Observed differences in virulence-associated phenotypes between a human clinical isolate and a veterinary isolate of Mycobacterium avium.

Mycobacterium avium, the most common opportunistic pathogen in patients with AIDS, is frequently isolated from a variety of environmental sources, but rarely can these environmental isolates be epidemiologically linked with isolates known to cause human disease. Using a number of in vitro tissue culture assays, we found significant pathogenic differences between a serotype 4 human clinical M. avium isolate and a serotype 2 veterinary isolate. Cell association of the patient strain with a human intestinal cell line was 1.7 times that of the veterinary strain. Growth of this clinical strain in human peripheral blood mononuclear cell-derived macrophages increased from 12-fold higher than that of the veterinary isolate after 2 days to 200-fold higher after 4 days. By the conclusion of each experiment, lysis of all examined host cell types and accumulation of cell debris were observed in infections with the human isolate, but monolayers remained relatively intact in the presence of the animal isolate. The two strains also differed in the ability to stimulate human immunodeficiency virus replication in coinfected host cells, with p24 antigen levels after 6 days threefold higher in the cells coinfected with the clinical strain than in those infected with the veterinary strain. If the genetic differences responsible for the phenotypes observed in these assays can be identified and characterized, it may be possible to determine which M. avium strains in the environment are potential human pathogens.

An in vitro tissue culture bilayer model to examine early events in Mycobacterium tuberculosis infection.

A tissue culture bilayer system that mimics some aspects of early alveolar infection by Mycobacterium tuberculosis was developed. This model incorporates human lung epithelial type II pneumocyte (A549) (upper chamber) and endothelial cell (lower chamber) layers separated by a microporous membrane. This construction makes it possible to observe and quantify the passage of bacteria through the two layers, to observe the interaction of the bacteria with the various cell types, and to examine the basic mechanisms of immune cell recruitment to the site of infection. After 10(7) organisms were added to the upper chamber we microscopically observed large numbers of bacteria attached to and within the pneumocytes and we determined by viable-cell counting that a small percentage of the inoculum (0.02 to 0.43%) passed through the bilayer into the lower chamber. When peripheral blood mononuclear cells were added to the lower chamber, microscopic examination indicated a migration of the mononuclear cells through the bilayer to the apical surface, where they were seen associated with the mycobacteria on the pneumocytes. The added complexity of the bilayer system offers an opportunity to define more precisely the roles of the various lung cell types in the pathogenesis of early tuberculosis.

Comparison of in vitro models for the study of Mycobacterium tuberculosis invasion and intracellular replication.

We recently evaluated several tissue culture model systems for the study of invasion and intracellular multiplication of Mycobacterium tuberculosis. These model systems include a human alveolar pneumocyte epithelial cell line, a murine macrophage cell line (J774), and fresh human peripheral blood-derived macrophages. Our data indicated that the initial level of association of M. tuberculosis with human alveolar pneumocyte cells (2%) was less than that observed with fresh human peripheral blood macrophages (9%) or J774 murine macrophages (13%) within 6 h of the addition of the bacteria. M. tuberculosis replicated in association with the pneumocyte cells by more than 55-fold by day 7 postinfection. In contrast, total bacteria] growth in the J774 cells and human macrophages was considerably less, with increases of only fourfold and threefold, respectively, over the same 7-day period. Amikacin, an aminoglycoside antimicrobial agent, was added to inhibit the growth of extracellular bacteria after the initial 6-h infection period. Decreases in viable counts were observed in all three cell cultures within the first 3 days after infection. However, unlike the case with either macrophage culture, intracellular bacterial CFU obtained from the infected pneumocytes increased by fourfold by day 7 after the addition of amikacin. These data indicate that M. tuberculosis infects and multiplies intracellularly in human lung epithelial cells and that these cells may be an alternative in vitro model for the study of intracellular multiplication of M. tuberculosis in the human lung.

Intracellular growth and cytotoxicity of Mycobacterium haemophilum in a human epithelial cell line (Hec-1-B).

We developed an in vitro model to study the temperature-regulated cytotoxicity and intracellular growth of Mycobacterium haemophilum in cultured human epithelial and endothelial cells. M. haemophilum associated with human epithelial and endothelial cells at similar rates when incubated at 33 and 37 degrees C, but only the epithelial cell line supported the multiplication of this organism. M. haemophilum grew equally well with epithelial cells at both temperatures. The aminoglycoside antibiotic amikacin was used to study the intracellular growth of M. haemophilum in the epithelial cells at 33 and 37 degrees C. Although an approximately equal number of bacteria were found within cells after 2 days of incubation at both temperatures, intracellular replication of M. haemophilum was 1,000-fold greater at 33 than at 37 degrees C. This intracellular multiplication was associated with destruction of the monolayers at 33 but not at 37 degrees C, and only culture filtrates from infected monolayers incubated at 33 degrees C were cytotoxic to fresh epithelial cell monolayers. This strain of M. haemophilum also produced contact-dependent hemolysis of sheep erythrocytes, demonstrating the possible presence of a cytolysin. These studies suggest that M. haemophilum has a preference for growth with cultured human epithelial cells. In addition, intracellular growth is best at 33 degrees C in epithelial cells, and this correlated with cytotoxicity at this temperature. This phenotype may be caused by induction of a soluble cytotoxic component, possibly a hemolytic cytolysin.

Inhibition of trypsin with active-site-directed enzyme-activated nitrosoamide substrates.

A series of active-site-directed enzyme-activated nitrosoamide inhibitors of trypsin has been designed, synthesized, and tested. The inhibitors contain an N-nitrosoamide group that can generate an alkylating agent and a positively charged ammonium ion group at the end of an aliphatic carbon chain that provides specificity. The half-lives of inhibition under normal conditions were 0.6 to 2 min for compounds in series 1. One of the compounds, N-(4-amino-1-butyl)-N-nitrosobenzamide (1b), is a very efficient inhibitor; its partition ratio, k2/kinact, is zero suggesting that it may be a useful titrant for trypsin and related enzymes. The extent of inhibition is substantially decreased by the competitive inhibitor benzamidine, indicating that the inhibitors were operating in the active site. Two modes of inhibition were noted: reversible and irreversible. The N-nitrosoamide inhibitors bind to the trypsin binding pocket guided by the primary specificity. They then acylate the enzyme (at Ser-195), producing a leaving group that generates diazonium ions (or) carbocations in the active site; these react with a proximal carboxylic acid side chain of the enzyme to form a carboxylic acid ester, presumably that of Asp-194. If primary amino groups are present on the alkyl group, the ester (13C NMR delta 67.2 ppm for R1COO13CH2-(CH2)nNH2) rearranges into the amide form (13C NMR delta 62.9 ppm for R1CONH(CH2)n13CH2OH) through an O-->N acyl migration; an irreversibly inhibited enzyme results. A model based on the orientation of the site-specific group of N-(4-amino-1-butyl)-N-nitroso-N'-isobutyryl-D-alaninamide (D2a) and its L antipode in the active site of trypsin is proposed to explain the preferential inhibition of trypsin by the D-isomer. Analysis of the structure--inhibition relationship revealed four factors that determine the inhibition modes of the inhibitors: 1, length of the alkyl group; 2, stability of the acyl-enzyme; 3, the option O--N acyl migration; and 4, chirality.

Intracellular multiplication and toxic destruction of cultured macrophages by Capnocytophaga canimorsus.

Capnocytophaga canimorsus is a gram-negative rod that causes opportunistic infections resulting in bacteremia, septicemia, meningitis, and death in immunocompromised, splenectomized, and alcoholic individuals. Infections caused by a related species, Capnocytophaga cynodegmi, remain localized at the site of the wound where the organism is introduced. Both organisms are part of the normal canine oral flora and are introduced through puncture wounds via dog bites. We found that both C. canimorsus and C. cynodegmi attach, are phagocytized, and multiply intracellularly in J774 mouse macrophage cells. After 48 h of infection by C. canimorsus, large sections of the macrophage cell layer were observed to detach and lyse, while the monolayer infected with C. cynodegmi demonstrated no cytotoxic effects. Tissue culture supernatants from the C. canimorsus-infected J774 cells filtered through a 0.22-micron-pore membrane produced a similar effect on fresh monolayers, while filtrates from C. cynodegmi and uninfected controls produced no effect. No endotoxin release was observed in these supernatants. We conclude that the cytotoxic phenotype of C. canimorsus is the likely result of a toxin produced by this organism.

Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis.

The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis. Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD50 of 10(3) colony forming units (CFU) or greater (low virulence) from those having an LD50 of approximately 10(1) or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica, a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD50 of 10(2) were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive.

Inhibition of alpha-chymotrypsin with an enzyme-activated n-nitrosoamide: active-site labeling by the naphthylmethyl cation.

alpha-Chymotrypsin was irreversibly inhibited with an enzyme-activated N-nitrosamide inhibitor, N-nitroso-N-(1-naphthylmethyl)-N'-isobutyrylalanine; alkylation of the active-site residues by the naphthylmethyl cation produced in the enzymatic reaction occurred. The inhibited enzyme was reduced and aminoethylated and then subjected to tryptic and chymotryptic digestion. Separation of the digest by reversed-phase HPLC revealed one major new peak relative to that of a control run from the native enzyme. Subsequent amino acid analysis and sequencing along with fast atom bombardment-mass spectrometry measurements indicated that this new peak stemmed from an active-site peptide, Met-192-Leu-199, and that the naphthylmethyl label was attached to the side-chain oxygen of Ser-195. The general approach employed can be applied to labeling active sites of a variety of hydrolytic enzymes.

Human microvascular endothelial tissue culture cell model for studying pathogenesis of Brazilian purpuric fever.

Brazilian purpuric fever (BPF) is a fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. All known BPF cases have been caused by three clones of Haemophilus influenzae biogroup aegyptius and have occurred in either Brazil or Australia. Using an immortalized line of human vascular endothelial cells, we developed an in vitro assay that identifies all known BPF-causing H. influenzae biogroup aegyptius strains (R. S. Weyant, F. D. Quinn, E. A. Utt, M. Worley, V. G. George, F. J. Candal, and E. W. Ades, J. Infect. Dis. 169:430-433, 1994). With multiplicities of infection (MOIs) as low as one bacterium per 1,000 tissue culture cells, BPF-associated strains produce a unique cytotoxic effect in which the tissue culture cells detach and aggregate in large floating masses after 48 h of incubation. In this study, using a BPF-associated strain and a non-BPF-associated control, we demonstrated that strains which produce the cytotoxic phenotype were able to replicate intracellularly whereas non-BPF-associated strains, with MOIs of > or = 1,000 did not replicate and did not produce the phenotype. We also showed that this phenotype is not caused by the activity of an endotoxin or the release of some other compound from the bacterial cell, since neither gamma irradiation-killed whole BPF clone bacteria nor bacterial cell fractions at MOIs of > 1,000 produced the cytotoxic effect. Furthermore, bacteria in numbers equal to MOIs of > 1,000 treated with chloramphenicol did not produce the cytotoxic phenotype, suggesting a requirement for bacterial protein synthesis. In addition, viable bacteria separated from the tissue culture monolayer by a 0.2-micron-pore-size membrane also failed to produce the phenotype. The ability of the bacterium to invade, replicate, and produce the phenotype appears to be primarily parasite directed since phagocytosis, pinocytosis, and eukaryotic protein synthesis inhibitors, including cycloheximide, cytochalasin D, and methylamine, had no effect on the ability of the bacterium to invade and cause a cytotoxic response. Understanding the basic mechanisms involved in this tissue-destructive process should enhance our knowledge of the general pathogenesis of BPF.

A tissue culture bilayer model to study the passage of Neisseria meningitidis.

A tissue culture bilayer system has been developed as a model to study the mechanisms of attachment and invasion involved in the pathogenesis of Neisseria meningitidis. The model incorporates epithelial and endothelial cell layers separated by a microporous membrane and makes it possible to observe and quantify the passage of bacteria through the multiple layers and to study the mechanisms by which they make this passage. This model is adaptable to a wide variety of microbial pathogens and can be modified by substituting any physiologically relevant eucaryotic cells for the component layers. The system's makeup of cells of human origin and its reproducibility give it advantages over animal and primary organ culture models, while the added complexity of multiple layers allowing cell-to-cell communication makes it a more realistic human tissue model than standard cell monolayers.

Fluorogenic N-nitrosoamides: active-site labeling reagents for chymotrypsin-like proteases.

Two fluorogenic N-nitrosoamides, N-nitroso-N-((7-methoxycoumarin-4-yl)methyl)-N'-isobutyrylalaninamide (6a) and N-nitroso-N-((6-methoxyquinolin-2-yl)methyl)-N'-isobutyrylalani namide (6b), were synthesized. Both N-nitrosoamides inhibited alpha-chymotrypsin irreversibly; they show promise as labeling reagents for the active sites of chymotrypsin-like proteases.

Labeling of the active site of alpha-chymotrypsin with N-nitroso-N-benzylacetamide and related compounds.

alpha-Chymotrypsin (CT) is slowly inhibited by N-nitroso-N-benzylacetamide (1) at 25 degrees C. The 13C-NMR spectrum of the hydrolysate of 13C-enriched 1-inhibited CT shows five new signals at 52.33, 44.55, 43.72, 36.79, and 32.90 ppm resulting from alkylation of the side chains and also of amide linkages of the enzyme backbone by benzyl carbocations produced in the active site. The alkylation pattern is different from those observed in the inhibitions of CT with D-N-nitroso-N-[alpha-13C]-benzyl-N'- isobutyrylalaninamide (2a) and D-N-nitroso-N-[alpha-13-C]-benzyl-N'-isobutyrylphenylalaninamide (2b) (White et al. JACS 1990, 112, 1956-1961). The chemical shift data suggest that the 52.33 ppm signal stems from N-benzylglycine, the 36.79 ppm signal from S-benzylcysteine, and the 32.90 ppm signal from 2-benzyltryptophan. A synthesis of the latter compound was developed.

Attachment and entry of Legionella pneumophila in Hartmannella vermiformis.

Legionella pneumophila is an intracellular parasite of Hartmannella vermiformis. Attachment to the amebae and entry of L. pneumophila were studied by two quantitative assays: One used plate counts to measure the number of bacteria attaching to amebae at 4 degrees C; the other determined the number of intracellular bacteria by use of transmission electron microscopy (TEM). The attachment assay showed that L. pneumophila are inefficient in attachment to amebae. About 0.05% of the bacteria were bound after 1 h with a 10- to 40-fold increase over the next 11 h. Attachment of both virulent and avirulent strains of L. pneumophila occurred at a similar rate. Uptake of L. pneumophila was measured by counting intracellular bacteria using TEM. Limited numbers of virulent L. pneumophila were found intracellularly before 4 h, but the numbers increased logarithmically after this time. The number of amebae containing virulent L. pneumophila increased linearly during the 12-h co-incubation. Avirulent L. pneumophila were rarely detected within amebae throughout the 12-h incubation. Results indicate that entry, not attachment, of virulent L. pneumophila is the limiting step in infection of axenically grown H. vermiformis.

Intracellular growth of Afipia felis, a putative etiologic agent of cat scratch disease.

The organism Afipia felis, which is though to be an etiologic agent of cat scratch disease, is a gram-negative rod that is clearly seen in infected tissue but is very difficult to isolate from clinical specimens; there has been only one report to date of the successful isolation and maintenance of the bacterium on artificial medium. We have found that A. felis will attach, invade via phagocytosis, and multiply intracellularly within the phagosomes of primary human monocytes and HeLa cells. Once in the cell, the bacterium appears to change morphologically, becoming longer and more pleomorphic, and loses its ability to grow on an artificial medium. Unique proteins have been identified in both the intra- and extracellular variants of A. felis. Convalescent-phase sera from patients with cat scratch disease react poorly with intracellular and extracellular bacteria, suggesting a poor humoral response. The tissue culture protocol presented has been used to isolate 14 new strains of A. felis and has for the first time permitted study of the pathogenesis of this unique organism.

Morphologic and staining characteristics of a cyanobacterium-like organism associated with diarrhea.

A spherical organism 9-10 microns in diameter, seen in three outbreaks of diarrhea in Southeast Asia and the United States during the past 2 years, bore characteristics of a cyanobacterium when observed in formalin-preserved stool specimens and by electron microscopy. Organisms in freshly passed stool specimens showed an internal morula of lipid-containing globules. In fresh water, the morula divided into two sausage-shaped structures resembling the sporocysts of an isosporid coccidian. After 7 months, the organisms had not developed the crescentic sporozoites seen in the Coccidia but had begun to multiply slowly in culture. It was impossible to stain the internal structures of the organisms because the outer cyst wall ruptured during desiccation, releasing the contents of the cysts. The organisms were readily identified by their intense blue autofluorescence under UV light, but they were also recognizable by bright-field microscopy and by a modified acid-fast stain. Almost all infected persons suffered intermittent diarrhea for 2-3 weeks and many emphasized a feeling of intense fatigue during the course of their illness.

Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells.

A cloned and axenically cultured strain of Hartmannella vermiformis was used as a model to study intracellular multiplication of Legionella pneumophila in amoebae. The growth of L. pneumophilia in both H. vermiformis and a human monocyte-like cell line (U937) was investigated with cytoskeletal and metabolic inhibitors. L. pneumophila replicated only intracellularly in these cellular models, and electron microscopy showed ultrastructural similarities in the initial phase of multiplication. Treatment of amoebae with an inhibitor of microfilament-dependent phagocytosis (cytochalasin D, 0.5 or 1.0 micrograms/ml) did not inhibit intracellular growth of L. pneumophila; however, intracellular multiplication was inhibited by treatment of U937 monocytes with the same concentrations of cytochalasin D. Methylamine (10 to 100 mM), an inhibitor of adsorptive pinocytosis, inhibited the replication of L. pneumophila in amoebae in a dose-dependent manner. All doses of methylamine tested (10 to 50 mM) inhibited growth of L. pneumophila in U937 monocytes. Cytochalasin D and methylamine had no effect on the multiplication of L. pneumophila in culture medium or on the viability of amoebae or U937 monocytes. Intracellular replication of L. pneumophila in H. vermiformis may be accomplished by a cytochalasin D-independent mechanism, such as adsorptive pinocytosis. In contrast, both cytochalasin D- and methylamine-sensitive mechanisms may be essential for the intracellular multiplication of L. pneumophila in U937 monocytes.

Analogs and derivatives of firefly oxyluciferin, the light emitter in firefly bioluminescence.

The 5-methyl analog of firefly oxyluciferin, two isomeric O-methyl ether derivatives of it and an O,O'-dimethyl ether derivative were synthesized and their UV absorption and fluorescence emission spectra were determined. Comparisons of the emission data with the emission wavelength in bioluminescence indicate that the mono-anions of firefly oxyluciferin are candidates for the light-emitters in bioluminescence. Further, we have found that the chemiluminescence of active esters of firefly luciferin produces (from the keto form of oxyluciferin) only red light emission under a variety of conditions; a yellow-green light emission (from the enolic forms of the oxyluciferin product) could not be elicited.