lipid-a and Klebsiella-Infections

Topics: Adult; Anti-Bacterial Agents; Bacterial Proteins; Colistin; Drug Resistance, Bacterial; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Microbial Sensitivity Tests

Colistin resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) poses health challenges. To investigate the prevalence and molecular characteristics of colistin-resistant CRKP, 708 isolates were collected consecutively from 28 tertiary hospitals in China from 2018 to 2019, and 14 colistin-resistant CRKP were identified. Two-component systems (TCSs) related to colistin resistance (PmrA/B, PhoP/Q, and CrrA/B), the negative regulator mgrB gene and mcr genes, were analysed using genomic sequencing. The relative expression of TCSs genes along with their downstream pmrC and pmrK genes was determined using quantitative real-time PCR (qRT‒PCR). A novel point mutation in PhoQ was confirmed by site-directed mutagenesis, and the subsequent transcriptome changes were analysed by RNA sequencing (RNA-Seq). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect modifications in lipid A. The results showed that only one isolate carried the mcr-8.1 gene, nine exhibited MgrB inactivation or absence, and three exhibited mutations in PmrB. One novel point mutation, L247P, in PhoQ was found to lead to a 64-fold increase in the minimum inhibitory concentration (MIC) of colistin. qRT‒PCR revealed overexpression of phoP/Q and pmrK in isolates with or without MgrB inactivation, and pmrB mutation resulted in overexpression of pmrA and pmrC. Furthermore, transcriptome analysis revealed that the PhoQ L247P novel point mutation caused upregulated expression of phoP/Q and its downstream operon pmrHFIJKLM. Meanwhile, the pmrA/B regulatory pathway did not evolve colistin resistance. Mass spectrometry analysis showed the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A in colistin-resistant isolates with absence of MgrB. These findings illustrate that the molecular mechanisms of colistin resistance in CRKP isolates are complex, and that MgrB inactivation or absence is the predominant molecular mechanism. Interventions should be initiated to monitor and control colistin resistance.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Carbapenems; Colistin; Drug Resistance, Bacterial; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Microbial Sensitivity Tests; Prevalence

Polymyxins are last-line antibiotics employed against multidrug-resistant (MDR) Klebsiella pneumoniae. Worryingly, polymyxin resistance is rapidly on the rise globally. Polymyxins initially target lipid A of lipopolysaccharides (LPSs) in the cell outer membrane (OM), causing disorganization and cell lysis. While most studies focus on how genetic variations confer polymyxin resistance, the mechanisms of membrane remodeling and metabolic changes in polymyxin-resistant strains remain unclear, thus hampering the development of effective therapies to treat severe K. pneumoniae infections. In the present study, lipid A profiling, OM lipidomics, genomics, and metabolomics were integrated to elucidate the global mechanisms of polymyxin resistance and metabolic adaptation in a polymyxin-resistant strain (strain S01R; MIC of >128 mg/L) obtained from K. pneumoniae strain S01, a polymyxin-susceptible (MIC of 2 mg/L), New Delhi metallo-β-lactamase (NDM)-producing MDR clinical isolate. Genomic analysis revealed a novel in-frame deletion at position V258 of PhoQ in S01R, potentially leading to lipid A modification with 4-amino-4-deoxy-l-arabinose (L-Ara4N) despite the absence of polymyxin B. Comparative metabolomic analysis revealed slightly elevated levels of energy production and amino acid metabolism in S01R compared to their levels in S01. Exposure to polymyxin B (4 mg/L for S01 and 512 mg/L for S01R) substantially altered energy, nucleotide, and amino acid metabolism and resulted in greater accumulation of lipids in both strains. Furthermore, the change induced by polymyxin B treatment was dramatic at both 1 and 4 h in S01 but only significant at 4 h in S01R. Overall, profound metabolic adaptation was observed in S01R following polymyxin B treatment. These findings contribute to our understanding of polymyxin resistance mechanisms in problematic NDM-producing K. pneumoniae strains and may facilitate the discovery of novel therapeutic targets.

Topics: Anti-Bacterial Agents; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Lipid Metabolism; Microbial Sensitivity Tests; Polymyxin B; Polymyxins

Klebsiella pneumoniae ST258 is a human pathogen associated with poor outcomes worldwide. We identify a member of the acyltransferase superfamily 3 (atf3), enriched within the ST258 clade, that provides a major competitive advantage for the proliferation of these organisms in vivo. Comparison of a wild-type ST258 strain (KP35) and a Δatf3 isogenic mutant generated by CRISPR-Cas9 targeting reveals greater NADH:ubiquinone oxidoreductase transcription and ATP generation, fueled by increased glycolysis. The acquisition of atf3 induces changes in the bacterial acetylome, promoting lysine acetylation of multiple proteins involved in central metabolism, specifically Zwf (glucose-6 phosphate dehydrogenase). The atf3-mediated metabolic boost leads to greater consumption of glucose in the host airway and increased bacterial burden in the lung, independent of cytokine levels and immune cell recruitment. Acquisition of this acyltransferase enhances fitness of a K. pneumoniae ST258 isolate and may contribute to the success of this clonal complex as a healthcare-associated pathogen.

Topics: Acetylation; Acyltransferases; Animals; Bacterial Proteins; Carbapenems; Citric Acid Cycle; Gene Deletion; Glucose; Glycolysis; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Lung; Lysine; Male; Metabolome; Metabolomics; Mice, Inbred C57BL; Phylogeny; Protein Processing, Post-Translational; Respiratory Tract Infections

The increased incidence of polymyxin-resistant MDR Klebsiella pneumoniae has become a major global health concern.. To characterize the lipid A profiles and metabolome differences between paired polymyxin-susceptible and -resistant MDR K. pneumoniae clinical isolates.. Three pairs of K. pneumoniae clinical isolates from the same patients were examined [ATH 7 (polymyxin B MIC 0.25 mg/L) versus ATH 8 (64 mg/L); ATH 15 (0.5 mg/L) versus ATH 16 (32 mg/L); and ATH 17 (0.5 mg/L) versus ATH 18 (64 mg/L)]. Lipid A and metabolomes were analysed using LC-MS and bioinformatic analysis was conducted.. The predominant species of lipid A in all three paired isolates were hexa-acylated and 4-amino-4-deoxy-l-arabinose-modified lipid A species were detected in the three polymyxin-resistant isolates. Significant metabolic differences were evident between the paired isolates. Compared with their corresponding polymyxin-susceptible isolates, the levels of metabolites in amino sugar metabolism (UDP-N-acetyl-α-d-glucosamine and UDP-N-α-acetyl-d-mannosaminuronate) and central carbon metabolism (e.g. pentose phosphate pathway and tricarboxylic acid cycle) were significantly reduced in all polymyxin-resistant isolates [fold change (FC) > 1.5, P < 0.05]. Similarly, nucleotides, amino acids and key metabolites in glycerophospholipid metabolism, namely sn-glycerol-3-phosphate and sn-glycero-3-phosphoethanolamine, were significantly reduced across all polymyxin-resistant isolates (FC > 1.5, P < 0.05) compared with polymyxin-susceptible isolates. However, higher glycerophospholipid levels were evident in polymyxin-resistant ATH 8 and ATH 16 (FC > 1.5, P < 0.05) compared with their corresponding susceptible isolates.. To our knowledge, this study is the first to reveal significant metabolic perturbations associated with polymyxin resistance in K. pneumoniae.

Topics: Anti-Bacterial Agents; Colistin; Drug Resistance, Bacterial; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Metabolomics; Microbial Sensitivity Tests; Polymyxins

In the present report we characterized the outer membrane proteome, genomic, and lipid A remodelling changes following the evolution of a colistin-susceptible K. pneumoniae ATCC 13883 strain into an extremely colistin-resistant strain. Lipid A profiling revealed the outer membrane of the colistin-susceptible strain is decorated primarily by hexa- and hepta-acylated lipid A species and a minor tetra-acylated species. In the lipid A profile of the extremely colistin-resistant strain, in addition to the aforementioned lipid A species, the obligatory 4-amino-4-deoxy-l-arabinose modification of the hexa-acylated lipid A was detected. Comparative genomic analysis revealed that the mgrB gene of the colistin-resistant strain is inactivated by a single nucleotide insertion which produces a frame-shift, resulting in premature termination. We also detected two synonymous mutations in the two-component system genes phoP and phoQ. Comparative profiling of the outer membrane proteome of each strain revealed that outer membrane proteins from bacterial stress response, glutamine degradation, pyruvate, aspartate, and asparagine metabolic pathways were over-represented in the extremely colistin-resistant K. pneumoniae ATCC 13883 strain. In comparison, in the sensitive strain, outer membrane proteins from carbohydrate metabolism, H

Topics: Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Colistin; Drug Resistance, Bacterial; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Microbial Sensitivity Tests; Proteome

Topics: Acyltransferases; Animals; Bacterial Proteins; Cells, Cultured; Disease Models, Animal; Gene Deletion; Gene Expression Regulation, Bacterial; Klebsiella Infections; Klebsiella pneumoniae; Lepidoptera; Lipid A; Macrophages; Mass Spectrometry; Mice; Phagocytosis; Virulence; Virulence Factors

Colistin resistance in Klebsiella pneumoniae typically involves inactivation or mutations of chromosomal genes mgrB, pmrAB or phoPQ, but data regarding consequent modifications of LPS are limited.. To examine the sequences of chromosomal loci implicated in colistin resistance and the respective LPS-derived lipid A profiles using 11 pairs of colistin-susceptible and -resistant KPC-producing K. pneumoniae clinical strains.. The strains were subjected to high-throughput sequencing with Illumina HiSeq. The mgrB gene was amplified by PCR and sequenced. Lipid profiles were determined using MALDI-TOF MS.. All patients were treated with colistimethate prior to the isolation of colistin-resistant strains (MIC >2 mg/L). Seven of 11 colistin-resistant strains had deletion or insertional inactivation of mgrB. Three strains, including one with an mgrB deletion, had non-synonymous pmrB mutations associated with colistin resistance. When analysed by MALDI-TOF MS, all colistin-resistant strains generated mass spectra containing ions at m/z 1955 and 1971, consistent with addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A, whereas only one of the susceptible strains displayed this lipid A phenotype.. The pathway to colistin resistance in K. pneumoniae primarily involves lipid A modification with Ara4N in clinical settings.

Topics: Adult; Aged; Amino Sugars; Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamases; Chromosomes, Bacterial; Colistin; Drug Resistance, Bacterial; Female; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Lipopolysaccharides; Male; Membrane Proteins; Microbial Sensitivity Tests; Middle Aged; Mutagenesis, Insertional

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Load; Bacterial Proteins; Colistin; Disease Models, Animal; Drug Resistance, Bacterial; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lepidoptera; Lipid A; Lung; Membrane Proteins; Mice, Inbred C57BL; Polymyxins; Survival Analysis; Virulence

The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiella pneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.

Topics: Animals; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Lung; Mice; Molecular Structure; Organ Specificity

The impact of under-acylation of lipid A on the interaction between Klebsiella pneumoniae LPS and polymyxins B and E was examined with fluorometric and calorimetric methods, and by (1)H NMR, using a paired wild type (WT) and the ΔlpxM mutant strains B5055 and B5055ΔlpxM, which predominantly express LPS with hexa- and penta-acylated lipid A structures respectively. LPS from B5055ΔlpxM displayed a fourfold increased binding affinity for polymyxins B and E compared with the B5055 WT LPS. EC50 values were consistent with polymyxin minimum inhibitory concentration (MIC) values for each strain. Accordingly, polymyxin exposure considerably enhanced the permeability of the B5055ΔlpxM OM. Analysis of the melting profiles of isolated LPS aggregates suggested that bactericidal polymyxin activity may relate to the acyl chains' phase of the outer membrane (OM). The enhanced polymyxin susceptibility of B5055ΔlpxM may be attributable to the favorable insertion of polymyxins into the more fluid OM compared with B5055. Molecular models of the polymyxin B-lipid A complex illuminate the key role of the lipid A acyl chains for complexation of polymyxin. The data provide important insight into the molecular basis for the increased polymyxin susceptibility of K. pneumoniae strains with under-acylated lipid A. Under-acylation appears to facilitate the integration of the N-terminal fatty-acyl chain of polymyxin into the OM resulting in an increased susceptibility to its antimicrobial activity/activities.

Topics: Acylation; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Cell Membrane Permeability; Colistin; Computer Simulation; Fatty Acids; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Mutation; Polymyxin B; Structure-Activity Relationship

Antimicrobial peptides (APs) impose a threat to the survival of pathogens, and it is reasonable to postulate that bacteria have developed strategies to counteract them. Polymyxins are becoming the last resort to treat infections caused by multidrug-resistant Gram-negative bacteria and, similar to APs, they interact with the anionic lipopolysaccharide. Given that polymyxins and APs share the initial target, it is possible that bacterial defense mechanisms against polymyxins will be also effective against host APs. We sought to determine whether exposure to polymyxin will increase Klebsiella pneumoniae resistance to host APs. Indeed, exposure of K. pneumoniae to polymyxin induces cross-resistance not only to polymyxin itself but also to APs present in the airways. Polymyxin treatment upregulates the expression of the capsule polysaccharide operon and the loci required to modify the lipid A with aminoarabinose and palmitate with a concomitant increase in capsule and lipid A species containing such modifications. Moreover, these surface changes contribute to APs resistance and also to polymyxin-induced cross-resistance to APs. Bacterial loads of lipid A mutants in trachea and lungs of intranasally infected mice were lower than those of wild-type strain. PhoPQ, PmrAB, and the Rcs system govern polymyxin-induced transcriptional changes, and there is a cross talk between PhoPQ and the Rcs system. Our findings support the notion that Klebsiella activates a defense program against APs that is controlled by three signaling systems. Therapeutic strategies directed to prevent the activation of this program could be a new approach worth exploring to facilitate the clearance of the pathogen from the airways.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Arabinose; Bacterial Capsules; Bacterial Load; Bacterial Proteins; Drug Resistance, Multiple, Bacterial; Female; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Lung; Mass Spectrometry; Mice; Mice, Inbred C57BL; Microbial Sensitivity Tests; Palmitic Acid; Polymyxins; Trachea; Virulence Factors

Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055DeltalpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055DeltalpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins.

Topics: Acylation; Animals; Anti-Bacterial Agents; Bacterial Capsules; Blood Bactericidal Activity; Defensins; Disease Models, Animal; Drug Resistance, Microbial; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Mice; Mice, Inbred BALB C; Phagocytosis; Pneumonia, Bacterial; Sepsis

To prepare monoclonal antibodies (MAbs) directed against the core-lipid A fractions of smooth lipopoly-saccharide (LPS) from Klebsiella pneumoniae O1:K2, we immunized BALB/c mice with the LPS-associated proteins plus LPS. This preparation exposed the core-lipid A moiety, which is normally hidden in the micellar structure of classical LPS preparations. Among 10 MAbs selected for their reactivity with LPS-associated proteins plus LPS from K. pneumoniae O1:K2, 6 (3A3, 3C2, 3C4, 7D2, 11C3, and 12B6) were directed against the core fraction and 2 (6C5 and 10A5) were directed against the lipid A fraction. Only one (2A4) recognized the O antigen, and one (6D5) had an undefined specificity. When injected before challenge with K. pneumoniae O1:K2 LPS in galactosamine-sensitized mice, five of the MAbs (3C4, 6D5, 7D2, 11C3, and 12B6) provided protection in this model of lethal endotoxemia. MAb 7D2 was also protective in an experimental infection with capsulated K. pneumoniae O1:K2.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Bacterial; Bacterial Proteins; Cross Reactions; Endotoxins; Epitopes; Escherichia coli; Female; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C; O Antigens