American Journal of Veterinary Research 46 2 332 335 UNITED STATES

Water extracts of virulent Brucella abortus were able to inhibit phagosome-lysosome fusion in unelicited murine peritoneal macrophages following ingestion of yeast. Extracts from an avirulent strain were unable to produce a similar effect. Lipopolysaccharide from B abortus did not appear to be involved with the ability of the extracts to inhibit fusion

Brucella suis-Impaired Specific Recognition of Phagosomes by Lysosomes due to Phagosomal Membrane Modifications

Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments

Identification of the Perosamine Synthetase Gene of Brucella melitensis 16M and Involvement of Lipopolysaccharide O Side Chain in Brucella Survival in Mice and in Macrophages

Brucella organisms are facultative intracellular bacteria that may infect many species of animals as well as humans. The smooth lipopolysaccharide (S-LPS) has been reported to be an important virulence factor of these organisms, but the genetic basis of expression of the S-LPS O antigen has not yet been described. Likewise, the role of the O side chain of S-LPS in the survival of Brucella has not been clearly defined. A mini-Tn5 transposon mutant library of Brucella melitensis 16M was screened by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (MAbs) directed against the O side chain of Brucella. One mutant, designated B3B2, failed to express any O side chain as confirmed by ELISA, Western blot analysis, and colony coloration with crystal violet. Nucleotide sequence analysis demonstrated that the transposon disrupted an open reading frame with significant homology to the putative perosamine synthetase genes of Vibrio cholerae O1 and Escherichia coli O157:H7. The low G+C content of this DNA region suggests that this gene may have originated from a species other than a Brucella sp. The survival of B. melitensis mutant strain B3B2 in the mouse model and in bovine macrophages was examined. The results suggested that S-LPS or, more precisely, its O side chain is essential for survival in mice but not in macrophages

Identification of Brucella spp. genes involved in intracellular trafficking.

After uptake by host cells, the pathogen Brucella transits through early endosomes, evades phago-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum (ER). The molecular mechanisms underlying these processes are still poorly understood. To identify new bacterial factors involved in these processes, a library of 1800 Brucella melitensis 16M mini-Tn5catkm mutants was screened for intracellular survival and multiplication in HeLa cells and J774A.1 macrophages. Thirteen mutants were identified as defective for their intracellular survival in both cell types. In 12 of them, the transposon had inserted in the virB operon, which encodes a type IV-related secretion system. The preponderance of virB mutants demonstrates the importance of this secretion apparatus in the intracellular multiplication of B. melitensis. We also examined the intracellular fate of three virB mutants (virB2, virB4 and virB9) in HeLa cells by immunofluorescence. The three VirB proteins are not necessary for penetration and the inhibition of phago-lysosomal fusion within non-professional phagocytes. Rather, the virB mutants are unable to reach the replicative niche and reside in a membrane-bound vacuole expressing the late endosomal marker, LAMP1, and the sec61beta protein from the ER membrane, proteins that are present in autophagic vesicles originating from the ER

Interruption of the cydB Locus in Brucella abortus Attenuates Intracellular Survival and Virulence in the Mouse Model of Infection

Brucellosis is characterized by abortion in ruminants and a protracted undulant fever in humans, which often results in severe pathological manifestations. Scant information exists about the molecular mechanisms employed by Brucella abortus to combat host defenses or to persist and replicate within host cells. Transposon (Tn5) mutagenesis of B. abortus and the subsequent screening of mutants for sensitivity to killing in murine macrophages and in the mouse model led to the identification of mutants which were severely attenuated for intracellular survival. One group of mutants was interrupted in cydB, a gene that is part of the cydAB operon encoding cytochrome bd oxidase, which catalyzes an alternate terminal electron transport step in bacterial respiration. The elevated affinity for molecular oxygen of this enzyme in Escherichia coli has suggested that it is involved in the protection of sensitive enzymatic activities such as those of hydrogenases and nitrogenases from damage. B. abortus cydB::Tn5 strains exhibited heightened sensitivity to the respiratory inhibitors zinc and azide, highly reactive oxygen species such as hydrogen peroxide, low pH, and attenuated virulence in the mouse model of infection. Virulence was restored by an intact copy of cydAB or by B. abortus genes encoding the oxidative radical-scavenging enzyme Cu/Zn superoxide dismutase or catalase. These results suggest a bifunctional role for the products of the cydAB operon, both in preventing the buildup of oxidative free radicals and in detoxifying the intracellular compartment, thus indicating the importance of these products in preventing intracellular destruction. Intracellular conditions that favor expression of the cydAB operon are under investigation and may be linked to the acid sensitivity also observed in this strain

Intracellular Survival of Brucella spp. in Human Monocytes Involves Conventional Uptake but Special Phagosomes

Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated how B. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca(2+)- and Mg(2+)-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall