Different roles of the two high-oxygen-affinity terminal oxidases of Brucella suis: Cytochrome c oxidase, but not ubiquinol oxidase, is required for persistence in mice

The survival of Brucella suis mutant strains in mice demonstrated different roles of the two high-oxygen-affinity terminal oxidases. The cbb3-type cytochrome c oxidase was essential for chronic infection in oxygen-deficient organs. Lack of the cytochrome bd ubiquinol oxidase led to hypervirulence of bacteria, which could rely on nitrite accumulation inhibiting the inducible nitric oxide synthase of the host.     

Opsonized virulent Brucella abortus replicates within nonacidic, endoplasmic reticulum-negative, LAMP-1-positive phagosomes in human monocytes

Cells in the Brucella spp. are intracellular pathogens that survive and replicate within host monocytes. Brucella maintains persistent infections in animals despite the production of high levels of anti-Brucella-specific antibodies. To determine the effect of antibody opsonization on the ability of Brucella to establish itself within monocytes, the intracellular trafficking of virulent Brucella abortus 2308 and attenuated hfq and bacA mutants was followed in the human monocytic cell line THP-1. Early trafficking events of B. abortus 2308-containing phagosomes (BCP) were indistinguishable from those seen for control particles (heat-killed B. abortus 2308, live Escherichia coli HB101, or latex beads). All phagosomes transiently communicated the early-endosomal compartment and rapidly matured into LAMP-1(+), cathepsin D(+), and acidic phagosomes. By 2 h postinfection, however, the number of cathepsin D(+) BCP was significantly lower for live B. abortus 2308-infected cells than for either Brucella mutant strains or control particles. B. abortus 2308 persisted within these cathepsin D(-), LAMP-1(+), and acidic vesicles; however, at the onset of intracellular replication, the numbers of acidic B. abortus 2308 BCP decreased while remaining cathepsin D(-) and LAMP-1(+). In contrast to B. abortus 2308, the isogenic hfq and bacA mutants remained in acidic, LAMP-1(+) phagosomes and failed to initiate intracellular replication. Notably, markers specific for the host endoplasmic reticulum were absent from the BCPs throughout the course of the infection. Thus, opsonized B. abortus in human monocytes survives within phagosomes that remain in the endosomal pathway and replication of virulent B. abortus 2308 within these vesicles corresponds with an increase in intraphagosomal pH.     

Differential use of the two high-oxygen-affinity terminal oxidases of Brucella suis for in vitro and intramacrophagic multiplication

Expression of the high-oxygen-affinity cytochrome cbb3 and cytochrome bd ubiquinol oxidases of Brucella suis was studied in vitro and in the intramacrophagic niche, which was previously proposed to be oxygen limited. The cytochrome cbb3 oxidase was exclusively expressed in vitro, whereas the cytochrome bd oxidase was preferentially used inside macrophages and contributed to intracellular bacterial replication.     

Bacterial Terminal Oxidases

Abstract

Oxidases, as such, regardless of their source, represent a diverse and complex series of enzymes. What they have in common is the ability to react with molecular oxygen, activate it chemically (in a manner which is still not understood), and utilize the “activated atoms of oxygen” primarily as electron acceptors. Should the “activated oxygen atoms” be used directly for oxygenating substrate molecules, such as hydrocarbons, then according to the conventions used today, the enzyme is termed an oxygenase rather than an oxidase. The subject of oxygenases is far too complex to be reviewed in any detailed treatment of oxidases. All oxidases serve as electron acceptors for specific dehydrogenation reactions that are carried out by the multitude of dehydrogenases that are found in tissues as well as in bacteria. The major end product that results from the oxidase reaction is either H₂O or H₂O₂. The oxidases can be (1) simple flavoprotein-containing enzymes, such as the glucose oxidase or the D- and L-amino acid oxidases, (2) metalloflavoprotein-containing enzymes, such as the xanthine oxidase which in addition to flavin contains both molybdenum and nonheme iron, (3) the heme-containing oxidases which essentially are free of flavoprotein yet carry out a peroxidase-oxidase type of reaction, and finally (4) the heme-containing and flavoprotein-free cytochrome (or terminal) oxidase molecule which may contain as many as two heme “a” components (multiple heme iron), copper protein, and the active form of which requires an enzyme complex containing some phospholipid.^{1 4 5} It is this latter group, the cytochrome or terminal oxidases, that will be considered in this review, particularly what is known about them in bacterial systems. The cytochrome or terminal oxidases are usually membrane-bound entities, play a major role in electron transport, and are very important in the bioenergetic mechanism of aerobic cells that allow for respiration.     

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.     

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.     

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.     

Cytochromes c-dependent aerobic respiration of Paracoccus denitrificans

Terminal parts of the respiratory chain of Paracoccus denitrificans containing cytochromes c have been investigated through the use of NNN′Nprime;-tetramethyl-p-phenylenediamine (TMPD) as an electron donor. The cells have been shown to possess two major membrane bound TMPD oxidases characterized by their different sensitivities to cyanide. The more sensitive one, identified as cytochrome aa₃, appears to be produced under oxygen-rich growth conditions, whereas a less sensitive and as yet uncharacterized enzyme accompanied with only moderate amounts of cytochrome aa₃ functions when oxygen becomes limiting. The soluble periplasmic cytochrome cd₁ does not contribute significantly to the total oxygen consumption rate, although its maximal catalytic capacity may be high. The fact thap the activity of alternative TMPD oxidase is not proportional to cytochrome o content is discussed in relation to possible occurrence of two distinct cytochrome o-type terminal oxidases.     

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.     

Brucella abortus deficient in copper/zinc superoxide dismutase is virulent in BALB/c mice.

The gene encoding the Cu/Zn superoxide dismutase (SOD) of Brucella abortus strain 2308 was identified in a Brucella genomic library utilizing a combination of Western blotting and native gel electrophoresis. The Cu/Zn SOD gene was inactivated in vitro by ligation of a kanamycin resistance gene into the open reading frame encoding SOD. The plasmid born construct was introduced back into B. abortus by electroporation. Replacement of the wild-type Cu/Zn SOD by recombination was demonstrated by showing that both the KnR gene and the Cu/Zn SOD gene hybridized to the same band in a Southern analysis of genomic DNA. In addition, KnR strains were deficient in Cu/Zn SOD activity as assessed by lack of Cu/Zn SOD activity on a native gel and by lack of reactivity with specific serum in a Western analysis. Either strain 2308 or the Cu/Zn SOD deficient mutant injected intraperitoneally into BALB/c mice, exhibited no differences in their ability to colonize the spleen at 7 and 28 days post-inoculation. Thus, the inability to produce Cu/Zn SOD by B. abortus does not significantly impair its virulence in mice.     

Identification and characterization of a Brucella abortus ATP-binding cassette transporter homolog to Rhizobium meliloti ExsA and its role in virulence and protection in mice

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively. Additionally, B. abortus ExsA, like R. meliloti and M. loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placed B. abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. In R. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed. Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA. Decreased survival in mice of the Brucella DeltaexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, the B. abortus exsA deletion mutant was used as a live vaccine. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51.     

Biochemical, genetic and ultrastructural defects in a mitochondrial mutant (ER-3) of Neurospora crassa with senescence phenotype

The structural and functional abnormalities in a new respiratory deficient, mitochondrial senescence mutant ER-3 of Neurospora crassa are described. The mitochondrial mutant, which grows at a rate of only 10% of that of the wild type, was found deficient in all three cytochromes, and completely lacking in cytochromes aa3. Cytochrome oxidase activity in the mutant mitochondria was only about 5% of the wild type mitochondria. However, the total whole cell respiration rate of the mutant was 33% greater than that of the wild type, while the cyanide-resistant respiration rates were equal. The results of inhibitor studies clearly demonstrate that the mutant possesses a defect in one or more components of the terminal oxidase. Electron microscopic examination of whole cell sections and subsequent morphometric analysis revealed a significant (33%) reduction in membrane surface density of mitochondrial cristae in the mutant as compared with the wild type. Results of genetic and heterokaryon analyses indicate the location of mutation (ER-3) in the mitochondrial DNA. It is concluded that the senescence mutant ER-3 possesses a defect in the terminal portion of the mitochondrial respiratory apparatus. These results are consistent with previous analyses of mitochondrial DNA populations, and support the notion that obligately aerobic eukaryotic cells deficient in mitochondrial respiration necessarily exist as a result of stable heteroplasmosis and that defects in mitochondria lead to senescence in Neurospora mutant ER-3.     

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.     

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.     

Respiration of Escherichia coli in the mouse intestine

Mammals are aerobes that harbor an intestinal ecosystem dominated by large numbers of anaerobic microorganisms. However, the role of oxygen in the intestinal ecosystem is largely unexplored. We used systematic mutational analysis to determine the role of respiratory metabolism in the streptomycin-treated mouse model of intestinal colonization. Here we provide evidence that aerobic respiration is required for commensal and pathogenic Escherichia coli to colonize mice. Our results showed that mutants lacking ATP synthase, which is required for all respiratory energy-conserving metabolism, were eliminated by competition with respiratory-competent wild-type strains. Mutants lacking the high-affinity cytochrome bd oxidase, which is used when oxygen tensions are low, also failed to colonize. However, the low-affinity cytochrome bo(3) oxidase, which is used when oxygen tension is high, was found not to be necessary for colonization. Mutants lacking either nitrate reductase or fumarate reductase also had major colonization defects. The results showed that the entire E. coli population was dependent on both microaerobic and anaerobic respiration, consistent with the hypothesis that the E. coli niche is alternately microaerobic and anaerobic, rather than static. The results indicate that success of the facultative anaerobes in the intestine depends on their respiratory flexibility. Despite competition for relatively scarce carbon sources, the energy efficiency provided by respiration may contribute to the widespread distribution (i.e., success) of E. coli strains as commensal inhabitants of the mammalian intestine.     

N-terminal-capturing screening system for the isolation of Brucella abortus genes encoding surface exposed and secreted proteins

Secreted as well as surface exposed proteins are assumed to play major roles in bacterial virulence. In this report we describe the construction of an N-terminal protein-capturing system and its use for the isolation of Brucella abortus S2308 genes coding for putative surface exposed or secreted proteins. For this purpose, a cloning vector that generates gene fusions to a ribosome binding site and start codon deficient Chloramphenicol Acetyl Transferase (CAT) reporter gene was constructed and the resulting library introduced into B. abortus S2308 and virB mutant strains. Secreted translational fusions were identified by determining CAT activity in culture supernatants. Secretion was confirmed by Western Blot using a polyclonal anti-CAT antibody. A total of 864 clones were screened and 10 genes encoding putative secreted/surface exposed proteins were identified. Seven are Brucella proteins with an assigned function, whereas three are hypothetical proteins. The number of amino acid residues that promotes CAT secretion varies from 5 to 386 and no conserved motifs were detected. Secretion in a virB mutant background of some of the isolated fusion proteins was also determined. Interestingly, some hybrid proteins seemed to require a full VirB system for their secretion.     

Brucella abortus requires the heme transporter BhuA for maintenance of chronic infection in BALB/c mice

The gene annotated BAB2_1150 in the Brucella abortus 2308 genome sequence is predicted to encode a homolog of the well-characterized heme transporter ShuA of Shigella dysenteriae and accordingly has been given the designation bhuA (Brucella heme utilization). Phenotypic analysis of an isogenic bhuA mutant derived from B. abortus 2308 verified that there is a link between BhuA and the ability of the parent strain to use heme as an iron source in in vitro assays. Maximum expression of bhuA in B. abortus 2308 is observed during stationary phase when this strain in cultivated in low-iron minimal medium, and a comparison of the growth characteristics of the B. abortus bhuA mutant and 2308 in this medium suggested that heme serves as an important iron source for the parent strain during stationary phase. The B. abortus bhuA mutant HR1703 exhibits significant attenuation in cultured murine macrophages compared to strain 2308, and unlike its parent strain, the B. abortus bhuA mutant is unable to maintain a chronic spleen infection in experimentally infected BALB/c mice. These experimental findings suggest that heme and/or heme-containing proteins represent important iron sources for B. abortus 2308 during its residence in the mammalian host and that BhuA is required for efficient utilization of these iron sources.     

Evidence for a branched electron transport chain in Trypanosoma brucei

The flow of electrons the terminal oxidases present in the bloodstream and procyclic trypomastigotes of Trypanosoma brucei LUMP 1026 has been investigated by the use of salicylhydroxamic acid (SHAM) and cyanide. Respiration in bloodstream trypomastigotes was completely inhibited by 0.5 mM SHAM with a Ki below 10 microM. The Ki for SHAM in procyclic trypomastigotes was 70 microM. In procyclic trypomastigotes there are at least three terminal oxidases of which the two major ones are cytochrome aa3 oxidase, sensitive to cyanide inhibition, and alpha-glycerophosphate oxidase (GPO), sensitive to SHAM inhibition. These two oxidases contribute 60 and 30%, respectively, to total cell respiration. Inhibition of the cytochrome system with cyanide causes an increase in the flow of electrons through the GPO system, and inhibition of the GPO system with SHAM stimulates electron flow in the cytochrome system. Succinate oxidation in the mitochondrial fraction is partially inhibited by SHAM and this SHAM-sensitive respiration is not inhibited by antimycin A. The kinetic data of respiration by procyclic trypomastigotes fit a model proposed by Bahr and Bonner to determine the maximum rates of two competing electron transport pathways. It is concluded that the electron transport chain in T. brucei is branched.