamma, TNF-alpha, or lipopolysaccharide as a secondary signal did not demonstrate increased microbicidal activity against N. asteroides even though these phagocytes were effective at killing the fungus Coccidioides immitis and the protozoan Toxoplasma gondii. Preincubation of phagocytes for 24 h with these compounds resulted in an enhancement of nocardial growth. In contrast, coincubation of these factors with the nocardiae and mononuclear cells during phagocytosis resulted in inhibition of nocardial growth even though this bacterium was not killed. Therefore, the specific timing of the exposure of the phagocyte in vitro to IFN-gamma or TNF-alpha has a significant effect on its ability to alter nocardial growth.

Effects of recombinant gamma interferon and tumor necrosis factor on in vitro interactions of human mononuclear phagocytes with Coccidioides immitis.

Human peripheral blood monocytes readily phagocytized Coccidioides immitis endospores (2 to 5 microns) in vitro. Within 24 to 30 h at 37 degrees C, the phagocytized endospores started developing into immature spherules. However, when the monocytes were incubated with recombinant human gamma interferon (rIFN-gamma) or recombinant human tumor necrosis factor alpha (rTNF-alpha) and then infected, fewer endospores developed into spherules. Treatment with rIFN-gamma or rTNF-alpha activated the fungicidal capabilities of the monocytes as evidenced by the significant reduction in CFU that could be recovered from rIFN-gamma- or rTNF-alpha-activated monocytes compared with nontreated controls.

Monoclonal antibodies demonstrate that superoxide dismutase contributes to protection of Nocardia asteroides within the intact host.

The importance of superoxide dismutase (SOD) in protecting cells of Nocardia asteroides from the oxidative killing mechanisms within the intact murine host was determined. Murine monoclonal antibodies specific for nocardial SOD and for another nocardial antigen were prepared. Both antibodies adhered to cell surface antigens, as shown by fluorescence-labeled-antibody staining. The anti-nocardial SOD antibody inhibited the effect of nocardial SOD on superoxide generated in vitro. Cells of N. asteroides GUH-2 in log phase of growth were incubated with monoclonal anti-nocardial SOD, another monoclonal antinocardial antibody (not reactive with SOD), or phosphate-buffered saline and then injected intravenously into mice. Total recovery of CFU and inhibition of growth were determined at 3, 24, and 48 h after infection. The brains, kidneys, spleens, lungs, and livers were weighed, homogenized, and plated in order to quantitate the number of organisms in each organ at each time period. There was an initial killing followed by enhanced clearance of N. asteroides from the lungs and livers of mice which had received anti-SOD antibody-treated nocardiae. There was also enhanced early killing in the spleen. At 48 h, there were fewer organisms recovered from the brains, kidneys, and livers of mice which had received anti-SOD antibody-treated nocardia. This was not true for mice which had received antinocardial antibody not specific for SOD. The data demonstrate that surface-associated SOD protects N. asteroides for oxidative killing in vivo during all stages of infection.

Acid phosphatase stimulation of the growth of Nocardia asteroides and its possible relationship to the modification of lysosomal enzymes in macrophages.

Lysosomal acid phosphatase levels are reduced in murine macrophages by virulent strains of Nocardia asteroides. At the same time, other lysosomal enzymes either remain unchanged or increase in activity, indicating that acid phosphatase is not lost because of degranulation or membrane leakage. This study shows that acid phosphatase was utilized as a sole carbon source by Nocardia asteroides and that acid phosphatase combined with glutamate as a carbon source enhanced nocardial growth. As a consequence, the inverse relationship that was observed between acid phosphatase activity and the bactericidal capacity of macrophages infected with N. asteroides appears to be due to the ability of N. asteroides to preferentially metabolize this lysosomal enzyme during growth within phagocytes.

Fungicidal activation of murine macrophages by recombinant gamma interferon.

Alveolar macrophages from BALB/c mice readily phagocytized endospores (2 to 5 micron) and arthroconidia of Coccidioides immitis in vitro. Within 24 to 30 h at 37 degrees C, the phagocytized endospores started developing into spherules, and the arthroconidia formed germ tubes and hyphae. However, these processes did not occur if the macrophages were incubated with murine recombinant gamma interferon (rIFN-gamma) during infection with C. immitis. Treatment with rIFN-gamma activated the fungicidal capabilities of the alveolar macrophages, as evidenced by the 50% reduction in the CFU which could be recovered from macrophages infected in the presence of gamma interferon compared with alveolar macrophages infected without gamma interferon (P less than 0.05). Similar results were seen with peritoneal macrophages incubated with rIFN-gamma and infected with C. immitis. As little as 10 U of rIFN-gamma per ml reduced by half the number of C. immitis CFU which could be recovered from the phagocytes 8 h after infection with arthroconidia, although interferon alone did not affect the viability of the fungi.

Role of superoxide dismutase and catalase as determinants of pathogenicity of Nocardia asteroides: importance in resistance to microbicidal activities of human polymorphonuclear neutrophils.

The roles of nocardial superoxide dismutase (SOD) and catalase in the resistance of Nocardia asteroides to the microbicidal properties of human polymorphonuclear leukocytes were determined in vitro. The neutrophils killed ca. 80% of the cells of the less virulent N. asteroides 10905 and ca. 50% of the log phase of the more virulent N. asteroides GUH-2 after 180 min of incubation. These phagocytes were not able to kill early-stationary-phase cells of strain GUH-2 that contained 10 times more intracytoplasmic catalase than log-phase cells of the same culture. However, the polymorphonuclear leukocytes were able to kill more than 50% of the cells of early-stationary-phase strain GUH-2 after treatment with purified antibody specific for surface-associated SOD. No killing was observed when the bacteria were treated with normal rabbit immunoglobulin G or with serum obtained from rabbits immunized against whole nocardial cells (containing little or no activity against SOD). These phagocytes killed more than 99% of Listeria monocytogenes used as a control. Chlorpromazine-treated polymorphonuclear leukocytes killed L. monocytogenes (70%) but they were not able to kill antibody-treated cells of N. asteroides GUH-2. Exogenously added SOD partially protected strain 10905, which lacked surface-associated enzyme, but it had no effect on the killing of strain GUH-2, which already possessed significant amounts of surface-bound SOD. In contrast, catalase added to the nocardiae provided almost complete protection to the log-phase cells of strain GUH-2, but strain 10905 was only partially protected. SOD combined with catalase had additive activity which completely protected the cells of strain 10905. A mutant of N. asteroides GUH-2 (SCII-C) is more virulent during the log phase than is the parental strain. This mutant contained at least 7 times more catalase at this stage of growth than did the parent. No other differences between these two strains were observed during the log phase. In sharp contrast to those of the parent, log-phase cells of this high-catalase mutant were not killed by polymorphonuclear phagocytes. These data indicate a role for both SOD and catalase in the resistance of Nocardia spp. to human neutrophils, and they represent at least two factors associated with virulence.

Activation of macrophages by lymphokines: enhancement of phagosome-lysosome fusion and killing of Coccidioides immitis.

Previously, it was shown that arthroconidia of Coccidioides immitis appear to inhibit phagosome-lysosome fusion and survive within normal mouse peritoneal macrophages. However, when these macrophages are exposed to antigen-stimulated T lymphocytes from immune mice, activation occurs, leading to enhanced phagosome-lysosome fusion and killing of C. immitis. Results indicate that the activation of macrophages can be effected after incubation with soluble lymphocyte product(s) (lymphokines). The activation of macrophages results if the macrophages are exposed to the lymphokine before, but not after, infection. The results indicate that the lymphocyte population responsible for the elaboration of the lymphokine is phenotypically Lyt1+2- and that activation of macrophages by the lymphokine can occur across H-2 histocompatibility barriers.

Role of lymphocytes in macrophage-induced killing of Coccidioides immitis in vitro.

Peritoneal macrophages from normal mice phagocytized arthroconidia and endospores of Coccidioides immitis without affecting the viability of the spores within 4 h after infection. In contrast, macrophages, when infected in the presence of lymphocytes from immune mice, significantly reduced the viability of phagocytized endospores and arthroconidia. The inability of macrophages from normal mice to kill C. immitis may in part be explained by the observation that C. immitis appeared to inhibit fusion of the phagosomes containing fungal spores with the lysosomes within the macrophages. However, fusion of phagosomes containing spores and lysosomes was observed in macrophages infected in the presence of lymphocytes from immune mice.

Interaction of nonhuman primate peripheral blood leukocytes and Coccidioides immitis in vitro.

The leukocytes from rhesus macaques could not kill either endospores or arthrospores of Coccidioides immitis even in the presence of immune serum and complement, although these leukocytes were able to kill Candida and Listeria organisms.

In vitro response of alveolar macrophages to infection with Coccidioides immitis.

Alveolar macrophages obtained from rhesus macaques (Macaca mulatta) by bronchial lavage were observed to phagocytize endospores and arthrospores of Coccidioides immitis. When the macrophages were subsequently maintained in vitro, the phagocytized spores developed into spherules. There was no significant reduction in the viability of C. immitis after phagocytosis by macrophages from normal macaques, nor was killing induced by the addition of immune serum, complement, or lung lining material obtained from the bronchial lavage fluid. The inability of the macrophages to kill C. immitis may in part be explained by the observation that C. immitis appeared to inhibit fusion of the phagosomes containing the fungal spores with the lysosomes within the macrophages.

The incidence of coccidioidomycosis among nonhuman primates housed outdoors at the California Primate Research Center.

Serum from 191 nonhuman primates housed outdoors from October 1977 to October 1978 at the California Primate Research Center was assayed for the presence of antibody specific for coccidioidin. One Macaca mulatta had coccidioidal antibody which was found to be associated with disseminated coccidioidomycosis. One Macaca radiata had complement-fixing antibody specific for coccidioidin and a pulmonary lesion. Additionally, two other animals had coccidioidal antibody but lacked clinical evidence of disease. The source of infection is suspected to be a severe dust storm originating in southern California.