Klebsiella pneumoniae is a multidrug-resistant opportunistic human pathogen related to various infections. As such, synthetic peptides have emerged as potential alternative molecules. Mo-CBP₃-PepI has presented great activity against K. pneumoniae by presenting an MIC₅₀ at a very low concentration (31.25 µg mL⁻¹). Here, fluorescence microscopy and proteomic analysis revealed the alteration in cell membrane permeability, ROS overproduction, and protein profile of K. pneumoniae cells treated with Mo-CBP₃-PepI. Mo-CBP₃-PepI led to ROS overaccumulation and membrane pore formation in K. pneumoniae cells. Furthermore, the proteomic analysis highlighted changes in essential metabolic pathways. For example, after treatment of K. pneumoniae cells with Mo-CBP₃-PepI, a reduction in the abundance of protein related to DNA and protein metabolism, cytoskeleton and cell wall organization, redox metabolism, regulation factors, ribosomal proteins, and resistance to antibiotics was seen. The reduction in proteins involved in vital processes for cell life, such as DNA repair, cell wall turnover, and protein turnover, results in the accumulation of ROS, driving the cell to death. Our findings indicated that Mo-CBP₃-PepI might have mechanisms of action against K. pneumoniae cells, mitigating the development of resistance and thus being a potent molecule to be employed in producing new drugs against K. pneumoniae infections.

C. albicans and C. parapsilosis are biofilm-forming yeasts responsible for bloodstream infections that can cause death. Synthetic antimicrobial peptides (SAMPs) are considered to be new weapons to combat these infections, alone or combined with drugs. Here, two SAMPs, called Mo-CBP₃-PepI and Mo-CBP₃-PepIII, were tested alone or combined with nystatin (NYS) and itraconazole (ITR) against C. albicans and C. parapsilosis biofilms. Furthermore, the mechanism of antibiofilm activity was evaluated by fluorescence and scanning electron microscopies. When combined with SAMPs, the results revealed a 2- to 4-fold improvement of NYS and ITR antibiofilm activity. Microscopic analyses showed cell membrane and wall damage and ROS overproduction, which caused leakage of internal content and cell death. Taken together, these results suggest the potential of Mo-CBP₃-PepI and Mo-CBP₃-PepIII as new drugs and adjuvants to increase the activity of conventional drugs for the treatment of clinical infections caused by C. albicans and C. parapsilosis.

Staphylococcus aureus is a human pathogen known to be resistant to antibiotics since the mid-20th century and is constantly associated with hospital-acquired infections. S. aureus forms biofilms, which are complex surface-attached communities of bacteria held together by a self-produced polymer matrix consisting of proteins, extracellular DNA, and polysaccharides. Biofilms are resistance structures responsible for increasing bacterial resistance to drugs by 1000 times more than the planktonic lifestyle. Therefore, studies have been conducted to discover novel antibacterial molecules to prevent biofilm formation and/or degrade preformed biofilms. Synthetic antimicrobial peptides (SAMPs) have appeared as promising alternative agents to overcome increasing antibiotic resistance. Here, the antibiofilm activity of eight SAMPs, in combination with the antibiotic ciprofloxacin, was investigated in vitro. Biofilm formation by S. aureus was best inhibited (76%) by the combination of Mo-CBP₃-PepIII (6.2 µg mL⁻¹) and ciprofloxacin (0.39 µg mL⁻¹). In contrast, the highest reduction (60%) of the preformed biofilm mass was achieved with RcAlb-PepII (1.56 µg mL⁻¹) and ciprofloxacin (0.78 µg mL⁻¹). Fluorescence microscopy analysis reinforced these results. These active peptides formed pores in the cellular membrane of S. aureus, which may be related to the enhanced ciprofloxacin’s antibacterial activity. Our findings indicated that these peptides may act with ciprofloxacin and are powerful co-adjuvant agents for the treatment of S. aureus infections.

Multidrug-resistant Cryptococcus neoformans is an encapsulated yeast causing a high mortality rate in immunocompromised patients. Recently, the synthetic peptide Mo-CBP₃-PepII emerged as a potent anticryptococcal molecule with an MIC₅₀ at low concentration. Here, the mechanisms of action of Mo-CBP₃-PepII were deeply analyzed to provide new information about how it led C. neoformans cells to death. Light and fluorescence microscopies, analysis of enzymatic activities, and proteomic analysis were employed to understand the effect of Mo-CBP₃-PepII on C. neoformans cells. Light and fluorescence microscopies revealed Mo-CBP₃-PepII induced the accumulation of anion superoxide and hydrogen peroxide in C. neoformans cells, in addition to a reduction in the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) in the cells treated with Mo-CBP₃-PepII. In the presence of ascorbic acid (AsA), no reactive oxygen species (ROS) were detected, and Mo-CBP₃-PepII lost the inhibitory activity against C. neoformans. However, Mo-CBP₃-PepII inhibited the activity of lactate dehydrogenase (LDH) ergosterol biosynthesis and induced the decoupling of cytochrome c (Cyt c) from the mitochondrial membrane. Proteomic analysis revealed a reduction in the abundance of proteins related to energetic metabolism, DNA and RNA metabolism, pathogenicity, protein metabolism, cytoskeleton, and cell wall organization and division. Our findings indicated that Mo-CBP₃-PepII might have multiple mechanisms of action against C. neoformans cells, mitigating the development of resistance and thus being a potent molecule to be employed in the production of new drugs against C. neoformans infections.