A Thermophilic Bacterial Esterase for Scavenging Nerve Agents: A Kinetic, Biophysical and Structural Study.

Organophosphorous nerve agents (OPNA) pose an actual and major threat for both military and civilians alike, as an upsurge in their use has been observed in the recent years. Currently available treatments mitigate the effect of the nerve agents, and could be vastly improved by means of scavengers of the nerve agents. Consequently, efforts have been made over the years into investigating enzymes, also known as bioscavengers, which have the potential either to trap or hydrolyze these toxic compounds. We investigated the previously described esterase 2 from Thermogutta terrifontis (TtEst2) as a potential bioscavenger of nerve agents. As such, we assessed its potential against G-agents (tabun, sarin, and cyclosarin), VX, as well as the pesticide paraoxon. We report that TtEst2 is a good bioscavenger of paraoxon and G-agents, but is rather slow at scavenging VX. X-ray crystallography studies showed that TtEst2 forms an irreversible complex with the aforementioned agents, and allowed the identification of amino-acids, whose mutagenesis could lead to better scavenging properties for VX. In conjunction with its cheap production and purification processes, as well as a robust structural backbone, further engineering of TtEst2 could lead to a stopgap bioscavenger useful for in corpo scavenging or skin decontamination.

A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene of Kingella kingae and Application to 201 Pediatric Clinical Specimens.

Kingella kingae is a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting the groEL gene and the RTX locus of K. kingae However, recent studies showed that real-time PCR (RT-PCR) assays targeting the Kingella sp. RTX locus that are currently available for the diagnosis of K. kingae infection lack specificity because they could not distinguish between K. kingae and the recently described Kingella negevensis species. Furthermore, in silico analysis of the groEL gene from a large collection of 45 K. kingae strains showed that primers and probes from K. kingaegroEL-based RT-PCR assays display a few mismatches with K. kingae groEL variations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative to groEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, a K. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of the K. kingae mdh gene from 20 distinct sequence types of K. kingae This novel K. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnose K. kingae infections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

Molecular Tests That Target the RTX Locus Do Not Distinguish between Kingella kingae and the Recently Described Kingella negevensis Species.

Kingella kingae is an important invasive pathogen in early childhood. The organism elaborates an RTX toxin presumably restricted to this species. Consequently, real-time quantitative PCR (qPCR) assays targeting the RTX locus have been developed in recent years and are gaining increasing use for the molecular diagnosis of K. kingae infections. However, the present study shows that Kingella negevensis, a Kingella species newly identified in young children, harbors an identical Kingella RTX locus, raising the question of whether K. negevensis can be misidentified as K. kingae by clinical microbiology laboratories. In silico comparison of Kingella sp. RTX and groEL genes and in vitro studies provided evidence that targeting the rtxA and rtxB genes could not differentiate between strains of K. kingae and K. negevensis, whereas targeting the groEL gene could. This prompted the design of a highly specific and sensitive qPCR assay targeting K. negevensis groEL (kngroEL). Ninety-nine culture-negative osteoarticular specimens from 99 children younger than 4 years of age were tested with a conventional 16S rRNA gene-based broad-range PCR assay and Kingella-specific rtxB, K. kingae-specific groEL (kkgroEL), and kngroEL qPCR assays. Forty-two specimens were rtxB positive, including 41 that were also kkgroEL positive and 1 (the remaining one) that was kngroEL positive. Thus, this study discloses an invasive infection caused by K. negevensis in humans and demonstrates that targeting the RTX locus cannot be used for the formal diagnosis of K. kingae infections. These findings stress the need for further studies on the epidemiology of asymptomatic carriage and invasive infections caused by K. negevensis in humans.

A modified multilocus sequence typing protocol to genotype Kingella kingae from oropharyngeal swabs without bacterial isolation.

BACKGROUND: Outbreaks of Kingella kingae infection are an emerging public health concern among daycare attendees carrying epidemic clones in the oropharynx. However, genotyping of such epidemic clones from affected cases is limited by the low performance of current methods to detect K. kingae from blood samples and lack of specimens available from infected sites. We aimed at developing a modified multilocus sequence typing (MLST) method to genotype K. kingae strains from oropharyngeal samples without prior culture. We designed in silico MLST primers specific for K. kingae by aligning whole nucleotide sequences of abcZ, adk, aroE, cpn60, recA, and gdh/zwf genes from closely related species belonging to the Kingella and Neisseria genera. We tested our modified MLST protocol on all Kingella species and N. meningitidis, as well as 11 oropharyngeal samples from young children with sporadic (n = 10) or epidemic (n = 1) K. kingae infection. RESULTS: We detected K. kingae-specific amplicons in the 11 oropharyngeal samples, corresponding to sequence-type 6 (ST-6) in 6 children including the epidemic cases, ST-25 in 2 children, and 3 possible novel STs (ST-67, ST-68, and ST-69). No amplicon was obtained from other Kingella species and N. meningitidis. CONCLUSIONS: We herein developed a specific MLST protocol that enables genotyping of K. kingae by MLST directly from oropharyngeal samples. This discriminatory tool, with which we identified the first K. kingae outbreak caused by ST-6 in Europe, may be used in further epidemiological investigations.

Isolation and characterization of Kingella negevensis sp. nov., a novel Kingella species detected in a healthy paediatric population.

We herein report the isolation and characterization of 21 Gram-stain-negative strains cultivated from the oropharynx of healthy children in Israel and Switzerland. Initially described as small colony variants of Kingella kingae, phenotypic analysis, biochemical analysis, phylogenetic analysis based on sequencing of the partial 16S rRNA gene and five housekeeping genes (abcZ, adk, G6PD, groEL and recA), and whole genome sequencing and comparison between members of the genera Kingella and Neisseria provided evidence for assigning them to the genus Kingella. Cellular fatty acids included important amounts of C12 : 0, C14 : 0, C16 : 0 and C16 : 1n7. Digital DNA-DNA hybridization between the isolates Sch538T and K. kingae ATCC 23330T revealed relatedness of 19.9 %. Comparative analysis of 16S rRNA gene sequences available in GenBank allowed matches to strains isolated in the USA, suggesting a wider geographical distribution. A novel species named Kingella negevensis sp. nov. is proposed, as most strains have been isolated in the Negev, a desert region of southern Israel. The type strain is Sch538T (=CCUG 69806T=CSUR P957).

Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems.

The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

Warfare Nerve Agents and Paraoxonase-1 as a Potential Prophylactic Therapy against Intoxication.

Nerve agents are a class of lethal neurotoxic chemicals used in chemical warfare. In this review, we have briefly discussed a brief history of chemical warfare, followed by an exploration of the historical context surrounding nerve agents. The article explores the classification of these agents, their contemporary uses, their toxicity mechanisms, and the disadvantages of the current treatment options for nerve agent poisoning. It then discusses the possible application of enzymes as prophylactics against nerve agent poisoning, outlining the benefits and drawbacks of paraoxonase-1. Finally, the current studies on paraoxonase-1 are reviewed, highlighting that several challenges need to be addressed in the use of paraoxonase-1 in the actual field and that its potential as a prophylactic antidote against nerve agent poisoning needs to be evaluated. The literature used in this manuscript was searched using various electronic databases, such as PubMed, Google Scholar, Web of Science, Elsevier, Springer, ACS, Google Patent, and books using the keywords chemical warfare agent, Butyrylcholinesterase, enzyme, nerve agent, prophylactic, and paraoxonase- 1, with the time scale for the analysis of articles between 1960 to 2023, respectively. The study has suggested that concerted efforts by researchers and agencies must be made to develop effective countermeasures against NA poisoning and that PON1 has suitable properties for the development of efficient prophylaxis against NA poisoning.

Biotechnological applications of quorum quenching enzymes.

Numerous bacteria use quorum sensing (QS) to synchronize their behavior and monitor their population density. They use signaling molecules known as autoinducers (AI's) that are synthesized and secreted into their local environment to regulate QS-dependent gene expression. Among QS-regulated pathways, biofilm formation and virulence factor secretion are particularly problematic as they are involved in surface-attachment, antimicrobial agent resistance, toxicity, and pathogenicity. Targeting QS represents a promising strategy to inhibit undesirable bacterial traits. This strategy, referred to as quorum quenching (QQ), includes QS-inhibitors and QQ enzymes. These approaches are appealing because they do not directly challenge bacterial survival, and consequently selection pressure may be low, yielding a lower occurrence of resistance. QQ enzymes are particularly promising because they act extracellularly to degrade AI's and can be used in catalytic quantities. This review draws an overview of QQ enzyme related applications, covering several economically important fields such as agriculture, aquaculture, biofouling and health issues. Finally, the possibility of resistance mechanism occurrence to QQ strategies is discussed.

Effect of Quorum Quenching Lactonase in Clinical Isolates of Pseudomonas aeruginosa and Comparison with Quorum Sensing Inhibitors.

Pseudomonas aeruginosa is a Gram negative pathogenic bacterium involved in many human infections including otitis, keratitis, pneumonia, and diabetic foot ulcers. P. aeruginosa uses a communication system, referred to as quorum sensing (QS), to adopt a group behavior by synchronizing the expression of certain genes. Among the regulated traits, secretion of proteases or siderophores, motility and biofilm formation are mainly involved in the pathogenicity. Many efforts have been dedicated to the development of quorum sensing inhibitors (QSI) and quorum quenching (QQ) agents to disrupt QS. QQ enzymes have been particularly considered as they may act in a catalytic way without entering the cell. Here we focus on the lactonase SsoPox which was previously investigated for its ability to degrade the signaling molecules, acyl-homoserine lactones, in particular on the engineered variant SsoPox-W263I. We highlight the potential of SsoPox-W263I to inhibit the virulence of 51 clinical P. aeruginosa isolates from diabetic foot ulcers by decreasing the secretion of two virulence factors, proteases and pyocyanin, as well as biofilm formation. We further compared the effect of SsoPox-W263I to the comprehensively described QSI, 5-fluorouracil and C-30. We found the lactonase SsoPox-W263I to be significantly more effective than the tested QSI at their respective concentration optimum and to retain its activity after immobilization steps, paving the way for future therapeutic applications.

An Outbreak of Kingella Kingae Infections Complicating a Severe Hand, Foot, And Mouth Disease Outbreak in Nice, France, 2016.

We report the investigation methods for the diagnosis of an epidemic and culture-negative Kingella kingae endocarditis complicating a severe outbreak of hand, foot and mouth disease in a childcare center. The diagnosis was confirmed by polymerase chain reaction testing performed from cardiac tissue. Our findings argue for the systematic investigation of K. kingae outbreaks by using molecular tools in such context.

Current and emerging strategies for organophosphate decontamination: special focus on hyperstable enzymes.

Organophosphorus chemicals are highly toxic molecules mainly used as pesticides. Some of them are banned warfare nerve agents. These compounds are covalent inhibitors of acetylcholinesterase, a key enzyme in central and peripheral nervous systems. Numerous approaches, including chemical, physical, and biological decontamination, have been considered for developing decontamination methods against organophosphates (OPs). This work is an overview of both validated and emerging strategies for the protection against OP pollution with special attention to the use of decontaminating enzymes. Considerable efforts have been dedicated during the past decades to the development of efficient OP degrading biocatalysts. Among these, the promising biocatalyst SsoPox isolated from the archaeon Sulfolobus solfataricus is emphasized in the light of recently published results. This hyperthermostable enzyme appears to be particularly attractive for external decontamination purposes with regard to both its catalytic and stability properties.

Crystal structure of VmoLac, a tentative quorum quenching lactonase from the extremophilic crenarchaeon Vulcanisaeta moutnovskia.

A new representative of the Phosphotriesterase-Like Lactonases (PLLs) family from the hyperthermophilic crenarchaeon Vulcanisaeta moutnovskia has been characterized and crystallized. VmoLac is a native, proficient lactonase with promiscuous, low phosphotriesterase activity. VmoLac therefore represents an interesting candidate for engineering studies, with the aim of developing an efficient bacterial quorum-quenching agent. Here, we provide an extensive biochemical and kinetic characterization of VmoLac and describe the X-ray structures of the enzyme bound to a fatty acid and to its cognate substrate 3-oxo-C10 AHL (Acyl-Homoserine Lactone). The structures highlight possible structural determinants that may be involved in its extreme thermal stability (Tm = 128 °C). Moreover, the structure reveals that the substrate binding mode of VmoLac significantly differs from those of its close homologues, possibly explaining the substrate specificity of the enzyme. Finally, we describe the specific interactions between the enzyme and its substrate, and discuss the possible lactone hydrolysis mechanism of VmoLac.

SacPox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase.

BACKGROUND: SacPox, an enzyme from the extremophilic crenarchaeal Sulfolobus acidocaldarius (Sac), was isolated by virtue of its phosphotriesterase (or paraoxonase; Pox) activity, i.e. its ability to hydrolyze the neurotoxic organophosphorus insecticides. Later on, SacPox was shown to belong to the Phosphotriesterase-Like Lactonase family that comprises natural lactonases, possibly involved in quorum sensing, and endowed with promiscuous, phosphotriesterase activity. RESULTS: Here, we present a comprehensive and broad enzymatic characterization of the natural lactonase and promiscuous organophosphorus hydrolase activities of SacPox, as well as a structural analysis using a model. CONCLUSION: Kinetic experiments show that SacPox is a proficient lactonase, including at room temperature. Moreover, we discuss the observed differences in substrate specificity between SacPox and its closest homologues SsoPox and SisLac together with the possible structural causes for these observations.

Inhaled lactonase reduces Pseudomonas aeruginosa quorum sensing and mortality in rat pneumonia.

RATIONALE: The effectiveness of antibiotic molecules in treating Pseudomonas aeruginosa pneumonia is reduced as a result of the dissemination of bacterial resistance. The existence of bacterial communication systems, such as quorum sensing, has provided new opportunities of treatment. Lactonases efficiently quench acyl-homoserine lactone-based bacterial quorum sensing, implicating these enzymes as potential new anti-Pseudomonas drugs that might be evaluated in pneumonia. OBJECTIVES: The aim of the present study was to evaluate the ability of a lactonase called SsoPox-I to reduce the mortality of a rat P. aeruginosa pneumonia. METHODS: To assess SsoPox-I-mediated quorum quenching, we first measured the activity of the virulence gene lasB, the synthesis of pyocianin, the proteolytic activity of a bacterial suspension and the formation of biofilm of a PAO1 strain grown in the presence of lactonase. In an acute lethal model of P. aeruginosa pneumonia in rats, we evaluated the effects of an early or deferred intra-tracheal treatment with SsoPox-I on the mortality, lung bacterial count and lung damage. MEASUREMENTS AND PRIMARY RESULTS: SsoPox-I decreased PAO1 lasB virulence gene activity, pyocianin synthesis, proteolytic activity and biofilm formation. The early use of SsoPox-I reduced the mortality of rats with acute pneumonia from 75% to 20%. Histological lung damage was significantly reduced but the lung bacterial count was not modified by the treatment. A delayed treatment was associated with a non-significant reduction of mortality. CONCLUSION: These results demonstrate the protective effects of lactonase SsoPox-I in P. aeruginosa pneumonia and open the way for a future therapeutic use.