Bacterial Cell Wall Structure Research Articles

Antibacterial effects of iron oxide (Fe3O4) nanoparticles: distinguishing concentration-dependent effects with different bacterial cells growth and membrane-associated mechanisms.

Nowadays, the influence of nanoparticles (NPs) on microorganisms attracts a great deal of attention as an alternative to antibiotics. Iron oxide (Fe3O4) NPs' effects on Gram-negative Escherichia coli BW 25113 and Gram-positive Enterococcus hirae ATCC 9790 growth and membrane-associated mechanisms have been investigated in this study. Growth specific rate of E. coli was decreased, indicating the bactericidal effect of Fe3O4 NPs. This inhibitory effect of NPs had a concentration-dependent manner. The reactive oxygen species together with superoxide radicals and singlet oxygen formed by Fe3O4 NPs could be the inhibition cause. Fe3O4 NPs showed opposite effects on E. hirae: the growth stimulation or inhibition was observed depending on NPs concentration used. Addition of NPs altered redox potential kinetics and inhibited H2 yield in E. coli; no change in intracellular pH was determined. Fe3O4 NPs decreased H+-fluxes through bacterial membrane more in E. coli than in E. hirae even in the presence of DCCD and increased ATPase activity more in E. hirae than in E. coli. Our results showed that the Fe3O4 NPs demonstrate differentiating effects on Gram-negative and Gram-positive bacteria likely due to the differences in bacterial cell wall structure and metabolic peculiarities. Fe3O4 NPs of different concentrations have no hemolytic (cytotoxic) activity against erythrocytes. Therefore, they can be proposed as antibacterial agents in biomedicine, biotechnology, and pharmaceutics.

In vitro treatment of Enterococcus faecalis with calcium hydroxide impairs phagocytosis by human macrophages

Objective: Monocyte-derived macrophages (MDMs) ability to phagocytize and produce nitric oxide (NO) was tested against root-canal strains of Enterococcus faecalis submitted to alkaline stress. Root-canal strains were also compared with urine Enterococci.Materials and Methods: Enterococcus faecalis were stressed with alkaline-BHI broth and incubated in vitro at a cell/bacteria ratio of 1:5. Phagocytosis was analyzed by fluorescence microscopy using acridine orange stain, and NO concentration was measured in supernatants.Results and Conclusions: Alkaline-stress significantly impaired MDMs phagocytosis of E. faecalis strains analyzed, except in ATCC4083 isolated from a pulpless tooth, but NO production was unchanged. Comparison of different strains showed the urine isolate had higher NO levels than root canal strains. Alterations in the bacterial cell wall structures after alkaline-stress possibly made bacteria less recognizable and phagocytized by MDMs but did not affect their ability to activate NO production. Furthermore, root canal strains elicited different responses by immune cells compared with strains from urine. Clinically, impaired phagocytosis of E. faecalis could contribute to their persistence in root canal systems previously treated with calcium hydroxide.

Antimicrobial activity of PIT-fabricated cinnamon oil nanoemulsions: Effect of surfactant concentration on morphology of foodborne pathogens

The impact of surfactant concentration (10 to 20 wt%) on the antimicrobial activity of cinnamon oil nanoemulsions formed by the phase inversion temperature (PIT) method was studied against a number of foodborne pathogens: Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and Vibrio parahaemolyticus. Minimum inhibitory concentration (MIC), dynamic time kill, and changes in bacteria morphology were determined. Increasing non-ionic surfactant (Tween®80) concentration from 10 to 20 wt% increased the MIC values of the nanoemulsions. However, dynamic time kill plots revealed that nanoemulsions with higher surfactant concentrations (15 and 20 wt%) led to faster or more prolonged inhibition of bacteria compared to those with lower concentration (10 wt%) or with bulk cinnamon oil. Morphological changes of the bacteria were more promoted for nanoemulsions containing higher surfactant concentrations as shown by field emission scanning electron microscopy (FE-SEM). The antimicrobial activity of the cinnamon oil nanoemulsions was attributed to their ability to disrupt bacterial cell wall structures and promote expulsion of internal cellular material.

Insights into Key Interactions between Vancomycin and Bacterial Cell Wall Structures.

Vancomycin is a glycopeptide antibiotic used for the treatment of serious infections by Gram-positive pathogens. Vancomycin inhibits cell wall biosynthesis by targeting the d-Ala-d-Ala terminus of peptidoglycan (PG). The highly cross-linked heptapeptide aglycon structure of vancomycin is the d-Ala-d-Ala binding site. The first residue of vancomycin is N-methyl-leucine, which is crucial for the dipeptide binding. The removal of N-methyl-leucine by Edman degradation results in desleucyl-vancomycin devoid of antimicrobial activities. To investigate the function of N-methyl-leucine for the dipeptide binding in vancomycin, molecular dynamics simulations of vancomycin and three N-terminus-modified vancomycin derivatives: desleucyl-vancomycin, vancomycinNtoC, and vancomycinSar, binding to a PG unit of the sequence l-Ala-d-iso-Gln-l-Lys-d-Ala-d-Ala with an intact pentaglycine bridge structure attached to the bridge link of l-Lys were carried out. Glycopeptide–PG binding interactions were characterized by root-mean-square-deviation contour analysis of atomic positions in vancomycin and its three analogues bound to a PG unit. The overall sampling space for four glycopeptide–PG complexes shows four distinct distributions with a continuous change between the conformational spaces. The hydrogen bond analyses show that multiple hydrogen bonds between the d-Ala-d-Ala and the vancomycin aglycon structure strengthened the dipeptide binding. The simulations revealed that the removal or chemical modification of N-methyl-leucine significantly weakens the dipeptide binding to the aglycon structure and provides interesting structural insights into glycopeptide–PG binding interactions.

The disruptive effect of lysozyme on the bacterial cell wall explored by an in-silico structural outlook.

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall and disrupt the bacterial life cycle by cleaving the linkage between the NAG and NAM carbohydrates. Lab exercises focused on the effects of lysozyme on the bacterial cell wall are frequently incorporated in biochemistry classes designed for undergraduate students in diverse fields as biology, microbiology, chemistry, agronomy, medicine, and veterinary medicine. Such exercises typically do not include structural data. We describe here a sequence of computer tasks designed to illustrate and reinforce both physiological and structural concepts involved in lysozyme effects on the bacterial cell-wall structure. This lab class usually lasts 3.5 hours. First, the instructor presents introductory concepts of the bacterial cell wall and the effect of lysozyme on its structure. Then, students are taught to use computer modeling to visualize the three-dimensional structure of a lysozyme in complex with bacterial cell-wall fragments. Finally, the lysozyme inhibitory effect on a bacterial culture is optionally proposed as a simple microbiological assay. The computer lab exercises described here give students a realistic understanding of the disruptive effect of lysozymes on the bacterial cell wall, a crucial component in bacterial survival. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):83-90, 2018.

Interaction of graphene family materials with Listeria monocytogenes and Salmonella enterica.

Graphene family materials have unique properties, which make them valuable for a range of applications. The antibacterial properties of graphene have been reported; however, findings have been contradictory. This study reports on the antimicrobial proprieties of three different graphene materials (pristine graphene (pG), graphene oxide (GO), and reduced graphene oxide (rGO)) against the food-borne bacterial pathogens Listeria monocytogenes and Salmonella enterica. A high concentration (250 μg/mL) of all the analyzed graphenes completely inhibited the growth of both pathogens, despite their difference in bacterial cell wall structure. At a lower concentration (25 μg/mL), similar effects were only observed with GO, as growth inhibition decreased with pG and rGO at the lower concentration. Interaction of the nanoparticles with the pathogenic bacteria was found to differ depending on the form of graphene. Microscopic imaging demonstrated that bacteria were arranged at the edges of pG and rGO, while with GO, they adhered to the nanoparticle surface. GO was found to have the highest antibacterial activity.

Antibiotic resistance: Origins, mechanisms, approaches to counter

Microbial resistance is emerging faster than we are replacing our armamentarium of antimicrobial agents. Resistance to penicillin developed soon after it was introduced into clinical practice in 1940s. Now resistance developed to every major class of antibiotics. In healthcare facilities around the world, bacterial pathogens that express multiple resistance mechanisms are becoming common. The origins of antibiotic resistance genes can be traced to the environmental microbiota. Mechanisms of antibiotic resistance include alterations in bacterial cell wall structure, growth in biofilms, efflux pump expression, modification of an antibiotic target or acquisition of a new target and enzymatic modification of the antibiotic itself. Specific examples of each mechanism are discussed in this review. Some approaches to counter resistance include antibiotic stewardship, co-administration with resistance inhibitors, exploiting genome data in search of new targets and use of non-antibiotic antimicrobials for topical indications. A coordinated effort from government, public and industry is needed to deal with antibiotic resistance health care crisis.

Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly

The peptidoglycan (PG) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. A quantitative understanding of the relationships between PG architecture, morphogenesis, immune system activation and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell wall synthesis and cell growth. High-performance liquid chromatography (HPLC) has played an important role in our understanding of the structural and chemical complexity of the cell wall by providing an analytical method to quantify differences in chemical composition. Here, we present a primer on the basic chemical features of wall structure that can be revealed through HPLC, along with a description of the applications of HPLC PG analyses for interpreting the effects of genetic and chemical perturbations to a variety of bacterial species in different environments. We describe the physical consequences of different PG compositions on cell shape, and review complementary experimental and computational methodologies for PG analysis. Finally, we present a partial list of future targets of development for HPLC and related techniques.

A Small-Group Activity Introducing the Use and Interpretation of BLAST

As biological sequence data are generated at an ever increasing rate, the role of bioinformatics in biological research also grows. Students must be trained to complete and interpret bioinformatic searches to enable them to effectively utilize the trove of sequence data available. A key bioinformatic tool for sequence comparison and genome database searching is BLAST (Basic Local Alignment Search Tool). BLAST identifies sequences in a database that are similar to the entered query sequence, and ranks them based on the length and quality of the alignment. Our goal was to introduce sophomore and junior level undergraduate students to the basic functions and uses of BLAST with a small group activity lasting a single class period. The activity provides students an opportunity to perform a BLAST search, interpret the data output, and use the data to make inferences about bacterial cell envelope structure. The activity consists of two parts. Part 1 is a handout to be completed prior to class, complete with video tutorial, that reviews cell envelope structure, introduces key terms, and allows students to familiarize themselves with the mechanics of a BLAST search. Part 2 consists of a hands-on, web-based small group activity to be completed during the class period. Evaluation of the activity through student performance assessments suggests that students who complete the activity can better interpret the BLAST output parameters % query coverage and % max identity. While the topic of the activity is bacterial cell wall structure, it could be adapted to address other biological concepts.

Antibacterial activity of hypocrellin A against Staphylococcus aureus

The antibacterial activity and acting mechanism of hypocrellin A (HA) were conducted regarding in vitro activity of HA on Staphylococcus aureus GZ86 by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with HA. The experimental results indicated 1.5 mg/l HA could completely inhibit the growth of 10⁷ CFU/ml S. aureus cells in liquid beef extract-peptone medium under a halogen-tungsten lamp for 120 min. Meanwhile, HA resulted in the leakage of reducing sugars and proteins and induced the respiratory chain dehydrogenases into inactive state, suggesting that HA were able to destroy the permeability of the bacterial membranes. When the cells of S. aureus were exposed to 2.5 mg/l HA under a halogen-tungsten lamp for 120 min, many pits and gaps were observed in bacterial cells by scanning electron microscopy, and the cell wall was fragmentary, indicating the bacterial cells were damaged severely. The experiments strongly confirmed the contribution of multiform reactive oxygen species (ROS) to bactericidal effect. In conclusion, the combined results suggested that ROS may damage the structure of bacterial cell wall and depress the activity of some membranous enzymes, which cause S. aureus bacteria to die eventually.

Two-Component Systems Are Involved in the Regulation of Botulinum Neurotoxin Synthesis in Clostridium botulinum Type A Strain Hall

Clostridium botulinum synthesizes a potent neurotoxin (BoNT) which associates with non-toxic proteins (ANTPs) to form complexes of various sizes. The bont and antp genes are clustered in two operons. In C. botulinum type A, bont/A and antp genes are expressed during the end of the exponential growth phase and the beginning of the stationary phase under the control of an alternative sigma factor encoded by botR/A, which is located between the two operons. In the genome of C. botulinum type A strain Hall, 30 gene pairs predicted to encode two-component systems (TCSs) and 9 orphan regulatory genes have been identified. Therefore, 34 Hall isogenic antisense strains on predicted regulatory genes (29 TCSs and 5 orphan regulatory genes) have been obtained by a mRNA antisense procedure. Two TCS isogenic antisense strains showed more rapid growth kinetics and reduced BoNT/A production than the control strain, as well as increased bacterial lysis and impairment of the bacterial cell wall structure. Three other TCS isogenic antisense strains induced a low level of BoNT/A and ANTP production. Interestingly, reduced expression of bont/A and antp genes was shown to be independent of botR/A. These results indicate that BoNT/A synthesis is under the control of a complex network of regulation including directly at least three TCSs.

Biochemical and immunological properties of the fractions of tubercle bacilli

In 1954, Dr. Yuichi Yamamura and his coassociates, National Sanatorium Toneyama Hospital, has reported the formation of experimental tuberculous cavity in rabbits. This work was a milestone of the researches in the field of cellular immunology. In 1960, I have started my scientific carrier in Kyushu University Medical School as the post-graduate student under the guidance of Prof. Yamamura. Since then, I have worked on the field of biochemical and immunological properties of tubercle bacilli. The arabinose mycolate, polysaccharides, cell-wall skeleton (CWS) and trehalose dimycolate (TDM) were purified from tubercle bacilli and their Chemical structures and biological activities were investigated in detail in our laboratory. In 1971, we have reported that adjuvant active principle of tubercle bacilli was a CWS fraction, and the details of chemical structure and adjuvantity of mycobacterial CWS were investigated in our research group. The application of BCG-CWS to cancer immunotherapy was also studied. In 1974, Prof. E. Lederer and his coworkers, University of Paris, have reported that N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP) was the minimum adjuvant-active structure of bacterial cell wall. We have synthesized several hundreds of MDP derivatives and selected, MDP-Lys(L18), romurtide, as the candidate of cytokine inducer for the clinical application. Romurtide is applied in cancer patients for the recovery of the number and functions of monocytes, neutrophils and platelets. These results suggest that the tubercle bacilli, especially, CWS and related synthetic MDP derivatives, are effective for the potentiation of host resistance against infectious diseases and cancer.

Sir James Baddiley. 15 May 1918 — 19 November 2008

James Baddiley was a biochemist who used the methods and insight of the organic chemist to answer important questions in biology, notably coenzyme structure and the structure and function of bacterial cell walls. A graduate of Manchester University, he moved to Cambridge in 1944 with A. R. Todd, where he synthesized adenosine triphosphate, the nucleotide concerned with essential energy transformations in all forms of life. As an independent researcher at the Lister Institute in London he elucidated the structure of coenzyme A and other coenzymes. He was appointed Professor of Organic Chemistry in Newcastle, where the exploration of the structures of two cytidine nucleotides led to the discovery of the teichoic acids, major components of the cell walls and membranes of Gram-positive bacteria. These discoveries were extended to cover the structures, biosynthesis, function and immunology of the teichoic acids. Baddiley became Professor of Chemical Microbiology in 1977. Moving to Cambridge after his retirement, he was able to continue his researches in the Department of Biochemistry. He was elected a Fellow of Pembroke College and as an elder statesman undertook extensive committee work, often as chairman, both in Cambridge University and nationally. He was knighted in 1977.

Bacteria survive multiple puncturings of their cell walls.

A bacterial cell wall is a highly dynamic multilayer structure interfacing the cytoplasm to the outside environment. It supports a multitude of chemical and biological processes necessary for life. It is therefore postulated that damage to the structure of bacterial cell wall would threaten cell integrity and result in cell death. We tested this hypothesis by repeatedly puncturing the cell wall of a live Gram negative bacterium Salmonella typhimurium at different locations using a sharp atomic force microscope nanotip and conducting multiple viability tests. Our study demonstrated that a S. typhimurium survives repeated puncturings of its cell wall and retains its integrity, viability, and ability to divide. The results are explained on the basis of the concept of the self-repairing of lipid bilayers and the peptidoglycan layer.

Impact of cell wall structure on the behavior of bacterial cells in the binding of copper and cadmium

In this study, Fourier transform infrared spectroscopy, potentiometric titration, along with sorption experiments using chemically modified bacteria were conducted to compare the behavior of Gram-positive Bacillus thuringiensis and Gram-negative Escherichia coli as sorbents for Cu 2+ and Cd 2+ ions. The IR spectra showed that there were obvious changes connected with the–COOH groups for Cu(II)- and Cd(II)-loaded B. thuringiensis. A three site non-electrostatic model provides an excellent fit to the titration curves of both E. coli and B. thuringiensis with the first corresponding p K a values of 4.16 ± 0.18 and 3.30 ± 0.24, respectively, implying that B. thuringiensis contains more carboxyl groups than E. coli. Chemical modification and metal sorption experiments further confirmed that carboxyl groups may play a more important role in the binding Cd(II) and Cu(II) for B. thuringiensis than for E. coli, which could be attributed to the higher concentration of carboxyl sites of B. thuringiensis than E. coli. These results show that the main chemical functional group responsible for the binding of metal ions on B. thuringiensis and E. coli is obviously different because of the different composition and structure of bacterial cell walls. The concentrations of the chemical functional groups on bacterial cell walls are regarded to govern their role in the binding of metal ions and affect the affinity between the bacterial cells and metal ions.

Microcalorimetric Study on the Inhibition of Escherichia coli by Some Novel Pyridine Amide Schiff Base Derivatives

AbstractThe antibacterial effect of a series of novel pyridine amide Schiff base compounds on Escherichia coli was investigated by a microcalorimetric method at 37 °C. The metabolic power‐time curves of the bacteria treated by the compounds were obtained, and the thermokinetic parameters were analyzed, from which the antibacterial activities of these compounds were evaluated. The results show that two compounds F and G have good activity on aerobic multiplying metabolism of E. coli, with the value of IC50 106 and 113 mg/L respectively, but have no effective action on the fermentation metabolism of E. coli. The action of the compounds on the non‐multiplying metabolism was investigated by taking the heat output of E. coli in the stationary phase as the guideline of the activity. The experimental results revealed that the hydrophilicity of these Schiff bases had a great influence on their antibacterial activity, which results from the bacterial cell wall structure. The antibacterial structure‐activity relationship of these Schiff base derivatives was also briefly discussed. The antibacterial activity of the compounds against E. coli was as follows: compound F>G>C>D>E>B>A.

The effect of low-intensity pulsed sound waves delivered by the Exogen device on Staphylococcus aureus morphology and genetics

We investigated the effect of low-intensity pulsed sound waves delivered by the Exogen device, which is recommended for the treatment of delayed union and nonunion in orthopedic surgery, on the colony number, antimicrobial susceptibility, bacterial morphology, and genetics of Staphylococcus aureus, which is a frequent pathogen in orthopedic infections. Thirty tubes containing 0.5 McFarland suspensions of S. aureus (ATCC 25923) were used. Fifteen tubes forming the test group were subjected to low-intensity sound waves by the Exogen device for 20 minutes. The remaining 15 tubes were untreated as controls. The two groups were then compared with respect to colony number, antibiotic susceptibility, and genotypic properties. The tubes were examined histologically by electron microscopy. The test tubes treated with sound waves showed a significantly lower number of bacteria colonies compared to the control tubes (p<0.001). The two groups were similar with respect to antibiotic susceptibility and genotypic properties. Bacterial cell wall structure in the control group was of normal appearance, whereas partial destruction and break-up were observed in test samples. Bacterial cell wall thickness was significantly higher in the test group compared to the control group (41.54 nm and 24.27 nm, respectively; p<0.001). Low-intensity sound waves may be beneficial as a prophylactic measure to prevent infections in primary orthopedic operations and as an adjuvant therapy for infected nonunions.

Multidimensional HRMAS NMR: a platform for in vivo studies using intact bacterial cells

In vivo analysis in whole cell bacteria, especially the native tertiary structures of the bacterial cell wall, remains an unconquered frontier. The current understanding of bacterial cell wall structures has been based on destructive analysis of individual components. These in vitro results may not faithfully reflect the native structural and conformational information. Multidimensional High Resolution Magic Angle Spinning NMR (HRMAS NMR) has evolved to be a powerful technique in a variety of in vivo studies, including live bacterial cells. Existing studies of HRMAS NMR in bacteria, technical consideration of its successful application, and current limitations in studying true human pathogens are briefly reviewed in this report.

Induction of pro-inflammatory cytokine release by human macrophages during exposure of Streptococcus pneumoniae to penicillin is influenced by minimum inhibitory concentration ratio

β-Lactam antibiotics cause release of pro-inflammatory bacterial cell wall structures. We determined the effect of penicillin treatment of Streptococcus pneumoniae on the induction of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) genes by human macrophages and the influence of antibiotic concentration and bacterial growth phase upon this induction. Gene expression was measured by real-time polymerase chain reaction (PCR) and protein was measured by enzyme-linked immunosorbent assay (ELISA). Treatment of lag phase S. pneumoniae with one-eighth minimum inhibitory concentration (MIC) penicillin resulted in enhanced expression of TNF-α messenger RNA (mRNA), but not TNF-α protein at 6 h compared with untreated bacteria. IL-1β mRNA and protein were not affected by these bacteria. MIC treatment of lag or early log phase bacteria induced both protein and mRNA for IL-1β. Bacteria exposed to concentrations of penicillin that cause lysis (MIC) or no lysis with morphological changes (sub-MIC) induce differential patterns of pro-inflammatory cytokine expression by human macrophages.

Methods for Acquisition and Assignment of Multidimensional High-Resolution Magic Angle Spinning NMR of Whole Cell Bacteria

High-resolution magic-angle spinning (HR-MAS) NMR was developed in late 1990s, and it has evolved quickly for the study of a variety of biological matrixes. Recently, it has been used as an effective means to study the cell wall structures of intact bacteria. (1)H-(13)C heteronuclear single quantum coherence (HSQC) HR-MAS NMR can provide rapid analysis of the cell wall structure in live bacterial cells, thus allowing observation of drug effects, gene mutation, species differentiation, and environmental effects. However, this rapid analysis is dependent on having an established framework of HR-MAS NMR experiments and a detailed assignment of the whole-cell NMR spectra. This study examines parameters and describes strategies for the effective application of 2D and 3D HR-MAS NMR techniques to assign and study bacterial cell wall structures using Mycobacterium smegmatis as a model organism. Important parameters for successful whole-cell HR-MAS NMR studies, including pulse sequences, rotor synchronization, acquisition times, labeling strategies, temperature, number of cells, and cell viability, are described. A four-prong approach is presented for assignment of the complex whole-cell spectra, including the use of 3D HCCH-TOCSY and HCCH-COSY HR-MAS NMR.