Effects of nutritional and ambient oxygen condition on biofilm formation in Mycobacterium avium subsp. hominissuis via altered glycolipid expression.

Mycobacterium avium subsp. hominissuis (MAH) is the major causative agent of nontuberculous mycobacteriosis, the representative case of environment-related infectious diseases the incidence of which is increasing in industrialized countries. MAH is found in biofilm in drinking water distribution system and residential environments. We investigated the effect of gaseous and nutritional conditions, and the role of glycopeptidolipids (GPLs) on biofilm-like pellicle formation in MAH. Pellicle formation was observed under 5% oxygen in Middlebrook 7H9 broth containing 0.2% glycerol and 10% albumin-dextrose-catalase enrichment but not under normoxia or in nutrient-poor media. An analysis of 17 environmental isolates revealed that hypoxia (5% oxygen) preferentially enhanced pellicle formation both in plastic plates and in glass tubes, compared with hypercapnia (5% carbon dioxide). Wild-type strains (WT) developed much thicker pellicles than GPL-deficient rough mutants (RM). WT bacterial cells distributed randomly and individually in contrast to that RM cells positioned linearly in a definite order. Exogenous supplementation of GPLs thickened the pellicles of RM, resulting in a similar morphological pattern to WT. These data suggest a significant implication of eutrophication and hypoxia in biofilm-like pellicle formation, and a functional role of GPLs on development of pellicles in MAH.

Reversible lipid accumulation and associated division arrest of Mycobacterium avium in lipoprotein-induced foamy macrophages may resemble key events during latency and reactivation of tuberculosis.

During the dormant phase of tuberculosis, Mycobacterium tuberculosis persists in lung granulomas by residing in foamy macrophages (FM) that contain abundant lipid bodies (LB) in their cytoplasm, allowing bacilli to accumulate lipids as intracytoplasmic lipid inclusions (ILI). An experimental model of FM is presented where bone marrow-derived mouse macrophages are infected with M. avium and exposed to very-low-density lipoprotein (VLDL) as a lipid source. Quantitative analysis of detailed electron microscope observations showed the following results. (i) Macrophages became foamy, and mycobacteria formed ILI, for which host triacylglycerides, rather than cholesterol, was essential. (ii) Lipid transfer occurred via mycobacterium-induced fusion between LB and phagosomes. (iii) Mycobacteria showed a thinned cell wall and became elongated but did not divide. (iv) Upon removal of VLDL, LB and ILI declined within hours, and simultaneous resumption of mycobacterial division restored the number of mycobacteria to the same level as that found in untreated control macrophages. This showed that the presence of ILI resulted in a reversible block of division without causing a change in the mycobacterial replication rate. Fluctuation between ILI either partially or fully extending throughout the mycobacterial cytoplasm was suggestive of bacterial cell cycle events. We propose that VLDL-driven FM constitute a well-defined cellular system in which to study changed metabolic states of intracellular mycobacteria that may relate to persistence and reactivation of tuberculosis.

Ocorrência de paratuberculose em búfalos (Bubalus bubalis) em Pernambuco / Occurrence of paratuberculosis in buffaloes (Bubalus bubalis) in Pernambuco

A paratuberculose (doença de Johne) é uma das doenças de maior importância econômica para ruminantes em vários países e pode representar uma ameaça ao desenvolvimento da pecuária brasileira. É uma doença infecto-contagiosa que provoca enterocolite granulomatosa crônica, incurável e de difícil controle, cujo agente é o Mycobacterium avium subsp. paratuberculosis (MAP). Descreve-se a ocorrência de paratuberculose em um rebanho de búfalos no Estado de Pernambuco, Brasil. Não foi encontrado registro, na literatura, da ocorrência de paratuberculose em búfalos no país. De 100 búfalos, cinco mostravam sinais clínicos característicos da doença. À necropsia de dois animais as lesões estavam restritas ao intestino delgado com evidente espessamento da mucosa, aumento de linfonodos mesentéricos e vasos linfáticos proeminentes e dilatados. À microscopia, observaram-se na mucosa do intestino, infiltrado inflamatório granulomatoso com numerosos macrófagos epitelióides e células gigantes de Langhans, além de bacilos álcool-ácido resistentes (BAAR) visualizados através da coloração de Ziehl-Neelsen (ZN). Nos linfonodos mesentéricos, havia espessamento da cápsula e marcada inflamação granulomatosa. O exame direto pela técnica de ZN para pesquisa do bacilo em esfregaços de fezes, raspado de mucosa intestinal e imprint de linfonodos mesentéricos resultou positivo. A PCR IS900 específico de linfonodo mesentérico e mucosa intestinal revelou amplificação de um fragmento de aproximadamente 110pb, confirmada pela comparação com outras sequências de M. avium subsp. paratuberculosis disponíveis no GenBank.

Paratuberculosis (PTB) is a disease of great economical importance for ruminant in several countries and represents a threat to the development of Brazilian livestock. The contagious disease caused by chronic PTB leads to incurable granulomatous enterocolitis of difficult control. PTB is caused by the Mycobacterium avium subsp. paratuberculosis (MAP). No record on the occurrence of paratuberculosis in buffaloes in Brazil could be found. Five of 100 buffaloes in a herd in Pernambuco-Brazil showed clinical signs characteristic of PTB. At necropsy, of two animals the lesions were restricted to the small intestine with thickening and corrugation of the mucosa, increase of mesenteric lymph nodes and prominent lymph vessels. Histopathology revealed granulomatous inflammation infiltrated with numerous epithelioid macrophages, Langhans type giant cells, and clusters of Ziehl-Neelsen (ZN) positive organisms within the intestinal mucosa. In the mesenteric lymph nodes there was thickening of the capsule and marked granulomatous inflammation. Smears of feces and scrapping smears were prepared from intestinal mucosa and cut surface of mesenteric lymph nodes and, stained by the Ziehl-Neelsen method for research of acid fast bacilli, with positive results. Lymph nodes and intestinal mucosa revealed at IS900 specific polymerase chain reaction amplification of a fragment of about 110pb, confirmed by the comparison with other sequences of M. avium subsp. paratuberculosis available in GenBank.

Cholesterol depletion in Mycobacterium avium-infected macrophages overcomes the block in phagosome maturation and leads to the reversible sequestration of viable mycobacteria in phagolysosome-derived autophagic vacuoles.

Phagocytic entry of mycobacteria into macrophages requires the presence of cholesterol in the plasma membrane. This suggests that pathogenic mycobacteria may require cholesterol for their subsequent intra-cellular survival in non-maturing phagosomes. Here we report on the effect of cholesterol depletion on pre-existing phagosomes in mouse bone marrow-derived macrophages infected with Mycobacterium avium. Cholesterol depletion with methyl-beta-cyclodextrin resulted in a loosening of the close apposition between the phagosome membrane and the mycobacterial surface, followed by fusion with lysosomes. The resulting phagolysosomes then autonomously executed autophagy, which did not involve the endoplasmic reticulum. After 5 h of depletion, intact mycobacteria had accumulated in large auto-phagolysosomes. Autophagy was specific for phagolysosomes that contained mycobacteria, as it did not involve latex bead-containing phagosomes in infected cells. Upon replenishment of cholesterol, mycobacteria became increasingly aligned to the lysosomal membrane, from where they were individually sequestered in phagosomes with an all-around closely apposed phagosome membrane and which no longer fused with lysosomes. These observations indicate that, cholesterol depletion (i) resulted in phagosome maturation and fusion with lysosomes and (ii) caused mycobacterium-containing phagolysosomes to autonomously undergo autophagy. Furthermore, (iii) mycobacteria were not killed in auto-phagolysosomes, and (iv) cholesterol replenishment enabled mycobacterium to rescue itself from autophagic phagolysosomes to again reside individually in phagosomes which no longer fused with lysosomes.

Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system.

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with (59)Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-gamma or TNF-alpha before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.

Mycobacterium tuberculosis and Mycobacterium avium modify the composition of the phagosomal membrane in infected macrophages by selective depletion of cell surface-derived glycoconjugates.

The growth of pathogenic mycobacteria in phagosomes of the host cell correlates with their ability to prevent phagosome maturation. The underlying molecular mechanism remains elusive. In a previous study, we have shown that Mycobacterium avium depletes the phagosome membrane of cell surface-derived glycoconjugates (de Chastellier and Thilo, Eur. J. Cell Biol. 81, 17-25, 2002). We now extended these quantitative observations to the major human pathogen, Mycobacterium tuberculosis (H37Rv). At increasing times after infection of mouse bone marrow-derived macrophages, cell-surface glycoconjugates were labelled enzymatically with [3H]galactose. Subsequent endocytic membrane traffic resulted in a redistribution of this label from the cell surface to endocytic membranes, including phagosomes. The steady-state distribution was measured by quantitative autoradiography at the electron microscope level. Relative to early endosomes, with which phagosomes continued to fuse and rapidly exchange membrane constituents, the phagosome membrane was depleted about 3-fold, starting during infection and in the course of 9 days thereafter. These results were in quantitative agreement with our previous observations for Mycobacterium avium. For the latter case, we now showed by cell fractionation that the depletion was selective, mainly involving glycoproteins in the 110-210 kDa range. Together, these results indicated that pathogenic mycobacteria induced and maintained a bulk change in phagosome membrane composition that could be of special relevance for survival of pathogenic mycobacteria within phagosomes.

Mycobacteria-containing phagosomes associate less annexins I, VI, VII and XI, but not II, concomitantly with a diminished phagolysosomal fusion.

We have studied the intracellular localization of annexins I,II, VI, VII, and XI in cells containing latex beads or Mycobacterium avium at different times after ingestion in order to establish whether a correlation existed between the association of annexins to phagosomes and phagolysosomal fusion, since the intracellular survival of mycobacteria is linked to an impairment of phagosome maturation. We demonstrate an important decrease in the levels of association of annexins I, VI, VII and XI, but not II to phagosomes containing either live or killed mycobacteria compared with phagosomes containing inert latex particles. The reduced association of annexins observed was detected only on M. avium-containing phagosomes and not in other cell membrane nor in cytosolic fractions from infected cells, and was apparent from 8 hours through to 4 days after phagocytosis. These findings add elements to the present knowledge of the phagosomal modifications that accompany the survival of intracellular pathogens, suggesting that annexins I, VI, VII, and XI play a secondary role in phagosomal fusion events while annexin II does not seem to be related to the mechanism of regulation of endolysosomal fusion.

Effect of Nramp1 on bacterial replication and on maturation of Mycobacterium avium-containing phagosomes in bone marrow-derived mouse macrophages.

Pathogenic mycobacteria prevent maturation of the phagosomes in which they reside inside macrophages and this is thought to be a major strategy allowing them to survive and multiply within macrophages. The molecular basis for this inhibition is only now beginning to emerge with the molecular characterization of the phagosome membrane enclosing these pathogens. We have used here several electron microscopy approaches in combination with counts of bacterial viability to analyse how expression of Nramp1 at the phagosomal membrane may influence survival of Mycobacterium avium and affect its ability to modulate the fusogenic properties of the phagosome in which it resides. The experiments were carried out in bone marrow-derived macrophages from wild-type 129sv (Nramp1(G169)) mice and from isogenic 129sv carrying a null mutation at Nramp1 (Nramp(1-/-)) following infection with a virulent strain of M. avium. We show here that Nramp1 expression has a bacteriostatic effect and that abrogation of Nramp1 restores the bacteria's capacity to replicate within macrophages. The combined analyses of the acquisition of endocytic contents markers delivered to early endosomes and/or lysosomes either prior to or after phagocytic uptake showed that in Nramp1-positive macrophages, M. avium was unable to prevent phagosome maturation and fusion with lysosomes but that in Nramp1-negative macrophages this capacity was restored. Several hypotheses are proposed to explain how Nramp1 could affect survival of M. avium. We also propose how the present observations could relate to the model according to which mycobacteria can prevent phagosome maturation by establishing a tight interaction with constituents of the phagosome membrane. Furthermore, these results show the importance of the choice of macrophages used as a model to study intracellular survival strategies of pathogens.

Pathogenic Mycobacterium avium remodels the phagosome membrane in macrophages within days after infection.

As part of their strategy for intracellular survival, mycobacteria prevent maturation of the phagosomes in which they reside inside macrophages. The molecular basis for this inhibition is only now beginning to emerge, by way of the molecular characterisation of the phagosome membrane when it encloses virulent mycobacteria. Our own work has shown that at 15 days after the phagocytic uptake of Mycobacterium avium by mouse bone marrow-derived macrophages, the phagosome membrane is depleted about 4-fold for cell surface-derived membrane glycoconjugates, labelled by exogalactosylation, in comparison to the membrane of early endosomes with which it continues to interact. Here we asked whether this depletion occurred at early or late stages after infection. We found that only about half of the depletion had occurred at about 5 hours after the beginning of phagocytic uptake, with the remainder becoming established thereafter, with a half-time of about 2.5 days. Phagosomes became depleted in relation to early endosomes with which they continued to exchange membrane constituents. Early endosomes themselves became gradually depleted by about 30% during the 15-day post-infection period. In contrast, late endosomes/lysosomes remained unchanged, with a concentration of surface-derived glycoconjugates between that of early endosomes and of phagosomes at day 15 post infection. In view of the slowness of the post-infection change of phagosome membrane composition, we proposed that this change did not play a role in preventing maturation immediately after phagosome formation, but rather correlated with the process of maintaining the phagosomes in an immature state.

Killing of Mycobacterium avium subspecies paratuberculosis within macrophages.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular pathogen that resides within host macrophages during infection of ruminant animals. We examined survival of M. paratuberculosis infections within cultured macrophages to better understand the interplay between bacterium and host. RESULTS: Serial plating of M. paratuberculosis infected macrophage lysates on Herold's egg yolk medium showed that mycobacterial replication takes place between 0 and 24 hours post-infection. This initial growth phase was followed by a steady decline in viability over the next six days. Antibodies against M. paratuberculosis were affinity purified and used in conjunction with transmission electron microscopy to track the development of intracellular bacilli. Immunogold labeling of infected macrophages with antibody against M. paratuberculosis showed degraded intracellular mycobacteria that were unrecognizable by morphology alone. Conversely, when macrophages were heavily infected with M. paratuberculosis, no degraded forms were observed and macrophages were killed. CONCLUSIONS: We present a general description of M. paratuberculosis survival within cultured macrophages using transmission electron microscopy and viability counts. The results of this study provides further insight surrounding M. paratuberculosis-macrophage infections and have implications in the pathogenesis of M. paratuberculosis, a pathogen known to persist inside cattle for many years.

Macrophage control of mycobacterial growth induced by picolinic acid is dependent on host cell apoptosis.

The effects of picolinic acid (PA) on the intramacrophagic growth of Mycobacterium avium were studied. PA reduced M. avium growth inside mouse macrophages and led to a complete control of mycobacterial growth when added together with IFN-gamma. The mechanism involved did not require TNF-alpha, NO, or the respiratory burst, and was not dependent on either iron or zinc withholding. The mycobacteriostatic activity of the macrophages was associated with the induction of morphological changes that culminated in apoptosis at day 4 of treatment. PA alone induced apoptosis in macrophages, and this effect was increased by IFN-gamma treatment. Apoptosis at day 4 of infection was reduced by inhibiting macrophage activation with the prostaglandin 15 deoxy-prostaglandin J2 or by treating the cells with the antioxidant N-acetylcysteine. Mycobacterial growth was partially restored in macrophages treated with PA and IFN-gamma when 15 deoxy-prostaglandin J2 was added, concomitant with a delay in apoptosis. N-Acetylcysteine or glutathione could also completely revert the mycobacteriostatic effects of PA or PA plus IFN-gamma.

Direct delivery of procathepsin D to phagosomes: implications for phagosome biogenesis and parasitism by Mycobacterium.

Phagosome maturation is characterized by the sequential acquisition and loss of proteins by the phagocytic vacuole during the formation of an acidic and hydrolytic compartment where degradation of the phagocytosed particle occurs. Transfer of proteins to the maturing phagosome occurs by fusion with a range of vesicles. Here we describe direct fusion of early phagosomes with vesicles that appear to be derived from the biosynthetic pathway. In mouse bone marrow macrophages, the 51 kDa proform of cathepsin D was found in vesicles of the ER/Golgi network that could be discriminated from endosomal vesicles which in turn contained the 46 and 30 kDa processed forms of the enzyme. Procathepsin D was acquired by phagosomes formed around inert particles such as IgG-coated beads and could be "protected" by blocking acidification with Bafilomycin A1. Mycobacterium avium-containing vacuoles from established infections possessed both pro- and processed cathepsin D similar to early bead-containing phagosomes. In contrast phagosomes harboring dead mycobacteria demonstrated markedly enhanced acquisition of the 46kDa form within 4 h post internalization and only low levels of procathepsin D.

Construction of chimeric phagosomes that shelter Mycobacterium avium and Coxiella burnetii (phase II) in doubly infected mouse macrophages: an ultrastructural study.

Dual infection of cells may divert pathogens to intracellular compartments different from those occupied in mono-infected cells. In the present studies, mouse bone marrow in vitro-derived macrophages were first infected with virulent Mycobacterium avium, which are normally singly lodged within tight phagosomes. These phagosomes do not mature; they undergo homotypic fusion with early endosomes and do not fuse with lysosomes. Seven days later, the cultures were superinfected with phase II (non-virulent) Coxiella burnetii, organisms sheltered in lysosome- (or prelysosome)-like, multi-occupancy phagosomes. The latter can attain large size and engage in efficient homo- and heterotypic fusion with other phagosomes. Cultures were fixed for transmission electron microscopy 6, 12, 24, and 48 h later. Other M. avium-infected cultures were superinfected with amastigotes of the trypanosomatid flagellate Leishmania amazonensis, which are also sheltered in lysosome- (or prelysosome)-like multi-occupancy vacuoles, and fixed at the same time periods. Chimeric phagosomes containing both M. avium and C. burnetii, were found already at 6 h and the proportion of M. avium that colocalized with C. burnetii in the same phagosomes reached over 90% after 48 h. In such phagosomes, both organisms were ultrastructurally well preserved. In contrast, colocalization of M. avium and L. amazonensis was rarely found. Speculative scenarios that could underlie the formation of chimeric phagosomes could involve delayed maturation of C. burnetii-containing phagosomes in presence of M. avium, which would allow for fusion of C. burnetii- and M. avium-containing phagosomes; the production, by C. burnetii, of molecules that upregulate the fusion of M. avium-containing phagosomes with those that contain C. burnetii; and the secretion of factors that could favour the survival of M. avium within chimeric vacuoles.

Mechanisms involved in the intrinsic isoniazid resistance of Mycobacterium avium.

Isoniazid (INH), which acts by inhibiting mycolic acid biosynthesis, is very potent against the tuberculous mycobacteria. It is about 100-fold less effective against Mycobacterium avium. This difference has often been attributed to a decreased permeability of the cell wall. We measured the rate of conversion of radiolabelled INH to 4-pyridylmethanol by whole cells and cell-free extracts and estimated the permeability barrier imposed by the cell wall to INH influx in Mycobacterium tuberculosis and M. avium. There was no significant difference in the relative permeability to INH between these two species. However, the total conversion rate in M. tuberculosis was found to be four times greater. Examination of in vitro-generated mutants revealed that the major resistance mechanism for both species is loss of the catalase-peroxidase KatG. Analysis of lipid and protein biosynthetic profiles demonstrated that the molecular target of activated INH was identical for both species. M. avium, however, formed colonies at INH concentrations inhibitory for mycolic acid biosynthesis. These mycolate-deficient M. avium exhibited altered colony morphologies, modified cell wall ultrastructure and were 10-fold more sensitive to treatment with hydrophobic antibiotics, such as rifampin. These findings may significantly impact the design of new therapeutic regimens for the treatment of infections with atypical mycobacteria.

Subcellular localization of the 65-kDa heat shock protein in mycobacteria by immunoblotting and immunogold ultracytochemistry.

The 65-kDa heat shock protein (hsp65) is an immunodominant antigen in mycobacterial infections and also the key etiologic factor in mycobacteria-induced autoimmune arthritis. Because the subcellular distribution of hsp65 in the mycobacteria may be relevant to understand its immunoreactivity, we have investigated the presence of hsp65 in the envelope and cytoplasmic compartments of the bacilli. Anti-hsp65 antibodies were used in western blottings to investigate the presence of hsp65 in cell fractions (membrane, envelope and cytosol) of Mycobacterium avium and M. smegmatis, and also to label hsp65 in situ by the immunogold method on thin-sectioned mycobacteria, including the non-cultivable M. leprae, that were studied by transmission electron microscopy. All of the three subcellular mycobacterial fractions showed significant labelling by anti-hsp65 antibodies. Immunogold ultracytochemistry revealed the presence of hsp65 in both the cytoplasm and the envelope of mycobacteria. The data indicate that hsp65 molecules are commonly present not only in the cytoplasm but also in the envelope of mycobacteria. The latter topography of hsp65 may contribute to the strong immunogenicity of hsp65 since it may correspond to export hsp65 molecules captured before being secreted into the extracellular milieu, thus making hsp65 a mycobacterial antigen readily available for presentation to the immune system of infected hosts.

Stazyme, a mycobacteriolytic preparation from a Staphylococcus strain, is able to break the permeability barrier in multiple drug resistant Mycobacterium avium.

As a strategy to augment the potential of existing drugs against Mycobacterium avium we investigated a mycobacteriolytic preparation (stazyme) from the Staphylococcus strain Clavelis, which results in significant mycobacterial growth inhibition. A total of 10 specific protein bands were characterized in the stazyme preparation: three bands within a major 40-60 kDa fraction, five bands within the range of 30-90 kDa, and two bands of about 12 and 14 kDa respectively. Tested at concentrations of 50 and 200 microg ml(-1) of total protein, stazyme was highly bactericidal against M. smegmatis, and bacteriostatic against M. tuberculosis and M. avium. Stazyme was able to break the permeability barrier of M. avium isolates, significantly enhancing the activity of other antituberculous drugs (ethambutol, rifampicin, and amikacin), used at sub-MIC level. Stazyme essentially possessed a lytic activity as evidenced by its ability to lyse purified M. smegmatis cell walls. This lytic activity was also confirmed on intact M. smegmatis and M. avium bacilli by transmission electron microscopy. Precise identification of this mycobacteriolytic determinant(s) in stazyme may be helpful to define novel drug targets in mycobacteria.

Formulation and efficacy of liposome-encapsulated antibiotics for therapy of intracellular Mycobacterium avium infection.

Mycobacterium avium is an intracellular pathogen that can invade and multiply within macrophages of the reticuloendothelial system. Current therapy is not highly effective. Particulate drug carriers that are targeted to the reticuloendothelial system may provide a means to deliver antibiotics more efficiently to M. avium-infected cells. We investigated the formulation of the antibiotics ciprofloxacin and azithromycin in liposomes and tested their antibacterial activities in vitro against M. avium residing within J774, a murine macrophage-like cell line. A conventional passive-entrapment method yielded an encapsulation efficiency of 9% for ciprofloxacin and because of aggregation mediated by the cationic drug, was useful only with liposomes containing < or = 50 mol% negatively charged phospholipid. In contrast, ciprofloxacin was encapsulated with > 90% efficiency, regardless of the content of negatively charged lipids, by a remote-loading technique that utilized both pH and potential gradients to drive drug into preformed liposomes. Both the cellular accumulation and the antimycobacterial activity of ciprofloxacin increased in proportion to the liposome negative charge; the maximal enhancement of potency was 43-fold in liposomes of distearoylphosphatidylglycerol-cholesterol (DSPG-Chol) (10:5). Azithromycin liposomes were prepared as a freeze-dried preparation to avoid chemical instability during storage, and drug could be incorporated at 33 mol% (with respect to phospholipid). Azithromycin also showed enhanced antimycobacterial effect in liposomes, and the potency increased in parallel to the moles percent of negatively charged lipids; azithromycin in DSPG-Chol (10:5) liposomes inhibited intracellular M. avium growth 41-fold more effectively than did free azithromycin. Thus, ciprofloxacin or azithromycin encapsulated in stable liposomes having substantial negative surface charge is superior to nonencapsulated drug in inhibition of M.avium growth within cultured macrophages and may provide more effective therapy of M.avium infections.

Mycobacterium avium rough-to-smooth colony conversion resulting from growth in Tween 80 without presence of type-specific glycopeptidolipid antigens.

Growth of a Mycobacterium avium complex serotype 20 rough-colony variant in 1.0% Tween 80 resulted in a smooth-colony morphological conversion that was reversible upon removal of Tween and was not associated with the presence of serotype-specific glycopeptidolipid antigens. Electron microscopic examination suggested the role of an outer layer in the Tween-related morphological modification.

Mycobacterial growth and ultrastructure in mouse L-929 fibroblasts and bone marrow-derived macrophages: evidence that infected fibroblasts secrete mediators capable of modulating bacterial growth in macrophages.

The intracellular growth kinetics of Mycobacterium avium and H37Rv (virulent) and H37Ra (avirulent) strains of Mycobacterium tuberculosis were compared by use of both the professional (mouse bone marrow-derived macrophages, BMM phi) and nonprofessional (mouse L-929 fibroblast cell line) phagocytes. The results obtained showed that all the mycobacterial strains grew more actively in fibroblasts than in BMM phi. This difference was paralleled by lesser acid phosphatase (AcP) labeling of noninfected fibroblasts and the observation that upon infection both the proportion of AcP-positive cells and AcP content were higher in BMM phi than in L-cells during the 7 days of infection. In parallel experiments, intracellular growth of M. tuberculosis H37Rv and M. avium was compared inside BMM phi from both the Bcgs (C57BL/6) and Bcgr (DBA-2) mice, which were matured and differentiated with either an L-cell-conditioned medium (LCM) obtained from control, noninfected L-929 cells, or a LCM obtained with M. tuberculosis- or M. avium-infected L-cells. Upon mycobacterial infection, fibroblasts were able to secrete mediators that stimulated the BMM phi to better control the infection by pathogenic mycobacteria. These results are discussed in terms of the mycobacteria-fibroblast interactions and their eventual role in the immune modulation of the host's response to invading mycobacteria.

Intramacrophagic Mycobacterium avium bacilli are coated by a multiple lamellar structure: freeze fracture analysis of infected mouse liver.

We used freeze fracture electron microscopy to study the fine structure of Mycobacterium avium inside phagosomes of murine macrophages. M. avium-susceptible C57BL/6 mice were infected with M. avium by intraperitoneal inoculation of 10(8) viable bacilli. We studied the microanatomy of the mycobacteria in 3-month infections of mice, a situation in which bacillary multiplication is extensive. In these samples, freeze fracture revealed that intraphagosomal bacilli were surrounded by a multilamellar coat that was apposed to the cell wall. In thin sections, in contrast, the area corresponding to the coat showed no substructure and was electron transparent (the so-called electron-transparent zone that has been previously reported by others). The multiple lamellae resembled an onionlike assembly that was inserted in between the mycobacterial wall outer surface and the phagosomal membrane. Each lamella of the M. avium coat was made up of parallel straight fibrils with a width of 5 nm. A variable number of lamellae, sometimes up to 10 or more elements, coated individual bacilli. The multilamellar coat was absent around both extracellular M. avium and intramacrophagic M. avium after short-term (45-min) inoculation of mice. The supramolecular organization of the M. avium lamellar coat as viewed here by freeze fracture is similar to that of purified mycoside C (P. Draper, J. Gen. Microbiol. 83:431-433, 1974; K.-S. Kim, M.R.J. Salton, and L. Barksdale, J. Bacteriol. 125:739-743, 1976), a mycobacterial component currently known as glycopeptidolipid (W.W. Barrow and P.J. Brennan, J. Bacteriol. 150:381-384, 1982). We conclude that M. avium bacilli growing in macrophages are surrounded by multilamellar capsulelike structures that contain glycopeptidolipid molecules.