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1.
Res Microbiol ; 170(8): 374-380, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31376483

RESUMO

FtsEX is a member of a small subclass of ABC transporters that uses mechano-transmission to perform work in the periplasm. FtsEX controls periplasmic peptidoglycan (PG) hydrolase activities in many Gram negative and positive organisms to ensure the safe separation of daughter cells during division. In these organisms FtsEX localizes to the Z ring and uses its ATPase activity to regulate its periplasmic effectors. In Escherichia coli, FtsEX also participates in building the divisome and coordinates PG synthesis with PG hydrolysis. This review discusses studies that are beginning to elucidate the mechanisms of FtsEX's various roles in cell division.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Fenômenos Biomecânicos/fisiologia , Parede Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Peptidoglicano/metabolismo
2.
PLoS Genet ; 15(8): e1008296, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31437162

RESUMO

The peptidoglycan (PG) sacculus is composed of long glycan strands cross-linked together by short peptides forming a covalently closed meshwork that protects the bacterial cell from osmotic lysis and specifies its shape. PG hydrolases play essential roles in remodeling this three-dimensional network during growth and division but how these autolytic enzymes are regulated remains poorly understood. The FtsEX ABC transporter-like complex has emerged as a broadly conserved regulatory module in controlling cell wall hydrolases in diverse bacterial species. In most characterized examples, this complex regulates distinct PG hydrolases involved in cell division and is intimately associated with the cytokinetic machinery called the divisome. However, in the gram-positive bacterium Bacillus subtilis the FtsEX complex is required for cell wall elongation where it regulates the PG hydrolase CwlO that acts along the lateral cell wall. To investigate whether additional factors are required for FtsEX function outside the divisome, we performed a synthetic lethal screen taking advantage of the conditional essentiality of CwlO. This screen identified two uncharacterized factors (SweD and SweC) that are required for CwlO activity. We demonstrate that these proteins reside in a membrane complex with FtsX and that amino acid substitutions in residues adjacent to the ATPase domain of FtsE partially bypass the requirement for them. Collectively our data indicate that SweD and SweC function as essential co-factors of FtsEX in controlling CwlO during cell wall elongation. We propose that factors analogous to SweDC function to support FtsEX activity outside the divisome in other bacteria.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Divisão Celular/genética , Parede Celular/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Elementos de DNA Transponíveis/genética , Mutação , Peptidoglicano/metabolismo
3.
Int J Biol Macromol ; 138: 881-889, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31356938

RESUMO

Quantitative measurement of cell lysis against a given microbial strain is essential to calculate the antimicrobial potency of protein/peptide/nanomaterial based formulations. Fluorescence spectroscopy based measurements offer precise quantification of a process via selected flurophore emission profile. In this context, we elucidate a reliable and robust green fluorescent protein (GFP) based fluorescence spectroscopy protocol to evaluate the antimicrobial activity of proteins. The technique is based on the fact that the intensity of the GFP emission released from cells correlates with cell lysis and henceforth the antimicrobial potential of the chosen agent. The technique was demonstrated with two different families of bacteriophage endolysins (T7 and T4 endolysins) using GFP expressing E. coli cells. The GFP based method allowed the absolute quantification of T4 and T7 endolysins cell lysis characteristics at different pH, salt concentrations, and metal ions. The results obtained from GFP based fluorimetric assay were substantiated with turbidimetric assay and fluorescence microscopy. This fluorimetric method in conjugation with different GFP expressing microbial strains and antimicrobial agents can be efficiently applied as a quantification technique to precisely measure cell lysis.


Assuntos
Escherichia coli/metabolismo , Fluorometria/métodos , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Análise Espectral
4.
Microbiology ; 165(9): 1013-1023, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31264955

RESUMO

Mycobacteriophages are viruses that infect and kill mycobacteria. The peptidoglycan hydrolase, lysin A (LysA), coded by one of the most potent mycobacteriophages, D29, carries two catalytic domains at its N-terminus and a cell wall-binding domain at its C-terminus. Here, we have explored the importance of the centrally located lysozyme-like catalytic domain (LD) of LysA in phage physiology. We had previously identified an R198A substitution that causes inactivation of the LD when it is present alone on a polypeptide. Here, we show that upon incorporation of the same mutation (i.e. R350A) in full-length LysA, the protein demonstrates substantially reduced activity in vitro, even in the presence of the N-terminal catalytic domain, and has less efficient mycobacterial cell lysis ability when it is expressed in Mycobacterium smegmatis. These data suggest that an active LD is required for the full-length protein to function optimally. Moreover, a mutant D29 phage harbouring this substitution (D29R350A) in its LysA protein shows significantly delayed host M. smegmatis lysis. However, the mutant phage demonstrates an increase in burst size and plaque diameter. Taken together, our data show the importance of an intact LD region in D29 LysA PG hydrolase, and indicate an evolutionary advantage over other phages that lack such a domain in their endolysins.


Assuntos
Endopeptidases/genética , Micobacteriófagos , Mycobacterium smegmatis/virologia , N-Acetil-Muramil-L-Alanina Amidase/genética , Domínio Catalítico/genética , Parede Celular/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Mutação , Micobacteriófagos/genética , Micobacteriófagos/crescimento & desenvolvimento , Micobacteriófagos/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
5.
Vet Microbiol ; 234: 92-100, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213278

RESUMO

Streptococcus suis (SS) is a major pathogen in the swine industry, and also an important zoonotic agent for humans. The novel SS cell surface protein, AtlASS, comprising the special GW module and N-acetylmuramidases domain, was designated as a putative autolysin. Indeed, the atlASS deletion mutant almost completely lost its activity in Triton X-100 induced bacterial autolysis, while the wild-type and CΔatlASS strains showed significant decrease, to less than 20% of the initial OD600 values. Unexpectedly, both immunofluorescence and immunogold electron microscopy confirmed that AtlASS is mainly located in the cell division septum, suggesting autolytic activity in peptidoglycan hydrolysis may be required for cell separation, thus modulating and truncating bacterial chain length. The biofilm capacity of the AtlASS mutation was reduced ˜ 40%, as compared to the wild-type strain. The ΔatlASS strain also attenuated bacterial adherence in human brain microvessel endothelial cells (HBMECs). Furthermore, we confirmed that AtlASS has fibrinogen/fibronectin binding capacities. In mouse infection model, the AtlASS inactivation also significantly attenuated bacterial virulence and proliferation in vivo. In conclusion, these results indicate that AtlASS autolysin modulates bacterial chain length, and contributes to the full virulence of SS during infection.


Assuntos
Divisão Celular , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Streptococcus suis/química , Streptococcus suis/patogenicidade , Animais , Autólise , Biofilmes/crescimento & desenvolvimento , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , N-Acetil-Muramil-L-Alanina Amidase/genética , Organismos Livres de Patógenos Específicos , Virulência
6.
Proc Natl Acad Sci U S A ; 116(16): 7825-7830, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30940749

RESUMO

Bacteria are surrounded by a protective exoskeleton, peptidoglycan (PG), a cross-linked mesh-like macromolecule consisting of glycan strands interlinked by short peptides. Because PG completely encases the cytoplasmic membrane, cleavage of peptide cross-links is a prerequisite to make space for incorporation of nascent glycan strands for its successful expansion during cell growth. In most bacteria, the peptides consist of l-alanine, d-glutamate, meso-diaminopimelic acid (mDAP) and d-alanine (d-Ala) with cross-links occurring either between d-Ala and mDAP or two mDAP residues. In Escherichia coli, the d-Ala-mDAP cross-links whose cleavage by specialized endopeptidases is crucial for expansion of PG predominate. However, a small proportion of mDAP-mDAP cross-links also exist, yet their role in the context of PG expansion or the hydrolase(s) capable of catalyzing their cleavage is not known. Here, we identified an ORF of unknown function, YcbK (renamed MepK), as an mDAP-mDAP cross-link cleaving endopeptidase working in conjunction with other elongation-specific endopeptidases to make space for efficient incorporation of nascent PG strands into the sacculus. E. coli mutants lacking mepK and another d-Ala-mDAP-specific endopeptidase (mepS) were synthetic sick, and the defects were abrogated by lack of l,d-transpeptidases, enzymes catalyzing the formation of mDAP cross-links. Purified MepK was able to cleave the mDAP cross-links of soluble muropeptides and of intact PG sacculi. Overall, this study describes a PG hydrolytic enzyme with a hitherto unknown substrate specificity that contributes to expansion of the PG sacculus, emphasizing the fundamental importance of cross-link cleavage in bacterial peptidoglycan synthesis.


Assuntos
Bactérias/enzimologia , Bactérias/metabolismo , Proteínas de Bactérias , Parede Celular , N-Acetil-Muramil-L-Alanina Amidase , Aminoácidos/química , Aminoácidos/metabolismo , Bactérias/química , Bactérias/citologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/química , Parede Celular/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Especificidade por Substrato
7.
Elife ; 82019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30969170

RESUMO

We discovered that Enterococcus faecium (E. faecium), a ubiquitous commensal bacterium, and its secreted peptidoglycan hydrolase (SagA) were sufficient to enhance intestinal barrier function and pathogen tolerance, but the precise biochemical mechanism was unknown. Here we show E. faecium has unique peptidoglycan composition and remodeling activity through SagA, which generates smaller muropeptides that more effectively activates nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mammalian cells. Our structural and biochemical studies show that SagA is a NlpC/p60-endopeptidase that preferentially hydrolyzes crosslinked Lys-type peptidoglycan fragments. SagA secretion and NlpC/p60-endopeptidase activity was required for enhancing probiotic bacteria activity against Clostridium difficile pathogenesis in vivo. Our results demonstrate that the peptidoglycan composition and hydrolase activity of specific microbiota species can activate host immune pathways and enhance tolerance to pathogens.


Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Enterococcus faecium/enzimologia , Enterococcus faecium/imunologia , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Proteína Adaptadora de Sinalização NOD2/metabolismo , Peptidoglicano/metabolismo , Conformação Proteica
8.
Metallomics ; 11(4): 799-809, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30869729

RESUMO

Peptidoglycan hydrolase of bacteriophage T5 (EndoT5) is a Ca2+-dependent l-alanyl-d-glutamate peptidase, although the mode of Ca2+ binding and its physiological significance remain obscure. Site-directed mutagenesis was used to elucidate the role of the polar amino acids of the mobile loop of EndoT5 (111-130) in Ca2+ binding. The mutant proteins were purified to electrophoretic homogeneity, the overall structures were characterized by circular dichroism, and the calcium dissociation constants were determined via NMR spectroscopy. The data suggest that polar amino acids D113, N115, and S117 of EndoT5 are involved in the coordination of calcium ions by forming the core of the EF-like Ca2+-binding loop while the charged residues D122 and E123 of EndoT5 contribute to maintaining the loop net charge density. The results suggest that Ca2+ binding to the EndoT5 molecule could be essential for the stabilization of the long mobile loop in the catalytically active "open" conformation. The possible mechanism of Ca2+ regulation of EndoT5 activity during bacteriophage T5's life cycle through the Ca2+ concentration difference between the cytoplasm and the periplasm of the host bacteria cell has been discussed. The study reveals valuable insight into the role of calcium in the regulation of phage-induced bacterial lysis.


Assuntos
Cálcio/metabolismo , Escherichia coli/virologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Fagos T/enzimologia , Proteínas Virais/metabolismo , Ativação Enzimática , Escherichia coli/citologia , Modelos Moleculares , Fagos T/metabolismo
9.
J Med Microbiol ; 68(4): 667-677, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30888314

RESUMO

PURPOSE: To determine the effect of allicin on Staphylococcus aureus cell wall peptidoglycans by the application of MALDI-TOF mass spectrometry on whole cells and to relate this to current knowledge of wall-processing enzymes. METHODOLOGY: Two different S. aureus strains were grown for 48 h after which period each culture was split into two, one part was then treated with sub-inhibitory levels of allicin while the other part left untreated as a control. After a further 24 h whole cells were recovered and analysed by MALDI-TOF mass spectrometry. RESULTS: Changes in the mass spectra between the treated and untreated cells revealed fragmented peptidoglycans identified by mass calculation only in the treated cells. These peptidoglycan fragments where identified as the products of specific peptidoglycan hydrolases. CONCLUSIONS: Allicin is known to target cysteine thiol groups. These are absent in peptidoglycan hydrolases and we might have expected identical results in both of the treated and untreated cells. Peptidoglycan synthesis enzymes such as the Fem family of enzymes do contain cysteines. Fem enzymes A, B and X all have a conserved conformation of 99 % for over 100 S. aureus strains and are therefore potential targets for allicin. Examination of FemA structure showed that cysteine102 is accessible from the surface. We propose that allicin has an inhibitory mechanism alongside others of targeting FemA and possibly other Fem enzymes by curtailing glycine bridging and leading to fragmentation. This study provided an insight into yet another antimicrobial mechanism of allicin.


Assuntos
Parede Celular/efeitos dos fármacos , Peptidoglicano/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia , Parede Celular/química , Hidrólise , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Staphylococcus aureus/enzimologia
10.
Arch Microbiol ; 201(5): 639-647, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30778632

RESUMO

Many bacteria exploit host proteins for their colonization. Vitronectin (Vn), present in the blood and extracellular matrix, is one such protein that acts as a bridge between the bacteria and the host tissues leading to infection. In this study, Vn binding protein of Staphylococcus aureus (COL strain) (SaVnBP) has been characterized as autolysin(s) based on mass spectrometry data and the ability of these proteins to degrade S. aureus substratum. Deletion of the heparin-binding domain (residues 341-380) from the Vn did not affect its ability to interact with SaVnBP. Similarly, change of R to A or D to A in the second arginine-glycine-aspartic (RGD2) motif of Vn had no negative effect on protein-protein interaction. These results imply that the primary heparin-binding site and the second RGD motif of caprine Vn may not be involved in the initial step of S. aureus colonization.


Assuntos
Proteínas de Transporte/metabolismo , Heparina/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Oligopeptídeos/metabolismo , Staphylococcus aureus/metabolismo , Vitronectina/metabolismo , Animais , Arginina/metabolismo , Ácido Aspártico/metabolismo , Proteínas de Bactérias/metabolismo , Matriz Extracelular , Glicina/metabolismo , Cabras/metabolismo , Humanos , Ligação Proteica/fisiologia , Somatomedinas/metabolismo
11.
Adv Virus Res ; 103: 33-70, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30635077

RESUMO

The first steps in phage lysis involve a temporally controlled permeabilization of the cytoplasmic membrane followed by enzymatic degradation of the peptidoglycan. For Caudovirales of Gram-negative hosts, there are two different systems: the holin-endolysin and pinholin-SAR endolysin pathways. In the former, lysis is initiated when the holin forms micron-scale holes in the inner membrane, releasing active endolysin into the periplasm to degrade the peptidoglycan. In the latter, lysis begins when the pinholin causes depolarization of the membrane, which activates the secreted SAR endolysin. Historically, the disruption of the first two barriers of the cell envelope was thought to be necessary and sufficient for lysis of Gram-negative hosts. However, recently a third functional class of lysis proteins, the spanins, has been shown to be required for outer membrane disruption. Spanins are so named because they form a protein bridge that connects both membranes. Most phages produce a two-component spanin complex, composed of an outer membrane lipoprotein (o-spanin) and an inner membrane protein (i-spanin) with a predominantly coiled-coil periplasmic domain. Some phages have a different type of spanin which spans the periplasm as a single molecule, by virtue of an N-terminal lipoprotein signal and a C-terminal transmembrane domain. Evidence is reviewed supporting a model in which the spanins function by fusing the inner membrane and outer membrane. Moreover, it is proposed that spanin function is inhibited by the meshwork of the peptidoglycan, thus coupling the spanin step to the first two steps mediated by the holin and endolysin.


Assuntos
Bacteriólise/fisiologia , Bacteriófagos/fisiologia , Bactérias Gram-Negativas/virologia , Proteínas Virais/genética , Bacteriófagos/genética , Parede Celular/metabolismo , Parede Celular/virologia , DNA/genética , DNA/metabolismo , Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Fusão de Membrana/fisiologia , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Transdução de Sinais/genética , Proteínas Virais/metabolismo
12.
mBio ; 10(1)2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696736

RESUMO

Streptococcus pneumoniae is a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically by targeting proteins that are essential for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises a cell division-regulating integral membrane protein (FtsX) and a cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present nuclear magnetic resonance (NMR) solution structural and crystallographic models of the large extracellular domain of FtsX, denoted extracellular loop 1 (ECL1). The structure of ECL1 reveals an upper extended ß-hairpin and a lower α-helical lobe, each extending from a mixed α-ß core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB via the coiled-coil domain of PcsB (PscBCC). Characterization of S. pneumoniae strain D39-derived strains harboring mutations in the α-helical lobe shows that this subdomain is essential for cell viability and required for proper cell division of S. pneumoniae IMPORTANCE FtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as "mechanotransmitters," proteins that relay signals from the inside to the outside of the cell. Here, we present a structural characterization of the large extracellular loop, ECL1, of FtsX from the opportunistic human pathogen S. pneumoniae We show the molecular nature of the direct interaction between the peptidoglycan hydrolase PcsB and FtsX and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Streptococcus pneumoniae/enzimologia , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Análise Mutacional de DNA , Genes Essenciais , Espectroscopia de Ressonância Magnética , Viabilidade Microbiana , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/fisiologia
13.
Int J Med Microbiol ; 309(1): 73-83, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30563740

RESUMO

Tuberculosis is a highly infectious disease and of high incidence in low-income countries that is caused by Mycobacterium tuberculosis (M. tuberculosis). M. tuberculosis can form biofilms in vitro and in vivo, and the cells in the biofilm can survive at high concentrations of antibiotics. CwlM is a peptidoglycan hydrolase (amidase) and can hydrolyze bacterial cell walls, and the effects of CwlM on autolysis and biofilms is worthy of in-depth study. In this study, we successfully constructed an in vitro biofilm model of M. tuberculosis and Mycobacterium smegmatis (M. smegmatis). Reverse transcription followed by real-time quantitative PCR (qPCR) revealed that the expression of cwlM in M. tuberculosis and M. smegmatis was significantly up-regulated during the middle stage of biofilm formation. Treatment with recombinant CwlM enhanced the autolytic ability of M. tuberculosis and M. smegmatis and reduced the formation of their biofilms. As M. smegmatis is a model bacterium of M. tuberculosis, we built the M. smegmatis cwlM-deletion strain MSΔ6935, whose autolytic ability, biofilm production, and eDNA and eRNA content were determined to be lower than those of its parental strain. In conclusion, the cwlM gene plays a key regulatory role in biofilm formation in M. tuberculosis and M. smegmatis. This study provided a theoretical basis for using peptidoglycan hydrolase as a target for the inhibition of biofilms.


Assuntos
Bacteriólise/genética , Biofilmes/crescimento & desenvolvimento , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Parede Celular/metabolismo , Humanos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Tuberculose/microbiologia
14.
mBio ; 9(6)2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538181

RESUMO

The human eukaryotic pathogen Trichomonas vaginalis causes trichomoniasis, a prevalent sexually transmitted infection. This extracellular protozoan is intimately associated with the human vaginal mucosa and microbiota, but key aspects of the complex interactions between the parasite and the vaginal bacteria remain elusive. We report that T. vaginalis has acquired, by lateral gene transfer from bacteria, genes encoding peptidoglycan hydrolases of the NlpC/P60 family. Two of the T. vaginalis enzymes were active against bacterial peptidoglycan, retaining the active-site fold and specificity as dl-endopeptidases. The endogenous NlpC/P60 genes are transcriptionally upregulated in T. vaginalis in the presence of bacteria. The overexpression of an exogenous copy enables the parasite to outcompete bacteria from mixed cultures, consistent with the biochemical activity of the enzyme. Our study results highlight the relevance of the interactions of this eukaryotic pathogen with bacteria, a poorly understood aspect of the biology of this important human parasite.IMPORTANCE Trichomonas vaginalis is a parasitic protozoan of the human urogenital tract that causes trichomoniasis, a very common sexually transmitted disease. Despite residing extracellularly and in close association with the vaginal bacteria (i.e., the microbiota), very little is known about the nature of the parasite-bacterium interactions. Our study showed that this parasite had acquired genes from bacteria which retained their original function. They produce active enzymes capable of degrading peptidoglycan, a unique polymer of the bacterial cell envelope, helping the parasite to outcompete bacteria in mixed cultures. This study was the first to show that a laterally acquired group of genes enables a eukaryotic mucosal pathogen to control bacterial population. We highlight the importance of understanding the interactions between pathogens and microbiota, as the outcomes of these interactions are increasingly understood to have important implications on health and disease.


Assuntos
Antibiose , Bactérias/efeitos dos fármacos , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/metabolismo , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/fisiologia , Feminino , Regulação da Expressão Gênica , Humanos , N-Acetil-Muramil-L-Alanina Amidase/genética , Trichomonas vaginalis/genética , Vagina/microbiologia , Vagina/parasitologia
15.
Cell Rep ; 25(1): 57-67.e5, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30282038

RESUMO

Tuberculosis claims >1 million lives annually, and its causative agent Mycobacterium tuberculosis is a highly successful pathogen. Protein kinase B (PknB) is reported to be critical for mycobacterial growth. Here, we demonstrate that PknB-depleted M. tuberculosis can replicate normally and can synthesize peptidoglycan in an osmoprotective medium. Comparative phosphoproteomics of PknB-producing and PknB-depleted mycobacteria identify CwlM, an essential regulator of peptidoglycan synthesis, as a major PknB substrate. Our complementation studies of a cwlM mutant of M. tuberculosis support CwlM phosphorylation as a likely molecular basis for PknB being essential for mycobacterial growth. We demonstrate that growing mycobacteria produce two forms of CwlM: a non-phosphorylated membrane-associated form and a PknB-phosphorylated cytoplasmic form. Furthermore, we show that the partner proteins for the phosphorylated and non-phosphorylated forms of CwlM are FhaA, a fork head-associated domain protein, and MurJ, a proposed lipid II flippase, respectively. From our results, we propose a model in which CwlM potentially regulates both the biosynthesis of peptidoglycan precursors and their transport across the cytoplasmic membrane.


Assuntos
Mycobacterium tuberculosis/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sequência de Aminoácidos , Parede Celular/enzimologia , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fosforilação , Proteínas Proto-Oncogênicas c-akt/deficiência
16.
Can J Microbiol ; 64(9): 589-599, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30169125

RESUMO

All Gram-negative bacteria release membrane vesicles. These vesicles contain a cargo of proteins and enzymes that include one or more autolysins. Autolysins are a group of enzymes with specificity for the different linkages within peptidoglycan sacculi that if uncontrolled cause bacteriolysis. This minireview, written in honor and memory of Terry Beveridge, presents an overview of autolytic activity and focuses on Beveridge's important original observations regarding predatory membrane vesicles and their associated autolysin cargo.


Assuntos
Bacteriólise/fisiologia , Membrana Celular/metabolismo , Bactérias Gram-Negativas/citologia , Vesículas Transportadoras/metabolismo , Bactérias Gram-Negativas/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/metabolismo , Vesículas Transportadoras/química
17.
Artigo em Inglês | MEDLINE | ID: mdl-30181369

RESUMO

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia. Over the past 2 decades, macrolide resistance among S. pneumoniae organisms has been increasing steadily and has escalated at an alarming rate worldwide. However, the use of macrolides in the treatment of community-acquired pneumonia has been reported to be effective regardless of the antibiotic susceptibility of the causative pneumococci. Although previous studies suggested that sub-MICs of macrolides inhibit the production of the pneumococcal pore-forming toxin pneumolysin by macrolide-resistant S. pneumoniae (MRSP), the underlying mechanisms of the inhibitory effect have not been fully elucidated. Here, we show that the release of pneumococcal autolysin, which promotes cell lysis and the release of pneumolysin, was inhibited by treatment with azithromycin and erythromycin, whereas replenishing with recombinant autolysin restored the release of pneumolysin from MRSP. Additionally, macrolides significantly downregulated ply transcription followed by a slight decrease of the intracellular pneumolysin level. These findings suggest the mechanisms involved in the inhibition of pneumolysin in MRSP, which may provide an additional explanation for the benefits of macrolides on the outcome of treatment for pneumococcal diseases.


Assuntos
Farmacorresistência Bacteriana/efeitos dos fármacos , Macrolídeos/farmacologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/metabolismo , Estreptolisinas/metabolismo , Antibacterianos/farmacologia , Azitromicina/farmacologia , Proteínas de Bactérias/metabolismo , Infecções Comunitárias Adquiridas/tratamento farmacológico , Infecções Comunitárias Adquiridas/microbiologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia
18.
Mol Microbiol ; 110(6): 879-896, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30230642

RESUMO

Bacteriophage-encoded endolysins can recognize and bind specific bacteria, and act to cleave the glycosidic and/or amide bonds in the peptidoglycan (PG) bacterial cell wall. Cleavage of the cell wall generally results in the death of the bacteria. Their utility as bacteriolytic agents could be exploited for human and veterinary medicines as well as various biotechnological applications. As interest grows in the commercial uses of these proteins, there has been much effort to successfully employ rational design and engineering to produce endolysins with bespoke properties. In this review, we interrogate the current structural data and identify structural features that would be of benefit to engineering the activity and specificity of phage endolysins. We show that the growing body of structural data can be used to predict catalytic residues and mechanism of action from sequences of hypothetical endolysins, and probe the importance of secondary structure repeats in bacterial cell wall-binding domains.


Assuntos
Antibacterianos/química , Bacteriófagos/enzimologia , Biocatálise , N-Acetil-Muramil-L-Alanina Amidase/química , Proteínas Virais/química , Bacteriólise , Parede Celular/metabolismo , Simulação por Computador , Cinética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Especificidade por Substrato
19.
Viruses ; 10(8)2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30110929

RESUMO

Mycobacteriophages are viruses that specifically infect mycobacteria, which ultimately culminate in host cell death. Dedicated enzymes targeting the complex mycobacterial cell envelope arrangement have been identified in mycobacteriophage genomes, thus being potential candidates as antibacterial agents. These comprise lipolytic enzymes that target the mycolic acid-containing outer membrane and peptidoglycan hydrolases responsive to the atypical mycobacterial peptidoglycan layer. In the recent years, a remarkable progress has been made, particularly on the comprehension of the mechanisms of bacteriophage lysis proteins activity and regulation. Notwithstanding, information about mycobacteriophages lysis strategies is limited and is mainly represented by the studies performed with mycobacteriophage Ms6. Since mycobacteriophages target a specific group of bacteria, which include Mycobacterium tuberculosis responsible for one of the leading causes of death worldwide, exploitation of the use of these lytic enzymes demands a special attention, as they may be an alternative to tackle multidrug resistant tuberculosis. This review focuses on the current knowledge of the function of lysis proteins encoded by mycobacteriophages and their potential applications, which may contribute to increasing the effectiveness of antimycobacterial therapy.


Assuntos
Membrana Celular/química , Parede Celular/química , Lisogenia , Micobacteriófagos/genética , Mycobacterium tuberculosis/virologia , Proteínas Virais/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Expressão Gênica , Hidrólise , Lipase/química , Lipase/genética , Lipase/metabolismo , Micobacteriófagos/enzimologia , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
20.
J Enzyme Inhib Med Chem ; 33(1): 1239-1247, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30141354

RESUMO

Autolysin E (AtlE) is a cell wall degrading enzyme that catalyzes the hydrolysis of the ß-1,4-glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid units of the bacterial peptidoglycan. Using our recently determined crystal structure of AtlE from Staphylococcus aureus and a combination of pharmacophore modeling, similarity search, and molecular docking, a series of (Phenylureido)piperidinyl benzamides were identified as potential binders and surface plasmon resonance (SPR) and saturation-transfer difference (STD) NMR experiments revealed that discovered compounds bind to AtlE in a lower micromolar range. (phenylureido)piperidinyl benzamides are the first reported non-substrate-like compounds that interact with this enzyme and enable further study of the interaction of small molecules with bacterial AtlE as potential inhibitors of this target.


Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , N-Acetil-Muramil-L-Alanina Amidase/antagonistas & inibidores , Piperidinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Piperidinas/síntese química , Piperidinas/química , Staphylococcus aureus/enzimologia , Relação Estrutura-Atividade
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