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Antibiotic drug combination therapy is critical for the successful treatment of infections caused by multidrug resistant pathogens. We investigated the efficacy of β-lactam and β-lactam/β-lactamase inhibitor combinations with other antibiotics, against the hypervirulent, ceftazidime/avibactam resistant Pseudomonas aeruginosa Liverpool epidemic strain (LES) B58. Although minimum inhibitory concentrations in vitro differed by up to eighty-fold between standard and host-mimicking media, combinatorial effects only marginally changed between conditions for some combinations. Effective combinations in vitro were further tested in a chronic, high-density murine infection model. Colistin and azithromycin demonstrated combinatorial effects with ceftazidime and ceftazidime/avibactam both in vitro and in vivo. Conversely, while tobramycin and tigecycline exhibited strong synergy in vitro, this effect was not observed in vivo. Our approach of using host-mimicking conditions and a sophisticated animal model to evaluate drug synergy against bacterial pathogens represents a promising approach. This methodology may offer insights into the prediction of combination therapy outcomes and the identification of potential treatment failures.

Abstract Background Rheumatoid arthritis (RA) is an autoimmune disease that can lead to joint destruction and deformity. Curculigo orchioides Gaertn (CO) was previously revealed to play a significant role in RA treatment. However, the main active ingredients and molecular mechanisms of CO in regulating RA are still unclear. Methods The active ingredients of CO were obtained from the Traditional Chinese Medicine Systems Pharmacology database and published literature. The targets corresponding to these compounds and the targets linked to RA were collected from public databases. The “ingredient-target” and “protein–protein interaction” networks were constructed to screen the main active ingredients and hub targets of CO in the treatment of RA. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment assays were used to elucidate the potential pharmacological mechanism of CO in RA. Molecular docking was performed to detect the binding between the main active ingredients and hub targets. Collagen-induced arthritis rats were used to validate the hub targets of CO against RA. Results Network pharmacological topology analysis showed that caffeine, 2,4-dichloro-5-methoxy-3-methylphenol, curculigoside, orcinol glucoside, and orcin were the main active ingredients of CO, and matrix metalloproteinase 9 (MMP9), transcription factor AP-1 (JUN), prostaglandin-endoperoxide synthase 2 (PTGS2), brain-derived neurotrophic factor, and receptor-type tyrosine-protein phosphatase C were the hub targets of CO for RA treatment. Molecular docking revealed that curculigoside and orcinol glucoside had effective binding potential with MMP9, JUN, and PTGS2, respectively. In vivo experiments demonstrated that CO alleviated RA symptoms and inhibited the expression of MMP9, JUN, and PTGS2 proteins. Conclusions Our study demonstrates the main active ingredients and potential targets of CO against RA, laying an experimental foundation for the development and application of CO as an anti-RA drug.

Pseudomonas aeruginosa is a multidrug-resistant Gram-negative pathogen and one of the leading causes of ventilator-associated pneumonia and infections in patients with chronic obstructive pulmonary disease and cystic fibrosis. Murepavadin is a peptidomimetic that specifically targets outer-membrane lipopolysaccharide transport protein LptD of P. aeruginosa. In this study, we find that murepavadin enhances the bactericidal efficacy of ciprofloxacin. We further demonstrate that murepavadin increases intracellular accumulation of ciprofloxacin by suppressing drug efflux. In addition, the murepavadin–ciprofloxacin combination exhibits a synergistic bactericidal effect in an acute murine pneumonia model. In conclusion, our results identify an effective drug combination for the treatment of P. aeruginosa infections.

Drying involves the evaporation of moisture, accompanied by simultaneous heat transfer, mass transfer, and momentum transfer. While the diffusion law is considered an applicable model for explaining the drying phenomenon, the actual drying process cannot be accurately predicted using an analytical solution with a constant diffusion coefficient. Energy efficiency in the drying process is low due to an insufficient understanding of the mechanisms governing moisture migration from solids to air. The development of drying theory has stalled due to an unsolvable discrepancy between experimental results and analytical results. This study analyzes the effect of the binding energy of moisture in paddy rice on the diffusion coefficient. The theoretical relationship between water activity and drying rate in paddy rice was investigated, and the drying process was successfully explained by analyzing free energy transfer and transition theory. The mechanism of rice grain drying was described using a new theoretical solution for the drying process. These results provide new insights into the development of a scientific evaluation standard for assessing the efficiency of actual drying processes.

In order to improve the efficiency of fine segmentation and obstacle removal in the road of tea plantation in hilly areas, a lightweight and high-precision DR-YOLO instance segmentation algorithm is proposed to realize environment awareness. Firstly, the road data of tea gardens in hilly areas were collected under different road conditions and light conditions, and data sets were generated. YOLOv8n-seg, which has the highest operating efficiency, was selected as the basic model. The MSDA-CBAM and DR-Neck feature fusion network were added to the YOLOv8-seg model to improve the feature extraction capability of the network and the feature fusion capability and efficiency of the model. Experimental results show that, compared with the YOLOv8-seg model, the DR-YOLO model proposed in this study has 2.0% improvement in AP@0.5 and 1.1% improvement in Precision. In this study, the DR-YOLO model is pruned and quantitatively compressed, which greatly improves the model inference speed with little reduction in AP. After deploying on Jetson, compared with the YOLOv8n-seg model, the Precision of DR-YOLO is increased by 0.6%, the AP@0.5 is increased by 1.6%, and the inference time is reduced by 17.1%, which can effectively improve the level of agricultural intelligent automation and realize the efficient operation of the instance segmentation model at the edge.

Ehrlichia chaffeensis infects and proliferates inside monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. After internalization, E. chaffeensis resides in specialized membrane-bound inclusions, E. chaffeensis-containing vesicles (ECVs), to evade from host cell innate immune responses and obtain nutrients. However, mechanisms exploited by host cells to inhibit E. chaffeensis growth in ECVs are still largely unknown. Here we demonstrate that host cells recognize E. chaffeensis Ech_1067, a penicillin-binding protein, and then upregulate the expression of PIT1, which is a phosphate transporter and transports phosphate from ECVs to the cytosol to inhibit bacterial growth. We found that host cells upregulate the PIT1 expression upon E. chaffeensis infection using transcriptome sequencing, qRT-PCR and Western blotting, and PIT1 is localized on the ECV membrane in infected THP-1 cells using confocal microscopy. Silence of PIT1 using shRNA enhances E. chaffeensis intracellular growth. Finally, we found that E. chaffeensis Ech_1067 induces the upregulation of PIT1 expression through the MyD88-NF-κB pathway using recombinant protein for stimulation and siRNA for silence. Our findings deepen the understanding of the innate immune responses of host cells to inhibit bacterial intracellular growth and facilitate the development of new therapeutics for HME.

Water scarcity poses a significant challenge for people living in arid areas. Despite the effectiveness of many bioinspired surfaces in promoting vapor condensation, their water-harvesting efficiency is insufficient. This is often exacerbated by overheating, which decreases the performance in terms of the micro-droplet concentration and movement on surfaces. In this study, we used a spotted amphiphilic surface to enhance the surfaces’ water-harvesting efficiency while maintaining their heat emissivity. Through hydrophilic particle screening and hydrophobic groove modifying, the coalescence and sliding characteristics of droplets on the amphiphilic surfaces were improved. The incorporation of boron nitride (BN) nanoparticles further enhanced the surfaces’ ability to harvest energy from condensation. To evaluate the water-harvesting performance of these amphiphilic surfaces, we utilized a real-time recording water-harvesting platform to identify microscopic weight changes on the surfaces. Our findings indicated that the inclusion of glass particles in hydrophobic grooves, combined with 1.0 wt.% BN nanoparticles, enhanced the water-harvesting efficiency of the amphiphilic surfaces by more than 20%.

In order to manufacture non-ferromagnetic heterogeneous material by Selective Laser Melting (SLM), it is necessary to solve the separation problem of non-ferromagnetic mixed powders after SLM forming. This research presents how to resolve this problem using TiNi/Ta mixed powders by ultrasonic vibration screening method and the analysis of SLM manufacturing experiment for non-ferromagnetic heterogeneous material. The purity of TiNi and Ta powders after separation could reach 99.7087%wt% and 98.8501wt% respectively, which both reached a relatively high purity. A TiNi alloy sample with Ta coating and TiNi/Ta gradient transition was manufactured successfully by SLM. The material interface of the sample achieves metallurgical bonding, and the color of the sample profile shows a gradient transition. The EDS analysis shows that the material composition changes from Ta ->Ta/TiNi gradient ->TiNi from surface to inside of the sample. The Ta coating contains over 92.5wt% Ta, and the TiNi matrix contains over 98.69wt% TiNi. Along the powder laying direction, it is difficult to clean the small powder near the solid-powder or solid-solid interfaces made of two different materials, which causes micro polluted areas near the interfaces. This study also provides a new method for integrated manufacturing of TiNi alloy part with Ta coating.

Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a variety of acute and chronic infections. Its type III secretion system (T3SS) plays a critical role in pathogenesis during acute infection. ExsA is a master regulator that activates the expression of all T3SS genes. Transcription of exsA is driven by two distinct promoters, its own promoter PexsA and its operon promoter PexsC. Here, in combination with a DNA pull-down assay and mass spectrometric analysis, we found that a histone-like nucleoid-structuring (H-NS) family protein MvaT can bind to the PexsC promoter. Using EMSA and reporter assays, we further found that MvaT directly binds to the PexsC promoter to repress the expression of T3SS genes. The repression of MvaT on PexsC is independent of ExsA, with MvaT binding to the -429 to -380 bp region relative to the transcription start site of the exsC gene. The presented work further reveals the complex regulatory network of the T3SS in P. aeruginosa.

Ehrlichia chaffeensis infects human monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening zoonosis. After internalization, E. chaffeensis resides in membrane-bound inclusions, E. chaffeensis-containing vesicles (ECVs), which have early endosome-like characteristics and fuse with early autophagosomes but not lysosomes, to evade host innate immune microbicidal mechanisms and obtain nutrients for bacterial intracellular growth. The mechanisms exploited by E. chaffeensis to modulate intracellular vesicle trafficking in host cells have not been comprehensively studied. Here, we demonstrate that E. chaffeensis type IV secretion system (T4SS) effector Etf-3 induces RAB15 upregulation in host cells and that RAB15, which is localized on ECVs, inhibits ECV fusion with lysosomes and induces autophagy. We found that E. chaffeensis infection upregulated RAB15 expression using qRT-PCR, and RAB15 was colocalized with E. chaffeensis using confocal microscopy. Silence of RAB15 using siRNA enhanced ECV maturation to late endosomes and fusion with lysosomes, as well as inhibited host cell autophagy. Overexpression of Etf-3 in host cells specifically induced RAB15 upregulation and autophagy. Our findings deepen the understanding of E. chaffeensis pathogenesis and adaptation in hosts as well as the function of RAB15 and facilitate the development of new therapeutics for HME.

ABSTRACT Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that can cause a variety of acute and chronic infections. The bacterium is highly resistant to numerous antibiotics. Murepavadin is a peptidomimetic antibiotic that blocks the function of P. aeruginosa lipopolysaccharide (LPS) transport protein D (LptD), thus inhibiting the insertion of LPS into the outer membrane. In this study, we demonstrated that sublethal concentrations of murepavadin enhance the bacterial outer membrane permeability. Proteomic analyses revealed the alteration of protein composition in bacterial inner and outer membranes following murepavadin treatment. The antisigma factor MucA was upregulated by murepavadin. In addition, the expression of the sigma E factor gene algU and the alginate synthesis gene algD was induced by murepavadin. Deletion of the algU gene reduces bacterial survival following murepavadin treatment, indicating a role of the envelope stress response in bacterial tolerance. We further demonstrated that murepavadin enhances the bactericidal activities of β-lactam antibiotics by promoting drug influx across the outer membrane. In a mouse model of acute pneumonia, the murepavadin–ceftazidime/avibactam combination showed synergistic therapeutic effect against P. aeruginosa infection. In addition, the combination of murepavadin with ceftazidime/avibactam slowed down the resistance development. In conclusion, our results reveal the response mechanism of P. aeruginosa to murepavadin and provide a promising antibiotic combination for the treatment of P. aeruginosa infections. Importance The ever increasing resistance of bacteria to antibiotics poses a serious threat to global public health. Novel antibiotics and treatment strategies are urgently needed. Murepavadin is a novel antibiotic that blocks the assembly of lipopolysaccharide (LPS) into the Pseudomonas aeruginosa outer membrane by inhibiting LPS transport protein D (LptD). Here, we demonstrated that murepavadin impairs bacterial outer membrane integrity, which induces the envelope stress response. We further found that the impaired outer membrane integrity increases the influx of β-lactam antibiotics, resulting in enhanced bactericidal effects. In addition, the combination of murepavadin and a β-lactam/β-lactamase inhibitor mixture (ceftazidime/avibactam) slowed down the resistance development of P. aeruginosa. Overall, this study demonstrates the bacterial response to murepavadin and provides a new combination strategy for effective treatment.

Abstract The increasing prevalence of antibiotic resistance by Pseudomonas aeruginosa (PA) raises an urgent need for an effective vaccine. The outer membrane proteins of PA, especially those that are upregulated during infection, are ideal vaccine targets. However, the strong hydrophobicity of these proteins hinders their application for this purpose. In this study, we selected eight outer membrane proteins from PA with the most significantly upregulated expression. Their extracellular loops were analyzed and screened by using sera from patients who had recovered from PA infection. As a result, a novel immunogenic epitope (Ep167-193) from PilY1 (PA4554) was found. Moreover, we constructed a macrophage membrane-coated PLGA (poly lactic-co-glycolic acid) nanoparticle vaccine carrying PilY1 Ep167-193 (PNPs@M-Ep167-193) that elicits a Th2 immune response and confers adequate protection in mice. Our data furnished the promising vaccine candidate PNPs@M-Ep167-193 while providing additional evidence for structure-based epitope identification and vaccine design. Graphical Abstract

ABSTRACT Polymyxins are currently the last-resort antibiotics for the treatment of multidrug-resistant Gram-negative bacterial infections. To expand the understanding of the intrinsic resistance mechanism against polymyxins, a laboratory strain of Pseudomonas aeruginosa PAO1 was subjected to serial passage in the presence of sublethal doses of polymyxin B over a period of 30 days. By whole-genome sequencing of successively isolated polymyxin B-resistant isolates, we identified a frameshift mutation (L183fs) in the mvfR gene that further increased polymyxin resistance in the pmrB mutant background. A ΔmvfR mutation alone showed higher tolerance to polymyxin B due to altered lipopolysaccharide (LPS) on the surface of bacterial cells, which decreases its outer membrane permeability. In the ΔmvfR mutant, polymyxin B treatment caused the upregulation of rfaD, the gene involved in LPS core oligosaccharide synthesis, which is responsible for polymyxin tolerance. To the best of our knowledge, this is the first report of mvfR mutation conferring polymyxin resistance in P. aeruginosa via increased integrity of bacterial outer membrane. IMPORTANCE Antibiotic resistance imposes a considerable challenge for the treatment of P. aeruginosa infections. Polymyxins are the last-resort antibiotics for the treatment of multidrug-resistant P. aeruginosa infections. Understanding the development and mechanisms of bacterial resistance to polymyxins may provide clues for the development of new or improved therapeutic strategies effective against P. aeruginosa. In this study, using an in vitro evolution assay in combination with whole-genome sequencing, we demonstrated that MvfR controls tolerance to polymyxin B by regulating the rfaD gene in P. aeruginosa. Our results reveal a novel mechanism employed by P. aeruginosa in the defense against polymyxin antibiotics.

COVID-19 pandemic continues to spread throughout the world with an urgent demand for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-CoV-2. Here, we report the development of a bacterial vector COVID-19 vaccine (aPA-RBD) that carries the gene for the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Live-attenuated strains of Pseudomonas aeruginosa (aPA) were constructed which express the recombinant RBD and effectively deliver RBD protein into various antigen presenting cells through bacterial type 3 secretion system (T3SS) in vitro. In mice, two-dose of intranasal aPA-RBD vaccinations elicited the development of RBD-specific serum IgG and IgM. Importantly, the sera from the immunized mice were able to neutralize host cell infections by SARS-CoV-2 pseudovirus as well as the authentic virus variants potently. T-cell responses of immunized mice were assessed by enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) assays. aPA-RBD vaccinations can elicit RBD-specific CD4+and CD8+T cell responses. T3SS-based RBD intracellular delivery heightens the efficiency of antigen presentation and enables the aPA-RBD vaccine to elicit CD8+T cell response. Thus, aPA vector has the potential as an inexpensive, readily manufactured, and respiratory tract vaccination route vaccine platform for other pathogens

ABSTRACT Carbohydrate metabolism plays essential roles in energy generation and providing carbon skeletons for amino acid syntheses. In addition, carbohydrate metabolism has been shown to influence bacterial susceptibility to antibiotics and virulence. In this study, we demonstrate that citrate synthase gltA mutation can increase the expression of the type III secretion system (T3SS) genes and antibiotic tolerance in Pseudomonas aeruginosa. The stringent response is activated in the gltA mutant, and deletion of the (p)ppGpp synthetase gene relA restores the antibiotic tolerance and expression of the T3SS genes to wild-type level. We further demonstrate that the intracellular level of cAMP is increased by the stringent response in the gltA mutant, which increases the expression of the T3SS master regulator gene exsA. Overall, our results reveal an essential role of GltA in metabolism, antibiotic tolerance, and virulence, as well as a novel regulatory mechanism of the stringent response-mediated regulation of the T3SS in P. aeruginosa. IMPORTANCE Rising antimicrobial resistance imposes a severe threat to human health. It is urgent to develop novel antimicrobial strategies by understanding bacterial regulation of virulence and antimicrobial resistance determinants. The stringent response plays an essential role in virulence and antibiotic tolerance. Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in humans. The bacterium produces an arsenal of virulence factors and is highly resistant to a variety of antibiotics. In this study, we provide evidence that citrate synthase GltA plays a critical role in P. aeruginosa metabolism and influences the antibiotic tolerance and virulence. We further reveal a role of the stringent response in the regulation of the antibiotic tolerance and virulence. The significance of this work is in elucidation of novel regulatory pathways that control both antibiotic tolerance and virulence in P. aeruginosa.

Laser powder bed fusion (LPBF) additive manufacturing has been advancing in the fabrication of metallic multi-material structures with intricate structures and refined material layouts. Herein, a comprehensive review of the recent achievements of multi-material structures via LPBF is provided in terms of interface characteristics and strengthening methods, critical technical issues and potential applications. It begins with the introduction of multi-material structures and the scope of the review. The interface characteristics (including representative multi-material types printed by LPBF, interfacial microstructure, defects, etc.) and strengthening methods of multi-material structures are then presented. Thereafter, the critical technical issues in LPBF for multi-material structures are discussed with regard to equipment development, data preparation, thermodynamic calculation and process simulation, and powder cross-contamination and recycling. Moreover, the potential applications (particularly in biomedical, electronic, aerospace) are illustrated and discussed. Finally, the outlook is outlined to provide guidance for future research.

ABSTRACT Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, the leading cause of acute and chronic infections in immunocompromised patients, frequently with high morbidity and mortality rates. The xenobiotic response element (XRE) family proteins are the second most common transcriptional regulators (TRs) in P. aeruginosa. However, only a few XRE-like TRs have been reported to regulate multiple bacterial cellular processes, encompassing virulence, metabolism, antibiotic synthesis or resistance, stress responses, and phage infection, etc. Our understanding of what roles these XRE-like small regulatory proteins play in P. aeruginosa remains limited. Here, we aimed to decipher the role of a putative XRE-type transcriptional regulator (designated LfsT) from a prophage region on the chromosome of a clinical P. aeruginosa isolate, P8W. Southern blot and reverse transcription quantitative PCR (RT-qPCR) assays demonstrated that LfsT controlled host sensitivity to the phage PP9W2 and was essential for efficient phage replication. In addition, electrophoretic mobility shift assays (EMSAs) and transcriptional lacZ fusion analyses indicated that LfsT repressed the lysogenic development and promoted the lytic cycle of phage PP9W2 by binding to the promoter regions of the gp71 gene (encoding a CI-like repressor) and several vital phage genes. Combined with RNA-seq and a series of phenotypic validation tests, our results showed that LfsT bound to the flexible palindromic sites within the promoters upstream of several genes in the bacterial genome, regulating fatty acid (FA) metabolism, spermidine (SPD) transport, as well as the type III secretion system (T3SS). Overall, this study reveals novel regulatory roles of LfsT in P. aeruginosa, improving our understanding of the molecular mechanisms behind bacterium-phage interactions. IMPORTANCE This work elucidates the novel roles of a putative XRE family TR, LfsT, in the intricate regulatory systems of P. aeruginosa. We found that LfsT bound directly to the core promoter regions upstream of the start codons of numerous genes involved in various processes, including phage infection, FA metabolism, SPD transport, and the T3SS, regulating as the repressor or activator. The identified partial palindromic motif NAACN(5,8)GTTN recognized by LfsT suggests extensive effects of LfsT on gene expression by maintaining preferential binding to nucleotide sites under evolutionary pressure. In summary, these findings indicate that LfsT enhances metabolic activity in P. aeruginosa, while it reduces host resistance to the phage. This study helps us better understand the coevolution of bacteria and phages (e.g., survival comes at a cost) and provides clues for designing novel antimicrobials against P. aeruginosa infections.

Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis (HME), is a Gram-negative obligatory intracellular bacterium, which infects and multiplies in human monocytes and macrophages. Host immune cells produce reactive oxygen species (ROS) to eliminate E. chaffeensis upon infection. E. chaffeensis global transcriptional regulator CtrA activates the expression of GshA and GshB to synthesize glutathione (GSH), the most potent natural antioxidant, upon oxidative stress to combat ROS damage. However, the mechanisms exploited by E. chaffeensis to utilize GSH are still unknown. Here, we found that in E. chaffeensis CtrA activated the expression of glutathione S-transferase (GST) upon oxidative stress, and E. chaffeensis GST utilizes GSH to eliminate ROS and confers the oxidative stress resistance to E. chaffeensis. We found that CtrA bound to the promoter regions of 211 genes, including gst, in E. chaffeensis using chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq). Recombinant E. chaffeensis CtrA directly bound to the gst promoter region determined with electrophoretic mobility shift assay (EMSA), and activated the gst expression determined with reporter assay. Recombinant GST showed GSH conjugation activity towards its typical substrate 2,4-dinitrochlorobenzene (CDNB) in vitro and peptide nucleic acid (PNA) transfection of E. chaffeensis, which can knock down the gst transcription level, reduced bacterial survival upon oxidative stress. Our results demonstrate that E. chaffeensis CtrA regulates GSH utilization, which plays a critical role in resistance to oxidative stress, and aid in the development of new therapeutics for HME.

ABSTRACT The evolution of bacterial antibiotic resistance is exhausting the list of currently used antibiotics and endangers those in the pipeline. The combination of antibiotics is a promising strategy that may suppress resistance development and/or achieve synergistic therapeutic effects. Eravacycline is a newly approved antibiotic that is effective against a variety of multidrug-resistant (MDR) pathogens. However, the evolution of resistance to eravacycline and strategies to suppress the evolution remain unexplored. Here, we demonstrated that a carbapenem-resistant Klebsiella pneumoniae clinical isolate quickly developed resistance to eravacycline, which is mainly caused by mutations in the gene encoding the Lon protease. The evolved resistant mutants display collateral sensitivities to β-lactam/β-lactamase inhibitor (BLBLI) combinations aztreonam/avibactam and ceftazidime-avibactam. Proteomic analysis revealed upregulation of the multidrug efflux system AcrA-AcrB-TolC and porin proteins OmpA and OmpU, which contributed to the increased resistance to eravacycline and susceptibility to BLBLIs, respectively. The combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam suppresses resistance development. We further demonstrated that eravacycline-resistant mutants evolved from an NDM-1-containing K. pneumoniae strain display collateral sensitivity to aztreonam/avibactam, and the combination of eravacycline with aztreonam/avibactam suppresses resistance development. In addition, the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam displayed synergistic therapeutic effects in a murine cutaneous abscess model. Overall, our results revealed mechanisms of resistance to eravacycline and collateral sensitivities to BLBLIs and provided promising antibiotic combinations in the treatment of multidrug-resistant K. pneumoniae infections. IMPORTANCE The increasing bacterial antibiotic resistance is a serious threat to global public health, which demands novel antimicrobial medicines and treatment strategies. Eravacycline is a newly approved antibiotic that belongs to the tetracycline antibiotics. Here, we found that a multidrug-resistant Klebsiella pneumoniae clinical isolate rapidly developed resistance to eravacycline and the evolved resistant mutants displayed collateral sensitivity to antibiotics aztreonam/avibactam and ceftazidime-avibactam. We demonstrated that the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam repressed resistance development and improved the treatment efficacies. We also elucidated the mechanisms that contribute to the increased resistance to eravacycline and susceptibility to aztreonam/avibactam and ceftazidime-avibactam. This work demonstrated the mechanisms of antibiotic resistance and collateral sensitivity and provided a new therapeutically option for effective antibiotic combinations.

Summary: Novel treatment strategies are in urgent need to deal with the rapid development of antibiotic-resistant superbugs. Combination therapies and targeted drug delivery have been exploited to promote treatment efficacies. In this study, we loaded neutrophils with azithromycin and colistin to combine the advantages of antibiotic combinations, targeted delivery, and immunomodulatory effect of azithromycin to treat infections caused by Gram-negative pathogens. Delivery of colistin into neutrophils was mediated by fusogenic liposome, while azithromycin was directly taken up by neutrophils. Neutrophils loaded with the drugs maintained the abilitity to generate reactive oxygen species and migrate. In vitro assays demonstrated enhanced bactericidal activity against multidrug-resistant pathogens and reduced inflammatory cytokine production by the drug-loaded neutrophils. A single intravenous administration of the drug-loaded neutrophils effectively protected mice from Pseudomonas aeruginosa infection in an acute pneumonia model. This study provides a potential effective therapeutic approach for the treatment of bacterial infections.

ABSTRACT Pseudomonas aeruginosa is a ubiquitous pathogenic bacterium that can adapt to a variety environments. The ability to effectively sense and respond to host local nutrients is critical for the infection of P. aeruginosa. However, the mechanisms employed by the bacterium to respond to nutrients remain to be explored. CspA family proteins are RNA binding proteins that are involved in gene regulation. We previously demonstrated that the P. aeruginosa CspA family protein CspC regulates the type III secretion system in response to temperature shift. In this study, we found that CspC regulates the quorum-sensing (QS) systems by repressing the translation of a QS negative regulatory gene, rsaL. Through RNA immunoprecipitation coupled with real-time quantitative reverse transcription-PCR (RIP-qRT-PCR) and electrophoretic mobility shift assays (EMSAs), we found that CspC binds to the 5′ untranslated region of the rsaL mRNA. Unlike glucose, itaconate (a metabolite generated by macrophages during infection) reduces the acetylation of CspC, which increases the affinity between CspC and the rsaL mRNA, leading to upregulation of the QS systems. Our results revealed a novel regulatory mechanism of the QS systems in response to a host-generated metabolite. IMPORTANCE Bacterial infectious diseases impose a severe threat to human health. The ability to orchestrate virulence determinant in response to the host environment is critical for the pathogenesis of bacterial pathogens. Pseudomonas aeruginosa is a leading pathogen that causes various infections in humans. In P. aeruginosa, the quorum-sensing (QS) systems play an important role in regulating the production of virulence factors. In this study, we find that a small RNA binding protein, CspC, regulates the QS systems by repressing the expression of a QS negative regulator. We further demonstrate that CspC is acetylated in response to a host-derived metabolite, itaconate, which alters the function of CspC in regulating the QS system. The importance of this work is in elucidation of a novel regulatory pathway that regulates virulence determinants in P. aeruginosa in response to a host signal.

Li, Cebrian et al. show that a combination of peptide D-11 and vancomycin exhibits a potent antimicrobial activity against a panel of Gram-negative pathogens without apparent toxicity. They find that this combination is also effective in clearing Pseudomonas aeruginosa in mice, providing a potential antimicrobial therapy for humans.

Ehrlichia chaffeensis causes human monocytic ehrlichiosis (HME), which is one of the most prevalent, life-threatening emerging infectious zoonoses. The life cycle of E. chaffeensis includes ticks and mammals, in which E. chaffeensis proteins are expressed differentially contributing to bacterial survival and infection. Among the E. chaffeensis P28-OMP outer membrane proteins, OMP-1B and P28 are predominantly expressed in tick cells and mammalian macrophages, respectively. The mechanisms regulating this differential expression have not been comprehensively studied. Here, we demonstrate that the transcriptional regulators EcxR and Tr1 regulate the differential expression of omp-1B and p28 in E. chaffeensis. Recombinant E. chaffeensis Tr1 bound to the promoters of omp-1B and p28, and transactivated omp-1B and p28 promoter-EGFP fusion constructs in Escherichia coli. The consensus sequence of Tr1 binding motifs was AC/TTATA as determined with DNase I footprint assay. Tr1 showed a higher affinity towards the p28 promoter than the omp-1B promoter as determined with surface plasmon resonance. EcxR activated the tr1 expression in response to a temperature decrease. At 37°C low level of Tr1 activated the p28 expression. At 25°C high level of Tr1 activated the omp-1B expression, while repressing the p28 expression by binding to an additional site upstream of the p28 gene. Our data provide insights into a novel mechanism mediated by Tr1 regulating E. chaffeensis differential gene expression, which may aid in the development of new therapeutics for HME.

Ehrlichia chaffeensis, a small Gram-negative obligatory intracellular bacterium, infects human monocytes or macrophages, and causes human monocytic ehrlichiosis, one of the most prevalent, life-threatening emerging zoonoses. Reactive oxygen species are produced by the host immune cells in response to bacterial infections. The mechanisms exploited by E. chaffeensis to resist oxidative stress have not been comprehensively demonstrated. Here, we found that E. chaffeensis encodes two functional enzymes, GshA and GshB, to synthesize glutathione that confers E. chaffeensis the oxidative stress resistance, and that the expression of gshA and gshB is upregulated by CtrA, a global transcriptional regulator, upon oxidative stress. We found that in E. chaffeensis, the expression of gshA and gshB was upregulated upon oxidative stress using quantitative RT-PCR. Ehrlichia chaffeensis GshA or GshB restored the ability of Pseudomonas aeruginosa GshA or GshB mutant to cope with oxidative stress, respectively. Recombinant E. chaffeensis CtrA directly bound to the promoters of gshA and gshB, determined with electrophoretic mobility shift assay, and activated the expression of gshA and gshB determined with reporter assay. Peptide nucleic acid transfection of E. chaffeensis, which reduced the CtrA protein level, inhibited the oxidative stress-induced upregulation of gshA and gshB. Our findings provide insights into the function and regulation of the two enzymes critical for E. chaffeensis resistance to oxidative stress and may deepen our understanding of E. chaffeensis pathogenesis and adaptation in hosts.

ABSTRACT NrtR is a Nudix-related transcriptional regulator that is distributed among diverse bacteria and plays an important role in modulating bacterial intracellular NAD homeostasis. Previously, we showed that NrtR influences the T3SS expression and pathogenesis of Pseudomonas aeruginosa and demonstrated that NrtR mediates T3SS regulation through the cAMP/Vfr pathway. In the present study, we found that mutation of the nrtR gene leads to upregulation of the Hcp secretion island-I type VI secretion system (H1-T6SS). Further analysis revealed that mutation of the nrtR gene results in upregulation of regulatory RNAs (RsmY/RsmZ) that are known to control the H1-T6SS by sequestration of RsmA or RsmN. Simultaneous deletion of rsmY/rsmZ reduced the expression of H1-T6SS in the ΔnrtR mutant. In addition, overexpression of either rsmA or rsmN in ΔnrtR decreased H1-T6SS expression. Chromatin immunoprecipitation (ChIP)-Seq and electrophoretic mobility shift assay (EMSA) analyses revealed that NrtR directly binds to the promoters of rsmY, rsmZ and tssA1 (first gene of the H1-T6SS operon). Overall, the results from this study reveal the molecular details of NrtR-mediated regulation of H1-T6SS in P. aeruginosa. IMPORTANCE NrtR is a Nudix-related transcriptional regulator and controls the NAD cofactor biosynthesis in bacteria. P. aeruginosa NrtR binds to the intergenic region between nadD2 and pcnA to repress the expression of the two operons, therefore controlling the NAD biosynthesis. We have previously reported that NrtR controls T3SS expression via the cAMP/Vfr pathway in P. aeruginosa. However, the global regulatory function and direct binding targets of the NrtR remain elusive in P. aeruginosa. This study reveals novel direct regulatory targets of the NrtR in P. aeruginosa, elucidating the molecular mechanism of NrtR-mediated regulation of H1-T6SS.

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa often adapts to its host environment and causes recurrent nosocomial infections. The extracytoplasmic function (ECF) sigma factor enables bacteria to alter their gene expression in response to host environmental stimuli. Here, we report an ECF sigma factor, HxuI, which is rapidly induced once P. aeruginosa encounters the host. Host stresses such as iron limitation, oxidative stress, low oxygen, and nitric oxide induce the expression of hxuI. By combining RNA-seq and promoter-lacZ reporter fusion analysis, we reveal that HxuI can activate the expression of diverse metabolic and virulence pathways which are critical to P. aeruginosa infections, including iron acquisition, denitrification, pyocyanin synthesis, and bacteriocin production. Most importantly, overexpression of the hxuI in the laboratory strain PAO1 promotes its colonization in both murine lung and subcutaneous infections. Together, our findings show that HxuI, a key player in host stress-response, controls the in vivo adaptability and virulence of P. aeruginosa during infection. IMPORTANCE P. aeruginosa has a strong ability to adapt to diverse environments, making it capable of causing recurrent and multisite infections in clinics. Understanding host adaptive mechanisms plays an important guiding role in the development of new anti-infective agents. Here, we demonstrate that an ECFσ factor of P. aeruginosa response to the host-inflicted stresses, which promotes the bacterial in vivo fitness and pathogenicity. Furthermore, our findings may help explain the emergence of highly transmissible strains of P. aeruginosa and the acute exacerbations during chronic infections.

ABSTRACT WCK 5222 (cefepime/zidebactam) is a β-lactam/β-lactamase inhibitor combination that is effective against a broad range of highly drug-resistant bacterial pathogens, including those producing metallo-β-lactamase. In this study, we isolated a multidrug-resistant Pseudomonas aeruginosa clinical strain that is resistant to a variety of β-lactam antibiotics and the ceftazidime-avibactam combination. A metallo-β-lactamase gene blaDIM-2 was identified on a self-transmissible megaplasmid in the strain, which confers the resistance to β-lactam antibiotics, leaving WCK 5222 potentially one of the last treatment resorts. In vitro passaging assay combined with whole-genome sequencing revealed mutations in the pbpA gene (encoding the zidebactam target protein PBP2) in the evolved resistant mutants. Among the mutations, a V516M mutation increased the bacterial virulence in a murine acute pneumonia model. Reconstitution of the mutations in the reference strain PAO1 verified their roles in the resistance to zidebactam and revealed their influences on cell morphology in the absence and presence of zidebactam. Microscale thermophoresis (MST) assays demonstrated that the mutations reduced the affinity between PBP2 and zidebactam to various extents. Overall, our results revealed that mutations in the pbpA gene might be a major cause of evolved resistance to WCK 5222 in clinical settings. IMPORTANCE Antibiotic resistance imposes a severe threat on human health. WCK 5222 is a β-lactam/β-lactamase inhibitor combination that is composed of cefepime and zidebactam. It is one of the few antibiotics in clinical trials that are effective against multidrug-resistant Pseudomonas aeruginosa, including those producing metallo-β-lactamases. Understanding the mechanisms and development of bacterial resistance to WCK 5222 may provide clues for the development of strategies to suppress resistant evolvement. In this study, we performed an in vitro passaging assay by using a multidrug-resistant P. aeruginosa clinical isolate. Our results revealed that mutations in the zidebactam target protein PBP2 play a major role in the bacterial resistance to WCK 5222. We further demonstrated that the mutations reduced the affinities between PBP2 and zidebactam and resulted in functional resistance of PBP2 to zidebactam.

Pseudomonas aeruginosa is an important opportunistic pathogen capable of causing variety of infections in humans. The type III secretion system (T3SS) is a critical virulence determinant of P. aeruginosa in the host infections. Expression of the T3SS is regulated by ExsA, a master regulator that activates the expression of all known T3SS genes. Expression of the exsA gene is controlled at both transcriptional and posttranscriptional levels. Here, we screened a P. aeruginosa transposon (Tn5) insertional mutant library and found rplI, a gene coding for the ribosomal large subunit protein L9, to be a repressor for the T3SS gene expression. Combining real-time quantitative PCR (qPCR), western blotting and lacZ fusion assays, we show that RplI controls the expression of exsA at the posttranscriptional level. Further genetic experiments demonstrated that RplI mediated control of the exsA translation involves 5’ untranslated region (5’ UTR). A ribosome immunoprecipitation assay and qPCR revealed higher amounts of a 24 nt fragment from exsA mRNA being associated with ribosomes in the ΔrplI mutant. An interaction between RplI and exsA mRNA harboring its 24 nt, but not 12 nt, 5’ UTR was confirmed by RNA Gel Mobility Shift and Microscale Thermophoresis assays. Overall, this study identifies the ribosomal large subunit protein L9 as a novel T3SS repressor that inhibits ExsA translation in P. aeruginosa. Author summary Ribosomes provide all living organisms the capacity to synthesize proteins. The production of many ribosomal proteins is often controlled by an autoregulatory feedback mechanism. P. aeruginosa is an opportunistic human pathogen and its type III secretion system (T3SS) is a critical virulence determinant in host infections. In this study, by screening a Tn5 mutant library, we identified rplI, encoding ribosomal large subunit protein L9, as a novel repressor for the T3SS. Further exploring the regulatory mechanism, we found that the RplI protein interacts with the 5’ UTR (5’ untranslated region) of exsA, a gene coding for transcriptional activator of the T3SS. Such an interaction likely blocks ribosome loading on the exsA 5’ UTR, inhibiting the initiation of exsA translation. The significance of this work is in the identification of a novel repressor for the T3SS and elucidation of its molecular mechanism. Furthermore, this work provides evidence for individual ribosomal protein regulating mRNA translation beyond its autogenous feedback control.

Pseudomonas aeruginosa is an important nosocomial pathogen which frequently becomes resistant to most antibiotics used in chemotherapy, resulting in treatment failure among infected individuals. Although the evolutionary trajectory and molecular mechanisms for becoming β-lactam resistant have been well established for P. aeruginosa, the molecular basis of reversion from β-lactam resistant to susceptible is largely unexplored. In this study, we investigated the molecular mechanisms by which a ceftazidime-resistant clinical strain is converted to a ceftazidime-susceptible isolate under the clinical setting. RNA sequencing and genomic DNA reference mapping were conducted to compare the transcriptional profiles and chromosomal mutations between these two isolates. Our results demonstrate that a gain-of-function mutation in ampD, via deletion of a 53 bp duplicated nucleotide sequence, is the contributory factor for the conversion. Furthermore, we show for the first time that AmpD is involved in intraspecies competitiveness in P. aeruginosa. We also found that AmpD is not responsible for phenotypic changes between R1 and S2, including growth rate, motilities, pyocyanin, rhamnolipid, and biofilm production. This finding provides novel insights into the alteration of β-lactam sensitivity in P. aeruginosa under the clinical setting.

Pseudomonas aeruginosa is a ubiquitous pathogen that causes a wide range of acute and chronic infections. Ciprofloxacin, one of the first-line fluoroquinolone class antibiotics, is commonly used for the treatment of P. aeruginosa infections. However, ciprofloxacin-resistant P. aeruginosa is increasingly reported worldwide, making treatment difficult. To determine resistance-related mutations, we conducted an experimental evolution using a previously identified ciprofloxacin-resistant P. aeruginosa clinical isolate, CRP42. The evolved mutants could tolerate a 512-fold higher concentration of ciprofloxacin than CRP42. Genomic DNA reference mapping was performed, which revealed mutations in genes known to be associated with ciprofloxacin resistance as well as in those not previously linked to ciprofloxacin resistance, including the ParER586W substitution and PA0625 frameshift insertion. Simulation of the ParER586W substitution and PA0625 frameshift insertion by gene editing in CRP42 and the model strain PAO1 demonstrated that while the PA0625 mutation does contribute to resistance, mutation in the ParER586W does not contribute to resistance but rather affects tolerance against ciprofloxacin. These findings advance our understanding of ciprofloxacin resistance in P. aeruginosa.

The emergence and rapid spread of multi-drug resistant (MDR) bacteria pose a serious threat to the global healthcare. There is an urgent need for new antibacterial substances or new treatment strategies to deal with the infections by MDR bacterial pathogens, especially the Gram-negative pathogens. In this study, we show that a number of synthetic cationic peptides display strong synergistic antimicrobial effects with multiple antibiotics against the Gram-negative pathogen Pseudomonas aeruginosa. We found that an all-D amino acid containing peptide called D-11 increases membrane permeability by attaching to LPS and membrane phospholipids, thereby facilitating the uptake of antibiotics. Subsequently, the peptide can dissipate the proton motive force (PMF) (reducing ATP production and inhibiting the activity of efflux pumps), impairs the respiration chain, promotes the production of reactive oxygen species (ROS) in bacterial cells and induces intracellular antibiotics accumulation, ultimately resulting in cell death. By using a P. aeruginosa abscess infection model, we demonstrate enhanced therapeutic efficacies of the combination of D-11 with various antibiotics. In addition, we found that the combination of D-11 and azithromycin enhanced the inhibition of biofilm formation and the elimination of established biofilms. Our study provides a realistic treatment option for combining close-to-nature synthetic peptide adjuvants with existing antibiotics to combat infections caused by P. aeruginosa.

Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a great challenge to clinical treatment. In this study, we characterized a ST768 MDR P. aeruginosa strain, Pa150, that was isolated from a diabetic foot patient. The minimum inhibitory concentration (MIC) assay showed that Pa150 was resistant to almost all kinds of antibiotics, especially aminoglycosides. Whole genome sequencing revealed multiple antibiotic resistant genes on the chromosome and a 437-Kb plasmid (named pTJPa150) that harbors conjugation-related genes. A conjugation assay verified its self-transmissibility. On the pTJPa150 plasmid, we identified a 16S rRNA methylase gene, rmtB, that is flanked by mobile genetic elements (MGEs). The transfer of the pTJPa150 plasmid or the cloning of the rmtB gene into the reference strain, PAO1, significantly increased the bacterial resistance to aminoglycoside antibiotics. To the best of our knowledge, this is the first report of an rmtB-carrying conjugative plasmid isolated from P. aeruginosa, revealing a novel possible transmission mechanism of the rmtB gene.

Therapy for Pseudomonas aeruginosa infections is hard due to its high natural and acquirable antibiotic resistance. After colonization in the hosts, P. aeruginosa commonly accumulates genomic mutations which confer them antibiotic resistance and better adaptations to the host environment. Deciphering the mechanisms of antibiotic resistance development in the clinical setting may provide critical insights into the design of effective combinatory antibiotic therapies to treat P. aeruginosa infections. In this work, we demonstrate a resistance mechanism to aztreonam of a clinical isolate (ARP36) in comparison with a sensitive one (CSP18). RNAseq and genomic DNA resequencing were carried out to compare the global transcriptional profiles and in the clinical setting genomic profiles between these two isolates. The results demonstrated that hyperexpression of an efflux pump MexAB-OprM caused by a R70Q substitution in MexR, contributed to the increased resistance to aztreonam in the isolate ARP36. Simulation of mexR of ARP36 by gene editing in CSP18 conferred CSP18 an ARP36-like susceptibility to the aztreonam. The R70Q substitution prevented MexR from binding to the intergenic region between mexR and mexAB-oprM operon, with no impact on its dimerization. The presented experimental results explain for the first time why the clinically relevant R70Q substitution in the MexR derepresses the expression of mexAB-oprM in P. aeruginosa.

Treatment of infections by Pseudomonas aeruginosa is difficult due to its high intrinsic and acquired antibiotic resistance. Upon colonization in the human hosts, P. aeruginosa accumulates genetic mutations that confer the bacterium antibiotic resistance and ability to better live in the host environment. Characterizing the evolutionary traits would provide important insights into the development of effective combinatory antibiotic therapies to cure P. aeruginosa infections. In this work, we performed a detailed analysis of the molecular mechanisms by which a clinical isolate (CSP18) yields a ciprofloxacin-resistant derivative (CRP42). Genomic DNA re-sequencing and RNAseq were carried out to compare the genomic mutational signature and transcriptional profiles between the two isolates. The results indicated that D87G mutation in GyrA, together with MexEF-OprN hyper-expression caused by F7S mutation in MexS, was responsible for the increased resistance to ciprofloxacin in the isolate CRP42. Further simulation of CRP42 by gene editing in CSP18 demonstrated that D87G mutation in GyrA rendered CSP18 a fourfold increase in minimum inhibitory concentration against ciprofloxacin, while F7S mutation in MexS conferred an additional eightfold increase. Our experimental results demonstrate for the first time that the clinically relevant F7S point mutation in MexS results in hyper-expression of the mexEF-oprN and thus confers P. aeruginosa resistance to ciprofloxacin.

Animal infection models play significant roles in studying bacterial pathogenic mechanisms, host pathogen interaction as well as evaluating drug and vaccine efficacies. We have been utilizing an acute pneumonia model to study bacterial colonization in lungs and assess virulence to the host by determination of bacterial loads and survival assays, as well as examine the bacterial gene expression in vivo. Additionally, the host's immune response to the pathogen can be explored through this infection model.

Carbon metabolism plays an important role in bacterial physiology and pathogenesis. The type III secretion system (T3SS) of Pseudomonas aeruginosa is a virulence factor that contributes to acute infections. It has been demonstrated that bacterial metabolism affects the T3SS. Meanwhile, expression of T3SS genes is negatively regulated by the small RNAs RsmY and RsmZ. In this study, we studied the relationship between the dihydrolipoamide acetyltransferase gene aceF and the T3SS. Our results reveal an upregulation of RsmY and RsmZ in the aceF mutant, which represses the expression of the T3SS genes. Meanwhile, the aceF mutant is more tolerant to hydrogen peroxide. We demonstrate that the expression levels of the catalase KatB and the alkyl hydroperoxide reductase AhpB are increased in the aceF mutant. The simultaneous deletion of rsmY and rsmZ in the aceF mutant restored the expression levels of katB and ahpB, as well as bacterial susceptibility to hydrogen peroxide. Thus, we identify a novel role of AceF in the virulence and oxidative response of P. aeruginosa.

ABSTRACT Posttranscriptional regulation plays an essential role in the quick adaptation of pathogenic bacteria to host environments, and RNases play key roles in this process by modifying small RNAs and mRNAs. We find that the Pseudomonas aeruginosa endonuclease YbeY is required for rRNA processing and the bacterial virulence in a murine acute pneumonia model. Transcriptomic analyses reveal that knocking out the ybeY gene results in downregulation of oxidative stress response genes, including the catalase genes katA and katB. Consistently, the ybeY mutant is more susceptible to H2O2 and neutrophil-mediated killing. Overexpression of katA restores the bacterial tolerance to H2O2 and neutrophil killing as well as virulence. We further find that the downregulation of the oxidative stress response genes is due to defective expression of the stationary-phase sigma factor RpoS. We demonstrate an autoregulatory mechanism of RpoS and find that ybeY mutation increases the level of a small RNA, ReaL, which directly represses the translation of rpoS through the 5′ UTR of its mRNA and subsequently reduces the expression of the oxidative stress response genes. In vitro assays demonstrate direct degradation of ReaL by YbeY. Deletion of reaL or overexpression of rpoS in the ybeY mutant restores the bacterial tolerance to oxidative stress and the virulence. We also demonstrate that YbeZ binds to YbeY and is involved in the 16S rRNA processing and regulation of reaL and rpoS as well as the bacterial virulence. Overall, our results reveal pleiotropic roles of YbeY and the YbeY-mediated regulation of rpoS through ReaL. IMPORTANCE The increasing bacterial antibiotic resistance imposes a severe threat to human health. For the development of effective treatment and prevention strategies, it is critical to understand the mechanisms employed by bacteria to grow in the human body. Posttranscriptional regulation plays an important role in bacterial adaptation to environmental changes. RNases and small RNAs are key players in this regulation. In this study, we demonstrate critical roles of the RNase YbeY in the virulence of the pathogenic bacterium Pseudomonas aeruginosa. We further identify the small RNA ReaL as the direct target of YbeY and elucidate the YbeY-regulated pathway on the expression of bacterial virulence factors. Our results shed light on the complex regulatory network of P. aeruginosa and indicate that inference with the YbeY-mediated regulatory pathway might be a valid strategy for the development of a novel treatment strategy.

Infections by Pseudomonas aeruginosa are difficult to cure due to its high intrinsic and acquired antibiotic resistance. Once colonized the human host, and thanks to antibiotic treatment pressure, P. aeruginosa usually acquires genetic mutations which provide bacteria with antibiotic resistance as well as ability to better adapt to the host environment. Deciphering the evolutionary traits may provide important insights into the development of effective combinatory antibiotic therapy to treat P. aeruginosa infections. In this study, we investigated the molecular mechanisms by which a clinical isolate (ISP50) yields a carbapenem-resistant derivative (IRP41). RNAseq and genomic DNA reference mapping were conducted to compare the transcriptional profiles and in vivo evolutionary trajectories between the two isolates. Our results demonstrated that oprD mutation together with ampC hyper-expression contributed to the increased resistance to carbapenem in the isolate IRP41. Furthermore, a ldcA (PA5198) gene, encoding murein tetrapeptide carboxypeptidase, has been demonstrated for the first time to negatively influence the ampC expression in P. aeruginosa.

ABSTRACT Carbon metabolism plays an essential role in bacterial pathogenesis and susceptibility to antibiotics. In Pseudomonas aeruginosa, Crc, Hfq, and a small RNA, CrcZ, are central regulators of carbon metabolism. By screening mutants of genes involved in carbon metabolism, we found that mutation of the tpiA gene reduces the expression of the type III secretion system (T3SS) and bacterial resistance to aminoglycoside antibiotics. TpiA is a triosephosphate isomerase that reversibly converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate, a key step connecting glucose metabolism with glycerol and phospholipid metabolisms. We found that mutation of the tpiA gene enhances the bacterial carbon metabolism, respiration, and oxidative phosphorylation, which increases the membrane potential and promotes the uptake of aminoglycoside antibiotics. Further studies revealed that the level of CrcZ is increased in the tpiA mutant due to enhanced stability. Mutation of the crcZ gene in the tpiA mutant background restored the expression of the T3SS genes and the bacterial resistance to aminoglycoside antibiotics. Overall, this study reveals an essential role of TpiA in the metabolism, virulence, and antibiotic resistance in P. aeruginosa. IMPORTANCE The increase in bacterial resistance against antibiotics imposes a severe threat to public health. It is urgent to identify new drug targets and develop novel antimicrobials. Metabolic homeostasis of bacteria plays an essential role in their virulence and resistance to antibiotics. Recent studies demonstrated that antibiotic efficacies can be improved by modulating the bacterial metabolism. Pseudomonas aeruginosa is an important opportunistic human pathogen that causes various infections. The bacterium is intrinsically resistant to antibiotics. In this study, we provide clear evidence that TpiA (triosephosphate isomerase) plays an essential role in the metabolism of P. aeruginosa and influences bacterial virulence and antibiotic resistance. The significance of this work is in identifying a key enzyme in the metabolic network, which will provide clues as to the development of novel treatment strategies against infections caused by P. aeruginosa.

You et al. show that SrpA, a small protein widely conserved among bacteria, controls core cellular processes in response to phage infection and environmental signals in Pseudomonas aeruginosa, including cell motility, chemotaxis, biofilm formation, and virulence.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes various acute and chronic infections. It is intrinsically resistant to a variety of antibiotics. However, production of pyocins during SOS response sensitizes P. aeruginosa to quinolone antibiotics by inducing cell lysis. The polynucleotide phosphorylase (PNPase) is a conserved phosphate-dependent 3′–5′ exonuclease that plays an important role in bacterial response to environmental stresses and pathogenesis by influencing mRNA and small RNA stabilities. Previously, we demonstrated that PNPase controls the type III and type VI secretion systems in P. aeruginosa. In this study, we found that mutation of the PNPase coding gene (pnp) increases the bacterial resistance to ciprofloxacin. Gene expression analyses revealed that the expression of pyocin biosynthesis genes is decreased in the pnp mutant. PrtR, a negative regulator of pyocin biosynthesis genes, is upregulated in the pnp mutant. We further demonstrated that PNPase represses the expression of PrtR on the post-transcriptional level. A fragment containing 43 nucleotides of the 5′ untranslated region was found to be involved in the PNPase mediated regulation of PrtR. Overall, our results reveled a novel layer of regulation on the pyocin biosynthesis by the PNPase in P. aeruginosa.

The type III secretion system (T3SS) plays an important role in the pathogenesis of Pseudomonas aeruginosa. Expression of the T3SS is controlled under a complicate regulatory network. In this study, we demonstrate that NrtR (PA4916) is involved in the T3SS expression and pathogenesis of P. aeruginosa in a mouse acute pneumonia model. Overexpression of the T3SS central activator ExsA or exogenous supplementation of cAMP restored the expression of T3SS in the ΔnrtR mutant, suggesting that NrtR might regulate T3SS through the cAMP-Vfr signaling pathway. Further experiments demonstrated that the decrease of cAMP content is not due to the expression change of adenylate cyclases or phosphodiesterase in the ΔnrtR mutant. As it has been shown that nadD2 is upregulated in the ΔnrtR mutant, we overexpressed nadD2 in wild type PAK, which reduced the intracellular cAMP level and the expression of the T3SS genes. Meanwhile, deletion of nadD2 in the ΔnrtR mutant restored the expression and secretion of the T3SS. Co-immunoprecipitation assay revealed an interaction between NadD2 and the catalytic domain of the adenylate cyclase CyaB. Further in vitro assay indicated that NadD2 repressed the enzymatic activity of CyaB. Therefore, we have identified a novel regulatory mechanism of T3SS in P. aeruginosa.

A new model is presented for studying the effects of crack parameters on the dynamics of a cracked beam structure. The model is established by the finite element displacement method. In particular, the stiffness matrix of the cracked beam element is firstly derived by the displacement method, which does not need the flexibility matrix inversion calculation compared with the previous local flexibility approaches based on the force method. Starting with a finite element model of cracked beam element, the equation of strain energy of a cracked beam element is formed by the displacement method combined with the linear fracture mechanics. Then, based on the finite element method, the dynamic model of the cracked beam structure is obtained. The results show that the dynamic model discovers the internal relation between the dynamic characteristics of cracked beam structure and structural parameters, material parameters, and crack parameters. Finally, an example is presented to validate the proposed dynamic model.

Bacterial persisters are slow-growing or dormant cells that are highly tolerant to bactericidal antibiotics and contribute to recalcitrant and chronic infections. Toxin/antitoxin (TA) systems play important roles in controlling persister formation. Here, we examined the roles of seven predicted type II TA systems in the persister formation of a Pseudomonas aeruginosa wild-type strain PA14. Overexpression of a toxin gene PA14_51010 or deletion of the cognate antitoxin gene PA14_51020 increased the bacterial tolerance to antibiotics. Co-overexpression of PA14_51010 and PA14_51020 or simultaneous deletion of the two genes resulted in a wild-type level survival rate following antibiotic treatment. The two genes were located in the same operon that was repressed by PA14_51020. We further demonstrated the interaction between PA14_51010 and PA14_51020. Sequence analysis revealed that PA14_51010 contained a conserved RES domain. Overexpression of PA14_51010 reduced the intracellular level of nicotinamide adenine dinucleotide (NAD+). Mutation of the RES domain abolished the abilities of PA14_51010 in reducing NAD+ level and promoting persister formation. In addition, overproduction of NAD+ by mutation in an nrtR gene counteracted the effect of PA14_51010 overexpression in promoting persister formation. In combination, our results reveal a novel TA system that contributes to persister formation through reducing the intracellular NAD+ level in P. aeruginosa.

Polymyxin B and E (colistin) are the last resorts to treat multidrug-resistant Gram-negative pathogens. Pseudomonas aeruginosa is intrinsically resistant to a variety of antibiotics. The PhoP-PhoQ two-component regulatory system contributes to the resistance to polymyxins by regulating an arnBCADTEF-pmrE operon that encodes lipopolysaccharide modification enzymes. To identify additional PhoP-regulated genes that contribute to the tolerance to polymyxin B, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) assay and found novel PhoP binding sites on the chromosome. We further verified that PhoP directly controls the expression of PA14_46900, PA14_50740 and PA14_52340, and the operons of PA14_11970-PA14_11960 and PA14_52350-PA14_52370. Our results demonstrated that mutation of PA14_46900 increased the bacterial binding and susceptibility to polymyxin B. Meanwhile, mutation of PA14_11960 (papP), PA14_11970 (mpl), PA14_50740 (slyB), PA14_52350 (ppgS), and PA14_52370 (ppgH) reduced the bacterial survival rates and increased ethidium bromide influx under polymyxin B or Sodium dodecyl sulfate (SDS) treatment, indicating roles of these genes in maintaining membrane integrity in response to the stresses. By 1-N-phenylnaphthylamine (NPN) and propidium iodide (PI) staining assay, we found that papP and slyB are involved in maintaining outer membrane integrity, and mpl and ppgS-ppgH are involved in maintaining inner membrane integrity. Overall, our results reveal novel PhoP-PhoQ regulated genes that contribute to polymyxin B tolerance.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen, which causes infectious disease in patients with cystic fibrosis and compromised immunity. P. aeruginosa is difficult to eradicate because of its intrinsic resistance to most traditional antibiotics as well as acquired resistance mechanisms after decades of antibiotic usage. A full understanding of the P. aeruginosa pathogenesis mechanisms is necessary for the development of novel prevention and treatment strategies. To identify novel vaccine candidates, here we comprehensively examined the expression levels of all the known outer membrane proteins in two P. aeruginosa strains in a murine acute pneumonia model. OprH was one of the most highly expressed proteins during infection. In addition, OprH is known to be highly immunogenic and accessible by host proteins. Thus, it was chosen as a vaccine candidate. To further identify vaccine candidates, 34 genes highly expressed during infection were evaluated for their contributions in virulence by testing individual transposon insertion mutants. Among them, fpvA, hasR, and foxA were found essential for bacterial virulence and therefore included in vaccine construction. Immunization with a mixture of FpvA, HasR, and FoxA rendered no protection, however, while immunization by OprH refolded in liposomes elicited specific opsonic antibodies and conferred protection against two lipopolysaccharide-heterologous P. aeruginosa strains (PA14 and PA103). Overall, by studying the expression profile of the P. aeruginosa outer membrane proteins during infection, we identified OprH as a potential vaccine candidate for the prevention of lung infection by P. aeruginosa.

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogenic bacterium, causes acute and chronic infections. Upon entering the host, P. aeruginosa alters global gene expression to adapt to host environment and avoid clearance by the host immune system. Proline utilization A (PutA) is a bifunctional enzyme, which converts proline to glutamate. Here we report that PutA was required for the virulence of P. aeruginosa in a murine acute pneumonia model. A putA mutant was more susceptible to oxidative stress compared to the wild type strain. An AraC/XylS family protein, PruR, directly bound to the upstream of −35 box in the putA promoter and activated putA expression. High concentration of proline in bacteria up-regulated pruR expression, which led to the activation of putA expression. As a feedback regulation, glutamate produced by PutA released PruR from the putA promoter and turned off the putA expression. PruR affected bacterial virulence through the regulation of the putA expression. Altogether, these data are the first to reveal that PutA plays an important role in the pathogenesis of P. aeruginosa, as well as to describe the genetic regulation of PutA in P. aeruginosa.

Virus-mediated expression of defined transcription factor (TF) genes can effectively induce cellular reprogramming. However, sustained expression of the TFs often hinders pluripotent stem cell (PSC) differentiation into specific cell types, as each TF exerts its effect on PSCs for a defined period of time during differentiation. Here, we applied a bacterial type III secretion system (T3SS)-based protein delivery tool to directly translocate TFs in the form of protein into human PSCs. This transient protein delivery technique showed high delivery efficiency for hPSCs, and it avoids potential genetic alterations caused by the introduction of transgenes. In an established cardiomyocyte de novo differentiation procedure, five transcriptional factors, namely GATA4, MEF2C, TBX5, ESRRG and MESP1 (abbreviated as GMTEM), were translocated at various time points. By detecting the expression of cardiac marker genes (Nkx2.5 and cTnT), we found that GMTEM proteins delivered on mesoderm stage of the cardiomyocytes lineage differentiation significantly enhanced both the human ESC and iPSC differentiation into cardiomyocytes, while earlier or later delivery diminished the enhancing effect. Furthermore, all of the five factors were required to enhance the cardiac differentiation. This work provides a virus-free strategy of transient transcription factors delivery for directing human stem cell fate without jeopardizing genome integrity, thus safe for biomedical applications.

Many Pseudomonas aeruginosa virulence traits that contribute to human infections are accepted as being associated with its environmental lifestyle. Therefore, identifying the molecular mechanisms that govern the lifestyle choice is of high significance. We previously reported that a mutation in suhB results in a decrease in swimming motility and increased biofilm formation compared to the wild-type strain. Yet, little is known about how this occurs. In this study, we demonstrated that SuhB inversely regulates motility and biofilm formation through the GacA-RsmY/Z-RsmA cascade. Mutations in gacA or the two small RNAs rsmY/rsmZ, or overproduction of the RsmA protein essentially rescued the motility defect of the suhB mutant. Additionally, we identified a c-di-GMP mediated mechanism for SuhB regulation of motility and biofilm formation. We showed that the ΔsuhB mutant displayed elevated levels of c-di-GMP, and the ΔsuhB motility and biofilm phenotypes could be switched by artificially decreasing c-di-GMP levels. Further experiments led to the identification of the diguanylate cyclase GcbA responsible for regulating the c-di-GMP concentration in ΔsuhB and hence the switch between planktonic and surface-associated growth. Together, our results demonstrate a novel mechanism for SuhB regulation of the lifestyle transition via the Gac/Rsm and c-di-GMP signaling networks in P. aeruginosa.