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The effect of iron on resistance to Salmonella typhimurium was investigated in mice inoculated with vaccines prepared from live and avirulent (SL3770) or killed and virulent (SR11 or LT2) bacteria. It has been found that mice vaccinated with SL3770 vaccine develop an immunity which can be neutralized with iron. Iron promoted the development of lethal infections by serving as a growth-essential nutrilite for infecting bacteria and by neutralizing the acquired immunity. The titration of this dual effect of iron showed that more iron was needed to neutralize the immunity in vaccinated animals than to promote bacterial growth in normal animals. In the presence of a sufficient amount of exogenous iron, as few as 10 bacteria caused lethal infections in normal and immune mice with the same effectiveness. This iron-sensitive immunity could be changed to iron-resistant immunity by the immunological stimulation of SL3770-vaccinated mice with a sonicated vaccine prepared from heat-killed SR11 or LT2 bacteria. In distinction to iron-sensitive immunity, iron-resistant immunity could be transferred from SR11- or LT2-stimulated to normal mice with serum. Although effective in the transfer of antibacterial immunity, sera of SR11- or LT2-stimulated mice supported the growth of virulent bacteria as well as did sera of normal mice. The absorption of immune serum with either SR11 or LT2 bacteria removed its protective quality, but the sensitized bacteria remained as infectious as untreated bacteria for iron-treated normal mice. Only in SL3770-vaccinated mice were the immune serum-sensitized bacteria not able to cause the infection in spite of daily treatment with iron. These results suggest that iron-resistant immunity is due to the synergistic action of specific antibody and phagocytes of immunologically stimulated animals.

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Bacterial ability to obtain iron in bovine serum or in media containing transferrin (Tr) or conalbumin (Ca) was investigated by using serum-resistant (virulent) and serum-sensitive (avirulent) strains of Escherichia coli and Salmonella typhimurium. Bacteria growing in bovine serum enriched with radioactive iron-saturated Tr or with radioactive iron-saturated enterobactin (E) did not acquire radioactive iron. It has been found that the passage of siderophore (Si)-iron complexes into bacteria is blocked in serum by Tr and in Ca-containing medium by Ca. The investigation of bacterial ability to take iron in synthetic media showed that bacteria take in Si-bound but no Tr-bound radioactive iron. In the absence of free iron, the growth of serum-exposed virulent bacteria was supported by their stored iron. Virulent bacteria passaged in medium void of usable iron became depleted in stored iron and did not grow in animal sera unless sera were enriched by the addition of exogenous iron. Experiments with serum-exposed avirulent bacteria showed that their growth in Si-enriched serum should not be attributed to the iron-providing activity of Si but to the stimulating effect of Si which facilitates the use of stored iron. As distinct from avirulent bacteria, virulent bacteria used stored iron without the stimulating activity of extracellular Si.

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The fate of virulent and avirulent strains of Salmonella typhimurium in untreated and iron-injected mice and in transferrin-containing media demonstrated a direct relationship between bacterial virulence and the ability of bacteria to acquire transferrin-bound iron. Effects of injected iron on the development of infections with virulent and avirulent bacterial strains were determined in normal and immune mice by determinations of bacterial numbers in tissue homogenates and the mortality of infected animals. Results showed that infected and iron-injected mice died much more rapidly and frequently from overwhelming infections than infected and saline-injected mice. The infection-promoting effect of iron varied with the degree of bacterial virulence; the more virulent the bacteria, the more helpful was iron for the development of lethal infections. Siderophores promoted lethal infections in mice infected with virulent but not with avirulent bacteria. Experiments with vaccinated animals showed that iron exerted a deleterious effect on acquired immunity. Immune mice infected with virulent bacteria and injected with iron developed lethal infections as rapidly and nearly as frequently as similarly treated normal mice. Siderophores did not promote the development of lethal infections in immune mice. The effectiveness of iron, but not of siderophores, to promote bacterial infections in vaccinated mice revealed that acquired immunity is dependent upon the activity of an iron-neutralizable antibacterial system.

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Cellular resistance to facultative intracellular parasites has been studied by determining the antimycobacterial activity and the amount of fatty acids in sera and in heptane extracts of freshly collected and 24-h-cultured normal and activated guinea pig alveolar macrophages and liver cells. The quantity and the antimycobacterial activity of extractable fatty acids were determined by gas chromatography and the agar plate diffusion test, respectively. These determinations showed that heptane extracts of activated cells inhibited the growth of BCG much more effectively than fractions prepared from normal cells; chromatographic analyses showed that extracts of activated cells contained six times more C(16) and C(18) long-chain fatty acids than did fractions of normal cells. Heptane extracts of 24-h-cultured cells and of their media showed that during incubation normal and activated cells release fatty acids into the culture media without apparent cell injury; in all experiments liver cells produced larger amounts of fatty acids than alveolar macrophages. Sera collected from activated guinea pigs inhibited the growth of BCG and contained two to five times more total fatty acids than did the growth-supporting normal serum. That bactericidal fatty acids are excreted into the tissue culture medium of incubated cells or into the blood of immunologically stimulated animals suggests that macrophages can exert antibacterial effects without phagocytosis.

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Two smooth and six rough strains of Salmonella typhimurium with progressively smaller amounts of sugar and protein in their outer membrane were tested for degree of virulence in normal and iron-injected mice and for ability to acquire iron in mammalian sera. The rate of mortality showed that bacterial virulence for mice was lowered with progressive decrease of outer-membrane sugar and protein. Iron injections increased the rate of mortality in mice infected either with smooth strains or with superficially rough strains but were without effect in mice infected with deep rough strains. In in vitro experiments, iron promoted with equal effectiveness the growth of all serum-exposed bacterial strains, whereas enterobactin (E) was much more effective in promoting the growth of smooth and superficial rough than in promoting that of deep rough strains. Various experiments showed that deep rough strains cannot grow in E-supplemented serum because they are not able to use the transferrin-iron-E complexes that E forms with transferrin-iron. This failure to use transferrin-iron-E complexes by deep rough strains was found to be due to the inability of these strains to absorb iron containing complexes to their outer membrane. Adsorption studies with chemically treated bacteria showed that the receptor of transferrin-iron-E or E-iron complexes is a protein of the outer membrane of bacterial cells.

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Effects of iron on the growth of avirulent and virulent strains of Escherichia coli were tested in mice and in mammalian sera. Infection of the animals with iron increased mortality rates in mice infected with the avirulent strain to levels found in mice infected with the virulent strain. In vitro experiments showed that bacteria deprived of iron in bovine or human sera or milk or in chicken egg white stopped miltiplication and died in a very short time. These antibacterial effects were neutralized effectively with the addition of exogenous iron or the iron-binding bacterial product, enterochelin. In contrast to avirulent bacteria, which were effectively inhibited in mammalian serum, virulent bacteria were able to obtain iron and multiply. The ability of virulent bacteria to grow in mammalian serum is being attributed to the presence of iron-binding enterochelin and lipopolysaccharide in large amounts on the cell walls of virulent bacteria.

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Heptane-extractable fractions (HEF) prepared from immune-activated macrophages (IA-M) of tubercle bacilli or bovine gamma globulin-sensitized and -challenged guinea pigs inhibited the growth of tubercle bacilli whereas HEF of normal macrophages exerted no antibacterial activity. In distinction to the strong antibacterial activity of HEF of IA-M, HEF of immune macrophages exerted weak or no antimycobacterial activity. HEF of alveolar macrophages exerted stronger antibacterial activity than HEF of peritoneal macrophages. The degree of the antibacterial activity of HEF was determined primarily by the time of macrophage collection from antigenically stimulated animals. The antibacterial activity gradually increased and peaked at 2 weeks after the antigenic stimulation of sensitized animals; subsequently, the activity declined and disappeared in about 5 weeks. Similar to other immunological reactions, the stimulation of sensitive animals with specific antigen induced an anamnestic reaction which was characterized by a rapid recall of the macrophage antimycobacterial phenomenon (MAP). The antibacterial strength of the recalled phenomenon in sensitized animals was dependent upon the intensity of the sensitizing regimen; the phenomenon was much stronger in three times-sensitized animals than in once- and twice-sensitized animals. The time of appearance and the specificity of induction and of recall of the MAP indicate that the phenomenon is associated with the activated state in macrophages and, as a consequence of this association, it has a well-defined immunological nature.

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Tubercle bacilli failed to grow in iron-void media enriched with solutions of iron-containing transferrin (Tr) or ferritin (F) because these substances do not provide the bacilli with iron, which is essential for their growth. Animal serum and macrophages possessed no iron carrier with an ability to satisfy the need of the bacteria for the metal. Mycobactin (M), the growth-product of tubercle bacilli, removed iron from Tr and F and supplied the metal for bacillary utilization. The role of M in the growth of tubercle bacilli was influenced by nonionic surfactants which inhibited bacillary growth by removing M from the bacillary cells and interfering with the absorption of M-iron complexes. Experiments with Tween 80, Triton WR-1339, and lecithin showed that avirulent bacilli lose M at lower concentrations of the surfactants than virulent bacilli. Since avirulent and virulent bacilli possess the same amount of M, these findings indicate that M is bound more firmly to lipid-rich virulent than lipid-poor avirulent cells. These findings indicate that the resistance of virulent bacilli to the M-removing activity of the surfactants is an indicator of their ability to multiply in the infected host and may be used as a measure of bacillary virulence.

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Lysates or heptane extracts of peritoneal (P) and alveolar (A) normal macrophages (N-M), immune macrophages (I-M), and immune-activated macrophages (IA-M) were examined for antimycobacterial activity by the agar-plate diffusion test. This test has been found suitable to reveal the antibacterial activity in 3-day incubated, but not in freshly prepared, lysates. Results showed that materials of IA-AM or I-AM and of IA-PM exerted antimycobacterial effects, whereas materials of N-PM, I-PM, and of N-AM were usually inactive. Antimycobacterial activity of lysates of AM was stronger than that of PM. The formation of antibacterial factors during an incubation of M lysates, the solubility of the factors in heptane, and various other characteristics suggested that the antimycobacterial effect was caused by the formation of toxic levels of non-esterified fatty acids. M lysates exerted equal activities against BCG, H(37)Ra, and H(37)Rv strains of tubercle bacilli. The presence of antimycobacterial activity in lysates prepared from IA-M of either BCG- or BCG-sensitized animals indicated that the potential to generate antimycobacterial activity is associated with the state of delayed hypersensitivity and the state of activation of M.

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Fractions prepared from normal and activated liver cells were tested for the antimycobacterial activity by the agar plate diffusion test. Results showed that lysosome extracts of normal and activated cells exerted no antibacterial activity, cell extracts and lysosome membranes exerted some activity, and cell membranes exerted the strongest activity. The active materials of activated cells exerted stronger antituberculous activity than the corresponding materials of normal cells. Degrees of the antimycobacterial activity of various cell fractions showed a close correlation with the amounts of nonesterified fatty acids. This correlation, as well as other data, suggested that the antimycobacterial activity of cell fractions was caused by toxic fatty acids which were produced during the hydrolysis of lipoporteins or phospholipids by the activity of tissue lipases. The relationship of these findings to the mechanism of cellular immunity is discussed.

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Mycobactin (M), an iron-chelating product of tubercle bacilli, neutralized serum tuberculostasis by removing growth-essential iron from transferrin (Tr) and supplying the metal to the bacteria. The competition for iron between Tr and M has been demonstrated by the agar-plate diffusion test. This test is suitable not only for the study of Tr-iron-M interplay but also for the evaluation of serum tuberculostasis. Extremely poor solubility of M in water and consequently its association with lipoidal cell wall of tubercle bacillus was overcome by the use of water-dispersible and surface-active Tween 80. The addition of Tween 80 to culture media insured the presence of M in spent media; otherwise M was extracted from bacillary cells with a solution of Tween 80 or a mixture of ethanol and Tween 80. Although M was produced irrespective of the amount of iron present in culture medium, its production in iron-poor medium was more prolific than in iron-rich medium. M extracted from BCG or H(37)Rv cells neutralized serum tuberculostasis as effectively for the homologous as for heterologous strains. However, the extract of virulent bacilli was much more active in the neutralization than similar extract prepared from attenuated cells; whether this difference is of quantitative or qualitative nature remains to be determined.

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Tubercle bacilli exposed to an iron-poor medium multiplied at a slower rate but released more of the serum-tuberculostasis neutralizing factor (TNF) than the bacilli in an iron-rich medium. This growth-promoting factor found in spent medium exhibited characteristics which suggested its relationship to or identity with mycobactin. The identity of these two bacillary products was established by showing that both iron-free mycobactin and TNF promoted bacillary multiplication in tuberculostatic serum. This study resolved a long-standing controversy as to whether mycobactin serves as a growth factor or as a carrier of iron for tubercle bacilli. It was found that the tuberculostasis in mycobactin-neutralized serum was reconstituted by the addition of iron-free transferrin (Tr). The investigation of the interplay between mycobactin and Tr revealed that mycobactin does not serve as a growth factor but as a carrier of growth-essential iron which mycobactin (as contrasted to Tr) provides to tubercle bacilli in a utilizable form.

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The growth of tubercle bacilli in serum samples of untreated animals depends upon the availability of ionic iron which serves as a growth factor in supporting bacillary multiplication. The amount of available iron in serum is determined by the ratio between iron-saturated and iron-free transferrin; a low value for the ratio is associated with tuberculostasis (e.g., human serum, 0.4), whereas a high value is associated with the growth-supporting quality (e.g., guinea pig serum, 5.6). The treatment of guinea pigs with lipopolysaccharide of Escherichia coli or tuberculous cell wall material consistently and significantly reduced serum iron levels; a similar but less striking effect was observed in BCG-vaccinated animals. Pronounced differences were observed in the time of appearance and duration of serum hypoferremia; in lipopolysaccharide-treated animals, it appeared in 1 day and lasted for several days, whereas in BCG-vaccinated animals it appeared in about 2 weeks and lasted for much longer time periods. The induced hypoferremia was always associated with the concomitant development of serum tuberculostasis which could be neutralized by the addition of iron. These results indicate, therefore, that the mechanism of induced serum tuberculostasis in lipopolysaccharide- or tuberculous cell wall-treated and BCG-vaccinated guinea pigs is the same as that present in tuberculostatic sera of untreated animals.

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