Lactoferricin-B

Nitric oxide (NO) is a potentially powerful weapon against Candida albicans, and the regulation of intracellular NO levels is therefore important for controlling its physiological functions. Lactoferricin B like peptide (LBLP) is a 23-mer antimicrobial peptide (AMP) derived from the Scolopendra subspinipes mutilans. We confirmed that LBLP treatment led to the generation of endogenous NO in C. albicans, which was associated with the NO synthase pathway. Here, we examined the antifungal activity of LBLP with focus on intracellular NO. Total glutathione levels were measured to evaluate cellular defense capacity against NO. LBLP decreased total glutathione levels, leading to nitrosative stress. LBLP also inhibited mitochondrial respiration and altered the NAD+/ NADH ratios. Inhibition of mitochondrial respiration induced mitochondrial membrane depolarization, thus leading to calcium homeostasis disruption and mitochondrial superoxide anion accumulation. Consequently, treatment of C. albicans with LBLP resulted in apoptosis. These physiological changes were attenuated when NO generation was inhibited. Our data strongly indicate that LBLP mediates apoptosis by affecting intracellular NO homeostasis. These results on antifungal activity of LBLP and its mechanism indicate the therapeutic promise of this AMP and support the role of NO in cell death regulation.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder that is characterized by recurrent and chronic infections of the lung predominantly by the opportunistic pathogens, Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa. While S. aureus is the main colonizing bacteria of the CF lungs during infancy and early childhood, its incidence declines thereafter and infections by P. aeruginosa become more prominent with increasing age. The competitive and cooperative interactions exhibited by these two pathogens influence their survival, antibiotic susceptibility, persistence and, consequently the disease progression. For instance, P. aeruginosa secretes small respiratory inhibitors like hydrogen cyanide, pyocyanin and quinoline N-oxides that block the electron transport pathway and suppress the growth of S. aureus. However, S. aureus survives this respiratory attack by adapting to respiration-defective small colony variant (SCV) phenotype. SCVs cause persistent and recurrent infections and are also resistant to antibiotics, especially aminoglycosides, antifolate antibiotics, and to host antimicrobial peptides such as LL-37, human β-defensin (HBD) 2 and HBD3; and lactoferricin B. The interaction between P. aeruginosa and S. aureus is multifaceted. In mucoid P. aeruginosa strains, siderophores and rhamnolipids are downregulated thus enhancing the survival of S. aureus. Conversely, protein A from S. aureus inhibits P. aeruginosa biofilm formation while protecting both P. aeruginosa and S. aureus from phagocytosis by neutrophils. This review attempts to summarize the current understanding of the molecular mechanisms that drive the competitive and cooperative interactions between S. aureus and P. aeruginosa in the CF lungs that could influence the disease outcome.

Natural antimicrobial peptides provide fundamental protection for multicellular organisms from microbes, such as Lactoferricin B (Lfcin B). Many studies have shown that Lfcin B penetrates the cell membrane and has intracellular activities. To elucidate the intracellular behavior of Lfcin B, we first used Escherichia coli K12 proteome chips to identify the intracellular targets of Lfcin B. The results showed that Lfcin B binds to two response regulators, BasR and CreB, of the two-component system. For further analysis, we conducted several in vitro and in vivo experiments and utilized bioinformatics methods. The electrophoretic mobility shift assays and kinase assays indicate that Lfcin B inhibits the phosphorylation of the response regulators (BasR and CreB) and their cognate sensor kinases (BasS and CreC). Antibacterial assays showed that Lfcin B reduced E. coli's tolerance to environmental stimuli, such as excessive ferric ions and minimal medium conditions. This is the first study to show that an antimicrobial peptide inhibits the growth of bacteria by influencing the phosphorylation of a two-component system directly.