Intracellular trafficking of Brucella abortus in J774 macrophages

Brucella abortus is a facultative intracellular bacterium capable of surviving inside professional and nonprofessional phagocytes. The microorganism remains in membrane-bound compartments that in several cell types resemble modified endoplasmic reticulum structures. To monitor the intracellular transport of B. abortus in macrophages, the kinetics of fusion of phagosomes with preformed lysosomes labeled with colloidal gold particles was observed by electron microscopy. The results indicated that phagosomes containing live B. abortus were reluctant to fuse with lysosomes. Furthermore, newly endocytosed material was not incorporated into these phagosomes. These observations indicate that the bacteria strongly affect the normal maturation process of macrophage phagosomes. However, after overnight incubation, a significant percentage of the microorganisms were found in large phagosomes containing gold particles, resembling phagolysosomes. Most of the Brucella bacteria present in phagolysosomes were not morphologically altered, suggesting that they can also resist the harsh conditions prevalent in this compartment. About 50% colocalization of B. abortus with LysoSensor, a weak base that accumulates in acidic compartments, was observed, indicating that the B. abortus bacteria do not prevent phagosome acidification. In contrast to what has been described for HeLa cells, only a minor percentage of the microorganisms were found in compartments labeled with monodansylcadaverine, a marker for autophagosomes, and with DiOC6 (3,3'-dihexyloxacarbocyanine iodide), a marker for the endoplasmic reticulum. These results indicate that B. abortus bacteria alter phagosome maturation in macrophages. However, acidification does occur in these phagosomes, and some of them can eventually mature to phagolysosomes.     

Brucella abortus Genes Identified following Constitutive Growth and Macrophage Infection

The chronicity of Brucella abortus infection in humans and animals depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage. Although no human vaccine exists for Brucella, vaccine development in other bacteria has been based on deletions of selective nutritional as well as regulatory systems. Our goal is to develop a vaccine for Brucella. To further this aim, we have used a green fluorescent protein (GFP) reporter system to identify constitutively and intracellularly induced B. abortus genes. Constitutively producing gfp clones exhibited sequence homology with genes associated with protein synthesis and metabolism (initiation factor-1 and tRNA ribotransferase) and detoxification (organic hydroperoxidase resistance). Of greater interest, clones negative for constitutively produced gfp in agar were examined by fluorescence microscopy to detect promoter activity induced within macrophages 4 and 24 h following infection. Bacterial genes activated in macrophages 4 h postinfection appear to be involved in adapting to intracellular environmental conditions. Included in this group were genes for detoxification (lactoglyglutathione lyase gene), repair (formamidopyrimidine-DNA glycosylase gene), osmotic protection (K(+) transport gene), and site-specific recombination (xerD gene). A gene involved in metabolism and biosynthesis (deoxyxylulose 5' phosphate synthase gene) was also identified. Genes activated 24 h following infection were biosynthesis- and metabolism-associated genes (iron binding protein and rhizopine catabolism). Identification of B. abortus genes that are activated following macrophage invasion provides insight into Brucella pathogenesis and thus is valuable in vaccine design utilizing selective targeted deletions of newly identified Brucella genes.     

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.     

Effects of cytokines on intracellular growth of Brucella abortus.

Interleukin 1 alpha (IL-1 alpha), IL-2, IL-4, IL-6, gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and granulocyte macrophage colony-stimulating factor (GM-CSF) were tested for their abilities to alter the growth of Brucella abortus in BALB/c J774A.1 murine macrophages. IL-1 alpha, IL-4, IL-6, tumor necrosis factor alpha, and granulocyte macrophage-colony-stimulating factor had no consistent or significant effect on the growth of the avirulent B. abortus strain 19. In contrast, the addition of either IFN-gamma or IL-2 at 100 U/ml to the macrophage cultures resulted in a significant reduction in the number of intracellular bacteria that was not attributable to decreased infection rates. With IL-2, the reduction was most often apparent only during the first 24 h after infection, while inhibition with IFN-gamma was apparent throughout the culture period of 48 h. The addition of either IL-2 or IFN-gamma to macrophage cultures also resulted in reduced intracellular CFU of the virulent B. abortus strain 2308 and the attenuated rough mutant B. abortus strain RB51. Inhibition of intracellular growth was not augmented by combinations of cytokines. Additional studies with IFN-gamma and IL-2 indicated that they could mediate the inhibition of intracellular growth of B. abortus in resident and thioglycolate broth-induced BALB/c peritoneal macrophages and in splenic macrophages. IFN-gamma also inhibited bacterial growth when added after infection of the macrophages, although the magnitude of the antibrucellae effects was less than that when it was added before infection. Furthermore, the maximal inhibitory effect was sustained only when IFN-gamma remained in the cultures after infection of the macrophages.     

Residual virulence of Brucella abortus in the absence of the cytochrome bc(1)complex in a murine model in vitro and in vivo

To maintain survival in macrophages, Brucella must overcome a hostile phagosomal environment defined as low pH, limited nutrition and low oxygen tension. The specific mechanisms utilized by Brucella to surmount such unfavorable environmental factors in phagosomes are not well understood. In general, to adapt to a change in environmental oxygen tension, bacteria use different terminal oxidases that have different oxygen affinity. To survive in phagosomes where low oxygen tension exists, Brucella, like other bacteria, may require high oxygen affinity terminal oxidases that can accept electrons through a cytochrome bc(1)complex dependent or independent pathway. Using a Brucella abortus cytochrome bc(1)complex deficient mutant, delta fbcF, the requirement for a high oxygen affinity terminal oxidase governed by the cytochrome bc(1)complex dependent pathway was tested. The number of cfu from RAW 264.7 macrophage cells and spleens of BALB/c mice infected with wild-type or the cytochrome bc(1)complex deficient mutant was similar during the course of infection. These results suggest that B. abortus contains no essential terminal oxidase utilized at low oxygen tension in phagosomes requiring the cytochrome bc(1)complex. Alternatively, other branched cytochrome bc(1)complex independent respiratory mechanisms that contain the high oxygen affinity terminal oxidases likely exist to facilitate Brucella survival in phagosomes. This is the first investigation regarding the Brucella respiratory system at the molecular level and the involvement of a respiratory system in Brucella pathogenesis.     

Effects of gamma interferon and indomethacin in preventing Brucella abortus infections in mice.

Increased resistance to infection with Brucella abortus 2308 resulted when recombinant murine gamma interferon (rMuIFN-gamma) was given to mice both before and during infection but not when given only before infection. Mice given rMuIFN-gamma had enhanced peritoneal and splenic macrophage bactericidal activity against B. abortus. Treatment of mice with rMuIFN-gamma plus indomethacin did not further enhance resistance to infection or macrophage bactericidal activity compared with that after treatment of mice with rMuIFN-gamma alone.     

Interleukin-10 downregulates protective immunity to Brucella abortus.

In vivo neutralization of interleukin-10 (IL-10) with an anti-IL-10 monoclonal antibody resulted in up to 10-fold fewer bacteria in the spleens of BALB/c mice infected with the virulent Brucella abortus strain 2308. In vitro neutralization of endogenous IL-10 in brucella antigen-stimulated cultures of splenocytes from infected mice resulted in increased gamma interferon production in these cultures, whereas exogenous recombinant IL-10 inhibited the ability of peptone-elicited peritoneal macrophages to control intracellular brucellae. These data suggest that IL-10 may be downregulating the immune response to B. abortus by affecting both macrophage effector function and the production of the protective Th1 cytokine gamma interferon.     

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.     

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.     

Protein synthesis in Brucella abortus induced during macrophage infection.

Brucella abortus is a facultative, intracellular, pathogenic bacterium that replicates within macrophages and resists macrophage microbicidal mechanisms. To study gene expression and to elucidate the defense mechanisms used by B. abortus to resist destruction within macrophages, protein synthesis by B. abortus was examined by pulse-labeling techniques during intracellular growth within J774A.1, a macrophage-like cell line. Prominent changes observed include increased synthesis of Brucella proteins with estimated molecular masses of 62, 28, 24, and 17 kDa. The 62-kDa protein was identified by immunoprecipitation analysis as Hsp62, a GroEL homolog. A protein of 60 kDa was expressed during acid shock and may represent a modified form of Hsp62. The 28- and 17-kDa proteins have not been observed under any in vitro stress condition and presumably represent macrophage-specific induction. The 24-kDa protein comigrates with an unidentified protein induced by acid shock, designated Asp24. Expression of Asp24 is optimal at pH values below 4.0 and within the first 3 h following a shift from pH 7.3 to 3.8. This corresponds directly with a period of optimal bacterial survival at a reduced pH and suggests an active role for this protein in resistance to such environments. The identification of these gene products and the mechanisms controlling their expression is an important step in understanding the resistance of Brucella spp. to intracellular destruction within macrophages.     

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.     

Identification and comparison of macrophage-induced proteins and proteins induced under various stress conditions in Brucella abortus.

Brucella abortus is a facultative intracellular pathogen of cattle and humans that is capable of survival inside macrophages. In order to understand how B. abortus copes with the conditions during intracellular growth in macrophages, the protein synthesis pattern of the bacteria grown inside bovine macrophages has been compared with that of bacteria grown in the cell culture medium by two-dimensional polyacrylamide gel electrophoresis. Approximately 24 new proteins that are not detected in the bacteria grown in the cell culture medium have been induced during intracellular growth in macrophages. In contrast, approximately 50 proteins that were expressed during growth in cell culture medium were completely repressed during intracellular growth. The level of expression of 19 proteins increases while that of 54 proteins decreases during intracellular growth. To understand these results, the protein synthesis pattern of B. abortus during intracellular growth was compared with those during other stress conditions. Under each stress condition studied, several new proteins were induced that were not present during regular growth conditions. Comparison of the protein synthesis pattern of B. abortus during intracellular growth with those obtained under various stress conditions has indicated that the response to intracellular growth was not just a simple sum of stress conditions studied so far.     

A beneficial aspect of a CB1 cannabinoid receptor antagonist: SR141716A is a potent inhibitor of macrophage infection by the intracellular pathogen Brucella suis

The psychoactive component of marijuana, delta9-tetrahydrocannabinol (THC) suppresses different functions of immunocytes, including the antimicrobicidal activity of macrophages. The triggering of cannabinoid receptors of CB1 and CB2 subtypes present on leukocytes may account for these effects. We investigated the influence of specific CB1 or CB2 receptor antagonists (SR141716A and SR144528, respectively) and nonselective CB1/CB2 cannabinoid receptor agonists (CP55,940 or WIN 55212-2) on macrophage infection by Brucella suis, an intracellular gram-negative bacteria. None of the compounds tested affected bacterial phagocytosis. By contrast, the intracellular multiplication of Brucella was dose-dependently inhibited in cells treated with 10-500 nM SR141716A and 1 microM SR141716A-induced cells exerted a potent microbicidal effect against the bacteria. SR144528, CP55,940, or WIN 55212-2 did not affect (or slightly potentiated) the growth of phagocytized bacteria. However, CP55,940 or WIN 55212-2 reversed the SR141716A-mediated effect, which strongly suggested an involvement of macrophage CB1 receptors in the phenomenon. SR141716A was able to pre-activate macrophages and to trigger an activation signal that inhibited Brucella development. The participation of endogenous cannabinoid ligand(s) in Brucella infection was discussed. Finally, our data show that SR141716A up-regulates the antimicrobial properties of macrophages in vitro and might be a pharmaceutical compound useful for counteracting the development of intramacrophagic gram-negative bacteria.     

virB-Mediated survival of Brucella abortus in mice and macrophages is independent of a functional inducible nitric oxide synthase or NADPH oxidase in macrophages

The Brucella abortus virB locus is required for establishing chronic infection in the mouse. Using in vitro and in vivo models, we investigated whether virB is involved in evasion of the bactericidal activity of NADPH oxidase and the inducible nitric oxide synthase (iNOS) in macrophages. Elimination of NADPH oxidase or iNOS activity in macrophages in vitro increased recovery of wild-type B. abortus but not recovery of a virB mutant. In mice lacking either NADPH oxidase or iNOS, however, B. abortus infected and persisted to the same extent as it did in congenic C57BL/6 mice up until 60 days postinfection, suggesting that these host defense mechanisms are not critical for limiting bacterial growth in the mouse. A virB mutant did not exhibit increased survival in either of the knockout mouse strains, indicating that this locus does not contribute to evasion of nitrosative or oxidative killing mechanisms in vivo.     

MyD88, but not toll-like receptors 4 and 2, is required for efficient clearance of Brucella abortus

It is not clear how the host initially recognizes and responds to infection by gram-negative pathogenic Brucella spp. It was previously shown (D. S. Weiss, B. Raupach, K. Takeda, S. Akira, and A. Zychlinsky, J. Immunol. 172:4463-4469, 2004) that the early macrophage response against gram-negative bacteria is mediated by Toll-like receptor 4 (TLR4), which signals in response to lipopolysaccharide (LPS). Brucella, however, has a noncanonical LPS which does not have potent immunostimulatory activity. We evaluated the kinetics of TLR4 activation and the cytokine response in murine macrophages after Brucella infection. We found that during infection of macrophages, Brucella avoids activation of TLR4 at 6 h but activates TLR4, TLR2, and myeloid differentiation factor 88 (MyD88) at 24 h postinfection. Interestingly, even though its activation is delayed, MyD88 is important for host defense against Brucella infection in vivo, since MyD88(-/-) mice do not clear the bacteria as efficiently as wild-type, TLR4(-/-), TLR2(-/-), or TLR4/TLR2(-/-) mice.     

Absence of evidence for the participation of the macrophage cellular prion protein in infection with Brucella suis

Brucella spp. are stealthy bacteria that enter host cells without major perturbation. The molecular mechanism involved is still poorly understood, although numerous studies have been published on this subject. Recently, it was reported that Brucella abortus utilizes cellular prion protein (PrP(C)) to enter the cells and to reach its replicative niche. The molecular mechanisms involved were not clearly defined, prompting us to analyze this process using blocking antibodies against PrP(C). However, the behavior of Brucella during cellular infection under these conditions was not modified. In a next step, the behavior of Brucella in macrophages lacking the prion gene and the infection of mice knocked out for the prion gene were studied. We observed no difference from results obtained with the wild-type control. Although some contacts between PrP(C) and Brucella were observed on the surface of the cells by using confocal microscopy, we could not show that Brucella specifically bound recombinant PrP(C). Therefore, we concluded from our results that prion protein (PrP(C)) was not involved in Brucella infection.     

Deletion of znuA virulence factor attenuates Brucella abortus and confers protection against wild-type challenge

znuA is known to be an important factor for survival and normal growth under low Zn(2+) concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 (DeltaznuA) was constructed and found to be lethal in low-Zn(2+) medium. When used to infect macrophages, DeltaznuA B. abortus showed minimal growth. Further study with DeltaznuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the DeltaznuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain.     

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.     

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.