Identification and characterization of in vivo attenuated mutants of Brucella melitensis

Abstract

SummaryBrucella melitensis 16M is a Gram-negative a 2 -proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades into and survives within both professional and non-professional phagocytes. Signature-tagged mutagenesis (STM) was used to identify genes required for the in vivo pathogenesis of Brucella. A library of transposon mutants was screened in a murine infection model. Out of 672 mutants screened, 20 were not recovered after a 5 day passage in BALB/c mice. The attenuation of 18 mutants was confirmed using an in vivo competition assay against the wild-type strain. The 18 mutants were characterized further for their ability to replicate in murine macrophages and in HeLa cells. The sequences disrupted by the transposon in the mutants have homology to genes coding for proteins of different functional classes: transport, amino acid and DNA metabolism, transcriptional regulation, peptidoglycan synthesis, a chaperone-like protein and proteins of unknown function. The mutants selected in this study provide new insights into the molecular basis of Brucella virulence.

The analysis of the intramacrophagic virulome ofBrucella suisdeciphers the environment encountered by the pathogen inside the macrophage host cell

“…inside the macrophage is a prerequisite to the understanding of the relation with the macrophage. Signature-tagged mutagenesis and differential fluorescence induction approaches were recently used to identify genes required for virulence in a murine model of infection and in human and murine macrophages (5)(6)(7)(8)(9). A large proportion of these genes encode enzymes of various metabolic pathways.…”

Genomic analysis of the original Elberg Brucella melitensis Rev.1 vaccine strain reveals insights into virulence attenuation

“…Notably, we found that the ORF BMEI1759 possesses a missense mutation (c.523G> C) in the Rev.1 genome. This ORF encodes the vitamin B12-dependent methyltransferase MetH, which is involved in methionine biosynthesis and was previously screened in a murine infection model aimed at identifying pathogenic Brucella genes expressed in vivo [28,42]. Thus, we suggest that mutations in Rev.1 genes that encode enzymes involved in amino acid biosynthesis may contribute to the attenuation of this strain.…”

The two-component system PrlS/PrlR of Brucella melitensis is required for persistence in mice and appears to respond to ionic strength

“…We described previously (Lestrate et al, 2000) that the G4 mutant bearing a miniTn5 inserted between prlS and prlR is attenuated in mice. Therefore, we started to characterize this TCS and its putative function further.…”

“…Several of these systems have been characterized previously in some Brucella species: BvrSR (Sola-Landa et al, 1998), FeuQP (Dorrell et al, 1998), NtrBC (Dorrell et al, 1999), NtrXY (Carrica et al, 2012;Foulongne et al, 2000), the flagellar master regulator FtcR (Léonard et al, 2007), the CtrA phosphorelay (Hallez et al, 2007), the blue-light-activated LOV HKs (Swartz et al, 2007) and CenR (Zhang et al, 2009). Some other TCSs have been identified by transpositional mutagenesis during global screening for virulence factors (Lestrate et al, 2000;Wu et al, 2006) but left uncharacterized. This is the case for the Brucella melitensis transpositional mutant (G4) that we identified in a previous study (Lestrate et al, 2000).…”

“…Some other TCSs have been identified by transpositional mutagenesis during global screening for virulence factors (Lestrate et al, 2000;Wu et al, 2006) but left uncharacterized. This is the case for the Brucella melitensis transpositional mutant (G4) that we identified in a previous study (Lestrate et al, 2000). Indeed, a mini-Tn5 was inserted between genes encoding a sensor (BMEI1606) and a regulator (BMEI1607) of a TCS pair that was later, based on homology, called PrlS and PrlR, for probable proline sensor and regulator (Lavín et al, 2010;Letesson & De Bolle, 2004).…”