Virulence Regulator Research Articles

A novel small RNA regulates Locus of Enterocyte Effacement and site-specific colonization of enterohemorrhagic Escherichia coli O157:H7 in gut

Enterohemorrhagic Escherichia coli (EHEC) is a contagious foodborne pathogen that specifically colonizes the human large intestine, which is regulated by different environmental stimuli within the gut. Transcriptional regulation of EHEC virulence and infection has been extensively studied, while the posttranscriptional regulation of these processes by small RNAs (sRNAs) remains not fully understood. Here we present a virulence-regulating pathway in EHEC O157:H7, in which the sRNA EvrS binds to and destabilizes the mRNA of Z2269, a novel transcriptional regulator. In turn, Z2269 indirectly activates the expression of LEE (locus of enterocyte effacement) pathogenicity island through the master regulator Ler. Importantly, the expression of EvrS is modulated by environmental oxygen levels. EvrS also exhibits lower expression in the colon compared to the ileum, influencing the site-specific colonization of EHEC O157:H7 in mice. These results indicate that the oxygen status within the intestine may regulate the expression of EvrS, thereby modulating virulence factors of EHEC and contributing to successful infection in vivo. This study has broader implications for understanding sRNA functions in spatiotemporal virulence control of EHEC and may provide novel strategies to prevent EHEC infections.

Pathogenic potential of ornithogenic <i>Escherichia coli</i> strains detected in the Earth's polar regions

Introduction. Pathogenic strains of Escherichia coli are an important object of surveillance within the One Health concept in the wild, agriculture and human society. Migratory bird colonies and high latitude avian colonies may be points of active intraspecies and interspecies contact between different animal species, accompanied by the spread of pathogens. At the same time, the phylogeography of E. coli in relation to the presence of natural foci of colibacillosis in polar regions remains virtually unstudied. The aim of this study was to assess the pathogenic potential of E. coli strains from the polar regions of the Earth, based on the analysis of the genomes of these bacteria from typical ornithogenic ecosystems of the Arctic and Antarctic. Materials and methods. The study used collections of E. coli isolated from ornithogenic biological material during expeditions to high latitude areas of the Arctic (archipelagos of Novaya Zemlya, Franz Josef Land, Svalbard) and Antarctic (Haswell Archipelago). 16 cultures associated with avian E. coli (12 polar and 4 temperate strains) were selected for genome-wide sequencing using BGI technology. The annotation of the genomes focused on the identification of genes for pathogenicity factors and antimicrobial resistance, as well as the identification of strains belonging to individual genetic lineages using the cgMLST method. Results. The annotation of the genomes allowed their assignment to different sequence types in the multilocus sequencing typing and genome-wide sequencing typing schemes. The analysis of the geographical distribution of the sequence types of polar E. coli strains determined by the cgMLST method showed their global representation in geographically distant regions of the planet. For example, cgST 133718 was observed in Antarctica (strain 17_1myr) and in the UK, and sequence 11903, to which strain 32-1 from the northernmost point of Novaya Zemlya belonged, was previously identified in the USA. All strains studied were characterized by the presence of extensive virulence. Among the pathogenicity factors identified were haemolysins A, E, F, siderophores, including the yersiniabactin gene cluster, a number of adhesion, colonization and invasion factors, as well as the thermostable enterotoxin EAST-1 and genes that characterize enteroaggregative strains of E. coli (the virulence regulator gene eilA and enteroaggregative protein (air)). One of the Arctic strains (33-1) had determinants of antibiotic resistance, in particular the extended-spectrum beta-lactamase gene TEM-1b and the Tn1721 transposon, including tetracycline resistance genes (tetA-TetR), were detected in its genome. Conclusion. The results of the study indicate the circulation of E. coli strains with strong pathogenic potential in high-latitude Arctic and Antarctic ornithogenic ecosystems. The analysis of genomic data indicates the presence of geographically widespread genetic lineages in these regions, which justifies the importance of monitoring epidemic clones of E. coli, along with monitoring for other pathogens, in bird colonies in high-latitude areas.

Distinct Virulence Mechanisms of Burkholderia gladioli in Onion Foliar and Bulb Scale Tissues.

Slippery skin of onion caused by Burkholderia gladioli pv. alliicola (Bga) is a common bacterial disease reported from onion growing regions around the world. Despite the increasing attention in recent years, our understanding of the virulence mechanisms of this pathogen remains limited. In this study, we characterized the predicted genetic determinants of virulence in Bga strain 20GA0385 using reverse genetics approach. Using the closely related rice pathogen, B. glumae as a reference, comparative genomics analysis was performed to identify Bga candidate virulence factors and regulators. Marked and unmarked deletion mutants were generated using allelic exchange and the mutants were functionally validated using in vitro and in vivo assays. The role of mutants in pathogenic phenotypes was analyzed using onion foliar/seedling necrosis assays, the Red Scale Necrosis (RSN) assay and in planta bacterial population counts. The phytotoxin toxoflavin was a major contributor to foliar necrosis and bacterial populations whereas the type II and type III secretion system (T2SS/T3SS) were dispensable for foliar symptoms. In onion scale tissue, the T2SS single mutant gspC and its double and triple mutant derivatives all contributed to scale lesion area. Neither the lipocyclopeptide icosalide, toxoflavin, nor T3SS were required for scale symptoms. Our results suggest the quorum sensing tofIMR system in Bga regulates, toxoflavin, T2SS, and T3SS, contributing to onion symptom production. We show different virulence factors contribute to onion tissue-specific virulence patterns in Bga and that decreases in scale symptoms often do not result in decreased Bga populations in onion tissue.

Systematic characterization of the bZIP gene family in Colletotrichum siamense and functional analysis of three family members

The basic leucine zipper (bZIP) transcription factors (TFs) play important roles in many physiological processes of plant-pathogenic fungi, especially concerning fungal development, fungicide resistance, and pathogenicity. Colletotrichum siamense is the predominant species causing Colletotrichum leaf disease (CLD) in rubber trees. However, little is known about the bZIP genes in C. siamense. In this study, 25 bZIP genes were systematically identified in the genome of C. siamense, and molecular features were characterized. Evolutionarily, the CsbZIP genes were divided into 11 groups, with the members in the same group sharing similar gene structures and conserved protein motif organizations. Furthermore, protein-protein interaction (PPI) analysis revealed that 15 bZIP proteins had functional partners in common or interacted with other CsbZIP proteins. Additionally, the expression of 23 CsbZIP genes changed in response to the antifungal chemicals melatonin, prochloraz, and thymol, and the genes could be divided into three clusters based on their expression patterns. Finally, gene deletion mutants of CsbZIP01/09/17 were constructed and functional analysis indicated that these genes operated as important regulators of mycelial growth, fungicide resistance, ergosterol biosynthesis, and virulence in C. siamense. This study provided the foundations crucial for further investigation of the functions of CsbZIP TFs in fungicide resistance and virulence.

Metarhizium acridum transcription factor MaFTF1 negatively regulates virulence of the entomopathogenic fungus by controlling cuticle penetration of locusts.

The entomopathogenic fungus (EPF) Metarhizium acridum, a typical filamentous fungus, has been utilized for the biological control of migratory locusts (Locusta migratoria manilensis). Fungal-specific transcription factors (TFs) play a crucial role in governing various cellular processes in fungi, although TFs with only the Fungal_trans domain remain poorly understood. In this study, we identified a unique fungal-specific TF in M. acridum, named MaFTF1, which contains only a Fungal_trans domain and functions as a negative regulator of M. acridum virulence by influencing cuticle penetration. The virulence of the MaFTF1 knockout strain (ΔMaFTF1) against L. migratoria was increased, with a median lethal time (LT50) ~0.91 days shorter than that of the wild-type (WT) strain when inoculated topically, mimicking natural infection conditions. Correspondingly, ΔMaFTF1 penetrated the cuticle earlier than did the WT strain. Our investigation revealed that the development of appressoria was accelerated in ΔMaFTF1 compared with the WT strain. Furthermore, the appressoria of the ΔMaFTF1 displayed higher turgor pressure and an upregulated expression of fungal hydrolases active toward the insect cuticle. RNA sequencing analysis indicated that the differences in appressorium behavior between the strains were due to MaFTF1 regulating a complex metabolism pathway. This study revealed that MaFTF1 acts as a negative regulator of virulence, impacting the process of cuticle penetration by slowing the formation of appressoria, decreasing their turgor pressure, and reducing the expression of hydrolases in appressoria, revealing an unexpected strategy in the EPFs. © 2024 Society of Chemical Industry.

ChIP-seq and structural analyses delineating the regulatory mechanism of master regulator EsrB in Edwardsiella piscicida.

As a crucial virulence regulator, EsrB possesses a LuxR-like superfamily domain at the C-terminal, which is conserved within the canonical NarL family regulators. Due to its critically important role in virulence and pathogenicity in fish hosts, the DNA binding ability has been believed to allow EsrB to regulate genes associated with the invasion process of host cells and basal metabolism in response to environmental stimuli. The lack of EsrB's crystal structure has been a major obstacle in understanding the molecular mechanisms of EsrB-DNA interaction which choreographs EsrB-mediated pathogenic behavior. Here, we conducted ChIP-seq and solved the crystal structure of the C-terminal of EsrB (EsrBC) at 2.20-Å resolution, which revealed that EsrB preferred to bind to virulence-associated promoters with a distinct 7'-4-7' pseudopalindromic DNA motif and interacted with metabolic-related promoters with a high AT DNA motif in Dulbecco's Modified Eagle's Medium (DMEM) (mimicking in vivo environments). Our results facilitate a detailed understanding of EsrB's regulatory role in Edwardsiella piscicida pathogenesis and expand our knowledge of virulence regulators in the family Hafniaceae.

A Gene Cassette Vd276-280 in Verticillium dahliae Contains Two Genes that Affect Melanized Microsclerotium Formation and Virulence.

Verticillium dahliae is a soilborne phytopathogenic fungus causing Verticillium wilt on hundreds of plant species. Several sequenced genomes of V. dahliae are available, but functional characterization of most genes has just begun. Based on our previous comparison of the transcriptome from the wild-type and ΔVdCf2 strains, a significant upregulation of the gene cassette, Vd276-280, in the ΔVdCf2 strain was observed. In this study, the functional characterization of the Vd276-280 gene cassette was performed. Agrobacterium-mediated knockout of this gene cassette in V. dahliae significantly inhibited conidiation, melanized microsclerotium formation in the mutant strains, and their virulence toward cotton. Furthermore, deletion of individual genes in the Vd276-280 gene cassette identified that the disruption of VDAG_07276 and VDAG_07280 delayed microsclerotium formation, inhibited conidiation, and reduced virulence toward cotton. Our data suggest that VDAG_07276 and VDAG_07280 in the Vd276-280 gene cassette mainly act as positive regulators of development and virulence in V. dahliae.

Modulation of physiological functions and metabolome of Vibrio alginolyticus by quorum-regulatory sRNA, Qrr1.

Vibrio alginolyticus, the causative agent of aquatic vertebrates and invertebrates, can cause severe infections (e.g. septicemia, gill necrosis, and surface ulcers) and high mortality in aquatic organisms, leading to serious economic losses in global aquaculture. Small non-coding RNAs (sRNAs), emerging modulators of gene expression, played vital regulatory roles in virulence, pathogenicity, and physiological metabolism of bacteria. In this work, the modulation of physiological functions and metabolome of V. alginolyticus by the quorum-regulatory sRNA, Qrr1, was figured out. We found that the deletion of qrr1 induced significant cell shape elongation. Meanwhile, Qrr1 could inhibit the production of alkaline serine protease by weakening the expression of main regulator LuxR in the quorum sensing (QS) system. Moreover, the untargeted metabolomics and lipidomics approaches showed that most of nucleotides, organic acids, carbohydrates, and lipidome (both lipid content and category) were significantly altered in response to the qrr1 deletion. Spearman correlation analysis demonstrated that most of the intermediates involved in glutamate metabolism, sphingolipid metabolism, and glycerolipid metabolism displayed high correlations with cell virulence factors. These findings illuminate the mechanism of bacterial virulence regulation and further exploit potential therapeutic targets for virulence prevention in V. alginolyticus.

Protein PARylation: a novel regulator of fungal virulence.

Protein PARylation is a reversible post-translational modification; however, its role in fungal virulence has remained elusive. Recently, Gao et al. demonstrated that PARylation of two 14-3-3 regulatory proteins by poly(ADP-ribose) polymerase is essential for the virulence of rice blast fungus, highlighting the critical regulatory function of PARylation in fungal pathogenicity.

Beyond protein folding: The pleiotropic functions of PPIases in cellular processes and microbial virulence.

Peptidyl prolyl cis/trans isomerases (PPIases), a ubiquitously distributed superfamily of enzymes, associated with signal transduction, trafficking, assembly, biofilm formation, stress tolerance, cell cycle regulation, gene expression and tissue regeneration, is a key regulator of metabolic disorders and microbial virulence. This review assumes an integrative approach, to provide a holistic overview of the structural and functional diversity of PPIases, examining their conformational dynamics, cellular distribution, and physiological significance. We explore their intricate involvement in cellular processes and virulence modulation in both eukaryotic and prokaryotic systems. Additionally, we evaluate the potential of these molecular chaperones as drug targets and vaccine candidates, emphasizing their relevance in therapeutic development. By synthesizing recent findings and providing a broader perspective on these proteins, this review aims to enhance our understanding of their multifaceted roles in biology and their potential applications in medicine.

Antibiofilm Activities of Multiple Halogenated Pyrimidines Against Staphylococcus aureus.

Staphylococcus aureus, prevalent in hospital and community settings, forms biofilms that are highly resistant to antibiotics and immune responses, complicating treatment and contributing to chronic infections. These challenges underscore the need for novel treatments that target biofilm formation and effectively reduce bacterial virulence. This study investigates the antibiofilm and antimicrobial efficacy of novel halogenated pyrimidine derivatives against S. aureus, focusing on three compounds identified as potent biofilm inhibitors: 2,4-dichloro-5-fluoropyrimidine (24DC5FP), 5-bromo-2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (24DC5BPP), and 2,4-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (24DC5IPP). The three active compounds are bacteriostatic. In particular, 24DC5FP at 5 µg/mL achieved a 95% reduction in hemolysis with a minimum inhibitory concentration (MIC) of 50 µg/mL. Interestingly, 24DC5FP increased cell size and produced wrinkled colonies. qRT-PCR analysis showed that 24DC5FP suppressed the gene expressions of agrA and RNAIII (quorum sensing regulator and effector), hla (α-hemolysin), nuc1 (nucleases nuc1), and saeR (S. aureus virulence regulator). These findings suggest that extensive halogenation enhances the antibiofilm and antivirulence activities of pyrimidine derivatives, offering a promising strategy for combatting S. aureus infections, including those resistant to conventional treatments.

The association of dextran sodium sulfate to the bioactive agent I-modulia® attenuates Staphylococcus aureus virulence expression and δ-toxin production

As a part of the human skin commensal bacterial community, Staphylococcus aureus contributes to the host's immune system education. Nevertheless, it is also considered as an opportunistic pathogen involved in cutaneous infections or skin pathologies, in particular atopic dermatitis. To switch to a pathogenic behavior, S. aureus uses regulatory mechanisms to collectively produce virulence factors. Deprivation of these factors has emerged as a promising way to prevent or treat Staphylococcal diseases in facilitating the role of the immune system, while preserving the protective one of the commensal communities. This study focuses on the anti-virulent effect of dextran sodium sulfate (DSS) and I-modulia®, two natural products that have already proven their value in skincare. The anti-virulent capacity of DSS was first demonstrated by a dose-dependent inhibition of δ-toxin release, a virulence factor known to be a potent inducer of mast cell degranulation, on in vitro S. aureus cultures at high and low virulent states. A transcriptomic study was then implemented for a comprehensive overview of the anti-virulent impact. The results have shown the downregulation of many transcripts related to host immune evasion (scn, sbi), as well as exotoxins (α,γ-toxin) and adhesins production (map, emp), mostly under the control of SaeRS Two-Component System (TCS), one of the two major virulence regulators in S. aureus. Interestingly, genes related to secretion systems and the synthesis of exo-proteases were significantly downregulated when DSS was used in combination with I-modulia®. The repression of these genes was not previously observed and reflects a broader inhibitory action. We have also demonstrated that the inhibition of virulence factors didn't affect S. aureus viability. Our findings suggest that combining DSS and I-modulia® could be a promising therapeutic strategy to counteract microbial dysbiosis in the treatment of S. aureus skin pathologies in re-empowering the host's natural immune defenses.

SarZ inhibits the hemolytic activity through regulation of phenol soluble modulins in Staphylococcus epidermidis.

Staphylococcus epidermidis is an important conditionally pathogenic bacterium. SarZ, belonging to the SarA family protein, has been demonstrated in S. aureus to promote the expression of invasive virulence factors while inhibiting biofilm formation. However, the regulatory role of SarZ on S. epidermidis virulence is not completely understood. In this study, we successfully deleted the sarZ gene by allelic replacement in S. epidermidis. The sarZ mutant strain exhibited remarkably increased hemolytic activity and drastically impaired biofilm formation, suggesting that SarZ is key regulator of virulence in S. epidermidis. Through butanol extraction of the spent medium and HPLC-MS/MS analysis, production of phenol soluble modulins (PSMs) possessing cytolytic effect was found to be elevated significantly in the mutant. Subsequent qRT-PCR experiments demonstrated that expression of the psm genes, especially the β-type, was upregulated dramatically in the mutant. Meanwhile, transcription of icaA gene responsible for biofilm formation was sharply diminished. The sarZ psmβ double mutant was further generated and displayed a significantly decreased hemolytic activity compared with the sarZ mutant. EMSA assays implied that recombinant SarZ protein can directly bind to the promoter regions of the psmβ and ica operon. DNase I footprinting assays further pinpointed two SarZ-binding sites on the psmβ operon promoter. Taken together, the results confirmed that SarZ is a pivotal regulator of virulence in S. epidermidis and might respectively regulate the hemolytic activity and biofilm formation mainly by directly controlling the transcription of psm genes, particularly the β-type, and the ica operon.

Key roles of two-component systems in intestinal signal sensing and virulence regulation in enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that infects humans by colonizing the large intestine. Upon reaching the large intestine, EHEC mediates local signal recognition and the transcriptional regulation of virulence genes to promote adherence and colonization in a highly site-specific manner. Two-component systems (TCSs) represent an important strategy used by EHEC to couple external stimuli with the regulation of gene expression, thereby allowing EHEC to rapidly adapt to changing environmental conditions. An increasing number of studies published in recent years have shown that EHEC senses a variety of host- and microbiota-derived signals present in the human intestinal tract and coordinates the expression of virulence genes via multiple TCS-mediated signal transduction pathways to initiate the disease-causing process. Here, we summarize how EHEC detects a wide range of intestinal signals and precisely regulates virulence gene expression through multiple signal transduction pathways during the initial stages of infection, with a particular emphasis on the key roles of TCSs. This review provides valuable insights into the importance of TCSs in EHEC pathogenesis, which has relevant implications for the development of antibacterial therapies against EHEC infection.

The Cryptococcus neoformans STRIPAK complex controls genome stability, sexual development, and virulence.

The eukaryotic serine/threonine protein phosphatase PP2A is a heterotrimeric enzyme composed of a scaffold A subunit, a regulatory B subunit, and a catalytic C subunit. Of the four known B subunits, the B"' subunit (known as striatin) interacts with the multi-protein striatin-interacting phosphatase and kinase (STRIPAK) complex. Orthologs of STRIPAK components were identified in Cryptococcus neoformans, namely PP2AA/Tpd3, PP2AC/Pph22, PP2AB/Far8, STRIP/Far11, SLMAP/Far9, and Mob3. Structural modeling, protein domain analysis, and detected protein-protein interactions suggest C. neoformans STRIPAK is assembled similarly to the human and fungal orthologs. Here, STRIPAK components Pph22, Far8, and Mob3 were functionally characterized. Whole-genome sequencing revealed that mutations in STRIPAK complex subunits lead to increased segmental and chromosomal aneuploidy, suggesting STRIPAK functions in maintaining genome stability. We demonstrate that PPH22 is a haploinsufficient gene: heterozygous PPH22/pph22Δ mutant diploid strains exhibit defects in hyphal growth and sporulation and have a significant fitness disadvantage when grown in competition against a wild-type diploid. Deletion mutants pph22Δ, far8Δ, and mob3Δ exhibit defects in mating and sexual differentiation, including impaired hyphae, basidia, and basidiospore production. Loss of either PPH22 or FAR8 in a haploid background leads to growth defects at 30°C, severely reduced growth at elevated temperature, abnormal cell morphology, and impaired virulence. Additionally, pph22Δ strains frequently accumulate suppressor mutations that result in overexpression of another putative PP2A catalytic subunit, PPG1. The pph22Δ and far8Δ mutants are also unable to grow in the presence of the calcineurin inhibitors cyclosporine A or FK506, and thus these mutations are synthetically lethal with loss of calcineurin activity. Conversely, mob3Δ mutants display increased thermotolerance, capsule production, and melanization, and are hypervirulent in a murine infection model. Taken together, these findings reveal that the C. neoformans STRIPAK complex plays an important role in genome stability, vegetative growth, sexual development, and virulence in this prominent human fungal pathogen.

Novel motif associated with carbon catabolite repression in two major Gram-positive pathogen virulence regulatory proteins.

Carbon catabolite repression (CCR) is a widely conserved regulatory process that ensures enzymes and transporters of less-preferred carbohydrates are transcriptionally repressed in the presence of a preferred carbohydrate. This phenomenon can be regulated via a CcpA-dependent or CcpA-independent mechanism. The CcpA-independent mechanism typically requires a transcriptional regulator harboring a phosphotransferase regulatory domain (PRD) that interacts with phosphotransferase system (PTS) components. PRDs contain a conserved histidine residue that is phosphorylated by the PTS-associated HPr-His15~P protein. PRD-containing regulators often harbor additional domains that resemble PTS-associated EIIB protein domains with a conserved cysteine residue that can be phosphorylated by cognate PTS components. We noted that Mga, the PRD-containing central virulence regulator of Streptococcus pyogenes, has an EIIBGat domain containing a cysteine that, based on the presence of a similar motif in glycerol kinase, could be a target for phosphorylation. Using site-directed mutagenesis, we constructed phospho-ablative and phospho-mimetic substitutions of this cysteine and found that these substitutions modify the CCR of the Rgg2/3 quorum-sensing system. Moreover, we provide genetic evidence that the phospho-donor of this cysteine residue is likely to be ManL, the EIIA/B subunit of the mannose PTS system. Interestingly, a structurally distinct virulence gene regulator, PrfA of Listeria monocytogenes, harbors a similar cysteine-containing motif, and phospho-ablative and phospho-mimetic substitutions of the cysteine-altered CCR of PrfA-dependent virulence gene expression. Collectively, our data suggest that phosphorylation of a cysteine within the shared novel motif in Mga and PrfA may be a heretofore missing link between cellular metabolism and virulence.IMPORTANCEIn this study, we identified a novel cysteine-containing motif within the amino acid sequence of two structurally distinct transcriptional regulators of virulence in two Gram-positive pathogens that appears to link carbon metabolism with virulence gene expression. The results also highlight the potential post-translational modification of cysteine in bacterial species, a rare and understudied modification.

Pleiotropic regulatory function of the RNA chaperone Hfq in the Pseudomonas protegens FD6

The rhizosphere bacterium Pseudomonas protegens FD6, which is associated with the production of antibiotics such as pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), has strong antagonistic effects on phytopathogens. Hfq is a conserved RNA chaperone involved in the regulation of stress tolerance, antibiotic production, and bacterial virulence. Here, we determined the regulatory effects of Hfq on biocontrol traits in P. protegens FD6. Mutation of hfq in FD6 reduced the growth rate, swimming and swarming ability, and production of proteases, siderophores, and hydrogen cyanide (HCN). Transmission electron microscopy revealed that disruption of hfq led to the loss of flagella. Furthermore, Hfq exerted a suppressive effect on biofilm formation and PLT production while not affecting antagonistic activity and control effect against tomato bacterial wilt. However, Hfq positively regulated the production of 2,4-DAPG and activated the phlD expression. Additionally, mutation of hfq abolished the function of the type VI secretion system (T6SS) due to a decrease in the expression of T6SS-related genes. Overall, these results suggest that Hfq plays a pleiotropic role in modulating the expression of biocontrol trait-related genes in P. protegens FD6.

Cyclic Diguanylate G-Quadruplex Inducer-Quorum Sensing Inhibitor Hybrids as Bifunctional Anti-biofilm and Anti-virulence Agents Against Pseudomonas aeruginosa.

The release of virulence factors and biofilm formation by Pseudomonas aeruginosa are pivotal drivers of its severe pathogenicity and antibiotic resistance. Based on our prior findings, cyclic di-GMP (c-di-GMP) G-quadruplex inducers are promising biofilm inhibitors and that quorum sensing systems are central regulators of virulence, we aimed to design and synthesize c-di-GMP G-quadruplex inducer-quorum sensing inhibitor hybrids. These hybrids were envisioned as bifunctional agents with both antibiofilm and antivirulence capabilities. Hybrids A7 and A11, characterized by their quinoline and 3-indole rings, emerged as potent inhibitors. They achieve this dual action by inducing c-di-GMP G-quadruplex formation and disrupting the las and pqs signaling system. Additionally, hybrids A7 and A11 attenuated virulence factors and inhibited the motility phenotypes of P. aeruginosa. Furthermore, when tested in in vivo Caenorhabditis elegans infection models, these hybrids, in combination with antibiotics such as tetracycline, improved survival rates, all while maintaining a favorable biosafety profile.

Vibrio cholerae virulence is blocked by chitosan oligosaccharide-mediated inhibition of ChsR activity.

Vibrio cholerae causes cholera, an important cause of death worldwide. A fuller understanding of how virulence is regulated offers the potential for developing virulence inhibitors, regarded as efficient therapeutic alternatives for cholera treatment. Here we show using competitive infections of wild-type and mutant bacteria that the regulator of chitosan utilization, ChsR, increases V. cholerae virulence in vivo. Mechanistically, RNA sequencing, chromatin immunoprecipitation with sequencing and molecular biology approaches revealed that ChsR directly upregulated the expression of the virulence regulator, TcpP, which promoted expression of the cholera toxin and the toxin co-regulated pilus, in response to low O2 levels in the small intestine. We also found that chitosan degradation products inhibit the ChsR-tcpP promoter interaction. Consistently, administration of chitosan oligosaccharide, particularly when delivered via sodium alginate microsphere carriers, reduced V. cholerae intestinal colonization and disease severity in mice by blocking the chsR-mediated pathway. These data reveal the potential of chitosan oligosaccharide as supplemental therapy for cholera treatment and prevention.

Exploring Phytochemical Compounds Against Pseudomonas Aeruginosa Using QSAR, Molecular Dynamics, and Free Energy Landscape

AbstractPseudomonas aeruginosa is a versatile opportunistic bacterium that presents a considerable risk in medical environments because of its strong adaptability and resistance to multiple medications. Targeting the LasR quorum sensing system, which plays a crucial role in controlling virulence factors and biofilm formation, is a key intervention point. In this study, in silico molecular docking, machine learning‐based Quantitative Structure‐Activity Relationship (QSAR) techniques along with molecular dynamics simulation were employed to screen phytochemical compounds for their ability to inhibit the LasR QS system, a key regulator of virulence in Pseudomonas aeruginosa. This study screened 1652 phytochemicals using the ML‐based QSAR model to identify 52 phytochemicals that had better activity than the control (N‐{[3,5‐dibromo‐2‐(methoxymethoxy)phenyl]methyl}‐2‐nitrobenzamide). The in silico molecular docking approach that targeted LasR identified compounds 5281647, 57331045, and 5281672 with high binding affinity and hydrogen bonds that were comparable to the control (docking score=−10.3 kcal/mol and hydrogen bonds=4). In the 200 ns post‐molecular dynamics simulation, 5281647 exhibited a stable RMSD of 0.25 nm, which was comparable to the control. The maximum number of hydrogen bonds was exhibited by 57331045, while 5281647 and 5281672 consistently exhibited four hydrogen bonds. Overall, the Principal component analysis (PCA) and Free Energy Landscape (FEL) analyses of the complexes demonstrated that these three compounds were in stable states. In comparison to the control (ΔGTOTAL=−39.58 kcal/mol), the cumulative binding free energy (ΔGTOTAL) for 5281647 and 57331045 was −39.95 kcal/mol and −39.25 kcal/mol, respectively. This further confirms the superior binding affinity of the two compounds. Both 5281647 and 57331045 were identified as potent inhibitors of the LasR transcription factor, which is essential for the quorum sensing of Pseudomonas aeruginosa, in the present investigation. These findings underscore the importance of further exploration and optimization of phytochemicals for combating bacterial infections, offering promising avenues for future drug discovery efforts targeting this resilient pathogen.