Acid-Fastness of pine pollen

This is a study of the acid-fast staining characteristics of pine pollen and an investigation of factors causing loss of acid-fastness after pine pollen has been in contact with tisues or mucous membranes. Intact loblolly pine pollen was readily stained with cold carbol-fuchsin, and retained its acid-fastness after decolorization with 3% HC1 in 95% ethyl alcohol for 2 min, followed by methylene blue counterstain. Pine pollen resembles spermatozoa in ease of staining and resistance to decolorization. Acid-fastness was destroyed by crushing or by germination of the pollen grain, and by contact for several hours with serum or saline solutions, but was unchanged by exposure to 0.1% solution of streptomycin in water. Nonviable pine pollen did not lose acid-fastness after suspension for several days in serum or water. When counterstain was omitted, crushed or germinated pollen appeared red to pink after staining with carbol-fuchsin and decolorization with acid alcohol, thus indicating that lipids of an acid-fast nature were still present. The large size and biologic properties of pine pollen provide a unique means of studying the chemical and physical aspects of the acid-fast phenomenon.     

Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4–6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.     

Mycobacterial Agglutination-Inhibition Test

The mycobacterial agglutination-inhibition test (MAIT) is described as a modification of the mycobacterial agglutination of Schaefer (MATS). It is an organized assemblage of absorption and agglutination tests which is especially useful in the identification of Mycobacterium avium isolates normally excluded from serological identification because of unstable cell suspension characteristics. The identity of 100 unstable M. avium and 3 unstable Mycobacterium intracellulare isolates was determined by the MAIT method and by animal pathogenicity tests. The comparability of the MAIT and MATS methods was demonstrated by testing 10 stable M. avium and 10 stable M. intracellulare isolates by both methods and obtaining the same serological identification for each culture. The reproducibility of the MAIT was confirmed when the same result was obtained in three consecutive tests on each of the 123 cultures used in this evaluation.     

Preservation of Mycobacterial Cultures

Storage of cultures of mycobacteria by freezing in skimmed milk was determined to be an easier and more reliable method of maintaining viability and stability than lyophilization.     

Specificity of Acquired Resistance Produced by Immunization with Mycobacterial Cells and Mycobacterial Fractions

Mice were immunized intraperitoneally with 5.0 mg of living and 5.0 mg of heat-killed H37Ra cells of the attenuated strain Mycobacterium tuberculosis and challenged intraperitoneally with Listeria monocytogenes and Klebsiella pneumoniae. The period of protection provided by the living and heat-killed H37Ra cells against both heterologous infections was the same. When mice were immunized intraperitoneally with graded doses of living and heat-killed H37Ra and challenged intraperitoneally with listeria or klebsiella, the lowest immunizing dose providing protection against both klebsiella and listeria challenge was the same for living and heat-killed cells. Living and heat-killed cells also immunized equally effectively when the routes of immunization and challenge were different. Mice also were immunized intraperitoneally with mycobacterial ribosomal fraction, mycobacterial cell walls, and several nonspecific agents (Escherichia coli endotoxin, mineral oil emulsion, and Freund's incomplete adjuvant). The mice were challenged intraperitoneally with listeria or klebsiella at varying times after immunization. The mycobacterial components and all the nonspecific agents provided transitory protection lasting no longer than 4 days after immunization. Only the mycobacterial cell walls and the endotoxin provided protection against listeria challenge. It was concluded that the protection provided by the mycobacterial ribosomal fraction is specific for tuberculosis infection, since this fraction provided no protection against listeria infection and only transitory protection against klebsiella. It was also concluded that the mycobacterial component providing protection against heterologous infections is heat stable and probably is found in the cell wall.     

Wild Animal Mycobacterial Isolates

Thirteen strains of mycobacteria isolated from deer and various species of wild birds were analysed by gas chromatography (GG) for cellular fatty acids and by thin-layer chromatography (TLG) for polar lipids. These strains were compared to reference strains of Mycobacterium avium, M. para tuberculosis and M. mal-moense. All the examined strains exhibited a generally similar fatty acid pattern characterized by relatively large amounts of hexadenca-noate (16:0), octadecenoate (18:1), octadecanoate (18:0) and 10-me-thyl-octadecanoate (tuberculostearic acid, 10-Me-18:0). Several additional acids were also generally present but in smaller amounts. By means of small but distinct differences in fatty acid composition, the wild animal isolates could be distinguished from both M. paratuber-culosis and M. malmoense but not from M. avium. The TLG polar lipid patterns on the other hand separated the wild animal isolates into 2 distinct groups of complex and simple polar lipid composition which corresponded to the morphologically smooth and rough types, respectively. The complex patterns of the smooth strains were comparable to those of the M. avium serovars whereas both the rough wild animal isolates and all the M. paratuber-culosis strains showed a simple pattern of polar lipids. Both fatty acid profiles and TLG polar lipid patterns support allocation of the wild animal isolates to the MAIS complex. Moreover, the 2 chemical techniques, particularly the GC procedure, are very useful for a more rapid and precise identification of the slow-growing wild animal mycobacterial isolates which have hitherto been characterized on basis of vague criteria.     

Mycobacterial RNase E-associated proteins

RNase E and its complex with other proteins ('degradosome') play an important role in RNA processing and decay in Escherichia coli and in many other bacteria. To identify the proteins which can potentially interact with this enzyme in mycobacteria, Mycobacterium tuberculosis H37Rv RNase E was cloned and expressed as a 6HisFLAG-tagged fusion protein. Analysis of the mycobacterial RNase E overexpressed and purified from M. bovis BCG revealed the presence of GroEL and two other copurified proteins, products of the Mb1721 (inorganic polyphosphate/ATP-NAD kinase) and Mb0825c (acetyltransferase) genes. Identical copies of these two genes can be found in M. tuberculosis H37Rv.     

Adjuvant Activity of Mycobacterial Fractions

A quantitative assay and characterization of oil-attached cell wall of Mycobacterium bovis BCG (BCG-CWS) which stimulates cell-mediated immunity of spleen cells to alloantigens in mice were carried out by an in vitro cell-mediated cytotoxicity test using ⁵¹Cr-labeled target cells. C57BL/6J mice (H-2^b) were immunized intraperitoneally with mastocytoma cells (H-2^d) with or without oil-attached BCG-CWS. The cytotoxicity, comparable to that of spleen cells from mice immunized with mastocytoma cells (3 × 10⁷), could be induced in spleens of mice immunized with a mixture of mastocytoma cells (10⁴) and oil-attached BCG-CWS. The enhancing effect persisted for 55 days or more after the alloantigenic immunization. Oil-attached BCG-CWS enhanced cell-mediated cytotoxicity of T cells in the spleen and the mesenteric lymph node, but not in the thymus. The cytotoxicity showed specificity toward the alloantigen used for immunization. In addition to BCG-CWS, the cell walls of Nocardia rubra and Corynebacterium diphtheriae PW8 and the peptidoglycolipids of Mycobacterium tuberculosis Aoyama B were found to be potent stimulants of cell-mediated cytotoxicity in mice. Oil-attached BCG-CWS did not enhance humoral response to mastocytoma cells but enhanced cell-mediated cytotoxicity when viable mastocytoma cells were used as antigen. The above result was supported by the fact that anti-hapten antibody response induced by viable trinitrophenyl (TNP)-mastocytoma cells (10⁴) plus oil-attached BCG-CWS did not increase to the maximum level as was observed in mice immunized with a larger number of mastocytoma cells (3 × 10⁷) alone, while cell-mediated cytotoxicity induced by the same treatment increased to the maximum level obtained by immunization with mastocytoma cells (3 × 10⁷) alone.     

Adjuvant Activity of Mycobacterial Fractions

The addition of adjuvant (Bacillus Calmette–Guérin-cell wall skeleton; BCG-CWS) to the culture medium substantially increased the immune response of mouse spleen cells to immunization with heterologous erythrocytes and hapten–protein conjugate in vitro. Cell walls of mycobacteria, nocardia and corynebacteria and their cell wall constituents were used as adjuvants. In the present case, it was found that cell walls of mycobacteria, nocardia and corynebacteria markedly facilitated primary humoral response of mouse spleen cells to heterologous erythrocytes in vitro. The adjuvant effect of BCG-CWS was present only in the formation of 19 S antibody in both primary and secondary responses, but not in that of 7 S antibody in vitro. Primary antihapten response of mouse spleen cells against dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) also succeeded when BCG-CWS was added to the culture medium, and it was found that BCG-CWS increased the helper activity of carrier specific helper T cells in vitro where a double chamber system, separated by a cell-impermeable nucleopore membrane, was used. This result suggested that BCG-CWS acts on T cells, resulting in the release of soluble factor(s) from T cells capable of exerting an adjuvant effect. Furthermore, mucopeptide moiety of BCG-CWS retained some adjuvant activity, but other cell wall constituents, such as mycolic acid, arabinose mycolate, and arabinogalactan, did not show any adjuvant effect in vitro. These results strongly imply that mucopeptide moiety of BCG-CWS plays an important role in the development of adjuvanticity.     

Purification of Mycobacterial Deoxyribonucleic Acid

Impurities believed to be polysaccharides have been found in mycobacterial deoxyribonucleic acid (DNA) preparations. Agar-gel diffusion of the DNA preparations against concanavalin A indicated the presence of three polysaccharides and was used to follow the purification procedures. The polysaccharides appeared to be the same for all strains studied. Precipitation of DNA with cetyltrimethylammonium bromide was used to separate impurities from some DNA preparations. The presence of the contaminants was found to affect markedly the determination of the guanine plus cytosine content according to a method dependent on the ratio of absorbancies at 260 and 280 nm; the impurities did not affect the determination by the method of thermal denaturation. The presence of a DNA-polysaccharide complex is suggested.     

Acid-Fastness of Nocardia asteroides GUH-2 Cultured in Brain Heart Infusion Broth Supplemented with Paraffin

Nocardia asteroides GUH-2 was not acid-fast when grown in brain heart infusion (BHI) broth. When grown in BHI broth supplemented with paraffin, many filamentous cells showed acid-fastness after treatment with 1% acid-alcohol as the decolorizing agent. When treated with 3% acid-alcohol, filamentous cells were not acid-fast. In addition to the acid-fast filamentous cells of nocardiae, unknown acid-fast spherical bodies were observed in the paraffin-supplemented BHI broth cultures.     

Mycobacterial Ecology of the Rio Grande

This is the first study to characterize the environmental conditions which contribute to the presence and proliferation of environmental mycobacteria in a major freshwater river. Over 20 different species of environmental mycobacteria were isolated, including the pathogenic M. avium and M. kansasii. Species of the rapidly growing M. fortuitum complex were the most commonly isolated mycobacteria, and one-third of all isolates were not identified at the species level, even by 16S sequencing. PCR restriction analysis of the hsp65 gene was more accurate and rapid than biochemical tests and as accurate as yet less expensive than 16S sequencing, showing great promise as a new tool for species identification of environmentally isolated mycobacteria. Total environmental mycobacteria counts positively correlated with coliform and Escherichia coli counts and negatively correlated with chemical toxicity and water temperature. Environmental mycobacteria can survive in the alkaline conditions of the river despite previous reports that especially acidic conditions favor their presence. A representative river isolate (M. fortuitum) survived better than E. coli O157:H7 at pHs below 7 and above 8 in nutrient broth. The river strain also retained viability at 8 ppm of free chlorine, while E. coli was eliminated at 2 ppm and above. Thus, in vitro studies support environmental observations that a variety of extreme conditions favor the hardy environmental mycobacteria.     

Mycobacterial manipulation of the host cell

Phagosome biogenesis, the process by which macrophages neutralize ingested pathogens and initiate antigen presentation, has entered the field of cellular mycobacteriology research largely owing to the discovery 30 years ago that phagosomes harboring mycobacteria are refractory to fusion with lysosomes. In the past decade, the use of molecular genetics and biology in different model systems to study phagosome biogenesis have made significant advances in understanding subtle mechanisms by which mycobacteria inhibit the maturation of its phagosome. Thus, we are beginning to appreciate the extent to which these pathogens are able to interfere with innate immune responses and manipulate defense mechanisms to enhance their survival within the human host cell. Here, we summarize current knowledge about phagosome maturation arrest in infected macrophages and the subsequent attenuation of the macrophage-initiated adaptive anti-mycobacterial immune defenses.