ABSTRACT
The circular RNA (circRNA) family is a group of endogenous non-coding RNAs (ncRNAs) that have critical functions in multiple physiological and pathological processes, including inflammation, cancer, and cardiovascular diseases. However, their roles in regulating innate immune responses remain unclear. Here, we define Cell division cycle 42 (CDC42)-165aa, a protein encoded by circRNA circCDC42, which is overexpressed in Klebsiella pneumoniae (KP)-infected alveolar macrophages. High levels of CDC42-165aa induces the hyperactivation of Pyrin inflammasomes and aggravates alveolar macrophage pyroptosis, while the inhibition of CDC42-165aa reduces lung injury in mice after KP infection by inhibiting Pyrin inflammasome-mediated pyroptosis. Overall, these results demonstrate that CDC42-165aa stimulates Pyrin inflammasome by inhibiting CDC42 GTPase activation and provides a potential clinical target for pathogenic bacterial infection in clinical practice.
Subject(s)
Inflammasomes , Klebsiella Infections , Klebsiella pneumoniae , Mice, Inbred C57BL , Pyroptosis , cdc42 GTP-Binding Protein , Animals , Pyroptosis/genetics , Klebsiella Infections/immunology , Klebsiella Infections/microbiology , Klebsiella Infections/metabolism , Mice , Inflammasomes/metabolism , cdc42 GTP-Binding Protein/metabolism , cdc42 GTP-Binding Protein/genetics , Macrophages, Alveolar/metabolism , Macrophages, Alveolar/immunology , Macrophages, Alveolar/microbiology , Male , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Humans , Immunity, Innate , Macrophages/metabolism , Macrophages/immunology , Macrophages/microbiology , CARD Signaling Adaptor ProteinsABSTRACT
The continuous emergence of multidrug-resistant bacterial pathogens poses a major global healthcare challenge, with Klebsiella pneumoniae being a prominent threat. We conducted a comprehensive study on K. pneumoniae's antibiotic resistance mechanisms, focusing on outer membrane vesicles (OMVs) and polymyxin, a last-resort antibiotic. Our research demonstrates that OMVs protect bacteria from polymyxins. OMVs derived from Polymyxin B (PB)-stressed K. pneumoniae exhibited heightened protective efficacy due to increased vesiculation, compared to OMVs from unstressed Klebsiella. OMVs also shield bacteria from different bacterial families. This was validated ex vivo and in vivo using precision cut lung slices (PCLS) and Galleria mellonella. In all models, OMVs protected K. pneumoniae from PB and reduced the associated stress response on protein level. We observed significant changes in the lipid composition of OMVs upon PB treatment, affecting their binding capacity to PB. The altered binding capacity of single OMVs from PB stressed K. pneumoniae could be linked to a reduction in the lipid A amount of their released vesicles. Although the amount of lipid A per vesicle is reduced, the overall increase in the number of vesicles results in an increased protection because the sum of lipid A and therefore PB binding sites have increased. This unravels the mechanism of the altered PB protective efficacy of OMVs from PB stressed K. pneumoniae compared to control OMVs. The lipid A-dependent protective effect against PB was confirmed in vitro using artificial vesicles. Moreover, artificial vesicles successfully protected Klebsiella from PB ex vivo and in vivo. The findings indicate that OMVs act as protective shields for bacteria by binding to polymyxins, effectively serving as decoys and preventing antibiotic interaction with the cell surface. Our findings provide valuable insights into the mechanisms underlying antibiotic cross-protection and offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug-resistant bacterial infections.
Subject(s)
Anti-Bacterial Agents , Klebsiella pneumoniae , Polymyxin B , Klebsiella pneumoniae/metabolism , Klebsiella pneumoniae/drug effects , Anti-Bacterial Agents/pharmacology , Animals , Polymyxin B/pharmacology , Bacterial Outer Membrane/metabolism , Polymyxins/pharmacology , Extracellular Vesicles/metabolism , Klebsiella Infections/microbiology , Klebsiella Infections/metabolism , Microbial Sensitivity Tests , Drug Resistance, Multiple, Bacterial/drug effectsABSTRACT
Successful microbial colonization of the gastrointestinal (GI) tract hinges on an organism's ability to overcome the intense competition for nutrients in the gut between the host and the resident gut microbiome. Enteric pathogens can exploit ethanolamine (EA) in the gut to bypass nutrient competition. However, Klebsiella pneumoniae (K. pneumoniae) is an asymptomatic gut colonizer and, unlike well-studied enteric pathogens, harbors two genetically distinct ethanolamine utilization (eut) loci. Our investigation uncovered unique roles for each eut locus depending on EA utilization as a carbon or nitrogen source. Murine gut colonization studies demonstrated the necessity of both eut loci in the presence of intact gut microbiota for robust GI colonization by K. pneumoniae. Additionally, while some Escherichia coli gut isolates could metabolize EA, other commensals were incapable, suggesting that EA metabolism likely provides K. pneumoniae a selective advantage in gut colonization. Molecular and bioinformatic analyses unveiled the conservation of two eut loci among K. pneumoniae and a subset of the related taxa in the K. pneumoniae species complex, with the NtrC-RpoN regulatory cascade playing a pivotal role in regulation. These findings identify EA metabolism as a critical driver of K. pneumoniae niche establishment in the gut and propose microbial metabolism as a potential therapeutic avenue to combat K. pneumoniae infections.
Subject(s)
Ethanolamine , Gastrointestinal Microbiome , Klebsiella Infections , Klebsiella pneumoniae , Klebsiella pneumoniae/metabolism , Klebsiella pneumoniae/genetics , Mice , Animals , Ethanolamine/metabolism , Gastrointestinal Microbiome/physiology , Klebsiella Infections/microbiology , Klebsiella Infections/metabolism , Gastrointestinal Tract/microbiology , Gastrointestinal Tract/metabolism , Mice, Inbred C57BL , FemaleABSTRACT
BACKGROUND: Outer membrane vesicles (OMVs) derived from bacteria are known to play a crucial role in the interactions between bacteria and their environment, as well as bacteria-bacteria and bacteria-host interactions.Specifically, OMVs derived from Klebsiella pneumoniae have been implicated in contributing to the pathogenesis of this bacterium.Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a global pathogen of great concern due to its heightened virulence compared to classical K. pneumoniae (cKp), and its ability to cause community-acquired infections, even in healthy individuals.The objective of this study was to investigate potential differences between hvKp-derived OMVs and cKp-derived OMVs in their interactions with microorganisms and host cells. METHODS: Four strains of K. pneumoniae were used to produce OMVs: hvKp strain NTUH-K2044 (K1, ST23), hvKp clinical strain AP8555, and two cKP clinical strains C19 and C250. To examine the morphology and size of the bacterial OMVs, transmission electron microscopy (TEM) was utilized. Additionally, dynamic light scattering (DLS) was used to analyze the size characterization of the OMVs.The normal pulmonary bronchial cell line HBE was exposed to OMVs derived from hvKp and cKP. Interleukin 8 (IL-8) messenger RNA (mRNA) expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR), while IL-8 secretion was analyzed using enzyme-linked immunosorbent assay (ELISA).Furthermore, the activation of nuclear factor kappa B (NF-κB) was evaluated using both Western blotting and confocal microscopy. RESULTS: After purification, OMVs appeared as electron-dense particles with a uniform spherical morphology when observed through TEM.DLS analysis indicated that hvKp-derived OMVs from K2044 and AP8555 measured an average size of 116.87 ± 4.95 nm and 96.23 ± 2.16 nm, respectively, while cKP-derived OMVs from C19 and C250 measured an average size of 297.67 ± 26.3 nm and 325 ± 6.06 nm, respectively. The average diameter of hvKp-derived OMVs was smaller than that of cKP-derived OMVs.A total vesicular protein amount of 47.35 mg, 41.90 mg, 16.44 mg, and 12.65 mg was generated by hvKp-K2044, hvKp-AP8555, cKP-C19, and cKP-C250, respectively, obtained from 750 mL of culture supernatant. Both hvKp-derived OMVs and cKP-derived OMVs induced similar expression levels of IL-8 mRNA and protein. However, IL-8 expression was reduced when cells were exposed to BAY11-7028, an inhibitor of the NF-κB pathway.Western blotting and confocal microscopy revealed increased phosphorylation of p65 in cells exposed to OMVs. CONCLUSIONS: Klebsiella pneumoniae produces outer membrane vesicles (OMVs) that play a key role in microorganism-host interactions. HvKp, a hypervirulent strain of K. pneumoniae, generates more OMVs than cKP.The average size of OMVs derived from hvKp is smaller than that of cKP-derived OMVs.Despite these differences, both hvKp-derived and cKP-derived OMVs induce a similar level of expression of IL-8 mRNA and protein.OMVs secreted by K. pneumoniae stimulate the secretion of interleukin 8 by activating the nuclear factor NF-κB.
Subject(s)
Bacterial Outer Membrane , Host-Pathogen Interactions , Interleukin-8 , Klebsiella Infections , Klebsiella pneumoniae , NF-kappa B , Humans , Bronchi/cytology , Bronchi/microbiology , Cell Line , Interleukin-8/immunology , Interleukin-8/metabolism , Klebsiella Infections/immunology , Klebsiella Infections/metabolism , Klebsiella Infections/microbiology , Klebsiella pneumoniae/chemistry , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/cytology , Klebsiella pneumoniae/pathogenicity , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , PhosphorylationABSTRACT
Despite the importance of encapsulation in bacterial pathogenesis, the biochemical mechanisms and forces that underpin retention of capsule by encapsulated bacteria are poorly understood. In Gram-negative bacteria, there may be interactions between lipopolysaccharide (LPS) core and capsule polymers, between capsule polymers with retained acyl carriers and the outer membrane, and in some bacteria, between the capsule polymers and Wzi, an outer membrane protein lectin. Our transposon studies in Klebsiella pneumoniae B5055 identified additional genes that, when insertionally inactivated, resulted in reduced encapsulation. Inactivation of the gene waaL, which encodes the ligase responsible for attaching the repeated O antigen of LPS to the LPS core, resulted in a significant reduction in capsule retention, measured by atomic force microscopy. This reduction in encapsulation was associated with increased sensitivity to human serum and decreased virulence in a murine model of respiratory infection and, paradoxically, with increased biofilm formation. The capsule in the WaaL mutant was physically smaller than that of the Wzi mutant of K. pneumoniae B5055. These results suggest that interactions between surface carbohydrate polymers may enhance encapsulation, a key phenotype in bacterial virulence, and provide another target for the development of antimicrobials that may avoid resistance issues associated with growth inhibition. IMPORTANCE Bacterial capsules, typically comprised of complex sugars, enable pathogens to avoid key host responses to infection, including phagocytosis. These capsules are synthesized within the bacteria, exported through the outer envelope, and then secured to the external surface of the organism by a force or forces that are incompletely described. This study shows that in the important hospital pathogen Klebsiella pneumoniae, the polysaccharide capsule is retained by interactions with other surface sugars, especially the repeated sugar molecule of the LPS molecule in Gram-negative bacteria known as "O antigen." This O antigen is joined to the LPS molecule by ligation, and loss of the enzyme responsible for ligation, a protein called WaaL, results in reduced encapsulation. Since capsules are essential to the virulence of many pathogens, WaaL might provide a target for new antimicrobial development, critical to the control of pathogens like K. pneumoniae that have become highly drug resistant.
Subject(s)
Klebsiella Infections , Klebsiella pneumoniae , Animals , Bacterial Capsules/metabolism , Capsules/analysis , Capsules/metabolism , Humans , Klebsiella Infections/metabolism , Klebsiella Infections/microbiology , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/metabolism , Lipopolysaccharides/metabolism , Mice , O Antigens/analysis , O Antigens/metabolism , Polymers/analysis , Polymers/metabolism , Sugars/metabolismABSTRACT
Human surfactant protein (SP)-A1 and SP-A2 exhibit differential qualitative and quantitative effects on the alveolar macrophage (AM), including a differential impact on the AM miRNome. Moreover, SP-A rescue (treatment) of SP-A-knockout (KO) infected mice impoves survival. Here, we studied for the first time the role of exogenous SP-A protein treatment on the regulation of lung alveolar cell (LAC) miRNome, the miRNA-RNA targets, and gene expression of SP-A-KO infected mice of both sexes. Toward this, SP-A-KO mice of both sexes were infected with Klebsiella pneumoniae, and half of them were also treated with SP-A2 (1A0). After 6 h of infection/SP-A treatment, the expression levels and pathways of LAC miRNAs, genes, and target miRNA-mRNAs were studied in both groups. We found 1) significant differences in the LAC miRNome, genes, and miRNA-mRNA targets in terms of sex, infection, and infection plus SP-A2 (1A0) protein rescue; 2) an increase in the majority of miRNA-mRNA targets in both study groups in KO male vs. female mice and involvement of the miRNA-mRNA targets in pathways of inflammation, antiapoptosis, and cell cycle; 3) genes with significant changes to be involved in TP-53, tumor necrosis factor (TNF), and cell cycle signaling nodes; 4) when significant changes in the expression of molecules from all analyses (miRNAs, miRNA-mRNA targets, and genes) were considered, two signaling pathways, the TNF and cell cycle, referred to as "integrated pathways" were shown to be significant; 5) the cell cycle pathway to be present in all comparisons made. Because SP-A could be used therapeutically in pulmonary diseases, it is important to understand the molecules and pathways involved in response to an SP-A acute treatment. The information obtained contributes to this end and may help to gain insight especially in the case of infection.
Subject(s)
Alveolar Epithelial Cells , Klebsiella Infections , MicroRNAs , Pulmonary Surfactant-Associated Protein A , Alveolar Epithelial Cells/metabolism , Animals , Female , Humans , Klebsiella Infections/genetics , Klebsiella Infections/metabolism , Klebsiella pneumoniae , Lung/metabolism , Male , Mice , Mice, Knockout , MicroRNAs/genetics , MicroRNAs/metabolism , Pulmonary Surfactant-Associated Protein A/biosynthesis , Pulmonary Surfactant-Associated Protein A/genetics , Pulmonary Surfactant-Associated Protein A/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , TranscriptomeABSTRACT
Neutrophil extracellular traps (NETs) are implicated in host defense and inflammatory pathologies alike. A wide range of pathogen- and host-derived factors are known to induce NETs, yet the knowledge about specific receptor-ligand interactions in this response is limited. We previously reported that macrophage-inducible C-type lectin (Mincle) regulates NET formation. In this article, we identify glycosphingolipid ß-glucosylceramide (ß-GlcCer) as a specific NET-inducing ligand of Mincle. We found that purified ß-GlcCer induced NETs in mouse primary neutrophils in vitro and in vivo, and this effect was abrogated in Mincle deficiency. Cell-free ß-GlcCer accumulated in the lungs of pneumonic mice, which correlated with pulmonary NET formation in wild-type, but not in Mincle-/-, mice infected intranasally with Klebsiella pneumoniae Although leukocyte infiltration by ß-GlcCer administration in vivo did not require Mincle, NETs induced by this sphingolipid were important for bacterial clearance during Klebsiella infection. Mechanistically, ß-GlcCer did not activate reactive oxygen species formation in neutrophils but required autophagy and glycolysis for NET formation, because ATG4 inhibitor NSC185058, as well as glycolysis inhibitor 2-deoxy-d-glucose, abrogated ß-GlcCer-induced NETs. Forced autophagy activation by tamoxifen could overcome the inhibitory effect of glycolysis blockage on ß-GlcCer-mediated NET formation, suggesting that autophagy activation is sufficient to induce NETs in response to this metabolite in the absence of glycolysis. Finally, ß-GlcCer accumulated in the plasma of patients with systemic inflammatory response syndrome, and its levels correlated with the extent of systemic NET formation in these patients. Overall, our results posit ß-GlcCer as a potent NET-inducing ligand of Mincle with diagnostic and therapeutic potential in inflammatory disease settings.
Subject(s)
Extracellular Traps , Klebsiella Infections , Animals , Extracellular Traps/metabolism , Glucosylceramides , Glycolipids , Inflammation/metabolism , Klebsiella Infections/metabolism , Ligands , Mice , Neutrophils/metabolismABSTRACT
Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections. Therefore, strategies for preventing and controlling the infections are urgently needed. Phage depolymerase, often found in the tail fiber protein or the tail spike protein, is reported to have antibiofilm activity. In this study, phage P560 isolated from sewage showed specific for capsule locus type KL47 K. pneumoniae, and the enlarged haloes around plaques indicated that P560 encoded a depolymerase. The capsule depolymerase, ORF43, named P560dep, derived from phage P560 was expressed, purified, characterized and evaluated for enzymatic activity as well as specificity. We reported that the capsule depolymerase P560dep, can digest the capsule polysaccharides on the surface of KL47 type K. pneumoniae, and the depolymerization spectrum of P560dep matched to the host range of phage P560, KL47 K. pneumoniae. Crystal violet staining assay showed that P560dep was able to significantly inhibit biofilm formation. Further, a single dose (50 µg/mouse) of depolymerase intraperitoneal injection protected 90%-100% of mice from lethal challenge before or after infection by KL47 carbapenem-resistant K. pneumoniae. And pathological changes were alleviated in lung and liver of mice infected by KL47 type K. pneumoniae. It is demonstrated that depolymerase P560dep as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.
Subject(s)
Bacteriophages , Klebsiella Infections , Animals , Anti-Bacterial Agents/pharmacology , Bacteriophages/genetics , Klebsiella Infections/drug therapy , Klebsiella Infections/metabolism , Klebsiella pneumoniae , MiceABSTRACT
AIM: Carbapenem-resistant Klebsiella pneumoniae- (CR-Kp-) mediated infections represent a challenge for clinical practitioners due to their expanding prevalence in hospital environments and antibiotic resistance. However, few studies have shown metabolic changes of carbapenem-resistant Klebsiella pneumoniae and CR-Kp-negative patients, and relevant studies are urgently needed. METHODS: In this study, we comprehensively profile the metabolites of 20 CR-Kp-positive and 18 CR-Kp-negative patients in plasma by using 2D gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS). RESULTS: We identified 58 metabolites that were carbapenem-resistant Klebsiella pneumoniae-associated. N-Acetyl glucosamine, butanedioic acid, and myoinositol play a significant character in CR-Kp infection. CONCLUSIONS: Our study provides valuable data to serve as potential targets for developing therapies against CR-Kp infection.
Subject(s)
Biomarkers/blood , Carbapenems/pharmacology , Drug Resistance, Bacterial , Klebsiella Infections/metabolism , Klebsiella pneumoniae/metabolism , Metabolome , Adult , Aged , Case-Control Studies , China/epidemiology , Female , Follow-Up Studies , Humans , Klebsiella Infections/epidemiology , Klebsiella Infections/microbiology , Klebsiella pneumoniae/isolation & purification , Male , Middle Aged , PrognosisABSTRACT
Chlorogenic acid (CGA) possesses a wide variety of bioactive properties, such as antioxidation, anti-inflammation and anti-bacteria. This study was aimed at exploring the effects of CGA of anti-inflammation and anti-bacteria on mouse pneumonia prepared by immunosuppressed mice infected with Klebsiella pneumoniae (K. pneumoniae) in vivo and the cellular inflammasomes through lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced RAW 264.7 murine macrophages in vitro. Mice received CGA treatment (30 and 90 mg kg-1) for 8 consecutive days and on the fourth day immunosuppression in mice was induced by cyclophosphamide (40 mg kg-1) for 5 days before inoculation of K. pneumoniae. Immunosuppressed mice infected with K. pneumoniae developed severe pneumonia, with marked interstitial vascular congestion, widened alveolar intervals, infiltration of monocytes, lymphocytes and macrophages as well as the damage of epithelial architecture, with growing mortality and count forming unit (CFU). CGA treatment significantly decreased the ratio of lung/body weight, reduced the severity of pneumonia induced by K. pneumoniae, decreased the lung injury, inflammatory cell infiltration scores and CD68 protein expression, inhibited the expression of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, and elevated the expression of IL-10. Meanwhile, we investigated the mechanism of CGA to counter K. pneumoniae-induced pneumonia and found that CGA remarkably repressed the activation of nucleotide-binding domain like receptor protein 3 (NLRP3) inflammasome. Altogether, our results indicate that the dietary intake of CGA or its rich foods ameliorates K. pneumonia-induced pneumonia by inhibiting the activation of NLRP3 inflammasomes.
Subject(s)
Chlorogenic Acid/therapeutic use , Immune Tolerance , Inflammasomes/metabolism , Klebsiella Infections/drug therapy , Klebsiella pneumoniae , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pneumonia, Bacterial/drug therapy , Animals , Chlorogenic Acid/pharmacology , Cytokines/genetics , Cytokines/metabolism , Klebsiella Infections/immunology , Klebsiella Infections/metabolism , Klebsiella Infections/pathology , Macrophages/immunology , Mice , Mice, Inbred BALB C , Pneumonia, Bacterial/immunology , Pneumonia, Bacterial/metabolism , Pneumonia, Bacterial/pathology , RAW 264.7 Cells , Signal Transduction/drug effectsABSTRACT
Despite their protective antimicrobial function, neutrophil extracellular traps (NETs) have been implicated in propagation of inflammatory responses in several disease conditions including sepsis. Highly diffusible exogenous ROS produced under such inflammatory conditions, can induce exuberant NETs, thus making inhibition of NETs desirable in inflammatory diseases. Here we report that helminth parasite excretory/secretory factors termed as parasitic ligands (PL) inhibit ROS-induced NETs by blocking the activation of nonselective calcium permeable channel Transient Receptor Potential Melastatin 2 (TRPM2). Therapeutic implication of PL mediated blockage of NET formation was tested in preclinical model of septic peritonitis, where PL treatment regulated neutrophil cell death modalities including NET formation and mitigated neutrophil mediated inflammatory response. This translated into improved survival and reduced systemic and local bacterial load in infected mice. Overall, our results posit PL as an important biological regulator of neutrophil functions with implications to a variety of inflammatory diseases including peritonitis.
Subject(s)
Extracellular Traps/metabolism , Klebsiella Infections/immunology , Klebsiella Infections/therapy , Mesocestoides , Peritonitis/immunology , Peritonitis/therapy , Animals , Female , Klebsiella Infections/metabolism , Klebsiella pneumoniae , Ligands , Male , Mice , Mice, Inbred C57BL , Neutrophils/immunology , Peritonitis/metabolism , Peritonitis/microbiology , Reactive Oxygen Species/metabolism , Sepsis , TRPM Cation Channels/metabolismABSTRACT
Klebsiella pneumoniae is a common pathogen in human sepsis. The emergence of multidrug-resistant K. pneumoniae strains represents a major clinical challenge in nosocomial and community acquired infections. The long pentraxin PTX3, a key component of humoral innate immunity, is involved in resistance to selected pathogens by promoting opsonophagocytosis. We investigated the relevance of PTX3 in innate immunity against K. pneumoniae infections using Ptx3-/- mice and mouse models of severe K. pneumoniae infections. Local and systemic PTX3 expression was induced following K. pneumoniae pulmonary infection, in association with the up-regulation of TNF-α and IL-1ß. PTX3 deficiency in mice was associated with higher bacterial burden and mortality, release of pro-inflammatory cytokines as well as IL-10 in the lung and systemically. The analysis of the mechanisms responsible of PTX3-dependent control of K. pneumoniae infection revealed that PTX3 did not interact with K. pneumoniae, or promote opsonophagocytosis. The comparison of susceptibility of wild-type, Ptx3-/-, C3-/- and Ptx3-/- /C3-/- mice to the infection showed that PTX3 acted in a complement-independent manner. Lung histopathological analysis showed more severe lesions in Ptx3-/- mice with fibrinosuppurative, necrotizing and haemorrhagic bronchopneumonia, associated with increased fibrin deposition in the lung and circulating fibrinogen consumption. These findings indicate that PTX3 contributes to the control of K. pneumoniae infection by modulating inflammatory responses and tissue damage. Thus, this study emphasizes the relevance of the role of PTX3 as regulator of inflammation and orchestrator of tissue repair in innate responses to infections.
Subject(s)
C-Reactive Protein/immunology , Klebsiella Infections/immunology , Klebsiella pneumoniae/pathogenicity , Serum Amyloid P-Component/immunology , Animals , Bacterial Load/immunology , C-Reactive Protein/deficiency , C-Reactive Protein/metabolism , Cytokines/metabolism , Fibrin/metabolism , Fibrinogen/metabolism , Immunity, Innate , Inflammation , Klebsiella Infections/metabolism , Klebsiella Infections/microbiology , Klebsiella Infections/pathology , Klebsiella pneumoniae/immunology , Lung/immunology , Lung/metabolism , Lung/microbiology , Lung/pathology , Mice , Sepsis/immunology , Sepsis/metabolism , Sepsis/microbiology , Sepsis/pathology , Serum Amyloid P-Component/deficiency , Serum Amyloid P-Component/metabolism , Stromal Cells/metabolismABSTRACT
Gram-negative pathogens, such as Klebsiella pneumoniae, remodel their outer membrane (OM) in response to stress to maintain its integrity as an effective barrier and thus to promote their survival in the host. The emergence of carbapenem-resistant K. pneumoniae (CR-Kp) strains that are resistant to virtually all antibiotics is an increasing clinical problem and OM impermeability has limited development of antimicrobial agents because higher molecular weight antibiotics cannot access sites of activity. Here, we demonstrate that TAM (translocation and assembly module) deletion increases CR-Kp OM permeability under stress conditions and enhances sensitivity to high-molecular weight antimicrobials. SILAC-based proteomic analyses revealed mis-localization of membrane proteins in the TAM deficient strain. Stress-induced sensitization enhances clearance of TAM-deficient CR-Kp from the gut lumen following fecal microbiota transplantation and from infection sites following pulmonary or systemic infection. Our study suggests that TAM, as a regulator of OM permeability, represents a potential target for development of agents that enhance the effectiveness of existing antibiotics.
Subject(s)
Adaptation, Physiological , Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/metabolism , Carbapenem-Resistant Enterobacteriaceae/drug effects , Klebsiella Infections/drug therapy , Klebsiella pneumoniae/drug effects , Proteome/metabolism , Animals , Bacterial Outer Membrane Proteins/genetics , Carbapenems/pharmacology , Cell Membrane Permeability , Female , Klebsiella Infections/genetics , Klebsiella Infections/metabolism , Klebsiella Infections/microbiology , Mice , Mice, Inbred C57BL , Stress, PhysiologicalABSTRACT
Evolution of multi-drug resistant bacteria has led to worldwide research to better understand the various resistance mechanisms in these strains. Every year, novel information on carbapenem resistance and its mechanisms is being discovered. In this study, radiation-mediated mutagenesis was used to transform a carbapenem-resistant Klebsiella pneumoniae strain to a carbapenem-susceptible bacterium. Through this process, we proved three conditions of loss of the OmpK35 and the OmpK36 genes and acquisition of blaCMY-10 worked together to produce carbapenem resistance in K. pneumoniae. Loss of only one of the porins did not evoke carbapenem resistance. This is the first report on the essential contribution of these three components of carbapenem resistance in K. pneumoniae.
Subject(s)
Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , Drug Resistance, Bacterial , Klebsiella Infections/metabolism , Klebsiella Infections/microbiology , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/radiation effects , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Humans , Klebsiella pneumoniae/drug effects , Mutagenesis/radiation effects , Porins/genetics , Porins/metabolism , Radiation , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
Background: Multi-drug resistant organisms, especially Vancomycin-Resistant Enterococcus (VRE) and Carbapenam Resistant Klebsiella pneumoniae (KPC), are serious health threat. Early detection of resistant bacteria colonization among patients in intensive care units (ICUs) not only enables effective treatment but more importantly prevents disease and limits transmission. Therefore, we aimed to to assess the frequency of VRE and KPC colonization via rectal swab sampling. Methods: The study was carried out in ICUs of a tertiary hospital. Two rectal swab samples were collected within the first 24 hours of admission and another one was taken every subsequent 15 days to test for for VRE and KPC carriage. Results: A total 316 rectal swab samples taken from 230 patients. Forty-seven patients were screened at least 2 times. 183 patients were not further screened due to discharge, exitus or transfer to other wards. Thirty-six patients (16%) were determined to be VRE (+). The most frequently isolated strain was E. faecium (80.5%) and its most common genotype was VanA (87.5%). Seven patients (3%) were identified as KPC (+). OXA-48 type crbapenamase was confirmed in all KPC isolates. Conclusion: This study shows that VRE and KPC colonization continues to be a serious threat in ICUs.
Subject(s)
Carbapenem-Resistant Enterobacteriaceae , Klebsiella Infections , Vancomycin Resistance , Vancomycin-Resistant Enterococci , Carbapenem-Resistant Enterobacteriaceae/genetics , Carbapenem-Resistant Enterobacteriaceae/isolation & purification , Carbapenem-Resistant Enterobacteriaceae/metabolism , Carbapenems/metabolism , Carbapenems/pharmacology , Carbapenems/therapeutic use , Carrier State/diagnosis , Carrier State/microbiology , Hospitalization , Humans , Intensive Care Units , Klebsiella/genetics , Klebsiella/isolation & purification , Klebsiella/metabolism , Klebsiella Infections/diagnosis , Klebsiella Infections/metabolism , Klebsiella Infections/microbiology , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/metabolism , Rectum/microbiology , Vancomycin Resistance/genetics , Vancomycin-Resistant Enterococci/genetics , Vancomycin-Resistant Enterococci/isolation & purification , beta-Lactam Resistance/geneticsABSTRACT
Bacterial pneumonia is a significant cause of morbidity, mortality, and health care expenditures. Optimum neutrophil recruitment and their function are critical defense mechanisms against respiratory pathogens. The nucleotide-binding oligomerization domain-like receptor (NLRP) 6 controls gut microbiota and immune response to systemic and enteric infections. However, the importance of NLRP6 in neutrophil homeostasis following lung infection remains elusive. To investigate the role of NLRs in neutrophil homeostasis, we used Nlrp6 gene-deficient (Nlrp6-/-) mice in a model of Klebsiella pneumoniae-induced pneumonia-derived sepsis. We demonstrated that NLRP6 is critical for host survival, bacterial clearance, neutrophil influx, and CXC-chemokine production. Kp-infected Nlrp6-/- mice have reduced numbers of hematopoietic stem cells and granulocyte-monocyte progenitors but increased retention of matured neutrophils in bone marrow. Neutrophil extracellular trap (NET) formation and NET-mediated bacterial killing were also impaired in Nlrp6-/- neutrophils in vitro. Furthermore, recombinant CXCL1 rescued the impaired host defense, granulopoietic response, and NETosis in Kp-infected Nlrp6-/- mice. Using A/J background mice and co-housing experiments, our findings revealed that the susceptible phenotype of Nlrp6-/- mice is not strain-specific and gut microbiota-dependent. Taken together, these data unveil NLRP6 as a central regulator of neutrophil recruitment, generation, and function during bacterial pneumonia followed by sepsis.
Subject(s)
Klebsiella Infections/metabolism , Klebsiella pneumoniae/physiology , Macrophages/immunology , Neutrophils/immunology , Pneumonia, Bacterial/metabolism , Receptors, Cell Surface/metabolism , Sepsis/metabolism , Animals , Chemokine CXCL1/genetics , Chemokine CXCL1/metabolism , Extracellular Traps/metabolism , Homeostasis , Humans , Klebsiella Infections/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophil Activation , Pneumonia, Bacterial/immunology , Receptors, Cell Surface/genetics , Sepsis/immunology , THP-1 CellsABSTRACT
In the current scenario, frontline antibiotics are losing effectiveness against multidrug-resistant (MDR) bacteria because of the single mode of action. The accumulation of mutations and spread of antibiotic resistance markers among the bacteria results into the severe threat to community health. Now, there is an urgent requirement for the development of an alternate and as well as multiple-targeted action of drugs to stop the spread of resistance in bacteria. Here, we showed an alternative nanoparticle based photodynamic therapy (PDT) targeting the bacterial efflux pumps and its cell wall. The dextran capped gold nanoparticles (GNPDEX) were localized to the bacterial surface by nanoparticle attached Concanavalin-A (ConA), where GNPDEX attached methylene blue (MB) photosensitizer as an MB@GNPDEX-ConA formulation induced the killing of MDR Klebsiella pneumoniae clinical isolates in no time. The intervention of efflux pump inhibitor (EPI) further improved the MB@GNPDEX-ConA treatment modality and displayed the maximum bactericidal cytoplasmic phototoxicity. The CCCP EPI (carbonyl cyanide m-chlorophenylhydrazone) with the PDT increased the bacterial killing by>3 log10 as compared with or without EPI intervention. Further, the fractionated (two light treatment after long dark phase) PDT treatment modality decreased the bacterial biofilm growth up to ~90%. The microscopic as well as ROS fluorescent probes showed the singlet oxygen mediated cytotoxicity. The mode of interactions and genomic DNA photo-toxicity confirmed that EPI enhanced the killing mediated by singlet oxygen generation. The multi-targeted (Cell wall, DNA and efflux pump) modality of MB@GNPDEX-ConA in presence of EPI is an effective and alternative therapeutic approach against most potent Klebsiella MDR infections.
Subject(s)
Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Drug Resistance, Multiple, Bacterial , Gold/chemistry , Klebsiella Infections/drug therapy , Klebsiella pneumoniae/drug effects , Membrane Transport Modulators/pharmacology , Metal Nanoparticles , Methylene Blue/pharmacology , Photochemotherapy , Photosensitizing Agents/pharmacology , Biofilms/drug effects , Biofilms/growth & development , Carbonyl Cyanide m-Chlorophenyl Hydrazone/chemistry , Concanavalin A/chemistry , Drug Compounding , Klebsiella Infections/metabolism , Klebsiella Infections/microbiology , Klebsiella pneumoniae/growth & development , Klebsiella pneumoniae/metabolism , Membrane Transport Modulators/chemistry , Methylene Blue/chemistry , Microbial Viability/drug effects , Nanomedicine , Photosensitizing Agents/chemistryABSTRACT
It has been reported that high mobility group nucleosomal binding domain 2 (HMGN2) is a nucleus-related protein that regulates gene transcription and plays a critical role in bacterial clearance. An elevated level of HMGN2 reduced integrin α5/ß1 expression of human pulmonary epithelial A549 cells was demonstrated during Klebsiella pneumoniae infection, thus weakening bacterial adhesion and invasion. However, the mechanism by which HMGN2 regulates integrin expression remains unclear. This study found that a transcription factor-nuclear factor I (NFI), which serves as the potential target of HMGN2 regulated integrin expression. The results showed that HMGN2 was able to promote NFIA and NFIB expression by increasing H3K27 acetylation of NFIA/B promoter regions. The integrin α5/ß1 expression was significantly enhanced by knockdown of NFIA/B via a siRNA approach. Meanwhile, NFIA/B silence could also compromise the inhibition effect of HMGN2 on the integrin α5/ß1 expression. Mechanistically, it was demonstrated that HMGN2 facilitated the recruitment of NFI on the promoter regions of integrin α5/ß1 according to the chromatin immunoprecipitation assay. In addition, it was further demonstrated that the knockdown of NFIA/B induced more adhesion of Klebsiella pneumoniae on pulmonary epithelial A549 cells, which could be reversed by the application of an integrin inhibitor RGD. The results revealed a regulatory role of HMGN2 on the transcription level of integrin α5/ß1, indicating a potential treatment strategy against Klebsiella pneumoniae-induced infectious lung diseases.
Subject(s)
Bacterial Adhesion/physiology , Epithelial Cells/microbiology , HMGN2 Protein/metabolism , Integrin alpha5beta1/metabolism , Klebsiella pneumoniae/metabolism , NFI Transcription Factors/metabolism , A549 Cells , Gene Expression Regulation , HMGN2 Protein/genetics , Humans , Integrin alpha5/genetics , Integrin alpha5/metabolism , Integrin alpha5beta1/genetics , Integrin beta1/genetics , Integrin beta1/metabolism , Klebsiella Infections/metabolism , Klebsiella pneumoniae/genetics , Lung , RNA, Small Interfering/metabolism , TranscriptomeABSTRACT
Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis in human and is associated with high morbidity and mortality. The microbiota promotes and maintains host immune homeostasis during bacterial infections. However, the mechanisms by which the gut microbiota affects immune responses in the lung still remain poorly understood. Here, we performed cecal metabolomics sequencing and fecal 16s rRNA sequencing in K. pneumoniae-infected mice and uninfected controls and showed that K. pneumoniae infection led to profound alterations in the gut microbiome and thus the cecal metabolome. We observed that the levels of Lactobacillus reuteri and Bifidobacterium pseudolongum were significantly decreased in K. pneumoniae-infected mice. Spearman correlation analysis showed that alterations in the richness and composition of the gut microbiota were associated with profound changes in host metabolite concentrations. Further, short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, were detected in cecal contents and serum by gas chromatography-mass spectrometry (GC-MS). We observed that the concentrations of these three SCFAs were all lower in the infected groups than in the untreated controls. Lastly, oral supplementation with these three SCFAs reduced susceptibility to K. pneumoniae infections, as indicated by lower bacterial burdens in the lung and higher survival rates. Our data highlight the protective roles of gut microbiota and certain metabolites in K. pneumoniae-pneumonia and suggests that it is possible to intervene in this bacterial pneumonia by targeting the gut microbiota.
Subject(s)
Cecum/metabolism , Gastrointestinal Microbiome/physiology , Klebsiella Infections , Pneumonia, Bacterial , Sepsis , Animals , Fatty Acids, Volatile/metabolism , Feces/microbiology , Klebsiella Infections/complications , Klebsiella Infections/metabolism , Klebsiella Infections/microbiology , Klebsiella pneumoniae , Metabolome , Mice , Mice, Inbred C57BL , Pneumonia, Bacterial/complications , Pneumonia, Bacterial/metabolism , Pneumonia, Bacterial/microbiology , Sepsis/metabolism , Sepsis/microbiologyABSTRACT
The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem. K. pneumoniae possesses numerous resistant genes in its genome. We isolated mutants resistant to various antimicrobials in vitro and investigated the importance of intrinsic genes in acquired resistance. The isolation frequency of the mutants was 10-7-10-9. Of the multidrug-resistant mutants, hyper-multidrug-resistant mutants (EB256-1, EB256-2, Nov1-8, Nov2-2, and OX128) were identified, and accelerated efflux activity of ethidium from the inside to the outside of the cells was observed in these mutants. Therefore, we hypothesized that the multidrug efflux pump, especially RND-type efflux pump, would be related to changes of the phenotype. We cloned all RND-type multidrug efflux pumps from the K. pneumoniae genome and characterized them. KexEF and KexC were powerful multidrug efflux pumps, in addition to AcrAB, KexD, OqxAB, and EefABC, which were reported previously. It was revealed that the expression of eefA was increased in EB256-1 and EB256-2: the expression of oqxA was increased in OX128; the expression of kexF was increased in Nov2-2. It was found that a region of 1,485 bp upstream of kexF, was deleted in the genome of Nov2-2. K. pneumoniae possesses more potent RND-multidrug efflux systems than E. coli. However, we revealed that most of them did not contribute to the drug resistance of our strain at basic levels of expression. On the other hand, it was also noted that the overexpression of these pumps could lead to multidrug resistance based on exposure to antimicrobial chemicals. We conclude that these pumps may have a role to maintain the intrinsic resistance of K. pneumoniae when they are overexpressed. The antimicrobial chemicals selected many resistant mutants at the same minimum inhibitory concentration (MIC) or a concentration slightly higher than the MIC. These results support the importance of using antibiotics at appropriate concentrations at clinical sites.