Livnat Afriat-Jurnou

Microorganisms use diverse small‐molecule signals to coordinate behavior, supporting interspecies and interkingdom communication. These chemical dialogs shape fundamental biological processes and define interactions from cooperation to antagonism. Quorum‐quenching (QQ) enzymes, which degrade or modify quorum sensing (QS) molecules such as N‐acyl‐homoserine lactones (AHLs), may play a pivotal role in shaping these networks. While bacterial QQ lactonases are well‐characterized, fungal lactonases remain undercharacterized, including their potential to target both bacterial QS molecules and fungal secondary metabolites. Using phylogenetic and structural analysis, we identified putative lactonases from the metallo‐β‐lactamase‐like lactonase family in fungi and biochemically validated the activity of one of them, from Aspergillus pseudonomiae . Structural alignment between bacterial lactonases and the AlphaFold‐predicted structure of the fungal homolog further supports that the fungal protein is indeed a lactonase. We then applied Protein Repair One‐Stop Shop algorithm for stability engineering to design a variant with improved expression in Escherichia coli and thermal stability. Purified enzyme demonstrated broad substrate specificity, effectively degrading various bacterial AHLs, with k _cat / K _M values of up to 1.4 × 10 ⁵ s ⁻¹ M ⁻¹ . Both docking analysis and in vitro assays indicated that the enzyme could accommodate and act on the fungal secondary metabolite patulin, a mycotoxin associated with post‐harvest disease, showing high catalytic efficiency (1.1 × 10 ⁴ s ⁻¹ M ⁻¹ ). When added exogenously to microbial cultures, the enzyme reduced bacterial biofilms and impaired fungal hyphal development, with the hyphal inhibition accompanied by a concentration‐dependent increase in ROS. Together, these results show that this fungal enzyme functions as a dual‐acting lactonase, positioning it as an interference enzyme in chemical signaling of fungi and bacteria and as a means to suppress pathogens and reduce patulin contamination in food.

The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some xenobiotics, several enzymatic steps are needed to obtain nontoxic products. Another challenge is the low durability and stability of many native enzymes in their purified form. Herein, we coupled peptide-based encapsulation of bacterial phosphotriesterase with soil-originated bacteria, Arthrobacter sp. 4H beta as an efficient system capable of biodegradation of paraoxon, a neurotoxin pesticide. Specifically, recombinantly expressed and purified methyl parathion hydrolase (MPH), with high hydrolytic activity toward paraoxon, was encapsulated within peptide nanofibrils, resulting in increased shelf life and retaining similar to 50% activity after 132 days since purification. Next, the addition of Arthrobacter sp. 4H beta, capable of degrading para-nitrophenol (PNP), the hydrolysis product of paraoxon, which is still toxic, resulted in nondetectable levels of PNP. These results present an efficient one-pot system that can be further developed as an environmentally friendly solution, coupling purified enzymes and native bacteria, for pesticide bioremediation. We further suggest that this system can be tailored for different xenobiotics by encapsulating the rate-limiting key enzymes followed by their combination with environmental bacteria that can use the enzymatic step products for full degradation without the need to engineer synthetic bacteria.

Bacteria, fungi, and mammals contain lactonases that can degrade the Gram-negative bacterial quorum sensing (QS) molecules N-acyl homoserine lactones (AHLs). AHLs are critical for bacteria to coordinate gene expression and pathogenicity with population density. However, AHL-degrading lactonases present variable substrate ranges, including degradation of the Pencillium expansum lactone mycotoxin patulin. We selected Erwinia spp. as our model bacteria to further investigate this interaction. We find both native apple microbiome Erwinia spp. and the fruit tree pathogen Erwinia amylovora to be inhibited by patulin. At patulin concentrations that inhibited E. amylovora growth, expression of E. amylovora lactonase encoded by EaaiiA was increased. EaAiiA demonstrated the ability to degrade patulin in vitro, as well, as in vivo where it reduced apple disease and patulin production by P. expansum . Fungal-bacterial co-cultures revealed that the E. amylovora Δ eaaiia strain failed to protect apples from P. expansum infections, which contained significant amounts of patulin. Our results suggest that bacterial lactonase production can modulate the pathogenicity of P. expansum in response to the secretion of toxic patulin. IMPORTANCE: Chemical signaling in the microbial world facilitates the regulation of gene expression as a function of cell population density. This is especially true for the Gram-negative bacterial signal N-acyl homoserine lactone (AHL). Lactonases that deactivate AHLs have attracted a lot of attention because of their antibacterial potential. However, the involvement of these enzymes in inhibiting fungal pathogens and the potential role of these enzymes in bacterial-fungal interactions are unknown. Here, we find that a bacterial enzyme involved in the degradation of AHLs is also induced by and degrades the fungal lactone mycotoxin, patulin. This work supports the potential use of bacterial enzymes and/or the producing bacteria in controlling the post-harvest fruit disease caused by the patulin-producing fungus Penicillium expansum .

Our results emphasize the potential of sequence and structure-based identification of new QQ enzymes from environmental metagenomes, such as from the ocean, with improved stability or activity. The findings also suggest that purified QQ enzymes can present new strategies against food spoilage, in addition to their recognized involvement in inhibiting bacterial pathogen virulence factors.

Penicillium expansum is a necrotrophic wound fungal pathogen that secrets virulence factors to kill host cells including cell wall degrading enzymes (CWDEs), proteases, and mycotoxins such as patulin. During the interaction between P. expansum and its fruit host, these virulence factors are strictly modulated by intrinsic regulators and extrinsic environmental factors. In recent years, there has been a rapid increase in research on the molecular mechanisms of pathogenicity in P. expansum; however, less is known regarding the bacteria–fungal communication in the fruit environment that may affect pathogenicity. Many bacterial species use quorum-sensing (QS), a population density-dependent regulatory mechanism, to modulate the secretion of quorum-sensing signaling molecules (QSMs) as a method to control pathogenicity. N-acyl homoserine lactones (AHLs) are Gram-negative QSMs. Therefore, QS is considered an antivirulence target, and enzymes degrading these QSMs, named quorum-quenching enzymes, have potential antimicrobial properties. Here, we demonstrate that a bacterial AHL lactonase can also efficiently degrade a fungal mycotoxin. The mycotoxin is a lactone, patulin secreted by fungi such as P. expansum. The bacterial lactonase hydrolyzed patulin at high catalytic efficiency, with a k_cat value of 0.724 ± 0.077 s⁻¹ and K_M value of 116 ± 33.98 μM. The calculated specific activity (k_cat/K_M) showed a value of 6.21 × 10³ s⁻¹M⁻¹. While the incubation of P. expansum spores with the purified lactonase did not inhibit spore germination, it inhibited colonization by the pathogen in apples. Furthermore, adding the purified enzyme to P. expansum culture before infecting apples resulted in reduced expression of genes involved in patulin biosynthesis and fungal cell wall biosynthesis. Some AHL-secreting bacteria also express AHL lactonase. Here, phylogenetic and structural analysis was used to identify putative lactonase in P. expansum. Furthermore, following recombinant expression and purification of the newly identified fungal enzyme, its activity with patulin was verified. These results indicate a possible role for patulin and lactonases in inter-kingdom communication between fungi and bacteria involved in fungal colonization and antagonism and suggest that QQ lactonases can be used as potential antifungal post-harvest treatment.

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.

Quorum quenching (QQ) is the ability to interfere with bacterial cell to cell communication, known as quorum sensing (QS). QQ enzymes that degrade or modify acyl homoserine lactones (AHLs) have been attracting increasing interest as promising agents for inhibiting QS-mediated bacterial pathogenicity. Plant pathogens from the genus Erwinia cause diseases in several economically important crops. Fire blight is a devastating plant disease caused by Erwinia amylovora, affecting a wide range of host species within the Rosaceae and posing a major global threat for commercial apple and pear production. While QS has been described in Erwinia species, no AHL-degrading enzymes were identified and characterized. Here, phylogenetic analysis and structural modeling were applied to identify an AHL lactonase in E. amylovora (dubbed EaAiiA). Following recombinant expression and purification, the enzyme was biochemically characterized. EaAiiA lactonase activity was dependent on metal ions and effectively degraded AHLs with high catalytic efficiency. Its highest specific activity (k_cat/K_M value) was observed against one of the AHLs (3-oxo-C6-homoserine lactone) secreted from E. amylovora. Exogenous addition of the purified enzyme to cultures of E. amylovora reduced the formation of levan, a QS-regulated virulence factor, by 40% and the transcription level of the levansucrase-encoding gene by 55%. Furthermore, preincubation of E. amylovora cultures with EaAiiA inhibited the progress of fire blight symptoms in immature Pyrus communis fruits. These results demonstrate the ability of the identified enzyme from E. amylovora to act as a quorum-quenching lactonase.

The need to increase agricultural yield has led to an extensive use of antibiotics against plant pathogens, which has resulted in the emergence of resistant strains. Therefore, there is an increasing demand for new methods, preferably with lower chances of developing resistant strains and a lower risk to the environment or public health. Many Gram-negative bacterial pathogens use quorum sensing, a population-density-dependent regulatory mechanism, to monitor the secretion of N-acyl-homoserine lactones (AHLs) and pathogenicity. Therefore, quorum sensing represents an attractive antivirulence target. AHL lactonases hydrolyze AHLs and have potential antibacterial properties; however, their use is limited by thermal instability and durability, or low activity. Here, we demonstrate that an AHL lactonase from the phosphotriesterase-like lactonase family exhibits high activity with the AHL secreted from the plant pathogen Erwinia amylovora and attenuates infection in planta. Using directed enzyme evolution, we were able to increase the enzyme’s temperature resistance (T₅₀, the temperature at which 50% of the activity is retained) by 8 °C. Then, by performing enzyme encapsulation in nanospherical capsules composed of tertbutoxycarbonyl-Phe-Phe-OH peptide, the shelf life was extended for more than 5 weeks. Furthermore, the encapsulated and free mutant were able to significantly inhibit up to 70% blossom’s infection in the field, achieving the same efficacy as seen with antibiotics commonly used today to treat the plant pathogen. We conclude that specific AHL lactonase can inhibit E. amylovora infection in the field, as it degrades the AHL secreted by this plant pathogen. The combination of directed enzyme evolution and peptide nanostructure encapsulation significantly improved the thermal resistance and shelf life of the enzyme, respectively, increasing its potential in future development as antibacterial treatment.

Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F₄₂₀H₂-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance. Bacterial pathogens often evolve resistance to antibiotics via mutations in the coding sequences of genes–frequently the target, an enzyme that metabolizes or transports the active drug, or an enzyme that activates a prodrug. In the case of tuberculosis, antibiotic resistance is a growing problem, with the rapid emergence of multi-drug resistant strains. New nitroimidazole-based antibiotic prodrugs, such as pretomanid and delamanid have the potential to help infected individuals, but we must guard against the evolution of resistance to these new compounds in Mycobacterium tuberculosis. In this report we use protein engineering to identify mutations that could potentially result in antibiotic resistance by knocking out the prodrug activating activity of the deazaflavin dependent nitroreductase (DDN), without completely abolishing its native menaquinone reductase activity. The retention of its native activity is important as DDN appears to be required for M. tuberculosis to emerge from hypoxia. Strikingly, when we analysed >15,000 M. tuberculosis genomes from clinical strains, we identified several that harboured mutations that we identified as abolishing prodrug activation in vitro. A hypervirulent Beijing strain N0008 from Vietnam (which had not been exposed to pretomanid in the clinic) was predicted, and confirmed, to be resistant to pretomanid, revealing that resistance to this drug has arisen through genetic drift and not selective pressure in this instance. These data show that by testing potential resistant mutations in the laboratory before large-scale use of antibiotics, we should be able to use them more judiciously in order to slow the spread of resistance.

Type‐II water‐soluble chlorophyll (Chl) proteins (WSCPs) of Brassicaceae are promising models for understanding how protein sequence and structure affect Chl binding and spectral tuning in photosynthetic Chl–protein complexes. However, to date, their use has been limited by the small number of known WSCPs, which also limited understanding their physiological roles. To overcome these limitations, we performed a phylogenetic analysis to compile a more comprehensive and complete set of natural type‐II WSCP homologues. The identified homologues were heterologously expressed in Escherichia coli, purified, tested for assembly with chlorophylls, and spectroscopically characterized. The analyses led to the discovery of previously unrecognized type‐IIa and IIb subclass WSCPs, as well as of a new subclass that did not bind chlorophylls. Further analysis by ancestral sequence reconstruction yielded sequences of putative ancestors of the three subclasses, which were subsequently recombinantly expressed in E. coli, purified and characterized. Combining the phylogenetic and spectroscopic data with molecular structural information revealed distinct Chl‐binding motifs, and identified residues critically impacting spectral tuning. The distinct Chl‐binding properties of the WSCP archetypes suggest that the non‐Chl‐binding subclass evolved from a Chl‐binding ancestor that most likely lost its Chl‐binding capacity upon localization in the plant tissues with low Chl content. This dual evolutionary trajectory is consistent with WSCPs association with the Kunitz‐type protease inhibitors superfamily, and indications of their inhibitory activity in response to various forms of stress in plants. These findings suggest new directions for exploring the physiological roles of WSCPs and the correlation, if any, between Chl‐binding and protease inhibition functionality.