BUSCADOR RECOLECTA

Brucellosis is a zoonosis caused by Brucella species. Brucellosis research in natural hosts is often precluded by practical, economical and ethical reasons and mice are widely used. However, mice are not natural Brucella hosts and the course of murine brucellosis depends on bacterial strain virulence, dose and inoculation route as well as breed, genetic background, age, sex and physiological statu of mice. Therefore, meaningful experiments require a definition of these variables. Brucella spleen replication profiles are highly reproducible and course in four phases: i), onset or spleen colonization (first 48h); ii), acute phase, from the third day to the time when bacteria reach maximal numbers; iii), chronic steady phase, where bacterial numbers plateaus; and iv), chronic declining phase, during which brucellae are eliminated. This pattern displays clear physiopathological signs and is sensitive to small virulence variations, making possible to assess attenuation when fully virulent bacteria are used as controls. Similarly, immunity studies using mice with known defects are possible. Mutations affecting INF-gamma, TLR9, Myd88, Tgammadelta and TNF-beta favor Brucella replication; whereas IL-1beta, IL-18, TLR4, TLR5, TLR2, NOD1, NOD2, GM-CSF, IL/17r, Rip2, TRIF, NK or Nramp1 deficiencies have no noticeable effects. Splenomegaly development is also useful: it correlates with IFN-gamma and IL-12 levels and with Brucella strain virulence. The genetic background is also important: Brucella-resistant mice (C57BL) yield lower splenic bacterial replication and less splenomegaly than susceptible breeds. When inoculum is increased, a saturating dose above which bacterial numbers per organ do not augment, is reached. Unlike many gram-negative bacteria, lethal doses are large (greater than or equal to] 108 bacteria/mouse) and normally higher than the saturating dose. Persistence is a useful virulence/attenuation index and is used in vaccine (Residual Virulence) quality control. Vaccine candidates are also often tested in mice by determining splenic Brucella numbers after challenging with appropriate virulent brucellae doses at precise post-vaccination times. Since most live or killed Brucella vaccines provide some protection in mice, controls immunized with reference vaccines (S19 or Rev1) are critical. Finally, mice have been successfully used to evaluate brucellosis therapies. It is concluded that, when used properly, the mouse is a valuable brucellosis model., This work was performed under agreement contract
2010020113, subscribed by UNA from Costa Rica and, CSIC, CITA, and UN
from Spain. This work was funded by grants FIDA-2009 UNA, FS-CONARE
UNA/UCR, NeTropica 8, and MICIT/CONICIT, CSIC-CRUSA (2010CR0005) from
Costa Rica; and CICYT-MICINN (AGL2010-20247, AGL2008-04514-C03-00 and
AGL2011-30453-C04-00) projects from Spain and grant ANR2010BLAN1308
Brutir., form France. This work was done as part of the UCR/DAAD Humboldt
Fellow award 2012 to EM.

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines., This work was funded by grants from the Spanish Ministerio de Ciencia y Tecnología (AGL2008-04514-C03-01 to I.M.; SAF2009-07885 to J.A.B and
AGL2010-20247 to MJG), EU Grant agreement Nu 221948, FIDA, Universidad Nacional de Costa Rica; FS-Conare UNA/UCR IFEG29 Costa Rica; NeTropica P00059 and
F00013-02; MICIT/CONICIT IFDG12; Fundación CRUSA-CSIC 2008CR0006 and 2010CR0005; Centre National de la Recherche Scientifique, Institut National de la
Sante et de la Recherche Medicale. Cooperation between University of Navarra, PIET and CSIC has been favoured by the Collaboration Agreement reference
2010020113. Research and fellowship support for KVB from ANR 2010BLAN1308 BruTir, fellowship support for R. C.-A. and L. P.-C. from the Ministerio de Ciencia y
Tecnología of Spain, Gobierno de Navarra and Friends of the University of Navarra and for A.M. from the Ministry of Education in France are gratefully
acknowledged.