Rapid Colorimetric Detection of Pseudomonas aeruginosa in Clinical Isolates Using a Magnetic Nanoparticle Biosensor

A rapid, sensitive, and specific colorimetric biosensor based on the use of magnetic nanoparticles (MNPs) was designed for the detection of Pseudomonas aeruginosa in clinical samples. The biosensing platform was based on the measurement of P. aeruginosa proteolytic activity using a specific protease substrate. At the N-terminus, this substrate was covalently bound to MNPs and was linked to a gold sensor surface via cystine at the C-terminus of the substrates. The golden sensor appears black to naked eyes because of the coverage of the MNPs. However, upon proteolysis, the cleaved peptide–MNP moieties will be attracted by an external magnet, revealing the golden color of the sensor surface, which can be observed by the naked eye. In vitro, the biosensor was able to detect specifically and quantitatively the presence of P. aeruginosa with a detection limit of 102 cfu/mL in less than 1 min. The colorimetric biosensor was used to test its ability to detect in situ P. aeruginosa in clinical isolates from patients. This biochip is anticipated to be useful as a rapid point-of-care device for the diagnosis of P. aeruginosa-related infections

Evaluation of a FRET-peptide substrate to predict virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa produces a number of proteases that are associated with virulence and disease progression. A substrate able to detect P. aeruginosa-specific proteolytic activity could help to rapidly alert clinicians to the virulence potential of individual P. aeruginosa strains. For this purpose we designed a set of P. aeruginosa-specific fluorogenic substrates, comprising fluorescence resonance energy transfer (FRET)-labeled peptides, and evaluated their applicability to P. aeruginosa virulence in a range of clinical isolates. A FRET-peptide comprising three glycines (3xGly) was found to be specific for the detection of P. aeruginosa proteases. Further screening of 97 P. aeruginosa clinical isolates showed a wide variation in 3xGly cleavage activity. The absence of 3xGly degradation by a lasI knock out strain indicated that 3xGly cleavage by P. aeruginosa could be quorum sensing (QS)-related, a hypothesis strengthened by the observation of a strong correlation between 3xGly cleavage, LasA staphylolytic activity and pyocyanin production. Additionally, isolates able to cleave 3xGly were more susceptible to the QS inhibiting antibiotic azithromycin (AZM). In conclusion, we designed and evaluated a 3xGly substrate possibly useful as a simple tool to predict virulence and AZM susceptibility

Is TIMP-1 a biomarker for periodontal disease?: A systematic review and meta-analysis

Objective: One of the most important families of proteases associated with periodontal disease is the family of the matrix metalloproteinases (MMPs). Their activity is regulated by tissue inhibitors of metalloproteinases (TIMPs), and an imbalance between MMP activity and regulation by TIMPs has been associated with the progression of periodontal disease. This strong interaction between TIMPs and MMPs might be an indication that TIMPs can be used as a biomarker to monitor periodontal disease progression in oral fluids. In particular, TIMP-1 is a frequently studied biomarker for periodontal diseases. Therefore, the aim of this systematic review was to evaluate the scientific literature regarding TIMP-1 concentrations in oral fluids of patients suffering from periodontitis or gingivitis in comparison to healthy individuals. Material and Methods: PubMed/ MedLine and Web of Science databases were searched electronically. Studies that met the inclusion criteria were systematically evaluated and assessed for eligibility and risk of bias. Meta-analysis was performed through the random effects model to assess the association between periodontitis/gingivitis and TIMP-1 concentration in stimulated saliva, unstimulated saliva, and gingival crevicular fluid (GCF). Results: The search strategy provided a total of 322 studies of which 10 studies met all inclusion criteria. Two studies investigated TIMP-1 concentrations in GCF, three studies in unstimulated saliva, and five studies investigated TIMP-1 concentrations in stimulated saliva. Three studies revealed that TIMP-1 levels in oral fluids were significantly decreased in periodontal disease. Meta-analysis revealed that there is no statistically significant difference between TIMP-1 concentration in oral fluids of periodontitis/gingivitis patients in comparison to healthy individuals. Conclusions: This systematic review with meta-analysis shows that periodontal diseases are not associated with a statistically significant change in TIMP-1 concentration in oral fluids

Gramicidin A is hydrolyzed by a d-stereospecific peptidase produced by Bacillus anthracis

Previously we described the discovery of a Bacillus spp. specific peptidase activity related to d-stereospecific peptidases (DSPs). The peptidase showed a strong preference for d-leucine and d-valine amino acids. These amino acids are present in the structure of the non-ribosomal peptide (NRP) antibiotics gramicidin A, B and C and polymyxin E. To examine if the Bacillus spp. DSP-related peptidase can hydrolyze these NRPs, the effect of gramicidin A and C and polymyxin E on peptidase activity in Bacillus anthracis culture supernatant was monitored. It was found that both gramicidins inhibited the DSP-related activity in a competitive manner. MALDI-TOF analysis revealed that upon incubation with B. anthracis culture supernatant gramicidin A hydrolyzation products appeared. This study shows that the Bacillus spp. specific DSP-like peptidase was potentially produced by the bacteria to gain intrinsic resistance against NRP antibiotics. These results are of utmost importance in research towards antimicrobial resistance, whereas transfer of DSP-related activity to other clinically relevant pathogens can be a serious threat to human health

Novel Antiamoebic Tyrocidine-Derived Peptide against Brain-Eating Amoebae

Acanthamoeba castellanii (A. castellanii) can cause Acanthamoeba keratitis, a sight-threatening infection, as well as a fatal brain infection termed granulomatous amoebic encephalitis, mostly in immunocompromised individuals. In contrast, Naegleria fowleri (N. fowleri) causes a deadly infection involving the central nervous system, recognized as primary amoebic encephalitis, mainly in individuals partaking in recreational water activities or those with nasal exposure to contaminated water. Worryingly, mortality rates due to these infections are more than 90%, suggesting the need to find alternative therapies. In this study, antiamoebic activity of a peptide based on the structure of the antibiotic tyrocidine was evaluated against A. castellanii and N. fowleri. The tyrocidine-derived peptide displayed significant amoebicidal efficacy against A. castellanii and N. fowleri. At 250 μg/mL, the peptide drastically reduced amoebae viability up to 13% and 21% after 2 h of incubation against N. fowleri and A. castellanii., whereas, after 24 h of incubation, the peptide showed 86% and 94% amoebicidal activity against A. castellanii and N. fowleri. Furthermore, amoebae pretreated with 100 μg/mL peptide inhibited 35% and 53% A. castellanii and N. fowleri, while, at 250 μg/mL, 84% and 94% A. castellanii and N. fowleri failed to adhere to human cells. Amoeba-mediated cell cytopathogenicity assays revealed 31% and 42% inhibition at 100 μg/mL, while at 250 μg/mL 75% and 86% A. castellanii and N. fowleri were inhibited. Assays revealed inhibition of encystation in both A. castellanii (58% and 93%) and N. fowleri (73% and 97%) at concentrations of 100 and 250 μg/mL respectively. Importantly, tyrocidine-derived peptide depicted minimal cytotoxicity to human cells and, thus, may be a potential candidate in the rational development of a treatment regimen against free-living amoebae infections. Future studies are necessary to elucidate the in vivo effects of tyrocidine-derived peptide against these and other pathogenic amoebae of importance

Proteolytic activity in <i>Nepenthes</i>.

<p><b>A</b> Samples of the pitcher fluid of ten different <i>Nepenthes</i> species were investigated for their PFU-093 cleaving activity. <b>B</b> Kinetics of proteolytic PFU-093 cleaving activity in pitcher fluid from open (grey line) and closed (black line) pitchers of <i>Nepenthes alata</i>. Relative fluorescence was measured over 12 h at 42°C.</p

Induction of nepenthesin protease activity in <i>Nepenthes mirabilis</i> pitcher fluid.

<p>Proteolytic activity was measured with PFU-093 substrate at different time points after various treatments: <b>A</b> supplementation with <i>D</i>. <i>melanogaster</i> (<i>n</i> = 3); <b>B</b> supplementation with <i>Nepenthes</i> leaf (<i>n</i> = 2); <b>C</b> injection of jasmonic acid (200 μM end concentration) (<i>n</i> = 3) <b>D</b> injection of salicylic acid (end concentration 200μM) (<i>n</i> = 4). Statistics employed one-way ANOVA, P<0.05, Post Hoc Tests_<i>SNK</i><b>(A, C, D),</b> Dunnett T3 <b>(B).</b></p

Proteolytic activity of recombinant <i>N</i>. <i>mirabilis</i> Nepenthesin II (<i>Nm</i>NepII/Sf9).

<p>PFU-093 fluorescence was measured over 6 h every 5 min after pre-incubation at pH 4 for 24 h for auto-activation. Different concentrations of <i>Nm</i>NepII/Sf9 were tested with constant concentrations of PFU-093 substrate. Line 1: 99 μl of <i>Nm</i>NepII/Sf9; line 2: 49 μl <i>Nm</i>NepII/Sf9; line 3: 24.5 μl <i>Nm</i>NepII/Sf9. All were mixed with pure H₂0 and 1 μl of 80 μM fluorescent substrate to a total volume of 100 μl per well. Controls: line 4: 99 μl <i>Nm</i>NepII/Sf9 and H₂O without the addition of fluorescent substrate; line 5: 99 μl <i>Nm</i>NepII/Sf9 and 1 μl of 80 μM PFU-093 without pre-incubation/ auto-activation in pH 4 glycine-buffer.</p