Brucella abortus Uses a Stealthy Strategy to Avoid Activation of the Innate Immune System during the Onset of Infection

Abstract

BackgroundTo unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity.Methodology/Principal Findings Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-α-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice.Conclusion/SignificanceWe propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections.

The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus

“…Based on the observation that the core defect in BaΔwadC generates attenuation and that the mutated LPS is more readily recognized by elements of innate immunity, we have proposed that the Brucella LPS core branch is a virulence-related structure accounting in part for the stealthy behavior of these bacteria [3,6]. Since mutation of another glycosyltransferase involved in the synthesis of the same structure also causes attenuation, our hypothesis is confirmed in the present work.…”

“…For Western blots, gels were electro transferred onto nitrocellulose sheets (Whatman, Dassel, Germany), blocked with 3% skim milk in PBS with 0.05% Tween 20 (PBST) overnight, and washed with the same buffer. Monoclonal antibodies A68/03F03/D05, A68/24D08/G09 and A68/24G12/A08 (all of which recognize R-LPS epitopes [3,16]) were diluted in PBST and blots were developed with peroxidase-conjugated goat anti-mouse immunoglobulin (Nordic immunological laboratories, Tilburg, Netherlands) or peroxidase labeled protein G and 4-chloro-1-naphtol-H 2 O 2 .…”

“…The affinity of Ba-parental and BaΔwadB LPS for polycationic peptides was measured using peptide 19.8-4 (derived from the Limulus anti-LPS factor) [1,3,17] in comparison with the LPS of the B. abortus wadC mutant (BaΔwadC) described previously [3,6], and E. coli ATCC 35218. For this purpose, ELISA plates were coated with the peptide (10 µg/mL in 20 mM in PBS; 100 µl/well) at 4ºC overnight and washed extensively with PBS-Tween 20.…”

“…The brucellae behave as facultative intracellular parasites, and their virulence results from a complex and not fully elucidated set of mechanisms that include the ability to escape prompt detection by innate immunity during the initial stages of infection [3]. Studies carried out mostly with B. abortus have shown that the outer membrane lipopolysaccharide (LPS) [1,3] lipoproteins [3] and ornithine lipids [4] induce a low proinflammatory response indicative of the absence of the marked pathogen-associated molecular patterns that are detected by innate immunity.…”

“…Studies carried out mostly with B. abortus have shown that the outer membrane lipopolysaccharide (LPS) [1,3] lipoproteins [3] and ornithine lipids [4] induce a low proinflammatory response indicative of the absence of the marked pathogen-associated molecular patterns that are detected by innate immunity. These outer membrane components structurally depart from counterparts molecules in most gram-negative bacteria, and the differences are particularly accentuated in the LPS.…”