Structural and biochemical analyses of the tetrameric carboxypeptidase S9Cfn from Fusobacterium nucleatum

2021 ◽  
Vol 77 (12) ◽  
Author(s):  
Xin Wang ◽  
Meng-Ting Cheng ◽  
Zhi-Peng Chen ◽  
Yong-Liang Jiang ◽  
Yu-Shu Ge ◽  
...  
Keyword(s):  
Oral Cavity ◽  
Enzymatic Activity ◽  
Conformational Changes ◽  
Fusobacterium Nucleatum ◽  
Site Directed Mutagenesis ◽  
Spectrometric Analysis ◽  
Oral Diseases ◽  
Unique Type ◽  
Human Oral Cavity ◽  
Arginine Residues

As one of the most abundant bacteria in the human oral cavity, Fusobacterium nucleatum is closely involved in various oral diseases and is also a risk factor for other diseases. The peptidases of F. nucleatum can digest exogenous peptides into amino acids to satisfy its nutrient requirements. Here, a putative F. nucleatum peptidase, termed S9Cfn, which belongs to the S9C peptidase family was identified. Enzymatic activity assays combined with mass-spectrometric analysis revealed that S9Cfn is a carboxypeptidase, but not an aminopeptidase as previously annotated. The crystal structure of the S9Cfn tetramer was solved at 2.6 Å resolution and was found to contain a pair of oligomeric pores in the center. Structural analysis, together with site-directed mutagenesis and enzymatic activity assays, revealed a substrate-entrance tunnel that extends from each oligomeric pore to the catalytic triad, adjacent to which three conserved arginine residues are responsible for substrate binding. Moreover, comparison with other S9 peptidase structures indicated drastic conformational changes of the oligomeric pores during the catalytic cycle. Together, these findings increase the knowledge of this unique type of tetrameric carboxypeptidase and provide insight into the homeostatic control of microbiota in the human oral cavity.

The distribution and frequency of Fusobacterium nucleatum subspecies in the human oral cavity

1990 ◽  
Vol 5 (6) ◽  
pp. 324-327 ◽  
Author(s):  
S. E. Gharbia ◽  
H. N. Shah ◽  
P. A. Lawson ◽  
M. Haapasalo
Keyword(s):  
Oral Cavity ◽  
Fusobacterium Nucleatum ◽  
Human Oral Cavity

scholarly journals Oral Bacteriophages

2021 ◽  
Author(s):  
Sonia Bhonchal Bhardwaj ◽  
Seema Kumari
Keyword(s):  
Oral Cavity ◽  
Bacterial Infections ◽  
Fusobacterium Nucleatum ◽  
Lactobacillus Species ◽  
Bacterial Cells ◽  
Oral Diseases ◽  
Neisseria Species ◽  
Oral Biofilms ◽  
Specific Polysaccharide ◽  
Therapeutic Tools

Bacteriophage or phage therapy involves using phages or their products as bio-agents for the treatment or prophylaxis of bacterial infections or diseases. Bacteriophages have the ability to regulate the oral microflora by lysing sensitive bacterial cells and releasing bacterial components with pro-inflammatory activity. Bacteriophages carry specific polysaccharide depolymerases that aid viral penetration and can disrupt the pathogenic process associated with biofilm and exopolysaccharide in the oral cavity. Oral diseases are mainly caused by biofilm forming microorganisms and phages are now being used for biocontrol of oral biofilms. Phages for Actinomyces species, Aggregatibacter actinomycetemcomitans, Enterococcus faecalis, Fusobacterium nucleatum, Lactobacillus species, Neisseria species, Streptococcus species, and Veillonella species have been isolated and characterized. Bacteriophages could be considered as potential therapeutic tools for the elimination of caries, periodontitis, and other diseases of the oral cavity.

scholarly journals Active-Site Conformational Fluctuations Promote the Enzymatic Activity of NDM-1

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Hongmin Zhang ◽  
Guixing Ma ◽  
Yifan Zhu ◽  
Lingxiao Zeng ◽  
Ashfaq Ahmad ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Bacterial Infections ◽  
Site Directed Mutagenesis ◽  
New Delhi ◽  
Native Form ◽  
Conformational Fluctuation ◽  
Almost All

ABSTRACT β-Lactam antibiotics are the mainstay for the treatment of bacterial infections. However, elevated resistance to these antibiotics mediated by metallo-β-lactamases (MBLs) has become a global concern. New Delhi metallo-β-lactamase-1 (NDM-1), a newly added member of the MBL family that can hydrolyze almost all β-lactam antibiotics, has rapidly spread all over the world and poses serious clinical threats. Broad-spectrum and mechanism-based inhibitors against all MBLs are highly desired, but the differential mechanisms of MBLs toward different antibiotics pose a great challenge. To facilitate the design of mechanism-based inhibitors, we investigated the active-site conformational changes of NDM-1 through the determination of a series of 15 high-resolution crystal structures in native form and in complex with products and by using biochemical and biophysical studies, site-directed mutagenesis, and molecular dynamics computation. The structural studies reveal the consistency of the active-site conformations in NDM-1/product complexes and the fluctuation in native NDM-1 structures. The enzymatic measurements indicate a correlation between enzymatic activity and the active-site fluctuation, with more fluctuation favoring higher activity. This correlation is further validated by structural and enzymatic studies of the Q123G mutant. Our combinational studies suggest that active-site conformational fluctuation promotes the enzymatic activity of NDM-1, which may guide further mechanism studies and inhibitor design.

scholarly journals Cleavage of Rhesus Rotavirus VP4 after Arginine 247 Is Essential for Rotavirus-Like Particle-Induced Fusion from Without

1998 ◽  
Vol 72 (6) ◽  
pp. 5323-5327 ◽  
Author(s):  
Joanna M. Gilbert ◽  
Harry B. Greenberg
Keyword(s):  
Cell Fusion ◽  
Conformational Changes ◽  
Viral Entry ◽  
Recombinant Baculovirus ◽  
Syncytium Formation ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Fusion Activity ◽  
Arginine Residues ◽  
Cell Cell

ABSTRACT We recently described our finding that recombinant baculovirus-produced virus-like particles (VLPs) can induce cell-cell fusion similar to that induced by intact rotavirus in our assay for viral entry into tissue culture cells (J. M. Gilbert and H. B. Greenberg, J. Virol. 71:4555–4563, 1997). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection. This VLP-mediated fusion activity was dependent on the presence of the outer-layer proteins, viral protein 4 (VP4) and VP7, and on the trypsinization of VP4. Fusion activity occurred only with cells that are permissive for rotavirus infection. Here we begin to dissect the role of VP4 in rotavirus entry by examining the importance of the precise trypsin cleavage of VP4 and the activation of VP4 function related to viral entry. We present evidence that the elimination of the three trypsin-susceptible arginine residues of VP4 by specific site-directed mutagenesis prevents syncytium formation. Two of the three arginine residues in VP4 are dispensable for syncytium formation, and only the arginine residue at site 247 appears to be required for activation of VP4 functions and cell-cell fusion. Using the recombinant VLPs in our syncytium assay will aid in understanding the conformational changes that occur in VP4 involved in rotavirus penetration into host cells.

A new approach to treatment of stomatitis in experiments on white rats (experimental research)

2018 ◽  
pp. 168-173
Author(s):  
Е.Б. Ганина ◽  
Ю.В. Червинец ◽  
Н.В. Грудинин ◽  
В.Г. Шестакова ◽  
В.М. Червинец ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Oral Cavity ◽  
Experimental Research ◽  
Quantitative Composition ◽  
Oral Diseases ◽  
New Approach ◽  
Highly Active ◽  
White Rats ◽  
Before And After ◽  
Intact Rats

Цель исследования - охарактеризовать микробиологическую картину стоматита до и после его лечения высокоактивными культурами лактобацилл, дать оценку эффективности их применения при экспериментальном стоматите. Методика. Опыты проводились на 32 самках беспородных белых крыс массой 230 г. Моделирование стоматита включало 2 этапа: моделирование травматического стоматита и воспроизведение бактериального стоматита на базе травматического. У крыс контрольной и опытных серий на всех этапах эксперимента брали мазки с поверхности десен для характеристики микробиоценоза ротовой полости. Результаты. После обработки слизистой оболочки полости рта (СОПР) культурой патогенного штамма Staphylococcus aureus существенно снизились частота встречаемости и количество условно-патогенных микроорганизмов. Лечение стоматита у крыс культурами лактобацилл ( Lactobacillus 11 зв., Lactobacillus 2 п.рта, Lactobacillus 24 д.ст.) и их комбинацией приводило к снижению распространенности S. aureus вплоть до его исчезновения, а также к нормализации количества условно-патогенной микробиоты. Заключение. Исследования показали эффективность использования отдельных высокоактивных штаммов лактобацилл или их комбинации при лечении заболеваний СОПР, в частности бактериального стоматита. Aim. To characterize the microbiological picture of stomatitis in intact rats before and after the treatment with highly active cultured lactobacilli and to evaluate the effectiveness of this treatment in experiments on white rats. Methods. Experiments were carried out on 32 mongrel female white rats weighing 230 g. Smears were taken from the gum surface of control and experimental rats at all stages of the experiment to characterize the oral cavity microbiocenosis. Results. After treatment of the oral cavity with cultured Staphylococcus aureus , the occurrence and quantity of opportunistic microorganisms significantly decreased. The treatment of stomatitis in rats with cultured lactobacillus ( Lactobacillus 11 zv., Lactobacillus 2 p.r., Lactobacillus 24 d.st. and their combination) led to a decrease in S. aureus prevalence up to its extinction and to normalization of the quantitative composition of opportunistic microbiota. Conclusions. The study showed the effieacy of highly active lactobacillus strains individually or their combinations in the treatment of oral diseases, in particular, bacterial stomatitis.

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

scholarly journals The occurrence of Entamoeba gingivalis among patients with periodontal disease

2016 ◽  
Vol 29 (2) ◽  
pp. 86-89 ◽  
Author(s):  
Joanna Luszczak ◽  
Michal Bartosik ◽  
Jolanta Rzymowska ◽  
Agnieszka Sochaczewska-Dolecka ◽  
Ewa Tomaszek ◽  
...  
Keyword(s):  
Oral Cavity ◽  
Periodontal Disease ◽  
Periodontal Diseases ◽  
Age Groups ◽  
Healthy People ◽  
Gingival Tissue ◽  
Pathological Changes ◽  
Oral Diseases

AbstractAccording to some studies, the Entamoeba gingivalis colonizing the gingival tissue is an important agent in bringing about periodontitis. Other studies, however, deem it an opportunist that is able to survive in the medium induced by periodontal disease. The aim of this study was to investigate the prevalence of Entamoeba gingivalis infection in patients from the Department of Periodontology, and compare this population with that of healthy people, so as to analyze the relationships between infection and patient sex and age. The result of this work is that in both groups, a correlation (p = 0,19) has been noted between the occurrence of amoebae and other diseases in the oral cavity. Indeed, 81,4% of all patients with some periodontal disease showed the presence of amoeba. Among those who are not afflicted with oral diseases, the presence of amoeba was indicated in 62,5% of the total. In addition, a correlation between the person's age and the presence of protozoa (p = 0,15) was strongly marked among women (p = 0,19). In the three age groups of women in this study (40-49, 60-69, and above 80 years), we observed a 100% presence of protozoa.Our study leads us to the conclusion that infections with Entamoeba gingivalis should be regarded as an factor that is associated with the pathological changes occurring in patients with periodontal diseases.

scholarly journals Campylobacter showae sp. nov., Isolated from the Human Oral Cavity

1993 ◽  
Vol 43 (4) ◽  
pp. 631-639 ◽  
Author(s):  
Y. ETOH ◽  
F. E. DEWHIRST ◽  
B. J. PASTER ◽  
A. YAMAMOTO ◽  
N. GOTO
Keyword(s):  
Oral Cavity ◽  
Human Oral Cavity
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