2-Deoxy-D-glucose inhibits intracellular multiplication and promotes intracellular killing of Legionella pneumophila in A/J mouse macrophages.

Legionella pneumophila can grow intracellularly in A/J mouse macrophages. 2-Deoxy-D-glucose (2dG) (0.1, 1, and 10 mM) inhibited intracellular multiplication and promoted intracellular killing of L. pneumophila dose dependently when it was added to the culture medium of macrophage monolayers, whereas it did not inhibit the bacterial growth in buffered yeast extract broth, which was used for an L. pneumophila culture. The effect of 2dG was reversible because the surviving bacteria resumed intracellular multiplication after the washing away of 2dG from the culture. The effect of 2dG was also competitively inhibited by high concentrations of glucose. The inhibitory effect of 2dG was not attributed to the inhibition of bacterial phagocytosis by macrophages. Furthermore, sodium fluoride (0.1 and 1 mM), cycloheximide (0.1 and 1 microgram/ml), and tunicamycin (1, 2, and 5 micrograms/ml) did not promote the killing of L. pneumophila in macrophages, implying that the inhibitory effect of 2dG cannot be attributed to the inhibition of glycolysis, protein synthesis, and protein glycosylation in macrophages. We suggest that 2dG promotes intracellular killing of L. pneumophila by activating some novel killing mechanism of macrophages.     

Genetic control of natural resistance in mouse macrophages regulating intracellular Legionella pneumophila multiplication in vitro.

It is known that Legionella pneumophila proliferates in peritoneal macrophage cultures derived from A/J mice but not in macrophage cultures derived from many other strains, including C57BL/6 mice. To analyze the genetic control of this trait and the location of the Legionella resistance-susceptibility gene, we prepared segregating progeny of A/J and C57BL/6 mice and determined the levels of susceptibility of individual mice. Peritoneal macrophages were collected by injecting thioglycolate medium, and macrophage monolayers were infected in vitro with L. pneumophila Philadelphia-1. Counting of colonies on buffered charcoal yeast extract agar plates and Gimenez staining of macrophage monolayers were carried out daily. There was a 10-fold increase in bacterial burden 1 day after infection and a 100-fold increase after 2 days in A/J (susceptible) macrophages. The increase in bacterial burden was always less than 10-fold in macrophages from C57BL/6 (resistant) progenitors, A/J x C57BL/6 F1 hybrids, and C57BL/6 x F1 backcross progeny. The ratios of resistant individuals to susceptible individuals were 22:6 for F2 progeny and 20:22 for A/J x F1 backcross progeny. The fact that the organism did not proliferate in macrophages from B10.A mice demonstrated that major histocompatibility antigens did not regulate the macrophage resistance of C57BL/6-derived mice. The sex and coat color genes of mice were not linked to the resistance-susceptibility gene. We suggest that resistance and susceptibility are controlled by a single gene or closely linked genes which are autosomal and that the resistance allele is dominant. The results of a comparison of the strain distribution pattern of this trait with the distribution pattern of 185 allelic markers in A/J x C57BL/6 and C57BL/6 x A/J recombinant inbred strains suggest that this susceptibility-resistance gene is located in the proximal part of chromosome 15.     

Isolation of Legionella pneumophila serogroup 14 from a human source.

A strain of Legionella pneumophila serogroup 14 was isolated during a retrospective study, after death from the sputum of a patient who had had acute leukaemia and pneumonia. This is the third strain of that serogroup to be isolated from a human source. This event emphasises the importance of performing culture as well as serological tests, so as to detect cases of legionellosis caused by strains which rarely cause fatal clinical illness.     

Involvement of tumor necrosis factor alpha in intracellular multiplication of Legionella pneumophila in human monocytes.

We investigated the role of tumor necrosis factor alpha (TNF-alpha) in human peripheral monocytes infected with Legionella pneumophila in vitro. Exogenous TNF-alpha significantly inhibited the intracellular multiplication of the bacterium. This effect was concentration and time dependent and was abrogated by anti-TNF antibodies. TNF-alpha levels in the culture supernatants were low but were enhanced by the addition of gamma interferon. When monocytes were cultured and infected in the presence of pentoxyphilline, a potent inhibitor of TNF-alpha synthesis, the intracellular bacterial growth was enhanced. The effect of pentoxyphilline was concentration and time dependent and was due to the inhibition of TNF-alpha production, as shown by Northern (RNA) blot hybridization of total RNA. In addition, the pentoxyphilline partially abolished the inhibitory effect of gamma interferon on bacterial intracellular multiplication. These results suggest that gamma interferon inhibits, at least partially, the intracellular multiplication of L. pneumophila by enhancing TNF-alpha synthesis.     

Identification of protein antigens of Legionella pneumophila serogroup 1.

Growth of Legionella pneumophila serogroup 1 in yeast extract broth was standardized, and protein profiles of detergent-solubilized cells were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Over 30 protein bands were identified, 6 of which were more prominent both in Coomassie brilliant blue-stained profiles and in fluorograms with intrinsically radiolabeled bacteria. These major proteins were 22,000 dalton (22K), 24K, 43K, 63K, 76K, and 78K. We found that the 24K and 63K major proteins were antigenic, as demonstrated both by radioimmunoprecipitation (RIP) of [35S]methionine-labeled organisms and by immunoblotting with rabbit hyperimmune sera. In addition, both techniques detected antigens migrating at 58K, 72K, 76K, and 78K. The major 22K and 43K major proteins and antigens migrating at 25.5K, 29K, and 46K were only detected by radioimmunoprecipitation, whereas antigens of 19K, 48K, 53K, and 68K were detected only by immunoblotting. Cell-surface localization of the proteins was determined by a modified radioimmunoprecipitation assay designed to react specifically with surface antigens and by the use of hyperimmune sera that had been extensively preabsorbed with intact cells to deplete the sera of antibodies directed against surface components. The 22K, 24K, 43K, 63K, and 78K major proteins and several minor proteins were found to be located on the surface of L. pneumophila cells.     

Differential nitric oxide (NO) production by macrophages from mice and guinea pigs infected with virulent and avirulent Legionella pneumophila serogroup 1

l-arginine-dependent reactive nitrogen intermediates have been identified as macrophage cytotoxic effector molecules against intracellular pathogens. To determine its role, ex vivo production of NO by peritoneal macrophages of C3H/HeN mice and Dunkin–Hartley guinea pigs infected intraperitoneally with a virulent and isogenic avirulent Legionella pneumophila serogroup 1 strain was compared with bacterial clearance from the lungs. While the virulent strain was cleared from mice lungs, the guinea pigs died within 96 h. In vivo infection with both strains resulted in the production of NO by mouse peritoneal macrophages ex vivo. In contrast, guinea pig macrophages did not produce detectable NO. In addition, infection by the avirulent strain led to the production of significantly more NO by mouse macrophages than the virulent parent strain, irrespective of stimulation with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-γ). These results suggest that resistance to Leg. pneumophila infection may depend on the production of NO by host macrophages.     

Crossed immunoelectrophoretic analysis of Legionella pneumophila serogroup 1 antigens.

By crossed immunoelectrophoresis, 85 different antigens were demonstrated in sonicated preparations of Legionella pneumophila serogroup 1 (Lp1). The precipitin patterns of 82 anodic-migrating antigens were numbered and were designated the Lp1 reference system. Eleven antigens were stable to boiling, and seven of these were shown to be surface antigens. One heat-stable surface antigen (antigen no. 61) was highly reactive with limulus amoebocyte lysates and formed a precipitin resembling lipopolysaccharide. Serum from an isolation confirmed case of Lp1 infection and serogroup-specific rabbit antiserum reacted specifically with antigen no. 61, which was designated the serogroup-specific antigen. Normal human and rabbit sera commonly had antibodies to antigen no. 66 of the Lp1 reference system. This antigen is antigenically related to the "common antigen" of Pseudomonas aeruginosa.     

Spectrum of Legionella species whose intracellular multiplication in murine macrophages is genetically controlled by Lgn1.

We examined the intracellular growth of 20 strains within six species of Legionella in thioglycolate-elicited peritoneal macrophages from A/J and C57BL/6 mice and a congenic strain derived from them (A.B Lgn1). With the exception of Legionella pneumophila Togus-1 and Bloomington-2, the intracellular growth of the 15 L. pneumophila strains tested was controlled by Lgn1. However, the intracellular growth of five Legionella species other than L. pneumophila was not under Lgn1's control.     

Subtyping of Legionella pneumophila serogroup 1 isolates by monoclonal antibody and plasmid techniques.

A group of environmental and clinical Legionella pneumophila serogroup 1 isolates was subtyped by monoclonal antibody dot immunoblotting and plasmid analysis. Monoclonal antibody analysis defined seven subtypes within three major groups. Plasmid analysis (including restriction endonuclease digestion) revealed 10 subtypes. By combining plasmid and monoclonal techniques, all 16 strains were shown to be distinct. Plasmid profiles and monoclonal antibody reactivities of selected strains were stable despite serial passage (greater than 100 times). No plasmid-associated antigen was defined by this panel of monoclonal antibodies. The observed dissociation of plasmid profiles and monoclonal antibody reactivity patterns suggests that accurate epidemiologic typing of L. pneumophila serogroup 1 strains will require use of both techniques.     

Heterogeneity of antibody response in infection with Legionella pneumophila serogroup 1.

The antibody response of patients infected with Legionella pneumophila serogroup 1 in a common source outbreak was investigated. Heat-killed antigens from L pneumophila serogroups 1-3 and 6-10, plus several other strains of L pneumophila, together with 13 other species of legionellas were used in an indirect fluorescence antibody test. Formolised yolk sac antigens made from L pneumophila serogroups 1, 6, and 7 were also used. Although antibodies were produced to several L pneumophila serogroups or Legionella species by individuals, there was no constant pattern, suggesting that the response is a characteristic of the infected individual and not of the infecting strain of Legionella. There is evidence that heat-killed antigen made from L pneumophila serogroup 7 may give unreliable results.     

Intracellular multiplication of Legionella pneumophila in cultured human embryonic lung fibroblasts.

Cell cultures of normal human embryonic lung fibroblasts were inoculated with two strains of Legionella pneumophila, the etiological agent of Legionnaires disease. Large numbers of intact organisms, including many dividing forms, were seen within fibroblasts by light and electron microscopy. Intracellular multiplication of organisms was demonstrated by the progressive increase in bacterial colony counts from plated extracts of infected fibroblast cultures in which extracellular multiplication had been eliminated by bactericidal concentrations of antibiotics. It is evident that L. pneumophila can thrive in an intracellular environment, and this property may be significant in the pathogenesis of Legionnaires disease.     

Isolation and characterization of a seventh serogroup of Legionella pneumophila.

An environmental isolate (Chicago 8) and a clinical isolate (Dallas 5) of Legionella pneumophila were shown to have similar serological characteristics; however, these characteristics were distinct from those of L. pneumophila serogroups 1 through 6. Chicago 8, ATCC 33823, was designated as the reference strain for L. pneumophila serogroup 7. The use of Mongolian gerbils for the isolation of L. pneumophila from the environment is described. Even though guinea pigs are the animals of choice in such studies, the isolation of Chicago 8 illustrates that the use of gerbils may be a viable option when cost is a major consideration in study design.     

Rapid and sensitive method for quantitation of Legionella pneumophila serogroup 1 antigen from human urine.

A reversed passive hemagglutination test was developed to assay relative concentrations of soluble antigen of Legionnaires disease (Legionella pneumophila serogroup 1) in human urine samples. The test is highly sensitive, being able to detect as little as 0.0002 microgram of total antigen. Preliminary results with this test on serial urine and serum samples from a patient with legionellosis show that measurable amounts of antigen are present in urine during the course of the illness. However, no antigen could be detected in the serum of the patient.     

Evaluation of urinary antigen ELISA for diagnosing Legionella pneumophila serogroup 1 infection.

The enzyme linked immunosorbent assay (ELISA) described was developed to detect a soluble antigen in the urine of patients with Legionnaires' disease caused by Legionella pneumophila serogroup 1 (L.pn 1). The assay was evaluated and showed good specificity (100%) and intra-assay reproducibility. Antigen was detected in the urine of 93 (77%) of 120 patients, overall, and in 86% of patients from whom a specimen obtained within seven days of onset of illness was available. On all but one occasion the first urine sample taken from a patient for whom a positive ELISA result was obtained, was itself positive. In one case antigen was not detected at four days but was present on the fifth day after onset of symptoms. In two patients urinary antigen was detectable as early as two days after onset of symptoms. In another the antigen persisted for at least 60 days. More than half the patients, however, had stopped producing detectable antigen within 14 days of onset of symptoms. It is therefore important that where Legionnaires' disease is suspected urine is collected as early as possible in the course of the disease.     

Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies.

A panel of monoclonal antibodies to Legionella pneumophila serogroup 1 and a subclassification scheme were developed in a collaborative project among three laboratories. The seven most useful monoclonal antibodies were selected from three previously developed panels on the basis of indirect fluorescent antibody patterns with 83 strains of L. pneumophila serogroup 1 that were obtained from widely distributed geographic locations. The isolates were divided into 10 major subgroups on the basis of reactivity patterns that can be readily reproduced in any laboratory and are not subject to major inconsistencies of interpretation of staining intensity. A standard protocol for the indirect fluorescent antibody procedure was also developed.     

Phenotypic modulation by Legionella pneumophila upon infection of macrophages.

Since many pathogenic bacteria manifest a coordinate regulation of gene expression in response to different environmental stimuli, we examined the phenotypic response of Legionella pneumophila to infection of macrophage-like U937 cells. Intracellular L. pneumophila was radiolabeled, and cell extracts were subjected to two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At least 35 Legionella proteins were selectively induced during infection of macrophages, and one of these proteins was not detected in organisms grown in vitro. Expression of at least 32 proteins was selectively repressed during infection of macrophages, and 9 of these proteins were undetectable in intracellularly grown organisms. Thirteen of the macrophage-induced proteins were also induced by one or more of several stress conditions in vitro, and two of these proteins were the heat shock GroEL- and GroES-like proteins. Nineteen of the macrophage-repressed proteins were also repressed by one or more of the stress conditions in vitro. Our data showed that intracellular L. pneumophila manifested a phenotypic modulation and a global stress response to the intracellular environment of the macrophage. The data suggested that multiple regulons are involved in this modulation, which may contribute to the survival of L. pneumophila within alveolar macrophages.     

Virulence conversion of Legionella pneumophila serogroup 1 by passage in guinea pigs and embryonated eggs.

When virulent Legionella pneumophila is passaged on supplemented Mueller-Hinton agar, it remains virulent for guinea pigs and embryonated hen eggs for two passages. However, by the fifth passage the cultures become avirulent for guinea pigs. Flagella were not produced by L. pneumophila on the first passage on supplemented Mueller-Hinton agar. In contrast, 12 passages on charcoal-yeast extract agar did not result in the reduction of virulence or the loss of flagella of L. pneumophila. Growth in supplemented yeast extract broth or on Norit-A-filtered supplemented yeast extract agar also did not result in a reduction of the virulence of L. pneumophila. However, L. pneumophila did not produce flagella when grown on these two media. Thus, it appears that the production of flagella is not required for the virulence of L. pneumophila when administered by the intraperitoneal route of infection. A virulent flagellated form of L. pneumophila was recovered by passing an avirulent form six times in guinea pigs. When avirulent L. pneumophila was passaged 12 times in embryonated eggs, a nonflagellated form of the bacterium was recovered which had an increased virulence for guinea pigs and embryonated eggs. However, virulent forms were not recovered by passage of avirulent forms on commonly used laboratory media. These results support the suggestion that a suitable host is required for the selection of the virulent form of L. pneumophila from avirulent cultures.     

Interaction of Leishmania donovani promastigotes with human monocyte-derived macrophages: parasite entry, intracellular survival, and multiplication.

Leishmania donovani promastigotes were incubated with human monocyte-derived macrophages in vitro to assess the role of macrophages in the early stage of visceral leishmaniasis. Adherent mononuclear cells, obtained from nonimmune human donors, were cultivated on glass cover slips for 5 days and then incubated with axenically grown promastigotes in the presence of heat-inactivated autologous serum. Promastigotes attached to macrophages with either their flagellar or aflagellar ends, and macrophage pseudopodia formed around them. Intracellular parasites were identified within phagocytic vacuoles by electron microscopy, and the parasites assumed a form similar to that of amastigotes obtained from infected hamster spleens. Initially, 67 +/- 5% of the macrophages were infected with a mean of 4.2 +/- 0.7 parasites per infected cell. After 6 days of incubation, 79 +/- 7% of the macrophages were infected with 15.9 +/- 3.2 parasites per infected cell. The total number of parasites per monolayer increased from 4.8 +/- 0.8 x 10(5) to 1.8 +/- 0.4 x 10(6) (P less than 0.05). Dividing parasites were identified in macrophage vacuoles by electron microscopy. Human monocyte-derived macrophage vacuoles by electron microscopy. Human monocyte-derived macrophages can phagocytize promastigotes, allow the conversion of promastigotes to an amastigote-like state, and support intracellular multiplication.     

Inhibition by retinoic acid of multiplication of virulent tubercle bacilli in cultured human macrophages.

The immunologically active vitamin retinoic acid (RA) was tested for the ability to increase the resistance of cultured human macrophages (MP) to experimental infection with virulent Mycobacterium tuberculosis Erdman (tubercle bacilli [TB]). It was added to MP in various concentrations and addition regimens. Protection against TB was measured by counting live TB (CFU) in lysates of samples of MP taken at 0, 4, and 7 days after MP infection. RA was protective when added after infection at the pharmacologic concentration of 10(-5) M and when added before infection at the physiologic concentration of 10(-7) M. The protection lengthened intracellular generation times for TB, occasionally caused bacteriostasis, and regularly kept CFU counts at 7 days (end of the period of infection) 1 to 2 log10 CFU below control values. Significant protection was seen in a series of 16 experiments with MP from seven different donors, but the degree of protection varied considerably. The protection depended partly on and was inversely proportional to concentrations of a serum substitute or autologous serum used as a supplement in the RPMI 1640 MP culture medium. It was strongest at concentrations of serum below 1%. RA at concentrations used in the MP cultures did not inhibit TB in the absence of MP. These results suggest that RA (vitamin A), like vitamin D, may have some immunoprotective role against human tuberculosis, as historically intimated by the regular use of vitamin A- and D-rich cod liver oil for the treatment of tuberculosis before the introduction of modern chemotherapy.