In vivo single cell transcriptomics reveals Klebsiella pneumoniae rewiring of lung macrophages to promote infection
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ABSTRACT:
ORGANISM(S): Klebsiella pneumoniae
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PROVIDER: S-BSST892 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:The strategies deployed by antibiotic-resistant bacteria to counteract host defences are poorly understood. Here, we elucidate a novel host-pathogen interaction resulting in skewing lung macrophage polarisation by the human pathogen Klebsiella pneumoniae. We identify interstitial macrophages (IMs) as the main population of lung macrophages associated with Klebsiella. Single-cell transcriptomics and trajectory analysis of cells reveal type I IFN and IL10 signalling, and macrophage polarisation are characteristic of infected IMs, whereas Toll-like receptor (TLR) and Nod-like receptor signalling are features of infected alveolar macrophages. Klebsiella-induced macrophage polarisation is a singular M2-type we termed M(Kp). To rewire macrophages, Klebsiella hijacks a TLR-type I IFN-IL10-STAT6 axis. Absence of STAT6 limits Klebsiella intracellular survival and facilitates the clearance of the pathogen in vivo. Glycolysis characterises M(Kp) metabolism, and inhibition of glycolysis results in clearance of intracellular Klebsiella. Capsule polysaccharide governs M(Kp). Klebsiella also skews human macrophage polarisation towards M(Kp) in a type I IFN-IL10-STAT6-dependent manner. Klebsiella induction of M(Kp) represents a novel strategy to overcome host restriction, and identifies STAT6 as target to boost defences against Klebsiella.
| S-EPMC9727930 | biostudies-literature
Project description:The strategies deployed by antibiotic resistant bacteria to counteract host defences are poorly understood. Here, we elucidate a novel host-pathogen interaction resulting in skewing lung macrophage polarisation by the human pathogen Klebsiella pneumoniae. We identify interstitial macrophages (IMs) as the main population of lung macrophages associated with Klebsiella. Single cell transcriptomics and trajectory analysis of cells reveal type I IFN and IL10 signalling, and macrophage polarization are characteristic of infected IMs, whereas Toll-like receptor (TLR) and Nod-like receptor signalling are features of infected alveolar macrophages. Klebsiella-induced macrophage polarization is a singular M2-type we termed M(Kp). To rewire macrophages, Klebsiella hijacks a TLR-type I IFN-IL10-STAT6 axis. Absence of STAT6 limits Klebsiella intracellular survival and facilitates the clearance of the pathogen in vivo. Glycolysis characterises M(Kp) metabolism, and inhibition of glycolysis results in clearance of intracellular Klebsiella. Capsule polysaccharide governs M(Kp). Klebsiella also skews human macrophage polarization towards M(Kp) in a type I IFN-IL10-STAT6-dependent manner. Klebsiella induction of M(Kp) represents a novel strategy to overcome host restriction, and identifies STAT6 as target to boost defences against Klebsiella.
| S-SCDT-10_15252-EMMM_202216888 | biostudies-other
Project description:Multidrug-resistant Gram-negative bacteria are a rapidly growing public health threat, and the development of novel antimicrobials has failed to keep pace with their emergence. Synergistic combinations of individually ineffective drugs present a potential solution, yet little is understood about the mechanisms of most such combinations. Here, we show that the combination of colistin (polymyxin E) and minocycline has a high rate of synergy against colistin-resistant and minocycline-intermediate or -resistant strains of Klebsiella pneumoniae. Furthermore, using transcriptome sequencing (RNA-Seq), we characterized the transcriptional profiles of these strains when treated with the drugs individually and in combination. We found a striking similarity between the transcriptional profiles of bacteria treated with the combination of colistin and minocycline at individually subinhibitory concentrations and those of the same isolates treated with minocycline alone. We observed a similar pattern with the combination of polymyxin B nonapeptide (a polymyxin B analogue that lacks intrinsic antimicrobial activity) and minocycline. We also found that genes involved in polymyxin resistance and peptidoglycan biosynthesis showed significant differential gene expression in the different treatment conditions, suggesting possible mechanisms for the antibacterial activity observed in the combination. These findings suggest that the synergistic activity of this combination against bacteria resistant to each drug alone involves sublethal outer membrane disruption by colistin, which permits increased intracellular accumulation of minocycline.
| S-EPMC8923212 | biostudies-literature
Project description:A novel in vivo expression technology (IVET) was performed to identify Klebsiella pneumoniae CG43 genes that are specifically expressed during infection of BALB/c mice. The IVET employed a UDP glucose pyrophosphorylase (galU)-deficient mutant of K. pneumoniae which is incapable of utilizing galactose and synthesizing capsular polysaccharide, as demonstrated by its low virulence to BALB/c mice and a white nonmucoid colony morphology on MacConkey-galactose agar. By using a functional galU gene as the reporter, an IVE promoter could render the galU mutant virulent while maintaining the white nonmucoid colony phenotype. A total of 20 distinct sequences were obtained through the in vivo selection. Five of them have been identified previously as virulence-associated genes in other pathogens, while another five with characterized functions are involved in regulation and transportation of nutrient uptake, biosynthesis of isoprenoids, and protein folding. No known functions have been attributed to the other 10 sequences. We have also demonstrated that 2 of the 20 IVE genes turn on under iron deprivation, whereas the expression of another five genes was found to be activated in the presence of paraquat, a superoxide generator.
| S-EPMC100105 | biostudies-literature
Project description:Mice were infected with Klebsiella pneumoniae and interstitial and alveolar macrophages isolated and subjected to single cell RNA sequencing to investigate the transcriptomes of Klebseilla-associated and uninfected bystander cells.
2022-09-16 | GSE184290 | GEO
Project description:Antofloxacin is a novel broad-spectrum fluoroquinolone under development for the treatment of infections caused by a diverse group of bacterial species. We explored the pharmacodynamic (PD) profile and targets of antofloxacin against seven Klebsiella pneumoniae isolates by using a neutropenic murine lung infection model. Plasma and bronchopulmonary pharmacokinetic (PK) studies were conducted at single subcutaneous doses of 2.5, 10, 40, and 160 mg/kg of body weight. Mice were infected intratracheally with K. pneumoniae and treated using 2-fold-increasing total doses of antofloxacin ranging from 2.5 to 160 mg/kg/24 h administered in 1, 2, 3, or 4 doses. The Emax Hill equation was used to model the dose-response data. Antofloxacin could penetrate the lung epithelial lining fluid (ELF) with pharmacokinetics similar to those in plasma with linear elimination half-lives over the dose range. All study strains showed a 3-log10 or greater reduction in bacterial burden and prolonged postantibiotic effects (PAEs) ranging from 3.2 to 5.3 h. Dose fractionation response curves were steep, and the free-drug area under the concentration-time curve over 24 h (AUC0-24)/MIC ratio was the PD index most closely linked to efficacy (R2 = 0.96). The mean free-drug AUC0-24/MIC ratios required to achieve net bacterial stasis, a 1-log10 kill, and a 2-log10 kill for each isolate were 52.6, 89.9, and 164.9, respectively. When integrated with human PK data, these PD targets could provide a framework for further optimization of dosing regimens. This could make antofloxacin an attractive option for the treatment of respiratory tract infections involving K. pneumoniae.
| S-EPMC5404557 | biostudies-literature
Project description:UnlabelledKlebsiella pneumoniae is an urgent public health threat because of resistance to carbapenems, antibiotics of last resort against Gram-negative bacterial infections. Despite the fact that K. pneumoniae is a leading cause of pneumonia in hospitalized patients, the bacterial factors required to cause disease are poorly understood. Insertion site sequencing combines transposon mutagenesis with high-throughput sequencing to simultaneously screen thousands of insertion mutants for fitness defects during infection. Using the recently sequenced K. pneumoniae strain KPPR1 in a well-established mouse model of pneumonia, insertion site sequencing was performed on a pool of >25,000 transposon mutants. The relative fitness requirement of each gene was ranked based on the ratio of lung to inoculum read counts and concordance between insertions in the same gene. This analysis revealed over 300 mutants with at least a 2-fold fitness defect and 69 with defects ranging from 10- to >2,000-fold. Construction of 6 isogenic mutants for use in competitive infections with the wild type confirmed their requirement for lung fitness. Critical fitness genes included those for the synthesis of branched-chain and aromatic amino acids that are essential in mice and humans, the transcriptional elongation factor RfaH, and the copper efflux pump CopA. The majority of fitness genes were conserved among reference strains representative of diverse pathotypes. These results indicate that regulation of outer membrane components and synthesis of amino acids that are essential to its host are critical for K. pneumoniae fitness in the lung.ImportanceKlebsiella pneumoniae is a bacterium that commonly causes pneumonia in patients after they are admitted to the hospital. K. pneumoniae is becoming resistant to all available antibiotics, and when these infections spread to the bloodstream, over half of patients die. Since currently available antibiotics are failing, we must discover new ways to treat these infections. In this study, we asked what genes the bacterium needs to cause an infection, since the proteins encoded by these genes could be targets for new antibiotics. We identified over 300 genes that K. pneumoniae requires to grow in a mouse model of pneumonia. Many of the genes that we identified are found in K. pneumoniae isolates from throughout the world, including antibiotic-resistant forms. If new antibiotics could be made against the proteins that these genes encode, they may be broadly effective against K. pneumoniae.
| S-EPMC4462621 | biostudies-literature
Project description:Chitinases and chitinase-like proteins are an evolutionary conserved group of proteins. In the absence of chitin synthesis in mammals, the conserved presence of chitinases suggests their roles in physiology and immunity, but experimental evidence to prove these roles is scarce. Chitotriosidase (chit1) is one of the two true chitinases present in mammals and the most prevalent chitinase in humans. In this study, we investigated the regulation and the role of chit1 in a mouse model of Klebsiella pneumoniae lung infection. We show that chitinase activity in bronchoalveolar lavage fluid is significantly reduced during K. pneumoniae lung infection. This reduced activity is inversely correlated with the number of neutrophils. Further, instilling neutrophil lysates in lungs decreased chitinase activity. We observed degradation of chit1 by neutrophil proteases. In a mouse model, chit1 deficiency provided a significant advantage to the host during K. pneumoniae lung infection by limiting bacterial dissemination. This phenotype was independent of inflammatory changes in chit1-/- mice as they exerted a similar inflammatory response. The decreased dissemination resulted in improved survival in chit1-/- mice infected with K. pneumoniae in the presence or absence of antibiotic therapy. The beneficial effects of chit1 deficiency were associated with altered Akt activation in the lungs. Chit1-/- mice induced a more robust Akt activation postinfection. The role of the Akt pathway in K. pneumoniae lung infection was confirmed by using an Akt inhibitor, which impaired health and survival. These data suggest a detrimental role of chit1 in K. pneumoniae lung infections.
| S-EPMC6291403 | biostudies-literature
Project description:Carbapenemase-producing Enterobacterales (CPE) are spreading rapidly in hospital settings. Asymptomatic CPE gut colonisation may be associated with dysbiosis and gut-lung axis alterations, which could impact lung infection outcomes. In this study, in male C57BL/6JRj mice colonised by CPE, we characterise the resulting gut dysbiosis, and analyse the lung immune responses and outcomes of subsequent Pseudomonas aeruginosa lung infection. Asymptomatic gut colonisation by CPE leads to a specific gut dysbiosis and increases the severity of P. aeruginosa lung infection through lower numbers of alveolar macrophages and conventional dendritic cells. CPE-associated dysbiosis is characterised by a near disappearance of the Muribaculaceae family and lower levels of short-chain fatty acids. Faecal microbiota transplantation restores immune responses and outcomes of lung infection outcomes, demonstrating the involvement of CPE colonisation-induced gut dysbiosis in altering the immune gut-lung axis, possibly mediated by microbial metabolites such as short-chain fatty acids.
| S-EPMC9816093 | biostudies-literature
Project description:BackgroundHypervirulent Klebsiella pneumoniae (hvKp) strains of capsule type K1 and K2 cause invasive infections associated with hepatic abscesses, which can be difficult to treat and are frequently associated with relapsing infections. Other K pneumoniae strains (non-hvKp), including lineages that have acquired carbapenem resistance, do not manifest this pathology. In this work we aimed to test the hypothesis that within-macrophage replication is a key mechanism underpinning abscess formation in hvKp infections.MethodsIn this exploratory investigation, to study the pathophysiology of abscess formation, mice were intravenously infected with 106 colony forming units (CFU) of either hvKp isolates (six strains) or non-hvKp isolates (seven strains). Intracellular bacterial replication and neutrophil influx in liver and spleen was quantified by fluorescence microscopy of sliced cryopreserved organs of mice collected 30 min, 6 h, and 24 h after infection with the aim to provide data of bacterial association to Kupffer cells in the liver and to the different tissue macrophages in the spleen. Microbiological and microscopy analysis of an ex-vivo model of pig liver and spleen infection were used to confirm within-macrophage replication. Pig organs were perfused with heparinised, autologous pig's blood and injected with 6·5 × 107 CFU of hvKp K2 sequence type 25 strain GMR151. Blood and tissue biopsies collected before infection and 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h after infection were used to measure bacterial counts and to identify the subcellular localisation of bacteria by immunohistochemistry analysis.FindingsWe show that hvKp resisted phagocyte-mediated clearance and replicated in mouse liver macrophages to form clusters 6 h after infection, with a mean of 7·0 bacteria per Kupffer cell (SD 6·2); however, non-hvKp were efficiently cleared (mean 1·5 bacteria per cell [SD 1·1]). HvKp infection promoted neutrophil recruitment to sites of infection, which in the liver resulted in histopathological signs of abscess formation as early as 24 h post-infection. Experiments in pig organs which share a high functional and anatomical resemblance to human organs, provided strong evidence for the propensity of hvKp to replicate within the hepatic macrophages.InterpretationThese findings show subversion of innate immune processes in the liver by K pneumoniae and resistance to Kupffer cell mediated clearance as an explanation for the propensity of hvKp strains to cause hepatic abscesses.FundingUniversity of Oxford and a Royal Society Wolfson grant funded biosafety facility.
| S-EPMC8641047 | biostudies-literature