Bacterial Outer Layer Research Articles

Role of novel polysaccharide layers in assembly of the exosporium, the outermost protein layer of the Bacillus anthracis spore.

A fundamental question in cell biology is how cells assemble their outer layers. The bacterial endospore is a well-established model for cell layer assembly. However, the assembly of the exosporium, a complex protein shell comprising the outermost layer in the pathogen Bacillus anthracis, remains poorly understood. Exosporium assembly begins with the deposition of proteins at one side of the spore surface, followed by the progressive encirclement of the spore. We seek to resolve a major open question: the mechanism directing exosporium assembly to the spore, and then into a closed shell. We hypothesized that material directly underneath the exosporium (the interspace) directs exosporium assembly to the spore and drives encirclement. In support of this, we show that the interspace possesses at least two distinct layers of polysaccharide. Secondly, we show that putative polysaccharide biosynthetic genes are required for exosporium encirclement, suggesting a direct role for the interspace. These results not only significantly clarify the mechanism of assembly of the exosporium, an especially widespread bacterial outer layer, but also suggest a novel mechanism in which polysaccharide layers drive the assembly of a protein shell.

Insight into the Antibacterial Activity of Selected Metal Nanoparticles and Alterations within the Antioxidant Defence System in Escherichia coli, Bacillus cereus and Staphylococcus epidermidis.

The antimicrobial activity of nanoparticles (NPs) is a desirable feature of various products but can become problematic when NPs are released into different ecosystems, potentially endangering living microorganisms. Although there is an abundance of advanced studies on the toxicity and biological activity of NPs on microorganisms, the information regarding their detailed interactions with microbial cells and the induction of oxidative stress remains incomplete. Therefore, this work aimed to develop accurate oxidation stress profiles of Escherichia coli, Bacillus cereus and Staphylococcus epidermidis strains treated with commercial Ag-NPs, Cu-NPs, ZnO-NPs and TiO2-NPs. The methodology used included the following determinations: toxicological parameters, reactive oxygen species (ROS), antioxidant enzymes and dehydrogenases, reduced glutathione, oxidatively modified proteins and lipid peroxidation. The toxicological studies revealed that E. coli was most sensitive to NPs than B. cereus and S. epidermidis. Moreover, NPs induced the generation of specific ROS in bacterial cells, causing an increase in their concentration, which further resulted in alterations in the activity of the antioxidant defence system and protein oxidation. Significant changes in dehydrogenases activity and elevated lipid peroxidation indicated a negative effect of NPs on bacterial outer layers and respiratory activity. In general, NPs were characterised by very specific nano-bio effects, depending on their physicochemical properties and the species of microorganism.

Opposing Effects of PhoPQ and PmrAB on the Properties of Salmonella enterica serovar Typhimurium: Implications on Resistance to Antimicrobial Peptides.

The increasing number of resistant bacteria is a major threat worldwide, leading to the search for new antibiotic agents. One of the leading strategies is the use of antimicrobial peptides (AMPs), cationic and hydrophobic innate immune defense peptides. A major target of AMPs is the bacterial membrane. Notably, accumulating data suggest that AMPs can activate the two-component systems (TCSs) of Gram-negative bacteria. These include PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB), responsible for remodeling of the bacterial cell surface. To better understand this mechanism, we utilized bacteria deficient either in one system alone or in both and biophysical tools including fluorescence spectroscopy, single-cell atomic force microscopy, electron microscopy, and mass spectrometry (MoskowitzS. M.;Antimicrob. Agents Chemother.2012, 56, 1019−103022106224; ChengH. Y.;J. Biomed. Sci.2010, 17, 6020653976). Our data suggested that the two systems have opposing effects on the properties of Salmonella enterica. The knockout of PhoPQ made the bacteria more susceptible to AMPs by making the surface less rigid, more polarized, and permeable with a slightly more negatively charged cell wall. In addition, the periplasmic space is thinner. In contrast, the knockout of PmrAB did not affect its susceptibility, while it made the bacterial outer layer very rigid, less polarized, and less permeable than the other two mutants, with a negatively charged cell wall similar to the WT. Overall, the data suggest that the coexistence of systems with opposing effects on the biophysical properties of the bacteria contribute to their membrane flexibility, which, on the one hand, is important to accommodate changing environments and, on the other hand, may inhibit the development of meaningful resistance to AMPs.

Choline-based ionic liquids for planktonic and biofilm growth control of Bacillus cereus and Pseudomonas fluorescens

Choline-based ionic liquids (ILs) emerge as promising alternatives for microbial growth control due to their relevant antimicrobial activity, in addition to eco-, bio-friendly and cost-effective properties. Nevertheless, data on choline-based ILs antibiofilm activity is still scarce. In this study, two choline-based ILs: choline alaninate - [Ch] [Ala] and choline glycinate - [Ch] [Gly] were evaluated in terms of antimicrobial and antibiofilm activity against Bacillus cereus and Pseudomonas fluorescens. Two quaternary ammonium compounds (QACs): benzalkonium chloride - BAC and cetyltrimethylammonium chloride – CTAC were used for comparison. [Ch] [Ala] and [Ch] [Gly] demonstrated antibacterial activity against B. cereus and P. fluorescens. However, their activity was lower than that of BAC and CTAC. The mode of action of the selected ILs on planktonic cells was found to be related to the dysfunction and disruption of the bacterial outer layer(s). Moreover, the ILs demonstrated an important action in biofilm control, causing significant removal of biomass (90%) and reduction of culturable population (3 log CFU/cm2). The dual combinations between the ILs and QACs caused additive antibiofilm effects. This study demonstrates the potential of choline-based ILs to be used alone or in combination with QACs, as a sustainable alternative for biofilm control.

Impact of an Engineered Copper-Titanium Dioxide Nanocomposite and Parent Substrates on the Bacteria Viability, Antioxidant Enzymes and Fatty Acid Profiling.

Due to the systematic increase in the production of nanomaterials (NMs) and their applications in many areas of life, issues associated with their toxicity are inevitable. In particular, the performance of heterogeneous NMs, such as nanocomposites (NCs), is unpredictable as they may inherit the properties of their individual components. Therefore, the purpose of this work was to assess the biological activity of newly synthesized Cu/TiO2-NC and the parent nanoparticle substrates Cu-NPs and TiO2-NPs on the bacterial viability, antioxidant potential and fatty acid composition of the reference Escherichia coli and Bacillus subtilis strains. Based on the toxicological parameters, it was found that B. subtilis was more sensitive to NMs than E. coli. Furthermore, Cu/TiO2-NC and Cu-NPs had an opposite effect on both strains, while TiO2-NPs had a comparable mode of action. Simultaneously, the tested strains exhibited varied responses of the antioxidant enzymes after exposure to the NMs, with Cu-NPs having the strongest impact on their activity. The most considerable alternations in the fatty acid profiles were found after the bacteria were exposed to Cu/TiO2-NC and Cu-NPs. Microscopic images indicated distinct interactions of the NMs with the bacterial outer layers, especially in regard to B. subtilis. Cu/TiO2-NC generally proved to have less distinctive antimicrobial properties on B. subtilis than E. coli compared to its parent components. Presumably, the biocidal effects of the tested NMs can be attributed to the induction of oxidative stress, the release of metal ions and specific electrochemical interactions with the bacterial cells.

Nutrition, Inflammation, and Infectious Disease

Pathogens and their related toxins can stimulate the production of endogenous mediators leading to dysregulated reactive oxidative stress, inflammatory responses, and alterations of organ functions. Nutrition support is necessary to protect against infectious and inflammatory conditions, but the optimal nutritional formulation is still under investigation. This special issue focuses on 5 distinct papers to deal with the pathogens and their products (i.e., H. pylori, LPS), specific nutrients (i.e., arginine, iron, plant extracts, antioxidants), and intervention and mechanism of interaction between various biological modifiers (i.e., superfamily of cytokines, inflammatory markers, adhesion molecules) involved in regulating the immune responses. H. pylori and Iron Loss — H. pylori is an important pathogen in the etiology of chronic gastritis and intestinal metaplasia. Studies suggest a link between iron deficiency anemia and H. pylori infection. However, the mechanism of action by which H. pylori causes anemia is yet to be defined. One possible mechanism is that lactoferrin captures the iron from transferrin mediated by the bacterial outer layer receptor to initiate iron loss in the feces. In a pilot clinical trial, Y. Dogan and his collogues seek to determine the lactoferrin levels as a marker of anemia in H. pylori patients by means of immunohistochemical staining. Magnolia to Preserve Bones — Skeletal structure turnover is organized by multifactorial regulators including cytokines and chemokines. In this process, osteoblasts release the osteoclastogenetic molecules such as RANKL and TNF-α and IL-6. Here, Magnolol isolated from Magnolia officinalis is reported to significantly block the osteoclastic differentiation in osteoblast MC3T3-E1 cultures. Therefore, E. J. Kwak et al. propose that Magnolol to preserve osteoblasts and possibly to prevent bone resorption and osteoporosis. Zerumbone and Pancreatitis — D. Wenhong et al. investigate the effects of zerumbone pretreatment on acute necrotizing pancreatitis. They suggest zerumbone to attenuate the severity of pancreatitis as well as pancreatitis-induced hepatic injury through NF-κB activation inhibition and downregulation of the ICAM-1 and IL-1β. Arginine Deiminase and IBD — Arginine, a conditional amino acid, is synthesized in the normal cells and tissues. Arginine deiminase (ADI) is a key enzyme player in the cell signaling pathways, apoptosis, differentiation, and transcriptional regulation. ADI function is dysregulated in multiple human inflammatory diseases and cancer. Since certain tumor cells and pathogens require arginine for growth, selective elimination of arginine may act as a cancer therapy and is being investigated in human trials. While ADI is very immunogenic with a short lifespan, when covalently modified by polyethylene glycol (ADI-PEG), it becomes more stable and less antigenic. H. S. Oz et al. challenge 2 diverse murine models representing Crohn's disease and ulcerative colitis to demonstrate the protective action of this novel microbial enzyme in attenuating inflammatory responses by suppressing macrophage infiltration and iNOS expression. Prediction of Survival in Sepsis — Finally, septic shock and multiple-organ dysfunction syndrome (MODS) are associated with 40–70% mortality rate in the intensive care units. T. Brenner et al. study the dysfunction of the arginine/nitric-oxide pathway in the pathogenesis of MODS and septic shock patients using an ELISA diagnostic technique. This simple technique may serve as a tool to predict the survival specifically in the severe sepsis patients with acute hepatic failure. Helieh S. Oz Sung-Ling Yeh

Modes of adherence of Helicobacter pylori to gastric surface epithelium in gastroduodenal disease: a possible sequence of events leading to internalisation.

We have investigated various modes of adherence of Helicobacter pylori to the human gastric epithelium, using transmission electron microscopy, in biopsies from nine patients with peptic ulcer disease and from four patients with chronic active gastritis. H. pylori was demonstrated in abundance in all cases within the surface mucous layer. In all ulcer- and in one out of four gastritis patients H. pylori was shown in close proximity to the gastric epithelium, with concurrent alterations in the configuration of microvilli and the apical cytoplasmic region of gastric cells. Previously described modes of H. pylori adherence were confirmed, such as loose attachment with fibrillar-like strands, firm attachment with pedestal formation, invasion in the intercellular spaces, and invagination with "cup" formation. Moreover, in many cases a fusion between the bacterial outer layer and gastric cell membranes was evident. In four cases (31%; three with active and one with past ulcer disease) viable H. pylori was found in the cytoplasm of gastric mucous cells. Our results support the hypothesis that the different modes of adherence of H. pylori represent a stepwise, possibly sequential, process which in a significant number of cases leads to internalisation of the organism. The invariable occurrence of adhesion and more frequent internalisation of H. pylori in ulcer patients may suggest a link with the pathogenesis of peptic ulcer disease.

Extracellular and intracellular activities of clarithromycin used alone and in association with ethambutol and rifampin against Mycobacterium avium complex

Mycobacterium avium complex bacteria are opportunistic human pathogens, and their chemotherapy remains a challenge since these organisms are resistant to a majority of routine antituberculous drugs. Recently, a wide range of new macrolide antibiotics has been developed, among which the drug clarithromycin appears to have a selective action against M. avium bacteria. In the present study, we have investigated the action of clarithromycin alone (MIC and MBC determinations) and in association with the routine antimycobacterial drugs ethambutol and rifampin at sublethal concentrations (1 micrograms/ml; below concentrations obtainable in human serum) against M. avium. Our viable count data showed that clarithromycin was bactericidal against all 10 strains of M. avium studied and that its activity was enhanced by ethambutol (in 8 of 9 strains) and rifampin (in 3 of 9 strains). The use of all three drugs in association resulted in higher bactericidal effects than found with any of the drugs used alone or in two-drug combinations in seven of nine strains. The bactericidal effects of various drugs used alone and in combination at concentrations obtainable in human serum were investigated against the type strain ATCC 15769 by using 7H9 broth and BACTEC radiometry (extracellular action) and a J-774 macrophage cell line (intracellular action). A good agreement between the extracellular and intracellular activities was found. Electron microscopy using a ruthenium red cytochemical staining of the bacteria showed that clarithromycin disorganized the outer wall layer and the cytoplasmic membrane in the mycobacterial cell envelope and resulted in formation of large vacuoles inside the cytoplasm, with solubilization of ribosomal structures and consequent plasmolysis. Its association with ethambutol and rifampin resulted in more drastic alterations in the bacterial morphology than were seen with any of the drugs used alone, leading to the removal of the bacterial outer layer, homogenization of cytoplasm, complete cell lysis, and formation of ghosts.

Action of 1-isonicotinyl-2-palmitoyl hydrazine against the Mycobacterium avium complex and enhancement of its activity by m-fluorophenylalanine

In the present work, we investigated whether resistance to isoniazid (INH) of organisms belonging to the Mycobacterium avium complex was caused by the bacterial cell envelope, with the cell wall and the outer layer acting as an exclusion barrier. We observed that this exclusion barrier was most efficient in excluding the hydrophilic drug INH, as this drug could not penetrate a wall matrix formed of various polymethylated lipidic or amphipathic substances. Two main strategies were proposed for circumventing this drug resistance: (i) synthesis of amphipathic derivatives of otherwise highly hydrophilic drugs and (ii) inhibition of synthesis of the bacterial outer layer. The purpose of this work was to demonstrate that attaching a palmitic acid side chain to INH rendered it growth inhibitory against M. avium complex bacteria and that the concomitant use of this amphipathic INH derivative with m-fluorophenylalanine (an inhibitor of mycoside C biosynthesis which causes the disruption of the bacterial outer layer) resulted in further enhancement of its activity, leading to a bactericidal effect.

Characterization of mycoside variants using glycoside residues substituted with acylated chains. Mycolic nature of mycoside G'

The hydrogenolysis of peracetylated mycoside G′ cleaves this mycoside between the aromatic nucleus and the acylated sugars. The mass spectrum of one of these sugars is typical of an ethyl 2‐O‐mycolyl‐3‐O‐methyl‐4‐O‐acetyl‐6‐deoxypyanoside (I): The highes peaks (m/e 1243, 1215 and 1187) are due to splitting on both sides of the methyl branching of the mycolic chain. Other important peaks prove that the sugar cycle is pyranic, that the mycolic chain is bound to the hydroxyl carried by C‐2 and that the methoxyl group is on C‐3. The acyl chain (IV) fragments (m/e 995, 967 and homologues) are typical of a mycolic acid with a methoxyl group (m/e 253, 281, 325) and a C22 side chain, as indicated by the +CH=6;Ċ–C22H45 peak.The structure of mycosides A, B and G comprising polar sugars bound through an aromatic nucleus to an apolar aglycone suggests that these substances might function as a carrier or cofactor in the vicinity of the bacterial membrane or close to the bacterial cell wall. That mycolic acids which esterify hydroxyl groups of mycoside sugars are found to exist favors this conclusion, the more so as mycolic substances are constituants of the cell walls (arabinogalactane mycolate) and of the bacterial outer layer (cord factor).