Comparative Genome-Wide Transcriptome Analysis of Brucella suis and Brucella microti Under Acid Stress at pH 4.5: Cold Shock Protein CspA and Dps Are Associated With Acid Resistance of B. microti

Abstract

Brucellae are facultative intracellular coccobacilli causing brucellosis, one of the most widespread bacterial zoonosis affecting wildlife animals, livestock and humans. The genus Brucella comprises classical and atypical species, such as Brucella suis and Brucella microti, respectively. The latter is characterized by increased metabolic activity, fast growth rates, and extreme acid resistance at pH 2.5, suggesting an advantage for environmental survival. In addition, B. microti is more acid-tolerant than B. suis at the intermediate pH of 4.5. This acid-resistant phenotype of B. microti may have major implications for fitness in soil, food products and macrophages. Our study focused on the identification and characterization of acid resistance determinants of B. suis and B. microti in Gerhardt's minimal medium at pH 4.5 and 7.0 for 20 min and 2 h by comparative RNA-Seq-based transcriptome analysis, validated by RT-qPCR. Results yielded a common core response in both species with a total of 150 differentially expressed genes, and acidic pH-dependent genes regulated specifically in each species. The identified core response mechanisms comprise proton neutralization or extrusion from the cytosol, participating in maintaining physiological intracellular pH values. Differential expression of 441 genes revealed species-specific mechanisms in B. microti with rapid physiological adaptation to acid stress, anticipating potential damage to cellular components and critical energy conditions. Acid stress-induced genes encoding cold shock protein CspA, pseudogene in B. suis, and stress protein Dps were associated with survival of B. microti at pH 4.5. B. suis response with 284 specifically regulated genes suggested increased acid stress-mediated protein misfolding or damaging, triggering the set-up of repair strategies countering the consequences rather than the origin of acid stress and leading to subsequent loss of viability. In conclusion, our work supports the hypothesis that increased acid stress resistance of B. microti is based on selective pressure for the maintenance of functionality of critical genes, and on specific differential gene expression, resulting in rapid adaptation.

Keywords: Brucella; Dps; acid stress; cold shock protein; transcriptome.

FIGURE 1

Survival and replication of B. microti and B. suis in minimal or complex medium at pH 4.5–5.5. Viability of B. microti (black bars) and B. suis (gray bars) in modified Gerhardt’s Minimal Medium (A–C) or Tryptic Soy broth (D–F) adjusted to pH 4.5, 5.0, and 5.5 was determined at different time points. Values are shown as means of 3 experiments ± standard deviations (SD).

FIGURE 2

Distribution of differentially expressed genes in B. microti and B. suis. Numbers of genes differentially expressed in both species at pH 4.5/pH 7 were determined either according to the rates of expression at both pH-values (upper panel), or according to the time points where differential expression was significant (lower panel). Bμ: B. microti; Bs: B. suis.

FIGURE 3

Validation of RNA-Seq data by RT-qPCR. Log2-values of fold-changes of gene expression at pH 4.5 and pH 7.0 detected by RNA-Seq were plotted against RT-qPCR data. Rates of validation are indicated as percentages in the graphs. (A) B. microti, 20 min; (B) B. microti, 120 min; (C) B. suis, 20 min; (D) B. suis, 120 min.

FIGURE 4

COG classification of Brucella genes regulated at pH 4.5. The differentially expressed core genes (A) and those specific to B. suis (B) and B. microti (C) cumulated for both 20 and 120 min were classified into Clusters of Orthologous Groups, and their percentages were determined with respect to the total number of genes affiliated to each group. Blue sections of the bars represent genes with higher expression at pH 7, red sections genes with higher expression at pH 4.5, and green sections correspond to genes with opposite expression over time. COGs: C: Energy production/conversion; D: Cell cycle control, cell division; E: amino acid transport/metabolism; F: Nucleotide transport/metabolism; G: Carbohydrate transport/metabolism; H: Coenzyme transport/metabolism; I: Lipid transport/metabolism; J: Translation/ribosomal structure/biogenesis; K: Transcription; L: Replication, recombination/repair; M: Cell wall/membrane/envelope biogenesis; N: Cell motility; O: Posttranslational modification/protein turnover/chaperones; P: Inorganic ion transport/metabolism; Q: Secondary metabolites synthesis/transport/catabolism; R: General function prediction; S: Function unknown; T: Signal transduction; U: Intracellular trafficking, secretion/vesicular transport; V: Defense mechanisms.

FIGURE 5

B. microti and B. suis genes of the denitrification pathway with increased expression at pH 4.5. Color codes indicate the species and the experimental time points (20 or 120 min) for each of the genes identified as being more expressed at pH 4.5 than at pH 7.

FIGURE 6

Participation of CspA in survival of B. microti (A) and B. suis (B) in GMM at pH 4.5. Survival of B. microti wild-type strain (black bars), B. microti ΔcspA (red bars), B. microti ΔcspA complemented with pBBR1-cspA (cyan bars), and of B. suis wild-type with pBBR1-AMP (black bars) or complemented with pBBR1-AMP-cspA (red bars) containing functional cspA of B. microti. Values are shown as means of 7 experiments ± SD. **: P < 0.01; ***: P < 0.001.

FIGURE 7

Participation of Dps in survival of B. microti in GMM at pH 4.5. Survival of B. microti wild-type strain (black bars), B. microti Δdps (red bars), and of B. microti Δdps complemented with pBBR1-dps (cyan bars). Values are shown as means of 6 experiments ± SD. ***: P < 0.005.

FIGURE 8

Morphological characterization of B. microti CCM4915 in GMM pH 7.0 and pH 4.5 by Atomic Force Microscopy (AFM). (A) AFM topographic images of B. microti at both pH values. (B) Topographical profile plots measured along the longitudinal axis of the cells, indicated by solid lines in (A). (C) Length, width and height measurements of bacteria at pH 7.0 (n = 46) and pH 4.5 (n = 53). (D) Average roughness R_a of the bacteria at pH 7.0 (n = 42) and pH 4.5 (n = 33), based on recorded surface roughness. Cell height analysis was carried out using the height (measured) channel of the QI mode, which corresponds to the height at 80% of the setpoint force determined on the reference force-distance curve. Height was calculated as the topographical maximal central height on each cell using the section tool of the analysis software. The average roughness (Ra) was calculated on the z channel values using a 400 × 400 nm area for each cell. Statistical differences were analyzed by t-test and yielded P values < 0.05, as indicated for panels (C) and (D).

FIGURE 9

Schematic representation of the highlighted common and species-specific responses for both Brucella species. For the experimental time points chosen, genes with higher expression at pH 4.5 are shown in the upper half, those with higher expression at pH 7 in the lower half of the scheme.