Role In Cell Wall Synthesis Research Articles

Identification of potential inhibitors against Escherichia coli Mur D enzyme to combat rising drug resistance: an in-silico approach

Indiscriminate use of anti-microbial agents has resulted in the inception, frequency, and spread of antibiotic resistance among targeted bacterial pathogens and the commensal flora. Mur enzymes, playing a crucial role in cell-wall synthesis, are one of the most appropriate targets for developing novel inhibitors against antibiotic-resistant bacterial pathogens. In the present study, in-silico high-throughput virtual (HTVS) and Standard-Precision (SP) screening was carried out with 0.3 million compounds from several small-molecule libraries against the E. coli Mur D enzyme (PDB ID 2UUP). The docked complexes were further subjected to extra-precision (XP) docking calculations, and highest Glide-score compound was further subjected to molecular simulation studies. The top six virtual hits (S1–S6) displayed a glide score (G-score) within the range of −9.013 to −7.126 kcal/mol and compound S1 was found to have the highest stable interactions with the Mur D enzyme (2UUP) of E. coli. The stability of compound S1 with the Mur D (2UUP) complex was validated by a 100-ns molecular dynamics simulation. Binding free energy calculation by the MM-GBSA strategy of the S1-2UUP (Mur D) complex established van der Waals, hydrogen bonding, lipophilic, and Coulomb energy terms as significant favorable contributors for ligand binding. The final lead molecules were subjected to ADMET predictions to study their pharmacokinetic properties and displayed promising results, except for certain modifications required to improve QPlogHERG values. So, the compounds screened against the Mur D enzyme can be further studied as preparatory points for in-vivo studies to develop potential drugs. HIGHLIGHTS E.coli is a common cause of urinary tract infections. E.coli MurD enzyme is a suitable target for drug development. Novel inhibitors against E.coli MurD enzyme were identified. Molecular dynamics studies identified in-silico potential of identified compound. ADMET predictions and Lipinski’s rule of five studies showed promising results. Communicated by Ramaswamy H. Sarma

Adhesion and Colonization of the Probiotic Lactobacillus plantarum HC-2 in the Intestine of Litopenaeus Vannamei Are Associated With Bacterial Surface Proteins.

Surface proteins are a type of proteins expressed on the surface of bacteria that play an important role in cell wall synthesis, maintenance of cell morphology, and signaling with the host. Our previous study showed that the probiotic Lactobacillus plantarum HC-2 improved the growth performance and immune response of Litopenaeus vannamei. To further investigate the probiotic mechanism, we determined the automatic aggregation ability of the bacteria and surface hydrophobicity of HC-2 after being treated with 5 M of lithium chloride (LiCl) and observed the morphology and adhesion of the bacteria to HCT116 cells. The results showed that with the removal of the HC-2 surface protein, the auto-aggregation ability and surface hydrophobicity of HC-2 decreased, and the crude mucus layer coated on the bacterial surface gradually dissociated. The adhesion rate of HC-2 to HCT116 cells decreased from 98.1 to 20.9%. Moreover, a total of 201 unique proteins were identified from the mixture of the surface proteins by mass spectrometry (MS). Several proteins are involved in transcription and translation, biosynthetic or metabolic process, cell cycle or division, cell wall synthesis, and emergency response. Meanwhile, a quantitative real-time PCR qPCR_ showed that HC-2 was mainly colonized in the midgut of shrimp, and the colonization numbers were 15 times higher than that in the foregut, while the colonization rate in the hindgut was lower. The adhesion activity measurement showed that the adhesion level of HC-2 to crude intestinal mucus of L. vannamei was higher than that of bovine serum albumin (BSA) and collagen, and the adhesion capacity of the bacterial cells decreased with the extension of LiCl-treatment time. Finally, we identified the elongation factor Tu, Type I glyceraldehyde-3-phosphate dehydrogenase, small heat shock protein, and 30S ribosomal protein from the surface proteins, which may be the adhesion proteins of HC-2 colonization in the shrimp intestine. The above results indicate that surface proteins play an important role in maintaining the cell structure stability and cell adhesion. Surface proteomics analysis contributes to describing potential protein-mediated probiotic-host interactions. The identification of some interacting proteins in this work may be beneficial to further understand the adhesion/colonization mechanism and probiotic properties of L. plantarum HC-2 in the shrimp intestine.

Evidence for a trap-and-flip mechanism in a proton-dependent lipid transporter

Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters take part in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acid synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a ‘trap-and-flip’ mechanism that might be shared among MFS lipid transporters.

Targeting UDP-Glycosyltransferase, Glucosamine-6-Phosphate Synthase and Chitin Synthase by Using Bioactive 1,8 Cineole for “Aspergillosis” Fungal Disease Mutilating COVID-19 Patients: Insights from Molecular Docking, Pharmacokinetics and In-vitro Studies

SARS-CoV-2 (COVID-19)-associated co-infections like “Aspergillosis”, has recently baffled the world. Due to its key role in cell wall synthesis, in the present study UDP-glycosyltransferase, glucosamine-6-phosphate synthase and chitin synthase have been chosen as appropriate targets for molecular docking. The objective of the present study was molecular docking of eucalyptus essential oil component 1,8 cineole against cell wall enzymes followed by in vitro validation. For molecular docking, patch-dock web based online tool was used. Ligand–Protein 2D and 3D Interactions were also studied. Drug likeliness, toxicity profile and cancer cell line toxicity were also studied. Molecular docking results indicated that 1,8 cineole form hydrogen bonding and hydrophobic interactions with UDP-glycosyltransferase, glucosamine-6-phosphate synthase and chitin synthase enzymes. 1,8 cineole also depicted drug likeliness by showing compliance with the LIPINSKY rule, sufficient level of bioactivity and cancer cell line toxicity thus signifying its role as a potent anti-fungal drug.

Structure-Based Identification of Potential Drugs Against FmtA of Staphylococcus aureus: Virtual Screening, Molecular Dynamics, MM-GBSA, and QM/MM.

Staphylococcus aureus is resistant to β-lactam antibiotics and causes several skin diseases to life-threatening diseases. FmtA is found to be one of the main factors involved in methicillin resistance in S. aureus. FmtA exhibits an esterase activity that removes the D-Ala from teichoic acid. Teichoic acids played a significant role in cell wall synthesis, cell division, colonization, biofilm formation, virulence, antibiotic resistance, and pathogenesis. The virtual screening of drug molecules against the crystal structure of FmtA was performed and the binding affinities of top three molecules (ofloxacin, roflumilast, and furazolidone) were predicted using molecular docking. The presence of positive potential and electron affinity regions in screened drug molecules by DFT analysis illustrated that these molecules are reactive in nature. The protein-ligand complexes were subjected to molecular dynamics simulation. Molecular dynamics analysis such as RMSD, RMSF, Rg, SASA, PCA, and FEL results suggested that FmtA-drug(s) complexes are stable. MM-GBSA binding affinity and QM/MM results (ΔG, ΔH, and ΔS) revealed that active site residues (Ser127, Lys130, Tyr211, Asp213, and Asn343) of FmtA played an essential for the binding of the drug(s) to form a lower energy stable protein-ligand complexes. FmtAΔ42 was purified using cation exchange and gel filtration chromatography. Fluorescence spectroscopy and circular dichroism results showed that interactions of drugs with FmtAΔ42 affect the tertiary structure and increase the thermostability of the protein. The screened molecules need to be tested and could be further modified to develop the antimicrobial compounds against S. aureus.

D-Methionine and d-Phenylalanine Improve Lactococcus lactis F44 Acid Resistance and Nisin Yield by Governing Cell Wall Remodeling.

Lactococcus lactis encounters various environmental challenges, especially acid stress, during its growth. The cell wall can maintain the integrity and shape of the cell under environmental stress, and d-amino acids play an important role in cell wall synthesis. Here, by analyzing the effects of 19 different d-amino acids on the physiology of L. lactis F44, we found that exogenously supplied d-methionine and d-phenylalanine increased the nisin yield by 93.22% and 101.29%, respectively, as well as significantly increasing the acid resistance of L. lactis F44. The composition of the cell wall in L. lactis F44 with exogenously supplied d-Met or d-Phe was further investigated via a vancomycin fluorescence experiment and a liquid chromatography-mass spectrometry assay, which demonstrated that d-Met could be incorporated into the fifth position of peptidoglycan (PG) muropeptides and d-Phe could be added to the fourth and fifth positions. Moreover, overexpression of the PG synthesis gene murF further enhanced the levels of d-Met and d-Phe involved in PG and increased the survival rate under acid stress and the nisin yield of the strain. This study reveals that the exogenous supply of d-Met or d-Phe can change the composition of the cell wall and influence acid tolerance as well as nisin yield in L. lactisIMPORTANCE As d-amino acids play an important role in cell wall synthesis, we analyzed the effects of 19 different d-amino acids on L. lactis F44, demonstrating that d-Met and d-Phe can participate in peptidoglycan (PG) synthesis and improve the acid resistance and nisin yield of this strain. murF overexpression further increased the levels of d-Met and d-Phe incorporated into PG and contributed to the acid resistance of the strain. These findings suggest that d-Met and d-Phe can be incorporated into PG to improve the acid resistance and nisin yield of L. lactis, and this study provides new ideas for the enhancement of nisin production.

Exploring Molecular Docking Studies of Alanine Racemase Inhibitors from Elettaria cardamomum

Background: Enterococcus faecalis has attracted much attention in recent times due to its increased virulence in hospital-acquired infections. Cardamom which is an exotic spice in food items can be proposed for its antimicrobial potential. In the present study, alanine racemase (AlaR) of the bacteria was considered as inhibitors’ target due to its crucial role in cell wall synthesis. Methods: GC-MS analysis of Cardamom extract was performed and the identified phytochemicals were docked against AlaR using AutoDock 4.0. Top score ligands were further subjected to Absorption, Distribution, Metabolism, Excretion (ADME) analysis. Results & Conclusion: Molecular docking studies reveal that among 85 phytoligands, ricinoleic acid, bombykol, 1,8- cineole, heptanoic acid, and linalool showed significant interaction to the enzyme with an energy of -7.81, -7.57, -7.03, -7.02 and -7 kcal/mol, respectively, as compared to its substrate (ΔG Alanine: -5.03 kcal/mol). Among all the five lead compounds, 1,8- cineole, heptanoic acid, and linalool exhibited high bioactivity score on druglikeliness. This enabled us to conclude that the compounds 1,8- cineole, heptanoic acid and linalool would be useful antibacterial agents against E. faecalis infections.

Molecular Insights into FaEG1, a Strawberry Endoglucanase Enzyme Expressed during Strawberry Fruit Ripening.

The endo-β-1,4-glucanases (EGs) that belong to the glycosyl hydrolase family 9 (GH9) have roles in cell wall synthesis, remodeling and degradation. Previous studies have suggested that EGs may play a key role in the ripening of different fruits including strawberries. In this study, we used reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assays to determine the transcript accumulation of an endo-β-1,4-glucanase (FaEG1) during fruit development in two different strawberry ‘Camarosa’ and ‘Monterey’ with contrasting softening ratios. Phylogenetic analyses suggest that FaEG1 belongs to the α group of the GH9 family with other proteins previously described with roles in elongation, abscission and ripening. Comparative modeling was used to obtain the FaEG1 structure. The model displays a α-barrel–type structure that is typical of the GH9 enzyme family, and comprises 12 α-helices, 2 310 helices and 6 β-sheets. The catalytic residues were oriented to the solvent in the middle of an open groove. Protein–ligand interactions were explored with cellulose and two xyloglucans as ligands; the results suggest that the FaEG1-cellulose and FaEG1-XXXGXXXG (the most abundant xyloglucan in strawberries) complexes were more stable complexes than XXFGXXFG. The cell wall degradation was observed by scanning electron microscopy (SEM). The data are congruent with the probable role of the FaEG1 protein in the dissembly of the cellulose-hemicellulose fraction during the ripening of strawberry fruit.

Expression profiling in a mammalian host reveals the strong induction of genes encoding LysM domain-containing proteins in Enterococcus faecium

Enterococcus faecium is an important health care-associated pathogen that is difficult to treat due to the high level of antibiotic resistance of clinical isolates. The identification of new potential therapeutic targets or vaccination strategies is therefore urgently needed. In this regard, we carried out a transcriptomic analysis of the E. faecium vancomycin-resistant strain AUS0004, comparing the gene expression of bacteria grown under laboratory conditions and bacteria isolated from an infection site. This analysis highlighted more than 360 genes potentially induced under infection conditions. Owing to their expression profiles, four LysM domain-containing proteins were characterized in more detail. The EFAU004_01059, 1150 and 494 proteins are highly homologous, whereas EFAU004_01209 has a unique domain-architecture and sequence. The analysis of corresponding mutants showed that all LysM proteins played relevant roles in the infection process of E. faecium in mice. The EFAU004_01209 mutant also displayed profound morphological modifications, suggesting it has a role in cell wall synthesis or cell division. Furthermore, the adhesion to kidney cells and growth of the mutant was affected in human urine. All these phenotypes and the surface exposure of EFAU004_01209 identify this protein as an interesting new drug target in E. faecium.

Gibberellin and auxin signals control scape cell elongation and proliferation in Agapanthus praecox ssp. orientalis

Plant height is determined by the processes of cell proliferation and elongation. Plant hormones play key roles in a species-dependent manner in these processes. We used paclobutrazol (PAC) at 400 mg·L-1 in this study to spray Agapanthus praecox ssp. orientalis plants in order to induce dwarf scape (inflorescence stem). Morphological examination showed that PAC reduced scape height by inhibiting the cell elongation by 54.56% and reducing cell proliferation by 10.45% compared to the control. Quantification and immunolocalization of endogenous gibberellins (GAs) and indole-3-acetic acid (IAA) showed that the GA1, GA3, and GA4 levels and the IAA gradient were reduced. Among these hormones, GA4 was the key component of GAs, which decreased 59.51-92.01% compared to the control in scape. The expression of cell wall synthesis related genes cellulose synthase (CESA) and UDP-glucuronic acid decarboxylase (UXS) were upregulated significantly, whereas cell wall loosening gene xyloglucan endotransglucosylase 2 (XET2) was downregulated by 99.99% surprisingly. Correlation analysis suggested GA regulated cell elongation and auxin modulated cell proliferation in Agapanthus scape. Additionally, the accumulation of sugars played roles in cell wall synthesis and cell expansion. These results indicated GA and IAA signals triggered a downstream signaling cascade, controlled cell expansion and proliferation during scape elongation.

Mechanical Genomics Identifies Diverse Modulators of Bacterial Cell Stiffness

Bacteria must maintain mechanical integrity to withstand the large osmotic pressure differential across the cell membrane and wall. Although maintaining mechanical integrity is critical for proper cellular function, a fact exploited by prominent cell-wall-targeting antibiotics, the proteins that contribute to cellular mechanics remain unidentified. Here, we describe a high-throughput optical method for quantifying cell stiffness and apply this technique to a genome-wide collection of ∼4,000 Escherichia coli mutants. We identify genes with roles in diverse functional processes spanning cell-wall synthesis, energy production, and DNA replication and repair that significantly change cell stiffness when deleted.We observe that proteins with biochemicallyredundant roles in cell-wall synthesis exhibit different stiffness defects when deleted. Correlating our data with chemical screens reveals that reducingmembrane potential generally increases cell stiffness. In total, our work demonstrates that bacterial cell stiffness is a property of both the cell wall andbroader cell physiology and lays the groundwork for future systematic studies of mechanoregulation.

Novel Drug Targets for Food-Borne Pathogen Campylobacter jejuni: An Integrated Subtractive Genomics and Comparative Metabolic Pathway Study.

Campylobacters are a major global health burden and a cause of food-borne diarrheal illness and economic loss worldwide. In developing countries, Campylobacter infections are frequent in children under age two and may be associated with mortality. In developed countries, they are a common cause of bacterial diarrhea in early adulthood. In the United States, antibiotic resistance against Campylobacter is notably increased from 13% in 1997 to nearly 25% in 2011. Novel drug targets are urgently needed but remain a daunting task to accomplish. We suggest that omics-guided drug discovery is timely and worth considering in this context. The present study employed an integrated subtractive genomics and comparative metabolic pathway analysis approach. We identified 16 unique pathways from Campylobacter when compared against H. sapiens with 326 non-redundant proteins; 115 of these were found to be essential in the Database of Essential Genes. Sixty-six proteins among these were non-homologous to the human proteome. Six membrane proteins, of which four are transporters, have been proposed as potential vaccine candidates. Screening of 66 essential non-homologous proteins against DrugBank resulted in identification of 34 proteins with drug-ability potential, many of which play critical roles in bacterial growth and survival. Out of these, eight proteins had approved drug targets available in DrugBank, the majority serving crucial roles in cell wall synthesis and energy metabolism and therefore having the potential to be utilized as drug targets. We conclude by underscoring that screening against these proteins with inhibitors may aid in future discovery of novel therapeutics against campylobacteriosis in ways that will be pathogen specific, and thus have minimal toxic effect on host. Omics-guided drug discovery and bioinformatics analyses offer the broad potential for veritable advances in global health relevant novel therapeutics.

Populus endo-β-1,4-glucanases gene family: genomic organization, phylogenetic analysis, expression profiles and association mapping.

Extensive characterization of the poplar GH9 gene family provides new insights into GH9 function and evolution in woody species, and may drive novel progress for molecular breeding in trees. In higher plants, endo-β-1,4-glucanases (cellulases) belonging to the glycosyl hydrolase family 9 (GH9) have roles in cell wall synthesis, remodeling and degradation. To increase the understanding of the GH9 family in perennial woody species, we conducted an extensive characterization of the GH9 family in the model tree species, Populus. We characterized 25 putative GH9 members in Populus with three subclasses (A, B, and C), using structures and bioinformatic analysis. Phylogenetic analyses of 114 GH9s from plant (dicot, monocot, and conifer) and bacterial species (outgroup) demonstrated that plant GH9s are monophyletic with respect to bacteria GH9s. Three subclasses, A, B, and C, of plant GH9 are formed before the divergence of angiosperms and gymnosperms. Chromosomal localization and duplications of GH9s in the Populus genome showed that eight paralogous pairs remained in conserved positions on segmental duplicated blocks, suggesting duplication of chromosomal segments has contributed to the family expansion. By examining tissue-specific expression profiles for all 25 members, we found that GH9 members exhibited distinct but partially overlapping expression patterns, while certain members have higher transcript abundance in mature or developing xylem. Based on our understanding of intraspecific variation and linkage disequilibrium of two KORRIGANs (PtoKOR1 and PtoKOR2) in natural population of Populus tomentosa, two non-synonymous SNPs in PtoKOR1 associated with fiber width and holocellulose content were obtained. Characterizations of the poplar GH9 family provide new insights into GH9 function and evolution in woody species, and may drive novel progress for molecular breeding in trees.

Cloning, expression and purification of Ag85A-ESAT6 antigens of Mycobacterium tuberculosis as a fusion protein

Aims and objectivesSecretory antigens of Ag85 complex and 6-kDa Early Secreted Antigenic Target (ESAT-6) play a role in cell wall synthesis and virulence of Mycobacterium tuberculosis (MTB), respectively. They could induce a potent immune response and showed protective effects in some studies in animal models. So, they are considered as potential candidate antigens in new vaccination strategies against MTB. The aim of this study includes cloning, expression and purification of recombinant Ag85A-ESAT6 as a fusion protein for further studies of vaccine designation. Materials and methodsA synthetic DNA fragment composed of coding sequences of Ag85A and ESAT6 was cloned into the BamHI and EcoRI restriction site of pET28a plasmid. Then the recombinant plasmid was transformed to Escherichia coli BL21 (DE3) and expressed in optimum condition. The expressed protein was analyzed and confirmed by SDS–PAGE and western blotting using anti-His tag and monoclonal anti-ESAT6 antibodies. Protein purification was performed by Ni–NTA spin column under denaturing conditions. ResultsThe results of SDS–PAGE and western blotting showed that Ag85A-ESAT6 recombinant fusion protein was successfully cloned and expressed with a His tag in its N-terminal. Considering the expression of this protein as an inclusion body, its purification was done by denaturing protocol. ConclusionThe Ag85A-ESAT6 fusion protein expressed in this study can be combined with a suitable adjuvant to induce the cellular and humoral immune responses, and its protective effects against MTB could be evaluated in subsequent studies.

The Great Wall Symposium.

The third Great Wall Symposium recently held in Paris, France, featured the latest advances in the field of peptidoglycan metabolism. We hosted two Keynote lectures by Kevin D. Young and Alexander Tomasz, as well as 24 speakers in six different sessions. The meeting attracted a great deal of interest from the scientific community, which has expanded dramatically in the last decade. The remerged interest in the field of peptidoglycan research occurred around the year 2000 with two important discoveries: (1) the characterization of bacterial cytoskeleton and their role in cell wall synthesis, and (2) the discovery of innate immune receptors in Drosophila (through peptidoglycan recognition proteins) and in mammals (through the Nod proteins) involved in sensing of peptidoglycan. As a consequence of the rediscovered interest in peptidoglycan by microbiologists, cell biologists, and immunologists, a first meeting exclusively focused on peptidoglycan research was organized in Baeza, Spain, October 4–6, 2010. This first small meeting answered the real need for a discussion platform of the peptidoglycan community, and a second meeting followed a year later in Lisbon, Portugal, September 28–30, 2011. To avoid overlapping with the Bacterial Cell Surface Gordon Conference, it was decided that this meeting would be organized every two years alternating with the Gordon Conference. In addition, given the fact that the Gordon conference is organized in the United States, the scientific community felt that this meeting should stay in Europe to favor diversity and opportunity for young scientists to meet with senior members of the field. The number of participants (128 in total) increased again with regard to the first and second meeting and was limited by the venue capacity. The meeting has rapidly become a reference in the field and a collective decision was made to pursue the adventure in two years in Florence, Italy, at the Villa Finaly on September 21–24, 2015. Microbial Drug Resistance has been an active promoter of the advances that have been made in the field of peptidoglycan metabolism during the last two decades, first with a special issue of the last Cell Wall meeting before the Great Wall Symposium era that took place at Lago de Garda, Italy, in 1995. Following this tradition, Alexander Tomasz has organized a special issue on the science presented at the Great Wall Symposium. A first special issue was published in June 2012 on the work presented at the second symposium. To maintain the visibility of the field, a second special issue on the third meeting is presented in this special issue, creating a great opportunity for young scientists to present their work to the scientific community.

Twitching Motility, Biofilm Communities in Cephalosporin Resistant <i>Proteus</i> spp and the Best <i>in vitro</i> Amoxicillin Susceptibility to Isolates

Proteus strains isolated from tap water are assessed for their twitching motility and biofilm formation on urethral catheter strips of two selected brands, Protos and Huaian, since these strains are the common cause of catheter associated urinary tract infection (CAUTI). Neither the strains of high swarming motility nor of high antimicrobial resistance enabled to twitch over the catheter strips. All of high antibiotic resistant strains were found to form biofilm on PVC strip, few strains on Huaian catheter strip and one strain (Pv) on both catheter strips. Based on biofilm quantification study, the urethral catheter from Protos brand was relatively safer than that of Huaian brand to prevent CAUTI. Moreover, Proteus isolates were strong resistant to cotrimoxazole (folate synthesis inhibitor) and cephalosporins (cell wall synthesis inhibitor). Underlying the β-lactam group, strong cephalosporin resistance and amoxicillin susceptibility in isolates indicated that the isolates were non β-lactamase producer. Tested antibiotics underlying the protein and DNA synthesis inhibitors were satisfactorily effective for the inhibition of Proteus isolates. The phenolic acid, –C6H4OH, present in the chemical formula of amoxicillin plays a crucial role in cell wall synthesis of Proteus isolates allowing more drugs to cell wall via porins.

Study on variability assessment and evolutionary relationships of glutamate racemase in Pseudomonas species

Pseudomonas species is known to cause multiple nosocomial infections in patients and results in high morbidity and mortality rates (10%). The greatest obstacle in treating patients infected with the Pseudomonas species is the widespread emergence of antibiotic resistance. Hence, there is an urgent need to develop new compounds which can be effective against Pseudomonas species and possibly remain tolerant to drug resistance. The enzyme glutamate racemase plays an important role in cell wall synthesis of bacteria and as a rate limiting step, thus it is an excellent target for the designing of new class of antibacterial agents. The objective of this study is to investigate the variations in sequences of glutamate racemase, a potential drug target across the all 31 species of Pseudomonas. Sequence variability and conservation for functional motif identification is helpful for identifying evolutionarily important residues with functional significance; subsequently these results of variable sites were supported by entropy profile obtained from protein variability server using Shannon entropy. Phylogenetic profile among the different Pseudomonas sp. having fully/highly conserved residues was observed, suggesting possible functional similarities between them. The variation analysis in conserved and non-conserved region of the sequence can be used to predict the binding site for target specific drug discovery.

Young's Modulus of B. Subtilis Cell Wall: Measuring and Modeling the Elasticity of Rod-Like Bacteria

The peptidoglycan layer is the principle structural component of load baring cell wall in rod-like bacteria. As a polymer cross-linked into a rigid scaffold, peptidoglycan is responsible for characteristic shape of bacterial cells, their mechanical strength and durability. Atomic force microscopy (AFM) is an ideal tool to make highly precise measurements, to analyze multiple isolated cells, and to compare the mechanical properties between the individual bacterial cells. We can then investigate how the biology of the cells (deletion of specific proteins) or treatment and stresses to the cells (exposure to antibiotics) can affect the mechanics of their cell walls. Although there have been many studies of the mechanics of bacteria using AFM, many of them treat the force-indentation relationship in the terms of the standard Hertz model (i.e. approximate the cell as a uniform elastic solid). Of the few studies that treat the compression of a cell as the deformation of a thin elastic shell, none treat bacteria as a rod-like structure, which it resembles. We used large radii colloidal probes to obtain force-compression curves on multiple individual cells of wild type B. Subtilis and a mutant deficient in the protein mbl (Δmbl) that plays a key role in cell wall synthesis. To interpret the data in a quantitative manner, we developed a variety of analytical models for a rod-like elastic shell filled with incompressible fluid. We applied these models to describe the stretching of the cell wall and to calculate the Young's modulus of peptidoglycan in hydrated rod-like bacteria. Compared to wild type cells, the Young's modulus of the peptidoglycan in mutant bacterial cells is reduced by a third.

Interaction of Platelets and Anidulafungin against Aspergillus fumigatus

The combination of platelets and anidulafungin at 0.03 μg/ml significantly (P < 0.05) reduced the germination rate and hyphal elongation in Aspergillus fumigatus compared to those with either anidulafungin only or an untreated control. Platelets decreased the expression of the fks gene, which plays an important role in cell wall synthesis. Our results suggest that human platelets plus anidulafungin might contribute to defense against A. fumigatus.

Fission yeast Cyk3p is a transglutaminase-like protein that participates in cytokinesis and cell morphogenesis

Cell morphogenesis is a complex process that relies on a diverse array of proteins and pathways. We have identified a transglutaminase-like protein (Cyk3p) that functions in fission yeast morphogenesis. The phenotype of a cyk3 knockout strain indicates a primary role for Cyk3p in cytokinesis. Correspondingly, Cyk3p localizes both to the actomyosin contractile ring and the division septum, promoting ring constriction, septation, and subsequent cell separation following ring disassembly. In addition, Cyk3p localizes to polarized growth sites and plays a role in cell shape determination, and it also appears to contribute to cell integrity during stationary phase, given its accumulation as dynamic puncta at the cortex of such cells. Our results and the conservation of Cyk3p across fungi point to a role in cell wall synthesis and remodeling. Cyk3p possesses a transglutaminase domain that is essential for function, even though it lacks the catalytic active site. In a wider sense, our work illustrates the physiological importance of inactive members of the transglutaminase family, which are found throughout eukaryotes. We suggest that the proposed evolution of animal transglutaminase cross-linking activity from ancestral bacterial thiol proteases was accompanied by the emergence of a subclass whose function does not depend on enzymatic activity.