Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196.

The Aspergillus niger CexA transporter belongs to the DHA1 (Drug-H+ antiporter) family. CexA homologs are exclusively found in eukaryotic genomes, and CexA is the sole citrate exporter to have been functionally characterized in this family so far. In the present work, we expressed CexA in Saccharomyces cerevisiae, demonstrating its ability to bind isocitric acid, and import citrate at pH 5.5 with low affinity. Citrate uptake was independent of the proton motive force and compatible with a facilitated diffusion mechanism. To unravel the structural features of this transporter, we then targeted 21 CexA residues for site-directed mutagenesis. Residues were identified by a combination of amino acid residue conservation among the DHA1 family, 3D structure prediction, and substrate molecular docking analysis. S. cerevisiae cells expressing this library of CexA mutant alleles were evaluated for their capacity to grow on carboxylic acid-containing media and transport of radiolabeled citrate. We also determined protein subcellular localization by GFP tagging, with seven amino acid substitutions affecting CexA protein expression at the plasma membrane. The substitutions P200A, Y307A, S315A, and R461A displayed loss-of-function phenotypes. The majority of the substitutions affected citrate binding and translocation. The S75 residue had no impact on citrate export but affected its import, as the substitution for alanine increased the affinity of the transporter for citrate. Conversely, expression of CexA mutant alleles in the Yarrowia lipolytica cex1Δ strain revealed the involvement of R192 and Q196 residues in citrate export. Globally, we uncovered a set of relevant amino acid residues involved in CexA expression, export capacity and import affinity.

Membrane transporters in the bioproduction of organic acids: state of the art and future perspectives for industrial applications.

Organic acids such as monocarboxylic acids, dicarboxylic acids or even more complex molecules such as sugar acids, have displayed great applicability in the industry as these compounds are used as platform chemicals for polymer, food, agricultural and pharmaceutical sectors. Chemical synthesis of these compounds from petroleum derivatives is currently their major source of production. However, increasing environmental concerns have prompted the production of organic acids by microorganisms. The current trend is the exploitation of industrial biowastes to sustain microbial cell growth and valorize biomass conversion into organic acids. One of the major bottlenecks for the efficient and cost-effective bioproduction is the export of organic acids through the microbial plasma membrane. Membrane transporter proteins are crucial elements for the optimization of substrate import and final product export. Several transporters have been expressed in organic acid-producing species, resulting in increased final product titers in the extracellular medium and higher productivity levels. In this review, the state of the art of plasma membrane transport of organic acids is presented, along with the implications for industrial biotechnology.

The use of selected phytochemicals with EDTA against Escherichia coli and Staphylococcus epidermidis single- and dual-species biofilms.

The aim of this study was the development of a novel and effective antibacterial formulation combining selected phytochemical compounds (quercetin, cuminaldehyde, indole-3-carbinol and vanillic acid) with ethylenediaminetetraacetic acid (EDTA), an aminopolycarboxylic acid. The antibacterial activity of the combinations was evaluated against Escherichia coli and Staphylococcus epidermidis in planktonic and sessile states as single and dual species. The compounds when applied individually demonstrated modest antibacterial activity. Nevertheless, synergy was observed when EDTA was combined with the selected phytochemicals, particularly with cuminaldehyde and indole-3-carbinol. These combinations were evaluated against single- and dual-species biofilms. An inactivation of 100% was obtained for almost all the biofilms, with E. coli biofilms showing the highest resistance. This study allowed the discovery of novel formulations of phytochemical compounds with antibacterial activity against E. coli and S. epidermidis single- and dual-species biofilms at concentrations close to the minimum bactericidal concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic combinations of EDTA and cuminaldehyde or indole-3-carbinol were effective against single- and dual-species E. coli and S. epidermidis planktonic cells and biofilms. The overall results highlight the role of phytochemical products as a green and sustainable source of antimicrobial potentiators to control bacteria in both planktonic and sessile states.

The Role of the Gut Microbiome on Chronic Kidney Disease.

Chronic kidney disease (CKD) is estimated to affect nearly 500 million people worldwide and cardiovascular (CV) disease is a major cause of death in this population. However, therapeutic interventions targeting traditional CV risks are not effective at lowering the incidence of CV events or at delaying the progression of the disease in CKD patients. In recent years, disturbances of normal gut microbiome were recognized in the pathogenesis of diverse chronic diseases. Gut dysbiosis is being unraveled in CKD and pointed as a nontraditional risk factor for CV risk and CKD progression. The most often reported changes in gut microbiome in CKD are related to the lower levels of Bifidobacteriaceae and Lactobacillaceae and to higher levels of Enterobacteriaceae. Although metagenomics brought us an amplified vision on the microbial world that inhabits the human host, it still lacks the sensitivity to characterize the microbiome up to species level, not revealing alterations that occur within specific genus. Here, we review the current state-of-the-art concerning gut dysbiosis in CKD and its role in pathophysiological mechanisms in CKD, particularly in relation with CV risk. Also, the strategies towards prevention and treatment of gut dysbiosis in CKD progression will be discussed.

LAMA2 gene analysis in a cohort of 26 congenital muscular dystrophy patients.

Congenital muscular dystrophy type 1A (MDC1A) is caused by mutations in the LAMA2 gene encoding laminin-alpha2. We describe the molecular study of 26 patients with clinical presentation, magnetic resonance imaging and/or laminin-alpha2 expression in muscle, compatible with MDC1A. The combination of full genomic sequencing and complementary DNA analysis led to the particularly high mutation detection rate of 96% (50/52 disease alleles). Besides 22 undocumented polymorphisms, 18 different mutations were identified in the course of this work, 14 of which were novel. In particular, we describe the first fully characterized gross deletion in the LAMA2 gene, encompassing exon 56 (c.7750-1713_7899-2153del), detected in 31% of the patients. The only two missense mutations detected were found in heterozygosity with nonsense or truncating mutations in the two patients with the milder clinical presentation and a partial reduction in muscle laminin-alpha2. Our results corroborate the previous few genotype/phenotype correlations in MDC1A and illustrate the importance of screening for gross rearrangements in the LAMA2 gene, which may be underestimated in the literature.