It has been proposed that NADP⁺-specificity of isocitrate dehydrogenase (ICDH) evolved as an adaptation of microorganisms to grow on acetate as the sole source of carbon and energy. In Escherichia coli, changing the cofactor specificity of ICDH from NADP⁺ to NAD⁺ (cofactor swapping) decreases the growth rate on acetate. However, the metabolic basis of this phenotype has not been analyzed. In this work, we used constraint-based modeling to investigate the effect of the cofactor swapping of ICDH in terms of energy production, response of alternative sources of NADPH, and partitioning of fluxes between ICDH and isocitrate lyase (ICL) -a crucial bifurcation when the bacterium grows on acetate-. We generated E. coli strains expressing NAD⁺-specific ICDH instead of the native enzyme, and bearing the deletion of the NADPH-producing transhydrogenase PntAB. We measured their growth rate and acetate uptake rate, modeled the distribution of metabolic fluxes by Flux Balance Analysis (FBA), and quantified the specific activities of NADPH-producing dehydrogenases in central pathways. The cofactor swapping of ICDH led to one-third decrease in biomass yield, irrespective of the presence of PntAB. According to our simulations, the diminution in growth rate observed upon cofactor swapping could be explained by one-half decrease in the total production of NADPH and a lower availability of carbon for biosynthesis because of a change in the partition at the isocitrate bifurcation. Together with an increased total ATP production, this scenario resulted in a 10-fold increment in the flux of ATP not used for growing purposes. PntAB was identified as the primary NADPH balancing response, with the dehydrogenases of the oxidative branch of the Pentose Phosphate Pathway and the malic enzyme playing a role in its absence. We propose that in the context of E. coli growing on acetate, the NADP⁺-specificity of ICDH is a trait that impacts not only NADPH production, but also the efficient allocation of carbon and energy.

Chile is one of the largest consumers of sugar-sweetened beverages (SSB) world-wide. However, it is unknown whether the effects from this highly industrialized food will mimic those reported in industrialized countries or whether they will be modified by local lifestyle or population genetics. Our goal is to evaluate the interaction effect between SSB intake and T2D susceptibility on fasting glucose. We calculated a weighted genetic risk score (GRSw) based on 16 T2D risk SNPs in 2828 non-diabetic participants of the MAUCO cohort. SSB intake was categorized in four levels using a food frequency questionnaire. Log-fasting glucose was regressed on SSB and GRSw tertiles while accounting for socio-demography, lifestyle, obesity, and Amerindian ancestry. Fasting glucose increased systematically per unit of GRSw (β = 0.02 ± 0.006, p = 0.00002) and by SSB intake (β[cat4] = 0.04 ± 0.01, p = 0.0001), showing a significant interaction, where the strongest effect was observed in the highest GRSw-tertile and in the highest SSB consumption category (β = 0.05 ± 0.02, p = 0.02). SNP-wise, SSB interacted with additive effects of rs7903146 (TCF7L2) (β = 0.05 ± 0.01, p = 0.002) and with the G/G genotype of rs10830963 (MTNRB1B) (β = 0.19 ± 0.05, p = 0.001). Conclusions: The association between SSB intake and fasting glucose in the Chilean population without diabetes is modified by T2D genetic susceptibility.

Avian pathogenic Escherichia coli (APEC) is a widespread pathogen that poses a significant threat to the poultry industry globally. A recent typing scheme has proposed several APEC pathotypes, including non-APEC, High Risk non-APEC (HR non-APEC), APEC, and High Risk APEC (HR APEC), based on the presence of the ompT and hlyF genes, sequence types (ST) ST131, ST23, ST428, ST355, and the O78 antigen. In Brazilian broiler farms, a higher prevalence of lesions in carcasses has been linked to the presence of HR APEC pathotypes. Due to the growing concern over antimicrobial resistance, bacteriophage-based formulations are emerging as a promising alternative for controlling APEC outbreaks. In this study, we isolated and sequenced 142 strains from Brazilian commercial broiler farms, classifying them as APEC (70), HR APEC (12), HR non-APEC (3) and non-APEC (57). Notably, 38 % of the isolates were classified as multi-drug resistant (MDR), with serotypes H10 and ST155 being the most frequently identified. Additionally, we isolated, sequenced, and classified 66 bacteriophages that exhibited lytic activity against these bacterial strains in both qualitative and quantitative assays. Eight of the bacteriophages demonstrated complementary host ranges against the bacterial collection. Bacteriophage cocktails were assembled, tested in quantitative assays, and shown to be effective against APEC. A cocktail consisting of four bacteriophages (AC-01) displayed a broad lytic spectrum in vitro, inhibiting the growth of 56.3 % (n = 80/142) of the isolates with a mean inhibition of 32.9 %. Remarkably, the in vitro lytic activity of the cocktail was significantly more effective against HR APEC isolates (12/12, 65.9 % mean inhibition) and HR non-APEC isolates (3/3, 58.0 % mean inhibition). Our results emphasize the importance of genetically characterizing target bacteria when developing an effective and specific bacteriophage cocktail against APEC.

Salmonella spp. is a prevalent pathogen that causes great public health concern worldwide. Bacteriophage-based cocktails have arisen as an alternative to antibiotics to inhibit the growth of Salmonella. However, the bactericidal effect of bacteriophage cocktails in vivo largely differs from their observed effect in vitro. This is partly because in vitro developments of cocktails do not always consider the bacterial diversity nor the environmental conditions where bacteriophages will have to replicate. Here, we isolated and sequenced 47 bacteriophages that showed variable degrees of lytic activity against 258 Salmonella isolates from a commercial broiler company in Brazil. Three of these bacteriophages were characterized and selected to assemble a cocktail. In vitro quantitative assays determined the cocktail to be highly effective against multiple serovars of Salmonella, including Minnesota and Heidelberg. Remarkably, the in vitro lytic activity of the cocktail was retained or improved in conditions that more closely resembled the chicken gut, such as anaerobiosis, 42°C, and Salmonella mono-strain biofilms. Analysis of bacterial cross-resistance between the 3 bacteriophages composing the cocktail revealed limited or no generation of cross-resistance. Our results highlight the relevance of an optimized flux of work to develop bacteriophage cocktails against Salmonella with high lytic efficacy and strong potential to be applied in vivo in commercial broiler farms.

Background: The enzyme 6-phosphogluconate dehydrogenase (6PGDH) is the central enzyme of the oxidative pentose phosphate pathway. Members of the 6PGDH family belong to different classes: either homodimeric enzymes assembled from long-chain subunits or homotetrameric ones assembled from short-chain subunits. Dimeric 6PGDHs bear an internal duplication absent in tetrameric 6PGDHs and distant homologues of the β-hydroxyacid dehydrogenase (βHADH) superfamily. Methods: We use X-ray crystallography to determine the structure of the apo form of the 6PGDH from Gluconobacter oxydans (Go6PGDH). We carried out a structural and phylogenetic analysis of short and long-chain 6PGDHs. We put forward an evolutionary hypothesis explaining the differences seen in oligomeric state vs. dinucleotide preference of the 6PGDH family. We determined the cofactor preference of Go6PGDH at different 6-phosphogluconate concentrations, characterizing the wild-type enzyme and three-point mutants of residues in the cofactor binding site of Go6PGDH. Results: The structural comparison suggests that the 6PG binding site initially evolved by exchanging C-terminal α-helices between subunits. An internal duplication event changed the quaternary structure of the enzyme from a tetrameric to a dimeric arrangement. The phylogenetic analysis suggests that 6PGDHs have spread from Bacteria to Archaea and Eukarya on multiple occasions by lateral gene transfer. Sequence motifs consistent with NAD+- and NADP+-specificity are found in the β2-α2 loop of dimeric and tetrameric 6PGDHs. Site-directed mutagenesis of Go6PGDH inspired by this analysis fully reverses dinucleotide preference. One of the mutants we engineered has the highest efficiency and specificity for NAD+ so far described for a 6PGDH. Conclusions: The family 6PGDH comprises dimeric and tetrameric members whose active sites are conformed by a C-terminal α-helix contributed from adjacent subunits. Dimeric 6PGDHs have evolved from the duplication-fusion of the tetrameric C-terminal domain before independent transitions of cofactor specificity. Changes in the conserved β2-α2 loop are crucial to modulate the cofactor specificity in Go6PGDH.

Although a lack of diversity in genetic studies is an acknowledged obstacle for personalized medicine and precision public health, Latin American populations remain particularly understudied despite their heterogeneity and mixed ancestry. This gap extends to COVID-19 despite its variability in susceptibility and clinical course, where ethnic background appears to influence disease severity, with non-Europeans facing higher hospitalization rates. In addition, access to high-quality samples and data is a critical issue for personalized and precision medicine, and it has become clear that the solution lies in biobanks. The creation of the Chilean COVID-19 Biorepository reported here addresses these gaps, representing the first nationwide multicentric Chilean initiative. It operates under rigorous biobanking standards and serves as one of South America’s largest COVID cohorts. A centralized harmonization strategy was chosen and included unified standard operating procedures, a sampling coding system, and biobanking staff training. Adults with confirmed SARS-CoV-2 infection provided broad informed consent. Samples were collected to preserve blood, plasma, buffy coat, and DNA. Quality controls included adherence to the standard preanalytical code, incident reporting, and DNA concentration and absorbance ratio 260/280 assessments. Detailed sociodemographic, health, medication, and preexisting condition data were gathered. In five months, 2262 participants were enrolled, pseudonymized, and sorted by disease severity. The average Amerindian ancestry considering all participant was 44.0% [SD 15.5%], and this value increased to 61.2% [SD 19.5%] among those who self-identified as Native South Americans. Notably, 279 participants self-identified with one of 12 ethnic groups. High compliance (>90%) in all assessed quality controls was achieved. Looking ahead, our team founded the COVID-19 Genomics Network (C19-GenoNet) focused on identifying genetic factors influencing SARS-CoV-2 outcomes. In conclusion, this bottom-up collaborative effort aims to promote the integration of Latin American populations into global genetic research and welcomes collaborations supporting this endeavor. Interested parties are invited to explore collaboration opportunities through our catalog, accessible online.