Brucella and Its Hidden Flagellar System

Abstract

Brucella, a Gram-negative bacterium with a high infective capacity and a wide spectrum of hosts in the animal world, is found in terrestrial and marine mammals, as well as amphibians. This broad spectrum of hosts is closely related to the non-classical virulence factors that allow this pathogen to establish its replicative niche, colonizing epithelial and immune system cells, evading the host's defenses and defensive response. While motility is the primary role of the flagellum in most bacteria, in Brucella, the flagellum is involved in virulence, infectivity, cell growth, and biofilm formation, all of which are very important facts in a bacterium that to date has been described as a non-motile organism. Evidence of the expression of these flagellar proteins that are present in Brucella makes it possible to hypothesize certain evolutionary aspects as to where a free-living bacterium eventually acquired genetic material from environmental microorganisms, including flagellar genes, conferring on it the ability to reach other hosts (mammals), and, under selective pressure from the environment, can express these genes, helping it to evade the immune response. This review summarizes relevant aspects of the presence of flagellar proteins and puts into context their relevance in certain functions associated with the infective process. The study of these flagellar genes gives the genus Brucella a very high infectious versatility, placing it among the main organisms in urgent need of study, as it is linked to human health by direct contact with farm animals and by eventual transmission to the general population, where flagellar genes and proteins are of great relevance.

Keywords: Brucella abortus; ORF; flagellin; flagellum; virulence factors.

Figure 1

Diagram of the bacterial flagellum of Brucella, according to the classical structure of the flagellum in Gram-negative bacteria and the most up-to-date literature. Brucella expresses its bacterial flagellum under specific and controlled in vitro growth conditions, and in the face of different environmental changes, the expression of the flagellum is involved in its survival in a hostile environment when it does not have a definitive host. (A) Under these specific conditions and after expression of the master regulators at gene level, FlgJ acts by drilling the membrane and the peptidoglycan (PG), a key protein in the construction of the flagellum base structure. (B) The correct functioning and conformation of this bacterial organelle depend on the perfect arrangement of the proteins that form the MS-ring, the transporter channel proteins, and the C-ring. Once the base of the flagellum has formed on the membrane, FlgJ is cleaved from the basal body. (C) This correct arrangement of previous proteins makes it possible to reach the end of the flagellum formation given by the cluster conglomerate of rod proteins, the LP-ring, followed by the hook, and finally, by the formation of the filament (FliC) that mediates the movement allowed by proton pumping from the periplasm. In Gram-negative bacteria, the final protein that “seals” the filament is called CapD (FliD); however, this has not yet been described in Brucella, even though Brucella presents the gene for its expression. LPS: lipopolysaccharide; OMP: outer membrane protein; IMP: inner membrane protein.

Figure 2

Transcriptional regulation of some of the flagellar genes described in Brucella at locus I. (A) Repression of flagellar expression in B. melitensis is linked by the repressor RpoE1 (and other unknown proteins) to ftcR. This master regulator allows FlaF to be expressed and inhibits the expression of fliC and fltB. (B) The expression of a unipolar flagellum in B. melitensis is linked to FtcR, a direct regulator of flagellar protein expression; this is partially activated by the regulator VjbR, which is involved in quorum sensing and the expression of the type-IV secretion system. (C) In Brucella abortus, when the ybeY gene (BAB2_1156), an endoribonuclease, is deleted, there is an overexpression of the FtcR and this results in an increase in flagellin.

Figure 3

p-value of conserved motifs related to flgJ gene of Brucella abortus 2308. Bioinformatics tool used: http://meme-suite.org (accessed on 15 November 2021).

Figure 4

Bacterial FliC amino-acid sequence analysis. (a) Multiple alignments of sequences of the FliC protein of Brucella abortus (Q2YJF1) against the FliC protein of Falsochrobactrum sp. HN4 (A0A316J970), Mesorhizobium sp. UASWS1009 (A0A1C2EA77) and Nitratireductor pacificus pht-3B (K2M4Y7) (b). There are 16 conserved motifs in relation to the fliC gene present in the abovementioned species. Bioinformatics tool used: http://meme-suite.org (accessed on 15 November 2021).

Figure 5

(A) In terms of its evolutionary origin, it is postulated that Brucella may have been a free-living bacterium. (B) Over time, and according to its evolution, it remained in contact with other microorganisms, such as soil bacteria and fungi, managing to acquire certain genes that, therefore, improved its metabolic resources. (C) Environmental changes helped give it the ability to adapt to new hosts, such as eukaryotic cells. In Brucella, certain virulence factors that are different from other bacteria, like its LPS, its type-four secretion system (T4SS) and the BvrR/BvrS system, enable Brucella to interact with the host cell surface and form an early Brucella-containing vacuole (BCV) for its subsequent interaction with the endoplasmic reticulum (ER), where bacteria multiply and reach their replicative niche. Likewise, the presence of flagellar genes and the active participation of flagellum proteins in different functions, such as the translocation of proteins to the outside and the formation of adhered biofilm-associated biomass gave Brucella the ability to be a successful organism in achieving its infectious chronicity and evading the immune response of the host organism.