29 research outputs found
GAMMA-PGA: Production and characterization of a versatile biopolymer by B. subtilis laboratory strains
No full textIn recent years the interest for potential applications of natural biopolymers has tremendously increased due to the growing demand of industrial processes based on safe raw materials. Poly γ-glutamic acid (γ-PGA) is a versatile unusual, water-soluble, anionic homopolyamide that is raising considerable industrial interests for the multitude of its potential applications. It is efficiently purified by natural bacterial isolates, mainly belonging to the genus Bacillus. Its cost is currently estimated to be much higher than that of conventional materials in current use and, for the moment, it is very difficult to prepare such high-molecular-weight polymer by chemical synthesis. For the industrial application of γ-PGA it is necessary to enhance its productivity and to find optimal fermentation conditions.
Recently, a new Bacillus subtilis producer strain has been derived from the fully genetically characterised laboratory strain 168 (1). The genetic, biochemical and physiological knowledge accumulated in such model organism allowed to devise genetic strategies for strain improvement and setting up of suitable growth conditions, overcoming the heuristic approach thus far applied with wild producers.
We have data showing the success of such strategic genetic engineering approaches: mutant strains with improved and sustained accumulation of the product have been obtained by introducing selected mutations.
The purification procedure has been carefully analysed and purity and size of the polymer have been checked by NMR and GPC. Copurification of enzymes and other by-products will be shown and discussed. The result is a competitive γ-PGA producer strain.
1) Osera, Amati, Calvio, Galizzi (2009). Microbiology, 155:2282-228
Burkholderia cenocepacia Infections in Cystic Fibrosis Patients: Drug Resistance and Therapeutic Approaches
No full textBurkholderia cenocepacia is an opportunistic pathogen particularly dangerous for cystic fibrosis (CF) patients. It can cause a severe decline in CF lung function possibly developing into a life-threatening systemic infection known as cepacia syndrome. Antibiotic resistance and presence of numerous virulence determinants in the genome make B. cenocepacia extremely difficult to treat. Better understanding of its resistance profiles and mechanisms is crucial to improve management of these infections. Here, we present the clinical distribution of B. cenocepacia described in the last 6 years and methods for identification and classification of epidemic strains. We also detail new antibiotics, clinical trials, and alternative approaches reported in the literature in the last 5 years to tackle B. cenocepacia resistance issue. All together these findings point out the urgent need of new and alternative therapies to improve CF patients’ life expectancy
Characterization of the dispirotripiperazine derivative PDSTP as antibiotic adjuvant and antivirulence compound against Pseudomonas aeruginosa
No full textPseudomonas aeruginosa is a major human pathogen, able to establish difficult-to-treat infections in immunocompromised and people with cystic fibrosis (CF). The high rate of antibiotic treatment failure is due to its notorious drug resistance, often mediated by the formation of persistent biofilms. Alternative strategies, capable of overcoming P. aeruginosa resistance, include antivirulence compounds which impair bacterial pathogenesis without exerting a strong selective pressure, and the use of antimicrobial adjuvants that can resensitize drug-resistant bacteria to specific antibiotics. In this work, the dispirotripiperazine derivative PDSTP, already studied as antiviral, was characterized for its activity against P. aeruginosa adhesion to epithelial cells, its antibiotic adjuvant ability and its biofilm inhibitory potential. PDSTP was effective in impairing the adhesion of P. aeruginosa to various immortalized cell lines. Moreover, the combination of clinically relevant antibiotics with the compound led to a remarkable enhancement of the antibiotic efficacy towards multidrug-resistant CF clinical strains. PDSTP-ceftazidime combination maintained its efficacy in vivo in a Galleria mellonella infection model. Finally, the compound showed a promising biofilm inhibitory activity at low concentrations when tested both in vitro and using an ex vivo pig lung model. Altogether, these results validate PDSTP as a promising compound, combining the ability to decrease P. aeruginosa virulence by impairing its adhesion and biofilm formation, with the capability to increase antibiotic efficacy against antibiotic resistant strains
The Crystal Structure of <i>Burkholderia cenocepacia</i> DfsA Provides Insights into Substrate Recognition and Quorum Sensing Fatty Acid Biosynthesis
No full textBurkholderia cenocepacia is a major concern among
respiratory tract infections in cystic fibrosis patients. This pathogen
is particularly difficult to treat because of its high level of resistance
to the clinically relevant antimicrobial agents. In B. cenocepacia, the quorum sensing cell–cell communication system is involved
in different processes that are important for bacterial virulence,
such as biofilm formation and protease and siderophore production.
Targeting the enzymes involved in this process represents a promising
therapeutic approach. With the aim of finding effective quorum sensing
inhibitors, we have determined the three-dimensional structure of B. cenocepacia diffusible factor synthase A, DfsA. This
bifunctional crotonase (dehydratase/thioesterase) produces the characteristic
quorum sensing molecule of B. cenocepacia, cis-2-dodecenoic acid or BDSF, starting from 3-hydroxydodecanoyl-acyl
carrier protein. Unexpectedly, the crystal structure revealed the
presence of a lipid molecule in the catalytic site of the enzyme,
which was identified as dodecanoic acid. Our biochemical characterization
shows that DfsA is able to use dodecanoyl-acyl carrier protein as
a substrate, demonstrating that dodecanoic acid, the product of this
reaction, is released very slowly from the DfsA active site, therefore
acting as a DfsA inhibitor. This molecule shows an unprecedented conformational
arrangement inside the DfsA active site. In contrast with previous
hypotheses, our data illustrate how DfsA and closely related homologous
enzymes can recognize long hydrophobic substrates without large conformational
changes or assistance by additional regulator molecules. The elucidation
of the substrate binding mode in DfsA provides the starting point
for structure-based drug discovery studies targeting B. cenocepacia quorum sensing-assisted virulence
Subfunctionalization influences the expansion of bacterial multidrug antibiotic resistance
No full textBackground: Antibiotic resistance is a major problem for human health. Multidrug resistance efflux pumps, especially those of the Resistance-Nodulation-Cell Division (RND) family, are major contributors to high-level antibiotic resistance in Gram-negative bacteria. Most bacterial genomes contain several copies of the different classes of multidrug resistance efflux pumps. Gene duplication and gain of function by the duplicate copies of multidrug resistance efflux pump genes plays a key role in the expansion and diversification of drug-resistance mechanisms.Results: We used two members of the Burkholderia RND superfamily as models to understand how duplication events affect the antibiotic resistance of these strains. First, we analyzed the conservation and distribution of these two RND systems and their regulators across the Burkholderia genus. Through genetic manipulations, we identified both the exact substrate range of these transporters and their eventual interchangeability. We also performed a directed evolution experiment, combined with next generation sequencing, to evaluate the role of antibiotics in the activation of the expression of these systems. Together, our results indicate that the first step to diversify the functions of these pumps arises from changes in their regulation (subfunctionalization) instead of functional mutations. Further, these pumps could rewire their regulation to respond to antibiotics, thus maintaining high genomic plasticity.Conclusions: Studying the regulatory network that controls the expression of the RND pumps will help understand and eventually control the development and expansion of drug resistance
ELONGATED MUCUS -PENETRATING NANOCRYSTALS FOR LUNG DELIVERY OF A NEW ANTI-BURKHOLDERIA AGENT IN CYSTIC FIBROSIS
No full textInvestigating the Mechanism of Action of Diketopiperazines Inhibitors of the Burkholderia cenocepacia Quorum Sensing Synthase CepI: A Site-Directed Mutagenesis Study
No full textQuorum sensing (QS) is a bacterial intercellular communication process which controls the production of major virulence factors, such as proteases, siderophores, and toxins, as well as biofilm formation. Since the inhibition of this pathway reduces bacterial virulence, QS is considered a valuable candidate drug target, particularly for the treatment of opportunistic infections, such as those caused by Burkholderia cenocepacia in cystic fibrosis patients. Diketopiperazine inhibitors of the acyl homoserine lactone synthase CepI have been recently described. These compounds are able to impair the ability of B. cenocepacia to produce proteases, siderophores, and to form biofilm, being also active in a Caenorhabditis elegans infection model. However, the precise mechanism of action of the compounds, as well as their effect on the cell metabolism, fundamental for candidate drug optimization, are still not completely defined. Here, we performed a proteomic analysis of B. cenocepacia cells treated with one of these inhibitors, and compared it with a cepI deleted strain. Our results demonstrate that the effects of the compound are similar to the deletion of cepI, clearly confirming that these molecules function as inhibitors of the acyl homoserine lactone synthase. Moreover, to deepen our knowledge about the binding mechanisms of the compound to CepI, we exploited previously published in silico structural insights about this enzyme structure and validated different candidate binding pockets on the enzyme surface using site-directed mutagenesis and biochemical analyses. Our experiments identified a region near the predicted S-adenosylmethionine binding site critically involved in interactions with the inhibitor. These results could be useful for future structure-based optimization of these CepI inhibitors
Discovery of new diketopiperazines inhibiting Burkholderia cenocepacia quorum sensing in vitro and in vivo
No full textBurkholderia cenocepacia, an opportunistic respiratory pathogen particularly relevant for cystic fibrosis patients, is difficult to eradicate due to its high level of resistance to most clinically relevant antimicrobials. Consequently, the discovery of new antimicrobials as well as molecules capable of inhibiting its virulence is mandatory. In this regard quorum sensing (QS) represents a good target for anti-virulence therapies, as it has been linked to biofilm formation and is important for the production of several virulence factors, including proteases and siderophores. Here, we report the discovery of new diketopiperazine inhibitors of the B. cenocepacia acyl homoserine lactone synthase CepI, and report their anti-virulence properties. Out of ten different compounds assayed against recombinant CepI, four were effective inhibitors, with IC50 values in the micromolar range. The best compounds interfered with protease and siderophore production, as well as with biofilm formation, and showed good in vivo activity in a Caenorhabditis elegans infection model. These molecules were also tested in human cells and showed very low toxicity. Therefore, they could be considered for in vivo combined treatments with established or novel antimicrobials, to improve the current therapeutic strategies against B. cenocepacia
Efflux-mediated resistance to a benzothiadiazol derivative effective against Burkholderia cenocepacia
No full textBurkholderia cenocepacia is a major concern for people suffering from Cystic Fibrosis as it contributes to serious respiratory tract infections. The lack of drugs effective against this opportunistic pathogen, along with the high level of resistance to multiple antibiotics, render the treatment of these infections particularly difficult.Here a new compound, belonging to the 2,1,3-benzothiadiazol-5-yl family (10126109), with a bactericidal effect and a MIC of 8 µg/ml against B. cenocepacia, is described. The compound is not cytotoxic and effective against B. cenocepacia clinical isolates and members of all the known Burkholderia cepacia complex species.Spontaneous mutants resistant to 10126109 were isolated and mutations in the MerR transcriptional regulator BCAM1948 were identified. In this way, a mechanism of resistance to this new molecule was described, which relies on the overexpression of the RND-9 efflux pump. Indeed, rnd-9 overexpression was confirmed by qRT-PCR, and RND-9 was identified in the membrane fractions of the mutant strains. Moreover, the increase in the MIC values of different drugs in the mutant strains, together with complementation experiments, suggested the involvement of RND-9 in the efflux of 10126109, thus indicating again the central role of efflux transporters in B. cenocepacia drug resistance
CodY Is a Global Transcriptional Regulator Required for Virulence in Group B Streptococcus
No full textGroup B Streptococcus (GBS) is a Gram-positive bacterium able to switch from a harmless commensal of healthy adults to a pathogen responsible for invasive infections in neonates. The signals and regulatory mechanisms governing this transition are still largely unknown. CodY is a highly conserved global transcriptional regulator that links nutrient availability to the regulation of major metabolic and virulence pathways in low-G+C Gram-positive bacteria. In this work, we investigated the role of CodY in BM110, a GBS strain representative of a hypervirulent lineage associated with the majority of neonatal meningitis. Deletion of codY resulted in a reduced ability of the mutant strain to cause infections in neonatal and adult animal models. The observed decreased in vivo lethality was associated with an impaired ability of the mutant to persist in the blood, spread to distant organs, and cross the blood-brain barrier. Notably, the codY null mutant showed reduced adhesion to monolayers of human epithelial cells in vitro and an increased ability to form biofilms, a phenotype associated with strains able to asymptomatically colonize the host. RNA-seq analysis showed that CodY controls about 13% of the genome of GBS, acting mainly as a repressor of genes involved in amino acid transport and metabolism and encoding surface anchored proteins, including the virulence factor Srr2. CodY activity was shown to be dependent on the availability of branched-chain amino acids, which are the universal cofactors of this regulator. These results highlight a key role for CodY in the control of GBS virulence
