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1.
Chembiochem ; 21(24): 3500-3503, 2020 12 11.
Article in English | MEDLINE | ID: mdl-32750193

ABSTRACT

One promising strategy to combat antibiotic-resistant bacteria is to develop compounds that block bacterial defenses against antibacterial conditions produced by the innate immune system. Salmonella enterica, which causes food-borne gastroenteritis and typhoid fever, requires histidine kinases (HKs) to resist innate immune defenses such as cationic antimicrobial peptides (CAMPs). Herein, we report that 2-aminobenzothiazoles block histidine kinase-dependent phenotypes in Salmonella enterica serotype Typhimurium. We found that 2-aminobenzothiazoles inhibited growth under low Mg2+ , a stressful condition that requires histidine kinase-mediated responses, and decreased expression of the virulence genes pagC and pagK. Furthermore, we discovered that 2-aminobenzothiazoles weaken Salmonella's resistance to polymyxin B and polymyxin E, which are last-line antibiotics and models for host defense CAMPs. These findings raise the possibilities that 2-aminobenzothiazoles can block HK-mediated bacterial defenses and can be used in combination with polymyxins to treat infections caused by Salmonella.


Subject(s)
Anti-Bacterial Agents/pharmacology , Benzothiazoles/pharmacology , Drug Resistance, Bacterial/drug effects , Gene Expression Regulation, Bacterial/drug effects , Polymyxins/pharmacology , Salmonella enterica/drug effects , Anti-Bacterial Agents/chemistry , Benzothiazoles/chemistry , Microbial Sensitivity Tests , Molecular Structure , Polymyxins/chemistry , Salmonella enterica/genetics , Virulence/drug effects
2.
Stud Fam Plann ; 51(2): 193-204, 2020 06.
Article in English | MEDLINE | ID: mdl-32538513

ABSTRACT

To accelerate their demographic transition, sub-Saharan African (SSA) countries must trigger significant and rapid fertility declines. These fertility declines will open a demographic window of opportunity and enable countries to capture a first demographic dividend. Despite some successes, many programs aimed at decreasing fertility in SSA have yielded disappointing results. This commentary argues that better integrated policies will help to accelerate fertility declines in SSA. Such an approach should harness the synergies between a set of four key policy levers: women's empowerment; female education; family planning, reproductive health services, and universal health coverage; and legal reforms. These policy levers should be coupled with renewed commitment from leaders on the continent and increased investments in both family planning and population institutions. By concomitantly investing in these priorities and harnessing the synergies of these policy levers, countries in SSA have a critical opportunity to accelerate their demographic transition, which could help them reach emerging economy status.


Subject(s)
Birth Rate , Family Planning Services/organization & administration , Policy , Africa South of the Sahara , Developing Countries , Empowerment , Health Services Accessibility/organization & administration , Humans , Reproductive Health Services/organization & administration , Socioeconomic Factors , Universal Health Care
3.
FEBS Lett ; 594(2): 337-357, 2020 01.
Article in English | MEDLINE | ID: mdl-31514245

ABSTRACT

Fumarase C (FumC) catalyzes the reversible conversion of fumarate to S-malate. Previous structural investigations within the superfamily have reported a dynamic structural segment, termed the SS Loop. To date, active-site asymmetry has raised the question of how SS Loop placement affects participation of key residues during the reaction. Herein, we report structural and kinetic analyses from Escherichia coli FumC variants to understand the contribution of SS Loop residues S318, K324, and N326. High-resolution X-ray crystallographic results reveal three distinct FumC active-site conformations; disordered-open, ordered-open, and the newly discovered ordered-closed. Surprisingly, each SS Loop variant has unaffected Michaelis constants coupled to reductions in turnover number. Based upon our structural and functional analyses, we propose structural and catalytic roles for each of the aforementioned residues.


Subject(s)
Catalysis , Fumarate Hydratase/ultrastructure , Protein Conformation , Amino Acid Sequence/genetics , Catalytic Domain/genetics , Crystallography, X-Ray , Escherichia coli/enzymology , Fumarate Hydratase/chemistry , Fumarate Hydratase/genetics , Kinetics , Models, Molecular
4.
J Struct Biol ; 204(3): 513-518, 2018 12.
Article in English | MEDLINE | ID: mdl-30339832

ABSTRACT

DcrB is an 18 kDa lipoprotein that contains a single domain of unknown function. DcrB is found within Enterobacteriaceae, a family of Gram-negative bacteria which includes pathogens that can cause food-borne illness and hospital-acquired infections. In Salmonella enterica serovar Typhimurium, DcrB is up-regulated by conditions that promote the production of known virulence factors. We determined the structure of a truncated form of DcrB from Salmonella to 1.92 Šresolution by X-ray crystallography. This truncated form, DcrBΔ37, contains the entire domain of unknown function but lacks the lipoprotein signal sequence (residues 1-20) as well as residues 21-37. The DcrBΔ37 monomer contains the Mog1p/PsbP-like fold, which is found in functionally diverse proteins in mammals, yeast, plants, and cyanobacteria. Interestingly, DcrBΔ37 crystallized as a domain-swapped homodimer in which the N-terminal ß-hairpin extends from one protomer to interact with the core of the second protomer. This domain-swapping indicates that the N-terminal portion of the Mog1p/PsbP-like fold likely has conformational flexibility. Overall, our results provide the first example of an enterobacterial protein that contains the Mog1p/PsbP-like fold and expands knowledge of the structural and phylogenetic diversity of Mog1p/PsbP-like proteins.


Subject(s)
Bacterial Proteins/genetics , Lipoproteins/genetics , Mutation , Salmonella enterica/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Circular Dichroism , Crystallography, X-Ray , Lipoproteins/chemistry , Lipoproteins/metabolism , Models, Molecular , Protein Conformation , Protein Structure, Secondary , Salmonella enterica/metabolism
6.
Mol Microbiol ; 88(5): 970-83, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23646936

ABSTRACT

Chemical modifications of components of the bacterial cell envelope can enhance resistance to antimicrobial agents. Why then are such modifications produced only under specific conditions? Here, we address this question by examining the role of regulated variations in O-antigen length in the lipopolysaccharide (LPS), a glycolipid that forms most of the outer leaflet of the outer membrane in Gram-negative bacteria. We determined that activation of the PmrA/PmrB two-component system, which is the major regulator of LPS alterations in Salmonella enterica serovar Typhimurium, impaired growth of Salmonella in bile. This growth defect required the PmrA-activated gene wzz(st), which encodes the protein that determines long O-antigen chain length and confers resistance to complement-mediated killing. By contrast, this growth defect did not require the wzz(fepE) gene, which controls production of very long O-antigen, or other PmrA-activated genes that mediate modifications of lipid A or core regions of the LPS. Additionally, we establish that long O-antigen inhibits growth in bile only in the presence of enterobacterial common antigen, an outer-membrane glycolipid that contributes to bile resistance. Our results suggest that Salmonella regulates the proportion of long O-antigen in its LPS to respond to the different conditions it faces during infection.


Subject(s)
O Antigens/metabolism , Salmonella typhimurium/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bile/metabolism , Bile/microbiology , Gene Expression Regulation, Bacterial , Salmonella typhimurium/drug effects , Salmonella typhimurium/growth & development , Salmonella typhimurium/metabolism , Stress, Physiological
7.
Biochemistry ; 52(25): 4391-8, 2013 Jun 25.
Article in English | MEDLINE | ID: mdl-23697711

ABSTRACT

Nematodes represent a diverse phylum of both free living and parasitic species. While the species Caenorhabditis elegans is a valuable model organism, parasitic nematodes or helminths pose a serious threat to human health. Indeed, helminths cause many neglected tropical diseases that afflict humans. Nematode glycoconjugates have been implicated in evasive immunomodulation, a hallmark of nematode infections. One monosaccharide residue present in the glycoconjugates of several human pathogens is galactofuranose (Galf). This five-membered ring isomer of galactose has not been detected in mammals, making Galf metabolic enzymes attractive therapeutic targets. The only known pathway for biosynthetic incorporation of Galf into glycoconjugates depends upon generation of the glycosyl donor UDP-Galf by the flavoenzyme uridine 5'-diphosphate (UDP) galactopyranose mutase (UGM or Glf). A putative UGM encoding gene (glf-1) was recently identified in C. elegans. We sought to assess the catalytic activity of the corresponding gene product (CeUGM). CeUGM catalyzes the isomerization of UDP-Galf and UDP-galactopyranose (UDP-Galp). In the presence of enzyme, substrate, and a hydride source, a galactose-N5-FAD adduct was isolated, suggesting the CeUGM flavin adenine dinucleotide (FAD) cofactor serves as a nucleophile in covalent catalysis. Homology modeling and protein variants indicate that CeUGM possesses an active site similar to that of prokaryotic enzymes, despite the low sequence identity (∼15%) between eukaryotic and prokaryotic UGM proteins. Even with the primary sequence differences, heterocyclic UGM inhibitors developed against prokaryotic proteins also inhibit CeUGM activity. We postulate that inhibitors of CeUGM can serve as chemical probes of Galf in nematodes and as anthelmintic leads. The available data suggest that CeUGM facilitates the biosynthetic incorporation of Galf into nematode glycoconjugates through generation of the glycosyl donor UDP-Galf.


Subject(s)
Caenorhabditis elegans/enzymology , Intramolecular Transferases/chemistry , Animals , Crystallography, X-Ray , Humans , Intramolecular Transferases/isolation & purification
8.
Biochemistry ; 51(6): 1148-59, 2012 Feb 14.
Article in English | MEDLINE | ID: mdl-22217153

ABSTRACT

Even in the absence of a template, glycosyltransferases can catalyze the synthesis of carbohydrate polymers of specific sequence. The paradigm has been that one enzyme catalyzes the formation of one type of glycosidic linkage, yet certain glycosyltransferases generate polysaccharide sequences composed of two distinct linkage types. In principle, bifunctional glycosyltransferases can possess separate active sites for each catalytic activity or one active site with dual activities. We encountered the fundamental question of one or two distinct active sites in our investigation of the galactosyltransferase GlfT2. GlfT2 catalyzes the formation of mycobacterial galactan, a critical cell-wall polymer composed of galactofuranose residues connected with alternating, regioisomeric linkages. We found that GlfT2 mediates galactan polymerization using only one active site that manifests dual regioselectivity. Structural modeling of the bifunctional glycosyltransferases hyaluronan synthase and cellulose synthase suggests that these enzymes also generate multiple glycosidic linkages using a single active site. These results highlight the versatility of glycosyltransferases for generating polysaccharides of specific sequence. We postulate that a hallmark of processive elongation of a carbohydrate polymer by a bifunctional enzyme is that one active site can give rise to two separate types of glycosidic bonds.


Subject(s)
Galactosyltransferases/chemistry , Amino Acid Substitution/genetics , Carbohydrate Conformation , Carbohydrate Sequence , Catalysis , Catalytic Domain , Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Glucuronosyltransferase/chemistry , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Hyaluronan Synthases , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerization , Sequence Homology, Amino Acid , Substrate Specificity/genetics
9.
Proc Natl Acad Sci U S A ; 106(29): 11851-6, 2009 Jul 21.
Article in English | MEDLINE | ID: mdl-19571009

ABSTRACT

Carbohydrate polymers are the most abundant organic substances on earth. Their degrees of polymerization range from tens to thousands of units, yet polymerases generate the relevant lengths without the aid of a template. To gain insight into template-independent length control, we investigated how the mycobacterial galactofuranosyl-transferase GlfT2 mediates formation of the galactan, a polymer of galactofuranose residues that is an integral part of the cell wall. We show that isolated recombinant GlfT2 can catalyze the synthesis of polymers with degrees of polymerization that are commensurate with values observed in mycobacteria, indicating that length control by GlfT2 is intrinsic. Investigations using synthetic substrates reveal that GlfT2 is processive. The data indicate that GlfT2 controls length by using a substrate tether, which is distal from the site of elongation. The strength of interaction of that tether with the polymerase influences the length of the resultant polymer. Thus, our data identify a mechanism for length control by a template-independent polymerase.


Subject(s)
Carbohydrates/chemistry , Models, Molecular , Polymers/chemistry , Bacterial Proteins/metabolism , Galactans/biosynthesis , Galactans/chemistry , Histidine/metabolism , Mycobacterium tuberculosis/enzymology , Oligopeptides/metabolism , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Uridine Diphosphate/metabolism
10.
J Am Chem Soc ; 130(21): 6706-7, 2008 May 28.
Article in English | MEDLINE | ID: mdl-18447352

ABSTRACT

Galactofuranose (Galf) residues are fundamental components of the cell wall of mycobacteria. A key enzyme, UDP-galactopyranose mutase (UGM), that participates in Galf incorporation mediates isomerization of UDP-Galf from UDP-galactopyranose (UDP-Galp). UGM is of special interest as a therapeutic target because the gene encoding it is essential for mycobacterial viability and there is no comparable enzyme in humans. We used structure-activity relationships and molecular design to devise UGM inhibitors. From a focused library of synthetic aminothiazoles, several compounds that block the UGM from Klebsiella pneumoniae or Mycobacterium tuberculosis were identified. These inhibitors block the growth of M. smegmatis.


Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Intramolecular Transferases/antagonists & inhibitors , Thiazoles/chemistry , Thiazoles/pharmacology , Intramolecular Transferases/metabolism , Models, Molecular , Molecular Conformation , Structure-Activity Relationship , Thiazolidines/chemistry , Thiazolidines/pharmacology
11.
Chem Biol ; 13(8): 825-37, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16931332

ABSTRACT

Many pathogenic prokaryotes and eukaryotes possess the machinery required to assemble galactofuranose (Galf)-containing glycoconjugates; these glycoconjugates can be critical for virulence or viability. Accordingly, compounds that block Galf incorporation may serve as therapeutic leads or as probes of the function of Galf-containing glycoconjugates. The enzyme UDP-galactopyranose mutase (UGM) is the only known generator of UDP-galactofuranose, the precursor to Galf residues. We previously employed a high-throughput fluorescence polarization assay to investigate the Klebsiella pneumoniae UGM. We demonstrate the generality of this assay by extending it to UGM from Mycobacterium tuberculosis. To identify factors influencing binding, we synthesized a directed library containing a 5-arylidene-2-thioxo-4-thiazolidinone core, a structure possessing features common to ligands for both homologs. Our studies offer a blueprint for identifying inhibitors of the growing family of UGM homologs and provide insight into UGM inhibition.


Subject(s)
Intramolecular Transferases/chemistry , Molecular Probes/chemistry , Thiazoles/chemistry , Binding Sites , Galactose/analogs & derivatives , Galactose/chemistry , Galactose/metabolism , Intramolecular Transferases/antagonists & inhibitors , Klebsiella pneumoniae/enzymology , Ligands , Models, Molecular , Molecular Probe Techniques , Molecular Probes/chemical synthesis , Molecular Probes/pharmacology , Molecular Structure , Mycobacterium tuberculosis/enzymology , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/pharmacology , Uridine Diphosphate/analogs & derivatives , Uridine Diphosphate/chemistry , Uridine Diphosphate/metabolism
12.
J Am Chem Soc ; 127(13): 4609-24, 2005 Apr 06.
Article in English | MEDLINE | ID: mdl-15796526

ABSTRACT

The principle of methyl scanning is proposed for determination of the sites of interaction between biologically active small molecules and their macromolecular target(s). It involves the systematic preparation of a family of methylated derivatives of a compound and their biological testing. As a functional assay, the method can identify the regions of a molecule that are important (and unimportant) for biological activity against even unknown targets, and thus provides an excellent complement to structural biology. Methyl scanning was applied to demethylasterriquinone B1, a small-molecule mimetic of insulin. A new, optimal total synthesis of this natural product was developed that enables the family of methyl scan derivatives to be concisely prepared for evaluation in a cellular assay. The results of this experiment were used to design a biotin-demethylasterriquinone conjugate for use as an affinity reagent. This compound was prepared in tens of milligram quantities in a four-step synthesis.


Subject(s)
Indoles/chemical synthesis , Receptors, Drug/chemistry , Animals , Binding Sites , Cell Line , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Fibroblasts/drug effects , Fibroblasts/enzymology , Indoles/chemistry , Indoles/pharmacology , Rats , Receptor, Insulin/metabolism , Receptors, Drug/isolation & purification , Structure-Activity Relationship
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