Mice lacking components of adaptive immunity show increased Brucella abortus virB mutant colonization

The Brucella abortus type IV secretion system (T4SS), encoded by the virB genes, is essential for survival in mononuclear phagocytes in vitro. In the mouse model, a B. abortus virB mutant was initially able to colonize the spleen at the level of the wild type for approximately 3 to 5 days, which coincided with the development of adaptive immunity. To investigate the relationship between survival in macrophages cultivated in vitro and persistence in tissues in vivo, we tested the ability of mutant mice lacking components of adaptive immunity to eliminate the virB mutant from the spleen during a mixed infection with the B. abortus wild type. Ifng(-/-) or beta(2)m(-/-) mice were able to clear the virB mutant to the same degree as control mice. However, spleens of Rag1(-/-) mice and Igh6(-/-) mice were more highly colonized by the virB mutant than control mice after 14 to 21 days, suggesting that, in these mice, there is not an absolute requirement for the T4SS to mediate persistence of B. abortus in the spleen. Macrophages isolated from Igh6(-/-) mice killed the virB mutant to the same extent as macrophages from control mice, showing that the reduced ability of these mice to clear the virB mutant from the spleen does not correlate with diminished macrophage function in vitro. These results show that in the murine model host, the T4SS is required for persistence beyond 3 to 5 days after infection and suggest that the T4SS may contribute to evasion of adaptive immune mechanisms by B. abortus.     

Inactivation of the type IV secretion system reduces the Th1 polarization of the immune response to Brucella abortus infection

The Brucella abortus type IV secretion system (T4SS), encoded by the virB operon, is essential for establishing persistent infection in the murine reticuloendothelial system. To gain insight into the in vivo interactions mediated by the T4SS, we compared host responses elicited by B. abortus with those of an isogenic mutant in the virB operon. Mice infected with the B. abortus virB mutant elicited smaller increases in serum levels of immunoglobulin G2a, gamma interferon (IFN-gamma), and interleukin-12p40 than did mice infected with wild-type B. abortus. Despite equal bacterial loads in the spleen, at 3 to 4 days postinfection, levels of IFN-gamma were higher in mice infected with wild-type B. abortus than in mice infected with the virB mutant, as shown by real-time PCR, intracellular cytokine staining, and cytokine levels. IFN-gamma-producing CD4(+) T cells were more abundant in spleens of mice infected with wild-type B. abortus than in virB mutant-infected mice. Similar numbers of IFN-gamma-secreting CD8(+) T cells were observed in the spleens of mice infected with B. abortus 2308 or a virB mutant. These results suggest that early differences in cytokine responses contribute to a stronger Th1 polarization of the immune response in mice infected with wild-type B. abortus than in mice infected with the virB mutant.     

The Brucella abortus Cu,Zn superoxide dismutase is required for optimal resistance to oxidative killing by murine macrophages and wild-type virulence in experimentally infected mice

Two-dimensional gel electrophoretic analysis of cell lysates from Brucella abortus 2308 and the isogenic hfq mutant Hfq3 revealed that the RNA binding protein Hfq (also known as host factor I or HF-I) is required for the optimal stationary phase production of the periplasmic Cu,Zn superoxide dismutase SodC. An isogenic sodC mutant, designated MEK2, was constructed from B. abortus 2308 by gene replacement, and the sodC mutant exhibited much greater susceptibility to killing by O(2)(-) generated by pyrogallol and the xanthine oxidase reaction than the parental 2308 strain supporting a role for SodC in protecting this bacterium from O(2)(-) of exogenous origin. The B. abortus sodC mutant was also found to be much more sensitive to killing by cultured resident peritoneal macrophages from C57BL6J mice than 2308, and the attenuation displayed by MEK2 in cultured murine macrophages was enhanced when these phagocytes were treated with gamma interferon (IFN-gamma). The attenuation displayed by the B. abortus sodC mutant in both resting and IFN-gamma-activated macrophages was alleviated, however, when these host cells were treated with the NADPH oxidase inhibitor apocynin. Consistent with its increased susceptibility to killing by cultured murine macrophages, the B. abortus sodC mutant also displayed significant attenuation in experimentally infected C57BL6J mice compared to the parental strain. These experimental findings indicate that SodC protects B. abortus 2308 from the respiratory burst of host macrophages. They also suggest that reduced SodC levels may contribute to the attenuation displayed by the B. abortus hfq mutant Hfq3 in the mouse model.     

Growth of Brucella abortus in macrophages from resistant and susceptible mouse strains

C57Bl/10 mice have a superior ability to control chronic infections with virulent strains of the intracellular bacteria Brucella abortus compared with BALB/c mice. While a number of differences in the cytokines produced by lymphocytes following infection of these two strains of mice have been shown, macrophages have not been evaluated for their role in conveying relative resistance. The importance of macrophages in control of brucella infections is demonstrated by the observations that intracellular survival of various strains of B. abortus directly correlates with their virulence in vivo, and the ability of macrophages to control brucellae in vitro has been shown to correlate with a brucella-resistant phenotype in ruminants. While both BALB/c and C57Bl are Nramp-susceptible mouse strains, additional differences in macrophage function outside of the Nramp1 gene effects could influence susceptibility to brucellosis. The studies conducted here comparing the ability of macrophages from C57Bl/10 and BALB/c mice indicate that the macrophages from resistant mice did not control intracellular growth of B. abortus strain 2308 more efficiently than those from the susceptible mice, either in the absence of, or following, interferon-gamma activation or iron supplementation. A number of different conditions for culturing macrophages were evaluated to rule out the influence of antibiotics on the conclusions drawn from the results.     

Brucella abortus virB12 is expressed during infection but is not an essential component of the type IV secretion system

The Brucella abortus virB operon, consisting of 11 genes, virB1 to virB11, and two putative genes, orf12 (virB12) and orf13, encodes a type IV secretion system (T4SS) that is required for intracellular replication and persistent infection in the mouse model. This study was undertaken to determine whether orf12 (virB12) encodes an essential part of the T4SS apparatus. The virB12 gene was found to encode a 17-kDa protein, which was detected in vitro in B. abortus grown to stationary phase. Mice infected with B. abortus 2308 produced an antibody response to the protein encoded by virB12, showing that this gene is expressed during infection. Expression of virB12 was not required for survival in J774 macrophages. VirB12 was also dispensable for the persistence of B. abortus, B. melitensis, and B. suis in mice up to 4 weeks after infection, since deletion mutants lacking virB12 were recovered from splenic tissue at wild-type levels. These results show that VirB12 is not essential for the persistence of the human-pathogenic Brucella spp. in the mouse and macrophage models of infection.     

Lipid raft microdomains mediate class A scavenger receptor-dependent infection of Brucella abortus

Brucella abortus is a facultative intracellular bacterium that can survive inside macrophages. Intracellular replication of B. abortus requires the VirB complex, which is highly similar to the conjugative DNA transfer system. In this study, we showed that a class A scavenger receptor (SR-A) of macrophages is required to internalize B. abortus and contributes to the establishment of bacterial infection in mice. Macrophages from SR-A-deficient mice inhibited internalization and intracellular replication of both wild type strain and the virB4 mutant, and that bacterial proliferation was inhibited in SR-A-deficient mice. Adding lipopolysaccharide from B. abortus and Salmonella enterica serovar Typhimurium, but not from Escherichia coli, to macrophages inhibited bacterial internalization. VirB-dependent bacterial internalization induced localization of SR-A into detergent-resistant membrane lipid rafts. These results indicate that B. abortus internalizes into macrophages by using SR-A as a receptor and that the VirB type IV secretion system of B. abortus regulates signal transduction dependent on SR-A to form replicative phagosomes, and which is mediated by lipid rafts.     

Brucella abortus HtrA functions as an authentic stress response protease but is not required for wild-type virulence in BALB/c mice

A second mutation has recently been identified in the previously described Brucella abortus htrA mutant PHE1. As a result of this finding, a new B. abortus htrA mutant, designated RWP11, was constructed to evaluate the biological function of the Brucella HtrA protease. RWP11 is more sensitive to oxidative killing in vitro and less resistant to killing by cultured murine neutrophils and macrophages than the virulent parental strain 2308 but is not attenuated in BALB/c mice through 4 weeks postinfection. The in vitro phenotype of B. abortus RWP11 is consistent with the proposed function of bacterial HtrA proteases as components of a secondary line of defense against oxidative damage. The in vivo phenotype of this mutant, however, indicates that, unlike the corresponding Salmonella and Yersinia proteins, Brucella HtrA does not play a critical role in virulence in the mouse model.     

Identification of genes required for chronic persistence of Brucella abortus in mice

The genetic basis for chronic persistence of Brucella abortus in lymphoid organs of mice, cows, and humans is currently unknown. We identified B. abortus genes involved in chronic infection, by assessing the ability of 178 signature-tagged mutants to establish and maintain persistent infection in mice. Each mutant was screened for its ability to colonize the spleens of mice at 2 and 8 weeks after inoculation. Comparison of the results from both time points identified two groups of mutants attenuated for chronic infection in mice. The first group was not recovered at either 2 or 8 weeks postinfection and was therefore defective in establishing infection. Mutants in this group carried transposon insertions in genes involved in lipopolysaccharide biosynthesis (wbkA), in aromatic amino acid biosynthesis, and in type IV secretion (virB1 and virB10). The second group, which was recovered at wild-type levels 2 weeks postinfection but not 8 weeks postinfection was able to establish infection but was unable to maintain chronic infection. One mutant in this group carried a transposon insertion in a gene with homology to gcvB of Mycobacterium tuberculosis, encoding glycine dehydrogenase, an enzyme whose activity is increased during the state of nonreplicating persistence. These results suggest that some mechanisms for long-term persistence may be shared among chronic intracellular pathogens. Furthermore, identification of two groups of genes, those required for initiating infection and those required only for long-term persistence, suggests that B. abortus uses distinct sets of virulence determinants to establish and maintain chronic infection in mice.     

Redox-sensitive regulation of macrophage-inducible nitric oxide synthase expression in vitro does not correlate with the failure of apocynin to prevent lung inflammation induced by endotoxin

Reactive oxygen and nitrogen species are among the crucial mediators in the development of the pathological inflammatory process in the lungs and contribute to the damage of lung epithelium. The aim of the present study was to evaluate the potential of selected antioxidants or inhibitors of NADPH oxidase (glutathione, N-acetyl cysteine, trolox, apocynin, and diphenyleneiodonium chloride) to modulate nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression by mouse macrophages induced by lipopolysaccharide (LPS) in vitro and to evaluate the potential of apocynin to modulate the course of LPS-induced lung inflammation in vivo. All the tested drugs revealed inhibitory effects on LPS-induced NO production and iNOS expression in RAW 264.7 macrophages. Further, apocynin significantly inhibited activation of nuclear factor kappa B induced by LPS. Ex vivo, diphenyleneiodonium chloride and apocynin significantly reduced ROS production by inflammatory cells isolated from bronchoalveolar lavage fluid. In contrast, in vivo intranasal application of apocynin did not exert any significant effect on the course of lung inflammation in mice induced by LPS that was evaluated based on the accumulation of cells, interleukine-6, interleukine-12, RANTES, tumor necrosis factor-alpha, and protein concentration in bronchoalveolar lavage fluid and expression of iNOS in lung tissue. Only effected were the levels of nitrites 36 h after induction of lung inflammation that were reduced in the apocynin-treated group. In conclusion, our data suggest that the inhibitors of NADPH oxidase possess inhibitory potential against LPS-induced NO production by mouse macrophages; however, apocynin failed to reduce LPS-induced lung inflammation in mice.     

Killing of Aspergillus fumigatus by alveolar macrophages is mediated by reactive oxidant intermediates

Phagocytosis and mechanisms of killing of Aspergillus fumigatus conidia by murine alveolar macrophages (AM), which are the main phagocytic cells of the innate immunity of the lung, were investigated. Engulfment of conidia by murine AM lasts 2 h. Killing of A. fumigatus conidia by AM begins after 6 h of phagocytosis. Swelling of the conidia inside the AM is a prerequisite for killing of conidia. The contributions of NADPH oxidase and inducible nitric oxide synthase to the conidicidal activity of AM were studied using AM from OF1, wild-type and congenic p47phox(-/-) 129Sv, and wild-type and congenic iNOS(-/-) C57BL/6 mice. AM from p47phox(-/-) mice were unable to kill A. fumigatus conidia. Inhibitors of NADPH oxidase that decreased the production of reactive oxidant intermediates inhibited the killing of A. fumigatus without altering the phagocytosis rate. In contrast to NADPH oxidase, nitric oxide synthase does not play a role in killing of conidia. Corticosteroids did not alter the internalization of conidia by AM but did inhibit the production of reactive oxidant intermediates and the killing of A. fumigatus conidia by AM. Impairment of production of reactive oxidant intermediates by corticosteroids is responsible for the development of invasive aspergillosis in immunosuppressed mice.     

Membrane sorting during swimming internalization of Brucella is required for phagosome trafficking decisions

Brucella infects macrophages by swimming internalization, after which it is enclosed in macropinosomes. We investigated the role of the uptake pathway in phagosome trafficking, which remains unclear. This study found membrane sorting during swimming internalization and is essential in intracellular replication of Brucella. The B. abortus virB mutant replicated intracellularly when it was in the macropinosome established by wild-type B. abortus that retained its ability to alter phagosome trafficking. Lipid rafts-associated molecules, such as GM1 ganglioside, were selectively included into macropinosomes, but Rab5 effector early endosome autoantigen (EEA1) and lysosomal glycoprotein LAMP-1 were excluded from macropinosomes containing B. abortus induced by swimming internalization. In contrast, when the swimming internalization was bypassed by phorbol myristate acetate (PMA)-induced macropinocytosis, lipid raft-associated molecules were excluded, and EEA1 and LAMP-1 were included into macropinosomes containing bacteria. The phosphatidylinositol 3-kinase inhibitor wortmannin that inhibits PMA-induced macropinocytosis blocked internalization of virB mutant, but not of wild-type of B. abortus and wortmannin treatment did not affect intracellular replication. Our results suggest that membrane sorting requires swimming internalization of B. abortus and decides the intracellular fate of the bacterium, and that Brucella -induced macropinosome formation is a different mechanism from PMA-induced macropinocytosis.     

Legionella pneumophila growth restriction in permissive macrophages cocultured with nonpermissive lipopolysaccharide-activated macrophages.

Macrophages can be activated by lipopolysaccharides (LPS) from gram-negative bacteria to evince a number of biological activities, including increased resistance to intracellular infection by opportunistic bacteria. In the present study, intraperitoneal injection of LPS into A/J mice activated peritoneal macrophages so that they resisted subsequent in vitro infection with Legionella pneumophila. Coculture of these macrophages with those from nontreated A/J mice converted the entire population of cells from permissive to nonpermissive. This effect did not appear to be mediated by soluble factors released from the LPS-treated macrophages, since the levels of interleukins-1 and -6 and tumor necrosis factor alpha produced by the macrophages were not found to be markedly elevated at the time when the macrophages from the LPS-treated mice were most effective in converting normal macrophages to nonpermissiveness. Furthermore, macrophages from mice injected intraperitoneally with either interferon or tumor necrosis factor alpha did not evince nonpermissiveness and also did not have the ability to convert normal spleen cells to nonpermissiveness. Polymyxin B, a known inactivator of LPS activity, did not inhibit the macrophages from the LPS-treated mice from inducing this resistance. It seemed unlikely that free LPS released from the macrophages mediated this effect. The results of this study thus showed that macrophages activated by LPS in vivo can evince nonpermissiveness for Legionella growth in vitro and also can induce macrophages from normal, permissive mice to become nonpermissive for Legionella growth in vitro.     

Differential requirements for VirB1 and VirB2 during Brucella abortus infection

The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.     

Strain-dependent cytotoxic effects of endotoxin for mouse peritoneal macrophages.

The cytotoxic effects of bacterial lipopolysaccharides (LPS) on mouse leukocytes have been examined in vivo and in vitro. Intraperitoneal injection of LPS into C57BL/6 mice greatly reduced the recovery of mononuclear cells; LPS was cytotoxic for macrophages, but had a mitogenic effect on lymphocytes. Similar effects of LPS on peritoneal leukocytes were observed in vitro. When monolayers of adherent peritoneal cells were studied in vitro, cytotoxicity was also observed, suggesting that the effect of LPS on macrophages is direct and does not require participation by lymphocytes. Entirely different results were obtained when peritoneal macrophages from LPS-resistant C3H/HeJ mice were studied. LPS failed to activate lymphocytes and was not cytotoxic for macrophages in vitro or in vivo. The effect of LPS on polymorphonuclear leukocytes appeared to be the same in all mouse stains studied. Lipid A was shown to be the most biologically active portion of the LPS molecule. Whereas polysaccharide-deficient endotoxins extracted from rough mutants of Salmonella typhimurium were cytotoxic for macrophages in vitro, polysaccharides that lacked esterified fatty acids did not exhibit this activity. Since LPS may mediate its effects through affinity for mammalian cell membranes, the cellular unresponsiveness of C3H/H3J mice to LPS may reflect an inability of cells from LPS-resistant strains to interact with LPS at the membrane level.     

Killing of Mycobacterium smegmatis by macrophages from genetically susceptible and resistant mice

The bactericidal function of macrophages was investigated in congenic mice expressing the phenotype of susceptibility (B10.A, Bcgs) or resistance (B10.ABcgr) to mycobacterial infection. When splenic and peritoneal macrophages from these two mouse strains were infected in vitro with Mycobacterium smegmatis, the Bcgr macrophages were shown to inactivate M. smegmatis more efficiently than their Bcgs congenic counterparts. The mechanisms of this superior antimycobacterial activity was studied further. Addition of catalase did not abolish killing to a significant degree in either allelic type of macrophage, suggesting that hydrogen peroxide production was not involved in the killing activity controlled by the Bcg gene. Activation of Bcgs macrophages by exposure to crude lymphokines rendered them equally as efficient as their Bcgr counterparts in their capacity to destroy M. smegmatis. This finding suggests that both the genetically resistant and susceptible macrophages have the potential to kill M. smegmatis in vitro. This potential is expressed constitutively by the Bcgr but not Bcgs macrophages and can be induced, by lymphokine treatment, in the Bcgs macrophages. In a final set of experiments, the macrophage killing of M. smegmatis was evaluated as a test system to type for the Bcg gene allelic type in vitro, using a set of AXB and BXA recombinant inbred strains of mice. Results obtained show that typing of AXB/BXA recombinant inbred strains for the trait of bactericidal activity vs. M. smegmatis in vitro revealed a perfect match with the strain distribution pattern of resistance/susceptibility to Mycobacterium bovis BCG in vivo.     

The role of integrase/recombinase xerD and monofunctional biosynthesis peptidoglycan transglycosylase genes in the pathogenicity of Brucella abortus infection in vitro and in vivo

Brucella abortus clones identified previously using a green fluorescence protein reporter system after 4h macrophage infection provided insight regarding possible genes involved in early host-pathogen interaction. Among identified genes were an integrase/recombinase (xerD) gene involved in cell division, and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) gene that catalyzes the final stages of the peptidoglycan membrane synthesis. Here, we evaluate the in vitro and in vivo survival of B. abortus xerD and mtgA insertional mutants. B. abortus xerD::kan and B. abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308. Also, neither gene was required for B. abortus S2308 replication in RAW 264.7 macrophages. However, experimental evidence using interferon regulatory factor 1 knockout mice, a mouse strain highly susceptible to virulent Brucella, revealed that mice infected with B. abortus xerD::kan or B. abortus mtgA::kan survived longer than mice infected with S2308. Additionally, in immunocompetent BALB/c mice, B. abortus xerD::kan had a significantly lower level of bacterial survival when compared to S2308. Together, these results suggest that B. abortus xerD and mtgA genes play a role during the initial phase of infection in mice.     

Brucella abortus tandem repeated ATP-binding proteins, BapA and BapB, homologs of haemophilus influenzae LktB, are not necessary for intracellular survival

Brucella abortus actively secretes materials and uptakes nutrients to maintain the survival and multiplication of the bacteria in host cells. ATP-binding cassette (ABC) transporters can uptake or secrete diverse materials across the bacterial membrane, and thus, ABC transporters may be important for survival of the pathogen in the host. In the present study, the B. abortus genes encoding tandem repeated Brucella ATP-binding proteins, BapA and BapB, were identified. The deduced amino acid sequences of these two genes place BapA and BapB into group 6 containing RTX toxin transporters and cyclic beta-1,2-glucan transporters, one of 25 ABC transporter ortholog groups. One of the ortholog group 6 proteins, Haemophilus influenzae LktB, shows the highest similarity and identity with these two Brucella proteins. To test the role of these putative tandem repeated ABC transporters in Brucella pathogenesis, a bap deletion mutant was constructed and used to infect murine RAW 264.7 macrophages and mice. The number of cfu from RAW 264.7 cells and spleens of BALB/c mice infected with wild type or the bap deletion mutant was similar during the course of infection, suggesting the bap genes are not necessary to maintain the pathogenesis of B. abortus, or alternative compensatory mechanisms may exist to permit the intracellular survival of B. abortus in vitro and in vivo. This is the first molecular approach to investigate the role of putative ABC transporters classified into ortholog group 6 in Brucella pathogenesis.     

Capacity of passively administered antibody to prevent establishment of Brucella abortus infection in mice.

In contrast to immunity against some other facultative intracellular parasites, protective immunity against Brucella abortus is mediated in mice by antibodies as well as by cell-mediated immune responses. It was the purpose of this study to determine whether antibody alone would prevent infection with B. abortus. The majority (82%) of CD-1 outbred mice infected with 100 CFU of virulent B. abortus 2308 preincubated with graded quantities of an O polysaccharide-specific IgG2a monoclonal antibody (MAb) were free of infection 1. 2, 4, and 6 weeks later, based on detection limits of 13 brucellae per spleen and 39 per liver. Infection was present in 95% of control animals. Similar results were obtained with a challenge dose of 500 CFU, but with a challenge dose of 5,000 CFU, infection became established even with the highest concentration of MAb used (50 micrograms of MAb per 5,000 brucellae). Pretreatment with an O polysaccharide-specific IgG1 MAb or with convalescent-phase serum diminished but did not prevent establishment of infection by 100 CFU of B. abortus. A majority of culture-negative mice tested 6 weeks after infection were serologically negative, which could have signified either the absence of previous infection or the early elimination of infection. In an in vitro test system, all of the antibody preparations were efficient in opsonizing B. abortus. Effective killing of the organism by unelicited mouse peritoneal macrophages occurred in conventional but not in endotoxin-free medium, suggesting that activated macrophages were required for killing of opsonized B. abortus. These results emphasize the potential importance of antibodies in the immunoprophylaxis of brucellosis and suggest that the design of a successful vaccine will require the induction of antibodies not only of appropriate specificity but also of the optimal isotype for mediating protective functions.     

Mutant of Salmonella typhimurium lacking the inhibitory function for phagosome-lysosome fusion in murine macrophages.

It has recently been described that Salmonella typhimurium is capable of inhibiting phagosome-lysosome fusion in murine macrophages after ingestion. We selected a mutant of S. typhimurium lacking the phagosome-lysosome fusion inhibitory function from a collection of Tn5-insertion mutants and examined its relevance to the pathogenesis in mice. The Tn5 insertion mutant which has a defect in fusion inhibitory function was found to be significantly sensitive to the intracellular killing by murine macrophages in vitro. However, the loss of the fusion inhibitory function did not reduce the level of virulence for mice in vivo. These results demonstrated that fusion inhibition did not play a critical role in the pathogenesis of S. typhimurium although it might contribute to at least a part of the resistance against macrophage killing mechanisms.     

High levels of nitric oxide production decrease early but increase late survival of Brucella abortus in macrophages.

Nitric oxide (NO), produced by the iNOS protein, is known as a defense mechanism against various pathogens and an apoptotic inducer of cells. Apoptosis can also be a host protective mechanism against intracellular bacteria. The intracellular survival of Brucella abortus in RAW264.7 macrophages was examined under conditions of the apoptotic inducer, NO. Since B. abortus does not induce high output of NO, Escherichia coli LPS and IFN-gamma, as potential therapeutic modalities, were added to increase the expression of iNOS, and thus NO. Using 10 ng/ml E. coli LPS and 25 U/ml IFN-gamma, nitrite production was as high as 140 microM by 72 h. However, when macrophages were infected with B. abortus, the nitrite concentration was 60 microM after 72 h post infection, greater than a two-fold decrease. The number of surviving bacteria decreased, from 6 to 24 h, in the presence of nitrite accumulation. In the absence of B. abortus there was an increase in apoptotic cells at 72 h with high nitrite accumulation. In contrast, the number of macrophage apoptotic bodies decreased in the presence of B. abortus. The data suggest that: (i) NO accelerates the killing of intracellular B. abortus, but not to completion during the first 24 h of infection; (ii) B. abortus can prevent apoptosis as an advantage for bacterial survival inside macrophages and (iii) surviving intracellular bacteria then replicate steadily after 24 h. B. abortus probably expresses genes that counteract the effect of a high NO environment or activates genes to utilize NO as a nitrogen source, as the Brucella genome codes for nitric and nitrous oxide reductase genes.