Charles V. Rice

Revisiting magnesium chelation by teichoic acid with phosphorus solid-state NMR and theoretical calculations.

Teichoic acids are essential components of the Gram-positive bacterial cell wall. One of their many functions is metal binding, a vital process for bacterial growth. With the combination of phosphorus-31 solid-state NMR spectroscopy and theoretical calculations using density functional theory (DFT), we have determined that the binding mode between teichoic acids and magnesium involves bidentate coordination by the phosphate groups of teichoic acid. Measurement of chemical shift anisotropy tensors gave a reduced anisotropy (delta) of 49.25 ppm and an asymmetry (eta) of 0.7. DFT calculations with diglycerol phosphate and triglycerol diphosphate model compounds were completed with Mg(2+) in anhydrous as well as partially hydrated bidentate and fully hydrated monodentate, bidentate, and bridging binding modes. (31)P CSA tensors were calculated from the energy-minimized model compounds using the combined DFT and GIAO methods, resulting in dramatically different tensor values for each binding mode. The anhydrous bidentate chelation mode was found to be a good approximation of the experimental data, an observation that alters the current monodentate paradigm for metal chelation by teichoic acids.

Conformation of the Phosphate d-Alanine Zwitterion in Bacterial Teichoic Acid from Nuclear Magnetic Resonance Spectroscopy

The conformation of d-alanine (d-Ala) groups of bacterial teichoic acid is a central, yet untested, paradigm of microbiology. The d-Ala binds via the C-terminus, thereby allowing the amine to exist as a free cationic NH(3)(+) group with the ability to form a contact ion pair with the nearby anionic phosphate group. This conformation hinders metal chelation by the phosphate because the zwitterion pair is charge neutral. To the contrary, the repulsion of cationic antimicrobial peptides (CAMPs) is attributed to the presence of the d-Ala cation; thus the ion pair does not form in this model. Solid-state nuclear magnetic resonance (NMR) spectroscopy has been used to measure the distance between amine and phosphate groups within cell wall fragments of Bacillus subtilis. The bacteria were grown on media containing (15)N d-Ala and beta-chloroalanine racemase inhibitor. The rotational-echo double-resonance (REDOR) pulse sequence was used to measure the internuclear dipolar coupling, and the results demonstrate (1) the metal-free amine-to-phosphate distance is 4.4 A and (2) the amine-to-phosphate distance increases to 5.4 A in the presence of Mg(2+) ions. As a result, the zwitterion exists in a nitrogen-oxygen ion pair configuration providing teichoic acid with a positive charge to repel CAMPs. Additionally, the amine of d-Ala does not prevent magnesium chelation in contradiction to the prevailing view of teichoic acids in metal binding. Thus, the NMR-based description of teichoic acid structure resolves the contradictory models, advances the basic understanding of cell wall biochemistry, and provides possible insight into the creation of new antibiotic therapies.

Water Behavior in Bacterial Spores by Deuterium NMR Spectroscopy

Dormant bacterial spores are able to survive long periods of time without nutrients, withstand harsh environmental conditions, and germinate into metabolically active bacteria when conditions are favorable. Numerous factors influence this hardiness, including the spore structure and the presence of compounds to protect DNA from damage. It is known that the water content of the spore core plays a role in resistance to degradation, but the exact state of water inside the core is a subject of discussion. Two main theories present themselves: either the water in the spore core is mostly immobile and the core and its components are in a glassy state, or the core is a gel with mobile water around components which themselves have limited mobility. Using deuterium solid-state NMR experiments, we examine the nature of the water in the spore core. Our data show the presence of unbound water, bound water, and deuterated biomolecules that also contain labile deuterons. Deuterium–hydrogen exchange experiments show that most of these deuterons are inaccessible by external water. We believe that these unreachable deuterons are in a chemical bonding state that prevents exchange. Variable-temperature NMR results suggest that the spore core is more rigid than would be expected for a gel-like state. However, our rigid core interpretation may only apply to dried spores whereas a gel core may exist in aqueous suspension. Nonetheless, the gel core, if present, is inaccessible to external water.

Sterilization of hydrogen peroxide resistant bacterial spores with stabilized chlorine dioxide

Bacillus pumilus SAFR-032 spores isolated from a clean room environment are known to exhibit enhanced resistance to peroxide, desiccation, UV radiation and chemical disinfection than other spore-forming bacteria. The survival of B. pumilus SAFR-032 spores to standard clean room sterilization practices requires development of more stringent disinfection agents. Here, we report the effects of a stabilized chlorine dioxide-based biocidal agent against spores of B. pumilus SAFR-032 and Bacillus subtilis ATCC 6051. Viability was determined via CFU measurement after exposure. Chlorine dioxide demonstrated efficacy towards sterilization of spores of B. pumilus SAFR-032 equivalent or better than exposure to hydrogen peroxide. These results indicate efficacy of chlorine dioxide delivered through a stabilized chlorine dioxide product as a means of sterilization of peroxide- and UV-resistant spores.

Sterilization Resistance of Bacterial Spores Explained with Water Chemistry.

Bacterial spores can survive for long periods without nutrients and in harsh environmental conditions. This survival is influenced by the structure of the spore, the presence of protective compounds, and water retention. These compounds, and the physical state of water in particular, allow some species of bacterial spores to survive sterilization schemes with hydrogen peroxide and UV light. The chemical nature of the spore core and its water has been a subject of some contention and the chemical environment of the water impacts resistance paradigms. Either the spore has a glassy core, where water is immobilized along with other core components, or the core is gel-like with mobile water diffusion. These properties affect the movement of peroxide and radical species, and hence resistance. Deuterium solid-state NMR experiments are useful for examining the nature of the water inside the spore. Previous work in our lab with spores of Bacillus subtilis indicate that, for spores, the core water is in a more immobilized state than expected for the gel-like core theory, suggesting a glassy core environment. Here, we report deuterium solid-state NMR observations of the water within UV- and peroxide-resistant spores from Bacillus pumilus SAFR-032. Variable-temperature NMR experiments indicate no change in the line shape after heating to 50 °C, but an overall decrease in signal after heating to 100 °C. These results show glass-like core dynamics within B. pumilus SAFR-032 that may be the potential source of its known UV-resistance properties. The observed NMR traits can be attributed to the presence of an exosporium containing additional labile deuterons that can aid in the deactivation of sterilizing agents.

Efficacy of Ampicillin Against Methicillin-Resistant Staphylococcus aureus Restored Through Synergy with Branched Poly(ethylenimine)

β-Lactam antibiotics kill Staphylococcus aureus bacteria by inhibiting the function of cell wall penicillin-binding proteins (PBPs) 1 and 3. However, β-lactams are ineffective against PBP2a, used by methicillin-resistant S. aureus (MRSA) to perform essential cell wall crosslinking functions. PBP2a requires teichoic acid to properly locate and orient the enzyme, and thus MRSA is susceptible to antibiotics that prevent teichoic acid synthesis in the bacterial cytoplasm. As an alternative, we have used branched poly(ethylenimine), BPEI, to target teichoic acid in the bacterial cell wall. The result is restoration of MRSA susceptibility to the β-lactam antibiotic ampicillin with a MIC of 1 μg ml−1, superior to that of vancomycin (MIC=3.7 μg ml−1). A checkerboard assay shows synergy of BPEI and ampicillin. NMR data show that BPEI alters the teichoic acid chemical environment. Laser scanning confocal microscopy images show BPEI residing on the bacterial cell wall, where teichoic acids and PBPs are located.

Cadmium Chelation by Bacterial Teichoic Acid from Solid-State Nuclear Magnetic Resonance Spectroscopy

An effective means of studying biological metal chemistry is through the use of cadmium NMR to probe the interaction between biomolecules, such as proteins and peptides, with divalent metals, such as zinc, copper, magnesium, or calcium. Gram-positive bacteria, such as S. aureus and B. subtilis , have peptidoglycan cell walls that contain teichoic acids, a poly(phosphodiester) biopolymer used for, among other things, metal chelation. Previous solid-state NMR and XAFS studies have shown that the cadmium ion binds in a bidentate manner to the phosphoryl centers of the dried teichoic acid backbone at physiological pH. However, current studies indicate that, when hydrated and at the low concentrations typically found in nature, the cadmium ions and phosphoryl sites interact through an extended solvent-separated ion pairing. These data reveal two unequal P-Cd interactions at distances of 4.2 and 4.9 A set approximately 180 degrees from each other in a linear arrangement.

Solid-state NMR studies of bacterial lipoteichoic acid adsorption on different surfaces

Teichoic acids are important to bacteria for surface adhesion, metal ion coordination, and other biological processes crucial to bacterial survival. In particular, the surface adhesion of teichoic acids plays a crucial role in the formation of Gram-positive biofilms. Biofilms have been implicated as the major cause of various chronic infections. Biofilm formation is essentially a four-step process beginning with the adhesion of bacteria to a surface, followed by the excretion of an extracellular polymeric substance (slime), development and maturation of the biofilm architecture, and finally biofilm spreading through bacterial release. Currently, there is very little molecular level information available for the initial adhesion of bacteria to solid surfaces. Solid-state NMR is ideally suited for the study of these samples, thus we use (31)P solid-state NMR experiments to study the initial adhesion of lipoteichoic acid (LTA) to various surfaces. (31)P CP-MAS spectra and T(1)(rho) data demonstrate that the structure of LTA changes when adhered to cellulose, cell wall peptidoglycan (PGN), or TiO(2). However, when LTA is simultaneously adhered to PGN and TiO(2) the observed structure is dependent on the amount of retained water. For LTA on TiO(2), we suggest that the alanine and glucosamine groups interact with the surface. However, during simultaneous adhesion to TiO(2) and PGN, the glucosamine groups bind to the PGN while the alanine groups bind to the surface. This arrangement traps water between the PGN and TiO(2) surface.

Equilibrium binding behavior of magnesium to wall teichoic acid

Peptidoglycan and teichoic acids are the major cell wall components of Gram-positive bacteria that obtain and sequester metal ions required for biochemical processes. The delivery of metals to the cytoplasmic membrane is aided by anionic binding sites within the peptidoglycan and along the phosphodiester polymer of teichoic acid. The interaction with metals is a delicate balance between the need for attraction and ion diffusion to the membrane. Likewise, metal chelation from the extracellular fluid must initially have strong binding energetics that weaken within the cell wall to enable ion release. We employed atomic absorption and equilibrium dialysis to measure the metal binding capacity and metal binding affinity of wall teichoic acid and Mg2+. Data show that Mg2+ binds to WTA with a 1:2Mg2+ to phosphate ratio with a binding capacity of 1.27 μmol/mg. The affinity of Mg2+ to WTA was also found to be 41×10(3) M(-1) at low metal concentrations and 1.3×10(3) M(-1) at higher Mg2+ concentrations due to weakening electrostatic effects. These values are lower than the values describing Mg2+ interactions with peptidoglycan. However, the binding capacity of WTA is 4 times larger than peptidoglycan. External WTA initially binds metals with positive cooperativity, but metal binding switches to negative cooperativity, whereas interior WTA binds metals with only negative cooperativity. The relevance of this work is to describe changes in metal binding behavior depending on environment. When metals are sparse, chelation is strong to ensure survival yet the binding weakens when essential minerals are abundant.

Targeting Wall Teichoic Acid in Situ with Branched Polyethylenimine Potentiates β-Lactam Efficacy against MRSA

Methicillin-resistant Staphylococcus aureus (MRSA) is a medical concern. Here, we show that branched polyethylenimine (BPEI), a nontoxic, cationic polymer, restores MRSAs susceptibility to β-lactam antibiotics. Checkerboard assays with MRSA demonstrated synergy between BPEI and β-lactam antibiotics. A time-killing curve showed BPEI to be bactericidal in combination with oxacillin. BPEI did not potentiate efficacy with vancomycin, chloramphenicol, or linezolid. When exposed to BPEI, MRSA increased in size and had difficulty forming septa. BPEI electrostatically binds to wall teichoic acid (WTA), a cell wall anionic polymer of Gram-positive bacteria that is important for localization of certain cell wall proteins. Lack of potentiation in a WTA knockout mutant supports the WTA-based mechanism. These data suggest that BPEI may prevent proper localization of cell wall machinery by binding to WTA; leading to cell death when administered in combination with β-lactam antibiotics. Negligible in vitro toxicity suggests the combination could be a viable treatment option.