Shigella induces stress granule formation by ADP-riboxanation of the eIF3 complex.

Under stress conditions, translationally stalled mRNA and associated proteins undergo liquid-liquid phase separation and condense into cytoplasmic foci called stress granules (SGs). Many viruses hijack SGs for their pathogenesis; however, whether pathogenic bacteria also exploit this pathway remains unknown. Here, we report that members of the OspC family of Shigella flexneri induce SG formation in infected cells. Mechanistically, the OspC effectors target multiple subunits of the host translation initiation factor 3 complex by ADP-riboxanation. The modification of eIF3 leads to translational arrest and thus the formation of SGs. Furthermore, OspC-mediated SGs are beneficial for S. flexneri replication within infected host cells, and bacterial strains unable to induce SGs are attenuated for virulence in a murine model of infection. Our findings reveal a mechanism by which bacterial pathogens induce SG assembly by inactivating host translational machinery and promote bacterial proliferation in host cells.

Cananga oil inhibits Salmonella infection by mediating the homeostasis of purine metabolism and the TCA cycle.

ETHNOPHARMACOLOGY RELEVANCE: Cananga oil (CO) is derived from the flowers of the traditional medicinal plant, the ylang-ylang tree. As a traditional antidepressant, CO is commonly utilized in the treatment of various mental disorders including depression, anxiety, and autism. It is also recognized as an efficient antibacterial insecticide, and has been traditionally utilized to combat malaria and acute inflammatory responses resulting from bacterial infections both in vitro and in vivo. AIM OF THE STUDY: The objective of this study is to comprehensively investigate the anti-Salmonella activity and mechanism of CO both in vitro and in vivo, with the expectation of providing feasible strategies for exploring new antimicrobial strategies and developing novel drugs. METHODS: The in vitro antibacterial activity of CO was comprehensively analyzed by measuring MIC, MBC, growth curve, time-killing curve, surface motility, biofilm, and Live/dead bacterial staining. The analysis of the chemistry and active ingredients of CO was conducted using GC-MS. To examine the influence of CO on the membrane homeostasis of Salmonella, we conducted utilizing diverse techniques, including ANS, PI, NPN, ONPG, BCECF-AM, DiSC3(5), and scanning electron microscopy (SEM) analysis. In addition, the antibacterial mechanism of CO was analyzed and validated through metabolomics analysis. Finally, a mouse infection model of Salmonella typhimurium was established to evaluate the toxic side effects and therapeutic effects of CO. RESULTS: The antibacterial effect of CO is the result of the combined action of the main chemical components within its six (palmitic acid, α-linolenic acid, stearic acid, benzyl benzoate, benzyl acetate, and myristic acid). Furthermore, CO disrupts the balance of purine metabolism and the tricarboxylic acid cycle (TCA cycle) in Salmonella, interfering with redox processes. This leads to energy metabolic disorders and oxidative stress damage within the bacteria, resulting in bacterial shock, enhanced membrane damage, and ultimately bacterial death. It is worth emphasizing that CO exerts an effective protective influence on Salmonella infection in vivo within a non-toxic concentration range. CONCLUSION: The outcomes indicate that CO displays remarkable anti-Salmonella activity both in vitro and in vivo. It triggers bacterial death by disrupting the balance of purine metabolism and the TCA cycle, interfering with the redox process, making it a promising anti-Salmonella medication.

Fisetin inhibits Salmonella Typhimurium type III secretion system regulator HilD and reduces pathology in vivo.

IMPORTANCE: Salmonella spp. remains a major worldwide health concern that causes significant morbidity and mortality in both humans and animals. The spread of antimicrobial resistant strains has declined the efficacy of conventional chemotherapy. Thus, novel anti-infection drugs or strategies are needed. Anti-virulence strategy represents one of the promising means for the treatment of bacterial infections. In this study, we found that the natural compound fisetin could inhibit Salmonella invasion of host cells by targeting SPI-1 regulation. Fisetin treatment impaired the interaction of the regulatory protein HilD with the promoters of its target genes, thereby suppressing the expression of T3SS-1 effectors as well as structural proteins. Moreover, fisetin treatment could reduce pathology in the Salmonella murine infection model. Collectively, our results suggest that fisetin may serve as a promising lead compound for the development of anti-Salmonella drugs.

Novel metallo-ß-lactamases inhibitors restore the susceptibility of carbapenems to New Delhi metallo-lactamase-1 (NDM-1)-harbouring bacteria.

BACKGROUND AND PURPOSE: The production of metallo-ß-lactamases is a major mechanisms adopted by bacterial pathogens to resist carbapenems. Repurposing approved drugs to restore the efficacy of carbapenems represents an efficient and cost-effective approach to fight infections caused by carbapenem resistant pathogens. EXPERIMENTAL APPROACH: The nitrocefin hydrolysis assay was employed to screen potential New Delhi metallo-lactamase-1 (NDM-1) inhibitors from a commercially available U.S. Food and Drug Administration (FDA) approved drug library. The mechanism of inhibition was clarified by metal restoration, inductively coupled plasma mass spectrometry (ICP-MS) and molecular dynamics simulation. The in vitro synergistic antibacterial effect of the identified inhibitors with meropenem was determined by the checkerboard minimum inhibitory concentration (MIC) assay, time-dependent killing assay and combined disc test. Three mouse infection models were used to further evaluate the in vivo therapeutic efficacy of combined therapy. KEY RESULTS: Twelve FDA-approved compounds were initially screened to inhibit the ability of NDM-1 to hydrolyse nitrocefin. Among these compounds, dexrazoxane, embelin, candesartan cilexetil and nordihydroguaiaretic acid were demonstrated to inhibit all tested metallo-ß-lactamases and showed an in vitro synergistic bactericidal effect with meropenem against metallo-ß-lactamases-producing bacteria. Dexrazoxane, embelin and candesartan cilexetil are metal ion chelating agents, while the inhibition of NDM-1 by nordihydroguaiaretic acid involves its direct binding to the active region of NDM-1. Furthermore, these four drugs dramatically rescued the treatment efficacy of meropenem in three infection models. CONCLUSIONS AND IMPLICATIONS: Our observations indicated that dexrazoxane, embelin, candesartan cilexetil and nordihydroguaiaretic acid are promising carbapenem adjuvants against metallo-ß-lactamases-positive carbapenem resistant bacterial pathogens.

Repurposing rabeprazole sodium as an anti-Clostridium perfringens drug by inhibiting perfringolysin O.

AIMS: Clostridium perfringens infections affect food safety, human health, and the development of the poultry feed industry. Anti-virulence is an alternative strategy to develop new drug. Perfringolysin O (PFO) is an exotoxin of C. perfringens that has been demonstrated to play critical roles in the pathogenesis of this organism, promising it an attractive target to explore drugs to combat C. perfringens infection. METHODS AND RESULTS: Based on an activity-based screening, we identified six PFO inhibitors from the Food and Drug Administration (FDA)-approved drug library, among which rabeprazole sodium (RS) showed an optimal inhibitory effect with an IC50 of 1.82 ± 0.746 µg ml-1. The GLY57, ASP58, SER190, SER193-194, ASN199, GLU204, ASN377, THR379, and ALA200 in PFO interacted with RS during binding based on an energy analysis and H-bond analysis. This interaction blocked the oligomer formation of PFO, thereby inhibiting its cytotoxicity. RS treatment significantly increased the survival rate and alleviated pathological damage in C. perfringens or PFO-treated Galleria mellonella. CONCLUSIONS: RS could potentially be used as a candidate drug for treating C. perfringens infection.

A new function of thymol nanoemulsion for reversing colistin resistance in Salmonella enterica serovar Typhimurium infection.

BACKGROUND: Adjuvant addition of approved drugs or foodborne additives to colistin might be a cost-effective strategy to overcome the challenge of plasmid-mediated mobile colistin resistance gene emergence, which poses a threat in the clinic and in livestock caused by infections with Gram-negative bacteria, especially carbapenem-resistant Enterobacteriaceae. METHODS: Chequerboard assay was applied to screen the colistin adjuvants from natural compounds. The killing-time curve, combined disc test and membrane permeation assay were conducted to identify the synergy efficacy of thymol and colistin in vitro. Thin-layer chromatography (TLC), LC-MS and fluorescence spectra were used to indicate the interaction of thymol and MCR-1. The potential binding sites were then investigated by molecular simulation dynamics. Finally, a thymol nanoemulsion was prepared with high-pressure homogenization as the clinical dosage form. RESULTS: Thymol presented an excellent synergistic effect in vitro with colistin against Salmonella enterica serovar Typhimurium and Escherichia coli bacteria. Thymol addition, forming a complex with MCR-1, might interfere with the efficacy of MCR-1. Moreover, thymol strengthened colistin activity associated with potentiating membrane damage, destroying the biofilm and enhancing reactive oxygen species-mediated oxidative damage. Thymol nanoemulsion combined with colistin remarkably prevented the intestinal damage caused by S. Typhimurium infection, resulting in a survival rate higher than 60%. CONCLUSIONS: This study achieved a promising thymol oral formulation as colistin adjuvant to combat S. Typhimurium infection, which could be used to extend the lifespan of colistin in clinical veterinary medicine.

Decreasing predictable DNA off-target effects and narrowing editing windows of adenine base editors by fusing human Rad18 protein variant.

Adenine base editors, enabling targeted A-to-G conversion in genomic DNA, have enormous potential in therapeutic applications. However, the currently used adenine base editors are limited by wide editing windows and off-target effects in genetic therapy. Here, we report human e18 protein, a RING type E3 ubiquitin ligase variant, fusing with adenine base editors can significantly improve the preciseness and narrow the editing windows compared with ABEmax and ABE8e by diminishing the abundance of base editor protein. As a proof of concept, ABEmax-e18 and ABE8e-e18 dramatically decrease Cas9-dependent and Cas9-independent off-target effects than traditional adenine base editors. Moreover, we utilized ABEmax-e18 to establish syndactyly mouse models and achieve accurate base conversion at human PCSK9 locus in HepG2 cells which exhibited its potential in genetic therapy. Furthermore, a truncated version of base editors-RING (ABEmax-RING or AncBE4max-RING), which fusing the 63 amino acids of e18 protein RING domain to the C terminal of ABEmax or AncBE4max, exhibited similar effect compared to ABEmax-e18 or AncBE4max-e18.In summary, the e18 or RING protein fused with base editors strengthens the precise toolbox in gene modification and maybe works well with various base editing tools with a more applicable to precise genetic therapies in the future.

Ubiquitination of Sec22b by a novel Legionella pneumophila ubiquitin E3 ligase.

Legionella pneumophila is a facultative intracellular pathogen that causes legionellosis. The key to its virulence is the delivery of hundreds of effector proteins into host cells via the defective in organelle trafficking/intracellular multiplication type IV secretion system. These effectors modulate numerous host signaling pathways to create a niche called the Legionella-containing vacuole (LCV) permissive for its intracellular replication. Previous investigation revealed that exploitation of the host ubiquitin system is among the most important strategies used by L. pneumophila to coopt host processes for its benefit. Here, we show that the effector Legionella ubiquitin ligase gene 15 (Lug15) (Lpg2327), which has no detectable homology with any enzyme involved in ubiquitin signaling, is an E3 ligase. In L. pneumophila-infected cells, Lug15 is localized on the LCV and impacts its association with polyubiquitinated proteins. We also demonstrate that Sec22b is ubiquitinated and recruited to the LCV by Lug15. Thus, our results establish Lug15 as a novel E3 ligase that functions to recruit a SNARE protein to remodel the L. pneumophila phagosome.IMPORTANCEProtein ubiquitination is one of the most important post-translational modifications that plays critical roles in the regulation of a wide range of eukaryotic signaling pathways. Many successful intracellular bacterial pathogens can hijack host ubiquitination machinery through the action of effector proteins that are injected into host cells by secretion systems. Legionella pneumophila is the etiological agent of legionellosis that is able to survive and replicate in various host cells. The defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IV secretion system of L. pneumophila injects over 330 effectors into infected cells to create an optimal environment permissive for its intracellular proliferation. To date, at least 26 Dot/Icm substrates have been shown to manipulate ubiquitin signaling via diverse mechanisms. Among these, 14 are E3 ligases that either cooperate with host E1 and E2 enzymes or adopt E1/E2-independent catalytic mechanisms. In the present study, we demonstrate that the L. pneumophila effector Legionella ubiquitin ligase gene 15 (Lug15) is a novel ubiquitin E3 ligase. Lug15 is involved in the remodeling of LCV with polyubiquitinated species. Moreover, Lug15 catalyzes the ubiquitination of host SNARE protein Sec22b and mediates its recruitment to the LCV. Ubiquitination of Sec22b by Lug15 promotes its noncanonical pairing with plasma membrane-derived syntaxins (e.g., Stx3). Our study further reveals the complexity of strategies utilized by L. pneumophila to interfere with host functions by hijacking host ubiquitin signaling.

Phosphoribosyl-linked serine ubiquitination of USP14 by the SidE family effectors of Legionella excludes p62 from the bacterial phagosome.

Xenophagy is an evolutionarily conserved host defensive mechanism to eliminate invading microorganisms through autophagic machinery. The intracellular bacterial pathogen Legionella pneumophila can avoid clearance by the xenophagy pathway via the actions of multiple Dot/Icm effector proteins. Previous studies have shown that p62, an adaptor protein involved in xenophagy signaling, is excluded from Legionella-containing vacuoles (LCVs). Such defects are attributed to the multifunctional SidE family effectors (SidEs) that exhibit classic deubiquitinase (DUB) and phosphoribosyl ubiquitination (PR-ubiquitination) activities, yet the mechanism remains elusive. In the present study, we demonstrate that the host DUB USP14 is PR-ubiquitinated by SidEs at multiple serine residues, which impairs its DUB activity and its interactions with p62. The exclusion of p62 from the bacterial phagosome requires the ubiquitin ligase but not the DUB activity of SidEs. These results reveal that PR-ubiquitination of USP14 by SidEs contributes to the evasion of xenophagic clearance by L. pneumophila.

Legionella longbeachae effector protein RavZ inhibits autophagy and regulates phagosome ubiquitination during infection.

Legionella organisms are ubiquitous environmental bacteria that are responsible for human Legionnaires' disease, a fatal form of severe pneumonia. These bacteria replicate intracellularly in a wide spectrum of host cells within a distinct compartment termed the Legionella-containing vacuole (LCV). Effector proteins translocated by the Dot/Icm apparatus extensively modulate host cellular functions to aid in the biogenesis of the LCV and intracellular proliferation. RavZ is an L. pneumophila effector that functions as a cysteine protease to hydrolyze lipidated LC3, thereby compromising the host autophagic response to bacterial infection. In this study, we characterized the RavZ (RavZLP) ortholog in L. longbeachae (RavZLLO), the second leading cause of Legionella infections in the world. RavZLLO and RavZLP share approximately 60% sequence identity and a conserved His-Asp-Cys catalytic triad. RavZLLO is recognized by the Dot/Icm systems of both L. pneumophila and L. longbeachae. Upon translocation into the host, it suppresses autophagy signaling in cells challenged with both species, indicating the functional redundancy of RavZLLO and RavZLP. Additionally, ectopic expression of RavZLLO but not RavZLP in mammalian cells reduces the levels of cellular polyubiquitinated and polyneddylated proteins. Consistent with this process, RavZLLO regulates the accumulation of polyubiquitinated species on the LCV during L. longbeachae infection.

Legionella longbeachae Regulates the Association of Polyubiquitinated Proteins on Bacterial Phagosome with Multiple Deubiquitinases.

Legionella spp. are the causative agents of a severe pneumonia known as Legionnaires' disease. Upon being engulfed by host cells, these environmental bacteria replicate intracellularly in a plasma membrane-derived niche termed Legionella-containing vacuole (LCV) in a way that requires the defective in organelle trafficking/intracellular multiplication (Dot/Icm) protein transporter. Our understanding of interactions between Legionella and its hosts was mostly based on studies of Legionella pneumophila. In this study, we found that the LCVs created by virulent Legionella longbeachae are similarly decorated by polyubiquitinated proteins to those formed by L. pneumophila and that the ubiquitin-proteasome system (UPS) is required for optimal intracellular growth of L. longbeachae. Furthermore, we utilized bioinformatics methods and the ubiquitin-vinylmethyl ester probe to obtain potential deubiquitinases (DUBs) encoded by L. longbeachae. These efforts led to the identification of 9 L. longbeachae DUBs that displayed distinct specificity toward ubiquitin chain types. Among these, LLO_1014 and LLO_2238 are associated with the LCVs and impact the accumulation of polyubiquitinated species on the bacterial phagosome. Moreover, LLO_1014 and LLO_2238 could fully restore the phenotypes associated with Δceg23 (lotB) and Δlem27 (lotC) mutants of L. pneumophila, indicating that these DUBs have similar functions. Together, these results reveal that L. longbeachae uses multiple DUBs to construct an intracellular niche for its replication. IMPORTANCE Legionella spp. are opportunistic intracellular bacterial pathogens that cause Legionnaires' disease. Legionella utilizes the Dot/Icm type IV secretion system to deliver effector protein into host cells to modulate various cellular functions. At least 26 L. pneumophila effectors are known to hijack the host ubiquitin system via diverse mechanisms. L. longbeachae is the second leading cause of Legionnaires' disease worldwide. However, our knowledge about the interactions between L. longbeachae and its hosts is very limited. Here, we found that, similar to L. pneumophila infection, the host ubiquitin proteasome system is also important for the intracellular replication of L. longbeachae. In addition, the bacterial phagosomes harboring L. longbeachae are enriched with polyubiquitinated proteins in a Dot/Icm system-dependent manner. We further identified 9 L. longbeachae proteins that function as DUBs with distinct ubiquitin chain specificity. Of note, several of the phagosome-associated L. longbeachae DUBs regulate the recruitment of polyubiquitinated proteins to the LCV.

Rapid and Visual Detection of SARS-CoV-2 RNA Based on Reverse Transcription-Recombinase Polymerase Amplification with Closed Vertical Flow Visualization Strip Assay.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was initially identified in 2019, after which it spread rapidly throughout the world. With the progression of the epidemic, new variants of SARS-CoV-2 with faster transmission speeds and higher infectivity have constantly emerged. The proportions of people asymptomatically infected or reinfected after vaccination have increased correspondingly, making the prevention and control of COVID-19 extremely difficult. There is therefore an urgent need for rapid, convenient, and inexpensive detection methods. In this paper, we established a nucleic acid visualization assay targeting the SARS-CoV-2 nucleoprotein (N) gene by combining reverse transcription-recombinase polymerase amplification with closed vertical flow visualization strip (RT-RPA-VF). This method had high sensitivity, comparable to that of reverse transcription-quantitative PCR (RT-qPCR), and the concordance between RT-RPA-VF and RT-qPCR methods was 100%. This detection method is highly specific and is not compatible with bat coronavirus HKU4, human coronaviruses 229E, OC43, and HKU1-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), or other respiratory pathogens. However, multiple SARS-CoV-2 variants are detectable within 25 min at 42°C using this visual method, including RNA transcripts of the Wuhan-Hu-1 strain at levels as low as 1 copy/µL, the Delta strain at 1 copy/µL, and the Omicron strain at 0.77 copies/µL. The RT-RPA-VF method is a simple operation for the rapid diagnosis of COVID-19 that is safe and free from aerosol contamination and could be an affordable and attractive choice for governments seeking to promote their emergency preparedness and better their responses to the continuing COVID-19 epidemic. In addition, this method also has great potential for early monitoring and warning of the epidemic situation at on-site-nursing points. IMPORTANCE The global COVID-19 epidemic, ongoing since the initial outbreak in 2019, has caused panic and huge economic losses worldwide. Due to the continuous emergence of new variants, COVID-19 has been responsible for a higher proportion of asymptomatic patients than the previously identified SARS and MERS, which makes early diagnosis and prevention more difficult. In this manuscript, we describe a rapid, sensitive, and specific detection tool, RT-RPA-VF. This tool provides a new alternative for the detection of SARS-CoV-2 variants in a range as low as 1 to 0.77 copies/µL RNA transcripts. RT-RPA-VF has great potential to ease the pressure of medical diagnosis and the accurate identification of patients with suspected COVID-19 at point-of-care.

Inhibition of the Type III Secretion System of Salmonella enterica Serovar Typhimurium via Treatment with Fraxetin.

The increasingly serious problem of bacterial drug resistance has led to the development of antivirulence agents. The Salmonella enterica serovar Typhimurium Salmonella pathogenicity island (SPI)-encoded type III secretion system (T3SS) and its effector proteins are important virulence factors for S. Typhimurium invasion and replication in host cells and for antivirulence drug screening. Fraxetin is isolated from Fraxinus spp. Extensive studies have reported its multiple pharmacological activities. However, it remains to be elucidated whether fraxetin affects the function of the S. Typhimurium T3SS. In this study, the anti-infection mechanism of fraxetin on S. Typhimurium and its T3SS was investigated. Fraxetin inhibited the S. Typhimurium invasion of HeLa cells without affecting the growth of bacteria in vitro. Further findings on the mechanism showed that fraxetin had an inhibitory effect on the S. Typhimurium T3SS by inhibiting the transcription of the pathogenesis-related SPI-1 transcriptional activator genes hilD, hilC, and rtsA. Animal experiments showed that fraxetin treatment protected mice against S. Typhimurium infection. Collectively, we provide the first demonstration that fraxetin may serve as an effective T3SS inhibitor for the development of treatments for Salmonella infection. IMPORTANCE The increasingly serious problem of bacterial antibiotic resistance limits the clinical application of antibiotics, which increases the need for the development of antivirulence agents. The type III secretion system (T3SS) plays a critical role in host cell invasion and pathogenesis of Salmonella and becomes a popular target for antivirulence agents screening. Our study found, for the first time, that fraxetin inhibited S. Typhimurium invasion by inhibiting the transcription of genes in a feed-forward regulatory loop. Further in vivo testing showed that fraxetin decreased bacterial burdens in the spleen and liver of S. Typhimurium-infected mice and improved survival outcomes in an in vivo mouse model of S. Typhimurium infection. Collectively, these results demonstrate that fraxetin inhibits S. Typhimurium infection by targeting the T3SS and may serve as a potential agent for the treatment of S. Typhimurium infection.

Harmine, an inhibitor of the type III secretion system of Salmonella enterica serovar Typhimurium.

New therapeutic strategies for clinical Salmonella enterica serovar Typhimurium (S. Typhimurium) infection are urgently needed due to the generation of antibiotic-resistant bacteria. Inhibition of bacterial virulence has been increasingly regarded as a potential and innovative strategy for the development of anti-infection drugs. Salmonella pathogenicity island (SPI)-encoded type III secretion system (T3SS) represents a key virulence factor in S. Typhimurium, and active invasion and replication in host cells is facilitated by the secretion of T3SS effector proteins. In this study, we found that harmine could inhibit T3SS secretion; thus, its potential anti-S. Typhimurium infection activity was elucidated. Harmine inhibits the secretion and expression of T3SS effector proteins and consequently attenuates the S. Typhimurium invasion function of HeLa cells. This inhibition may be implemented by reducing the transcription of pathogenesis-related SPI-1 transcriptional activator genes hilD, hilC, and rtsA. Harmine improves the survival rate and bacterial loads of mice infected with S. Typhimurium. In summary, harmine, an effective T3SS inhibitor, could be a leading compound for the development of treatments for Salmonella infection.

Emergence and Transmission of Plasmid-Mediated Mobile Colistin Resistance Gene mcr-10 in Humans and Companion Animals.

Mobile colistin resistance (mcr) genes mediated by plasmids have widely disseminated throughout the world. Recently, 10 mcr genes (mcr-1 to mcr-10) and a large number of variants have been identified in more than 60 countries. However, only a few instances of Enterobacter cloacae complex (ECC) bearing mcr-10 from animal origin have been reported globally. The aim of this study was to fill a knowledge gap in mcr-10-positive ECC of animal origin and analyze the potential transmission trend and different characteristics between human and companion animal isolates. The mcr-10 gene was identified on a self-transmissible plasmid in the human isolate and non-transmissible plasmids in other three animal strains. mcr-10 was adjacent to a XerC-type tyrosine recombinase-gene, and various insertion sequences were located on the downstream of core conservative structure xerC-mcr-10, thus indicating this region might be a candidate for insertions of mobile genetic elements and mcr-10 might be mobilized by IS-mediated mechanisms. Moreover, phylogenetic analysis found that mcr-10-positive isolates were mainly distributed in the clade of Enterobacter roggenkampii, exhibiting significant species specificity. These findings indicated that mcr-10 has emerged among Enterobacter spp. within humans and companion animals, highlighting that the importance of taking effective control measures to monitor the dissemination and evolution of mcr genes. IMPORTANCE Colistin was considered as the last-resort drug against severe clinical infections caused by multidrug-resistant Gram-negative pathogens. Mobile colistin resistance (mcr) genes and its variants carried by plasmids have been reported in diverse niches in recent years, and yet few studies reported carriage of mcr-10 in ECC strains of companion animal origin. How plasmid-borne mcr-10 transmitted in opportunistic pathogens and different characteristics of mcr-10-bearing strains isolated from humans and companion animals are not well understood. In this study, we discovered mcr-10-harboring strains in multidrug-resistant ECC isolates of companion animal origin for the first time and conducted a comprehensive analysis of the genetic environment of mcr-10 from multiple countries around the world, providing the potential basis for formulating control measures to slow down the spread of colistin resistance.

Piceatannol Alleviates Clostridium perfringens Virulence by Inhibiting Perfringolysin O.

Clostridium perfringens (C. perfringens) is an important foodborne pathogen that can cause diseases such as gas gangrene and necrotizing enteritis in a variety of economic animals, seriously affecting public health and the economic benefits and healthy development of the livestock and poultry breeding industry. Perfringolysin O (PFO) is an important virulence factor of C. perfringens and plays critical roles in necrotic enteritis and gas gangrene, rendering it an ideal target for developing new drugs against infections caused by this pathogen. In this study, based on biological activity inhibition assays, oligomerization tests and computational biology assays, we found that the foodborne natural component piceatannol reduced pore-forming activity with an inhibitory ratio of 83.84% in the concentration of 16 µg/mL (IC50 = 7.83 µg/mL) by binding with PFO directly and changing some of its secondary structures, including 3-Helix, A-helix, bend, and in turn, ultimately affecting oligomer formation. Furthermore, we confirmed that piceatannol protected human intestinal epithelial cells from the damage induced by PFO with LDH release reduced by 38.44% at 16 µg/mL, based on a cytotoxicity test. By performing an animal experiment, we found the C. perfringens clones showed an approximate 10-fold reduction in infected mice. These results suggest that piceatannol may be a candidate for anti-C. perfringens drug development.

Molecular mechanism of toxin neutralization in the HipBST toxin-antitoxin system of Legionella pneumophila.

Toxin-antitoxin (TA) systems are ubiquitous genetic modules in bacteria and archaea. Here, we perform structural and biochemical characterization of the Legionella pneumophila effector Lpg2370, demonstrating that it is a Ser/Thr kinase. Together with two upstream genes, lpg2370 constitutes the tripartite HipBST TA. Notably, the toxin Lpg2370 (HipTLp) and the antitoxin Lpg2369 (HipSLp) correspond to the C-terminus and N-terminus of HipA from HipBA TA, respectively. By determining crystal structures of autophosphorylated HipTLp, its complex with AMP-PNP, and the structure of HipTLp-HipSLp complex, we identify residues in HipTLp critical for ATP binding and those contributing to its interactions with HipSLp. Structural analysis reveals that HipSLp binding induces a loop-to-helix shift in the P-loop of HipTLp, leading to the blockage of ATP binding and inhibition of the kinase activity. These findings establish the L. pneumophila effector Lpg2370 as the HipBST TA toxin and elucidate the molecular basis for HipT neutralization in HipBST TA.

Nordihydroguaiaretic acid reverses the antibacterial activity of colistin against MCR-1-positive bacteria in vivo/in vitro by inhibiting MCR-1 activity and injuring the bacterial cell membrane.

BACKGROUND: Colistin (polymyxin E) is an effective antibiotic for the treatment of most multidrug-resistant Gram-negative bacteria. However, some bacteria, including bacterial spp. belonging to the Enterobacteriaceae family, have an acquired resistance against polymyxins, which is attributed to they possess plasmid-carried resistance genes (mcr-1 and its variants). So, there is an urgent need to develop new therapeutic strategies to target broad spectrum resistant spp. from Enterobacteriaceae family in response to the loss of the protective barrier of last-line antibiotics. Here, we report the adjuvant capacity of nordihydroguaiaretic acid (NDGA) for restoring the antibacterial activity of colistin against MCR-1-positive E. coli ZJ487 in vivo/in vitro. METHODS: A checkerboard assay, time-killing analysis, isobolograms, growth curves and inducible resistance test showed the effect of NDGA combined with colistin in vitro. TLC was used to detect the inhibitory effect of NDGA on MCR-1. Colony determination and hematoxylin and eosin (HE) staining were used to assess the synergistic effect of NDGA and colistin in mice. RESULTS: Our results showed that NDGA in combination with colistin showed a synergistic bactericidal action without inducing resistance. NDGA directly inhibited MCR-1 activity and resulted in measurable injury to the bacterial cell membrane to recover the antibacterial effect of colistin. Most importantly, NDGA in combination with colistin exhibited an in vivo synergistic effect in murine peritonitis infection models, as evidenced by the survival rate of MCR-1-positive E. coli ZJ487-infected mice which increased from 6.67 to 50.0%. CONCLUSION: Our study demonstrated that NDGA effectively rescues the efficiency of colistin against MCR-positive E. coli ZJ487 by simultaneously inhibiting both, the MCR activity and the injury to the cell membrane of bacteria.

Coxiella burnetii inhibits host immunity by a protein phosphatase adapted from glycolysis.

Coxiella burnetii is a bacterial pathogen that replicates within host cells by establishing a membrane-bound niche called the Coxiella-containing vacuole. Biogenesis of this compartment requires effectors of its Dot/Icm type IV secretion system. A large cohort of such effectors has been identified, but the function of most of them remain elusive. Here, by a cell-based functional screening, we identified the effector Cbu0513 (designated as CinF) as an inhibitor of NF-κB signaling. CinF is highly similar to a fructose-1,6-bisphosphate (FBP) aldolase/phosphatase present in diverse bacteria. Further study reveals that unlike its ortholog from Sulfolobus tokodaii, CinF does not exhibit FBP phosphatase activity. Instead, it functions as a protein phosphatase that specifically dephosphorylates and stabilizes IκBα. The IκBα phosphatase activity is essential for the role of CinF in C. burnetii virulence. Our results establish that C. burnetii utilizes a protein adapted from sugar metabolism to subvert host immunity.

Inhibitory effect of hederagenin on Streptococcus pneumoniae pneumolysin in vitro.

Streptococcus pneumoniae is an important pathogen that causes otitis media, pneumonia, meningitis and bacteremia. As an important virulence factors of S. pneumoniae, pneumolysin (PLY) can penetrate cell membranes and lead to cell lysis and inflammation, which is one of the main causes of infection and damage of S. pneumoniae. Therefore, using pneumolysin as a target to study its inhibitors can provide a new treatment strategy for pneumococcal disease. This study analyzed the inhibitory effect of the natural compound hederagenin on PLY in vitro. The results show that hederagenin has great potential as a new strategy for the treatment of pneumococcal diseases.