Comparative genomics of Tn6411 transposons carrying the blaIMP-1 gene in Pseudomonas aeruginosa.

We aimed to determine the molecular characteristics of carbapenem-resistant Pseudomonas aeruginosa strains 18081308 and 18083286, which were isolated from the urine and the sputum of two Chinese patients, respectively. Additionally, we conducted a comparative analysis between Tn6411 carrying blaIMP-1 in strain 18083286 and transposons from the same family available in GenBank. Bacterial genome sequencing was carried out on strains 18081308 and 18083286 to obtain their whole genome sequence. Average nucleotide identity (ANI) was used for their precise species identification. Serotyping and multilocus sequence typing were performed. Furthermore, the acquired drug resistance genes of these strains were identified. The carbapenem-resistant P. aeruginosa strains isolated in the present study were of sequence type ST865 and serotype O6. They all carried the same resistance genes (aacC2, tmrB, and blaIMP-1). Tn6411, a Tn7-like transposon carrying blaIMP-1, was found in strain 18083286 by single molecule real time (SMRT) sequencing. We also identified the presence of this transposon sequence in other chromosomes of P. aeruginosa and plasmids carried by Acinetobacter spp. in GenBank, indicating the necessity for heightening attention to the potential transferability of this transposon.

A Wohlfahrtiimonas chitiniclastica with a novel type of blaVEB-1-carrying plasmid isolated from a zebra in China.

Background: Wohlfahrtiimonas chitiniclastica is an emerging fly-borne zoonotic pathogen, which causes infections in immunocompromised patients and some animals. Herein, we reported a W. chitiniclastica BM-Y from a dead zebra in China. Methods: The complete genome sequencing of BM-Y showed that this isolate carried one chromosome and one novel type of blaVEB-1-carrying plasmid. Detailed genetic dissection was applied to this plasmid to display the genetic environment of blaVEB-1. Results: Three novel insertion sequence (IS) elements, namely ISWoch1, ISWoch2, and ISWoch3, were found in this plasmid. aadB, aacA1, and gcuG were located downstream of blaVEB-1, composing a gene cassette array blaVEB-1-aadB-aacA1-gcuG bracketed by an intact ISWoch1 and a truncated one, which was named the blaVEB-1 region. The 5'-RACE experiments revealed that the transcription start site of the blaVEB-1 region was located in the intact ISWoch1 and this IS provided a strong promoter for the blaVEB-1 region. Conclusion: The spread of the blaVEB-1-carrying plasmid might enhance the ability of W. chitiniclastica to survive under drug selection pressure and aggravate the difficulty in treating infections caused by blaVEB-1-carrying W. chitiniclastica. To the best of our knowledge, this is the first report of the genetic characterization of a novel blaVEB-1-carrying plasmid with new ISs from W. chitiniclastica.

Vibrio parahaemolyticus from Migratory Birds in China Carries an Extra Copy of tRNA-Gly and Plasmid-Mediated Quinolone Resistance Gene qnrD.

Vibrio parahaemolyticus is a marine bacterium coming from estuarine environments, where the migratory birds can easily be colonized by V. parahaemolyticus. Migratory birds may be important reservoirs of V. parahaemolyticus by growth and re-entry into the environment. To further explore the spreading mechanism of V. parahaemolyticus among marine life, human beings, and migratory birds, we aimed to investigate the characteristics of the genetic diversity, antimicrobial resistance, virulence genes, and a potentially informative gene marker of V. parahaemolyticus isolated from migratory birds in China. This study recovered 124 (14.55%) V. parahaemolyticus isolates from 852 fecal and environmental (water) samples. All of the 124 strains were classified into 85 known sequence types (STs), of which ST-2738 was most frequently identified. Analysis of the population structure using whole-genome variation of the 124 isolates illustrated that they grouped into 27 different clonal groups (CGs) belonging to the previously defined geographical populations VppX and VppAsia. Even though these genomes have high diversity, an extra copy of tRNA-Gly was presented in all migratory bird-carried V. parahaemolyticus isolates, which could be used as a potentially informative marker of the V. parahaemolyticus strains derived from birds. Antibiotic sensitivity experiments revealed that 47 (37.10%) isolates were resistant to ampicillin. Five isolates harbored the plasmid-mediated quinolone resistance (PMQR) gene qnrD, which has not previously been identified in this species. The investigation of antibiotic resistance provides the basic knowledge to further evaluate the risk of enrichment and reintroduction of pathogenic V. parahaemolyticus strains in migratory birds. IMPORTANCE The presence of V. parahaemolyticus in migratory birds' fecal samples implies that the human pathogenic V. parahaemolyticus strains may also potentially infect birds and thus pose a risk for zoonotic infection and food safety associated with re-entry into the environment. Our study firstly highlights the extra copy of tRNA as a potentially informative marker for identifying the bird-carried V. parahaemolyticus strains. Also, we firstly identify the plasmid-mediated quinolone resistance (PMQR) gene qnrD in V. parahaemolyticus. To further evaluate the risk of enrichment and reintroduction of pathogenic strains carried by migratory birds, we suggest conducting estuarine environmental surveillance to monitor the antibiotic resistance and virulence factors of bird-carried V. parahaemolyticus isolates.

Characteristics of Carbapenem-resistant Klebsiella pneumoniae in sewage from a tertiary hospital in Jilin Province, China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection is a serious problem in hospitals worldwide. We monitored a tertiary hospital in Changchun, Jilin Province, China, and found that CRKP was the major species among the carbapenem-resistant isolates in sewage. Subsequently, we evaluated the drug susceptibility, resistance genes, virulence genes, outer pore membrane protein-related genes (OmpK35 & OmpK 36), multi-locus sequence typing and replicons, biofilm formation capabilities, and resistance to chlorine-containing disinfectants among KP isolates. Identification of drug sensitivity, multiple resistance profiles were observed including 77 (82.80%) multidrug resistant (MDR), 16 (17.20%) extensive drug resistant (XDR). Some antibiotic resistance genes were detected, the most prevalent carbapenemase gene was blaKPC, and 16 resistance genes were associated with other antibiotics. In addition, 3 (3.23%) CRKP isolates demonstrated loss of OmpK-35 and 2 (2.15%) demonstrated loss of OmpK-36. In the detection of multi-locus sequence typing (MLST), 11 ST11 isolates carried virulence genes. The most common replicon type was IncFII. Biofilm-forming capabilities were demonstrated by 68.8% of the isolates, all of which were resistant to chlorine-containing disinfectants. The results of the study showed that antibiotic-resistant isolates, especially CRKP, could resist disinfectants in hospital wastewater, and improper treatment of hospital wastewater may lead to the spread of drug-resistant bacteria and their genes. Thus, these bacteria must be eliminated before being discharged into the municipal sewage system.

Anti-inflammatory, antioxidant and anti-virulence roles of atractylodin in attenuating Listeria monocytogenes infection.

Background: Listeria monocytogenes (L. monocytogenes), as a pandemic foodborne pathogen, severely threatens food security and public health care worldwide, which evolves multiple bacterial virulence factors (such as listeriolysin O, LLO) for manipulating the immune response of L. monocytogenes-host interactions. Methods: Hemolysis assay was employed to screen a potential LLO inhibitor and the underlying mechanisms were investigated using molecular dynamics (MD) simulation and oligomerization assay. The effects of candidates on immune response were examined by qRT-PCR and immunoblotting analysis. Histological analysis, ELISA assay and biochemistry detection were conducted to assess in vivo efficacy of candidates. Results: In the present study, natural terpenoid atractylodin was characterized as an alternative drug candidate for the treatment of L. monocytogenes by the regulation of LLO function and host Nrf2/NLRP3 signaling pathway. Notably, in vivo infection model by L. monocytogenes also highlighted that atractylodin treatment provided effective therapeutic benefits, as evidenced by decreased bacterial burden and diminished inflammation. Congruently, the survival rate of L. monocytogenes-infection mice increased significantly from 10.0% to 40.0% by atractylodin treatment. Conclusion: Collectively, our study showed for the first time that atractylodin has tremendous potential to attenuate L. monocytogenes pathogenicity by blocking LLO pore formation and mediating the suppression of inflammation and oxidative stress, providing a promising therapeutic strategy and broadening the applications of atractylodin against L. monocytogenes infection.

A clinical Pseudomonas juntendi strain with bla IMP-1 carried by an integrative and conjugative element in China.

Objective: To precisely determine the species of a carbapenem-resistant Pseudomonas strain 1809276 isolated from the urine of a Chinese patient and analyze its integrative and conjugative element (ICE) 1276 formation mechanism. Methods: Single-molecule real-time (SMRT) sequencing was carried out on strain 18091276 to obtain the complete chromosome and plasmid (pCN1276) sequences, and average nucleotide identity (ANI) was used for precise species identification. The ICEs in GenBank with the same integrase structure as ICE 1276 were aligned. At the same time, the transfer ability of bla IMP-1 and the antibiotic sensitivity of Pseudomonas juntendi 18091276 were tested. Results: This bacterium was P. juntendi, and its drug resistance mechanism is the capture of the accA4' gene cassette by the Tn402-like type 1 integron (IntI1-bla IMP-1) to form In1886 before its capture by the ΔTn4662a-carrying ICE 1276. The acquisition of bla IMP-1 confers carbapenem resistance to P. juntendi 18091276. Conclusion: The formation of bla IMP-1-carrying ICE 1276, its further integration into the chromosomes, and transposition and recombination of other elements promote bacterial gene accumulation and transmission.

Uncovering the efficacy and mechanisms of Genkwa flos and bioactive ingredient genkwanin against L. monocytogenes infection.

ETHNOPHARMACOLOGICAL RELEVANCE: Genkwa flos (yuanhua in Chinese), the dried flower buds of the plant Daphne genkwa Siebold & Zucc., as a traditional herb widely used for the treatment of inflammation-related symptoms and diseases, with the efficacies of diuretic, phlegm-resolving and cough suppressant. AIM OF THE STUDY: Traditional Chinese Medicine (TCM) is presumed to be of immense potential against pathogens infection. Whereas, the potential efficacy and mechanisms of Genkwa flos against L. monocytogenes infection has not been extensively explored. The present study aimed to identify the bioactive ingredients of Genkwa flos against L. monocytogenes infection and to delineate the underlying mechanisms of action. MATERIALS AND METHODS: Bioinformatics approach at protein network level was employed to investigate the therapeutic mechanisms of Genkwa flos against L. monocytogenes infection. And hemolysis inhibition assay, cytoprotection test, western blotting, oligomerization assay and molecular docking analysis were applied to substantiate the multiple efficacies of Genkwa flos and the bioactive ingredient genkwanin. Histopathological analysis and biochemistry detection were conducted to evaluate the in vivo protective effect of genkwanin. RESULTS: Network pharmacology and experimental validation revealed that Traditional Chinese Medicine (TCM) Genkwa flos exhibited anti-L. monocytogenes potency and was found to inhibit the hemolytic activity of LLO. Bioactive ingredient genkwanin interfered with the pore-forming activity of LLO by engaging the active residues Tyr414, Tyr98, Asn473, Val100, Tyr440 and Val438, and thereby attenuated LLO-mediated cytotoxicity. Consistent with the bioinformatics prediction, exposed to genkwanin could upregulate the Nrf2 level and promote the translocation of Nrf2. In vivo, genkwanin oral administration (80 mg/kg) significantly protected against systemic L. monocytogenes infection, as evidenced by reduced myeloperoxidase (MPO) and malondialdehyde (MDA) levels, increased mice survival rate by 30% and decreased pathogen colonization. CONCLUSION: Our study demonstrated that Genkwa flos is a potential anti-L. monocytogenes TCM, highlighted the therapeutic potential of Genkwa flos active ingredient genkwanin by targeting the pore-forming cytolysin LLO and acting as a promising antioxidative candidate against L. monocytogenes infection.

First Report of the Colistin Resistance Gene mcr-10.1 Carried by IncpA1763-KPC Plasmid pSL12517-mcr10.1 in Enterobacter cloacae in Sierra Leone.

Mobile colistin resistance (mcr) gene mcr-10.1 has been distributed widely since it was initially identified in 2020. The aim of this study was to report the first mcr-10.1 in Africa and the first mcr in Sierra Leone; furthermore, we presented diverse modular structures of mcr-10.1 loci. Here, the complete sequence of one mcr-10.1-carrying plasmid in one clinical Enterobacter cloacae isolate from Sierra Leone was determined. Detailed genetic dissection and comparison were applied to this plasmid, together with a homologous plasmid carrying mcr-10.1 from GenBank. Moreover, a genetic comparison of 19 mcr-10.1 loci was performed. In this study, mcr-10.1 was carried by an IncpA1763-KPC plasmid from one Enterobacter cloacae isolate. A total of 19 mcr-10.1 loci displayed diversification in modular structures through complex transposition and homologous recombination. A site-specific tyrosine recombinase XerC was located upstream of mcr-10.1, and at least one insertion sequence element was inserted adjacent to a conserved xerC-mcr-10.1-orf336-orf177 region. Integration of mcr-10.1 into a different gene context and carried by various Inc plasmids contributed to the wide distribution of mcr-10.1 and enhanced the ability of bacteria to survive under colistin selection pressure. IMPORTANCE Colistin is used as one of the last available choices of antibiotics for patients infected by carbapenem-resistant bacterial strains, but the unrestricted use of colistin aggravated the acquisition and dissemination of mobile colistin resistance (mcr) genes. So far, 10 mcr genes have been reported in four continents around the world. This study presented one mcr-10.1-carrying Enterobacter cloacae isolate from Sierra Leone. The mcr-10.1 gene was identified on an IncpA1763-KPC plasmid. According to the results of genetic comparison of 19 mcr-10.1 loci, the mcr-10.1 gene was found to be located in a conserved xerC-mcr-10.1-orf336-orf177 region, and at least one insertion sequence element was inserted adjacent to this region. To our knowledge, this is the first report of identifying the mcr-10.1 gene in Africa and the mcr gene in Sierra Leone.

Pan-Genome Analysis of Vibrio cholerae and Vibrio metschnikovii Strains Isolated From Migratory Birds at Dali Nouer Lake in Chifeng, China.

Migratory birds are recently recognized as Vibrio disease vectors, but may be widespread transporters of Vibrio strains. We isolated Vibrio cholerae (V. cholerae) and Vibrio metschnikovii (V. metschnikovii) strains from migratory bird epidemic samples from 2017 to 2018 and isolated V. metschnikovii from migratory bird feces in 2019 from bird samples taken from the Inner Mongolia autonomous region of China. To investigate the evolution of these two Vibrio species, we sequenced the genomes of 40 V. cholerae strains and 34 V. metschnikovii strains isolated from the bird samples and compared these genomes with reference strain genomes. The pan-genome of all V. cholerae and V. metschnikovii genomes was large, with strains exhibiting considerable individual differences. A total of 2,130 and 1,352 core genes were identified in the V. cholerae and V. metschnikovii genomes, respectively, while dispensable genes accounted for 16,180 and 9,178 of all genes for the two strains, respectively. All V. cholerae strains isolated from the migratory birds that encoded T6SS and hlyA were non-O1/O139 serotypes without the ability to produce CTX. These strains also lacked the ability to produce the TCP fimbriae nor the extracellular matrix protein RbmA and could not metabolize trimetlylamine oxide (TMAO). Thus, these characteristics render them unlikely to be pandemic-inducing strains. However, a V. metschnikovii isolate encoding the complete T6SS system was isolated for the first time. These data provide new molecular insights into the diversity of V. cholerae and V. metschnikovii isolates recovered from migratory birds.

Betulin efficiently suppresses the process of an experimental Listeria monocytogenes infection as an antagonist against listeriolysin O.

Listeria monocytogenes (Lm) is a widespread foodborne intracellular pathogen that invades a variety of cells, causing abortions and severe human diseases. After internalization into host cells, pore-forming cytolysin listeriolysin O (LLO) disrupts the phagosome, which allows the bacterium to survive and colonize the cytoplasm, providing the bacterium the chance to infect neighboring cells. Betulin is an extracted natural compound from birch bark with diverse pharmacological activities. Here, we showed that LLO-induced rabbit red blood cell lysis in vitro was inhibited by preincubation with betulin, which suppressed the oligomerization process. Infectious assays performed with human monocyte macrophages indicated that betulin significantly protected cells against Lm-induced cell injury. In addition, Balb/c mice were used to perform a general infection, and betulin administration obviously inhibited organ damage and bacterial burden in livers and spleens of infected mice. In conclusion, betulin obviously inhibited Lm-induced cell injury in vitro and protected against infection in vivo through an antivirulence effect. Our results showed betulin as a new candidate against listeriosis by targeting LLO and highlight the potential of natural product-based medicine to be applied in the treatment of pathogenic infections.

A potential bio-control agent from baical skullcap root against listeriosis via the inhibition of sortase A and listeriolysin O.

Listeria monocytogenes (LM) is a classical model intracellular pathogen and the leading cause of listeriosis, which has long been a global public health issue. The successful infection of LM is related to a series of virulence factors, such as the transpeptidase enzyme sortase A (SrtA) and listeriolysin O (LLO), which are crucial for bacterial internalization and escape from phagosomes respectively. It is speculated that targeting multiple virulence factors may be due to a synergistic effect on listeriosis therapy. In this study, an active flavonoids component of Scutellaria baicalensis Georgi, baicalein, was found to potently block both listerial SrtA catalyzed activity and LLO hemolytic activity within 16 µg/mL. After pretreatment with baicalein, 86.30 (±11.35) % of LM failed to associate with Caco-2 cells compared to the LM without preincubation (regarded as 100% internalization). Furthermore, baicalein addition may aid in bacterial degradation and clearance in macrophagocytes. During a 5 h observation, LM in cells incubated with baicalein showed significantly decreased vacuole escapes and sluggish endocellular growth. In addition, baicalein directly prevented LM-induced cells injury and mice fatality (survival rate from 10.00% to 54.55% in 4 days post-intraperitoneal injection). Taken together, as an antagonist against SrtA and LLO, baicalein can be further developed into a biotherapeutic agent for listeriosis.

Chalcone Attenuates Staphylococcus aureus Virulence by Targeting Sortase A and Alpha-Hemolysin.

Staphylococcus aureus (S.aureus) resistance, considered a dilemma for the clinical treatment of this bacterial infection, is becoming increasingly intractable. Novel anti-virulence strategies will undoubtedly provide a path forward in combating these resistant bacterial infections. Sortase A (SrtA), an enzyme responsible for anchoring virulence-related surface proteins, and alpha-hemolysin (Hla), a pore-forming cytotoxin, have aroused great scientific interest, as they have been regarded as targets for promising agents against S. aureus infection. In this study, we discovered that chalcone, a natural small compound with little anti-S. aureus activity, could significantly inhibit SrtA activity with an IC50 of 53.15 µM and Hla hemolysis activity with an IC50 of 17.63 µM using a fluorescence resonance energy transfer (FRET) assay and a hemolysis assay, respectively. In addition, chalcone was proven to reduce protein A (SpA) display in intact bacteria, binding to fibronectin, formation of biofilm and S. aureus invasion. Chalcone could down-regulate the transcriptional levels of the hla gene and the agrA gene, thus leading to a reduction in the expression of Hla and significant protection against Hla-mediated A549 cell injury; more importantly, chalcone could also reduce mortality in infected mice. Additionally, molecular dynamics simulations and mutagenesis assays were used to identify the mechanism of chalcone against SrtA, which implied that the inhibitory activity lies in the bond between chalcone and SrtA residues Val168, Ile182, and Arg197. Taken together, the in vivo and in vitro experiments suggest that chalcone is a potential novel therapeutic compound for S. aureus infection via targeting SrtA and Hla.

Curcumin Promotes the Clearance of Listeria monocytogenes both In Vitro and In Vivo by Reducing Listeriolysin O Oligomers.

The pore-forming toxin listeriolysin O (LLO), an essential virulence factor that is secreted by Listeria monocytogenes (L. monocytogenes), is responsible for bacterial breaching at the phagosomal membranes and subsequent release into the cytoplasm; it cannot be recognized by the host immune system. The vital role that LLO plays in bacterial pathogenicity and evading host immune clearance makes this virulence a promising target for addressing L. monocytogenes infection. In this study, we hypothesized that curcumin, a polyphenol derived from turmeric that could effectively inhibit LLO pore-forming activity, might be useful in the prevention or treatment of L. monocytogenes infection. Thus, the in vitro protective effects of curcumin against L. monocytogenes infection by targeting LLO were assessed via hemolytic activity assays, cytotoxicity tests, intracellular growth assays, and confocal microscopy. Our results revealed that treating infected macrophages with curcumin can lead to a decrease in LLO-mediated bacteria phagosomal escape and limit the intracellular growth of L. monocytogenes. Moreover, results from animal experiments show that this natural compound effectively increases protection against bacterial infection and helps the host to clear the invading pathogen completely from an animal model, establishing it as a potent antagonist of L. monocytogenes. The results from our molecular modeling and mutational analysis demonstrated that curcumin directly engages with domains 2 and 4 of LLO, thereby decreasing the hemolytic activity of LLO by influencing its oligomerization. Taken together, these results suggest that, as an antitoxin agent, curcumin can be further developed into a novel therapy against L. monocytogenes infections by targeting LLO.

Juglone alleviates pneumolysin-induced human alveolar epithelial cell injury via inhibiting the hemolytic activity of pneumolysin.

Streptococcus pneumoniae (the pneumococcus) is an opportunistic pathogen responsible for several human diseases, including acute otitis media, pneumonia, sepsis and bacterial meningitis, and possesses numerous virulence factors associated with pneumococcal infection and pathogenesis. With the capacity to form pores in cholesterol-rich membranes, pneumolysin (PLY) is a key virulence factor of S. pneumoniae and causes severe tissue damage during pneumococcal infection. Juglone (JG), a natural 1,4-naphthoquinone widely found in the roots, leaves, woods and fruits of Juglandaceae walnut trees, inhibits PLY-induced hemolysis via inhibition of the oligomerization of PLY and exhibits minimal anti-S. pneumoniae activity. In addition, when human alveolar epithelial (A549) cells were co-cultured with PLY and JG, PLY-mediated cell injury was significantly alleviated. These results indicate that JG directly interacts with PLY to reduce the cytotoxicity of the toxin in human alveolar epithelial cells. Hence, JG is an effective inhibitor of PLY and protects lung cells from PLY-mediated cell injury. This study also provides the basis for the development of anti-virulence drugs for the treatment of S. pneumoniae infections.

Morin Attenuates Streptococcus suis Pathogenicity in Mice by Neutralizing Suilysin Activity.

Streptococcus suis, a Gram-positive pathogen, is widely recognized as an important agent of swine infection, and it is also known to cause a variety of zoonoses, such as meningitis, polyarthritis and pneumonia. Suilysin (SLY), an extracellular pore-forming toxin that belongs to the cholesterol-dependent cytolysin family, is an essential virulence factor of S. suis capsular type 2 (SS2). Here, we found that morin hydrate (morin), a natural flavonoid that lacks anti-SS2 activity, inhibits the hemolytic activity of SLY, protects J774 cells from SS2-induced injury and protects mice from SS2 infection. Further, by molecular modeling and mutational analysis, we found that morin binds to the "stem" domain 2 in SLY and hinders its transformation from the monomer form to the oligomer form, which causes the loss of SLY activity. Our study demonstrates that morin hinders the cell lysis activity of SLY through a novel mechanism of interrupting the heptamer formation. These findings may lead to the development of promising therapeutic candidates for the treatment of SS2 infections.

Phloretin Attenuates Listeria monocytogenes Virulence Both In vitro and In vivo by Simultaneously Targeting Listeriolysin O and Sortase A.

The critical roles of sortase A (SrtA) and listeriolysin O (LLO) in Listeria monocytogenes pathogenicity render these two virulence factors as ideal targets for the development of anti-virulence agents against L. monocytogenes infection. Additionally, the structures of SrtA and LLO are highly conserved among the members of sortase enzyme family and cholesterol dependent toxin family. Here, phloretin, a natural polyphenolic compound derived from apples and pears that has little anti-L. monocytogenes activity, was identified to simultaneously inhibit LLO expression and neutralize SrtA catalytic activity. Phloretin neutralized SrtA activity by causing a conformational change in the protein's active pocket, which prevented engagement with its substrate. Treatment with phloretin simultaneously reduced L. monocytogenes invasion into host cells and blocked the escape of vacuole-entrapped L. monocytogenes into cytoplasm. Further, L. monocytogenes-infected mice that received phloretin showed lower mortality, decreased bacterial burden and reduced pathological injury. Our results demonstrate that phloretin is a promising anti-infective therapeutic for infections caused by L. monocytogenes due to its simultaneous targeting of SrtA and LLO, which may result in fewer side effects than those caused by other antibiotics.

Inhibition of listeriolysin O oligomerization by lutein prevents Listeria monocytogenes infection.

The foodborne pathogenic bacterial species Listeria monocytogenes (L. monocytogenes) has caused incalculable damages to public health, and its successful infection requires various virulence factors, including Listeriolysin O (LLO). By forming pores in phagosomal membranes and even in some organelles, LLO plays an indispensable role in the ability of L. monocytogenes to escape from host immune attacks. Because of its critical role, LLO offers an appropriate therapeutic target against L. monocytogenes infection. Here, lutein, a natural small molecule existing widely in fruits and vegetables, is demonstrated as an effective inhibitor of LLO that works by blocking its oligomerization during invasion without showing significant bacteriostatic activity. Further assays applying lutein in cell culture models of invasion and in animal models showed that lutein could effectively inhibit L. monocytogenes infection. Overall, our results indicate that lutein may represent a promising and novel therapeutic agent against L. monocytogenes infection.

Inhibition of sortase A by chalcone prevents Listeria monocytogenes infection.

The critical role of sortase A in gram-positive bacterial pathogenicity makes this protein a good potential target for antimicrobial therapy. In this study, we report for the first time the crystal structure of Listeria monocytogenes sortase A and identify the active sites that mediate its transpeptidase activity. We also used a sortase A (SrtA) enzyme activity inhibition assay, simulation, and isothermal titration calorimetry analysis to discover that chalcone, an agent with little anti-L. monocytogenes activity, could significantly inhibit sortase A activity with an IC50 of 28.41 ± 5.34 µM by occupying the active site of SrtA. The addition of chalcone to a co-culture of L. monocytogenes and Caco-2 cells significantly inhibited bacterial entry into the cells and L. monocytogenes-mediated cytotoxicity. Additionally, chalcone treatment decreased the mortality of infected mice, the bacterial burden in target organs, and the pathological damage to L. monocytogenes-infected mice. In conclusion, these findings suggest that chalcone is a promising candidate for the development of treatment against L. monocytogenes infection.

Isorhamnetin protects mice from lipopolysaccharide-induced acute lung injury via the inhibition of inflammatory responses.

OBJECTIVE AND DESIGN: Isorhamnetin (Isor), a 3-O-methylated metabolite of quercetin, has shown antioxidant and anti-proliferative effects in previous studies. In this study, we investigated the anti-inflammatory effect of Isor on LPS-induced acute lung injury (ALI). Accordingly, we evaluated the effect of Isor on cytokine production elevated by LPS (1 µg/ml) in vitro. An in vivo ALI murine model was also established via lipopolysaccharide inhalation (LPS, 20 mg/kg), and the cytokine levels and inflammatory cell count in bronchoalveolar lavage fluid (BALF) were evaluated. The observed lung injury was assessed using histopathologic sections via H&E straining. Furthermore, to investigate whether the anti-inflammatory effect of Isor is associated with NF-κB and MAPKs pathway activation, the phosphorylated levels of ERK, JNK, IκBa and NF-κB(p65) were determined. RESULTS: Isor significantly inhibited LPS-induced TNF-α, IL-1ß and IL-6 secretion both in vitro and in vivo. Neutrophil infiltration and edema in an ALI model were substantially alleviated. The histopathological changes induced by LPS were lessened by Isor. Additionally, Isor notably suppressed the phosphorylation of ERK, JNK, IκBa and NF-κB(p65) activated by LPS in vivo. CONCLUSIONS: Isor showed efficient protective effects on an LPS-induced ALI model. MAPKs and NF-κB pathways are critical for Isor to perform its protective effects.

Tenuigenin exhibits anti-inflammatory activity via inhibiting MAPK and NF-κB and inducing Nrf2/HO-1 signaling in macrophages.

Tenuigenin (TNG), isolated from the root of the Chinese herb Polygala tenuifolia, possesses various biological and pharmacological activities, including anti-oxidation and anti-inflammation activities. In this study, we aimed to further investigate whether its anti-inflammatory activity is associated with the inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Our results showed that TNG treatment dramatically reduced prostaglandin E2 (PGE2) and NO production, decreased iNOS and COX-2 gene expression, inhibited JNK1/2, ERK1/2, p38 and NF-κB (p65) phosphorylation, and blocked IκBα phosphorylation and degradation. Further studies revealed that TNG dramatically up-regulated heme oxygenase (HO)-1 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression, which was related to the induction of Nrf2 nuclear translocation and decreased Keap1 protein expression. Additionally, treatment with JNK1/2, ERK1/2 or p38 inhibitors had no effect on the TNG-induced HO-1 protein expression. Furthermore, the LPS-induced iNOS and COX-2 expression levels were inhibited by TNG, which was partially reversed by the HO-1-siRNA and HO-1 inhibitors. Together, these results showed that TNG's anti-inflammatory activity is related to the inhibition of iNOS and COX-2 expression via down-regulation of the MAPK and NF-κB, and up-regulation of the Nrf2/HO-1 signaling pathways.