Characterizing the transport and utilization of the neurotransmitter GABA in the bacterial pathogen Brucella abortus

Abstract

The neurotransmitter gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the human brain; however, it is becoming more evident that this non-proteinogenic amino acid plays multiple physiological roles in biology. In the present study, the transport and function of GABA is studied in the highly infectious intracellular bacterium Brucella abortus. The data show that 3H-GABA is imported by B. abortus under nutrient limiting conditions and that the small RNAs AbcR1 and AbcR2 negatively regulate this transport. A specific transport system, gts, is responsible for the transport of GABA as determined by measuring 3H-GABA transport in isogenic deletion strains of known AbcR1/2 regulatory targets; however, this locus is unnecessary for Brucella infection in BALB/c mice. Similar assays revealed that 3H-GABA transport is uninhibited by the 20 standard proteinogenic amino acids, representing preference for the transport of 3H-GABA. Metabolic studies did not show any potential metabolic utilization of GABA by B. abortus as a carbon or nitrogen source, and RNA sequencing analysis revealed limited transcriptional differences between B. abortus 2308 with or without exposure to GABA. While this study provides evidence for GABA transport by B. abortus, questions remain as to why and when this transport is utilized during Brucella pathogenesis.

Fig 1. Organization of putative GABA ABC-type transport systems in B. abortus 2308 and homology to the GABA transport systems in Rhizobium leguminosarum bv. viciae 3841 and Agrobacterium tumefaciens str. C58.

A. Genetic organization of bab1_1972-bab1_1799 located on chromosome I of Brucella melitensis biovar Abortus 2308. Putative functions for each gene and percent amino acid identity to bra genes in related organisms are located below the gene. Proteins encoded from this locus exhibit high amino acid identity to the bra locus in Agrobacterium tumefaciens str. C58. B. Genetic organization of bab2_0876-bab2_0879 located on chromosome II of Brucella melitensis biovar Abortus 2308. Putative functions for each gene and percent amino acid identity to gts genes in related organisms are located below the gene. Proteins encoded from this locus exhibit moderate amino acid identity to the gts locus in R. leguminosarum bv. viciae 3841.

Fig 2. ³H-GABA import is induced under nutrient limiting conditions.

³H-GABA uptake by B. abortus 2308 was assessed in minimal medium with (GMM) and without (-Glu) the addition of glutamate to the medium. Data is normalized to GMM(-Glu) at 100%. Controls include the addition of excess nonradiolabled GABA to competitively inhibit ³H-GABA uptake. The asterisks denote a statistically significant difference (** P<0.005, *** P<0.0005; Student’s t test) in uptake.

Fig 3. ³H-GABA import by B. abortus 2308 is not greatly inhibited by the presence of other amino acids in vitro.

³H-GABA uptake by B. abortus 2308 was assessed uninhibited and in the presence of 1,000-fold excess GABA or 20 proteinogenic amino acids. Data is normalized to the absence of inhibitor at 100%. The asterisk denotes a statistically significant difference (* P<0.05, ** P<0.005, *** P<0.0005; Student’s t test) in uptake of ³H-GABA between B. abortus 2308 uninhibited (no inhibitor) and in the presence of excess nonradiolabled GABA, asparagine, cysteine, lysine, methionine, and tryptophan.

Fig 4. ³H-GABA import is negatively regulated by the sRNAs AbcR1 and AbcR2 in B.

abortus. ³H-GABA uptake by B. abortus 2308 and B. abortus 2308:: ΔabcR1/2 was assessed in minimal medium, GMM(-Glu). Data is normalized to 2308 at 100%. Asterisks denote a statistically significant difference (* P<0.05, ** P<0.005; Student’s t test) in uptake.

Fig 5. Transport of ³H-GABA transport in B. abortus 2308 by AbcR regulated systems.

A. ³H-GABA uptake by B. abortus 2308, Δbab2_0612, Δbab2_0879, Δbab1_1792Δbab1_1794, and Δbab2_0879 in assessed in minimal medium, GMM(-Glu). Data is normalized to 2308 at 100%. Asterisks denote a statistically significant difference (* P<0.05, *** P<0.0005; Student’s t test) in uptake between B. abortus 2308 and deletion strains. B. ³H-GABA uptake by B. abortus 2308, Δbab2_0879, and Δbab2_0879-RCbab2_0879 in minimal medium, GMM(-Glu). Data is normalized to 2308 at 100%. Asterisks denote a statistically significant difference (*** P<0.0005; Student’s t test) in uptake between strains.

Fig 6. Virulence of B. abortus 2308 and Δbab2_0879 in peritoneally derived macrophages and BALB/c mice.

A. Macrophage survival and replication experiments. Cultured peritoneal macrophages from BALB/c mice were infected with B. abortus 2308 and the isogenic bab2_0879 deletion strain (Δbab2_0879). At the indicated times post-infection, macrophages were lysed, and the number of intracellular brucellae present in these phagocytes was determined by serial dilution and plating on agar medium. B. Oral mouse infection experiments. BALB/c mice (6 per strain) were infected intraperitoneally with B. abortus 2308 and Δbab2_0879. Mice were sacrificed 1, 2, and 4 weeks post-infection, and Log₁₀ brucellae/spleen were calculated. The data are presented as average numbers of brucellae ± standard deviations of results from the 6 mice (3 male and 3 female mice) colonized with a specific Brucella strain at each time point.

Fig 7. GABA is not utilized as a metabolite by B. abortus 2308 in vitro.

A. Growth of B. abortus 2308 in minimal medium (GMM), minimal medium lacking glutamate (GMM-Glu), and minimal medium lacking glutamate with the addition of GABA (0.15%) (GMM-Glu+GABA). The asterisk denotes a statistically significant difference (* P<0.05; Student’s t test) in uptake between B. abortus 2308 grown in GMM compared to B. abortus 2308 grown in either GMM-Glu or GMM-Glu+GABA. B. Oxygen consumption by B. abortus 2308 grown in TSB with the addition of either GABA, glutamate, or erythritol 300 seconds after inoculation measured via oxygraph machine.