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PloS One 2020The neurotransmitter gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the human brain; however, it is becoming more evident that this...
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.
Topics: Animals; Biological Transport; Brucella abortus; Glutamic Acid; Mice; Mice, Inbred BALB C; Neurotransmitter Agents; gamma-Aminobutyric Acid
PubMed: 32845904
DOI: 10.1371/journal.pone.0237371 -
MBio Apr 2021The ability to sense and respond to environmental cues is essential for adaptation and survival in living organisms. In bacteria, this process is accomplished by...
The ability to sense and respond to environmental cues is essential for adaptation and survival in living organisms. In bacteria, this process is accomplished by multidomain sensor histidine kinases that undergo autophosphorylation in response to specific stimuli, thereby triggering downstream signaling cascades. However, the molecular mechanism of allosteric activation is not fully understood in these important sensor proteins. Here, we report the full-length crystal structure of a blue light photoreceptor LOV histidine kinase (LOV-HK) involved in light-dependent virulence modulation in the pathogenic bacterium Joint analyses of dark and light structures determined in different signaling states have shown that LOV-HK transitions from a symmetric dark structure to a highly asymmetric light state. The initial local and subtle structural signal originated in the chromophore-binding LOV domain alters the dimer asymmetry via a coiled-coil rotary switch and helical bending in the helical spine. These amplified structural changes result in enhanced conformational flexibility and large-scale rearrangements that facilitate the phosphoryl transfer reaction in the HK domain. Bacteria employ two-component systems (TCSs) to sense and respond to changes in their surroundings. At the core of the TCS signaling pathway is the multidomain sensor histidine kinase, where the enzymatic activity of its output domain is allosterically controlled by the input signal perceived by the sensor domain. Here, we examine the structures and dynamics of a naturally occurring light-sensitive histidine kinase from the pathogen in both its full-length and its truncated constructs. Direct comparisons between the structures captured in different signaling states have revealed concerted protein motions in an asymmetric dimer framework in response to light. Findings of this work provide mechanistic insights into modular sensory proteins that share a similar modular architecture.
Topics: Bacterial Proteins; Brucella abortus; Color; Histidine Kinase; Light; Models, Molecular; Protein Domains; Signal Transduction
PubMed: 33879593
DOI: 10.1128/mBio.00264-21 -
Journal of Clinical Microbiology Apr 2018We compared the performances of various DNA extraction kits for their ability to recover strain 19 inoculated into -free bovine tissues. Tissues were homogenized in a... (Comparative Study)
Comparative Study
We compared the performances of various DNA extraction kits for their ability to recover strain 19 inoculated into -free bovine tissues. Tissues were homogenized in a FastPrep bead homogenizer and extracted in triplicate by using one of five kits (Qiagen DNeasy, GE Illustra, Omega Bio-tek E.Z.N.A., Quanta Extracta, and IBI Science DNA Tissue kit). Whole blood was also taken from animals prior to chemical euthanasia, aliquoted, and then fractioned into buffy coat, red blood cells, and plasma. DNA was extracted from whole blood, buffy coat, and plasma by using four kits (Qiagen DNeasy, Omega Bio-tek E.Z.N.A., IBI Science DNA Blood kit, and 5PRIME PerfectPure). Previously reported primers targeting strain 19 were used to amplify extracted DNA and identify the optimal extraction kit. Real-time PCR was performed, and kits were compared for statistical differences by using quantification cycles as an outcome measure. Omega Bio-tek E.Z.N.A. was superior ( < 0.0068) in its lower quantification cycle values across all tissue kits. The IBI Science DNA Blood kit was superior to Qiagen DNeasy, 5PRIME PerfectPure, and Quanta Extracta ( < 0.0001, = 0.0004, and = 0.0013, respectively) but was not different from Omega Bio-tek E.Z.N.A. ( = 1.0). In summary, the optimal extraction kit for strain 19 for tissues is Omega Bio-tek E.Z.N.A., and that for blood and its fractions is the IBI Science Mini Genomic DNA kit. Eluted DNA was also concentrated by using the Zymo Research DNA Clean & Concentrator-25 kit. Concentrated eluted DNA with the target was superior ( = <0.0001) to unconcentrated eluted DNA.
Topics: Animals; Brucella abortus; Brucellosis; Cattle; DNA Primers; DNA, Bacterial; Female; Lymph Nodes; Reagent Kits, Diagnostic; Real-Time Polymerase Chain Reaction; Spleen; Uterus
PubMed: 29436425
DOI: 10.1128/JCM.01894-17 -
Journal of Bacteriology Aug 2014The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular parasites and thus are able to...
The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular parasites and thus are able to adjust their metabolism to the extra- and intracellular environments encountered during an infectious cycle. However, this aspect of Brucella biology is imperfectly understood, and the nutrients available in the intracellular niche are unknown. Here, we investigated the central pathways of C metabolism used by Brucella abortus by deleting the putative fructose-1,6-bisphosphatase (fbp and glpX), phosphoenolpyruvate carboxykinase (pckA), pyruvate phosphate dikinase (ppdK), and malic enzyme (mae) genes. In gluconeogenic but not in rich media, growth of ΔppdK and Δmae mutants was severely impaired and growth of the double Δfbp-ΔglpX mutant was reduced. In macrophages, only the ΔppdK and Δmae mutants showed reduced multiplication, and studies with the ΔppdK mutant confirmed that it reached the replicative niche. Similarly, only the ΔppdK and Δmae mutants were attenuated in mice, the former being cleared by week 10 and the latter persisting longer than 12 weeks. We also investigated the glyoxylate cycle. Although aceA (isocitrate lyase) promoter activity was enhanced in rich medium, aceA disruption had no effect in vitro or on multiplication in macrophages or mouse spleens. The results suggest that B. abortus grows intracellularly using a limited supply of 6-C (and 5-C) sugars that is compensated by glutamate and possibly other amino acids entering the Krebs cycle without a critical role of the glyoxylate shunt.
Topics: Animals; Brucella abortus; Brucellosis; Carbon; Disease Models, Animal; Fructose-Bisphosphatase; Gene Deletion; Malate Dehydrogenase; Metabolic Networks and Pathways; Mice; Pyruvate, Orthophosphate Dikinase; Virulence
PubMed: 24936050
DOI: 10.1128/JB.01663-14 -
Revista Argentina de Microbiologia 2019The objective of this study was to identify twelve Brucella abortus isolates of bovine origin from the department of Nariño in Colombia up to the biovar level. These...
The objective of this study was to identify twelve Brucella abortus isolates of bovine origin from the department of Nariño in Colombia up to the biovar level. These isolates are included in the collection of the Germplasm Bank of Microorganisms of Animal Health Interest - Bacteria and Virus (BGSA-BV). The identification was carried out through conventional methods such as macro and microscopic morphological descriptions, enzymatic activity, biochemical profile, substrate use and sensitivity to dyes. Complementary genotypic characterization was carried out using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis-Erytritol (AMOS-ERY-PCR), RFLP-IS711, by southern blot hybridization, as well as by the multiple locus variable number of tandem repeat analysis (MLVA) using the ery gene and the insertion sequence IS711 and variable number of tandem repeats (VNTR) as molecular markers. The results of the phenotypic and molecular characterization allowed to identify twelve isolates as B. abortus biovar 4 as well as to differentiate field from vaccine strains. This is the first study on the phenotypic and molecular identification of B. abortus isolates in Colombia. It was concluded that the phenotypic and molecular identification of twelve isolates as B. abortus biovar 4 could be achieved using conventional and molecular techniques with enough resolution power. The identification of these isolates to the biovar level in taxonomic and epidemiological terms will allow the use of this genetic resource as reference strains in future research. This finding constitutes the basis for identifying biotypes not previously reported in the country that might be useful to support brucellosis survey programs in Colombia.
Topics: Animals; Bacteriological Techniques; Biological Specimen Banks; Biomarkers; Brucella abortus; Brucellosis, Bovine; Cattle; Colombia; DNA, Bacterial; Genes, Bacterial; Genotype; Minisatellite Repeats; Multiplex Polymerase Chain Reaction; Phenotype
PubMed: 30551811
DOI: 10.1016/j.ram.2018.08.002 -
PloS One 2013Brucellosis is a worldwide distributed zoonosis that causes important economic losses to animal production. In Brazil, information on the distribution of biovars and...
Brucellosis is a worldwide distributed zoonosis that causes important economic losses to animal production. In Brazil, information on the distribution of biovars and genotypes of Brucella spp. is scarce or unavailable. This study aimed (i) to biotype and genotype 137 Brazilian cattle isolates (from 1977 to 2008) of B. abortus and (ii) to analyze their distribution. B. abortus biovars 1, 2 and 3 (subgroup 3b) were confirmed and biovars 4 and 6 were first described in Brazil. Genotyping by the panel 1 revealed two groups, one clustering around genotype 40 and another around genotype 28. Panels 2A and 2B disclosed a high diversity among Brazilian B. abortus strains. Eighty-nine genotypes were found by MLVA16. MLVA16 panel 1 and 2 showed geographic clustering of some genotypes. Biotyping and MLVA16 genotyping of Brazilian B. abortus isolates were useful to better understand the epidemiology of bovine brucellosis in the region.
Topics: Animals; Bacterial Typing Techniques; Brazil; Brucella abortus; Brucellosis; Cattle; Cattle Diseases; DNA, Bacterial; Genotype; Minisatellite Repeats; Phylogeny; Zoonoses
PubMed: 24324670
DOI: 10.1371/journal.pone.0081152 -
British Medical Journal Feb 1977
Topics: Animals; Brucellosis; Brucellosis, Bovine; Cattle; Humans; United Kingdom
PubMed: 837164
DOI: No ID Found -
Journal of Bacteriology May 2015Cyclic β-1,2-glucans (CβG) are periplasmic homopolysaccharides that play an important role in the virulence and interaction of Brucella with the host. Once synthesized...
UNLABELLED
Cyclic β-1,2-glucans (CβG) are periplasmic homopolysaccharides that play an important role in the virulence and interaction of Brucella with the host. Once synthesized in the cytoplasm by the CβG synthase (Cgs), CβG are transported to the periplasm by the CβG transporter (Cgt) and succinylated by the CβG modifier enzyme (Cgm). Here, we used a bacterial two-hybrid system and coimmunoprecipitation techniques to study the interaction network between these three integral inner membrane proteins. Our results indicate that Cgs, Cgt, and Cgm can form both homotypic and heterotypic interactions. Analyses carried out with Cgs mutants revealed that the N-terminal region of the protein (Cgs region 1 to 418) is required to sustain the interactions with Cgt and Cgm as well as with itself. We demonstrated by single-cell fluorescence analysis that in Brucella, Cgs and Cgt are focally distributed in the membrane, particularly at the cell poles, whereas Cgm is mostly distributed throughout the membrane with a slight accumulation at the poles colocalizing with the other partners. In summary, our results demonstrate that Cgs, Cgt, and Cgm form a membrane-associated biosynthetic complex. We propose that the formation of a membrane complex could serve as a mechanism to ensure the fidelity of CβG biosynthesis by coordinating their synthesis with the transport and modification.
IMPORTANCE
In this study, we analyzed the interaction and localization of the proteins involved in the synthesis, transport, and modification of Brucella abortus cyclic β-1,2-glucans (CβG), which play an important role in the virulence and interaction of Brucella with the host. We demonstrate that these proteins interact, forming a complex located mainly at the cell poles; this is the first experimental evidence of the existence of a multienzymatic complex involved in the metabolism of osmoregulated periplasmic glucans in bacteria and argues for another example of pole differentiation in Brucella. We propose that the formation of this membrane complex could serve as a mechanism to ensure the fidelity of CβG biosynthesis by coordinating synthesis with the transport and modification.
Topics: Brucella abortus; Immunoprecipitation; Membrane Proteins; Protein Interaction Mapping; Protein Multimerization; Succinates; Two-Hybrid System Techniques; beta-Glucans
PubMed: 25733613
DOI: 10.1128/JB.00068-15 -
Microbes and Infection Jun 2000Brucella abortus is a facultative intracellular parasite that promotes its own internalization in nonphagocytic cells. The bacterium initially interacts with... (Review)
Review
Brucella abortus is a facultative intracellular parasite that promotes its own internalization in nonphagocytic cells. The bacterium initially interacts with compartments of the early endocytic cascade, then rapidly segregates from this intracellular pathway and associates with the autophagocytic cascade. During the late stages of infection, Brucella proliferates within the endoplasmic reticulum of host cells.
Topics: Animals; Brucella abortus; Cell Compartmentation; Endocytosis; Endoplasmic Reticulum; Endosomes; Humans
PubMed: 10955964
DOI: 10.1016/s1286-4579(00)90368-x -
Veterinary Medicine and Science Nov 2019Brucellosis is an infectious and contagious zoonotic bacterial disease of both humans and animals. In developing countries where brucellosis is endemic, baseline data on...
BACKGROUND
Brucellosis is an infectious and contagious zoonotic bacterial disease of both humans and animals. In developing countries where brucellosis is endemic, baseline data on the prevalence of brucellosis, using abattoir facilities, is important.
OBJECTIVES
The aim of this study was to determine the seroprevalence of antibodies against Brucella in slaughter cattle at Gauteng province, South Africa and to characterize isolates of Brucella spp.
METHODS
In this cross-sectional study, un-clotted blood samples with corresponding organ tissue samples were collected from slaughtered cattle. Serological [Rose Bengal test (RBT), complement fixation test (CFT) and indirect ELISA (iELISA)], molecular (PCR) and bacteriological methods were used to detect Brucella antibodies and Brucella spp. from 200 slaughtered cattle in 14 abattoirs.
RESULTS
The RBT revealed a seroprevalence of brucellosis as 11.0% (22 of 200) and iELISA confirmed 5.5% (11 of 200). The estimated seroprevalence from RBT and iELISA was 5.5% while RBT and CFT was 2.0% (4 of 200). Brucella melitensis (n = 6) and B. abortus (n = 5) were isolated from 11 cattle tissues (5.5%) as confirmed to species level with AMOS PCR and differentiated from vaccine strains with Bruce-ladder PCR. Seven of the 11 isolates originated from seropositive cattle of which five were biotyped as B. abortus bv 1 (n = 2) and B. melitensis bv 2 (n = 1) and B. melitensis bv 3 (n = 2).
CONCLUSIONS
This is the first documentation of B. melitensis in cattle in South Africa. The zoonotic risk of brucellosis posed by Brucella-infected slaughter cattle to abattoir workers and consumers of improperly cooked beef cannot be ignored.
Topics: Abattoirs; Animals; Antibodies, Bacterial; Brucella; Brucellosis, Bovine; Cattle; Cross-Sectional Studies; Female; Male; Prevalence; Seroepidemiologic Studies; South Africa
PubMed: 31414558
DOI: 10.1002/vms3.190