Characterization of Three Small Proteins in Brucella abortus Linked to Fucose Utilization

Abstract

Elucidating the function of proteins <50 amino acids in length is no small task. Nevertheless, small proteins can play vital roles in the lifestyle of bacteria and influence the virulence of pathogens; thus, the investigation of the small proteome is warranted. Recently, our group identified the Brucella abortus protein VtlR as a transcriptional activator of four genes, one of which is the well-studied small regulatory RNA AbcR2, while the other three genes encode hypothetical small proteins, two of which are highly conserved among the order Rhizobiales This study provides evidence that all three genes encode authentic small proteins and that all three are highly expressed under oxidative stress, low-pH, and stationary-phase growth conditions. Fractionation of the cells revealed that the proteins are localized to the membranes of B. abortus We demonstrate that the small proteins under the transcriptional control of VtlR are not accountable for attenuation observed with the B. abortusvtlR deletion strain. However, there is an association between VtlR-regulated genes and growth inhibition in the presence of the sugar l-fucose. Subsequent transcriptomic analyses revealed that B. abortus initiates the transcription of a locus encoding a putative sugar transport and utilization system when the bacteria are cultured in the presence of l-fucose. Altogether, our observations characterize the role of the VtlR-controlled small proteins BAB1_0914, BAB2_0512, and BAB2_0574 in the biology of B. abortus, particularly in the capacity of the bacteria to utilize l-fucose.IMPORTANCE Despite being one of the most common zoonoses worldwide, there is currently no human vaccine to combat brucellosis. Therefore, a better understanding of the pathogenesis and biology of Brucella spp., the causative agent of brucellosis, is essential for the discovery of novel therapeutics against these highly infectious bacteria. In this study, we further characterize the virulence-associated transcriptional regulator VtlR in Brucella abortus Our findings not only shed light on our current understanding of a virulence related genetic system in Brucella spp. but also increase our knowledge of small proteins in the field of bacteriology.

Keywords: Brucella; VtlR regulon; small protein.

FIG 1

Organization of bab1_0914, bab2_0512, and bab2_0574 in Brucella abortus 2308 and amino acid sequence similarity among BAB1_0914 and BAB2_0512. (A to C) Genetic contexts and encoding amino acid sequences of bab1_0914 (bab_rs20300) (A), bab2_0512 (bab_rs28790) (B), and bab2_0574 (bab_rs29075) (C). (D) Amino acid sequence alignment of BAB1_0914 and BAB2_0512 created by NCBI Align Sequences Protein Blast. Identical amino acids are depicted by abbreviated amino acid symbols, and similar amino acids are depicted by +. (E) PHYLIP ML phylogenetic tree of BAB1_0914 and BAB2_0512 homologs created using the “Determine sequence relationships” tool at https://dnasubway.cyverse.org/. Percentages represent amino acid identity to BAB1_0914.

FIG 2

bab1_0914, bab2_0512, and bab2_0574 are activated during stationary phase of growth, low pH, and in the presence of H₂O₂. (A) Western blot analyses for protein expression of B. abortus 2308, B. abortus 2308 bab1_0914-3×FLAG, B. abortus 2308 bab2_0512-3×FLAG, and B. abortus 2308 bab2_0574-3×FLAG. Probes include anti-FLAG and anti-Omp89. (B) Northern blot analyses for RNA expression of AbcR1, AbcR2, BAB1_0914, BAB2_0512, and BAB2_0574 during stationary phase and exponential phase of growth in brucella broth, GMM, and E medium, and in the presence of H₂O₂ (5 μM), pH 4.5, and deoxycholate (0.5%) in brucella broth. (C to E) Western blot analyses for protein expression of BAB1_0914-3×FLAG, BAB2_0512-3×FLAG, BAB2_0574-3×FLAG, respectively, during stationary phase and exponential phase of growth in brucella broth, GMM, and E medium, and in the presence of H₂O₂ (5 μM), pH 4.5, and deoxycholate (0.5%) in brucella broth. Probes include anti-FLAG and anti-Omp89.

FIG 3

Localization of BAB1_0914, BAB2_0512, and BAB2_0574. Western blot analysis of Brucella strains grown to stationary phase, followed by fractionation methods described in Materials and Methods for B. abortus 2308 bab1_0914-3×FLAG (A), B. abortus 2308 bab2_0512-3×FLAG (B), and B. abortus 2308 bab2_0574-3×FLAG (C). Antibodies utilized include anti-FLAG, anti-Omp89, anti-SodC, and anti-GroEL.

FIG 4

Characterization of B. abortus 2308 and VtlR regulon mutants using Biolog Phenotype MicroArray plates. (A) Growth of Brucella strains in plate 1, well B4, in minimal medium plus 24 mM l-fucose, over 84 h of growth. Shown are pictures of plate 1, well B4, (center well) after 84 h of growth. The graph shows the OD₅₉₀ of plate 1, well B4, over 84 h of incubation. (B) Growth of Brucella strains in plate 10, well D8, in minimal medium, pH 4.5, plus hydroxylysine over 84 h of growth. Shown are pictures of plate 10, well D8 (center well), after 84 h of growth. The graph shows the OD₅₉₀ of plate 10, well D8 over 84 h of incubation.

FIG 5

l-Fucose contribution to B. abortus growth and growth inhibition in VtlR regulon mutants. Cultures of Gerhardt's minimal medium (GMM) supplemented with and without the addition of 24 mM l-fucose were inoculated with Brucella strains at an initial concentration of 5 × 10⁴ CFU/ml and incubated at 37°C. (A) B. abortus 2308 grows to a higher cell density in GMM supplemented with 24 mM l-fucose than GMM without 24 mM l-fucose. (B) In the presence of l-fucose, B. abortus 2308 Δbab2_0512 and B. abortus 2308 ΔabcR2 Δbab1_0914 Δbab2_0512 Δbab2_0574 (Δquad) growth is significantly lower than that of B. abortus 2308. (C) B. abortus 2308 ΔvtlR is sensitive to the presence of 24 mM l-fucose in GMM. Statistical significance (*) was determined using one-way analysis of variance (ANOVA; P < 0.05).

FIG 6

bab1_0914, bab2_0512, and bab2_0574 do not contribute to the ability of B. abortus 2308 to colonize the spleens of BALB/c mice. BALB/c mice were infected intraperitoneally with 10⁵ CFU B. abortus 2308, B. abortus 2308 Δbab1_0914, B. abortus 2308 Δbab2_0512, B. abortus 2308 Δbab2_0574, B. abortus 2308 Δbab1_0914 Δbab2_0512 Δbab2_0574 (Δtriple), or B. abortus 2308 ΔabcR2 Δbab1_0914 Δbab2_0512 Δbab2_0574 (Δquad). Mice were sacrificed at 8 weeks postinfection, and the number of brucellae colonizing the spleens was determined. The data are presented as the average brucellae ± the standard deviation from the 5 mice colonized with a specific Brucella strain.