RESUMO
The bacterial cell wall is the validated target of mainstream antimicrobials such as penicillin and vancomycin. Penicillin and other ß-lactams act by targeting Penicillin-Binding Proteins (PBPs), enzymes that play key roles in the biosynthesis of the main component of the cell wall, the peptidoglycan. Despite the spread of resistance towards these drugs, the bacterial cell wall continues to be a major Achilles' heel for microbial survival, and the exploration of the cell wall formation machinery is a vast field of work that can lead to the development of novel exciting therapies. The sheer complexity of the cell wall formation process, however, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. New developments in genetic and biochemical screens, as well as different aspects of structural biology, have shed new light on the importance of complexes formed by PBPs, notably within the cell wall elongation machinery. This chapter summarizes structural and functional details of PBP complexes involved in the periplasmic and membrane steps of peptidoglycan biosynthesis with a focus on cell wall elongation. These assemblies could represent interesting new targets for the eventual development of original antibacterials.
Assuntos
Bactérias/citologia , Bactérias/metabolismo , Parede Celular/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Parede Celular/química , Peptidoglicano/biossínteseRESUMO
In this study, we revealed that OA and UA significantly inhibited the expression of most genes related to peptidoglycan biosynthesis in S. mutans UA159. To the best of our knowledge, this is the first report to introduce the antimicrobial mechanism of OA and UA against S. mutans.
Assuntos
Antibacterianos/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Ácido Oleanólico/farmacologia , Peptidoglicano/biossíntese , Streptococcus mutans/efeitos dos fármacos , Triterpenos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ácido UrsólicoRESUMO
In this study, we revealed that OA and UA significantly inhibited the expression of most genes related to peptidoglycan biosynthesis in S. mutans UA159. To the best of our knowledge, this is the first report to introduce the antimicrobial mechanism of OA and UA against S. mutans.
Assuntos
Antibacterianos/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Ácido Oleanólico/farmacologia , Peptidoglicano/biossíntese , Streptococcus mutans/efeitos dos fármacos , Triterpenos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacosRESUMO
In this study, we revealed that OA and UA significantly inhibited the expression of most genes related to peptidoglycan biosynthesis in S. mutans UA159. To the best of our knowledge, this is the first report to introduce the antimicrobial mechanism of OA and UA against S. mutans.(AU)
Assuntos
Antibacterianos/farmacologia , Vias Biossintéticas , Vias Biossintéticas/genética , Ácido Oleanólico/farmacologia , Peptidoglicano/biossíntese , Streptococcus mutans , Triterpenos/farmacologia , Regulação Bacteriana da Expressão GênicaRESUMO
Tuberculosis (TB) is the second leading cause of human mortality from infectious diseases worldwide. The WHO reported 1.3 million deaths and 8.6 million new cases of TB in 2012. Mycobacterium tuberculosis (M. tuberculosis), the infectious bacteria that causes TB, is encapsulated by a thick and robust cell wall. The innermost segment of the cell wall is comprised of peptidoglycan, a layer that is required for survival and growth of the pathogen. Enzymes that catalyse biosynthesis of the peptidoglycan are essential and are therefore attractive targets for discovery of novel antibiotics as humans lack similar enzymes making it possible to selectively target bacteria only. In this paper, we have reviewed the structures and functions of enzymes GlmS, GlmM, GlmU, MurA, MurB, MurC, MurD, MurE and MurF from M. tuberculosis that are involved in peptidoglycan biosynthesis. In addition, we report homology modelled 3D structures of those key enzymes from M. tuberculosis of which the structures are still unknown. We demonstrated that natural substrates can be successfully docked into the active sites of the GlmS and GlmU respectively. It is therefore expected that the models and the data provided herein will facilitate translational research to develop new drugs to treat TB.
Assuntos
Antituberculosos/farmacologia , Desenho de Fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Peptidoglicano/biossíntese , Proteínas de Bactérias/fisiologia , Humanos , Modelos Moleculares , Terapia de Alvo Molecular/métodos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Relação Quantitativa Estrutura-AtividadeRESUMO
Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa.
Assuntos
Proteínas de Bactérias/química , Escherichia coli , Lipoproteínas/química , Peptidoglicano/química , Redobramento de Proteína , Xylella , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Cromatografia em Gel , Lipoproteínas/biossíntese , Lipoproteínas/isolamento & purificação , Dados de Sequência Molecular , Peptidoglicano/biossíntese , Peptidoglicano/isolamento & purificação , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
The MurA enzyme from Pseudomonas aeruginosa was purified to homogeneity and found to be biologically active as a UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase in a coupled enzyme assay where ATPase activity was measured by the release of inorganic phosphate. A microtiter plate assay coupled to competitive biopanning using the UDP-N-acetylglucosamine was used to screen 10(9) C-7-C and 12-mers peptides from phage display libraries. From 60 phage-encoded peptides identified after the fourth round of biopanning, deduced amino acid sequences were aligned and two peptides were synthesized and tested for inhibition of the MurA-catalyzed reaction. The PEP 1354 peptide inhibited the ATPase activity of MurA with an IC(50) value of 200muM and was found to be a competitive inhibitor of UNAG. The pre-incubation of MurA with inhibitor indicated a time-independent inhibition. This time-dependent inhibition is the first report of peptide inhibitors of MurA, which represent the scaffold for the synthesis of inhibitory peptidomimetic molecules.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/metabolismo , Peptídeos/fisiologia , Peptidoglicano/biossíntese , Açúcares de Uridina Difosfato/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Pseudomonas aeruginosa/enzimologiaRESUMO
Chromobacterium violaceum is a versatile, Gram-negative beta-protebacterium that grows in a variety of ecosystems in tropical and subtropical areas, such as the water and borders of the Negro River, in the Amazon region of Brazil. Although it is a saprophyte and is generally considered non-pathogenic, sporadic cases of human infection have been described, mainly in young children and in immunodeficient individuals. Although rare, infections with C. violaceum are characterized by rapid dissemination and high mortality. With the complete genome sequence of C. violaceum now available, a detailed description of the molecular arsenal required for this bacterium's remarkable versatility has been revealed. Most importantly, a more detailed picture of its biotechnological properties, including the characteristic violacein pigment, has emerged. The complete genome sequence also enabled us to make a thorough examination of the repertoire of genes encoding probable virulence factors, which determine the potential for pathogenesis. We described a number of genes involved in infectious processes, such as host cell adhesion, "contact-dependent secretion" of factors that promote cell invasion, as well as other virulence factors, such as cytolytic proteins. We also described genes involved with the synthesis of lipopolysaccharides and proteoglycan, known to elicit the synthesis of pro-inflammatory cytokines and involved in the detoxification process, which may contribute to the evasion of the bacteria from the host immune response.
Assuntos
Chromobacterium/genética , Genoma Bacteriano , Lipopolissacarídeos/biossíntese , Peptidoglicano/biossíntese , Fatores de Virulência/genética , Aderência Bacteriana/genética , Chromobacterium/patogenicidade , Colicinas/biossíntese , Colicinas/genética , Proteínas Hemolisinas/biossíntese , Proteínas Hemolisinas/genética , Indóis , Peptidoglicano/genética , Virulência/genéticaRESUMO
The role of glutamate as osmoprotector was investigated through the study of a mutation in its biosynthetic pathway. A glt::Tn917-lacZ-cat insertion mutant (N1) conferring glutamate auxotrophy and enhanced beta-galactosidase expression on high-salt media was selected. Co-transformation experiments and PCR analysis allowed locating the insertion into the gltB gene corresponding to the small unit of the glutamate synthase (GOGAT). The N1 mutant strain presented a glutamate requirement for growth and a tenfold decrease in GOGAT activity. Transcriptional activity of GOGAT, measured as beta-galactosidase from the transposon fusion, correlated with enzymatic activity; expression was enhanced at the stationary phase and in high-ionic-strength media. However, osmotolerance of cultures of N1 mutant were as wild-type (wt), at least in semi-rich medium. In contrast, sporulation was slightly reduced (75% of wt), and spores were less resistant to UV, heat, and osmolarity, properties linked to the content of small, acid-soluble proteins (SASP). The content of these proteins was, in fact, reduced, in particular the SASP-gamma type. The peptidoglycan-cortex, however, was not impaired since spores maintained lysozyme resistance. Addition of glutamate during sporulation partially rescued spore resistance, but germination and outgrowth remained impaired. Deficiencies in germination and outgrowth were also observed with spores from a gltA mutant strain. Taken together, these results pointed to the importance of GOGAT activity during sporulation, in particular for the synthesis SASPs.