The Addition of a Synthetic LPS-Targeting Domain Improves Serum Stability While Maintaining Antimicrobial, Antibiofilm, and Cell Stimulating Properties of an Antimicrobial Peptide.
ABSTRACT: Multi-drug resistant (MDR) bacteria and their biofilms are a concern in veterinary and human medicine. Protegrin-1 (PG-1), a potent antimicrobial peptide (AMP) with antimicrobial and immunomodulatory properties, is considered a potential alternative for conventional antibiotics. AMPs are less stable and lose activity in the presence of physiological fluids, such as serum. To improve stability of PG-1, a hybrid peptide, SynPG-1, was designed. The antimicrobial and antibiofilm properties of PG-1 and the PG-1 hybrid against MDR pathogens was analyzed, and activity after incubation with physiological fluids was compared. The effects of these peptides on the IPEC-J2 cell line was also investigated. While PG-1 maintained some activity in 25% serum for 2 h, SynPG-1 was able to retain activity in the same condition for up to 24 h, representing a 12-fold increase in stability. Both peptides had some antibiofilm activity against Escherichia coli and Salmonella typhimurium. While both peptides prevented biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA), neither could destroy MRSA's pre-formed biofilms. Both peptides maintained activity after incubation with trypsin and porcine gastric fluid, but not intestinal fluid, and stimulated IPEC-J2 cell migration. These findings suggest that SynPG-1 has much better serum stability while maintaining the same antimicrobial potency as PG-1.
Project description:The increasing onset of multidrug-resistant bacteria has propelled microbiology research towards antimicrobial peptides as new possible antibiotics from natural sources. Antimicrobial peptides are short peptides endowed with a broad range of activity against both Gram-positive and Gram-negative bacteria and are less prone to trigger resistance. Besides their activity against planktonic bacteria, many antimicrobial peptides also show antibiofilm activity. Biofilms are ubiquitous in nature, having the ability to adhere to virtually any surface, either biotic or abiotic, including medical devices, causing chronic infections that are difficult to eradicate. The biofilm matrix protects bacteria from hostile environments, thus contributing to the bacterial resistance to antimicrobial agents. Biofilms are very difficult to treat, with options restricted to the use of large doses of antibiotics or the removal of the infected device. Antimicrobial peptides could represent good candidates to develop new antibiofilm drugs as they can act at different stages of biofilm formation, on disparate molecular targets and with various mechanisms of action. These include inhibition of biofilm formation and adhesion, downregulation of quorum sensing factors, and disruption of the pre-formed biofilm. This review focuses on the proprieties of antimicrobial and antibiofilm peptides, with a particular emphasis on their mechanism of action, reporting several examples of peptides that over time have been shown to have activity against biofilm.
Project description:Weaning stress renders piglets susceptible to pathogen infection, which leads to post-weaning diarrhea, a severe condition characterized by heavy diarrhea and mortality in piglets. Enterotoxigenic Escherichia coli (ETEC) is one of typical strains associated with post-weaning diarrhea. Thus, prevention and inhibition of ETEC infection are of great concern. Probiotics possess anti-pathogenic activity and can counteract ETEC infection; however, their underlying mechanisms and modes of action have not yet been clarified. In the present study, the direct and indirect protective effects of Lactobacillus plantarum ZLP001 against ETEC infection were investigated by different methods. We found that bacterial culture and culture supernatant of L. plantarum ZLP001 prevented ETEC growth by the Oxford cup method, and ETEC growth inhibition was observed in a co-culture assay as well. This effect was suggested to be caused mainly by antimicrobial metabolites produced by L. plantarum ZLP001. In addition, adhesion capacity of L. plantarum ZLP001 to IPEC-J2 cells were observed using microscopy and counting. L. plantarum ZLP001 also exhibited a concentration-dependent ability to inhibit ETEC adhesion to IPEC-J2 cells, which mainly occurred via exclusion and competition mode. Furthermore, quantitative real time polymerase chain reaction (qPCR) analysis showed that L. plantarum ZLP001 upregulated the expression of host defense peptides (HDPs) but did not trigger an inflammatory response. In addition, L. plantarum ZLP001 induced HDP secretion, which enhanced the potential antimicrobial activity of IPEC-J2 cell-culture supernatant after incubation with L. plantarum ZLP001. Our findings demonstrate that L. plantarum ZLP001, an intestinal Lactobacillus species associated with piglet health, possesses anti-ETEC activity. L. plantarum ZLP001 might prevent ETEC growth, inhibit ETEC adhesion to the intestinal mucosa, and activate the innate immune response to secret antimicrobial peptides. L. plantarum ZLP001 is worth investigation as a potential probiotics.
Project description:<h4>Background</h4>Streptococcus agalactiae (S. agalactiae) is a contagious pathogen of bovine mastitis. It has financial implications for the dairy cattle industry in certain areas of the world. Since antimicrobial resistance increases in dairy farms, natural antimicrobials from herbal origins and nanoparticles have been given more attention as an alternative therapy. Hence, this study reported the antimicrobial and antibiofilm potentials of cinnamon oil, silver nanoparticles (AgNPs), and their combination against multidrug-resistant (MDR) S. agalactiae recovered from clinical bovine mastitis in Egypt.<h4>Results</h4>Our findings revealed that 73% (146/200) of the examined milk samples collected from dairy cows with clinical mastitis were infected with Streptococci species. Of these, 9.59% (14/146) were identified as S. agalactiae and categorized as MDR. S. agalactiae isolates expressed four virulence genes (Hyl, cylE, scpB, and lmb) and demonstrated an ability to produce biofilms. Cinnamon oil showed high antimicrobial (MICs ≤0.063 μg /mL) and antibiofilm (MBIC<sub>50</sub> = 4 μg/mL) potentials against planktonic and biofilms of S. agalactiae isolates, respectively. However, AgNPs showed reasonable antimicrobial (MICs ≤16 μg/mL) and relatively low antibiofilm (MBIC<sub>50</sub> = 64 μg/mL) activities against screened isolates. Synergistic antimicrobial or additive antibiofilm interactions of cinnamon oil combined with AgNPs were reported for the first time. Scanning electron microscope (SEM) analysis revealed that biofilms of S. agalactiae isolates treated with cinnamon oil were more seriously damaged than observed in AgNPs cinnamon oil combination. Moreover, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) showed that cinnamon oil exerted a remarkable down-regulation of pili biosynthesis genes (pilA and pilB) and their regulator (rogB) against S. agalactiae biofilms, meanwhile the AgNPs cinnamon oil combination demonstrated a lower efficacy.<h4>Conclusions</h4>This is an in vitro preliminary approach that documented the antibiofilm potential of cinnamon oil and the inhibitory activity of cinnamon oil and its combination with AgNPs against MDR S. agalactiae recovered from clinical mastitis. Further in vivo studies should be carried out in animal models to provide evidence of concept for implementing these alternative candidates in the treatment of dairy farms infected by streptococcal mastitis in the future.
Project description:Escherichia coli is the most frequent agent of urinary tract infections in humans. The emergence of uropathogenic multidrug-resistant (MDR) E. coli strains that produce extended spectrum ?-lactamases (ESBL) has created additional problems in providing adequate treatment of urinary tract infections. We have previously reported the antimicrobial activity of 1,8-cineole, one of the main components of Rosmarinus officinalis volatile oil, against Gram negative bacteria during planktonic growth. Here, we evaluated the antibiofilm activity of 1,8-cineole against pre-formed mature biofilms of MDR ESBL-producing uropathogenic E. coli clinical strains by carrying out different technical approaches such as counting of viable cells, determination of biofilm biomass by crystal violet staining, and live/dead stain for confocal microscopy and flow cytometric analyses. The plant compound showed a concentration- and time-dependent antibiofilm activity over pre-formed biofilms. After a 1 h treatment with 1% (v/v) 1,8-cineole, a significant decrease in viable biofilm cell numbers (3-log reduction) was observed. Biofilms of antibiotic-sensitive and MDR ESBL-producing E. coli isolates were sensitive to 1,8-cineole exposure. The phytochemical treatment diminished the biofilm biomass by 48-65% for all four E. coli strain tested. Noteworthy, a significant cell death in the remaining biofilm was confirmed by confocal laser scanning microscopy after live/dead staining. In addition, the majority of the biofilm-detached cells after 1,8-cineole treatment were dead, as shown by flow cytometric assessment of live/dead-stained bacteria. Moreover, phytochemical-treated biofilms did not fully recover growth after 24 h in fresh medium. Altogether, our results support the efficacy of 1,8-cineole as a potential antimicrobial agent for the treatment of E. coli biofilm-associated infections.
Project description:Host defense peptides (HDPs) have antimicrobial and immunoregulatory activities and are involved in epithelial innate immune defense. Dietary modulation of endogenous HDP synthesis is an effective way to boost the host innate immune system. This study aimed to investigate the role of the probiotic Lactobacillus plantarum strain ZLP001 in porcine HDP induction and the underlying mechanism. To this end, we evaluated the stimulatory effect of L. plantarum ZLP001 on HDP expression in piglet intestinal tissue in vivo and porcine IPEC-J2 cells and 3D4/31 cells in vitro, and we examined the underlying intracellular signaling pathway in IPEC-J2 cells. L. plantarum ZLP001 treatment increased the mRNA expression of jejunal and ileal HDPs in weaned piglets. In IPEC-J2 and 3D4/31 cells, L. plantarum ZLP001 stimulated HDP expression, but different HDP induction patterns were observed, with the various HDPs exhibiting different relative mRNA levels in each cell line. L. plantarum ZLP001 induced porcine HDP expression through toll-like receptor (TLR)2 recognition as evidenced by the fact that HDP expression was suppressed in TLR2-knockdown IPEC-J2 cells. Furthermore, we found that L. plantarum ZLP001 activated the extracellular signal-regulated kinase (ERK)1/2 and c-jun N-terminal kinase (JNK) signaling pathways, as indicated by enhanced phosphorylation of both ERK1/2 and JNK and the fact that HDP expression was suppressed upon inhibition of ERK1/2 and JNK. Furthermore, L. plantarum ZLP001 activated c-fos and c-jun transcription factor phosphorylation and activity. We conclude that L. plantarum ZLP001 induces porcine HDP expression in vivo and in vitro, and the induction seems to be regulated via TLR2 as well as the ERK1/2/JNK and c-jun/c-fos signaling pathways. Modulation of endogenous HDPs mediated by L. plantarum ZLP001 might be a promising approach to improving intestinal health and enhancing diarrhea resistance in weaning piglets.
Project description:Enterotoxigenic Escherichia coli (ETEC) are an important cause of post-weaning diarrhea (PWD) in piglets. The IL-17 cytokine family is well known to play important roles in the host defense against bacterial infections at the mucosa. Previously, we reported the potential role of IL-17A in clearing an ETEC infection in piglets. IL-17C, another member of the IL-17 family, is highly expressed in the intestinal epithelium, however, its role during an ETEC infection is still unclear. In this study, we demonstrate that F4<sup>+</sup> ETEC induce IL-17C mRNA and protein expression in intestinal tissues as well as in porcine intestinal epithelial cells (IPEC-J2). This IL-17C production is largely dependent on TLR5 signaling in IPEC-J2 cells. Both F4<sup>+</sup> ETEC infection and exogenous IL-17C increased the expression of antimicrobial peptides and tight junction proteins, such as porcine beta-defensin (pBD)-2, claudin-1, claudin-2 and occludin in IPEC-J2 cells. Taken together, our data demonstrate that TLR5-mediated IL-17C expression in intestinal epithelial cells enhances mucosal host defense responses in a unique autocrine/paracrine manner in the intestinal epithelium against ETEC infection.
Project description:Defensins are small antimicrobial peptides (AMPs) that play an important role in the innate immune system of mammals. Since the effect of mycotoxin contamination of food and feed on the secretion of intestinal AMPs is poorly understood, the aim of this study was to elucidate the individual and combined effects of four common Fusarium toxins, deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEA), and fumonisin B1 (FB1), on the mRNA expression, protein secretion, and corresponding antimicrobial effects of porcine ?-defensins 1 and 2 (pBD-1 and pBD-2) using a porcine jejunal epithelial cell line, IPEC-J2. In general, upregulation of pBD-1 and pBD-2 mRNA expression occurred following exposure to Fusarium toxins, individually and in mixtures (P < 0.05). However, no significant increase in secreted pBD-1 and pBD-2 protein levels was observed, as measured by enzyme-linked immunosorbent assay (ELISA). Supernatants from IPEC-J2 cells exposed to toxins, singly or in combination, however, possessed significantly less antimicrobial activity against Escherichia coli than untreated supernatants. When single toxins and two-toxin combinations were assessed, toxicity effects were shown to be nonadditive (including synergism, potentiation, and antagonism), suggesting interactive toxin effects when cells are exposed to mycotoxin combinations. The results show that Fusarium toxins, individually and in mixtures, activate distinct antimicrobial defense mechanisms possessing the potential to alter the intestinal microbiota through diminished antimicrobial effects. Moreover, by evaluating toxin mixtures, this improved understanding of toxin effects will enable more effective risk assessments for common mycotoxin combinations observed in contaminated food and feed.
Project description:Interleukin-22 (IL-22), a member of the IL-10 superfamily, plays essential roles in fighting against mucosal microbial infection and maintaining mucosal barrier integrity within the intestine. However, little knowledge exists on the ability of porcine IL-22 (pIL-22) to fight against viral infection in the gut. In this study, we found that recombinant mature pIL-22 (mpIL-22) inhibited the infection of multiple diarrhea viruses, including alpha coronavirus, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine rotavirus (PoRV), in the intestinal porcine epithelial cell line J2 (IPEC-J2) cells. mpIL-22 up-regulated the expression of the antimicrobial peptide beta-defensin (BD-2), cytokine IL-18 and IFN-?. Furthermore, we found that mpIL-22 induced phosphorylation of STAT3 on Ser727 and Tyr705 in IPEC-J2 cells. Inhibition of STAT3 phosphorylation by S3I-201 abrogated the antiviral ability of mpIL-22 and the mpIL-22-induced expression of BD-2, IL-18, and IFN-?. Together, mpIL-22 inhibited the infection of PoRV and enteric coronaviruses, and up-regulated the expression of antimicrobial genes in IPEC-J2, which were mediated by the activation of the STAT3 signal pathway. The significant antiviral activity of IL-22 to curtail multiple enteric diarrhea viruses in vitro suggests that pIL-22 could be a novel therapeutic against devastating viral diarrhea in piglets.
Project description:Recurrent caries, the development of carious lesions at the interface between the restorative material and the tooth structure, is highly prevalent and represents the primary cause for failure of dental restorations. Correspondingly, we exploited the self-assembly and strong antibiofilm activity of amphipathic antimicrobial peptides (AAMPs) to form novel coatings on dentin that aimed to prevent recurrent caries at susceptible cavosurface margins. AAMPs are alternative to traditional antimicrobial agents and antibiotics with the ability to target the complex and heterogeneous organization of microbial communities. Unlike approaches that have focused on using these AAMPs in aqueous solutions for a transient activity, here we assess the effects on microcosm biofilms of a long-acting AAMPs-based antibiofilm coating to protect the tooth-composite interface. Genomewise, we studied the impact of AAMPs coatings on the dental plaque microbial community. We found that non-native all D-amino acids AAMPs coatings induced a marked shift in the plaque community and selectively targeted three primary acidogenic colonizers, including the most common taxa around Class II composite restorations. Accordingly, we investigated the translational potential of our antibiofilm dentin using multiphoton pulsed near infra-red laser for deep bioimaging to assess the impact of AAMPs-coated dentin on plaque biofilms along dentin-composite interfaces. Multiphoton enabled us to record the antibiofilm potency of AAMPs-coated dentin on plaque biofilms throughout exaggeratedly failed interfaces. In conclusion, AAMPs-coatings on dentin showed selective and long-acting antibiofilm activity against three dominant acidogenic colonizers and potential to resist recurrent caries to promote and sustain the interfacial integrity of adhesive-based interfaces.
Project description:Porcine NK-Lysine (PNKL) is a new antimicrobial peptide (AMP) identified in the small intestine. In this study, PNKL protein was obtained through heterologous expression in <i>Escherichia coli</i> and was estimated by SDS-PAGE at 33 kDa. The antibacterial activities of PNKL were determined using various bacterial strains and showed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, <i>E. coli</i> K88-challenged IPEC-J2 cells were used to determine PNKL influences on inflammatory responses. Hemolytic assays showed that PNKL had no detrimental impact on cell viability. Interestingly, PNKL elevated the viability of IPEC-J2 cells exposure to <i>E. coli</i> K88. PNKL significantly decreased the cell apoptosis rate, and improved the distribution and abundance of tight junction protein ZO-1 in IPEC-J2 cells upon <i>E. coli</i> K88-challenge. Importantly, PNKL not only down regulated the expressions of inflammatory cytokines such as the IL-6 and TNF-α, but also down regulated the expressions of NF-κB, Caspase3, and Caspase9 in the <i>E. coli</i> K88-challenged cells. These results suggest a novel function of natural killer (NK)-lysin, and the anti-bacterial and anti-inflammatory properties of PNKL may allow it a potential substitute for conventionally used antibiotics or drugs.