Optimizing Alternative Substrate for Tomato Production in Arid Zone: Lesson from Growth, Water Relations, Chlorophyll Fluorescence, and Photosynthesis.

Soilless culture is considered the mostpromising, intensive, and sustainable approach with various advantages for plant production in terms of saving water and nutrients. It can provide consumers with sufficient and high-quality food. However, the commonly used growing substrate for soilless cultivation, coconut fiber (CF), is usually imported and expensive or even unavailable. The objectives of this study were to investigate the impact of local organic farm resources substrates on tomato (Solanum lycopersicum L.) plant growth, water relations, photosynthesis, chlorophyll fluorescence, and phytochemical analysis of fruits in a hydroponics culture system. Two growth substrates were evaluated: date-palm waste composted with animal manure (7:3 w/w) (DPAM) and date-palm trunk compost (DPT). CF and local soil were utilized as positive and negative controls, respectively, in randomized blocks. The results revealed that DPAM substrate enhanced plant growth and physiology: shoot development, leaves tissues hydration, and photosynthetic parameters, as well as chlorophyll fluorescence. However, DPT and CF improved fruit quality: water, mineral, sugar, and protein content. The antioxidant activity of the fruit extract was the greater in DPAM, reaching 13.8 mg GAEg-1 DW. This value wasdecreased in soil by 40%. Photosynthesis activity was the most important in DPAM with 12 µmol CO2 m-2 s-1, and only 6.4 µmol CO2 m-2 s-1 in the soil condition. However, regarding the non-photochemical quenching, the dissipated light energy was greater in soil (0.096 ± 0.02) than in DPAM (0.025 ± 0.04). Date-palm waste-based substrates improved tomato vegetative growth and fruit quality as compared to soil-based culture. Date-palm waste-based substrates supplemented with manure appear to be promising and less expensive alternatives to the coconut fiber substrate extensively used in soilless crops in North Africa.

PLGA nanocapsules improve the delivery of clarithromycin to kill intracellular Staphylococcus aureus and Mycobacterium abscessus.

Drug delivery systems are promising for targeting antibiotics directly to infected tissues. To reach intracellular Staphylococcus aureus and Mycobacterium abscessus, we encapsulated clarithromycin in PLGA nanocapsules, suitable for aerosol delivery by nebulization of an aqueous dispersion. Compared to the same dose of free clarithromycin, nanoencapsulation reduced 1000 times the number of intracellular S. aureus in vitro. In RAW cells, while untreated S. aureus was located in acidic compartments, the treated ones were mostly situated in non-acidic compartments. Clarithromycin-nanocapsules were also effective against M. abscessus (70-80% killing efficacy). The activity of clarithromycin-nanocapsules against S. aureus was also confirmed in vivo, using a murine wound model as well as in zebrafish. The permeability of clarithromycin-nanocapsules across Calu-3 monolayers increased in comparison to the free drug, suggesting an improved delivery to sub-epithelial tissues. Thus, clarithromycin-nanocapsules are a promising strategy to target intracellular S. aureus and M. abscessus.

H-NS is the major repressor of Salmonella Typhimurium Pef fimbriae expression.

Fimbriae play an important role in adhesion and are therefore essential for the interaction of bacteria with the environments they encounter. Most of them are expressed in vivo but not in vitro, thus making difficult the full characterization of these fimbriae. Here, we characterized the silencing of plasmid-encoded fimbriae (Pef) expression, encoded by the pef operon, in the worldwide pathogen Salmonella Typhimurium. We demonstrated that the nucleoid-associated proteins H-NS and Hha, and their respective paralogs StpA and YdgT, negatively regulate at pH 5.1 and pH 7.1 the transcription of the pef operon. Two promoters, PpefB and PpefA, direct the transcription of this operon. All the nucleoid-associated proteins silence the PpefB promoter and H-NS also targets the PpefA promoter. While Hha and YdgT are mainly considered as acting primarily through H-NS to modulate gene transcription, our results strongly suggest that Hha and YdgT silence pef transcription at acidic pH either by interacting with StpA or independently of H-NS and StpA. We also confirmed the previously described post-transcriptional repression of Pef fimbriae by CsrA titration via the fim mRNA and CsrB and CsrC sRNA. Finally, among all these regulators, H-NS clearly appeared as the major repressor of Pef expression. These results open new avenues of research to better characterize the regulation of these bacterial adhesive proteins and to clarify their role in the virulence of pathogens.

An efficient system for intracellular delivery of beta-lactam antibiotics to overcome bacterial resistance.

The "Golden era" of antibiotics is definitely an old story and this is especially true for intracellular bacterial infections. The poor intracellular bioavailability of antibiotics reduces the efficency of many treatments and thereby promotes resistances. Therefore, the development of nanodevices coupled with antibiotics that are capable of targeting and releasing the drug into the infected-cells appears to be a promising solution to circumvent these complications. Here, we took advantage of two natural terpenes (farnesyl and geranyl) to design nanodevices for an efficient intracellular delivery of penicillin G. The covalent linkage between the terpene moieties and the antibiotic leads to formation of prodrugs that self-assemble to form nanoparticles with a high drug payload between 55-63%. Futhermore, the addition of an environmentally-sensitive bond between the antibiotic and the terpene led to an efficient antibacterial activity against the intracellular pathogen Staphylococcus aureus with reduced intracellular replication of about 99.9% compared to untreated infected cells. Using HPLC analysis, we demonstrated and quantified the intracellular release of PenG when this sensitive-bond (SB) was present on the prodrug, showing the success of this technology to deliver antibiotics directly into cells.

Direct regulation of the pefI-srgC operon encoding the Rck invasin by the quorum-sensing regulator SdiA in Salmonella†Typhimurium.

One important step for the pathogenesis of Salmonella is its ability to penetrate host cells. Recently, a new entry system involving the outer membrane protein Rck has been characterized. Previous studies have shown that the pefI-srgC locus, which contains rck, was regulated by the temperature and SdiA, the transcriptional regulator of quorum sensing in Salmonella. To decipher the regulation of rck by SdiA, we first confirmed the operon organization of the pefI-srgC locus. Using plasmid-based transcriptional fusions, we showed that only the predicted distal promoter upstream of pefI, PefIP2, displays an SdiA- and acyl-homoserine lactones-dependent activity while the predicted proximal PefIP1 promoter exhibits a very low activity independent on SdiA in our culture conditions. A direct and specific interaction of SdiA with this PefIP2 region was identified using electrophoretic mobility shift assays and surface plasmon resonance studies. We also observed that Rck expression is negatively regulated by the nucleoid-associated H-NS protein at both 25°C and 37°C. This work is the first demonstration of a direct regulation of genes by SdiA in Salmonella and will help further studies designed to identify environmental conditions required for Rck expression and consequently contribute to better characterize the role of this invasin in vivo.

Nanocarriers for antibiotics: a promising solution to treat intracellular bacterial infections.

In the field of antibiotherapy, intracellular infections remain difficult to eradicate mainly due to the poor intracellular penetration of most of the commonly used antibiotics. Bacteria have quickly understood that their intracellular localisation allows them to be protected from the host immune system, but also from the action of antimicrobial agents. In addition, in most cases pathogens nestle in professional phagocytic cells, and can even use them as a 'Trojan horse' to induce a secondary site of infection thereby causing persistent or recurrent infections. Thus, new strategies had to be considered in order to counteract these problems. Amongst them, nanocarriers loaded with antibiotics represent a promising approach. Nowadays, it is possible to encapsulate, incorporate or even conjugate biologically active molecules into different families of nanocarriers such as liposomes or nanoparticles in order to deliver antibiotics intracellularly and hence to treat infections. This review gives an overview of the variety of nanocarriers developed to deliver antibiotics directly into infected cells.

Calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII)-mediated intramolecular opening of integrin cytoplasmic domain-associated protein-1 (ICAP-1α) negatively regulates ß1 integrins.

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the ß integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of ß1A integrins, limits both talin and kindlin interaction with ß1 integrin, thereby restraining focal adhesion assembly. Here we provide evidence that the calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII) is an important regulator of ICAP-1α for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1α and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1α, unmasking the PTB domain, thereby permitting ICAP-1α binding onto the ß1 integrin tail. ICAP-1α direct interaction with the ß1 integrin tail and the modulation of ß1 integrin affinity state are required for down-regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1α function by CaMKII, allowing the dynamic control of ß1 integrin activation and cell adhesion.

Deciphering the roles of BamB and its interaction with BamA in outer membrane biogenesis, T3SS expression and virulence in Salmonella.

The folding and insertion of ß-barrel proteins in the outer membrane of Gram-negative bacteria is mediated by the BAM complex, which is composed of the outer membrane protein BamA and four lipoproteins BamB to BamE. In Escherichia coli and/or Salmonella, the BamB lipoprotein is involved in (i) ß-barrel protein assembly in the outer membrane, (ii) outer membrane permeability to antibiotics, (iii) the control of the expression of T3SS which are major virulence factors and (iv) the virulence of Salmonella. In E. coli, this protein has been shown to interact directly with BamA. In this study, we investigated the structure-function relationship of BamB in order to assess whether the roles of BamB in these phenotypes were inter-related and whether they require the interaction of BamB with BamA. For this purpose, recombinant plasmids harbouring point mutations in bamB were introduced in a ΔSalmonella bamB mutant. We demonstrated that the residues L173, L175 and R176 are crucial for all the roles of BamB and for the interaction of BamB with BamA. Moreover, the results obtained with a D229A BamB variant, which is unable to immunoprecipitate BamA, suggest that the interaction of BamB with BamA is not absolutely necessary for BamB function in outer-membrane protein assembly, T3SS expression and virulence. Finally, we showed that the virulence defect of the ΔbamB mutant is not related to its increased susceptibility to antimicrobials, as the D227A BamB variant fully restored the virulence of the mutant while having a similar antibiotic susceptibility to the ΔbamB strain. Overall, this study demonstrates that the different roles of BamB are not all inter-related and that L173, L175 and R176 amino-acids are privileged sites for the design of BamB inhibitors that could be used as alternative therapeutics to antibiotics, at least against Salmonella.

Heterogeneity of type III secretion system (T3SS)-1-independent entry mechanisms used by Salmonella Enteritidis to invade different cell types.

Salmonella causes a wide range of diseases from acute gastroenteritis to systemic typhoid fever, depending on the host. To invade non-phagocytic cells, Salmonella has developed different mechanisms. The main invasion system requires a type III secretion system (T3SS) known as T3SS-1, which promotes a Trigger entry mechanism. However, other invasion factors have recently been described in Salmonella, including Rck and PagN, which were not expressed under our bacterial culture conditions. Based on these observations, we used adhesion and invasion assays to analyse the respective roles of Salmonella Enteritidis T3SS-1-dependent and -independent invasion processes at different times of infection. Diverse cell lines and cell types were tested, including endothelial, epithelial and fibroblast cells. We demonstrated that cell susceptibility to the T3SS-1-independent entry differs by a factor of nine between the most and the least permissive cell lines tested. In addition, using scanning electron and confocal microscopy, we showed that T3SS-1-independent entry into cells was characterized by a Trigger-like alteration, as for the T3SS-1-dependent entry, and also by Zipper-like cellular alteration. Our results demonstrate for what is believed to be the first time that Salmonella can induce Trigger-like entry independently of T3SS-1 and can induce Zipper-like entry independently of Rck. Overall, these data open new avenues for discovering new invasion mechanisms in Salmonella.

A comparative analysis of perturbations caused by a gene knock-out, a dominant negative allele, and a set of peptide aptamers.

The study of protein function mostly relies on perturbing regulatory networks by acting upon protein expression levels or using transdominant negative agents. Here we used the Escherichia coli global transcription regulator Fur (ferric uptake regulator) as a case study to compare the perturbations exerted by a gene knock-out, the expression of a dominant negative allele of a gene, and the expression of peptide aptamers that bind a gene product. These three perturbations caused phenotypes that differed quantitatively and qualitatively from one another. The Fur peptide aptamers inhibited the activity of their target to various extents and reduced the virulence of a pathogenic E. coli strain in Drosophila. A genome-wide transcriptome analysis revealed that the "penetrance" of a peptide aptamer was comparable to that of a dominant negative allele but lower than the penetrance of the gene knock-out. Our work shows that comparative analysis of phenotypic and transcriptome responses to different types of perturbation can help decipher complex regulatory networks that control various biological processes.

Disruption of focal adhesions by integrin cytoplasmic domain-associated protein-1 alpha.

Regulation of integrin affinity and clustering plays a key role in the control of cell adhesion and migration. The protein ICAP-1 alpha (integrin cytoplasmic domain-associated protein-1 alpha) binds to the cytoplasmic domain of the beta(1A) integrin and controls cell spreading on fibronectin. Here, we demonstrate that, despite its ability to interact with beta(1A) integrin, ICAP-1 alpha is not recruited in focal adhesions, whereas it is colocalized with the integrin at the ruffling edges of the cells. ICAP-1 alpha induced a rapid disruption of focal adhesions, which may result from the ability of ICAP-1 alpha to inhibit the association of beta(1A) integrin with talin, which is crucial for the assembly of these structures. ICAP-1 alpha-mediated dispersion of beta(1A) integrins is not observed with beta(1D) integrins that do not bind ICAP. This strongly suggests that ICAP-1 alpha action depends on a direct interaction between ICAP-1 alpha and the cytoplasmic domain of the beta(1) chains. Altogether, these results suggest that ICAP-1 alpha plays a key role in cell adhesion by acting as a negative regulator of beta(1) integrin avidity.