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1.
PLoS One ; 17(9): e0270697, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36170255

RESUMO

Nicotinamide adenine dinucleotide (NAD+) is an essential co-factor for cellular metabolism and serves as a substrate in enzymatic processes. NAD+ is produced by de novo synthesis or salvage pathways in nearly all bacterial species. Haemophilus influenzae lacks the capacity for de novo synthesis, so it is dependent on import of NAD+ from the external environment or salvage biosynthetic pathways for recycling of NAD+ precursors and breakdown products. However, the actual sources of NAD+ utilized by H. influenzae in the respiratory tract are not well defined. In this study, we found that a variety of bacteria, including species found in the upper airway of humans, released NAD+ that was readily detectable in extracellular culture fluid, and which supported growth of H. influenzae in vitro. By contrast, certain strains of Streptococcus pyogenes (group A streptococcus or GAS) inhibited growth of H. influenzae in vitro by secreting NAD+-glycohydrolase (NADase), which degraded extracellular NAD+. Conversely, GAS strains that lacked enzymatically active NADase released extracellular NAD+, which could support H. influenzae growth. Our results suggest that many bacterial species, including normal flora of the upper airway, release NAD+ into the environment. GAS is distinctive in its ability to both release and degrade NAD+. Thus, colonization of the airway with H. influenzae may be promoted or restricted by co-colonization with GAS in a strain-specific manner that depends, respectively, on release of NAD+ or secretion of active NADase. We suggest that, in addition to its role as a cytotoxin for host cells, NADase may serve a separate function by restricting growth of H. influenzae in the human respiratory tract.


Assuntos
NAD , Streptococcus pyogenes , Citotoxinas/metabolismo , Haemophilus influenzae/metabolismo , Humanos , NAD/metabolismo , NAD+ Nucleosidase/metabolismo , Streptococcus pyogenes/metabolismo
2.
mBio ; 11(5)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934086

RESUMO

One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies.IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.


Assuntos
Antibacterianos/farmacologia , Sinergismo Farmacológico , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Salicilanilidas/metabolismo , Salicilanilidas/farmacologia , Animais , Desenho de Fármacos , Reposicionamento de Medicamentos , Farmacorresistência Bacteriana Múltipla , Quimioterapia Combinada/métodos , Feminino , Camundongos , Testes de Sensibilidade Microbiana , Niclosamida/metabolismo , Niclosamida/farmacologia
3.
Nat Chem Biol ; 15(5): 463-471, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30936502

RESUMO

Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. Here we identify gacH, a gene in the Streptococcus pyogenes group A carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA-secreted phospholipase A2. Subsequent structural and phylogenetic analysis of the GacH extracellular domain revealed that GacH represents an alternative class of glycerol phosphate transferase. We detected the presence of glycerol phosphate in the GAC, as well as the serotype c carbohydrate from Streptococcus mutans, which depended on the presence of the respective gacH homologs. Finally, nuclear magnetic resonance analysis of GAC confirmed that glycerol phosphate is attached to approximately 25% of the GAC N-acetylglucosamine side-chains at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design.


Assuntos
Glicerol/metabolismo , Fosfatos/metabolismo , Polissacarídeos Bacterianos/metabolismo , Streptococcus/metabolismo , Glicerol/química , Fosfatos/química , Polissacarídeos Bacterianos/química , Streptococcus/química
4.
Mol Microbiol ; 106(6): 891-904, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28971540

RESUMO

Alternative sigma (σ) factors govern expression of bacterial genes in response to diverse environmental signals. In Pseudomonas aeruginosa σPvdS directs expression of genes for production of a siderophore, pyoverdine, as well as a toxin and a protease. σFpvI directs expression of a receptor for ferripyoverdine import. Expression of the genes encoding σPvdS and σFpvI is iron-regulated and an antisigma protein, FpvR20 , post-translationally controls the activities of the sigma factors in response to the amount of ferripyoverdine present. Here we show that iron represses synthesis of σPvdS to a far greater extent than σFpvI . In contrast ferripyoverdine exerts similar effects on the activities of both sigma factors. Using a combination of in vivo and in vitro assays we show that σFpvI and σPvdS have comparable affinities for, and are equally inhibited by, FpvR20 . Importantly, in the absence of ferripyoverdine the amount of FpvR20 per cell is lower than the amount of σFpvI and σPvdS , allowing basal expression of target genes that is required to activate the signalling pathway when ferripyoverdine is present. This complex interplay of transcriptional and post-translational regulation enables a co-ordinated response to ferripyoverdine but distinct responses to iron.


Assuntos
Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Repressoras/metabolismo , Fator sigma/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Quelantes de Ferro , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ligação Proteica , Pseudomonas aeruginosa/genética , Elementos Reguladores de Transcrição , Proteínas Repressoras/genética , Sideróforos/genética , Sideróforos/metabolismo , Fator sigma/antagonistas & inibidores , Fator sigma/genética
5.
J Biol Chem ; 292(47): 19441-19457, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-29021255

RESUMO

In many Lactobacillales species (i.e. lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, Streptococcus pyogenes, synthesizes a key antigenic surface polymer, the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with N-acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry, and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphoryl-undecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltransferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function in Streptococcus agalactiae and Enterococcus faecalis In conclusion, the elucidation of GAC biosynthesis in S. pyogenes reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall.


Assuntos
Acetilglucosamina/metabolismo , Proteínas de Bactérias/metabolismo , Carboidratos/química , Fosfolipídeos/metabolismo , Ramnose/biossíntese , Streptococcus pyogenes/metabolismo , Amidoidrolases/metabolismo , Proteínas de Bactérias/química , Membrana Celular/metabolismo , Peptidoglicano/metabolismo , Streptococcus pyogenes/química
6.
Artigo em Inglês | MEDLINE | ID: mdl-27790410

RESUMO

Streptococcus pyogenes (Group A Streptococcus or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C3-like ADP-ribosyltransferase SpyA. In this study, we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to ß-lactams of the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Thus, our data suggest the possibility that SpyB activity is regulated by heme.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Parede Celular/química , Hemeproteínas/genética , Hemeproteínas/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Antibacterianos/farmacologia , Aderência Bacteriana , Farmacorresistência Bacteriana , Deleção de Genes , Glicosídeos/análise , Heme/metabolismo , Proteínas Ligantes de Grupo Heme , N-Acetil-Muramil-L-Alanina Amidase/análise , Peptidoglicano/análise , Ligação Proteica , Multimerização Proteica , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/fisiologia , beta-Lactamas/farmacologia
7.
BMC Microbiol ; 14: 287, 2014 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-25433393

RESUMO

BACKGROUND: Synthesis and uptake of pyoverdine, the primary siderophore of the opportunistic pathogen Pseudomonas aeruginosa, is dependent on two extra-cytoplasmic function (ECF) sigma factors, FpvI and PvdS. FpvI and PvdS are required for expression of the ferri-pyoverdine receptor gene fpvA and of pyoverdine synthesis genes respectively. In the absence of pyoverdine the anti-sigma factor FpvR that spans the cytoplasmic membrane inhibits the activities of both FpvI and PvdS, despite the two sigma factors having low sequence identity. RESULTS: To investigate the interactions of FpvR with FpvI and PvdS, we first used a tandem affinity purification system to demonstrate binding of PvdS by the cytoplasmic region of FpvR in P. aeruginosa at physiological levels. The cytoplasmic region of FpvR bound to and inhibited both FpvI and PvdS when the proteins were co-expressed in Escherichia coli. Each sigma factor was then subjected to error prone PCR and site-directed mutagenesis to identify mutations that increased sigma factor activity in the presence of FpvR. In FpvI, the amino acid changes clustered around conserved region four of the protein and are likely to disrupt interactions with FpvR. Deletion of five amino acids from the C-terminal end of FpvI also disrupted interactions with FpvR. Mutations in PvdS were present in conserved regions two and four. Most of these mutations as well as deletion of thirteen amino acids from the C-terminal end of PvdS increased sigma factor activity independent of whether FpvR was present, suggesting that they increase either the stability of PvdS or its affinity for core RNA polymerase. CONCLUSIONS: These data show that FpvR binds to PvdS in both P. aeruginosa and E. coli, inhibiting its activity. FpvR also binds to and inhibits FpvI and binding of FpvI is likely to involve conserved region four of the sigma factor protein.


Assuntos
Regulação Bacteriana da Expressão Gênica , Oligopeptídeos/metabolismo , Pseudomonas aeruginosa/fisiologia , Fator sigma/antagonistas & inibidores , Fator sigma/metabolismo , Transdução de Sinais , Análise Mutacional de DNA , Mutagênese Sítio-Dirigida , Ligação Proteica , Mapeamento de Interação de Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Deleção de Sequência
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