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1.
Mol Microbiol ; 120(2): 258-275, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37357823

RESUMO

Type VIIb secretion systems (T7SSb) in Gram-positive bacteria facilitate physiology, interbacterial competition, and/or virulence via EssC ATPase-driven secretion of small ɑ-helical proteins and toxins. Recently, we characterized T7SSb in group B Streptococcus (GBS), a leading cause of infection in newborns and immunocompromised adults. GBS T7SS comprises four subtypes based on variation in the C-terminus of EssC and the repertoire of downstream effectors; however, the intraspecies diversity of GBS T7SS and impact on GBS-host interactions remains unknown. Bioinformatic analysis indicates that GBS T7SS loci encode subtype-specific putative effectors, which have low interspecies and inter-subtype homology but contain similar domains/motifs and therefore may serve similar functions. We further identify orphaned GBS WXG100 proteins. Functionally, we show that GBS T7SS subtype I and III strains secrete EsxA in vitro and that in subtype I strain CJB111, esxA1 appears to be differentially transcribed from the T7SS operon. Furthermore, we observe subtype-specific effects of GBS T7SS on host colonization, as CJB111 subtype I but not CNCTC 10/84 subtype III T7SS promotes GBS vaginal colonization. Finally, we observe that T7SS subtypes I and II are the predominant subtypes in clinical GBS isolates. This study highlights the potential impact of T7SS heterogeneity on host-GBS interactions.


Assuntos
Infecções Estreptocócicas , Sistemas de Secreção Tipo VII , Recém-Nascido , Feminino , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Tipo VII/genética , Virulência , Óperon/genética , Genitália Feminina/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/metabolismo , Vagina/metabolismo , Vagina/microbiologia
2.
Nat Chem Biol ; 17(8): 878-887, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34045745

RESUMO

In ovoid-shaped, Gram-positive bacteria, MapZ guides FtsZ-ring positioning at cell equators. The cell wall of the ovococcus Streptococcus mutans contains peptidoglycan decorated with serotype c carbohydrates (SCCs). In the present study, we identify the major cell separation autolysin AtlA as an SCC-binding protein. AtlA binding to SCC is attenuated by the glycerol phosphate (GroP) modification. Using fluorescently labeled AtlA constructs, we mapped SCC distribution on the streptococcal surface, revealing enrichment of GroP-deficient immature SCCs at the cell poles and equators. The immature SCCs co-localize with MapZ at the equatorial rings throughout the cell cycle. In GroP-deficient mutants, AtlA is mislocalized, resulting in dysregulated cellular autolysis. These mutants display morphological abnormalities associated with MapZ mislocalization, leading to FtsZ-ring misplacement. Altogether, our data support a model in which maturation of a cell wall polysaccharide provides the molecular cues for the recruitment of cell division machinery, ensuring proper daughter cell separation and FtsZ-ring positioning.


Assuntos
Parede Celular/metabolismo , Polissacarídeos/metabolismo , Streptococcus mutans/metabolismo , Divisão Celular , Parede Celular/química , Polissacarídeos/química , Streptococcus mutans/citologia
3.
J Bacteriol ; 204(12): e0023322, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36448785

RESUMO

Pathogenic mycobacteria use the ESX-1 secretion system to escape the macrophage phagosome and survive infection. We demonstrated that the ESX-1 system is regulated by feedback control in Mycobacterium marinum, a nontuberculous pathogen and model for the human pathogen Mycobacterium tuberculosis. In the presence of a functional ESX-1 system, the WhiB6 transcription factor upregulates expression of ESX-1 substrate genes. In the absence of an assembled ESX-1 system, the conserved transcription factor, EspM, represses whiB6 expression by specifically binding the whiB6 promoter. Together, WhiB6 and EspM fine-tune the levels of ESX-1 substrates in response to the secretion system. The mechanisms underlying control of the ESX-1 system by EspM are unknown. Here, we conduct a structure and function analysis to investigate how EspM is regulated. Using biochemical approaches, we measured the formation of higher-order oligomers of EspM in vitro. We demonstrate that multimerization in vitro can be mediated through multiple domains of the EspM protein. Using a bacterial monohybrid system, we showed that EspM self-associates through multiple domains in Escherichia coli. Using this system, we performed a genetic screen to identify EspM variants that failed to self-associate. The screen yielded four EspM variants of interest, which we tested for activity in M. marinum. Our study revealed that the two helix-turn-helix domains are functionally distinct. Moreover, the helix bundle domain is required for wild-type multimerization in vitro. Our data support models where EspM monomers or hexamers contribute to the regulation of whiB6 expression. IMPORTANCE Pathogenic mycobacteria are bacteria that pose a large burden to human health globally. The ESX-1 secretion system is required for pathogenic mycobacteria to survive within and interact with the host. Proper function of the ESX-1 secretion system is achieved by tightly controlling the expression of secreted virulence factors, in part through transcriptional regulation. Here, we characterize the conserved transcription factor EspM, which regulates the expression of ESX-1 virulence factors. We define domains required for EspM to form multimers and bind DNA. These findings provide an initial characterization an ESX-1 transcription factor and provide insights into its mechanism of action.


Assuntos
Proteínas de Bactérias , Mycobacterium marinum , Sistemas de Secreção Tipo VII , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium marinum/genética , Mycobacterium tuberculosis/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sistemas de Secreção Tipo VII/metabolismo , Fatores de Virulência/genética
4.
Proc Natl Acad Sci U S A ; 116(8): 3202-3210, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30723150

RESUMO

The human pathogen Mycobacterium tuberculosis encodes a proteasome that carries out regulated degradation of bacterial proteins. It has been proposed that the proteasome contributes to nitrogen metabolism in M. tuberculosis, although this hypothesis had not been tested. Upon assessing M. tuberculosis growth in several nitrogen sources, we found that a mutant strain lacking the Mycobacterium proteasomal activator Mpa was unable to use nitrate as a sole nitrogen source due to a specific failure in the pathway of nitrate reduction to ammonium. We found that the robust activity of the nitrite reductase complex NirBD depended on expression of the groEL/groES chaperonin genes, which are regulated by the repressor HrcA. We identified HrcA as a likely proteasome substrate, and propose that the degradation of HrcA is required for the full expression of chaperonin genes. Furthermore, our data suggest that degradation of HrcA, along with numerous other proteasome substrates, is enhanced during growth in nitrate to facilitate the derepression of the chaperonin genes. Importantly, growth in nitrate is an example of a specific condition that reduces the steady-state levels of numerous proteasome substrates in M. tuberculosis.


Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Chaperonina 60/genética , Proteínas de Choque Térmico/genética , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Compostos de Amônio/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Humanos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Nitrogênio/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Tuberculose/genética , Tuberculose/metabolismo , Tuberculose/patologia
5.
J Biol Chem ; 295(36): 12706-12715, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675282

RESUMO

Mycobacterium tuberculosis has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. ESX-1, ESX-3, and ESX-5 systems have been shown to each secrete a distinct set of substrates, including PE and PPE families of proteins, named for conserved Pro-Glu and Pro-Pro-Glu motifs in their N termini. Proper secretion of the PE-PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems, and how each EspG recognizes its subset of PE-PPE proteins is currently unknown. The only current structural characterization of PE-PPE-EspG heterotrimers is from the ESX-5 system. Here we present the crystal structure of the PE5mt-PPE4mt-EspG3mm heterotrimer from the ESX-3 system. Our heterotrimer reveals that EspG3mm interacts exclusively with PPE4mt in a similar manner to EspG5, shielding the hydrophobic tip of PPE4mt from solvent. The C-terminal helical domain of EspG3mm is dynamic, alternating between "open" and "closed" forms, and this movement is likely functionally relevant in the unloading of PE-PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 heterotrimers, the PE-PPE heterodimer of our ESX-3 heterotrimer is interacting with its chaperone at a drastically different angle and presents different faces of the PPE protein to the chaperone. We conclude that the PPE-EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate among noncognate PE-PPE pairs.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Mycobacterium tuberculosis/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Mycobacterium tuberculosis/genética , Domínios Proteicos
6.
Nat Chem Biol ; 15(5): 463-471, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30936502

RESUMO

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


Assuntos
Glicerol/metabolismo , Fosfatos/metabolismo , Polissacarídeos Bacterianos/metabolismo , Streptococcus/metabolismo , Glicerol/química , Fosfatos/química , Polissacarídeos Bacterianos/química , Streptococcus/química
7.
J Biol Chem ; 293(4): 1106-1119, 2018 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-29229778

RESUMO

The ß-barrel assembly machinery (BAM) is a conserved multicomponent protein complex responsible for the biogenesis of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Given its role in the production of OMPs for survival and pathogenesis, BAM represents an attractive target for the development of therapeutic interventions, including drugs and vaccines against multidrug-resistant bacteria such as Neisseria gonorrhoeae The first structure of BamA, the central component of BAM, was from N. gonorrhoeae, the etiological agent of the sexually transmitted disease gonorrhea. To aid in pharmaceutical targeting of BAM, we expanded our studies to BamD and BamE within BAM of this clinically relevant human pathogen. We found that the presence of BamD, but not BamE, is essential for gonococcal viability. However, BamE, but not BamD, was cell-surface-displayed under native conditions; however, in the absence of BamE, BamD indeed becomes surface-exposed. Loss of BamE altered cell envelope composition, leading to slower growth and an increase in both antibiotic susceptibility and formation of membrane vesicles containing greater amounts of vaccine antigens. Both BamD and BamE are expressed in diverse gonococcal isolates, under host-relevant conditions, and throughout different phases of growth. The solved structures of Neisseria BamD and BamE share overall folds with Escherichia coli proteins but contain differences that may be important for function. Together, these studies highlight that, although BAM is conserved across Gram-negative bacteria, structural and functional differences do exist across species, which may be leveraged in the development of species-specific therapeutics in the effort to combat multidrug resistance.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Neisseria gonorrhoeae/química , Proteínas da Membrana Bacteriana Externa/genética , Humanos , Viabilidade Microbiana , Neisseria gonorrhoeae/genética , Domínios Proteicos , Relação Estrutura-Atividade
8.
J Biol Chem ; 292(47): 19441-19457, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-29021255

RESUMO

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


Assuntos
Acetilglucosamina/metabolismo , Proteínas de Bactérias/metabolismo , Carboidratos/química , Fosfolipídeos/metabolismo , Ramnose/biossíntese , Streptococcus pyogenes/metabolismo , Amidoidrolases/metabolismo , Proteínas de Bactérias/química , Membrana Celular/metabolismo , Peptidoglicano/metabolismo , Streptococcus pyogenes/química
9.
Artigo em Inglês | MEDLINE | ID: mdl-28584144

RESUMO

Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, which is highly prevalent worldwide and has a major impact on reproductive and neonatal health. The superbug status of N. gonorrhoeae necessitates the development of drugs with different mechanisms of action. Here, we focused on targeting the nitrite reductase AniA, which is a pivotal component of N. gonorrhoeae anaerobic respiration and biofilm formation. Our studies showed that gonococci expressing AniA containing the altered catalytic residues D137A and H280A failed to grow under anaerobic conditions, demonstrating that the nitrite reductase function is essential. To facilitate the pharmacological targeting of AniA, new crystal structures of AniA were refined to 1.90-Å and 2.35-Å resolutions, and a phage display approach with libraries expressing randomized linear dodecameric peptides or heptameric peptides flanked by a pair of cysteine residues was utilized. Biopanning experiments led to the identification of 29 unique peptides, with 1 of them, C7-3, being identified multiple times. Evaluation of their ability to interact with AniA using enzyme-linked immunosorbent assay and computational docking studies revealed that C7-3 was the most promising inhibitor, binding near the type 2 copper site of the enzyme, which is responsible for interaction with nitrite. Subsequent enzymatic assays and biolayer interferometry with a synthetic C7-3 and its derivatives, C7-3m1 and C7-3m2, demonstrated potent inhibition of AniA. Finally, the MIC50 value of C7-3 and C7-3m2 against anaerobically grown N. gonorrhoeae was 0.6 mM. We present the first peptide inhibitors of AniA, an enzyme that should be further exploited for antigonococcal drug development.


Assuntos
Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Gonorreia/tratamento farmacológico , Neisseria gonorrhoeae/efeitos dos fármacos , Oxigênio/metabolismo , Peptídeos/antagonistas & inibidores , Anaerobiose/efeitos dos fármacos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Técnicas de Visualização da Superfície Celular , Cristalização , Ensaio de Imunoadsorção Enzimática , Gonorreia/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Modelos Estruturais , Mutagênese Sítio-Dirigida , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Nitrito Redutases/metabolismo , Nitritos/metabolismo
10.
BMC Struct Biol ; 16: 5, 2016 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-26922638

RESUMO

BACKGROUND: The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system. RESULTS: This study characterizes the structures of the soluble domains of two conserved core ESX-1 components - EccB1 and EccD1. The periplasmic domain of EccB1 consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. The repeat domains of EccB1 are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD1has a ubiquitin-like fold and forms a dimer with a negatively charged groove. CONCLUSIONS: These structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies.


Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Mycobacterium tuberculosis/química , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência
11.
Acta Neuropathol ; 132(4): 563-76, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27481264

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Mutations in Cu/Zn superoxide dismutase (SOD1) are responsible for approximately 20 % of the familial ALS cases. ALS-causing SOD1 mutants display a gain-of-toxicity phenotype, but the nature of this toxicity is still not fully understood. The Ras GTPase-activating protein-binding protein G3BP1 plays a critical role in stress granule dynamics. Alterations in the dynamics of stress granules have been reported in several other forms of ALS unrelated to SOD1. To our surprise, the mutant G93A SOD1 transgenic mice exhibited pathological cytoplasmic inclusions that co-localized with G3BP1-positive granules in spinal cord motor neurons. The co-localization was also observed in fibroblast cells derived from familial ALS patient carrying SOD1 mutation L144F. Mutant SOD1, unlike wild-type SOD1, interacted with G3BP1 in an RNA-independent manner. Moreover, the interaction is specific for G3BP1 since mutant SOD1 showed little interaction with four other RNA-binding proteins implicated in ALS. The RNA-binding RRM domain of G3BP1 and two particular phenylalanine residues (F380 and F382) are critical for this interaction. Mutant SOD1 delayed the formation of G3BP1- and TIA1-positive stress granules in response to hyperosmolar shock and arsenite treatment in N2A cells. In summary, the aberrant mutant SOD1-G3BP1 interaction affects stress granule dynamics, suggesting a potential link between pathogenic SOD1 mutations and RNA metabolism alterations in ALS.


Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Transporte/genética , Corpos de Inclusão/metabolismo , Mutação/genética , Superóxido Dismutase-1/genética , Animais , DNA Helicases , Modelos Animais de Doenças , Corpos de Inclusão/patologia , Camundongos Transgênicos , Neurônios Motores/patologia , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Medula Espinal/metabolismo , Medula Espinal/patologia
12.
Trends Biochem Sci ; 36(8): 433-43, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21565514

RESUMO

Secretins form megadalton bacterial-membrane channels in at least four sophisticated multiprotein systems that are crucial for translocation of proteins and assembled fibers across the outer membrane of many species of bacteria. Secretin subunits contain multiple domains, which interact with numerous other proteins, including pilotins, secretion-system partner proteins, and exoproteins. Our understanding of the structure of secretins is rapidly progressing, and it is now recognized that features common to all secretins include a cylindrical arrangement of 12-15 subunits, a large periplasmic vestibule with a wide opening at one end and a periplasmic gate at the other. Secretins might also play a key role in the biogenesis of their cognate secretion systems.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Secretina/química , Secretina/fisiologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana Transportadoras/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico
13.
J Struct Biol ; 191(2): 236-44, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26051906

RESUMO

Mycobacterium tuberculosis secretes multiple virulence factors during infection via the general Sec and Tat pathways, and via specialized ESX secretion systems, also referred to as type VII secretion systems. The ESX-1 secretion system is an important virulence determinant because deletion of ESX-1 leads to attenuation of M. tuberculosis. ESX-1 secreted protein B (EspB) contains putative PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains, and a C-terminal domain, which is processed by MycP1 protease during secretion. We determined the crystal structure of PE-PPE domains of EspB, which represents an all-helical, elongated molecule closely resembling the structure of the PE25-PPE41 heterodimer despite limited sequence similarity. Also, we determined the structure of full-length EspB, which does not have interpretable electron density for the C-terminal domain confirming that it is largely disordered. Comparative analysis of EspB in cell lysate and culture filtrates of M. tuberculosis revealed that mature secreted EspB forms oligomers. Electron microscopy analysis showed that the N-terminal fragment of EspB forms donut-shaped particles. These data provide a rationale for the future investigation of EspB's role in M. tuberculosis pathogenesis.


Assuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis/química , Sistemas de Secreção Tipo VII/química , Fatores de Virulência/química , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Modelos Moleculares , Estrutura Terciária de Proteína
14.
J Biol Chem ; 289(12): 8288-98, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24459146

RESUMO

The chymotrypsin subfamily A of serine proteases consists primarily of eukaryotic proteases, including only a few proteases of bacterial origin. VesB, a newly identified serine protease that is secreted by the type II secretion system in Vibrio cholerae, belongs to this subfamily. VesB is likely produced as a zymogen because sequence alignment with trypsinogen identified a putative cleavage site for activation and a catalytic triad, His-Asp-Ser. Using synthetic peptides, VesB efficiently cleaved a trypsin substrate, but not chymotrypsin and elastase substrates. The reversible serine protease inhibitor, benzamidine, inhibited VesB and served as an immobilized ligand for VesB affinity purification, further indicating its relationship with trypsin-like enzymes. Consistent with this family of serine proteases, N-terminal sequencing implied that the propeptide is removed in the secreted form of VesB. Separate mutagenesis of the activation site and catalytic serine rendered VesB inactive, confirming the importance of these features for activity, but not for secretion. Similar to trypsin but, in contrast to thrombin and other coagulation factors, Na(+) did not stimulate the activity of VesB, despite containing the Tyr(250) signature. The crystal structure of catalytically inactive pro-VesB revealed that the protease domain is structurally similar to trypsinogen. The C-terminal domain of VesB was found to adopt an immunoglobulin (Ig)-fold that is structurally homologous to Ig-folds of other extracellular Vibrio proteins. Possible roles of the Ig-fold domain in stability, substrate specificity, cell surface association, and type II secretion of VesB, the first bacterial multidomain trypsin-like protease with known structure, are discussed.


Assuntos
Cólera/microbiologia , Serina Proteases/química , Serina Proteases/metabolismo , Vibrio cholerae/enzimologia , Sequência de Aminoácidos , Ativação Enzimática , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Sódio/metabolismo , Homologia Estrutural de Proteína , Trombina/química , Tripsina/química , Vibrio cholerae/química
15.
Biochim Biophys Acta ; 1843(8): 1707-16, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24263244

RESUMO

Mycobacteria use type VII secretion (T7S) systems to secrete proteins across their complex cell envelope. Pathogenic mycobacteria, such as the notorious pathogen Mycobacterium tuberculosis, have up to five of these secretion systems, named ESX-1 to ESX-5. At least three of these secretion systems are essential for mycobacterial virulence and/or viability. Elucidating T7S is therefore essential to understand the success of M. tuberculosis and other pathogenic mycobacteria as pathogens, and could be instrumental to identify novel targets for drug- and vaccine-development. Recently, significant progress has been achieved in the identification of T7S substrates and a general secretion motif. In addition, a start has been made with unraveling the mechanism of secretion and the structural analysis of the different subunits. This review summarizes these recent findings, which are incorporated in a working model of this complex machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Mycobacterium tuberculosis/patogenicidade , Transporte Proteico/genética , Proteínas de Bactérias/química , Parede Celular/genética , Parede Celular/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Estrutura Terciária de Proteína , Tuberculose/genética , Tuberculose/metabolismo , Tuberculose/microbiologia
16.
Mol Microbiol ; 94(2): 367-82, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25155747

RESUMO

The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX-1, ESX-3 and ESX-5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system-specific cytoplasmic chaperones for secretion. Here, we report the crystal structure of M. tuberculosis ESX-5-secreted PE25-PPE41 heterodimer in complex with the cytoplasmic chaperone EspG(5). EspG(5) represents a novel fold that is unrelated to previously characterized secretion chaperones. Functional analysis of the EspG(5) -binding region uncovered a hydrophobic patch on PPE41 that promotes dimer aggregation, and the chaperone effectively abolishes this process. We show that PPE41 contains a characteristic chaperone-binding sequence, the hh motif, which is highly conserved among ESX-1-, ESX-3- and ESX-5-specific PPE proteins. Disrupting the interaction between EspG(5) and three different PPE target proteins by introducing different point mutations generally affected protein secretion. We further demonstrate that the EspG(5) chaperone plays an important role in the ESX secretion mechanism by keeping aggregation-prone PE-PPE proteins in their soluble state.


Assuntos
Proteínas de Bactérias/química , Sistemas de Secreção Bacterianos , Chaperonas Moleculares/química , Mycobacterium tuberculosis/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Análise Mutacional de DNA , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Transporte Proteico
17.
PLoS Pathog ; 9(1): e1003117, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23326233

RESUMO

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Porinas/metabolismo , Secretina/metabolismo , Vibrio cholerae/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Sítios de Ligação/fisiologia , Biologia Computacional , Cristalização , Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Klebsiella/fisiologia , Dados de Sequência Molecular , Filogenia , Porinas/química , Ligação Proteica , Especificidade da Espécie , Vibrio cholerae/genética
18.
J Struct Biol ; 187(3): 223-235, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25092625

RESUMO

The type II secretion system (T2SS) is present in many Gram-negative bacteria and is responsible for secreting a large number of folded proteins, including major virulence factors, across the outer membrane. The T2SS consists of 11-15 different proteins most of which are present in multiple copies in the assembled secretion machinery. The ATPase GspE, essential for the functioning of the T2SS, contains three domains (N1E, N2E and CTE) of which the N1E domain is associated with the cytoplasmic domain of the inner membrane protein GspL. Here we describe and analyze the structure of the GspE•cyto-GspL complex from Vibrio vulnificus in the presence of an ATP analog, AMPPNP. There are three such ∼83 kDa complexes per asymmetric unit with essentially the same structure. The N2E and CTE domains of a single V. vulnificus GspE subunit adopt a mutual orientation that has not been seen before in any of the previous GspE structures, neither in structures of related ATPases from other secretion systems. This underlines the tremendous conformational flexibility of the T2SS secretion ATPase. Cyto-GspL interacts not only with the N1E domain, but also with the CTE domain and is even in contact with AMPPNP. Moreover, the cyto-GspL domains engage in two types of mutual interactions, resulting in two essentially identical, but crystallographically independent, "cyto-GspL rods" that run throughout the crystal. Very similar rods are present in previous crystals of cyto-GspL and of the N1E•cyto-GspL complex. This arrangement, now seen four times in three entirely different crystal forms, involves contacts between highly conserved residues suggesting a role in the biogenesis or the secretion mechanism or both of the T2SS.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Membrana/química , Estrutura Terciária de Proteína , Vibrio vulnificus/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Homologia de Sequência de Aminoácidos , Vibrio vulnificus/genética
19.
Proteins ; 82(1): 159-63, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23818233

RESUMO

EccA1 is an important component of the type VII secretion system (T7SS) that is responsible for transport of virulence factors in pathogenic mycobacteria. EccA1 has an N-terminal domain of unknown function and a C-terminal AAA+ (ATPases associated with various cellular activities) domain. Here we report the crystal structure of the N-terminal domain of EccA1 from Mycobacterium tuberculosis, which shows an arrangement of six tetratricopeptide repeats that may mediate interactions of EccA1 with secreted substrates. Furthermore, the size and shape of the N-terminal domain suggest its orientation in the context of a hexamer model of full-length EccA1.


Assuntos
Adenosina Trifosfatases/química , Sistemas de Secreção Bacterianos/genética , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Adenosina Trifosfatases/genética , Estrutura Terciária de Proteína/genética
20.
Bioorg Med Chem Lett ; 24(15): 3546-8, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24915878

RESUMO

Mycosin protease-1 (MycP1) cleaves ESX secretion-associated protein B (EspB) that is a virulence factor of Mycobacterium tuberculosis, and accommodates an octapeptide, AVKAASLG, as a short peptide substrate. Because peptidoboronic acids are known inhibitors of serine proteases, the synthesis and binding of a boronic acid analog of the pentapeptide cleavage product, AVKAA, was studied using MycP1 variants from Mycobacterium thermoresistible (MycP1mth), Mycobacterium smegmatis (MycP1msm) and M. tuberculosis (MycP1mtu). We synthesized the boropentapeptide, HAlaValLysAlaAlaB(OH)2 (1) and the analogous pinanediol PD-protected HAlaValLysAlaAlaBO2(PD) (2) using an Fmoc/Boc peptide strategy. The pinanediol boropentapeptide 2 displayed IC50 values 121.6±25.3 µM for MycP1mth, 93.2±37.3 µM for MycP1msm and 37.9±5.2 µM for MycP1mtu. Such relatively strong binding creates a chance for crystalizing the complex with 2 and finding the structure of the unknown MycP1 catalytic site that would potentially facilitate the development of new anti-tuberculosis drugs.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Ácidos Borônicos/farmacologia , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Subtilisinas/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Ácidos Borônicos/síntese química , Ácidos Borônicos/química , Relação Dose-Resposta a Droga , Conformação Molecular , Mycobacterium tuberculosis/enzimologia , Oligopeptídeos/síntese química , Oligopeptídeos/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-Atividade , Subtilisinas/metabolismo
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