Identification and Characterization of an HtrA Sheddase Produced by Coxiella burnetii.

Having previously shown that soluble E-cadherin (sE-cad) is found in sera of Q fever patients and that infection of BeWo cells by C. burnetii leads to modulation of the E-cad/ß-cat pathway, our purpose was to identify which sheddase(s) might catalyze the cleavage of E-cad. Here, we searched for a direct mechanism of cleavage initiated by the bacterium itself, assuming the possible synthesis of a sheddase encoded in the genome of C. burnetii or an indirect mechanism based on the activation of a human sheddase. Using a straightforward bioinformatics approach to scan the complete genomes of four laboratory strains of C. burnetii, we demonstrate that C. burnetii encodes a 451 amino acid sheddase (CbHtrA) belonging to the HtrA family that is differently expressed according to the bacterial virulence. An artificial CbHtrA gene (CoxbHtrA) was expressed, and the CoxbHtrA recombinant protein was found to have sheddase activity. We also found evidence that the C. burnetii infection triggers an over-induction of the human HuHtrA gene expression. Finally, we demonstrate that cleavage of E-cad by CoxbHtrA on macrophages-THP-1 cells leads to an M2 polarization of the target cells and the induction of their secretion of IL-10, which "disarms" the target cells and improves C. burnetii replication. Taken together, these results demonstrate that the genome of C. burnetii encodes a functional HtrA sheddase and establishes a link between the HtrA sheddase-induced cleavage of E-cad, the M2 polarization of the target cells and their secretion of IL-10, and the intracellular replication of C. burnetii.

Origin, Diversity, and Multiple Roles of Enzymes with Metallo-ß-Lactamase Fold from Different Organisms.

ß-lactamase enzymes have generated significant interest due to their ability to confer resistance to the most commonly used family of antibiotics in human medicine. Among these enzymes, the class B ß-lactamases are members of a superfamily of metallo-ß-lactamase (MßL) fold proteins which are characterised by conserved motifs (i.e., HxHxDH) and are not only limited to bacteria. Indeed, as the result of several barriers, including low sequence similarity, default protein annotation, or untested enzymatic activity, MßL fold proteins have long been unexplored in other organisms. However, thanks to search approaches which are more sensitive compared to classical Blast analysis, such as the use of common ancestors to identify distant homologous sequences, we are now able to highlight their presence in different organisms including Bacteria, Archaea, Nanoarchaeota, Asgard, Humans, Giant viruses, and Candidate Phyla Radiation (CPR). These MßL fold proteins are multifunctional enzymes with diverse enzymatic or non-enzymatic activities of which, at least thirteen activities have been reported such as ß-lactamase, ribonuclease, nuclease, glyoxalase, lactonase, phytase, ascorbic acid degradation, anti-cancer drug degradation, or membrane transport. In this review, we (i) discuss the existence of MßL fold enzymes in the different domains of life, (ii) present more suitable approaches to better investigating their homologous sequences in unsuspected sources, and (iii) report described MßL fold enzymes with demonstrated enzymatic or non-enzymatic activities.

clbP Gene, a Potential New Member of the ß-Lactamase Family.

The colibactin island (pks) of Escherichia coli formed by 19 genes (55-Kb), encodes non-ribosomal peptide (NRP) and polyketide (PK) synthases, which allow the synthesis of colibactin, a suspected hybrid PK-NRP compound that causes damage to DNA in eukaryotic cells. The clbP, an unusual essential gene, is found in the operon structure with the clbS gene in the pks-encoded machinery. Interestingly, the clbP gene has been annotated as a ß-lactamase but no previous study has reported its ß-lactamase characteristics. In this study, we (i) investigated the ß-lactamase properties of the clbP gene in silico by analysing its phylogenetic relationship with bacterial ß-lactamase and peptidase enzymes, (ii) compared its three-dimensional (3D) protein structure with those of bacterial ß-lactamase proteins using the Phyr2 database and PyMOL software, and (iii) evaluated in vitro its putative enzymatic activities, including ß-lactamase, nuclease, and ribonuclease using protein expression and purification from an E. coli BL21 strain. In this study, we reveal a structural configuration of toxin/antitoxin systems in this island. Thus, similar to the toxin/antitoxin systems, the role of the clbP gene within the pks-island gene group appears as an antitoxin, insofar as it is responsible for the activation of the toxin, which is colibactin. In silico, our analyses revealed that ClbP belonged to the superfamily of ß-lactamase, class C. Furthermore, in vitro we were unable to demonstrate its ß-lactamase activity, likely due to the fact that the clbP gene requires co-expression with other genes, such as the genes present in the pks-island (19 genes). More research is needed to better understand its actions, particularly with regards to antibiotics, and to discover whether it has any additional functions due to the importance of this gene and its toxicity.

New Beta-lactamases in Candidate Phyla Radiation: Owning Pleiotropic Enzymes Is a Smart Paradigm for Microorganisms with a Reduced Genome.

The increased exploitation of microbial sequencing methods has shed light on the high diversity of new microorganisms named Candidate Phyla Radiation (CPR). CPR are mainly detected via 16S rRNA/metabarcoding analyses or metagenomics and are found to be abundant in all environments and present in different human microbiomes. These microbes, characterized by their symbiotic/epiparasitic lifestyle with bacteria, are directly exposed to competition with other microorganisms sharing the same ecological niche. Recently, a rich repertoire of enzymes with antibiotic resistance activity has been found in CPR genomes by using an in silico adapted screening strategy. This reservoir has shown a high prevalence of putative beta-lactamase-encoding genes. We expressed and purified five putative beta-lactamase sequences having the essential domains and functional motifs from class A and class B beta-lactamase. Their enzymatic activities were tested against various beta-lactam substrates using liquid chromatography-mass spectrometry (LC-MS) and showed some beta-lactamase activity even in the presence of a beta-lactamase inhibitor. In addition, ribonuclease activity was demonstrated against RNA that was not inhibited by sulbactam and EDTA. None of these proteins could degrade single- and double-stranded-DNA. This study is the first to express and test putative CPR beta-lactamase protein sequences in vitro. Our findings highlight that the reduced genomes of CPR members harbor sequences encoding for beta-lactamases known to be multifunction hydrolase enzymes.

Incomplete tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: is it still a virus?

The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3 µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5 Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that eight putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all eight viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question "what is a virus?".

Spread of Mink SARS-CoV-2 Variants in Humans: A Model of Sarbecovirus Interspecies Evolution.

The rapid spread of SARS-CoV-2 variants has quickly spanned doubts and the fear about their ability escape vaccine protection. Some of these variants initially identified in caged were also found in humans. The claim that these variants exhibited lower susceptibility to antibody neutralization led to the slaughter of 17 million minks in Denmark. SARS-CoV-2 prevalence tests led to the discovery of infected farmed minks worldwide. In this study, we revisit the issue of the circulation of SARS-CoV-2 variants in minks as a model of sarbecovirus interspecies evolution by: (1) comparing human and mink angiotensin I converting enzyme 2 (ACE2) and neuropilin 1 (NRP-1) receptors; (2) comparing SARS-CoV-2 sequences from humans and minks; (3) analyzing the impact of mutations on the 3D structure of the spike protein; and (4) predicting linear epitope targets for immune response. Mink-selected SARS-CoV-2 variants carrying the Y453F/D614G mutations display an increased affinity for human ACE2 and can escape neutralization by one monoclonal antibody. However, they are unlikely to lose most of the major epitopes predicted to be targets for neutralizing antibodies. We discuss the consequences of these results for the rational use of SARS-CoV-2 vaccines.

Could ß-Lactam Antibiotics Block Humoral Immunity?

It has been reported that treatment with ß-lactam antibiotics induces leukopenia and candidemia, worsens the clinical response to anticancer immunotherapy and decreases immune response to vaccination. ß-lactamases can cleave ß-lactam antibiotics by blocking their activity. Two distincts superfamilies of ß-lactamases are described, the serine ß-lactamases and the zinc ion dependent metallo-ß-lactamases. In human, 18 metallo-ß-lactamases encoding genes (hMBLs) have been identified. While the physiological role of most of them remains unknown, it is well established that the SNM1A, B and C proteins are involved in DNA repair. The SNM1C/Artemis protein is precisely associated in the V(D)J segments rearrangement, that leads to immunoglobulin (Ig) and T-cell receptor variable regions, which have a crucial role in the immune response. Thus in humans, SNM1C/Artemis mutation is associated with severe combined immunodeficiency characterized by hypogammaglobulinemia deficient cellular immunity and opportunistic infections. While catalytic site of hMBLs and especially that of the SNM1 family is highly conserved, in vitro studies showed that some ß-lactam antibiotics, and precisely third generation of cephalosporin and ampicillin, inhibit the metallo-ß-lactamase proteins SNM1A & B and the SNM1C/Artemis protein complex. By analogy, the question arises as to whether ß-lactam antibiotics can block the SNM1C/Artemis protein in humans inducing transient immunodeficiency. We reviewed here the literature data supporting this hypothesis based on in silico, in vitro and in vivo evidences. Understanding the impact of ß-lactam antibiotics on the immune cell will offer new therapeutic clues and new clinical approaches in oncology, immunology, and infectious diseases.

A metallo-ß-lactamase enzyme for internal detoxification of the antibiotic thienamycin.

Thienamycin, the first representative of carbapenem antibiotics was discovered in the mid-1970s from soil microorganism, Streptomyces cattleya, during the race to discover inhibitors of bacterial peptidoglycan synthesis. Chemically modified into imipenem (N-formimidoyl thienamycin), now one of the most clinically important antibiotics, thienamycin is encoded by a thienamycin gene cluster composed of 22 genes (thnA to thnV) from S. cattleya NRRL 8057 genome. Interestingly, the role of all thn-genes has been experimentally demonstrated in the thienamycin biosynthesis, except thnS, despite its annotation as putative ß-lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo-ß-lactamase fold proteins. Compared to known ß-lactamases such as OXA-48 and NDM-1, ThnS exhibited a lower affinity and less efficiency toward penicillin G and cefotaxime, while imipenem is more actively hydrolysed. Moreover, like most MBL fold enzymes, additional enzymatic activities of ThnS were detected such as hydrolysis of ascorbic acid, single strand DNA, and ribosomal RNA. ThnS appears as a MBL enzyme with multiple activities including a specialised ß-lactamase activity toward imipenem. Thus, like toxin/antitoxin systems, the role of thnS gene within the thienamycin gene cluster appears as an antidote against the produced thienamycin.

Spreading of a new SARS-CoV-2 N501Y spike variant in a new lineage.

OBJECTIVES: Surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic epidemiology led us to detect several variants since summer 2020. We report the recent spread of a new SARS-CoV-2 spike 501Y variant. METHODS: SARS-CoV-2 sequences obtained from human nasopharyngeal samples by Illumina next-generation sequencing were analysed using Nextclade and an in-house Python script and were compared using BLASTn to the GISAID database. Phylogeny was investigated using the IQ-TREE software. RESULTS: We identified that SARS-CoV-2 genomes from four patients diagnosed in our institute harboured a new set of amino acid substitutions including L18F, L452R, N501Y, A653V, H655Y, D796Y, G1219V ± Q677H. These spike N501Y genomes are the first of Nextstrain clade 19B. We obtained partial spike gene sequences of this genotype for an additional 43 patients. All patients infected with this genotype were diagnosed since mid-January 2021. We detected 42 other genomes of this genotype in GISAID, which were obtained from samples collected in December 2020 in four individuals and in 2021 in 38 individuals. The 89 sequences obtained in our institute or other laboratories originated from the Comoros archipelago, western European countries (mostly metropolitan France), Turkey and Nigeria. CONCLUSION: These findings warrant further studies to investigate the spread, epidemiological and clinical features, and sensitivity to immune responses of this variant.

Emergence of Bat-Related Betacoronaviruses: Hazard and Risks.

The current Coronavirus Disease 2019 (COVID-19) pandemic, with more than 111 million reported cases and 2,500,000 deaths worldwide (mortality rate currently estimated at 2.2%), is a stark reminder that coronaviruses (CoV)-induced diseases remain a major threat to humanity. COVID-19 is only the latest case of betacoronavirus (ß-CoV) epidemics/pandemics. In the last 20 years, two deadly CoV epidemics, Severe Acute Respiratory Syndrome (SARS; fatality rate 9.6%) and Middle East Respiratory Syndrome (MERS; fatality rate 34.7%), plus the emergence of HCoV-HKU1 which causes the winter common cold (fatality rate 0.5%), were already a source of public health concern. Betacoronaviruses can also be a threat for livestock, as evidenced by the Swine Acute Diarrhea Syndrome (SADS) epizootic in pigs. These repeated outbreaks of ß-CoV-induced diseases raise the question of the dynamic of propagation of this group of viruses in wildlife and human ecosystems. SARS-CoV, SARS-CoV-2, and HCoV-HKU1 emerged in Asia, strongly suggesting the existence of a regional hot spot for emergence. However, there might be other regional hot spots, as seen with MERS-CoV, which emerged in the Arabian Peninsula. ß-CoVs responsible for human respiratory infections are closely related to bat-borne viruses. Bats are present worldwide and their level of infection with CoVs is very high on all continents. However, there is as yet no evidence of direct bat-to-human coronavirus infection. Transmission of ß-CoV to humans is considered to occur accidentally through contact with susceptible intermediate animal species. This zoonotic emergence is a complex process involving not only bats, wildlife and natural ecosystems, but also many anthropogenic and societal aspects. Here, we try to understand why only few hot spots of ß-CoV emergence have been identified despite worldwide bats and bat-borne ß-CoV distribution. In this work, we analyze and compare the natural and anthropogenic environments associated with the emergence of ß-CoV and outline conserved features likely to create favorable conditions for a new epidemic. We suggest monitoring South and East Africa as well as South America as these regions bring together many of the conditions that could make them future hot spots.

Implementation of an in-house real-time reverse transcription-PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant.

INTRODUCTION: The SARS-CoV-2 pandemic has been associated with the occurrence since summer 2020 of several viral variants that overlapped or succeeded each other in time. Those of current concern harbor mutations within the spike receptor binding domain (RBD) that may be associated with viral escape to immune responses. In our geographical area a viral variant we named Marseille-4 harbors a S477 N substitution in this RBD. MATERIALS AND METHODS: We aimed to implement an in-house one-step real-time reverse transcription-PCR (qPCR) assay with a hydrolysis probe that specifically detects the SARS-CoV-2 Marseille-4 variant. RESULTS: All 6 cDNA samples from Marseille-4 variant strains identified in our institute by genome next-generation sequencing (NGS) tested positive using our Marseille-4 specific qPCR, whereas all 32 cDNA samples from other variants tested negative. In addition, 39/42 (93 %) respiratory samples identified by NGS as containing a Marseille-4 variant strain and 0/26 samples identified as containing non-Marseille-4 variant strains were positive. Finally, 2018/3960 (51%) patients SARS-CoV-2-diagnosed in our institute, 10/277 (3.6 %) respiratory samples collected in Algeria, and none of 207 respiratory samples collected in Senegal, Morocco, or Lebanon tested positive using our Marseille-4 specific qPCR. DISCUSSION: Our in-house qPCR system was found reliable to detect specifically the Marseille-4 variant and allowed estimating it is involved in about half of our SARS-CoV-2 diagnoses since December 2020. Such approach allows the real-time surveillance of SARS-CoV-2 variants, which is warranted to monitor and assess their epidemiological and clinical characterics based on comprehensive sets of data.

Emergence and outcomes of the SARS-CoV-2 'Marseille-4' variant.

BACKGROUND: In Marseille, France, following a first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak in March-May 2020, a second epidemic phase occurred from June, involving 10 new variants. The Marseille-4 variant caused an epidemic that started in August and is still ongoing. METHODS: The 1038 SARS-CoV-2 whole genome sequences obtained in our laboratory by next-generation sequencing with Illumina technology were analysed using Nextclade and nextstrain/ncov pipelines and IQ-TREE. A Marseille-4-specific qPCR assay was implemented. Demographic and clinical features were compared between patients with the Marseille-4 variant and those with earlier strains. RESULTS: Marseille-4 harbours 13 hallmark mutations. One leads to an S477N substitution in the receptor binding domain of the spike protein targeted by current vaccines. Using a specific qPCR, it was observed that Marseille-4 caused 12-100% of SARS-CoV-2 infections in Marseille from September 2020, being involved in 2106 diagnoses. This variant was more frequently associated with hypoxemia than were clade 20A strains before May 2020. It caused a re-infection in 11 patients diagnosed with different SARS-CoV-2 strains before June 2020, suggesting either short-term protective immunity or a lack of cross-immunity. CONCLUSIONS: Marseille-4 should be considered as a major SARS-CoV-2 variant. Its sudden appearance points towards an animal reservoir, possibly mink. The protective role of past exposure and current vaccines against this variant should be evaluated.

A protein of the metallo-hydrolase/oxidoreductase superfamily with both beta-lactamase and ribonuclease activity is linked with translation in giant viruses.

Proteins with a metallo-beta-lactamase (MBL) fold have been largely studied in bacteria in the framework of resistance to beta-lactams, but their spectrum of activities is broader. We show here that the giant Tupanvirus also encodes a MBL fold-protein that has orthologs in other giant viruses, a deep phylogenetic root and is clustered with tRNases. This protein is significantly associated with translation components in giant viruses. After expression in Escherichia coli, it was found to hydrolyse nitrocefin, a beta-lactam, and penicillin G. This was inhibited by sulbactam, a beta-lactamase inhibitor. In addition, the tupanvirus MBL fold-protein was not active on single- or double-stranded DNA, but degraded RNAs from bacteria and Acanthamoeba castellanii, the tupanvirus amoebal host. This activity was not neutralized by sulbactam. Overall, our results still broaden the host range of MBL fold-proteins, showing dual beta-lactamase/nuclease activities in giant viruses.

Dual RNase and ß-lactamase Activity of a Single Enzyme Encoded in Archaea.

ß-lactam antibiotics have a well-known activity which disturbs the bacterial cell wall biosynthesis and may be cleaved by ß-lactamases. However, these drugs are not active on archaea microorganisms, which are naturally resistant because of the lack of ß-lactam target in their cell wall. Here, we describe that annotation of genes as ß-lactamases in Archaea on the basis of homologous genes is a remnant of identification of the original activities of this group of enzymes, which in fact have multiple functions, including nuclease, ribonuclease, ß-lactamase, or glyoxalase, which may specialized over time. We expressed class B ß-lactamase enzyme from Methanosarcina barkeri that digest penicillin G. Moreover, while weak glyoxalase activity was detected, a significant ribonuclease activity on bacterial and synthetic RNAs was demonstrated. The ß-lactamase activity was inhibited by ß-lactamase inhibitor (sulbactam), but its RNAse activity was not. This gene appears to have been transferred to the Flavobacteriaceae group especially the Elizabethkingia genus, in which the expressed gene shows a more specialized activity on thienamycin, but no glyoxalase activity. The expressed class C-like ß-lactamase gene, from Methanosarcina sp., also shows hydrolysis activity on nitrocefin and is more closely related to DD-peptidase enzymes. Our findings highlight the need to redefine the nomenclature of ß-lactamase enzymes and the specification of multipotent enzymes in different ways in Archaea and bacteria over time.

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.

Can ACE2 Receptor Polymorphism Predict Species Susceptibility to SARS-CoV-2?

A novel severe acute respiratory syndrome coronavirus, SARS-CoV-2, emerged in China in December 2019 and spread worldwide, causing more than 1.3 million deaths in 11 months. Similar to the human SARS-CoV, SARS-CoV-2 shares strong sequence homologies with a sarbecovirus circulating in Rhinolophus affinis bats. Because bats are expected to be able to transmit their coronaviruses to intermediate animal hosts that in turn are a source of viruses able to cross species barriers and infect humans (so-called spillover model), the identification of an intermediate animal reservoir was the subject of intense researches. It was claimed that a reptile (Ophiophagus hannah) was the intermediate host. This hypothesis was quickly ruled out and replaced by the pangolin (Manis javanica) hypothesis. Yet, pangolin was also recently exonerated from SARS-CoV-2 transmission to humans, leaving other animal species as presumed guilty. Guided by the spillover model, several laboratories investigated in silico the species polymorphism of the angiotensin I converting enzyme 2 (ACE2) to find the best fits with the SARS-CoV-2 spike receptor-binding site. Following the same strategy, we used multi-sequence alignment, 3-D structure analysis, and electrostatic potential surface generation of ACE2 variants to predict their binding capacity to SARS-CoV-2. We report evidence that such simple in silico investigation is a powerful tool to quickly screen which species are potentially susceptible to SARS-CoV-2. However, possible receptor binding does not necessarily lead to successful replication in host. Therefore, we also discuss here the limitations of these in silico approaches in our quest on the origins of COVID-19 pandemic.

Human metallo-ß-lactamase enzymes degrade penicillin.

Nonribosomal peptides are assemblages, including antibiotics, of canonical amino acids and other molecules. ß-lactam antibiotics act on bacterial cell walls and can be cleaved by ß-lactamases. ß-lactamase activity in humans has been neglected, even though eighteen enzymes have already been annotated such in human genome. Their hydrolysis activities on antibiotics have not been previously investigated. Here, we report that human cells were able to digest penicillin and this activity was inhibited by ß-lactamase inhibitor, i.e. sulbactam. Penicillin degradation in human cells was microbiologically demonstrated on Pneumococcus. We expressed a MBLAC2 human ß-lactamase, known as an exosome biogenesis enzyme. It cleaved penicillin and was inhibited by sulbactam. Finally, ß-lactamases are widely distributed, archaic, and have wide spectrum, including digesting anticancer and ß-lactams, that can be then used as nutriments. The evidence of the other MBLAC2 role as a bona fide ß-lactamase allows for reassessment of ß-lactams and ß-lactamases role in humans.

Evaluation of a robust engineered enzyme towards organophosphorus insecticide bioremediation using planarians as biosensors.

Organophosphorus compounds (OPs) are neurotoxic molecules developed as insecticides and chemical warfare nerve agents (CWNAs). They are covalent inhibitors of acetylcholinesterase (AChE), a key enzyme in central and peripheral nervous systems and are responsible for numerous poisonings worldwide. Many animal models have been studied over the years but finding a suitable in vivo model to account for both acute toxicity and long-term exposure remains a topical issue. Recently, an emerging aquatic animal model harboring a mammalian-like cholinergic nervous system, the freshwater planarian from Platyhelminthes, has been used to investigate neurotoxicity and developmental disruption. Given the tremendous toxicity of OPs, various bioremediation strategies have been considered over the years to counter their poisonous effects. Among these, enzymes have been particularly highlighted as they can degrade OPs in a fast, non toxic and environmentally friendly manner. In this article we investigated the biotechnological potential for decontaminating OPs of the previously reported variant SsoPox-αsD6 from the hyperstable enzyme SsoPox, isolated from the archaea Sulfolobus solfataricus. The capacity to hydrolyze 4 new substrates (methyl-pirimiphos, quinalphos, triazophos and dibrom) was demonstrated and the degradation products generated by enzymatic hydrolysis were characterized. We further evaluated the capacity of SsoPox-αsD6 for in vivo protection of freshwater planarians Schmidtea mediterranea (Smed). The use of SsoPox-αsD6 drastically decreased mortality and enhanced mobility of planarians. Then, an enzyme-based filtration device was developed by immobilizing intact Escherichia coli cells expressing SsoPox-αsD6 into alginate beads. The efficacy of the device was demonstrated using planarians as biosensors.

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.

Zafirlukast inhibits complexation of Lsr2 with DNA and growth of Mycobacterium tuberculosis.

The mycobacterial nucleoid-associated protein Lsr2 is a DNA-bridging protein that plays a role in condensation and structural organization of the genome and acts as a global repressor of gene transcription. Here we describe experiments demonstrating that zafirlukast inhibits the complexation between Lsr2 and DNA in vitro. Zafirlukast is shown to inhibit growth in two different species of mycobacteria tested but exhibits no growth inhibition of Escherichia coli. The Lsr2 inhibitory activity is reflected in vivo as determined by monitoring of transcription levels in Mycobacterium tuberculosis. These data suggest that zafirlukast inhibits Lsr2 function in vivo, promoting dysregulation of the expression of an array of genes typically bound by Lsr2 and hindering growth. Since zafirlukast likely operates by a mechanism distinct from current M. tuberculosis drugs and is currently used as a prophylactic treatment for asthma, it offers an intriguing lead for development of new treatments for tuberculosis.