Cytotoxicity and the effect of cationic peptide fragments against cariogenic bacteria under planktonic and biofilm conditions

This study evaluated the cytotoxicity and effect of fragments derived from three oral cationic peptides (CP): LL-37, D6-17 and D1-23 against cariogenic bacteria under planktonic and biofilm conditions. For cytotoxicity analysis, two epithelial cell lines were used. The minimum inhibitory concentration and the minimal bactericidal concentration were determined for the CP fragments and the control (chlorhexidine-CHX) against cariogenic bacteria. The fractional inhibitory concentration was obtained for the combinations of CP fragments on Streptococcus mutans. Biofilm assays were conducted with the best antimicrobial CP fragment against S. mutans. The results indicated that D6-17 was not cytotoxic. D1-23, LL-37 and CHX were not cytotoxic in low concentrations. D1-23 presented the best bactericidal activity against S. mutans, S. mitis and S. salivarius. Combinations of CP fragments did not show a synergic effect. D1-23 presented a higher activity against S. mutans biofilm than CHX. It was concluded that D1-23 showed a substantial effect against cariogenic bacteria and low cytotoxicity.     

The human antimicrobial peptide LL-37 and its fragments possess both antimicrobial and antibiofilm activities against multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii infections are difficult to treat due to multidrug resistance. Biofilm formation by A. baumannii is an additional factor in its ability to resist antimicrobial therapy. The antibacterial and antibiofilm activities of the human antimicrobial peptide LL-37 and its fragments KS-30, KR-20 and KR-12 against clinical isolates of multidrug-resistant (MDR) A. baumannii were evaluated. The minimal inhibitory concentration (MIC) of LL-37 against MDR A. baumannii isolates ranged from 16 to 32 μg/mL. The MIC of KS-30 fragment varied from 8.0 to16 μg/mL and the KR-20 fragment MIC ranged from 16 to 64 μg/mL. LL-37 and KS-30 fragment exhibited 100% bactericidal activity against five A. baumannii strains, including four MDR clinical isolates, within 30 min at concentrations of 0.25–1 μg/mL. By 0.5 h, the fragments KR-20 and KR-12 eliminated all tested strains at 8 and 64 μg/mL respectively. LL-37 and its fragments displayed anti-adherence activities between 32-128 μg/mL. A minimum biofilm eradication concentration (MBEC) biofilm assay demonstrated that LL-37 inhibited and dispersed A. baumannii biofilms at 32 μg/mL respectively. Truncated fragments of LL-37 inhibited biofilms at concentrations of 64–128 μg/mL. KS-30, the truncated variant of LL-37, effectively dispersed biofilms at 64 μg/mL. At 24 h, no detectable toxicity was observed at the efficacious doses when cytotoxicity assays were performed. Thus, LL-37, KS-30 and KR-20 exhibit significant antimicrobial activity against MDR A. baumannii. The prevention of biofilm formation in vitro by LL-37, KS-30 and KR-20 adds significance to their efficacy. These peptides can be potential therapeutics against MDR A. baumannii infections.     

Recombinant expression of human cathelicidin (hCAP18/LL-37) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.     

Modulation of antimicrobial potency of human cathelicidin peptides against the ESKAPE pathogens and in vivo efficacy in a murine catheter-associated biofilm model

Antimicrobial peptides are essential components of innate immune systems that protect hosts from infection. They are also useful candidates for developing a new generation of antibiotics to fight antibiotic-resistant pathogens. Human innate immune peptide LL-37 can inhibit biofilm formation, but suffers from high cost due to a long peptide length and rapid protease degradation. To improve the peptide, we previously identified the major active region and changed the peptide backbone structure. This study designed two families of new peptides by altering peptide side chains. Interestingly, these peptides displayed differential potency against various ESKAPE pathogens in vitro and substantially reduced hemolysis. Further potency test in vivo revealed that 17tF-W eliminated the burden of methicillin-resistant Staphylococcus aureus (MRSA) USA300 in both mouse-embedded catheters and their surrounding tissues. In addition, peptide treatment suppressed the level of chemokine TNFα, and boosted the levels of chemokines MCP-1, IL-17A and IL-10 in the surrounding tissues of the infected catheter embedded in mice. In conclusion, we have designed a set of new LL-37 peptides with varying antimicrobial activities, opening the door to potential topical treatment of infections involving different drug-resistant pathogens.     

High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli

The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter. The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms. Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 μM; for Staphylococcus aureus 2.31 μM, and for Enterococcus faecalis 5.54 μM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of β-galactosidase activity in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate quantities of active human antimicrobial peptide LL-37.     

Antimicrobial and anti-inflammatory activities of chemokine CXCL14-derived antimicrobial peptide and its analogs

CXCL14 is a CXC chemokine family that exhibits antimicrobial activity and contains an amphipathic cationic α-helical region in the C-terminus, a characteristic structure of antimicrobial peptides (AMPs). In this study, we designed three analogs of CXCL14_59–75 (named CXCL14-C17) corresponding to the C-terminal α-helix of CXCL14, which displayed potential antimicrobial activity against a wide variety of gram-negative and gram-positive bacteria with minimum inhibitory concentrations of 4?16?μM without mammalian cell toxicity. Furthermore, two CXCL14-C17 analogs (CXCL14-C17-a1 and CXCL14-C17-a3) with improved cell selectivity were engineered by introducing Lys, Arg, or Trp in CXCL14-C17. Additionally, CXCL14-C17 analogs showed much greater synergistic effect (FICI: 0.3125–0.375) with chloramphenicol and ciprofloxacin against multidrug-resistant Pseudomonas aeruginosa (MDRPA) than LL-37 did (FICI: 0.75–1.125). CXCL14-C17 analogs were more active against antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), MDRPA, and vancomycin-resistant Enterococcus faecium (VREF) than LL-37 and melittin. In particular, CXCL14-C17-a2 and CXCL14-C17-a3 completely inhibited the biofilm formation at sub-MIC and all of the peptides were able to eliminate pre-formed biofilm as well. Membrane depolarization, flow cytometry, sytox green uptake, ONPG hydrolysis and confocal microscopy revealed the possible target of the native peptide (CXCL14-C17) to likely be intracellular, and the amphipathic designed analogs targeted the bacterial membrane. CXCL14-C17 also showed DNA binding characteristic activity similar to buforin-2. Interestingly, CXCL14-C17-a2 and CXCL14-C17-a3 effectively inhibited the production and expression of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 from lipopolysaccharide (LPS)-stimulated RAW264.7 cells, suggesting that these peptides could be promising anti-inflammatory and antimicrobial agents.     

Activities of a broad-spectrum antimicrobial peptide analogue SAMP-A4-C8 and its combat against pneumonia in Staphylococcus aureus-infected mice

Antimicrobial peptides and their analogues have become substitutes for antibiotics in recent years. The antimicrobial peptide analogue SAMP-A4-C8 (n-octanoic-VRLLRRRI) with high antimicrobial activity was found in our lab. We speculate that it may kill pathogens by some lethal mechanism of action. In the present investigation, the microbicidal activities of SAMP-A4-C8 and its mechanism of action were investigated. The results demonstrated that SAMP-A4-C8 had lethal activities against Staphylococcus aureus and Candida albicans by cell disruption. Based on its microbicidal activities, we believe that it is worth further research for its potential as drug candidate. The results showed that SAMP-A4-C8, with low propensity to induce the resistance of S. aureus and C. albicans, could kill the persister cells of S. aureus and C. albicans, exhibited biofilm forming inhibition activity and preformed biofilm eradication ability against S. aureus and C. albicans, and displayed therapeutic potential on pneumonia in S. aureus-infected mice by reducing lung inflammation. The present study provided a promising drug candidate in the war against multidrug resistance.     

Sequence determinants in the cathelicidin LL-37 that promote inflammation via presentation of RNA to scavenger receptors

Cathelicidins such as the human 37-amino acid peptide (LL-37) are peptides that not only potently kill microbes but also trigger inflammation by enabling immune recognition of endogenous nucleic acids. Here, a detailed structure–function analysis of LL-37 was performed to understand the details of this process. Alanine scanning of 34-amino acid peptide (LL-34) showed that some variants displayed increased antimicrobial activity against Staphylococcus aureus and group A Streptococcus. In contrast, different substitutions clustered on the hydrophobic face of the LL-34 alpha helix inhibited the ability of those variants to promote type 1 interferon expression in response to U1 RNA or to present U1 to the scavenger receptor (SR) B1 on the keratinocyte cell surface. Small-angle X-ray scattering experiments of the LL-34 variants LL-34, F5A, I24A, and L31A demonstrated that these peptides form cognate supramolecular structures with U1 characterized by inter-dsRNA spacings of approximately 3.5 nm, a range that has been previously shown to activate toll-like receptor 3 by the parent peptide LL-37. Therefore, while alanine substitutions on the hydrophobic face of LL-34 led to loss of binding to SRs and the complete loss of autoinflammatory responses in epithelial and endothelial cells, they did not inhibit the ability to organize with U1 RNA in solution to associate with toll-like receptor 3. These observations advance our understanding of how cathelicidin mediates the process of innate immune self-recognition to enable inert nucleic acids to trigger inflammation. We introduce the term “innate immune vetting” to describe the capacity of peptides such as LL-37 to enable certain nucleic acids to become an inflammatory stimulus through SR binding prior to cell internalization.     

Antiviral Activity of the Human Cathelicidin,LL-37, and Derived Peptides on Seasonal and Pandemic Influenza A Viruses

Human LL-37, a cationic antimicrobial peptide, was recently shown to have antiviral activity against influenza A virus (IAV) strains in vitro and in vivo. In this study we compared the anti-influenza activity of LL-37 with that of several fragments derived from LL-37. We first tested the peptides against a seasonal H3N2 strain and the mouse adapted H1N1 strain, PR-8. The N-terminal fragment, LL-23, had slight neutralizing activity against these strains. In LL-23V9 serine 9 is substituted by valine creating a continuous hydrophobic surface. LL-23V9 has been shown to have increased anti-bacterial activity compared to LL-23 and we now show slightly increased antiviral activity compared to LL-23 as well. The short central fragments, FK-13 and KR-12, which have anti-bacterial activity did not inhibit IAV. In contrast, a longer 20 amino acid central fragment of LL-37 (GI-20) had neutralizing activity similar to LL-37. None of the peptides inhibited viral hemagglutination or neuraminidase activity. We next tested activity of the peptides against a strain of pandemic H1N1 of 2009 (A/California/04/09/H1N1 or “Cal09”). Unexpectedly, LL-37 had markedly reduced activity against Cal09 using several cell types and assays of antiviral activity. A mutant viral strain containing just the hemagglutinin (HA) of 2009 pandemic H1N1 was inhibited by LL-37, suggested that genes other than the HA are involved in the resistance of pH1N1. In contrast, GI-20 did inhibit Cal09. In conclusion, the central helix of LL-37 incorporated in GI-20 appears to be required for optimal antiviral activity. The finding that GI-20 inhibits Cal09 suggests that it may be possible to engineer derivatives of LL-37 with improved antiviral properties.     

Antimicrobial and antibiofilm activity of cathelicidins and short, synthetic peptides against Francisella

Francisella infects the lungs causing pneumonic tularemia. Focusing on the lung’s host defense, we have examined antimicrobial peptides as part of the innate immune response to Francisella infection. Interest in antimicrobial peptides, such as the cathelicidins, has grown due their potential therapeutic applications and the increasing problem of bacterial resistance to commonly used antibiotics. Only one human cathelicidin, LL-37, has been characterized. Helical cathelicidins have also been discovered in snakes including the Chinese King Cobra, Naja atra (NA-CATH). Four synthetic 11-residue peptides (ATRA-1, -2, -1A and -1P) containing variations of a repeated motif within NA-CATH were designed. We hypothesized that these smaller synthetic peptides could have excellent antimicrobial effectiveness with shorter length (and less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds. We tested the susceptibility of F. novicida to four ATRA peptides, LL-37, and NA-CATH. Two of the ATRA peptides had high antimicrobial activity (μM), while the two proline-containing ATRA peptides had low activity. The ATRA peptides did not show significant hemolytic activity even at high peptide concentration, indicating low cytotoxicity against host cells. NA-CATH killed Francisella bacteria more quickly than LL-37. However, LL-37 was the most effective peptide against F. novicida (EC50 = 50 nM). LL-37 mRNA was induced in A549 cells by Francisella infection. We recently demonstrated that F. novicida forms in vitro biofilms. LL-37 inhibited F. novicida biofilm formation at sub-antimicrobial concentrations. Understanding the properties of these peptides, and their endogenous expression in the lung could lead to potential future therapeutic interventions for this lung infection.     

The Host Defence Peptide LL-37 is Susceptible to Proteolytic Degradation by Wound Fluid Isolated from Foot Ulcers of Diabetic Patients

The pleiotropic effects of host defence peptides (HDPs), including the ability to kill microorganisms, enhance re-epithelialisation and increase angiogenesis, indicates a role for these important peptides as potential therapeutic agents in the treatment of chronic, non-healing wounds. However, the maintenance of peptide integrity, through resistance to degradation by the array of proteinases present at the wound site, is a prerequisite for clinical success. In this study we explored the degradation of exogenous LL-37, one such HDP, by wound fluid from diabetic foot ulcers to determine its susceptibility to proteolytic degradation. Our results suggest that LL-37 is unstable in the diabetic foot ulcer microenvironment. Following overnight treatment with wound fluid, LL-37 was completely degraded. Analysis of cleavage sites suggested potential involvement of both host- and bacterial-derived proteinases. The degradation products were shown to retain some antibacterial activity against Pseudomonas aeruginosa but were inactive against Staphylococcus aureus. In conclusion, our data suggest that stabilising selected peptide bonds within the sequence of LL-37 would represent an avenue for future research prior to clinical studies to address its potential as an exogenously-applied therapeutic in diabetic wounds.     

Snake Cathelicidin NA-CATH and Smaller Helical Antimicrobial Peptides Are Effective against Burkholderia thailandensis

Burkholderia thailandensis is a Gram-negative soil bacterium used as a model organism for B. pseudomallei, the causative agent of melioidosis and an organism classified category B priority pathogen and a Tier 1 select agent for its potential use as a biological weapon. Burkholderia species are reportedly “highly resistant” to antimicrobial agents, including cyclic peptide antibiotics, due to multiple resistance systems, a hypothesis we decided to test using antimicrobial (host defense) peptides. In this study, a number of cationic antimicrobial peptides (CAMPs) were tested in vitro against B. thailandensis for both antimicrobial activity and inhibition of biofilm formation. Here, we report that the Chinese cobra (Naja atra) cathelicidin NA-CATH was significantly antimicrobial against B. thailandensis. Additional cathelicidins, including the human cathelicidin LL-37, a sheep cathelicidin SMAP-29, and some smaller ATRA peptide derivatives of NA-CATH were also effective. The D-enantiomer of one small peptide (ATRA-1A) was found to be antimicrobial as well, with EC50 in the range of the L-enantiomer. Our results also demonstrate that human alpha-defensins (HNP-1 & -2) and a short beta-defensin-derived peptide (Peptide 4 of hBD-3) were not bactericidal against B. thailandensis. We also found that the cathelicidin peptides, including LL-37, NA-CATH, and SMAP-29, possessed significant ability to prevent biofilm formation of B. thailandensis. Additionally, we show that LL-37 and its D-enantiomer D-LL-37 can disperse pre-formed biofilms. These results demonstrate that although B. thailandensis is highly resistant to many antibiotics, cyclic peptide antibiotics such as polymyxin B, and defensing peptides, some antimicrobial peptides including the elapid snake cathelicidin NA-CATH exert significant antimicrobial and antibiofilm activity towards B. thailandensis.     

FK-16 Derived from the Anticancer Peptide LL-37 Induces Caspase-Independent Apoptosis and Autophagic Cell Death in Colon Cancer Cells

Host immune peptides, including cathelicidins, have been reported to possess anticancer properties. We previously reported that LL-37, the only cathelicidin in humans, suppresses the development of colon cancer. In this study, the potential anticancer effect of FK-16, a fragment of LL-37 corresponding to residues 17 to 32, on cultured colon cancer cells was evaluated. FK-16 induced a unique pattern of cell death, marked by concurrent activation of caspase-independent apoptosis and autophagy. The former was mediated by the nuclear translocation of AIF and EndoG whereas the latter was characterized by enhanced expression of LC3-I/II, Atg5 and Atg7 and increased formation of LC3-positive autophagosomes. Knockdown of Atg5 or Atg7 attenuated the cytotoxicity of FK-16, indicating FK-16-induced autophagy was pro-death in nature. Mechanistically, FK-16 activated nuclear p53 to upregulate Bax and downregulate Bcl-2. Knockdown of p53, genetic ablation of Bax, or overexpression of Bcl-2 reversed FK-16-induced apoptosis and autophagy. Importantly, abolition of AIF/EndoG-dependent apoptosis enhanced FK-16-induced autophagy while abolition of autophagy augmented FK-16-induced AIF−/EndoG-dependent apoptosis. Collectively, FK-16 induces caspase-independent apoptosis and autophagy through the common p53-Bcl-2/Bax cascade in colon cancer cells. Our study also uncovered previously unknown reciprocal regulation between these two cell death pathways.     

Antibacterial and anti-biofilm effects of cathelicidin peptides against pathogens isolated from cystic fibrosis patients

Six different cathelicidin-derived peptides were compared to tobramycin for antibacterial and anti-biofilm effects against S. aureus, P. aeruginosa, and S. maltophilia strains isolated from cystic fibrosis patients. Overall, SMAP-29, BMAP-28, and BMAP-27 showed relevant antibacterial activity (MIC₅₀ 4-8 μg/ml), and in some cases higher than tobramycin. In contrast, indolicidin, LL-37, and Bac7(1-35) showed no significant antimicrobial activity (MIC₅₀ > 32 μg/ml). Killing kinetics experiments showed that in contrast to tobramycin the active cathelicidin peptides exert a rapid bactericidal activity regardless of the species tested. All three peptides significantly reduced biofilm formation by S. maltophilia and P. aeruginosa strains at 1/2× MIC, although at a lower extent than tobramycin. In addition, BMAP-28, as well as tobramycin, was also active against S. aureus biofilm formation. Preformed biofilms were significantly affected by bactericidal SMAP-29, BMAP-27 and BMAP-28 concentrations, although at a lesser extent than tobramycin. Overall, our results indicate the potential of some cathelicidin-derived peptides for the development of novel therapeutic agents for cystic fibrosis lung disease.     

Antimicrobial activity of four cationic peptides immobilised to poly-hydroxyethylmethacrylate

The objective of this study was to immobilise and characterise a variety of antimicrobial peptides (AMPs) onto poly-hydroxyethylmethacrylate (pHEMA) surfaces to achieve an antibacterial effect. Four AMPs, viz. LL-37, melimine, lactoferricin and Mel-4 were immobilised on pHEMA by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) which assisted covalent attachment. Increasing concentrations of AMPs were immobilised to determine the effect on the adhesion of Pseudomonas aeruginosa and Staphylococcus aureus. The AMP immobilised pHEMAs were characterised by X-ray photoelectron spectroscopy (XPS) to determine the surface elemental composition and by amino acid analysis to determine the total amount of AMP attached. In vitro cytotoxicity of the immobilised pHEMA samples to mouse L929 cells was investigated. Melimine and Mel-4 when immobilised at the highest concentrations showed 3.1 ± 0.6 log and 1.3 ± 0.2 log inhibition against P. aeruginosa, and 3.9 ± 0.6 log and 2.4 ± 0.5 log inhibition against S. aureus, respectively. Immobilisation of LL-37 resulted in up to 2.6 ± 1.0 log inhibition against only P. aeruginosa, but no activity against S. aureus. LFc attachment showed no antibacterial activity. Upon XPS analysis, immobilised melimine, LL-37, LFc and Mel-4 had 1.57 ± 0.38%, 1.13 ± 1.36%, 0.66 ± 0.47% and 0.73 ± 0.32% amide nitrogen attached to pHEMA compared to 0.12 ± 0.14% in the untreated controls. Amino acid analysis determined that the total amount of AMP attachment to pHEMA was 44.3 ± 7.4 nmol, 3.8?±?0.2?nmol, 6.5 ± 0.6 nmol and 48.9 ± 2.3 nmol for the same peptides respectively. None of the AMP immobilised pHEMA surfaces showed any toxicity towards mouse L929 cells. The immobilisation of certain AMPs at nanomolar concentration to pHEMA is an effective option to develop a stable antimicrobial surface.     

The Human Antimicrobial Peptide LL-37 Binds Directly to CsrS,a Sensor Histidine Kinase of Group A Streptococcus,to Activate Expression of Virulence Factors

Group A Streptococcus (GAS) responds to subinhibitory concentrations of LL-37 by up-regulation of virulence factors through the CsrRS (CovRS) two-component system. The signaling mechanism, however, is unclear. To determine whether LL-37 signaling reflects specific binding to CsrS or rather a nonspecific response to LL-37-mediated membrane damage, we tested LL-37 fragments for CsrRS signaling and for GAS antimicrobial activity. We identified a 10-residue fragment (RI-10) of LL-37 as the minimal peptide that retains the ability to signal increased expression of GAS virulence factors, yet it has no detectable antimicrobial activity against GAS. Substitution of individual key amino acids in RI-10 reduced or abrogated signaling. These data do not support the hypothesis that CsrS detects LL-37-induced damage to the bacterial cell membrane but rather suggest that LL-37 signaling is mediated by a direct interaction with CsrS. To test whether LL-37 binds to CsrS, we used the purified CsrS extracellular domain to pull down LL-37 in vitro, a result that provides further evidence that LL-37 binds to CsrS. The dissociation of CsrS-mediated signaling from membrane damage by LL-37 fragments together with in vitro evidence for a direct LL-37-CsrS binding interaction constitute compelling evidence that signal transduction by LL-37 through CsrS reflects a direct ligand/receptor interaction.     

Synthesis and in vitro microbiological evaluation of novel diethyl 6,6′-(1,4-phenylene)bis(4-aryl-2-oxo-cyclohex-3-enecarboxylates)

Novel bis cyclohexenone ester derivatives 14–19 were synthesized and characterized by their spectral data. In vitro microbiological evaluations were carried out for all the novel compounds 14–19 against clinically isolated bacterial and fungal strains. Compounds 15, 16, 18 against Staphylococcus aureus, 14, 15 against β-Haemolytic streptococcus, 15, 19 against Micrococcus luteus, 17, 18 against Salmonella typhii, 14, 17 against Shigella flexneri, 15 against Escherichia coli, 16 against Pseudomonas aeruginosa, 15, 18, 19 against Klebsiella pneumonia exhibited potent antibacterial activity at an minimum inhibitory concentration (MIC) value of 6.25 μg/ml, whereas compound 16 against Aspergillus flavus, 17 against A. niger, 16, 18 against Mucor indicus, 15, 17–19 against Microsporum gypseum revealed excellent antifungal activity at an MIC value of 6.25 μg/ml.     

Interaction of sortase A and lipase 2 in the inhibition of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilm formation

Recombinant sortase A (SrtA) was used to immune rabbit, and the inhibitory activity of anti-SrtA serum on Staphylococcus aureus biofilm formation was tested. Biofilm formation was inhibited by anti-SrtA rabbit serum in S. aureus ATCC25923 and two clinical isolated strains. The antiserum was separated into two fractions, and the main component with the inhibitory activity was demonstrated to be the IgG fraction. Two proteins interact with the IgG fraction were identified by using an in vitro pull-down assay and were confirmed to be lipase 2 and γ-hemolysin by mass spectrometry. Cross-interaction between SrtA and lipase 2 was further confirmed by Western blotting. Addition of anti-lipase 2 serum in the culture medium also showed inhibitory effect against biofilm formation. Together, our study suggests anti-SrtA serum inhibits S. aureus biofilm formation and lipase 2 is one of the targets of anti-SrtA serum in this inhibition process. This is the first study to demonstrate the roles of antisera against SrtA and lipase 2 in the inhibition of biofilm formation in S. aureus.     

Uropathogenic Escherichia coli Modulates Immune Responses and Its Curli Fimbriae Interact with the Antimicrobial Peptide LL-37

Bacterial growth in multicellular communities, or biofilms, offers many potential advantages over single-cell growth, including resistance to antimicrobial factors. Here we describe the interaction between the biofilm-promoting components curli fimbriae and cellulose of uropathogenic E. coli and the endogenous antimicrobial defense in the urinary tract. We also demonstrate the impact of this interplay on the pathogenesis of urinary tract infections. Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys. Interestingly, LL-37 inhibited curli formation by preventing the polymerization of the major curli subunit, CsgA. Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro. Taken together, our data demonstrate that biofilm components are involved in the pathogenesis of urinary tract infections by E. coli and can be a target of local immune defense mechanisms.     

Antimicrobial efficacy of the alkaloid harmaline alone and in combination with chlorhexidine digluconate against clinical isolates of Staphylococcus aureus grown in planktonic and biofilm cultures

Aims: To investigate the antimicrobial efficacy of an alkaloid, harmaline alone and in combination with chlorhexidine digluconate (CHG) against clinical isolates of Staphylococcus aureus (S. aureus) grown in planktonic and biofilm cultures. Methods: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for each micro‐organism grown in suspension and in biofilm using microbroth dilution method. Chequerboard assays were used to determine synergistic, indifferent or antagonistic interactions between harmaline and CHG, and the some of results were verified by confocal laser scanning microscopy. Results: Harmaline and CHG showed effective antimicrobial activity against suspensions and biofilm cultures of S. aureus, respectively. As determined by fractional inhibitory concentration index (FICI), synergistic antimicrobial effects between harmaline and CHG were observed in nine and 11 of the 13 S. aureus strains when in suspension and in biofilm, respectively. FICI values were from 0·375 to 1·25 when in suspension and from 0·25 to 1·25 when in biofilm. Conclusions: Synergistic activity of harmaline and CHG against clinical isolates of S. aureus (in suspension and in biofilm) was observed in vitro. Significance and Impact of the Study: This study might provide alternative methods to overcome the problem of drug‐resistance of S. aureus both in suspension and in biofilm.