Legionella pneumophila cell surface RtxA release by LapD/LapG and its role in virulence.

BACKGROUND: Legionella pneumophila is a Gram-negative intracellular bacillus and is the causative agent of a severe form of pneumonia called Legionnaires' disease which accounts for 2-9% of cases of community acquired pneumonia. It produces an extremely large protein belonging to the RTX (Repeats in ToXin) family, called RtxA, and we previously reported that RtxA is transported by a dedicated type 1 secretion system (T1SS) to the cell surface. RTX proteins have been shown to participate in the virulence or biofilm formation of various bacteria, the most studied models being the pore forming hemolysin A (HlyA) of Escherichia coli and the biofilm associated protein LapA of P. fluorescens. LapA localization depends on the enzymatic release by LapD/LapG complex activity. This study aimed to elucidate the dual localization (cell surface associated or released state) of L. pneumophila RTX protein (RtxA) and whether this released versus sequestered state of RtxA plays a role in L. pneumophila virulence. RESULTS: The hereby work reveals that, in vitro, LapG periplasmic protease cleaves RtxA N-terminus in the middle of a di-alanine motif (position 108-109). Consistently, a strain lacking LapG protease maintains RtxA on the cell surface, whereas a strain lacking the c-di-GMP receptor LapD does not exhibit cell surface RtxA because of its continuous cleavage and release, as in the LapA-D-G model of Pseudomonas fluorescens. Interestingly, our data point out a key role of RtxA in enhancing the infection process of amoeba cells, regardless of its location (embedded or released); therefore, this may be the result of a secondary role of this surface protein. CONCLUSIONS: This is the first experimental identification of the cleavage site within the RTX protein family. The primary role of RtxA in Legionella is still questionable as in many other bacterial species, hence it sounds reasonable to propose a major function in biofilm formation, promoting cell aggregation when RtxA is embedded in the outer membrane and facilitating biofilm dispersion in case of RtxA release. The role of RtxA in enhancing the infection process may be a result of its action on host cells (i.e., PDI interaction or pore-formation), and independently of its status (embedded or released).

Antioxidant and Antisteatotic Activities of a New Fucoidan Extracted from Ferula hermonis Roots Harvested on Lebanese Mountains.

Fucoidan is a fucose-rich sulfated polysaccharide with attractive therapeutic potential due to a variety of biological activities, including antioxidant action. Fucoidan is typically found in the cell wall of marine brown algae, but extra-algal sources have also been discovered. In the present work, for the first time we extracted a water soluble fucoidan fraction from the roots of the terrestrial shrub Ferula hermonis. This fucoidan fraction was termed FUFe, and contained fucose, glucose, sulfate, smaller amounts of monosaccharides such as galactose and mannose, and a minor quantity of proteins. FUFe structural features were investigated by FTIR, 1H NMR and 13C NMR spectroscopy. The antioxidant property of FUFe was measured by DPPH, ABTS and FRAP assays, which revealed a high radical scavenging capacity that was confirmed in in vitro cellular models. In hepatic and endothelial cells, 50 µg/mL FUFe could reduce ROS production induced by intracellular lipid accumulation. Moreover, in hepatic cells FUFe exhibited a significant antisteatotic action, being able to reduce intracellular triglyceride content and to regulate the expression of key genes of hepatic lipid metabolism. Altogether, our results candidate FUFe as a possible bioactive compound against fatty liver disease and related vascular damage.

Antibacterial and antibiofilm activities of Scorzonera mackmeliana.

Scorzonera have been confirmed to have potent bioactivity. Scorzonera mackmeliana (Asteraceae), the endemic plant to Lebanon, has not yet been investigated. In the present study, we assessed the antibacterial activity of S. mackmeliana extracts against referenced bacterial strains. Extracts from different parts of the plant were evaluated against Staphylococcus, Enterococcus, Escherichia and Pseudomonas species. Phytochemical screening was done by standard biochemical tests and minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and minimal biofilm eradication concentration (MBEC) were determined by micro dilution method. The extracts possessed mainly alkaloids, phenols, flavonoids and coumarins. Gram-negative bacteria were most sensitive, whose MICs ranged between 48.98 and 341.85 mg/ml. Water stems extract, rich in phenols, was the most active with an MIC of 48.98 mg/ml. MBC was only recorded for water flowers extract, rich in resins, against P. aeruginosa and ethanolic roots extract, rich in terpenoids, against S. epidermidis with values of 160.85 mg/ml and 284.35 mg/ml, respectively. Furthermore, antibiofilm activity showed that the lowest MBEC was 0.1 mg/ml for water stems extract with an eradication ability of 91% (p <0.0001). Hence, this study suggests S. mackmeliana as a promising candidate for future investigations to elucidate the major bioactive compound behind the antibacterial and antibiofilm effect.

Carboxylic acids from brown algae Fucus vesiculosus and Padina pavonica.

Using the gas chromatographic-mass spectrometric method, the content of 28 carboxylic acids was determined in the thalli of Fucus vesiculosus, palmitic, oxalic and malic acids predominating. In the thalli of Padina pavonica, the content of 27 carboxylic acids was determined. Palmitic, oxalic and oleic acids prevail.Key words: brown algae Fucus vesiculosus Padina pavonica, carboxylic acids gas chromatography mass spectrometry.

Detection of SARS-CoV-2 B.1.1.529 (Omicron) variant by SYBR Green-based RT-qPCR.

The coronavirus disease 2019 (COVID-19) pandemic is unceasingly spreading across the globe, and recently a highly transmissible Omicron SARS-CoV-2 variant (B.1.1.529) has been discovered in South Africa and Botswana. Rapid identification of this variant is essential for pandemic assessment and containment. However, variant identification is mainly being performed using expensive and time-consuming genomic sequencing. In this study, we propose an alternative RT-qPCR approach for the detection of the Omicron BA.1 variant using a low-cost and rapid SYBR Green method. We have designed specific primers to confirm the deletion mutations in the spike (S Δ143-145) and the nucleocapsid (N Δ31-33) which are characteristics of this variant. For the evaluation, we used 120 clinical samples from patients with PCR-confirmed SARS-CoV-2 infections, and displaying an S-gene target failure (SGTF) when using TaqPath COVID-19 kit (Thermo Fisher Scientific, Waltham, USA) that included the ORF1ab, S, and N gene targets. Our results showed that all the 120 samples harbored S Δ143-145 and N Δ31-33, which was further confirmed by whole-genome sequencing of 10 samples, thereby validating our SYBR Green-based protocol. This protocol can be easily implemented to rapidly confirm the diagnosis of the Omicron BA.1 variant in COVID-19 patients and prevent its spread among populations, especially in countries with high prevalence of SGTF profile.

Scar-Free Genome Editing in Legionella pneumophila.

Studying bacterial physiology and pathogenesis often requires isolation of targeted mutants. From the early days of bacterial genetics, many genetic tools have been developed to achieve this goal in a lot of bacteria species, and a major key is to be able to manipulate the targeted genome region with a minimum impact on the rest of the genome. Here, we described a two-step protocol relevant in Legionella pneumophila. This efficient two-step protocol uses the natural transformability of L. pneumophila and linear DNA fragments as substrates for recombination without the necessity of intermediate hosts to amplify targeted DNA. Based on a suicide cassette strategy, this genetic toolbox enables to generate clean scar-free deletions, single-nucleotide mutation, transcriptional or translational fusions, as well as insertion at any chosen place in L. pneumophila chromosome, therefore enabling multiple mutations with no need of multiple selection markers.

Screening for antibacterial and antibiofilm activities in Astragalus angulosus.

AIM: In a search for finding novel therapeutic agents, extracts from an endemic Lebanese plant, Astragalus angulosus, were evaluated for their potential in-vitro antibacterial and antibiofilm activities against three Gram-positive bacterial strains; Staphylococcus epidermidis (CIP444), Staphylococcus aureus (ATCC25923), and Enterococcus faecalis (ATCC29212); in addition to two Gram-negative strains, Escherichia coli (ATCC35218) and Pseudomonas aeruginosa (ATCC27853). MATERIALS AND METHODS: The plant was collected in April of 2013 and divided into several different portions, then its extracts were obtained by maceration using two different solvents. Extract analysis followed directly where microtiter broth dilution method was employed to assess antibacterial activity, while antibiofilm potential was tested using colorimetric method. RESULTS: Whole plant ethanolic extract showed the highest bacteriostatic effect at a concentration of 12.78 mg/ml and also was the most versatile exerting its effect against 3 different strains. Other extracts also exhibited an effect but at higher concentrations and each against a single strain. Regarding antibiofilm activity, the majority of the extracts were able to eradicate >50% of S. epidermidis preformed biofilm, where the highest activity was obtained with flower fraction extracted in water, achieving 67.7% biofilm eradication at 0.2 mg/ml. CONCLUSIONS: This plant possesses a promising potential in regard to eradicating bacteria and their biofilms and it is the first contributing step of establishing a library for the endemic Lebanese plants in this domain.

Antibacterial and antibiofilm activities of polysaccharides, essential oil, and fatty oil extracted from Laurus nobilis growing in Lebanon.

OBJECTIVE: To evaluate the antibacterial activity of the extracts of Laurus nobilis against three Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212 and Staphylococcus epidermidis CIP 444) and two Gram-negative bacteria (Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853). Also, the antibiofilm activity has been investigated against the biofilm produced by Staphylococcus epidermidis CIP 444. MATERIALS: The polysaccharides, essential oil, and fatty oils extracted from the plant were used in broth microdilution methods to study the minimal inhibitory concentration, and then the minimal bactericidal concentration was determined. RESULTS: The results showed that alginate, fucoidan, fatty oils and essential oil have good antibacterial activities against the 5 bacterial strains, and a negligible biofilm eradication activity of fucoidan, laminaran, fatty oil, and essential oil was observed, but a promising biofilm eradication activity was obtained with alginate, which showed a reduced biofilm mass even at low concentration. CONCLUSIONS: The extracts obtained have promising antibacterial capacities which need further investigation for them to be incorporated in medical or nutritional applications.