Interactions of antimicrobial peptide chrysophsin-3 with Bacillus anthracis in sporulated, germinated, and vegetative states.

Bacillus anthracis spores contain on their surface multilayered protein coats that provide barrier properties, mechanical strength, and elasticity that aid in protecting the sporulated state and preventing germination, outgrowth, and transition into the virulent vegetative bacterial state. In this work, the antimicrobial peptide (AMP) chrysophsin-3 was tested against B. anthracis in each of the three distinct metabolic states (sporulated, germinated, and vegetative) for its bacteria-killing activity and its ability to modify the surface nanomechanical properties. Our results provide the first demonstration that chrysophsin-3 killed B. anthracis even in its sporulated state while more killing was observed for germinated and vegetative states. The elasticity of vegetative B. anthracis increased from 12 ± 6 to 84 ± 17 MPa after exposure to 0.22 mM chrysophsin-3. An increase in cellular spring constant was also observed for chrysophsin-3-treated vegetative B. anthracis. Atomic force microscopy images suggested that the changes in mechanical properties of vegetative B. anthracis after chrysophsin-3 treatment are due to loss of water content and cellular material from the cell, possibly caused by the disruption of the cell membrane by the AMP. In contrast, sporulated and germinated B. anthracis retained their innate mechanical properties. Our data indicate that chrysophsin-3 can penetrate the spore coat of B. anthracis spores and kill them without causing any significant mechanical changes on the spore surface. These results reveal a yet unrecognized role for chrysophsin-3 in the killing of B. anthracis spores without the need for complete germination or release of spore coats.

Oral consumption of cranberry juice cocktail inhibits molecular-scale adhesion of clinical uropathogenic Escherichia coli.

Cranberry juice cocktail (CJC) has been shown to inhibit the formation of biofilm by uropathogenic Escherichia coli. In order to investigate whether the anti-adhesive components could reach the urinary tract after oral consumption of CJC, a volunteer was given 16 oz of either water or CJC. Urine samples were collected at 0, 2, 4, 6, and 8 hours after consumption of a single dose. The ability of compounds in the urine to influence bacterial adhesion was tested for six clinical uropathogenic E. coli strains, including four P-fimbriated strains (B37, CFT073, BF1023, and J96) and two strains not expressing P-fimbriae but exhibiting mannose-resistant hemagglutination (B73 and B78). A non-fimbriated strain, HB101, was used as a control. Atomic force microscopy (AFM) was used to measure the adhesion force between a silicon nitride probe and bacteria treated with urine samples. Within 2 hours after CJC consumption, bacteria of the clinical strains treated with the corresponding urine sample demonstrated lower adhesion forces than those treated with urine collected before CJC consumption. The adhesion forces continued decreasing with time after CJC consumption over the 8-hour measurement period. The adhesion forces of bacteria after exposure to urine collected following water consumption did not change. HB101 showed low adhesion forces following both water and CJC consumption, and these did not change over time. The AFM adhesion force measurements were consistent with the results of a hemagglutination assay, confirming that oral consumption of CJC could act against adhesion of uropathogenic E. coli.

Direct adhesion force measurements between E. coli and human uroepithelial cells in cranberry juice cocktail.

SCOPE: Atomic force microscopy (AFM) was used to directly measure the nanoscale adhesion forces between P-fimbriated Escherichia coli (E. coli) and human uroepithelial cells exposed to cranberry juice, in order to reveal the molecular mechanisms by which cranberry juice cocktail (CJC) affects bacterial adhesion. METHODS AND RESULTS: Bacterial cell probes were created by attaching P-fimbriated E. coli HB101pDC1 or non-fimbriated E. coli HB101 to AFM tips, and the cellular probes were used to directly measure the adhesion forces between E. coli and uroepithelial cells in solutions containing: 0, 2.5, 5, 10, and 27 wt% CJC. Macroscale attachment of E. coli to uroepithelial cells was measured and correlated to nanoscale adhesion force measurements. The adhesion forces between E. coli HB101pDC1 and uroepithelial cells were dose-dependent, and decreased from 9.32±2.37 nN in the absence of CJC to 0.75±0.19 nN in 27 wt% CJC. Adhesion forces between E. coli HB101 and uroepithelial cells were low in buffer (0.74±0.18 nN), and did not change significantly in CJC (0.78±0.18 nN in 27 wt% CJC; P=0.794). CONCLUSION: Our study shows that CJC significantly decreases nanoscale adhesion forces between P-fimbriated E. coli and uroepithelial cells.

Effects of L-alanine and inosine germinants on the elasticity of Bacillus anthracis spores.

The surface of dormant Bacillus anthracis spores consists of a multilayer of protein coats and a thick peptidoglycan layer that allow the cells to resist chemical and environmental insults. During germination, the spore coat is degraded, making the spore susceptible to chemical inactivation by antisporal agents as well as to mechanical inactivation by high-pressure or mechanical abrasion processes. While chemical changes during germination, especially the release of the germination marker, dipicolinic acid (DPA), have been extensively studied, there is as yet no investigation of the corresponding changes in the mechanical properties of the spore. In this work, we use atomic force microscopy (AFM) to characterize the mechanical properties of the surface of Bacillus anthracis spores during germination. The Hertz model of continuum mechanics of contact was used to evaluate the Young's moduli of the spores before and after germination by applying the model to load-indentation curves. The highest modulus was observed for dormant spores, with average elasticity values of 197 +/- 81 MPa. The elasticity decreased significantly after incubation of the spores with the germinants L-alanine or inosine (47.5 +/- 41.7 and 35.4 +/- 15.8 MPa, respectively). Exposure of B. anthracis spores to a mixture of both germinants resulted in a synergistic effect with even lower elasticity, with a Young's modulus of 23.5 +/- 14.8 MPa. The elasticity of the vegetative B. anthracis cells was nearly 15 times lower than that of the dormant spores (12.4 +/- 6.3 MPa vs 197.0 +/- 80.5 MPa, respectively). Indeed from a mechanical strength point of view, the germinated spores were closer to the vegetative cells than to the dormant spores. Further, the decrease in the elasticity of the cells was accompanied by increasing AFM tip indentation depths on the cell surfaces. Indentation depths of up to 246.2 nm were observed for vegetative B. anthracis compared to 20.5 nm for the dormant spores. These results provide quantitative information on how the mechanical properties of the cell wall change during germination, which may explain how spores become susceptible to inactivation processes based on mechanical forces during germination and outgrowth. The study of spore elasticity may be a valuable tool in the design of improved antisporal treatments.

Atomic force microscopy study of germination and killing of Bacillus atrophaeus spores.

Bacterial spores such as Bacillus atrophaeus are one of the most resistant life forms known and are extremely resistant to chemical and environmental factors in the dormant state. During germination, as bacterial spores progress towards the vegetative state, they become susceptible to anti-sporal agents. B. atrophaeus spores were exposed to the non-nutritive germinant dodecylamine (DDA), a cationic surfactant that can also be used as a killing agent, for up to 60 min, or to the nutrient germinant L-alanine. In kinetic studies, 99% of the spores were killed within 5 min of exposure to DDA. Atomic force microscopy (AFM) can be used as a sensitive tool to assess how the structure of the spore coat changes upon exposure to germinants or killing agents. Changes in cell height and roughness over time of exposure to DDA were examined using AFM. DDA caused the spore height to decrease by >50%, which may have been due to a partial breakdown of the spore coat. Treatment of B. atrophaeus with the nutrient germinant resulted in a decrease in height of spores after 2 h of incubation, from 0.7 +/- 0.1 microm to 0.3 +/- 0.2 microm. However, treatment with L-alanine did not change the surface roughness of the spores, indicating that the changes that occur during germination take place underneath the spore coat. We propose that exposure to DDA at high concentrations causes pores to form in the coat layer, killing B. atrophaeus without the need to fully germinate spores.

Role of cranberry on bacterial adhesion forces and implications for Escherichia coli-uroepithelial cell attachment.

Previous clinical research has suggested that the consumption of cranberry products prevents the adhesion of Escherichia coli to uroepithelial cells by causing changes in bacterial fimbriae. Atomic force microscopy was used to probe the adhesion forces between E. coli (nonfimbriated strain HB101 and the P-fimbriated variant HB101pDC1) and a model surface (silicon nitride), to determine the effect of growth in cranberry products on bacterial adhesion. Bacteria were grown in tryptic soy broth supplemented with either light cranberry juice cocktail (L-CJC) or cranberry proanthocyanidins (PACs). Growth of E. coli HB101pDC1 and HB101 in L-CJC or PACs resulted in a decrease in adhesion forces with increasing number of cultures. In a macroscale bacteria-uroepithelial cell adhesion assay a decrease in bacterial attachment was observed for E. coli HB101pDC1 grown in L-CJC or PACs. This effect was reversible because bacteria that were regrown in cranberry-free medium regained their ability to attach to uroepithelial cells, and their adhesion forces reverted to the values observed in the control condition. Exposure to increasing concentrations of L-CJC resulted in a decrease of bacterial attachment to uroepithelial cells for the P-fimbriated strain after L-CJC treatment (27% by weight) and after PACs treatment (345.8 microg/mL). Cranberry products affect the surface properties, such as fimbriae and lipopolysaccharides, and adhesion of fimbriated and nonfimbriated E. coli. The concentration of cranberry products and the number of cultures the bacteria were exposed to cranberry determines how much the adhesion forces and attachment are altered.

Importance of LPS structure on protein interactions with Pseudomonas aeruginosa.

Atomic force microscopy (AFM) was used to quantify the adhesion forces between Pseudomonas aeruginosa PAO1 and AK1401, and a representative model protein, bovine serum albumin (BSA). The two bacteria strains differ in terms of the structure of their lipopolysaccharide (LPS) layers. While PAO1 is the wild-type expressing a complete LPS and two types of saccharide units in the O-antigen (A(+) B(+)), the mutant AK1401 expresses only a single unit of the A-band saccharide (A(+) B(-)). The mean adhesion force (F(adh)) between BSA and AK1401 was 1.12 nN, compared to 0.40 nN for F(adh) between BSA and PAO1. In order to better understand the fundamental forces that would control bacterial-protein interactions at equilibrium conditions, we calculated interfacial free energies using the van Oss-Chaudhury-Good (VCG) thermodynamic modeling approach. The hydrogen bond strength was also calculated using a Poisson statistical analysis. AK1401 has a higher ability to participate in hydrogen bonding with BSA than does PAO1, which may be because the short A-band and absence of B-band polymer allowed the core oligosaccharides and lipid A regions to be more exposed and to participate in hydrogen and chemical bonding. Interactions between PAO1 and BSA were weak due to the dominance of neutral and hydrophilic sugars of the A-band polymer. These results show that bacterial interactions with protein-coated surfaces will depend on the types of bonds that can form between bacterial surface macromolecules and the protein. We suggest that strategies to prevent bacterial colonization of biomaterials can focus on inhibiting these bonds.

Cranberry changes the physicochemical surface properties of E. coli and adhesion with uroepithelial cells.

Cranberries have been suggested to decrease the attachment of bacteria to uroepithelial cells (UC), thus preventing urinary tract infections, although the mechanisms are not well understood. A thermodynamic approach was used to calculate the Gibbs free energy of adhesion changes (DeltaG(adh)) for bacteria-UC interactions, based on measuring contact angles with three probe liquids. Interfacial tensions and DeltaG(adh) values were calculated for Escherichia coli HB101pDC1 (P-fimbriated) and HB101 (non-fimbriated) exposed to cranberry juice (0-27 wt.%). HB101pDC1 can form strong bonds with the Gal-Gal disaccharide receptor on uroepithelial cells, while HB101-UC interactions are only non-specific. For HB101 interacting with UC, DeltaG(adh) was always negative, suggesting favorable adhesion, and the values were insensitive to cranberry juice concentration. For the HB101pDC1-UC system, DeltaG(adh) became positive at 27wt.% cranberry juice, suggesting that adhesion was unfavorable. Acid-base (AB) interactions dominated the interfacial tensions, compared to Lifshitz-van der Waals (LW) interactions. Exposure to cranberry juice increased the AB component of the interfacial tension of HB101pDC1. LW interactions were small and insensitive to cranberry juice concentration. The number of bacteria attached to UC was quantified in batch adhesion assays and quantitatively correlated with DeltaG(adh). Since the thermodynamic approach should not agree with the experimental results when specific interactions are present, such as HB101pDC-UC ligand-receptor bonds, our results may suggest that cranberry juice disrupts bacterial ligand-UC receptor binding. These results help form the mechanistic explanation of how cranberry products can be used to prevent bacterial attachment to host tissue, and may lead to the development of better therapies based on natural products.