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1.
Nucleic Acids Res ; 48(3): 1583-1598, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31956908

RESUMO

Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.


Assuntos
Proteínas de Bactérias/genética , Bambermicinas/biossíntese , Produtos Biológicos/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/antagonistas & inibidores , GMP Cíclico/genética , GMP Cíclico/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/genética , Nucleotídeos/genética , Peptidoglicano Glicosiltransferase/antagonistas & inibidores , Fósforo-Oxigênio Liases/genética , Sistemas do Segundo Mensageiro/genética , Streptomycetaceae/genética , Streptomycetaceae/metabolismo , Fatores de Transcrição/antagonistas & inibidores
2.
mBio ; 10(4)2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387902

RESUMO

Carboxyl (C)-terminal processing proteases (CTPs) participate in protective and regulatory proteolysis in bacteria. The PDZ domain is central to the activity of CTPs but plays inherently different regulatory roles. For example, the PDZ domain inhibits the activity of the signaling protease CtpB by blocking the active site but is required for the activation of Prc (or Tsp), a tail-specific protease that degrades SsrA-tagged proteins. Here, by structural and functional analyses, we show that in the unliganded resting state of Prc, the PDZ domain is docked inside the bowl-shaped scaffold without contacting the active site, which is kept in a default misaligned conformation. In Prc, a hydrophobic substrate sensor distinct from CtpB engages substrate binding to the PDZ domain and triggers a structural remodeling to align the active-site residues. Therefore, this work reveals the structural basis for understanding the contrasting roles of the PDZ domain in the regulation of CTPs.IMPORTANCE Prc, also known previously as Tsp, is the founding member of the carboxyl-terminal processing protease (CTP) family of PDZ domain-containing proteases that include CtpA and CtpB. The substrate-binding PDZ domain is responsible for regulating the protease activity of CTP proteases; however, the regulatory role of PDZ domain is stimulatory in Prc but inhibitory in CtpA/B. By determining a series of crystal structures of Prc in the unliganded resting state, this study presents the structural basis for PDZ-dependent activation of Prc, the results of which explain the contrasting roles of the PDZ domain in the regulation of the protease activity of CTPs.


Assuntos
Endopeptidases/química , Endopeptidases/metabolismo , Escherichia coli/enzimologia , Sítios de Ligação , Endopeptidases/genética , Ativação Enzimática , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Domínios PDZ , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Proteólise , Especificidade por Substrato
3.
mBio ; 10(4)2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387909

RESUMO

Rod-shaped bacteria have two modes of peptidoglycan synthesis: lateral synthesis and synthesis at the cell division site. These two processes are controlled by two macromolecular protein complexes, the elongasome and divisome. Recently, it has been shown that the Bacillus subtilis RodA protein, which forms part of the elongasome, has peptidoglycan glycosyltransferase activity. The cell division-specific RodA homolog FtsW fulfils a similar role at the divisome. The human pathogen Listeria monocytogenes carries genes that encode up to six FtsW/RodA homologs; however, their functions have not yet been investigated. Analysis of deletion and depletion strains led to the identification of the essential cell division-specific FtsW protein, FtsW1. Interestingly, L. monocytogenes carries a gene that encodes a second FtsW protein, FtsW2, which can compensate for the lack of FtsW1, when expressed from an inducible promoter. L. monocytogenes also possesses three RodA homologs, RodA1, RodA2, and RodA3, and their combined absence is lethal. Cells of a rodA1 rodA3 double mutant are shorter and have increased antibiotic and lysozyme sensitivity, probably due to a weakened cell wall. Results from promoter activity assays revealed that expression of rodA3 and ftsW2 is induced in the presence of antibiotics targeting penicillin binding proteins. Consistent with this, a rodA3 mutant was more susceptible to the ß-lactam antibiotic cefuroxime. Interestingly, overexpression of RodA3 also led to increased cefuroxime sensitivity. Our study highlights that L. monocytogenes genes encode a multitude of functional FtsW and RodA enzymes to produce its rigid cell wall and that their expression needs to be tightly regulated to maintain growth, cell division, and antibiotic resistance.IMPORTANCE The human pathogen Listeria monocytogenes is usually treated with high doses of ß-lactam antibiotics, often combined with gentamicin. However, these antibiotics only act bacteriostatically on L. monocytogenes, and the immune system is needed to clear the infection. Therefore, individuals with a compromised immune system are at risk to develop a severe form of Listeria infection, which can be fatal in up to 30% of cases. The development of new strategies to treat Listeria infections is necessary. Here we show that the expression of some of the FtsW and RodA enzymes of L. monocytogenes is induced by the presence of ß-lactam antibiotics, and the combined absence of these enzymes makes bacteria more susceptible to this class of antibiotics. The development of antimicrobial agents that inhibit the activity or production of FtsW and RodA enzymes might therefore help to improve the treatment of Listeria infections and thereby lead to a reduction in mortality.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Listeria monocytogenes/citologia , Listeria monocytogenes/enzimologia , Proteínas de Membrana/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Proteínas de Bactérias/genética , Divisão Celular , Humanos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Proteínas de Membrana/genética , Peptidoglicano Glicosiltransferase/genética , Deleção de Sequência
4.
mSphere ; 4(4)2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270174

RESUMO

Penicillin-binding proteins (PBPs) are essential for bacterial cell wall biosynthesis, and several are clinically validated antibacterial targets of ß-lactam antibiotics. We identified mutations in the mrdA gene encoding the PBP2 protein in two Escherichia coli bla NDM-1 clinical isolates that reduce susceptibility to carbapenems and to the intrinsic antibacterial activity of a diazabicyclooctane (DBO) PBP2 and ß-lactamase inhibitor. These mutations coexisted with previously described mutations in ftsI (encoding PBP3) that reduce susceptibility to monobactams, penicillins, and cephalosporins. Clinical exposure to ß-lactams is driving the emergence of multifactorial resistance that may impact the therapeutic usefulness of existing antibacterials and novel compounds that target PBPs.IMPORTANCE Emerging antibacterial resistance is a consequence of the continued use of our current antibacterial therapies, and it is limiting their utility, especially for infections caused by multidrug-resistant isolates. ß-Lactams have enjoyed extensive clinical success, but their broad usage is linked to perhaps the most extensive and progressive example of resistance development for any antibacterial scaffold. In Gram-negative pathogens, this largely involves constant evolution of new ß-lactamases able to degrade successive generations of this scaffold. In addition, more recently, alterations in the targets of these compounds, penicillin-binding proteins (PBPs), are being described in clinical isolates, which often also have multiple ß-lactamases. This study underscores the multifactorial nature of ß-lactam resistance by uncovering alterations of PBP2 that reduce susceptibility to carbapenems in E. coli clinical isolates that also have alterations of PBP3 and express the NDM-1 ß-lactamase. The changes in PBP2 also reduced susceptibility to the intrinsic antibacterial activity of some diazabicyclooctane (DBO) compounds that can target PBP2. This may have implications for the development and use of the members of this relatively newer scaffold that are inhibitors of PBP2 in addition to their inhibition of serine-ß-lactamases.


Assuntos
Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Carbapenêmicos/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase/genética , Compostos Azabicíclicos/química , Testes de Sensibilidade Microbiana , Mutação , Resistência beta-Lactâmica , beta-Lactamases/genética , beta-Lactamas/farmacologia
5.
BMC Microbiol ; 19(1): 140, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234796

RESUMO

BACKGROUND: Persisters are rare phenotypic variants within a bacterial population that are capable of tolerating lethal antibiotic concentrations. Passage through stationary phase is associated with the formation of persisters (type I), and a major physiological response of Escherichia coli during stationary phase is cell wall restructuring. Given the concurrence of these processes, we sought to assess whether perturbation to cell wall synthesis during stationary phase impacts type I persister formation. RESULTS: We tested a panel of cell wall inhibitors and found that piperacillin, which primarily targets penicillin binding protein 3 (PBP3 encoded by ftsI), resulted in a significant reduction in both ß-lactam (ampicillin, carbenicillin) and fluoroquinolone (ofloxacin, ciprofloxacin) persister levels. Further analyses showed that piperacillin exposure through stationary phase resulted in cells with more ATP, DNA, RNA, and protein (including PBPs) than untreated controls; and that their physiology led to more rapid resumption of DNA gyrase supercoiling activity, translation, and cell division upon introduction into fresh media. Previously, PBP3 inhibition had been linked to antibiotic efficacy through the DpiBA two component system; however, piperacillin suppressed persister formation in ΔdpiA to the same extent as it did in wild-type, suggesting that DpiBA is not required for the phenomenon reported here. To test the generality of PBP3 inhibition on persister formation, we expressed FtsI Ser307Ala to genetically inhibit PBP3, and suppression of persister formation was also observed, although not to the same magnitude as that seen for piperacillin treatment. CONCLUSIONS: From these data we conclude that stationary phase PBP3 activity is important to type I persister formation in E. coli.


Assuntos
Farmacorresistência Bacteriana/efeitos dos fármacos , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/crescimento & desenvolvimento , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Peptidoglicano Glicosiltransferase/antagonistas & inibidores , Piperacilina/farmacologia , Parede Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoroquinolonas/farmacologia , Mutação , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase/genética , Fenótipo , Proteínas Quinases/metabolismo , Fatores de Transcrição/metabolismo , beta-Lactamas/farmacologia
6.
Nat Microbiol ; 4(8): 1368-1377, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31086309

RESUMO

Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure that is essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely, through the localization of late-stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in elongation and division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 probably constitute cognate pairs of interacting proteins. A lack of RodA-PBP3 resulted in more spherical cells due to deficient sidewall PGN synthesis, whereas depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilizing the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings or arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate sidewall and septal PGN incorporation, respectively, and that their activity must be balanced to maintain coccoid morphology.


Assuntos
Parede Celular/metabolismo , Peptidoglicano/metabolismo , Staphylococcus aureus/citologia , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular/fisiologia , Genes Bacterianos/genética , Proteínas de Membrana/metabolismo , Mutação , Oligossacarídeos/farmacologia , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Peptidil Transferases/metabolismo , Ligação Proteica , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Transcriptoma
7.
mBio ; 10(1)2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30723128

RESUMO

Gram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show that Escherichia coli cells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.IMPORTANCE In Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show that Escherichia coli cells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane.


Assuntos
Membrana Celular/metabolismo , Parede Celular/metabolismo , Escherichia coli/fisiologia , Viabilidade Microbiana , Peptidoglicano/metabolismo , Bacteriólise , Transporte Biológico , Proteínas de Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Peptidil Transferases/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo
8.
Nat Commun ; 10(1): 178, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30643125

RESUMO

Bacterial mutations predisposing pneumococcus to causing meningitis, a more severe form of invasive pneumococcal disease (IPD), are largely unknown. Knowledge of such mutations may improve our understanding of pathogenesis and inform preventive strategies. Here we report a pneumococcal pbp1b gene mutation (pbp1bA641C causing N214T change in PBP1b transglycosylase domain) that is associated with meningitis in an exploratory cohort of IPD patients (n = 2054, p = 6.8 × 10-6), in an independent confirmatory cohort (n = 2518, p = 2.3 × 10-6), and in a combined analysis (n = 4572, p = 3.0 × 10-10). Patients infected by the pbp1b641C genotype pneumococci show 2.8-fold odds (95% CI 1.7 to 4.8) of meningitis compared to those infected by non-pbp1b641C pneumococci, after controlling for pneumococcal serotype, antibiotic resistance, and patient age. The pbp1bA641C change results in longer time needed for bacterial killing by antibiotic treatment and shows evidence of being under positive selection. Thus, a pneumococcal mutation conferring increased antibiotic tolerance is associated with meningitis among IPD patients.


Assuntos
Proteínas de Bactérias/genética , Estudos de Associação Genética , Genoma Bacteriano/genética , Meningite Pneumocócica/microbiologia , Streptococcus pneumoniae/genética , Adolescente , Adulto , Idoso , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Criança , Pré-Escolar , Estudos de Coortes , Farmacorresistência Bacteriana/genética , Feminino , Genótipo , Humanos , Lactente , Masculino , Meningite Pneumocócica/tratamento farmacológico , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase , Domínios Proteicos/genética , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/isolamento & purificação , Adulto Jovem
9.
mBio ; 10(1)2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622193

RESUMO

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, PG synthesis localizes at midcell under the control of a multiprotein complex, the divisome, allowing the safe formation of two new cell poles and separation of daughter cells. Genetic studies in Escherichia coli pointed out that FtsBLQ and FtsN participate in the regulation of septal PG (sPG) synthesis; however, the underlying molecular mechanisms remained largely unknown. Here we show that FtsBLQ subcomplex directly interacts with the PG synthase PBP1b and with the subcomplex FtsW-PBP3, mainly via FtsW. Strikingly, we discovered that FtsBLQ inhibits the glycosyltransferase activity of PBP1b and that this inhibition was antagonized by the PBP1b activators FtsN and LpoB. The same results were obtained in the presence of FtsW-PBP3. Moreover, using a simple thioester substrate (S2d), we showed that FtsBLQ also inhibits the transpeptidase domain of PBP3 but not of PBP1b. As the glycosyltransferase and transpeptidase activities of PBP1b are coupled and PBP3 activity requires nascent PG substrate, the results suggest that PBP1b inhibition by FtsBLQ will block sPG synthesis by these enzymes, thus maintaining cell division as repressed until the maturation of the divisome is signaled by the accumulation of FtsN, which triggers sPG synthesis and the initiation of cell constriction. These results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which seems to counterbalance the inhibitory effect of FtsBLQ to restore PBP1b activity.IMPORTANCE Bacterial cell division is governed by a multiprotein complex called divisome, which facilitates a precise cell wall synthesis at midcell and daughter cell separation. Protein-protein interactions and activity studies using different combinations of the septum synthesis core of the divisome revealed that the glycosyltransferase activity of PBP1b is repressed by FtsBLQ and that the presence of FtsN or LpoB suppresses this inhibition. Moreover, FtsBLQ also inhibits the PBP3 activity on a thioester substrate. These results provide enzymatic evidence of the regulation of the peptidoglycan synthase PBP1b and PBP3 within the divisome. The results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which functions to relieve the repression on PBP1b by FtsBLQ and to initiate septal peptidoglycan synthesis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Divisão Celular , Parede Celular/metabolismo , Escherichia coli/genética , Escherichia coli/fisiologia , Peptidoglicano/biossíntese
10.
Curr Genet ; 65(1): 99-101, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30056491

RESUMO

Bacterial cells need to divide. This process requires more than 30 different proteins, which gather at the division site. It is widely assumed that these proteins assemble into a macromolecular complex (the divisome), but capturing the molecular layout of this complex has proven elusive. Super-resolution microscopy can provide spatial information, down to a few tens of nanometers, about how the division proteins assemble into complexes and how their activities are co-ordinated. Herein we provide insight into recent work from our laboratories, where we used super-resolution gSTED nanoscopy to explore the molecular organization of FtsZ, FtsI and FtsN. The resulting images show that all three proteins form discrete densities organised in patchy pseudo-rings at the division site. Significantly, two-colour imaging highlighted a radial separation between FtsZ and FtsN, indicating that there is more than one type of macromolecular complex operating during division. These data provide a first glimpse into the spatial organisation of PG-synthesising enzymes during division in Gram-negative bacteria.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Complexos Multiproteicos/metabolismo , Peptidoglicano/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Complexos Multiproteicos/genética , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/genética , Peptidoglicano Glicosiltransferase/metabolismo
11.
J Bacteriol ; 201(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30322854

RESUMO

The cell wall of Staphylococcus aureus is composed of peptidoglycan and the anionic polymers lipoteichoic acid (LTA) and wall teichoic acid. LTA is required for growth and normal cell morphology in S. aureus Strains lacking LTA are usually viable only when grown under osmotically stabilizing conditions or after the acquisition of compensatory mutations. LTA-negative suppressor strains with inactivating mutations in gdpP, which resulted in increased intracellular c-di-AMP levels, were described previously. Here, we sought to identify factors other than c-di-AMP that allow S. aureus to survive without LTA. LTA-negative strains able to grow in unsupplemented medium were obtained and found to contain mutations in sgtB, mazE, clpX, or vraT The growth improvement through mutations in mazE and sgtB was confirmed by complementation analysis. We also showed that an S. aureus sgtB transposon mutant, with the monofunctional peptidoglycan glycosyltransferase SgtB inactivated, displayed a 4-fold increase in the MIC of oxacillin, suggesting that alterations in the peptidoglycan structure could help bacteria compensate for the lack of LTA. Muropeptide analysis of peptidoglycans isolated from a wild-type strain and sgtB mutant strain did not reveal any sizable alterations in the peptidoglycan structure. In contrast, the peptidoglycan isolated from an LTA-negative ltaS mutant strain showed a significant reduction in the fraction of highly cross-linked peptidoglycan, which was partially rescued in the sgtB ltaS double mutant suppressor strain. Taken together, these data point toward an important function of LTA in cell wall integrity through its necessity for proper peptidoglycan assembly.IMPORTANCE The bacterial cell wall acts as a primary defense against environmental insults such as changes in osmolarity. It is also a vulnerable structure, as defects in its synthesis can lead to growth arrest or cell death. The important human pathogen Staphylococcus aureus has a typical Gram-positive cell wall, which consists of peptidoglycan and the anionic polymers LTA and wall teichoic acid. Several clinically relevant antibiotics inhibit the synthesis of peptidoglycan; therefore, it and teichoic acids are considered attractive targets for the development of new antimicrobials. We show that LTA is required for efficient peptidoglycan cross-linking in S. aureus and inactivation of a peptidoglycan glycosyltransferase can partially rescue this defect, together revealing an intimate link between peptidoglycan and LTA synthesis.


Assuntos
Lipopolissacarídeos/metabolismo , Viabilidade Microbiana , Peptidoglicano Glicosiltransferase/deficiência , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/metabolismo , Antibacterianos/farmacologia , Parede Celular/metabolismo , Meios de Cultura/química , Elementos de DNA Transponíveis , Farmacorresistência Bacteriana , Teste de Complementação Genética , Testes de Sensibilidade Microbiana , Mutagênese Insercional , Oxacilina/farmacologia , Peptidoglicano Glicosiltransferase/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Supressão Genética
12.
Appl Environ Microbiol ; 84(24)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30341076

RESUMO

Corynebacterium glutamicum is frequently engineered to serve as a versatile platform and model microorganism. However, due to its complex cell wall structure, transformation of C. glutamicum with exogenous DNA is inefficient. Although efforts have been devoted to improve the transformation efficiency by using cell wall-weakening agents, direct genetic engineering of cell wall synthesis for enhancing cell competency has not been explored thus far. Herein, we reported that engineering of peptidoglycan synthesis could significantly increase the transformation efficiency of C. glutamicum Comparative analysis of C. glutamicum wild-type strain ATCC 13869 and a mutant with high electrotransformation efficiency revealed nine mutations in eight cell wall synthesis-related genes. Among them, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA dramatically increased the electrotransformation of strain ATCC 13869 by 19.25-fold in the absence of cell wall-weakening agents, with no inhibition on growth. The Y489C mutation had no effect on the membrane localization of PonA but affected the peptidoglycan structure. Deletion of the ponA gene led to more dramatic changes to the peptidoglycan structure but only increased the electrotransformation by 4.89-fold, suggesting that appropriate inhibition of cell wall synthesis benefited electrotransformation more. Finally, we demonstrated that the PonAY489C mutation did not cause constitutive or enhanced glutamate excretion, making its permanent existence in C. glutamicum ATCC 13869 acceptable. This study demonstrates that genetic engineering of genes involved in cell wall synthesis, especially peptidoglycan synthesis, is a promising strategy to improve the electrotransformation efficiency of C. glutamicum IMPORTANCE Metabolic engineering and synthetic biology are now the key enabling technologies for manipulating microorganisms to suit the practical outcomes desired by humankind. The introduction of exogenous DNA into cells is an indispensable step for this purpose. However, some microorganisms, including the important industrial workhorse Corynebacterium glutamicum, possess a complex cell wall structure to shield cells against exogenous DNA. Although genes responsible for cell wall synthesis in C. glutamicum are known, engineering of related genes to improve cell competency has not been explored yet. In this study, we demonstrate that mutations in cell wall synthesis genes can significantly improve the electrotransformation efficiency of C. glutamicum Notably, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA increased electrotransformation efficiency by 19.25-fold by affecting peptidoglycan synthesis.


Assuntos
Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Mutação , Peptidoglicano/biossíntese , Peptidoglicano/genética , Aminoaciltransferases/genética , Proteínas de Transporte , Parede Celular/metabolismo , DNA Bacteriano/genética , Ácido Glutâmico/metabolismo , Engenharia Metabólica , Proteínas dos Microfilamentos , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano/química , Peptidoglicano Glicosiltransferase/genética
13.
Mol Microbiol ; 110(3): 335-356, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30044025

RESUMO

Bacteria surround their cytoplasmic membrane with an essential, stress-bearing peptidoglycan (PG) layer consisting of glycan chains linked by short peptides into a mesh-like structure. Growing and dividing cells expand their PG layer using inner-membrane anchored PG synthases, including Penicillin-binding proteins (PBPs), which participate in dynamic protein complexes to facilitate cell wall growth. In Escherichia coli, and presumably other Gram-negative bacteria, growth of the mainly single layered PG is regulated by outer membrane-anchored lipoproteins. The lipoprotein LpoB is required to activate PBP1B, which is a major, bi-functional PG synthase with glycan chain polymerising (glycosyltransferase) and peptide cross-linking (transpeptidase) activities. In this work we show how the binding of LpoB to the regulatory UB2H domain of PBP1B activates both activities. Binding induces structural changes in the UB2H domain, which transduce to the two catalytic domains by distinct allosteric pathways. We also show how an additional regulator protein, CpoB, is able to selectively modulate the TPase activation by LpoB without interfering with GTase activation.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/química , Peptidoglicano Glicosiltransferase/metabolismo , Peptidoglicano/biossíntese , D-Ala-D-Ala Carboxipeptidase Tipo Serina/química , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Regulação Alostérica , Proteínas de Membrana/metabolismo , Ligação Proteica , Conformação Proteica
14.
mBio ; 9(3)2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29895635

RESUMO

Clostridium difficile is the major etiologic agent of antibiotic-associated intestinal disease. Pathogenesis of C. difficile is mainly attributed to the production and secretion of toxins A and B. Unlike most clostridial toxins, toxins A and B have no signal peptide, and they are therefore secreted by unusual mechanisms involving the holin-like TcdE protein and/or autolysis. In this study, we characterized the cell surface protein Cwp19, a newly identified peptidoglycan-degrading enzyme containing a novel catalytic domain. We purified a recombinant His6-tagged Cwp19 protein and showed that it has lytic transglycosylase activity. Moreover, we observed that Cwp19 is involved in cell autolysis and that a C. difficilecwp19 mutant exhibited delayed autolysis in stationary phase compared to the wild type when bacteria were grown in brain heart infusion (BHI) medium. Wild-type cell autolysis is correlated to strong alterations of cell wall thickness and integrity and to release of cytoplasmic material. Furthermore, we demonstrated that toxins were released into the extracellular medium as a result of Cwp19-induced autolysis when cells were grown in BHI medium. In contrast, Cwp19 did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium. These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributions in vitro depending on growth conditions. Thus, Cwp19 is an important surface protein involved in autolysis of vegetative cells of C. difficile that mediates the release of the toxins from the cell cytosol in response to specific environment conditions.IMPORTANCEClostridium difficile-associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains of C. difficile generally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase in C. difficile, Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes to C. difficile cell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriólise , Clostridium difficile/enzimologia , Clostridium difficile/crescimento & desenvolvimento , Peptidoglicano Glicosiltransferase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Parede Celular/genética , Parede Celular/metabolismo , Infecções por Clostridium/microbiologia , Clostridium difficile/genética , Clostridium difficile/fisiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Peptidoglicano Glicosiltransferase/química , Peptidoglicano Glicosiltransferase/genética
15.
Biochimie ; 152: 1-5, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29909047

RESUMO

Peptidoglycan glycosyltransferases (GTase) of family 51 are essential enzymes for the synthesis of the glycan chains of the bacterial cell wall. They are considered potential antibacterial target, but discovery of inhibitors was hampered so far by the lack of efficient and affordable screening assay. Here we used Staphylococcus aureus MtgA to introduce a single tryptophan reporter residue in selected positions flanking the substrates binding cavity of the protein. We selected a mutant (Y181W) that shows strong fluorescence quenching in the presence of moenomycin A and two lipid II analogs inhibitors. The assay provides a simple method to study GTase-ligand interactions and can be used as primary high throughput screening of GTase inhibitors without the need for lipid II substrate or reporter ligands.


Assuntos
Ensaios de Triagem em Larga Escala , Peptidoglicano Glicosiltransferase/metabolismo , Staphylococcus aureus/enzimologia , Triptofano/metabolismo , Bambermicinas/metabolismo , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Ligantes , Mutagênese Sítio-Dirigida , Peptidoglicano Glicosiltransferase/antagonistas & inibidores , Peptidoglicano Glicosiltransferase/genética , Ligação Proteica , Espectrometria de Fluorescência , Especificidade por Substrato , Triptofano/genética , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
16.
FEMS Microbiol Lett ; 365(12)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29788206

RESUMO

High molecular weight penicillin-binding proteins (PBPs) are responsible for the biosynthesis of peptidoglycan. In Escherichia coli, PBP1a and PBP1b form multienzyme peptidoglycan-synthesizing complexes with outer membrane lipoproteins LpoA and LpoB, respectively. The two complexes appear to be largely redundant, although their distinct physiological roles remain unclear. PBP1a/LpoA and PBP1b/LpoB also exist in Shewanella oneidensis strain MR-1, but effects of the two complexes on aerobic growth and ß-lactam resistance are quite different. In this study, the phenotypes of strains lacking a certain complex in S. oneidensis were compared. Deletion of PBP1a/LpoA caused aberrant cell morphology (including branches and bulges), enhanced sensitivity to various envelope stresses and outer membrane permeability. On the contrary, strains lacking PBP1b/LpoB displayed phenotypes similar to the wild type.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/metabolismo , Peptidoglicano Glicosiltransferase/genética , Deleção de Sequência , Shewanella/citologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Permeabilidade da Membrana Celular , Parede Celular/metabolismo , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano/metabolismo , Fenótipo , Shewanella/genética
17.
Biochemistry ; 56(48): 6317-6320, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29131935

RESUMO

Formation of catenanes by proteins is rare, with few known examples. We report herein the X-ray structure of a catenane dimer of lytic transglycosylase SltB1 of Pseudomonas aeruginosa. The enzyme is soluble and exists in the periplasmic space, where it modifies the bacterial cell wall. The catenane dimer exhibits the protein monomers in a noncovalent chain-link arrangement, whereby a stretch of 51 amino acids (to become a loop and three helices) from one monomer threads through the central opening of the structure of the partner monomer. The protein folds after threading in a manner that leaves two helices (α1 and α2) as stoppers to impart stability to the dimer structure. The symmetric embrace by the two SltB1 molecules occludes both active sites entirely, an arrangement that is sustained by six electrostatic interactions between the two monomers. In light of the observation of these structural motifs in all members of Family 3 lytic transglycosylases, catenanes might be present for those enzymes, as well. The dimeric catenane might represent a regulated form of SltB1.


Assuntos
Cristalografia por Raios X , Peptidoglicano Glicosiltransferase/química , Peptidoglicano Glicosiltransferase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína
18.
Mol Microbiol ; 106(3): 419-438, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28833791

RESUMO

Most bacteria possess a peptidoglycan cell wall that determines their morphology and provides mechanical robustness during osmotic challenges. The biosynthesis of this structure is achieved by a large set of synthetic and lytic enzymes with varying substrate specificities. Although the biochemical functions of these proteins are conserved and well-investigated, the precise roles of individual factors and the regulatory mechanisms coordinating their activities in time and space remain incompletely understood. Here, we comprehensively analyze the autolytic machinery of the alphaproteobacterial model organism Caulobacter crescentus, with a specific focus on LytM-like endopeptidases, soluble lytic transglycosylases and amidases. Our data reveal a high degree of redundancy within each protein family but also specialized functions for individual family members under stress conditions. In addition, we identify two lytic transglycosylases and an amidase as new divisome components that are recruited to midcell at distinct stages of the cell cycle. The midcell localization of these proteins is affected by two LytM factors with degenerate catalytic domains, DipM and LdpF, which may serve as regulatory hubs coordinating the activities of multiple autolytic enzymes during cell constriction and fission respectively. These findings set the stage for in-depth studies of the molecular mechanisms that control peptidoglycan remodeling in C. crescentus.


Assuntos
Amidoidrolases/metabolismo , Caulobacter crescentus/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Divisão Celular , Parede Celular/metabolismo , Endopeptidases/metabolismo , Glicosiltransferases , Peptidoglicano/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-28559260

RESUMO

Carbapenemase-producing Enterobacteriaceae isolates (n = 110) from health care centers in central Indiana (from 2010 to 2013) were tested for susceptibility to combinations of avibactam (4 µg/ml) with ceftazidime, ceftaroline, or aztreonam. MIC50/MIC90 values were 1/2 µg/ml (ceftazidime-avibactam), 0.5/2 µg/ml (ceftaroline-avibactam), and 0.25/0.5 µg/ml (aztreonam-avibactam.) A ß-lactam MIC of 8 µg/ml was reported for the three combinations against one Escherichia coli isolate with an unusual TIPY insertion following Tyr344 in penicillin-binding protein 3 (PBP 3) as the result of gene duplication.


Assuntos
Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Aztreonam/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Ceftazidima/farmacologia , Cefalosporinas/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Elementos de DNA Transponíveis/genética , Combinação de Medicamentos , Duplicação Gênica/genética , Humanos , beta-Lactamases/genética , beta-Lactamases/metabolismo
20.
Plasmid ; 91: 9-18, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28219792

RESUMO

Conjugative transfer plays a major role in the transmission of antibiotic resistance in bacteria. pIP501 is a Gram-positive conjugative model plasmid with the broadest transfer host-range known so far and is frequently found in Enterococcus faecalis and Enterococcus faecium clinical isolates. The pIP501 type IV secretion system is encoded by 15 transfer genes. In this work, we focus on the VirB1-like protein TraG, a modular peptidoglycan metabolizing enzyme, and the VirB8-homolog TraM, a potential member of the translocation channel. By providing full-length traG in trans, but not with a truncated variant, we achieved full recovery of wild type transfer efficiency in the traG-knockout mutant E. faecalis pIP501ΔtraG. With peptidoglycan digestion experiments and tandem mass spectrometry we could assign lytic transglycosylase and endopeptidase activity to TraG, with the CHAP domain alone displaying endopeptidase activity. We identified a novel interaction between TraG and TraM in a bacterial-2-hybrid assay. In addition we found that both proteins localize in focal spots at the E. faecalis cell membrane using immunostaining and fluorescence microscopy. Extracellular protease digestion to evaluate protein cell surface exposure revealed that correct membrane localization of TraM requires the transmembrane helix of TraG. Thus, we suggest an essential role for TraG in the assembly of the pIP501 type IV secretion system.


Assuntos
Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/genética , Enterococcus faecalis/genética , Regulação Bacteriana da Expressão Gênica , Plasmídeos/química , Deleção de Sequência , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Proteínas de Transporte/metabolismo , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Conjugação Genética , Endopeptidases/genética , Endopeptidases/metabolismo , Enterococcus faecalis/metabolismo , Enterococcus faecalis/ultraestrutura , Peptidoglicano Glicosiltransferase/genética , Peptidoglicano Glicosiltransferase/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Sistemas de Secreção Tipo IV/metabolismo
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