Antimicrobial peptides (AMPs) are promising antimicrobials, however, the potential of bacterial resistance is a major concern. Here we systematically study the evolution of resistance to 14 chemically diverse AMPs and 12 antibiotics in Escherichia coli. Our work indicates that evolution of resistance against certain AMPs, such as tachyplesin II and cecropin P1, is limited. Resistance level provided by point mutations and gene amplification is very low and antibiotic-resistant bacteria display no cross-resistance to these AMPs. Moreover, genomic fragments derived from a wide range of soil bacteria confer no detectable resistance against these AMPs when introduced into native host bacteria on plasmids. We have found that simple physicochemical features dictate bacterial propensity to evolve resistance against AMPs. Our work could serve as a promising source for the development of new AMP-based therapeutics less prone to resistance, a feature necessary to avoid any possible interference with our innate immune system.

Microglial activation results in profound morphological, functional and gene expression changes that affect the pro- and anti-inflammatory mechanisms of these cells. Although statins have beneficial effects on inflammation, they have not been thoroughly investigated for their ability to affect microglial functions. Therefore the effects of rosuvastatin, one of the most commonly prescribed drugs in cardiovascular therapy, either alone or in combination with bacterial lipopolysaccharide (LPS), were profiled in pure microglial cultures derived from the forebrains of 18-day-old rat embryos. To reveal the effects of rosuvastatin on a number of pro- and anti-inflammatory mechanisms, we performed morphometric, functional and gene expression studies relating to cell adhesion and proliferation, phagocytosis, pro- and anti-inflammatory cytokine (IL-1β, tumor necrosis factor α (TNF-α) and IL-10, respectively) production, and the expression of various inflammation-related genes, including those related to the above morphological parameters and cellular functions. We found that microglia could be an important therapeutic target of rosuvastatin. In unchallenged (control) microglia, rosuvastatin inhibited proliferation and cell adhesion, but promoted microspike formation and elevated the expression of certain anti-inflammatory genes (Cxcl1, Ccl5, Mbl2), while phagocytosis or pro- and anti-inflammatory cytokine production were unaffected. Moreover, rosuvastatin markedly inhibited microglial activation in LPS-challenged cells by affecting both their morphology and functions as it inhibited LPS-elicited phagocytosis and inhibited pro-inflammatory cytokine (IL-1β, TNF-α) production, concomitantly increasing the level of IL-10, an anti-inflammatory cytokine. Finally, rosuvastatin beneficially and differentially affected the expression of a number of inflammation-related genes in LPS-challenged cells by inhibiting numerous pro-inflammatory and stimulating several anti-inflammatory genes. Since the microglia could elicit pro-inflammatory responses leading to neurodegeneration, it is important to attenuate such mechanisms and promote anti-inflammatory properties, and develop prophylactic therapies. By beneficially regulating both pro- and anti-inflammatory microglial functions, rosuvastatin may be considered as a prophylactic agent in the prevention of inflammation-related neurological disorders.

In Rhizobium-legume symbiosis, the bacteria are converted into nitrogen-fixing bacteroids. In many legume species, differentiation of the endosymbiotic bacteria is irreversible, culminating in definitive loss of their cell division ability. This terminal differentiation is mediated by plant peptides produced in the symbiotic cells. In Medicago truncatula more than ∼700 nodule-specific cysteine-rich (NCR) peptides are involved in this process. We have shown previously that NCR247 and NCR335 have strong antimicrobial activity on various pathogenic bacteria and identified interaction of NCR247 with many bacterial proteins, including FtsZ and several ribosomal proteins, which prevent bacterial cell division and protein synthesis. In this study we designed and synthetized various derivatives of NCR247, including shorter fragments and various chimeric derivatives. The antimicrobial activity of these peptides was tested on the ESKAPE bacteria; Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli as a member of Enterobacteriaceae and in addition Listeria monocytogenes and Salmonella enterica. The 12 amino acid long C-terminal half of NCR247, NCR247C partially retained the antimicrobial activity and preserved the multitarget interactions with partners of NCR247. Nevertheless NCR247C became ineffective on S. aureus, P. aeruginosa, and L. monocytogenes. The chimeric derivatives obtained by fusion of NCR247C with other peptide fragments and particularly with a truncated mastoparan sequence significantly increased bactericidal activity and altered the antimicrobial spectrum. The minimal bactericidal concentration of the most potent derivatives was 1.6 μM, which is remarkably lower than that of most classical antibiotics. The killing activity of the NCR247-based chimeric peptides was practically instant. Importantly, these peptides had no hemolytic activity or cytotoxicity on human cells. The properties of these NCR derivatives make them promising antimicrobials for clinical use.

Kynurenic acid is an endogenous modulator of ionotropic glutamate receptors and a suppressor of the immune system. Since glutamate and microglia are important in the pathogenesis of epilepsy, we investigated the possible action of the synthetic kynurenic acid analogue, SZR104, in epileptic mice and the action of kynurenic acid and SZR104 on the phagocytotic activity of cultured microglia cells. Pilocarpine epilepsy was used to test the effects of SZR104 on morphological microglia transformation, as evaluated through ionized calcium-binding adaptor molecule 1 (Iba1) immunohistochemistry. Microglia-enriched rat secondary cultures were used to investigate phagocytosis of fluorescent microbeads and Iba1 protein synthesis in control and lipopolysaccharide-challenged cultures. SZR104 inhibited microglia transformation following status epilepticus. Kynurenic acid and SZR104 inhibited lipopolysaccharide-stimulated phagocytotic activity of microglia cells. Although kynurenic acid and its analogues proved to be glutamate receptor antagonists, their immunosuppressive action was dominant in epilepsy. The inhibition of phagocytosis in vitro raised the possibility of the inhibition of genes encoding inflammatory cytokines in microglial cells.

Background and Aims: Crohn’s disease [CD] and ulcerative colitis [UC] require lifelong treatment and patient monitoring. Current biomarkers have several limitations; therefore, there is an unmet need to identify novel biomarkers in inflammatory bowel disease [IBD]. Previously, the role of plasminogen activator inhibitor 1 [PAI-1] was established in the pathogenesis of IBD and suggested as a potential biomarker. Therefore, we aimed to comprehensively analyse the selectivity of PAI-1 in IBD, its correlation with disease activity, and its potential to predict therapeutic response. Methods: Blood, colon biopsy, organoid cultures [OC], and faecal samples were used from active and inactive IBD patients and control subjects. Serpin E1 gene expressions and PAI-1 protein levels and localisation in serum, biopsy, and faecal samples were evaluated by qRT-PCR, ELISA, and immunostaining, respectively. Results: The study population comprised 132 IBD patients [56 CD and 76 UC] and 40 non-IBD patients. We demonstrated that the serum, mucosal, and faecal PAI-1 concentrations are elevated in IBD patients, showing clinical and endoscopic activity. In responders [decrease of eMayo ≥3 in UC; or SES-CD 50% in CD], the initial PAI-1 level decreased significantly upon successful therapy. OCs derived from active IBD patients produced higher concentrations of PAI-1 than the controls, suggesting that epithelial cells could be a source of PAI-1. Moreover, faecal PAI-1 selectively increases in active IBD but not in other organic gastrointestinal diseases. Conclusions: The serum, mucosal, and faecal PAI-1 concentration correlates with disease activity and therapeutic response in IBD, suggesting that PAI-1 could be used as a novel, non-invasive, disease-specific, faecal biomarker in patient follow-up.