A 6-Nucleotide Regulatory Motif within the AbcR Small RNAs of Brucella abortus Mediates Host-Pathogen Interactions

Abstract

In Brucella abortus, two small RNAs (sRNAs), AbcR1 and AbcR2, are responsible for regulating transcripts encoding ABC-type transport systems. AbcR1 and AbcR2 are required for Brucella virulence, as a double chromosomal deletion of both sRNAs results in attenuation in mice. Although these sRNAs are responsible for targeting transcripts for degradation, the mechanism utilized by the AbcR sRNAs to regulate mRNA in Brucella has not been described. Here, two motifs (M1 and M2) were identified in AbcR1 and AbcR2, and complementary motif sequences were defined in AbcR-regulated transcripts. Site-directed mutagenesis of M1 or M2 or of both M1 and M2 in the sRNAs revealed transcripts to be targeted by one or both motifs. Electrophoretic mobility shift assays revealed direct, concentration-dependent binding of both AbcR sRNAs to a target mRNA sequence. These experiments genetically and biochemically characterized two indispensable motifs within the AbcR sRNAs that bind to and regulate transcripts. Additionally, cellular and animal models of infection demonstrated that only M2 in the AbcR sRNAs is required for Brucella virulence. Furthermore, one of the M2-regulated targets, BAB2_0612, was found to be critical for the virulence of B. abortus in a mouse model of infection. Although these sRNAs are highly conserved among Alphaproteobacteria, the present report displays how gene regulation mediated by the AbcR sRNAs has diverged to meet the intricate regulatory requirements of each particular organism and its unique biological niche.

An account of evolutionary specialization: the AbcR small RNAs in the Rhizobiales

“…However, it stands to reason that one motif, M2, may be responsible for regulating targets that are directly or indirectly involved in pathogenesis. One of these M2‐regulated targets, BAB2_0612, was found to be involved in Brucella pathogenesis, strengthening this hypothesis (Sheehan and Caswell, ), and will be interesting to determine if any other M2 targets are linked to Brucella pathogenesis.…”

“…Quantitative reverse‐transcriptase PCR with the B. abortus single and double abcR mutants found that the sRNAs are truly redundant, with the isogenic abcR mutants resulting in gene expression levels comparable to the wild‐type strain, and a double abcR mutant leading to over‐expression of target genes (Sheehan and Caswell, ). Furthermore, AbcR1 and AbcR2 both physically bind to the 5' UTR of one of the targets, BAB2_0879, confirming the redundant roles these sRNAs have in Brucella .…”

“…After determining the redundancy of the B. abortus AbcR sRNAs; two motifs, named M1 and M2, were identified in both AbcR1 and AbcR2 (Sheehan and Caswell, ). Similar to the organization in A. tumefaciens AbcR1, M1 (CUUCCC) is located in the first hairpin, and M2 (GUUCCC) is located in a single‐stranded region between the first and second hairpin (Fig.…”

“…Only one target transcript, BAB1_1799, encoding a putative ABC transporter permease, was regulated by M1 alone. Conversely, four targets were regulated exclusively by M2, three of which encode putative ABC transporter periplasmic binding proteins (Sheehan and Caswell, ). Altogether, it seems that the AbcR sRNAs have retained almost identical motifs to carry out their regulatory roles in the Rhizobiales examined to date.…”

“…Due to the role M1 and M2 play in regulation of transcripts, the single and double abcR motif mutants were tested to assess the role of these regulatory motifs in Brucella virulence. Surprisingly, while the abcR‐M1 mutant exhibited wild‐type infection levels, the abcR‐M2 and abcR‐M1/2 mutant strains were attenuated in cells and mice (Sheehan and Caswell, ). This result was interesting, as both motifs in the sRNAs are necessary for the proper regulation of transcripts in Brucella .…”