Modified activities of macrophages' deubiquitinating enzymes after Francisella infection.

Francisella tularensis influences several host molecular/signaling pathways during infection. Ubiquitination and deubiquitination are among the most important regulatory mechanisms and respectively occur through attachment or removal of the ubiquitin molecule. The process is necessary not only to mark molecules for degradation, but also, for example, to the activation of signaling pathways leading to pro-inflammatory host response. Many intracellular pathogens, including Francisella tularensis, have evolved mechanisms of modifying such host immune responses to escape degradation. Here, we describe that F. tularensis interferes with the host's ubiquitination system. We show increased total activity of deubiquitinating enzymes (DUBs) in human macrophages after infection, while confirm reduced enzymatic activities of two specific DUBs (USP10 and UCH-L5), and demonstrate increased activity of USP25. We further reveal the enrichment of these three enzymes in exosomes derived from F. tularensis-infected cells. The obtained results show the regulatory effect on ubiquitination mechanism in macrophages during F. tularensis infection.

Aggregation-aided SERS: Selective detection of arsenic by surface-enhanced Raman spectroscopy facilitated by colloid cross-linking.

Arsenic of natural or industrial origin often occurs in water and makes it impotable. Due to its high toxicity, very sensitive detection is required. In the present study an ultra-sensitive arsenite (As3+) sensing is reported, based on aggregation-aided surface-enhanced Raman scattering (AA-SERS) of modified silver colloids. SERS intensity of mercapto-compounds attached to the colloidal silver nanoparticles surface is greatly increased in the presence of arsenic. Colloid aggregation is facilitated by cross-linking; a meshwork consisting of arsenic atoms and glutathione bridges is formed, as indicated by UV-Vis absorption spectroscopy, TEM and Raman imaging. The best 2-mercaptopyridine reporter molecule makes it possible to directly detect As3+ at concentrations as low as 0.5 ppb, which is better than achieved by the SERS technique so far.

Characterization of insulin crystalline form in isolated ß-cell secretory granules.

Insulin is stored in vivo inside the pancreatic ß-cell insulin secretory granules. In vitro studies have led to an assumption that high insulin and Zn2+ concentrations inside the pancreatic ß-cell insulin secretory granules should promote insulin crystalline state in the form of Zn2+-stabilized hexamers. Electron microscopic images of thin sections of the pancreatic ß-cells often show a dense, regular pattern core, suggesting the presence of insulin crystals. However, the structural features of the storage forms of insulin in native preparations of secretory granules are unknown, because of their small size, fragile character and difficult handling. We isolated and investigated the secretory granules from MIN6 cells under near-native conditions, using cryo-electron microscopic (Cryo-EM) techniques. The analysis of these data from multiple intra-granular crystals revealed two different rhomboidal crystal lattices. The minor lattice has unit cell parameters (a ≃ b ≃ 84.0 Å, c ≃ 35.2 Å), similar to in vitro crystallized human 4Zn2+-insulin hexamer, whereas the largely prevalent unit cell has more than double c-axis (a ≃ b ≃ c ≃ 96.5 Å) that probably corresponds to two or three insulin hexamers in the asymmetric unit. Our experimental data show that insulin can be present in pancreatic MIN6 cell granules in a microcrystalline form, probably consisting of 4Zn2+-hexamers of this hormone.

Non-thermal plasma causes Pseudomonas aeruginosa biofilm release to planktonic form and inhibits production of Las-B elastase, protease and pyocyanin.

The increasing risk of antibiotic failure in the treatment of Pseudomonas aeruginosa infections is largely related to the production of a wide range of virulence factors. The use of non-thermal plasma (NTP) is a promising alternative to antimicrobial treatment. Nevertheless, there is still a lack of knowledge about the effects of NTP on the virulence factors production. We evaluated the ability of four NTP-affected P. aeruginosa strains to re-form biofilm and produce Las-B elastase, proteases, lipases, haemolysins, gelatinase or pyocyanin. Highly strains-dependent inhibitory activity of NTP against extracellular virulence factors production was observed. Las-B elastase activity was reduced up to 82% after 15-min NTP treatment, protease activity and pyocyanin production by biofilm cells was completely inhibited after 60 min, in contrast to lipases and gelatinase production, which remained unchanged. However, for all strains tested, a notable reduction in biofilm re-development ability was depicted using spinning disc confocal microscopy. In addition, NTP exposure of mature biofilms caused disruption of biofilm cells and their dispersion into the environment, as shown by transmission electron microscopy. This appears to be a key step that could help overcome the high resistance of P. aeruginosa and its eventual elimination, for example in combination with antibiotics still highly effective against planktonic cells.

Correction: Krízová et al. Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein. Viruses 2021, 13, 2451.

The authors wish to make the following corrections to this paper [...].

Photochemical synthesis of pink silver and its use for monitoring peptide nitration via surface enhanced Raman spectroscopy (SERS).

Oxidative stress may cause extended tyrosine posttranslational modifications of peptides and proteins. The 3-nitro-L-tyrosine (Nit), which is typically formed, affects protein behavior during neurodegenerative processes, such as Alzheimer's and Parkinson's diseases. Such metabolic products may be conveniently detected at very low concentrations by surface enhanced Raman spectroscopy (SERS). Previously, we have explored the SERS detection of the Nit NO2 bending vibrational bands in a presence of hydrogen chloride (Niederhafner et al., Amino Acids 53:517-532, 2021, ibid). In this article, we describe performance of a new SERS substrate, "pink silver", synthesized photochemically. It provides SERS even without the HCl induction, and the acid further decreases the detection limit about 9 times. Strong SERS bands were observed in the asymmetric (1550-1475 cm-1) and symmetric (1360-1290 cm-1) NO stretching in the NO2 group. The bending vibration was relatively weak, but appeared stronger when HCl was added. The band assignments were supported by density functional theory modeling.

Cobalt Bis-Dicarbollide Enhances Antibiotics Action towards Staphylococcus epidermidis Planktonic Growth Due to Cell Envelopes Disruption.

The emergence of antibiotic resistance in opportunistic pathogens represents a huge problem, the solution for which may be a treatment with a combination of multiple antimicrobial agents. Sodium salt of cobalt bis-dicarbollide (COSAN.Na) is one of the very stable, low-toxic, amphiphilic boron-rich sandwich complex heteroboranes. This compound has a wide range of potential applications in the biological sciences due to its antitumor, anti-HIV-1, antimicrobial and antibiofilm activity. Our study confirmed the ability of COSAN.Na (in the concentration range 0.2-2.48 µg/mL) to enhance tetracycline, erythromycin, and vancomycin action towards Staphylococcus epidermidis planktonic growth with an additive or synergistic effect (e.g., the combination of 1.24 µg/mL COSAN.Na and 6.5 µg/mL TET). The effective inhibitory concentration of antibiotics was reduced up to tenfold most efficiently in the case of tetracycline (from 65 to 6.5 µg/mL). In addition, strong effect of COSAN.Na on disruption of the cell envelopes was determined using propidium iodide uptake measurement and further confirmed by transmission electron microscopy. The combination of amphiphilic COSAN.Na with antibiotics can therefore be considered a promising way to overcome antibiotic resistance in Gram-positive cocci.

Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein.

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

The efficiency of insulin production and its content in insulin-expressing model ß-cells correlate with their Zn2+ levels.

Insulin is produced and stored inside the pancreatic ß-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model ß-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 ß-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent ß-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model ß-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.

Characterization and in vitro assembly of tick-borne encephalitis virus C protein.

Tick-borne encephalitis virus (TBEV), a member of flaviviruses, represents a serious health threat by causing human encephalitis mainly in central and eastern Europe, Russia, and northeastern Asia. As no specific therapy is available, there is an urgent need to understand all steps of the TBEV replication cycle at the molecular level. One of the critical events is the packaging of flaviviral genomic RNA by TBEV C protein to form a nucleocapsid. We purified recombinant TBEV C protein and used a combination of physical-chemical approaches, such as size-exclusion chromatography, circular dichroism, NMR spectroscopies, and transmission electron microscopy, to analyze its structural stability and its ability to dimerize/oligomerize. We compared the ability of TBEV C protein to assemble in vitro into a nucleocapsid-like structure with that of dengue C protein.

PF74 and Its Novel Derivatives Stabilize Hexameric Lattice of HIV-1 Mature-Like Particles.

A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein-protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.

Cellular Localization of Carbonic Anhydrase Nce103p in Candida albicans and Candida parapsilosis.

Pathogenic yeasts Candida albicans and Candida parapsilosis possess a ß-type carbonic anhydrase Nce103p, which is involved in CO2 hydration and signaling. C. albicans lacking Nce103p cannot survive in low CO2 concentrations, e.g., in atmospheric growth conditions. Candida carbonic anhydrases are orthologous to the Saccharomyces cerevisiae enzyme, which had originally been detected as a substrate of a non-classical export pathway. However, experimental evidence on localization of C. albicans and C. parapsilosis carbonic anhydrases has not been reported to date. Immunogold labeling and electron microscopy used in the present study showed that carbonic anhydrases are localized in the cell wall and plasmatic membrane of both Candida species. This localization was confirmed by Western blot and mass spectrometry analyses of isolated cell wall and plasma membrane fractions. Further analysis of C. albicans and C. parapsilosis subcellular fractions revealed presence of carbonic anhydrases also in the cytosolic and mitochondrial fractions of Candida cells cultivated in shaken liquid cultures, under the atmospheric conditions.

A simple, high-throughput stabilization assay to test HIV-1 uncoating inhibitors.

Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.

Antifungal activity of analogues of antimicrobial peptides isolated from bee venoms against vulvovaginal Candida spp.

Candida albicans is the main causative agent of vulvovaginal candidiasis (VVC), a common mycosis in women, relapses of which are difficult to manage due to biofilm formation. This study aimed at developing novel non-toxic compounds active against Candida spp. biofilms. We synthesised analogues of natural antifungal peptides LL-III (LL-III/43) and HAL-2 (peptide VIII) originally isolated from bee venoms and elucidated their structures by nuclear magnetic resonance spectroscopy. The haemolytic, cytotoxic, antifungal and anti-biofilm activities of LL-III/43 and peptide VIII were then tested. LL-III/43 and VIII showed moderate cytotoxicity to HUVEC-2 cells and had comparable inhibitory activity against C. albicans and non-albicans spp. The lowest minimum inhibitory concentration (MIC90) of LL-III/43 was observed towards Candida tropicalis (0.8 µM). That was 8-fold lower than that of antimycotic amphotericin B. Both peptides can be used to inhibit Candida spp. bio film f ormation. Biofilm inhibitory concentrations (BIC50) ranged from 0.9 to 58.6 µM and biofilm eradication concentrations (BEC50) for almost all tested Candida spp. strains ranged from 12.8 to 200 µM. Als o pro ven were the peptides' abilities to reduce the area colonised by biofilms , inhibit hyphae formation and permeabilise cell membranes in biofil ms . LL-III/43 and VIII are promising candidates for further development as therapeutics against VVC.

Sex-Pairing Pheromones in Three Sympatric Neotropical Termite Species (Termitidae: Syntermitinae).

Termite colonies are almost always founded by a pair of winged dispersers, in spite of the high costs and low success rates inherent in independent colony foundation. The dispersal flights of imagoes from natal colonies are followed by mate search, mediated by sex-pairing pheromones. Here, we studied the chemistry of sex-pairing pheromones and the related aspects of mate search in winged imagoes of two facultatively parthenogenetic species, Embiratermes neotenicus and Silvestritermes minutus, and an additional species from the same subfamily, Silvestritermes heyeri. All three species are widespread in the Neotropics, including the rainforests of French Guiana. After the dispersal flight and spontaneous loss of wings, females expose their hypertrophied tergal glands situated under abdominal tergites VIII - X. The females are attractive to males and, upon direct contact, the two sexes form characteristic tandems. Chemical analyses indicated that the females secrete species-specific combinations of unbranched, unsaturated C12 primary alcohols from the tergal glands, (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (approx. 200 pg per female) and (3Z)-dodec-3-enol (185 pg) in E. neotenicus, (3Z,6Z)-dodeca-3,6-dien-1-ol (3500 pg) in S. heyeri, and (3Z,6Z)-dodeca-3,6-dien-1-ol (300 pg) and (3Z)-dodec-3-enol (50 pg) in S. minutus. (3Z,6Z,8E)-Dodeca-3,6,8-trien-1-ol and (3Z,6Z)-dodeca-3,6-dien-1-ol act as major pheromone components in the respective species and mimic the function of female tergal gland extracts in electrophysiological and behavioral experiments. Biologically relevant amounts of the third compound, (3Z)-dodec-3-enol, elicited non-significant reactions in males of E. neotenicus and S. minutus, and slight synergistic effects in males of S. minutus when tested in combination with the major component.

Mutations in the Basic Region of the Mason-Pfizer Monkey Virus Nucleocapsid Protein Affect Reverse Transcription, Genomic RNA Packaging, and the Virus Assembly Site.

In addition to specific RNA-binding zinc finger domains, the retroviral Gag polyprotein contains clusters of basic amino acid residues that are thought to support Gag-viral genomic RNA (gRNA) interactions. One of these clusters is the basic K16NK18EK20 region, located upstream of the first zinc finger of the Mason-Pfizer monkey virus (M-PMV) nucleocapsid (NC) protein. To investigate the role of this basic region in the M-PMV life cycle, we used a combination of in vivo and in vitro methods to study a series of mutants in which the overall charge of this region was more positive (RNRER), more negative (AEAEA), or neutral (AAAAA). The mutations markedly affected gRNA incorporation and the onset of reverse transcription. The introduction of a more negative charge (AEAEA) significantly reduced the incorporation of M-PMV gRNA into nascent particles. Moreover, the assembly of immature particles of the AEAEA Gag mutant was relocated from the perinuclear region to the plasma membrane. In contrast, an enhancement of the basicity of this region of M-PMV NC (RNRER) caused a substantially more efficient incorporation of gRNA, subsequently resulting in an increase in M-PMV RNRER infectivity. Nevertheless, despite the larger amount of gRNA packaged by the RNRER mutant, the onset of reverse transcription was delayed in comparison to that of the wild type. Our data clearly show the requirement for certain positively charged amino acid residues upstream of the first zinc finger for proper gRNA incorporation, assembly of immature particles, and proceeding of reverse transcription.IMPORTANCE We identified a short sequence within the Gag polyprotein that, together with the zinc finger domains and the previously identified RKK motif, contributes to the packaging of genomic RNA (gRNA) of Mason-Pfizer monkey virus (M-PMV). Importantly, in addition to gRNA incorporation, this basic region (KNKEK) at the N terminus of the nucleocapsid protein is crucial for the onset of reverse transcription. Mutations that change the positive charge of the region to a negative one significantly reduced specific gRNA packaging. The assembly of immature particles of this mutant was reoriented from the perinuclear region to the plasma membrane. On the contrary, an enhancement of the basic character of this region increased both the efficiency of gRNA packaging and the infectivity of the virus. However, the onset of reverse transcription was delayed even in this mutant. In summary, the basic region in M-PMV Gag plays a key role in the packaging of genomic RNA and, consequently, in assembly and reverse transcription.

How proteases from Enterococcus faecalis contribute to its resistance to short α-helical antimicrobial peptides.

HYL-20 (GILSSLWKKLKKIIAK-NH2) is an analogue of a natural antimicrobial peptide (AMP) previously isolated from the venom of wild bee. We examined its antimicrobial activity against three strains of Enterococcus faecalis while focusing on its susceptibility to proteolytic degradation by two known proteases-gelatinase (GelE) and serine protease (SprE)-which are secreted by these bacterial strains. We found that HYL-20 was primarily deamidated at its C-terminal which made the peptide susceptible to consecutive intramolecular cleavage by GelE. Further study utilising 1,10-phenanthroline, a specific GelE inhibitor and analogous peptide with D-Lys at its C-terminus (HYL-20k) revealed that the C-terminal deamidation of HYL-20 is attributed to not yet unidentified protease which also cleaves internal peptide bonds of AMPs. In contrast to published data, participation of SprE in the protective mechanism of E. faecalis against AMPs was not proved. The resistance of HYL-20k to C-terminal deamidation and subsequent intramolecular cleavage has resulted in increased antimicrobial activity against E. faecalis grown in planktonic and biofilm form when compared to HYL-20.

Sensitive Versatile Fluorogenic Transmembrane Peptide Substrates for Rhomboid Intramembrane Proteases.

Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.

Functional and Structural Characterization of Novel Type of Linker Connecting Capsid and Nucleocapsid Protein Domains in Murine Leukemia Virus.

The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single α-helices. This is the first single α-helix motif identified in viral proteins.

Antimicrobial Peptide from the Wild Bee Hylaeus signatus Venom and Its Analogues: Structure-Activity Study and Synergistic Effect with Antibiotics.

Venoms of hymenopteran insects have attracted considerable interest as a source of cationic antimicrobial peptides (AMPs). In the venom of the solitary bee Hylaeus signatus (Hymenoptera: Colletidae), we identified a new hexadecapeptide of sequence Gly-Ile-Met-Ser-Ser-Leu-Met-Lys-Lys-Leu-Ala-Ala-His-Ile-Ala-Lys-NH2. Named HYL, it belongs to the category of α-helical amphipathic AMPs. HYL exhibited weak antimicrobial activity against several strains of pathogenic bacteria and moderate activity against Candida albicans, but its hemolytic activity against human red blood cells was low. We prepared a set of HYL analogues to evaluate the effects of structural modifications on its biological activity and to increase its potency against pathogenic bacteria. This produced several analogues exhibiting significantly greater activity compared to HYL against strains of both Staphylococcus aureus and Pseudomonas aeruginosa even as their hemolytic activity remained low. Studying synergism of HYL peptides and conventional antibiotics showed the peptides act synergistically and preferentially in combination with rifampicin. Fluorescent dye propidium iodide uptake showed the tested peptides were able to facilitate entrance of antibiotics into the cytoplasm by permeabilization of the outer and inner bacterial cell membrane of P. aeruginosa. Transmission electron microscopy revealed that treatment of P. aeruginosa with one of the HYL analogues caused total disintegration of bacterial cells. NMR spectroscopy was used to elucidate the structure-activity relationship for the effect of amino acid residue substitution in HYL.