Microtubules are required for efficient epithelial tight junction homeostasis and restoration.

Epithelial tight junctions are critical for creating a barrier yet allowing paracellular transport. Although it is well established that the actin cytoskeleton is critical for preserving the dynamic organization of the tight junction and maintaining normal tight junction protein recycling, contributions of microtubules to tight junction organization and function remain undefined. The aim of this study is to determine the role of microtubules in tight junction homeostasis and restoration. Our data demonstrate that occludin traffics on microtubules and that microtubule disruption perturbs tight junction structure and function. Microtubules are also shown to be required for restoring barrier function following Ca(2+) chelation and repletion. These processes are mediated by proteins participating in microtubule minus-end-directed trafficking but not plus-end-directed trafficking. These studies show that microtubules participate in the preservation of epithelial tight junction structure and function and play a vital role in tight junction restoration, thus expanding our understanding of the regulation of tight junction physiology.

Saturation mutagenesis of selected residues of the α-peptide of the lantibiotic lacticin 3147 yields a derivative with enhanced antimicrobial activity.

The lantibiotic lacticin 3147 consists of two ribosomally synthesized and post-translationally modified antimicrobial peptides, Ltnα and Ltnß, which act synergistically against a wide range of Gram-positive microorganisms. We performed saturation mutagenesis of specific residues of Ltnα to determine their functional importance. The results establish that Ltnα is more tolerant to change than previously suggested by alanine scanning mutagenesis. One substitution, LtnαH23S, was identified which improved the specific activity of lacticin 3147 against one pathogenic strain, Staphylococcus aureus NCDO1499. This represents the first occasion upon which the activity of a two peptide lantibiotic has been enhanced through bioengineering.

Comparison of the Potency of the Lipid II Targeting Antimicrobials Nisin, Lacticin 3147 and Vancomycin Against Gram-Positive Bacteria.

While nisin (lantibiotic), lacticin 3147 (lantibiotic) and vancomycin (glycopeptides) are among the best studied lipid II-binding antimicrobials, their relative activities have never been compared. Nisin and lacticin 3147 have been employed/investigated primarily as food preservatives, although they do have potential in terms of veterinary and clinical applications. Vancomycin is used exclusively in clinical therapy. We reveal a higher potency for lacticin 3147 (MIC 0.95-3.8 µg/ml) and vancomycin (MIC 0.78-1.56 µg/ml) relative to that of nisin (MIC 6.28-25.14 µg/ml) against the food-borne pathogen Listeria monocytogenes. A comparison of the activity of the three antimicrobials against nisin resistance mutants of L. monocytogenes also reveals that their susceptibility to vancomycin and lacticin 3147 changed only slightly or not at all. A further assessment of relative activity against a selection of Bacillus cereus, Enterococcus and Staphylococcus aureus targets revealed that vancomycin MICs consistently ranged between 0.78 and 1.56 µg/ml against all but one strain. Lacticin 3147 was found to be more effective than nisin against B. cereus (lacticin 3147 MIC 1.9-3.8 µg/ml; nisin MIC 4.1-16.7 µg/ml) and E. faecium and E. faecalis targets (lacticin 3147 MIC from 1.9 to 3.8 µg/ml; nisin MIC ≥8.3 µg/ml). The greater effectiveness of lacticin 3147 is even more impressive when expressed as molar values. However, in agreement with the previous reports, nisin was the more effective of the two lantibiotics against S. aureus strains. This study highlights that in many instances the antimicrobial activity of these leading lantibiotics are comparable with that of vancomycin and emphasizes their particular value with respect to use in situations including foods and veterinary medicine, where the use of vancomycin is not permitted.