Effect of mycobacterial phospholipids on interaction of Mycobacterium tuberculosis with macrophages.

This study demonstrates that pretreatment of macrophages with phosphatidylinositol, of either soya bean or mycobacterial origin, results in a down-regulation of the binding and uptake of Mycobacterium tuberculosis by the phagocytes. We also describe the novel observation that cardiolipin induces an increase in the binding and uptake of M. tuberculosis by macrophages. Neither phospholipid interacts with macrophages via the 2F8 epitope of scavenger receptor A, and treatment of macrophages with either phospholipid results in a down-regulation of CR3 function and tumor necrosis factor alpha production by the phagocyte. We have also shown that the ability of macrophages to interact with mycobacteria is greatly affected by an as yet unidentified product from the interaction of chloroform and polypropylene tubes.

Two distinct receptors mediate nonopsonic phagocytosis of different strains of Pseudomonas aeruginosa.

Complement receptor 3 (CR3) mediates both opsonic and nonopsonic phagocytosis of bacteria. Leukocyte adhesion deficiency (LAD) allows for the study of CR3-dependent phagocyte-bacterial ingestion, since LAD phagocytes do not express this receptor. Phagocytes from an infant with LAD were unable to ingest 50% of the Pseudomonas aeruginosa strains studied, which indicates a requirement for CR3. However, the remaining strains were phagocytosed in the absence of CR3, and ingestion was blocked by monoclonal antibodies directed at CD14. This CR3/CD14 receptor bias was further confirmed by using thioglycollate-elicited murine peritoneal macrophages, which have nonfunctional CR3 before activation. Results indicate that either CR3 or CD14 is involved independently in nonopsonic phagocytosis of different P. aeruginosa strains. Clearance of P. aeruginosa from the endobronchial space may be facilitated by nonopsonic phagocytosis, since low levels of opsonins are present. The impact of lung infection with P. aeruginosa may be determined, in part, by the phagocytic receptor that mediates ingestion.

Utilization of CD11b knockout mice to characterize the role of complement receptor 3 (CR3, CD11b/CD18) in the growth of Mycobacterium tuberculosis in macrophages.

Using CD11b knockout mice as a source of macrophages (Mphi;), we show that complement receptor 3 (CR3) mediates approximately 40-50% of nonopsonic binding and 50-60% of serum-mediated binding of Mycobacterium tuberculosis to resident Mphi;. We demonstrate that opsonic binding of M. tuberculosis to Mphi; is mediated by an immunoglobulin-independent, heat-labile component of serum, in both the presence and the absence of CD11b. The survival and replication of M. tuberculosis in an in vitro Mphi; model and an in vivo mouse model of infection were not significantly affected by the absence of CD11b, indicating that CR3-mediated uptake of M. tuberculosis is not a major factor in controlling the subsequent intracellular survival of the mycobacteria. However, whether a mycobacterium will gain access to the intracellular environment, and the type of Mφ that the bacterium enters, is significantly affected by the presence or absence of CR3.

Interaction of Mycobacterium tuberculosis with MH-S, an immortalized murine alveolar macrophage cell line: a comparison with primary murine macrophages.

UNLABELLED: We are interested in identifying a suitable model for investigating mycobacteria interactions with alveolar macrophages. MH-S, a murine alveolar macrophage cell line, is a possible candidate. OBJECTIVE: To compare the receptor mediated interactions of mycobacteria with primary murine macrophages and MH-S. DESIGN: The association of MH-S monolayers with Mycobacterium tuberculosis (MTB) and other defined particles was compared to that of resident Day 1 peritoneal macrophage (PM) and Day 4 alveolar macrophage (AM) monolayers. RESULTS: In the absence of serum, the association of MTB with MH-S was comparable to that of AM, with approximately 35% of each macrophage type binding at least one bacterium. In contrast, almost 80% of PM bound at least one bacterium. MTB binding was enhanced for all macrophage types by a heat-labile component of normal mouse serum. Antibodies recognising CR3 inhibited the serum-mediated enhanced binding of MTB by MH-S. Binding of latex, immunoglobulin coated or complement coated SRBC by MH-S, AM and PM was comparable. Binding of zymosan by MH-S was greatly inferior to AM and PM. CONCLUSION: The receptor expression and particle binding properties of MH-S are similar to AM in many, but not all, ways. MH-S, therefore, has the potential to be used as a model for investigating MTB-macrophage interactions.

The receptor-mediated uptake, survival, replication, and drug sensitivity of Mycobacterium tuberculosis within the macrophage-like cell line THP-1: a comparison with human monocyte-derived macrophages.

We have compared the interaction of Mycobacterium tuberculosis with the human macrophage-like cell line THP-1 and with human monocyte-derived macrophages (MDM). The association of M. tuberculosis with THP-1 and MDM was comparable in both the presence and the absence of serum. For both cells, serum-mediated binding was much greater than nonopsonic binding and was mediated by a heat-labile serum component. Nonopsonic binding of M. tuberculosis to both cells could be inhibited by antibodies recognizing CD11b and by mannan and glucan. Intracellular M. tuberculosis grew progressively in infected MDM and THP-1 cells. Treatment of the infected MDM and THP-1 cells with the anti-mycobacterial isoniazid resulted in the rapid killing of the intracellular mycobacteria. Differentiated, adherent THP-1 cells bound IgG and complement-coated particles at levels similar to those of MDM. However, binding of zymosan by THP-1 cells was significantly lower than that seen for MDM.

Site-directed mutagenesis and virulence assessment of the katG gene of Mycobacterium intracellulare.

Mycobacterial catalases have been suggested as acting as virulence factors by protecting intracellular mycobacteria from reactive oxidative metabolites produced by host phagocytes. Mycobacterium intracellulare, like many other mycobacteria, produces two proteins with catalase activity: a heat-stable catalase (KatE) and an inducible, heat-labile catalase peroxidase (KatG). The M. intracellulare katG gene was cloned, and a plasmid derivative with a 4 bp insertion in the katG coding sequence was constructed and used for site-directed mutagenesis of M. intracellulare 1403 (ATCC 35761). The resulting katG mutant was highly resistant to isoniazid (INH), showed an increased sensitivity to H2O2 and had lost peroxidase and heat-sensitive catalase activity but retained heat-stable catalase activity. The plasmid carrying the katG frameshift allele was also used for mutagenesis of the mouse virulent M. intracellulare isolate D673. After intravenous injection into BALB/c mice, D673 and the isogenic katG mutant showed the same growth kinetics in the spleen, liver and lungs of the infected mice. Our results demonstrate that the KatG catalase peroxidase mediates resistance to H2O2 and susceptibility to INH but is not an essential virulence factor for the survival and growth of M. intracellulare in the mouse.

Site-directed mutagenesis of the 19-kilodalton lipoprotein antigen reveals No essential role for the protein in the growth and virulence of Mycobacterium intracellulare.

The mycobacterial 19-kilodalton antigen (19Ag) is a highly expressed, surface-associated glycolipoprotein which is immunodominant in infected patients and has little homology with other known proteins. To investigate the pathogenic significance of the 19Ag, site-directed mutagenesis of the Mycobacterium intracellulare 19Ag gene was carried out by using a suicide vector-based strategy. Allelic replacement of the 19Ag gene of a mouse-avirulent M. intracellulare strain, 1403, was achieved by double-crossover homologous recombination with a gentamicin resistance gene-mutated allele. Unfortunately, an isogenic 19Ag was not achievable in the mouse-virulent strain, D673. However, a 19Ag mutant was successfully constructed in M. intracellulare FM1, a chemically mutagenized derivative of strain D673. FM1 was more amenable to genetic manipulation and susceptible to site-directed mutagenesis of the 19Ag gene yet retained the virulent phenotype of the parental strain. No deleterious effects of 19Ag gene mutation were observed during in vitro growth of M. intracellulare. Virulence assessment of the isogenic 19Ag mutants in a mouse infection model demonstrated that the antigen plays no essential role in the growth of M. intracellulare in vivo. Site-directed mutagenesis of the 19Ag gene demonstrated that it plays no essential role in growth and pathogenicity of M. intracellulare; however, the exact nature of its biological function remains unknown.

Nonopsonic and opsonic association of Mycobacterium tuberculosis with resident alveolar macrophages is inefficient.

The association of Mycobacterium tuberculosis with alveolar macrophages (Mphi) in a serum-free environment is a crucial first step in the pathogenesis of this facultative intracellular pathogen. We present data demonstrating that freshly explanted alveolar Mphi do not efficiently bind M. tuberculosis in a serum-free system, although a small subpopulation of these Mphi (10-15%) can bind mycobacteria. In contrast, almost 100% of a peritoneal Mphi population bind mycobacteria under the same conditions. The poor binding of mycobacteria by alveolar Mphi does not reflect a general inability to associate with particles; binding and ingestion of latex beads and zymosan particles were comparable with that seen with peritoneal Mphi. Resident alveolar Mphi did not efficiently bind mycobacteria in the presence of serum and expressed poorly several Mphi surface receptors, including CR3. Furthermore, we demonstrate that bovine surfactant protein A does not enhance the association of M. tuberculosis with alveolar Mphi. Differentiation of alveolar Mphi in vitro resulted in increased expression of Mphi surface receptors and an increased capacity to bind mycobacteria in the presence and absence of serum. Evidence is presented that opsonic binding of M. tuberculosis by differentiated alveolar Mphi is mediated by complement and CR3, and that the poor binding by resident alveolar Mphi is due to their poor expression of CR3. The receptor mediating nonopsonic binding of M. tuberculosis to differentiated alveolar Mphi was not unequivocally identified in this study, but could also be CR3.

Antimycobacterial polyynes of Devil's Club (Oplopanax horridus), a North American native medicinal plant.

Two new (3 and 5), as well as three known (1, 2, and 4), polyynes were isolated from Devil's Club (Oplopanax horridus; Araliaceae), a medicinal plant of North America. The structures were established by 1H and 13C NMR. The absolute configurations of 2 and 5 were determined by application of Mosher's method. All the polyynes exhibited significant anti-Candida, antibacterial, and antimycobacterial activity, with an ability to kill Mycobacterium tuberculosis and isoniazid-resistant Mycobacterium avium at 10 micrograms/disk in a disk diffusion assay.

Mycobacterium tuberculosis efpA encodes an efflux protein of the QacA transporter family.

The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism analysis demonstrated that these upstream flanking sequences and the 231-bp, 5' coding region are highly conserved among both drug-sensitive and multiply-drug-resistant isolates of M. tuberculosis. The efpA gene was present in the slow-growing human pathogens M. tuberculosis, Mycobacterium leprae, and Mycobacterium bovis and in the opportunistic human pathogens Mycobacterium avium and Mycobacterium intracellular. However, efpA was not present in 17 other opportunistically pathogenic or nonpathogenic mycobacterial species.

Gene replacement through homologous recombination in Mycobacterium intracellulare.

Mycobacterium intracellulare is a slow-growing pathogenic mycobacterium closely related to Mycobacterium avium. In contrast to Mycobacterium tuberculosis and Mycobacterium bovis BCG, M. intracellulare has received little attention as a model species for studies of mycobacterial molecular biology and genetics. This study shows that M. intracellulare 1403 (ATCC 35761) can be transformed by electroporation with high frequencies (up to 10(6) transformants per microgram of DNA), using plasmids pYT937 and pMH94 as replicative and integrative vectors, respectively. We also describe an experimental system that we used to study DNA recombination in M. intracellulare. First, an integrative plasmid was introduced into M. intracellulare 1403. A nonreplicative, nonintegrative plasmid having homology with the integrated plasmid was then introduced, and the resultant recombinants were analyzed to distinguish between events of homologous and illegitimate recombination. No illegitimate recombination occurred; in all recombinants, a single crossover between homologous regions of the two plasmids was noted. During subsequent growth of a recombinant clone, a spontaneous deletion occurred that resulted in a gene replacement on the chromosome of M. intracellulare 1403. The ability to construct site-specific mutations in M. intracellulare will provide novel insights into the biology of slow-growing mycobacteria.

Lipoarabinomannan inhibits nonopsonic binding of Mycobacterium tuberculosis to murine macrophages.

The initial phagocytic interaction between Mycobacterium tuberculosis and macrophages (M phi) in the lung is probably nonopsonic, which would mean that M phi receptors will bind directly to bacterial ligands without the involvement of serum opsonins. Lipoarabinomannan (LAM) is a major component of the cell wall of mycobacteria. The possibility that LAM is involved in the nonopsonic binding of M. tuberculosis to M phi was investigated by using competitive inhibition assays. LAM inhibited binding of M. tuberculosis to murine peritoneal M phi in a dose-dependent manner. LAM also inhibited the binding of Mycobacterium avium and Mycobacterium bovis BCG to M phi. Phosphatidylinositol mannoside and lipomannan have the same phosphatidylinositol (PI) moiety as LAM, but differ in their glycosylation patterns. Both molecules inhibited binding of M. tuberculosis to M phi. Deacylation of LAM abrogated its capacity to inhibit binding of M. tuberculosis to M phi. These observations indicated that it was the PI moiety of LAM that was important in mediating its inhibitory properties. In support of this hypothesis, commercial PI was shown to inhibit the binding of M. tuberculosis to M phi. Our results suggest that cellfree LAM is able to inhibit the binding of mycobacteria to M phi, but that it does not do so by competing with any interaction between M phi receptors and cell-associated LAM, because the PI end of the molecule is believed to be anchored in the bacterial plasma membrane, and therefore not available as a ligand on the cell surface. However, LAM that is released from M. tuberculosis in the course of its active replication during infection may be able to interfere with the phagocytic clearance of mycobacteria.

Mycobacteria-macrophage interactions. Macrophage phenotype determines the nonopsonic binding of Mycobacterium tuberculosis to murine macrophages.

During tuberculosis, host defenses may be determined, in part, by the capacity of resident, elicited, and activated macrophages to bind and ingest Mycobacterium tuberculosis. We have investigated the mechanism by which macrophages bind M. tuberculosis and other mycobacteria in a serum-free system. The extent of binding of M. tuberculosis to macrophages was dependent on the phenotype of the macrophage; thioglycollate-elicited and immune-activated macrophages bound mycobacteria poorly, whereas resident macrophages bound mycobacteria efficiently. Within 'freshly' explanted macrophage populations (from 2 to 24 h in vitro) poor binding of mycobacteria correlated with poor binding of C3bi-coated particles, but not with variations in the level of complement receptor 3 (CR3) expression. Induction of C3bi-coated particle binding in thioglycollate-elicited macrophages by PMA was not accompanied by enhanced M. tuberculosis binding. Inhibition of M. tuberculosis binding by resident macrophages could only be achieved using a mAb recognizing an epitope within CR3 distinct from that which recognizes C3bi. Our results suggest that nonopsonic binding of M. tuberculosis is mediated by a site within CR3, which is distinct from the C3bi binding site. In addition, we show a variation in the capacity of different macrophage phenotypes to bind mycobacteria nonopsonically. These data suggest that heterogeneity in macrophage-mediated clearance of M. tuberculosis may be a significant factor in the progression of tuberculosis.

Tumor cells transfected with a bacterial heat-shock gene lose tumorigenicity and induce protection against tumors.

The gene encoding a highly immunogenic mycobacterial heat-shock protein (hsp65) was transfected into the murine macrophage tumor cell line J774. The resulting hsp65-expressing cells (J774-hsp65) were no longer able to produce tumors in syngeneic mice. This loss of tumorigenicity was not mediated through T cells since the transfected cells did not produce tumors in athymic mice. If mice are first immunized with the J774-hsp65 cells and then challenged with the parent J774 cells, the mice do not develop tumors, indicating that the presence of the mycobacterial hsp65 protein greatly enhances immunological recognition of unique structures expressed by the parent tumor cells. This is further confirmed by the demonstration in vitro of T cells derived from J774-hsp65-immunized mice that are cytotoxic for the parent J774 cells. The results provide the basis for a novel strategy for enhancing the immunological recognition and decreasing the tumorigenicity of transformed cells.

Passive transfer of immunity of Mycobacterium avium in susceptible and resistant strains of mice.

Naturally susceptible mice (C57BL/6) infected with M. avium (strain Weybridge) developed a population of splenic T cells which, on transfer to syngeneic recipient mice, conferred significant protection against a subsequent challenge inoculum of M. avium. Similar T cells from naturally resistant mice (A/J) did not protect syngeneic recipient mice. Growth of M. avium in donor mice only occurred in the C57BL/6 strain. Replication of M. avium in donor mice was necessary for the development of protective T cells. High numbers of killed mycobacterium did not induce immune T cells. In addition, A/J mice inoculated with increasing numbers of viable M. avium (which still did not replicate) failed to develop protective T lymphocytes. Further studies indicated that no protective T cells were present in the M. avium-infected A/J mouse, although evidence for non-specific immunity in these mice was obtained. In addition, BCG (which grows progressively in A/J mice) stimulated a population of splenic T cells which protected recipient mice from subsequent infection with M. tuberculosis.

Growth of Mycobacterium avium in activated macrophages harvested from inbred mice with differing innate susceptibilities to mycobacterial infection.

The growth of Mycobacterium avium in macrophages obtained from Mycobacterium bovis BCG-infected mice was compared with that in macrophages from uninfected mice. BCG vaccination resulted in substantial macrophage activation, measured as increased acid phosphatase and superoxide anion production, as well as enhanced leishmanicidal activity. However, the activated macrophages were only able to reduce the rate of intracellular growth by Listeria monocytogenes and M. avium in vivo and did not express detectable levels of mycobactericidal activity in vitro. Exposure of the macrophage monolayers to concanavalin A-stimulated spleen cell supernatant fluid and lipopolysaccharide did not further enhance the ability of the BCG-activated macrophages to control the intracellular replication of the M. avium. Macrophages from BCG-infected C57BL/6 (BCGs) mice were quantitatively better able to control the intracellular replication of the M. avium challenge than were similar phagocytes obtained from BCGr (A/J) mice. These findings have important implications with respect to the expression of acquired resistance to these atypical mycobacterial infections.

Mycobacterium avium-complex infections in normal and immunodeficient mice.

Specific pathogen-free C57Bl/6 X DBA/2 (B6D2) F1 hybrid mice were challenged aerogenically with M. avium, M. intracellulare and M. scrofulaceum and the resulting lung and spleen infections were followed over a period of several months. Growth of the mouse virulent M. avium 724 and M. intracellulare D673 was more extensive in the susceptible (Balb/c or C57Bl/6) mice than it was in the resistant A/J and B6D2 strains. Oral challenge of normal C57Bl/6 mice with virulent M. avium-complex serotypes resulted in substantial infection of the gut-associated lymphoid tissues, the lungs and spleen. No infection developed when the mice were infected orally with the avirulent MAC serotypes. T-cell depleted Balb/c (nude or Thxb) mice infected with virulent M. avium developed markedly enhanced lung and spleen infections compared to those seen in the immunocompetent controls. T-cell depletion did not potentiate the systemic growth of the avirulent MAC strains. The significance of these growth patterns (especially in the T-cell depleted mouse) is discussed in relation to the development of life-threatening M. avium-complex infections in patients with Acquired Immune Deficiency Syndrome.

A method for the purification of Leishmania promastigotes from infected phlebotomine sandflies.

We describe a method for the purification of Leishmania promastigotes, isolated from infected sandflies (Lutzomyia longipalpis) using a discontinuous density centrifugation gradient (Percoll/Homem). The sandflies, infected seven days previously with Leishmania donovani chagasi or Leishmania mexicana mexicana from culture, were homogenized and centrifuged on a Percoll discontinuous gradient. Five interface bands were formed, and most of the promastigotes settled out at the interface between the (30% and 40%) Percoll concentrations. An extraction of 3.5 x 10(4) promastigotes from 90 female flies was achieved using this technique.

Role of mononuclear phagocytes in expression of resistance and susceptibility to Mycobacterium avium infections in mice.

The growth of Mycobacterium avium 702 in the spleens and livers of four inbred strains of mice varied such that the mice could be separated into naturally susceptible (BALB/c and C57BL/6) and naturally resistant (A/Tru and DBA/2) strains. This phenomenon was independent of the size of the infecting inoculum of bacteria in that both low (10(4))- and high (10(7))-dose inocula of M. avium grew progressively in susceptible strains and were eliminated from the target organs of resistant strains. Resistance and susceptibility were also demonstrated in in vitro preparations of macrophages from these strains of mice. Over a 7-day period, replication of M. avium in susceptible mouse macrophages was far greater than that in resistant macrophages. Evidence was obtained to suggest that toxic oxygen metabolites were not responsible for this difference. Though no difference was found in the rate of clearance of M. avium from the blood of susceptible or resistant mice, resident macrophages from susceptible mice ingested more M. avium in vitro than did resident macrophages from resistant animals. Growth of M. avium in spleens of susceptible mice induced a large influx of phagocytes, whereas this was not observed in resistant mice. In contrast to this it was found that, after injection of a variety of inflammatory agents, influx of leukocytes into the peritoneal cavity could not be used to distinguish susceptible and resistant strains of mice.