PubMed · 14149376
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G PACHECO, M DE ANDRADE. 1963. [SUPPURATED BRUCELLAR THYROIDITIS].. https://pubmed.ncbi.nlm.nih.gov/14149376/
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The genus Brucella comprises important human and animal pathogens, as well as numerous environmental and symbiotic species. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, plays a crucial role in bacterial physiology and host interactions. In this study, the structures of lipid A, the hydrophobic anchor of lipopolysaccharide, isolated from two plant-associated strains, Brucella cytisi ESC1ᵀ and Brucella lupini LUP21ᵀ, were presented. Lipid A preparations were structurally characterized using chemical methods, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The obtained results indicated that both lipid A molecules have almost identical structures. Their sugar backbones consist exclusively of 2,3-diamino-2,3-dideoxy-d-glucose (d-GlcpN3N). Phosphate residues were connected to distal and proximal GlcpN3N in approximately half of the lipid A molecules. Fatty acid analysis revealed the presence of C14:0 (3-OH), C16:0 (3-OH), and traces of C18:0 (3-OH). All of these were primary fatty substituents of the sugar backbone and were amide-linked residues. Lactobacillic acid C19:0cyc and 27-hydroxyoctacosanoic acid (C28:0 (27-OH)) were found as ester-linked secondary acyl residues. In turn, C28:0 (27-OH) was partly esterified by a 3-hydroxybutyroyl residue. Two unsubstituted 3-hydroxyfatty acids were linked exclusively to the proximal d-GlcpN3N residue. It was pointed out that sequences of putative genes encoding enzymes required for lipid A biosynthesis and genes encoding specific enzymes involved in structural modifications of lipid A occurring in the genomes of both bacterial species are almost identical. The high sequence similarity of these proteins reflects the observed similarities in the lipid A structures in both investigated Brucella species.
In mice, nitric oxide (NO) production by inducible NO synthase (iNOS), is a component of the control of Brucella infection. In humans, the involvement of iNOS in infection is still a matter of debate. Based on in vitro experiments, it was recently postulated that in humans, Brucella infection tends to become chronic because NO cannot exert its deleterious effect. In fact, conditions allowing NO production by human macrophages in culture are poorly defined, rendering the in vitro study of NO function difficult. Using DFGiNOS U937 macrophagic cells engineered to produce NO and U937 cells activated by ligation of IgE receptors, we showed that the intracellular development of Brucella was impaired in human macrophages, which produced NO. Although Brucella-infected human macrophagic phagocytes did not release NO in commonly used models of infection, the machinery required to produce NO was expressed in these cells and could be triggered by cell membrane receptors present on the infected cells. Therefore, the lack of NO production in isolated human macrophages infected by Brucella under in vitro conditions did not exclude a possible involvement of NO in the control of human brucellosis.
Virulence of the intracellular pathogen Brucella for humans is mainly associated with its lipopolysaccharide (LPS) phenotype, with smooth LPS phenotypes generally being virulent and rough ones not. The reason for this association is not quite understood. We now demonstrate by flow cytometry, electron microscopy, and ELISA that human peripheral blood monocytes interact both quantitatively and qualitatively different with smooth and rough Brucella organisms in vitro. We confirm that considerably higher numbers of rough than smooth brucellae attach to and enter the monocytes in nonopsonic conditions; but only smooth brucellae replicate in the host cells. We show for the first time that rough brucellae induce higher amounts than smooth brucellae of several CXC (GRO-alpha, IL-8) and CC (MIP-1alpha, MIP-1beta, MCP-1, RANTES) chemokines, as well as pro- (IL-6, TNF-alpha) and anti-inflammatory (IL-10) cytokines released by challenged monocytes. Upon uptake, phagosomes containing rough brucellae develop selective fusion competence to form spacious communal compartments, whereas phagosomes containing smooth brucellae are nonfusiogenic. Collectively, our data suggest that rough brucellae attract and infect monocytes more effectively than smooth brucellae, but only smooth LPS phenotypes establish a specific host cell compartment permitting successful parasitism. These novel findings link the LPS phenotype of Brucella and its virulence for humans at the level of the infected host cells. Whether this is due to a direct effect of the LPS molecules or to upstream bacterial mechanisms remains to be established.