Lipoxin A4 promotes antibiotic and monocyte bacterial killing in established Pseudomonas aeruginosa biofilm formed under hydrodynamic conditions.

Pseudomonas aeruginosa is a gram-negative, opportunistic bacteria commonly found in wounds and in lungs of immunocompromised patients. These bacteria commonly form biofilms which encapsulate the bacteria, making it difficult for antibiotics or immune cells to reach the bacterial cells. We previously reported that Lipoxin A4 (LxA4 ), a Specialized Pro-resolving Mediator, has direct effects on P. aeruginosa where it reduced biofilm formation and promoted ciprofloxacin antibiotic efficacy in a static biofilm-forming system. In the current studies, we examined the actions of LxA4 on established biofilms formed in a biofilm reactor under dynamic conditions with constant flow and shear stress. These conditions allow for biofilm growth with nutrient replenishment and for examination of bacteria within the biofilm structure. We show that LxA4 helped ciprofloxacin reduction of live/dead ratio of bacteria within the biofilm. THP-1 monocytes interacted with the biofilm to increase the number of viable bacteria within the biofilm as well as TNF-α production in the biofilm milieu, suggesting that monocyte interaction with bacterial biofilm exacerbates the inflammatory state. Pre-treatment of the THP-1 monocytes with LxA4 abolished the increase in biofilm bacteria and reduced TNF-α production. The effect of decreased biofilm bacteria was associated with increased LxA4 -induced monocyte adherence to biofilm but not increased bacteria killing suggesting that the mechanism for the reduced biofilm bacteria was due to LxA4 -mediated increase in adherence to biofilm. These results suggest that LxA4 can help antibiotic efficacy and promote monocyte activity against established P. aeruginosa biofilm formed under hydrodynamic conditions.

Computational analysis of drug resistance of taxanes bound to human ß-tubulin mutant (D26E).

The single-point mutation D26E in human ß-tubulin is associated with drug resistance seen with two anti-mitotic taxanes (paclitaxel and docetaxel) when used to treat cancers. The molecular mechanism of this resistance remains elusive. However, docetaxel and a third-generation taxane, cabazitaxel, are thought to overcome this resistance. Here, structural models of both the wildtype (WT) and D26E mutant (MT) human ß-tubulin were constructed based on the crystal structure of pig ß-tubulin in complex with docetaxel (PDB ID: 1TUB). The three taxanes were docked into the WT and MT ß-tubulin, and the resulting complexes were submitted to three independent runs of 200 ns molecular dynamic simulations, which were then averaged. MM/GBSA calculations revealed the binding energy of paclitaxel with WT and MT ß-Tubulin to be -101.5 ± 8.4 and -90.4 ± 8.9 kcal/mol, respectively. The binding energy of docetaxel was estimated to be -104.7 ± 7.0 kcal/mol with the WT and -103.8 ± 5.5 kcal/mol with the MT ß-tubulin. Interestingly, cabazitaxel was found to have a binding energy of -122.8 ± 10.8 kcal/mol against the WT and -106.2 ± 7.0 kcal/mol against the MT ß-tubulin. These results show that paclitaxel and docetaxel bound to the MT less strongly than the WT, suggesting possible drug resistance. Similarly, cabazitaxel displayed a greater binding propensity against WT and MT ß-tubulin than the other two taxanes. Furthermore, the dynamic cross-correlation matrices (DCCM) analysis suggests that the single-point mutation D26E induces a subtle dynamical difference in the ligand-binding domain. Overall, the present study revealed how the single-point mutation D26E may reduce the binding affinity of the taxanes, however, the effect of the mutation does not significantly affect the binding of cabazitaxel.

Role of Specialized Pro-Resolving Mediators in Modifying Host Defense and Decreasing Bacterial Virulence.

Bacterial infection activates the innate immune system as part of the host's defense against invading pathogens. Host response to bacterial pathogens includes leukocyte activation, inflammatory mediator release, phagocytosis, and killing of bacteria. An appropriate host response requires resolution. The resolution phase involves attenuation of neutrophil migration, neutrophil apoptosis, macrophage recruitment, increased phagocytosis, efferocytosis of apoptotic neutrophils, and tissue repair. Specialized Pro-resolving Mediators (SPMs) are bioactive fatty acids that were shown to be highly effective in promoting resolution of infectious inflammation and survival in several models of infection. In this review, we provide insight into the role of SPMs in active host defense mechanisms for bacterial clearance including a new mechanism of action in which an SPM acts directly to reduce bacterial virulence.