AHL-Based Quorum Sensing Regulates the Biosynthesis of a Variety of Bioactive Molecules in Bacteria.

Bacteria are social microorganisms that use communication systems known as quorum sensing (QS) to regulate diverse cellular behaviors including the production of various secreted molecules. Bacterial secondary metabolites are widely studied for their bioactivities including antibiotic, antifungal, antiparasitic, and cytotoxic compounds. Besides playing a crucial role in natural bacterial niches and intermicrobial competition by targeting neighboring organisms and conferring survival advantages to the producer, these bioactive molecules may be of prime interest to develop new antimicrobials or anticancer therapies. This review focuses on bioactive compounds produced under acyl homoserine lactone-based QS regulation by Gram-negative bacteria that are pathogenic to humans and animals, including the Burkholderia, Serratia, Pseudomonas, Chromobacterium, and Pseudoalteromonas genera. The synthesis, regulation, chemical nature, biocidal effects, and potential applications of these identified toxic molecules are presented and discussed in light of their role in microbial interactions.

Yaravirus: A novel 80-nm virus infecting Acanthamoeba castellanii.

Here we report the discovery of Yaravirus, a lineage of amoebal virus with a puzzling origin and evolution. Yaravirus presents 80-nm-sized particles and a 44,924-bp dsDNA genome encoding for 74 predicted proteins. Yaravirus genome annotation showed that none of its genes matched with sequences of known organisms at the nucleotide level; at the amino acid level, six predicted proteins had distant matches in the nr database. Complimentary prediction of three-dimensional structures indicated possible function of 17 proteins in total. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially representing a divergent major capsid protein (MCP) with a predicted double jelly-roll domain. Structure-guided phylogeny of MCP suggests that Yaravirus groups together with the MCPs of Pleurochrysis endemic viruses. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists.

Tuning the Envelope Structure of Enzyme Nanoreactors for In Vivo Detoxification of Organophosphates.

Encapsulated phosphotriesterase nanoreactors show their efficacy in the prophylaxis and post-exposure treatment of poisoning by paraoxon. A new enzyme nanoreactor (E-nRs) containing an evolved multiple mutant (L72C/Y97F/Y99F/W263V/I280T) of Saccharolobus solfataricus phosphotriesterase (PTE) for in vivo detoxification of organophosphorous compounds (OP) was made. A comparison of nanoreactors made of three- and di-block copolymers was carried out. Two types of morphology nanoreactors made of di-block copolymers were prepared and characterized as spherical micelles and polymersomes with sizes of 40 nm and 100 nm, respectively. The polymer concentrations were varied from 0.1 to 0.5% (w/w) and enzyme concentrations were varied from 2.5 to 12.5 µM. In vivo experiments using E-nRs of diameter 106 nm, polydispersity 0.17, zeta-potential -8.3 mV, and loading capacity 15% showed that the detoxification efficacy against paraoxon was improved: the LD50 shift was 23.7xLD50 for prophylaxis and 8xLD50 for post-exposure treatment without behavioral alteration or functional physiological changes up to one month after injection. The pharmacokinetic profiles of i.v.-injected E-nRs made of three- and di-block copolymers were similar to the profiles of the injected free enzyme, suggesting partial enzyme encapsulation. Indeed, ELISA and Western blot analyses showed that animals developed an immune response against the enzyme. However, animals that received several injections did not develop iatrogenic symptoms.

MIMIVIRE is a defence system in mimivirus that confers resistance to virophage.

Since their discovery, giant viruses have revealed several unique features that challenge the conventional definition of a virus, such as their large and complex genomes, their infection by virophages and their presence of transferable short element transpovirons. Here we investigate the sensitivity of mimivirus to virophage infection in a collection of 59 viral strains and demonstrate lineage specificity in the resistance of mimivirus to Zamilon, a unique virophage that can infect lineages B and C of mimivirus but not lineage A. We hypothesized that mimiviruses harbour a defence mechanism resembling the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system that is widely present in bacteria and archaea. We performed de novo sequencing of 45 new mimivirus strains and searched for sequences specific to Zamilon in a total of 60 mimivirus genomes. We found that lineage A strains are resistant to Zamilon and contain the insertion of a repeated Zamilon sequence within an operon, here named the 'mimivirus virophage resistance element' (MIMIVIRE). Further analyses of the surrounding sequences showed that this locus is reminiscent of a defence mechanism related to the CRISPR-Cas system. Silencing the repeated sequence and the MIMIVIRE genes restores mimivirus susceptibility to Zamilon. The MIMIVIRE proteins possess the typical functions (nuclease and helicase) involved in the degradation of foreign nucleic acids. The viral defence system, MIMIVIRE, represents a nucleic-acid-based immunity against virophage infection.

AmyP53, a Therapeutic Peptide Candidate for the Treatment of Alzheimer's and Parkinson's Disease: Safety, Stability, Pharmacokinetics Parameters and Nose-to Brain Delivery.

Neurodegenerative disorders are a major public health issue. Despite decades of research efforts, we are still seeking an efficient cure for these pathologies. The initial paradigm of large aggregates of amyloid proteins (amyloid plaques, Lewis bodies) as the root cause of Alzheimer's and Parkinson's diseases has been mostly dismissed. Instead, membrane-bound oligomers forming Ca2+-permeable amyloid pores are now considered appropriate targets for these diseases. Over the last 20 years, our group deciphered the molecular mechanisms of amyloid pore formation, which appeared to involve a common pathway for all amyloid proteins, including Aß (Alzheimer) and α-synuclein (Parkinson). We then designed a short peptide (AmyP53), which prevents amyloid pore formation by targeting gangliosides, the plasma membrane receptors of amyloid proteins. Herein, we show that aqueous solutions of AmyP53 are remarkably stable upon storage at temperatures up to 45 °C for several months. AmyP53 appeared to be more stable in whole blood than in plasma. Pharmacokinetics studies in rats demonstrated that the peptide can rapidly and safely reach the brain after intranasal administration. The data suggest both the direct transport of AmyP53 via the olfactory bulb (and/or the trigeminal nerve) and an indirect transport via the circulation and the blood-brain barrier. In vitro experiments confirmed that AmyP53 is as active as cargo peptides in crossing the blood-brain barrier, consistent with its amino acid sequence specificities and physicochemical properties. Overall, these data open a route for the use of a nasal spray formulation of AmyP53 for the prevention and/or treatment of Alzheimer's and Parkinson's diseases in future clinical trials in humans.

Engineering acyl-homoserine lactone-interfering enzymes toward bacterial control.

Enzymes able to degrade or modify acyl-homoserine lactones (AHLs) have drawn considerable interest for their ability to interfere with the bacterial communication process referred to as quorum sensing. Many proteobacteria use AHL to coordinate virulence and biofilm formation in a cell density-dependent manner; thus, AHL-interfering enzymes constitute new promising antimicrobial candidates. Among these, lactonases and acylases have been particularly studied. These enzymes have been isolated from various bacterial, archaeal, or eukaryotic organisms and have been evaluated for their ability to control several pathogens. Engineering studies on these enzymes were carried out and successfully modulated their capacity to interact with specific AHL, increase their catalytic activity and stability, or enhance their biotechnological potential. In this review, special attention is paid to the screening, engineering, and applications of AHL-modifying enzymes. Prospects and future opportunities are also discussed with a view to developing potent candidates for bacterial control.

Complexin in ivermectin resistance in body lice.

Ivermectin has emerged as very promising pediculicide, particularly in cases of resistance to commonly used pediculicides. Recently, however, the first field-evolved ivermectin-resistance in lice was reported. To gain insight into the mechanisms underlying ivermectin-resistance, we both looked for mutations in the ivermectin-target site (GluCl) and searched the entire proteome for potential new loci involved in resistance from laboratory susceptible and ivermectin-selected resistant body lice. Polymorphism analysis of cDNA GluCl showed no non-silent mutations. Proteomic analysis identified 22 differentially regulated proteins, of which 13 were upregulated and 9 were downregulated in the resistant strain. We evaluated the correlation between mRNA and protein levels by qRT-PCR and found that the trend in transcriptional variation was consistent with the proteomic changes. Among differentially expressed proteins, a complexin i.e. a neuronal protein which plays a key role in regulating neurotransmitter release, was shown to be the most significantly down-expressed in the ivermectin-resistant lice. Moreover, DNA-mutation analysis revealed that some complexin transcripts from resistant lice gained a premature stop codon, suggesting that this down-expression might be due, in part, to secondary effects of a nonsense mutation inside the gene. We further confirmed the association between complexin and ivermectin-resistance by RNA-interfering and found that knocking down the complexin expression induces resistance to ivermectin in susceptible lice. Our results provide evidence that complexin plays a significant role in regulating ivermectin resistance in body lice and represents the first evidence that links complexin to insecticide resistance.

Enzymatic Decontamination of G-Type, V-Type and Novichok Nerve Agents.

Organophosphorus nerve agents (OPNAs) are highly toxic compounds inhibiting cholinergic enzymes in the central and autonomic nervous systems and neuromuscular junctions, causing severe intoxications in humans. Medical countermeasures and efficient decontamination solutions are needed to counteract the toxicity of a wide spectrum of harmful OPNAs including G, V and Novichok agents. Here, we describe the use of engineered OPNA-degrading enzymes for the degradation of various toxic agents including insecticides, a series of OPNA surrogates, as well as real chemical warfare agents (cyclosarin, sarin, soman, tabun, VX, A230, A232, A234). We demonstrate that only two enzymes can degrade most of these molecules at high concentrations (25 mM) in less than 5 min. Using surface assays adapted from NATO AEP-65 guidelines, we further show that enzyme-based solutions can decontaminate 97.6% and 99.4% of 10 g∙m-2 of soman- and VX-contaminated surfaces, respectively. Finally, we demonstrate that these enzymes can degrade ethyl-paraoxon down to sub-inhibitory concentrations of acetylcholinesterase, confirming their efficacy from high to micromolar doses.

Culture of salivary methanogens assisted by chemically produced hydrogen.

Methanogen cultures require hydrogen produced by fermentative bacteria such as Bacteroides thetaiotaomicron (biological method). We developed an alternative method for hydrogen production using iron filings and acetic acid with the aim of cultivating methanogens more efficiently and more quickly (chemical method). We developed this new method with a reference strain of Methanobrevibacter oralis, compared the method to the biological reference method with a reference strain of Methanobrevibacter smithii and finally applied the method to 50 saliva samples. Methanogen colonies counted using ImageJ software were identified using epifluorescence optical microscopy, real-time PCR and PCR sequencing. For cultures containing pure strains of M. oralis and M. smithii, colonies appeared three days postinoculation with the chemical method versus nine days with the biological method. The average number of M. smithii colonies was significantly higher with the chemical method than with the biological method. There was no difference in the delay of observation of the first colonies in the saliva samples between the two methods. However, the average number of colonies was significantly higher with the biological method than with the chemical method at six days and nine days postinoculation (Student's test, p = 0.005 and p = 0.04, respectively). The chemical method made it possible to isolate four strains of M. oralis and three strains of M. smithii from the 50 saliva samples. Establishing the chemical method will ease the routine isolation and culture of methanogens.

Promiscuous Enzyme Activity as a Driver of Allo and Iso Convergent Evolution, Lessons from the ß-Lactamases.

The probability of the evolution of a character depends on two factors: the probability of moving from one character state to another character state and the probability of the new character state fixation. The more the evolution of a character is probable, the more the convergent evolution will be witnessed, and consequently, convergent evolution could mean that the convergent character evolution results as a combination of these two factors. We investigated this phenomenon by studying the convergent evolution of biochemical functions. For the investigation we used the case of ß-lactamases. ß-lactamases hydrolyze ß-lactams, which are antimicrobials able to block the DD-peptidases involved in bacterial cell wall synthesis. ß-lactamase activity is present in two different superfamilies: the metallo-ß-lactamase and the serine ß-lactamase. The mechanism used to hydrolyze the ß-lactam is different for the two superfamilies. We named this kind of evolution an allo-convergent evolution. We further showed that the ß-lactamase activity evolved several times within each superfamily, a convergent evolution type that we named iso-convergent evolution. Both types of convergent evolution can be explained by the two evolutionary mechanisms discussed above. The probability of moving from one state to another is explained by the promiscuous ß-lactamase activity present in the ancestral sequences of each superfamily, while the probability of fixation is explained in part by positive selection, as the organisms having ß-lactamase activity allows them to resist organisms that secrete ß-lactams. Indeed, an organism that has a mutation that increases the ß-lactamase activity will be selected, as the organisms having this activity will have an advantage over the others.

Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P-S Bonded Organophosphorus as Monitored by Spectrofluorimetry.

Enzyme-catalyzed hydrolysis of echothiophate, a P-S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with Km = 0.20 ± 0.03 mM and kcat = 5.4 ± 1.6 min-1. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (Km = 2.6 ± 0.2 mM; kcat = 53400 min-1). With a kcat/Km = (2.6 ± 1.6) × 107 M-1min-1, GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P-S bonded OPs by thiol-free OP hydrolases.

Routine Culture-Resistant Mycobacterium tuberculosis Rescue and Shell-Vial Assay, France.

We used shell-vial assay with a medium that buffered rifampin to isolate routine culture-resistant Mycobacterium tuberculosis bacteria from cerebrospinal fluid and rifampin-containing intervertebral disc and vertebral corpus of a patient in treatment for Pott's disease and disseminated tuberculosis. Whole-genome sequencing confirmed M. tuberculosis lineage 4 (Euro-American) strain.

Direct Identification of Pathogens in Urine by Use of a Specific Matrix-Assisted Laser Desorption Ionization-Time of Flight Spectrum Database.

Urinary tract infections are among the most common reasons for antimicrobial treatment, and early diagnosis could have a significant impact by enabling rapid administration of the adapted antibiotic and preventing complications. The current delay between sample receipt and pathogen identification is about 24 to 48 h, which could be significantly shortened by use of an accurate direct method. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is already used for the identification of pathogens in clinical laboratories and constitutes a promising tool for direct diagnosis. A simple preparation protocol was established for the processing of urine samples prior to MS analysis. MALDI-TOF spectra collected directly from 1,000 infected urine samples were used to create a specific reference database (named Urinf). A prospective study was then carried out to evaluate the Urinf database and compare the results obtained with the standard database provided by Bruker on the Biotyper Real Time Classification software. Seven hundred eighty urine specimens were processed and analyzed according to our method. Among them, almost 90% of 500 infected monobacterial samples could be correctly diagnosed with the Urinf database, compared to 50% using the standard database. The identification of Enterobacteriaceae, Staphylococcus aureus, Staphylococcus saprophyticus, Pseudomonas aeruginosa, Enterococcus faecalis, and Enterococcus faecium was greatly improved but not for Staphylococcus epidermidis The creation of a database adapted to a particular type of clinical sample has great potential to increase both the rate and rapidity of pathogen identification. Sensitivity still remains to be improved for bacterial species that exhibit few specific peaks on mass spectra.

Rapid identification of microorganisms from platelet concentrates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry after short-term incubation on liquid medium.

BACKGROUND: Platelets (PLTs) are especially affected by the risk of bacterial contamination. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) is an accurate method for the routine identification of bacterial isolates in microbiology laboratories. We directly applied the MALDI-TOF method to bacterial detection in PLTs. In this study, we evaluated the sensitivity, specificity, and speed of a direct MALDI-TOF approach compared to the conventional method BACTEC. STUDY DESIGN AND METHODS: Eight bacteria associated with PLT contamination, cited by the ISBT on transfusion-transmitted infectious diseases, were spiked into PLTs for a final concentration of approximately 100 CFU/bag (n = 5 for each strain). The PLTs were then agitated for 24 hours. One milliliter of PLTs was incubated in a shaker incubator for 8 hours at 37°C with 1 mL of trypticase soy broth (TSB). The spectra were analyzed using the MALDI Biotyper software. As a control, 8 mL of PLTs incubated into BACTEC bottles and a positive bottle were subcultured to ensure identification of bacterial growth. RESULTS: Regardless of the strain of PLTs tested, MALDI-TOF analysis made detection and early identification possible at 8 hours. Analysis by BACTEC of PLTs infected with Escherichia coli, Bacillus cereus, and Providencia stuartii made early identification possible. For the remaining bacteria, the detection time by BACTEC was significantly longer than 8 hours. CONCLUSION: We demonstrated the possibility of detecting bacteria in PLTs using a standardized culture step in TSB with MALDI-TOF, regardless of the strain, with the same specificity and analytical sensitivity and with a time to results of 12 hours. This direct method presented rapid and reliable results.

The molecular basis of phosphate discrimination in arsenate-rich environments.

Arsenate and phosphate are abundant on Earth and have striking similarities: nearly identical pK(a) values, similarly charged oxygen atoms, and thermochemical radii that differ by only 4% (ref. 3). Phosphate is indispensable and arsenate is toxic, but this extensive similarity raises the question whether arsenate may substitute for phosphate in certain niches. However, whether it is used or excluded, discriminating phosphate from arsenate is a paramount challenge. Enzymes that utilize phosphate, for example, have the same binding mode and kinetic parameters as arsenate, and the latter's presence therefore decouples metabolism. Can proteins discriminate between these two anions, and how would they do so? In particular, cellular phosphate uptake systems face a challenge in arsenate-rich environments. Here we describe a molecular mechanism for this process. We examined the periplasmic phosphate-binding proteins (PBPs) of the ABC-type transport system that mediates phosphate uptake into bacterial cells, including two PBPs from the arsenate-rich Mono Lake Halomonas strain GFAJ-1. All PBPs tested are capable of discriminating phosphate over arsenate at least 500-fold. The exception is one of the PBPs of GFAJ-1 that shows roughly 4,500-fold discrimination and its gene is highly expressed under phosphate-limiting conditions. Sub-ångström-resolution structures of Pseudomonas fluorescens PBP with both arsenate and phosphate show a unique mode of binding that mediates discrimination. An extensive network of dipole-anion interactions, and of repulsive interactions, results in the 4% larger arsenate distorting a unique low-barrier hydrogen bond. These features enable the phosphate transport system to bind phosphate selectively over arsenate (at least 10(3) excess) even in highly arsenate-rich environments.

New Approaches to Prevent Healthcare-Associated Infection.

Healthcare-associated infection (HCAI) in hospitals mainly results from unsolved but well-identified causes such as hand hygiene, overuse of catheters, and to a lesser extent, the airborne transmission of infectious agents caused by the misuse of respiratory precautions. The aims of the Institut Hospitalo-Universitaire Méditerranée Infection are to develop new approaches to fight HCAIs. Among them, new technologies that allow for the traceability of care and good practices reminders have been developed concomitantly to an anthropological approach, facilitating acceptability by healthcare workers. While the automated continuous monitoring system is validated and commercially available, some other technologies are still under clinical evaluation or in the early development phase. Quorum sensing-based biotechnologies are developed with the aims to fight against wound colonization.

Clostridium butyricum Strains and Dysbiosis Linked to Necrotizing Enterocolitis in Preterm Neonates.

BACKGROUND: Necrotizing enterocolitis (NEC) is the most common and serious gastrointestinal disorder among preterm neonates. We aimed to assess a specific gut microbiota profile associated with NEC. METHODS: Stool samples and clinical data were collected from 4 geographically independent neonatal intensive care units, over a 48-month period. Thirty stool samples from preterm neonates with NEC (n = 15) and controls (n = 15) were analyzed by 16S ribosomal RNA pyrosequencing and culture-based methods. The results led us to develop a specific quantitative polymerase chain reaction (qPCR) assay for Clostridium butyricum, and we tested stool samples from preterm neonates with NEC (n = 93) and controls (n = 270). We sequenced the whole genome of 16 C. butyricum strains, analyzed their phylogenetic relatedness, tested their culture supernatants for cytotoxic activity, and searched for secreted toxins. RESULTS: Clostridium butyricum was specifically associated with NEC using molecular and culture-based methods (15/15 vs 2/15; P < .0001) or qPCR (odds ratio, 45.4 [95% confidence interval, 26.2-78.6]; P < .0001). Culture supernatants of C. butyricum strains from preterm neonates with NEC (n = 14) exhibited significant cytotoxic activity (P = .008), and we identified in all a homologue of the ß-hemolysin toxin gene shared by Brachyspira hyodysenteriae, the etiologic agent of swine dysentery. The corresponding protein was secreted by a NEC-associated C. butyricum strain. CONCLUSIONS: NEC was associated with C. butyricum strains and dysbiosis with an oxidized, acid, and poorly diversified gut microbiota. Our findings highlight the plausible toxigenic mechanism involved in the pathogenesis of NEC.

Fetuin is the key for nanon self-propagation.

"Nanobacteria", also known as nanons or calciprotein particles (CPP), are nano-sized protein mineral complexes which have been isolated from numerous biological sources. Nanons possess self-replication properties and contain only serum proteins (e.g. Fetuin-A, Albumin). Herein, we develop a simplified in vitro model of nanons propagation composed of only fetuin-A as a protein. Using this model, we demonstrate that fetuin from nanons possesses a different, non-native conformation. Moreover, we show that nanons induce soluble fetuin-A precipitation which could serve as a template for calcification. This phenomenon explains the observed self-propagating properties that mimic infectious behavior. We also demonstrate that renal calculi are capable of inducing a conformational change in fetuin-A, suggesting that the propagation phenomenon of nanons may occur in vivo.

The DING family of phosphate binding proteins in inflammatory diseases.

Human paraoxonase 1 (hPON-1) is a protein that has been studied in relation to its antioxidant and anti-atherosclerotic properties. Despite extensive studies, the molecular mechanisms responsible for its functional properties remain unclear. During the last decade, a new partner of hPON-1 has been identified. Hidden for a long time because of a similar molecular weight with hPON-1, this protein, termed human phosphate-binding protein (HPBP), may contribute to the biological functions of hPON-1. Belonging to the DING protein, a sub-family of phosphate binding proteins (PBP or pstS), HPBP stabilizes hPON-1 and might prevent calcification of arteries in case of advance atherosclerosis. The role of other DING proteins in some calcification processes (i.e. nephrolithiasis) and the identification of HPBP in the atheroma plaque support this hypothesis. Nevertheless, the relevance of hPON-1/HPBP as well as the molecular determinants in atherosclerosis remains to be elucidated.