Assigning a function to a conserved archaeal metallo-β-lactamase from Haloferax volcanii

The metallo-β-lactamase family of enzymes comprises a large group of proteins with diverse functions in the metabolism of the cell. Among others, this superfamily contains proteins which are involved in DNA and RNA metabolism, acting as nucleases in e.g. repair and maturation. Many proteins have been annotated in prokaryotic genomes as being potential metallo-β-lactamases, but very often the function has not been proven. The protein HVO_2763 from Haloferax volcanii is such a potential metallo-β-lactamase. HVO_2763 has sequence similarity to the metallo-β-lactamase tRNase Z, a tRNA 3′ processing endonuclease. Here, we report the characterisation of this metallo-β-lactamase HVO_2763 in the halophilic archaeon Haloferax volcanii. Using different in vitro assays with the recombinant HVO_2763, we could show that the protein does not have tRNA 3′ processing or exonuclease activity. According to transcriptome analyses of the HVO_2763 deletion strain, expression of proteins involved in membrane transport is downregulated in the mutant. Therefore, HVO_2763 might be involved directly or indirectly in membrane transport

Genomic organization and distribution of <i>sdiA</i> in the <i>Enterobacteriaceae</i>.

<p>a) Distribution of <i>sdiA</i> on a phylogenetic tree based on 16S rDNA sequences. Only bootstrap values of ≥50 are displayed. Blue lines indicate species that contain a solo <i>sdiA</i>. Green lines indicate species that contain <i>sdiA</i> and an adjacent <i>luxI</i> homolog. A maximum likelihood tree gave similar results (not shown). b) Map of the <i>sdiA</i> region in representative organisms that encode SdiA orthologs. Any additional LuxR/LuxI pairs in those organisms are also listed. Genes depicted in white are conserved in all genera and genes in gray are not conserved. <i>sdiA</i> is represented in black and <i>sirA</i> in gray hatched lines.</p

Integrated Use of Biochemical, Native Mass Spectrometry, Computational, and Genome-Editing Methods to Elucidate the Mechanism of a Salmonella deglycase

Salmonella is a foodborne pathogen that causes annually millions of cases of salmonellosis globally, yet Salmonella-specific antibacterials are not available. During inflammation, Salmonella utilizes the Amadori compound fructose-asparagine (F-Asn) as a nutrient through the successive action of three enzymes, including the terminal FraB deglycase. Salmonella mutants lacking FraB are highly attenuated in mouse models of inflammation due to the toxic build-up of the substrate 6-phosphofructose-aspartate (6-P-F-Asp). This toxicity makes Salmonella FraB an appealing drug target, but there is currently little experimental information about its catalytic mechanism. Therefore, we sought to test our postulated mechanism for the FraB-catalyzed deglycation of 6-P-F-Asp (via an enaminol intermediate) to glucose-6-phosphate and aspartate. A FraB homodimer model generated by RosettaCM was used to build substrate-docked structures that, coupled with sequence alignment of FraB homologs, helped map a putative active site. Five candidate active-site residues-including three expected to participate in substrate binding-were mutated individually and characterized. Native mass spectrometry and ion mobility were used to assess collision cross sections and confirm that the quaternary structure of the mutants mirrored the wild type, and that there are two active sites/homodimer. Our biochemical studies revealed that FraB Glu214Ala, Glu214Asp, and His230Ala were inactive in vitro, consistent with deprotonated-Glu214 and protonated-His230 serving as a general base and a general acid, respectively. Glu214Ala or His230Ala introduced into the Salmonella chromosome by CRISPR/Cas9-mediated genome editing abolished growth on F-Asn. Results from our computational and experimental approaches shed light on the catalytic mechanism of Salmonella FraB and of phosphosugar deglycases in general. (C) 2019 Elsevier Ltd. All rights reserved

Serendipitous Discovery of a Competitive Inhibitor of FraB, a Salmonella Deglycase and Drug Target

Although salmonellosis, an infectious disease, is a significant global healthcare burden, there are no Salmonella-specific vaccines or therapeutics for humans. Motivated by our finding that FraB, a Salmonella deglycase responsible for fructose-asparagine catabolism, is a viable drug target, we initiated experimental and computational efforts to identify inhibitors of FraB. To this end, our recent high-throughput screening initiative yielded almost exclusively uncompetitive inhibitors of FraB. In parallel with this advance, we report here how a separate structural and computational biology investigation of FrlB, a FraB paralog, led to the serendipitous discovery that 2-deoxy-6-phosphogluconate is a competitive inhibitor of FraB (KI ~ 3 &mu;M). However, this compound was ineffective in inhibiting the growth of Salmonella in a liquid culture. In addition to poor uptake, cellular metabolic transformations by a Salmonella dehydrogenase and different phosphatases likely undermined the efficacy of 2-deoxy-6-phosphogluconate in live-cell assays. These insights inform our ongoing efforts to synthesize non-hydrolyzable/-metabolizable analogs of 2-deoxy-6-phosphogluconate. We showcase our findings largely to (re)emphasize the role of serendipity and the importance of multi-pronged approaches in drug discovery

Additional file 4: of Chemical and pathogen-induced inflammation disrupt the murine intestinal microbiome

Figure S2. Non-metric multidimensional scaling (NMDS) ordination of all samples without Salmonella OTU. A NMDS of Bray-Curtis similarity metric among microbial communities in each pretreatment fecal, late fecal, and cecal sample (stress = 0.10) shows a statistically significant (mrpp, p < 0.001) separation of cecal microbial communities from control, DSS, low-responder, and high-responder groups. Each point represents one sample with colors denoting treatment. (PDF 136 kb

Additional file 2: of Chemical and pathogen-induced inflammation disrupt the murine intestinal microbiome

Sequences of all OTUs in fasta format. (FNA 10560 kb

Additional file 6: of Chemical and pathogen-induced inflammation disrupt the murine intestinal microbiome

Mapping file detailing time point, treatment, Shannon’s diversity and richness for each sample. (CSV 5 kb

Additional file 10: of Chemical and pathogen-induced inflammation disrupt the murine intestinal microbiome

FDR adjusted p-values. (CSV 88 kb

Fructose-Asparagine Is a Primary Nutrient during Growth of <i>Salmonella</i> in the Inflamed Intestine

<div><p><i>Salmonella enterica</i> serovar Typhimurium (<i>Salmonella</i>) is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that <i>Salmonella</i> encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn), which is essential for <i>Salmonella</i> fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10^−/− mice). The locus encoding F-Asn utilization, <i>fra</i>, provides an advantage only if <i>Salmonella</i> can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the <i>fra</i> phenotype is lost in <i>Salmonella</i> SPI1⁻ SPI2⁻ or <i>ttrA</i> mutants, respectively). The severe fitness defect of a <i>Salmonella fra</i> mutant suggests that F-Asn is the primary nutrient utilized by <i>Salmonella</i> in the inflamed intestine and that this system provides a valuable target for novel therapies.</p></div

Protection of mice against <i>Salmonella</i> serovar Typhimurium strain 14028 by <i>Enterobacter cloacae</i> strain JLD400.

<p>Germ-free C57BL/6 mice were divided into two groups. One group was colonized with 10⁷ cfu of <i>Enterobacter cloacae</i> via the intragastric route (i.g.) and one group was not. One day later both groups were challenged i.g. with 10⁷ cfu of <i>Salmonella</i>. After 24 hours, the cecum and spleen were homogenized and plated to enumerate <i>Salmonella</i>. Each point represents the CFU/g recovered from one mouse with the geometric mean shown by a horizontal line. Statistical significance between select groups was determined by using an unpaired two-tailed Student <i>t</i> test. ** = P value<0.01, *** = P value<0.001.</p