Chimeric bacteriocin S5-PmnH engineered by domain swapping efficiently controls Pseudomonas aeruginosa infection in murine keratitis and lung models.

Rampant rise of multidrug resistant strains among Gram-negative bacteria has necessitated investigation of alternative antimicrobial agents with novel modes of action including antimicrobial proteins such as bacteriocins. The main hurdle in the clinical development of bacteriocin biologics is their narrow specificity and limited strain activity spectrum. Genome mining of bacteria for broadly active bacteriocins have identified a number of promising candidates but attempts to improve these natural multidomain proteins further, for example by combining domains of different origin, have so far met with limited success. We have found that domain swapping of Pseudomonas bacteriocins of porin type, when carried out between phylogenetically related molecules with similar mechanism of activity, allows the generation of highly active molecules with broader spectrum of activity, for example by abolishing strain resistance due to the presence of immunity proteins. The most broadly active chimera engineered in this study, S5-PmnH, exhibits excellent control of Pseudomonas aeruginosa infection in validated murine keratitis and lung infection models.

Reduction of gastrointestinal tract colonization by Klebsiella quasipneumoniae using antimicrobial protein KvarIa.

BACKGROUND: Klebsiella quasipneumoniae is an opportunistic pathogen causing antibiotic-resistant infections of the gastrointestinal tract in many clinical cases. Orally delivered bioactive Klebsiella-specific antimicrobial proteins, klebicins, could be a promising method to eradicate Klebsiella species infecting the gut. METHODS: Mouse infection model was established based on infection of antibiotic-treated BALB/C mice with K. quasipneumoniae strain DSM28212. Four study groups were used (3 animals/group) to test the antimicrobial efficacy of orally delivered klebicin KvarIa: vehicle-only group (control, phosphate-buffered saline), and other three groups with bacteria, antibiotic therapy and 100 µg of uncoated Kvarla, 100 µg coated KvarIa, 1000 µg coated-KvarIa. Because of the general sensitivity of bacteriocins to gastroduodenal proteases, Kvarla doses were coated with Eudragit®, a GMP-certified formulation agent that releases the protein at certain pH. The coating treatment was selected based on measurements of mouse GI tract pH. The quantity of Klebsiella haemolysin gene (khe) in faecal samples of the study animals was used to quantify the presence of Klebsiella. RESULTS: GI colonization of K. quasipneumoniae was achieved only in the antibiotic-treated mice groups. Significant changes in khe marker quantification were found after the use of Eudragit® S100 formulated klebicin KvarIa, at both doses, with a significant reduction of K. quasipneumoniae colonization compared to the vehicle-only control group. CONCLUSIONS: Mouse GI tract colonization with K. quasipneumoniae can be achieved if natural gut microbiota is suppressed by prior antibiotic treatment. The study demonstrates that GI infection caused by K. quasipneumoniae can be significantly reduced using Eudragit®-protected klebicin KvarIa.

Broad and Efficient Control of Klebsiella Pathogens by Peptidoglycan-Degrading and Pore-Forming Bacteriocins Klebicins.

Gram-negative bacteria belonging to the genus Klebsiella are important nosocomial pathogens, readily acquiring resistance to all known antibiotics. Bacteriocins, non-antibiotic antibacterial proteins, have been earlier proposed as potential therapeutic agents for control of other Gram-negative species such as Escherichia, Pseudomonas and Salmonella. This study is the first report describing pore-forming and peptidoglycan-degrading bacteriocins klebicins from Klebsiella. We have identified, cloned, expressed in plants and characterized nine pore-forming and peptidoglycan-degrading bacteriocins from different Klebsiella species. We demonstrate that klebicins can be used for broad and efficient control of 101 of the 107 clinical isolates representing five Klebsiella species, including multi-drug resistant pathovars and pathovars resistant to carbapenem antibiotics.

Plant-expressed bacteriophage lysins control pathogenic strains of Clostridium perfringens.

The anaerobic spore-forming bacterium Clostridium perfringens is a source of one of the most common food-borne illnesses in the United States and Europe. The costs associated with disease management are high and interventions are limited; therefore, effective and safe antimicrobials are needed to control food contamination by C. perfringens. A viable solution to this problem could be bacteriophage lysins used as food additives or food processing aids. Such antimicrobials could be produced cost-effectively and in ample supply in green plants. By using edible plant species as production hosts the need for expensive product purification can be reduced or obviated. We describe the first successful expression in plants of C. perfringens-specific bacteriophage lysins. We demonstrate that six lysins belonging to two different families (N-acetylmuramoyl-L-alanine amidase and glycosyl hydrolase 25) are active against a panel of enteropathogenic C. perfringens strains under salinity and acidity conditions relevant to food preparation environments. We also demonstrate that plant-expressed lysins prevent multiplication of C. perfringens on cooked meat matrices far better than nisin, the only currently approved bacteriocin food preservative to control this pathogen.

Plant-expressed pyocins for control of Pseudomonas aeruginosa.

The emergence, persistence and spread of antibiotic-resistant human pathogenic bacteria heralds a growing global health crisis. Drug-resistant strains of gram-negative bacteria, such as Pseudomonas aeruginosa, are especially dangerous and the medical and economic burden they impose underscore the critical need for finding new antimicrobials. Recent studies have demonstrated that plant-expressed bacteriocins of the colicins family can be efficient antibacterials against all major enteropathogenic strains of E. coli. We extended our studies of colicin-like bacteriocins to pyocins, which are produced by strains of P. aeruginosa for ecological advantage against other strains of the same species. Using a plant-based transient expression system, we expressed six different pyocins, namely S5, PaeM, L1, L2, L3 and one new pyocin, PaeM4, and purified them to homogeneity. Among these pyocins, PaeM4 demonstrated the broadest spectrum of activity by controlling 53 of 100 tested clinical isolates of P. aeruginosa. The activity of plant-made pyocins was confirmed in the agar drop, liquid culture susceptibility and biofilm assays, and in the Galleria mellonella animal infection model.

Hydration of lysozyme studied by Raman spectroscopy.

Hydration plays a fundamental role in maintaining the three-dimensional structure and function of proteins. In this study, Raman spectroscopy was used to probe the hydration induced structural changes at various sites of lysozyme under isothermal conditions in the range of water contents from 0 to 44 wt %. Raman hydration curves were constructed from detailed analysis of marker bands. Transition inflection points (w(m)) and onsets determined from the hydration curves have shown that structural changes start at 7-10 and end at about 35 wt % water. The onset of structural changes coincides with the onset of the broad glass transition earlier observed in this system. The increase of α-helix content starts at very low concentrations of water with w(m) = 12 wt %. Monitoring the development of importance for enzymatic action hydrophobic clusters has revealed wm = 15 wt % and completion of the process at 25 wt %. The parameters of 621 cm(-1) (Phe) and 1448 cm(-1) (CH2 bending) modes were found to be sensitive to hydration, suggesting changes in organization of water molecules near the protein surface. The native structure of lysozyme was achieved at 35 wt % water where its content is high enough for filling the space between lysozyme molecules.

Horse heart cytochrome c entrapped into the hydrated liquid-crystalline phases of phytantriol: X-ray diffraction and Raman spectroscopic characterization.

Small angle X-ray diffraction (SAXD), resonance Raman (RR) spectroscopy with 413 nm excitation, and non-resonance Raman technique with 785 nm excitation were used to probe the influence of entrapped cytochrome c (Cyt c) on the structure of hydrated phytantriol (Phyt) liquid-crystalline phases as well as conformational changes of heme group and secondary structure of the protein. SAXD measurements indicated that incorporation of Cyt c affects both nanostructure dimensions and type of liquid-crystalline phases of hydrated Phyt. The unit cell dimensions decrease with increasing Cyt c concentration for all phases. In addition, protein perturbs the nanostructure of Q(230) and Q(224) liquid-crystalline phases of hydrated Phyt to such an extent that they transform into the Q(229) phase with the Im3m space group. RR data revealed that entrapment of oxidized Cyt c into the Q(230) phase at 1 wt.% content results in near complete reduction of central iron ion of the heme group, while its low-spin state and six-ligand coordination configuration are preserved. Based on the analysis of heme out-of-plane folding vibration near 568 cm(-1) (γ(21)) and ν(48) mode at 633 cm(-1), it was demonstrated that the protein matrix tension on the heme group is relaxed upon incorporation of protein into Q(230) phase. Non-resonant Raman bands of difference spectra showed the preservation of α-helix secondary structure of Cyt c in the liquid-crystalline phase at relatively high (5 wt.%) content. The Cyt c induced spectroscopic changes of Phyt bands were found to be similar as decrease in temperature.

Thermomyces lanuginosus lipase in the liquid-crystalline phases of aqueous phytantriol: X-ray diffraction and vibrational spectroscopic studies.

The influence of Thermomyces lanuginosus lipase (TLL) on the phase behaviour of liquid-crystalline phases of aqueous phytantriol as well as conformational changes of TLL entrapped in the cubic Q230 phase have been studied by small angle X-ray diffraction (SAXD), FT-Raman, and FT-IR techniques. It was found that the lipidic Q230 phase is able to accommodate up to 10 wt.% of TLL, and the temperature of phase transition to the inverted hexagonal phase H(II) increases indicating stabilizing effect of the protein. FT-Raman analysis of Trp amino acid marker band W3 revealed that the average rotation angle around the C3-Cbeta bond of four Trp residues of TLL in the Q230 phase increases. Reasoning from available TLL crystallographic data, this result is explained by structural transition of entrapped protein to so-called "open" and more related to the enzymatically-active conformation. TLL secondary structure analysis by amide I and amide III vibrational bands showed that content of alpha-helixes does not change, while a part of beta-sheet structures transforms to less ordered elements upon incorporation of protein into the Q230 phase of aqueous phytantriol.

"Sponge" nanoparticle dispersions in aqueous mixtures of diglycerol monooleate, glycerol dioleate, and polysorbate 80.

Lipid nanoparticles of nonlamellar lyotropic phases have a wide solubilizing and encapsulating spectrum for a range of substances thanks to their nanostructured interior featuring both lipophilic and hydrophilic domains. As a consequence, these systems have emerged as promising drug delivery systems in various pharmaceutical and diagnostic applications. Here we present the phase behavior and dispersion properties of a novel three-component lipid system composed of diglycerol monooleate (DGMO), glycerol dioleate (GDO), and polysorbate 80 (P80) which shows several advantageous features relating to drug delivery applications including: spontaneous dispersion formation with a narrow size distribution and tunable particle phase-structure. The obtained phase diagram shows the presence of lamellar (L(alpha)), hexagonal (H(2)), and reverse bicontinuous cubic (V(2)) liquid crystalline phases and an inverse micellar (L(2)) solution. A particularly interesting observation is the presence of a phase region where two liquid phases coexist, most likely the L(2) and L(3) ("sponge phase"). These two phase structures appear also to coexist in the submicron particles formed in the dilute water region, where the L(3) element appears to stabilize nanoparticles with inner L(2) structure. Increasing the fraction of the dispersing P80 component results in the growth of the more water rich L(3) "surface phase" at the expense of the size of the inner L(2) core.

Infrared and Raman bands of phytantriol as markers of hydrogen bonding and interchain interaction.

Hydrogen bonding and interchain interactions in phytantriol, 3,7,11,15-tetramethyl-1,2,3-hexadecanetriol, have been studied by Fourier transform infrared (FT-IR) and Raman spectroscopies. Assignments of the bands were performed based on the OH/OD isotopic substitution, molecular modeling, and measurements of polarized Raman spectra. Marker bands were evaluated from the temperature-dependent spectral changes. It is shown that Raman spectroscopy provides sensitive markers, namely I(delta(CH2))/I(deltas(CH3)), tau(CH)2, I(nus(CH3)(FR))/I(nus(CH2)), and nus(CH2) for probing the interactions between the hydrocarbon chains. Hydrogen bonding interaction might be studied through the difference Raman spectroscopy by the analysis of polarized band at 811 cm-1. Relationship is found between the frequencies of IR bands at 883-873 and 1097-1086 cm-1, associated with the vibrations localized at the primary COH site, and the frequencies of OH stretching mode, making these bands specific markers in the analysis of hydrogen bonding. Evaluated marker bands may be of utility to probe the interchain and hydrogen bonding interaction of phytantriol with guest molecules in the practically important aqueous liquid-crystalline phases of this lipid.