Characterisation of the secondary effects of sub-inhibitory concentrations of 1-(1-naphthylmethyl)-piperazine (NMP) in Klebsiella pneumoniae
ABSTRACT: Efflux of antimicrobial compounds from bacterial cells is one of the important mechanisms responsible for multi-drug resistance (MDR). Inhibiting the activity of efflux pumps using chemosensitizers like 1-(1-naphthylmethyl)-piperazine (NMP) is currently considered as a promising strategy to overcome MDR. However, additional effects of NMP other than inhibition are rarely if ever considered. Here, using phenotypic, phenotypic microarray and transcriptomic assays we show that NMP plays a role in membrane destabilization in MDR Klebsiella pneumoniae MGH 78578 strain. The observation of membrane destabilization was supported by RNA-seq data which showed that many up-regulated genes were either directly involved in responses to envelope stress or bacterial repair systems which are essential to maintain viability in an environment containing NMP. Membrane destabilization happens as early as 15 minutes post-NMP treatment. We postulate that the early membrane disruption leads to destabilization of inner membrane potential, impairing ATP production and consequently resulting in efflux pump inhibition. Overall design: β-Lactamase activity, membrane potential and Transmission Electron Microscopy assays were used to assay the NMP mediated membrane destabilisation in MDR Klebsiella pneumoniae MGH 78578 strain. We further used the Phenotypic Microarray (Biolog) and RNA-seq to elucidate the transcriptional and metabolic signals. Crystal violet-based assays were also used to assay the biofilm formation ability of NMP treated bacterial cells.
Project description:To investigate the whole-genome gene expression difference between the wild-type and capsule deletion mutant in Klebsiella pneumoniae MGH 78578. The mutants analyzed in this study are further described in Huang T.W., Stapleton J.C., Chang H.Y., Tsai S.F., Palsson B.O., Charusanti P. Capsule removal via lambda-Red knockout system perturbs biofilm formation and fimbriae extression in Klesiella pneumoniae MGH 78578 (manuscript submission) A six chip study using total RNA recovered from three separate wild-type cultures and three separate cultures of a capsule deltion mutant of Klebsiella pneumoniae MGH 78578. The capsule gene cluster (KPN_02493 to KPN_02515) was entirely removed in the capsule deletion mutant. Each chip measures the expression level of 5,305 genes from Klebsiella pneumoniae MGH 78578 and the associated five plasmids (pKPN3, pKPN4, pKPN5, pKPN6 and pKPN7) with 50-mer oligo tiling array with 30-mer spacer.
Project description:The interactions between Gram-negative respiratory pathogens and the host environment at the site of infection largely unknown. Pulmonary surfactant serves as an initial point of contact for inhaled bacteria entering the lung and is thought to contain molecular cues that aid colonization and pathogenesis. To gain insight into this ecological transition, we characterized the transcriptional responses of Pseudomonas aeruginosa PA14, Burkholderia thailandensis E264, Klebsiella pneumoniae MGH 78578, and Stenotrophomonas maltophilia K279A exposed to purified pulmonary surfactant (Survanta) through microarrays. This study provides novel insight into the interactions occurring between Gram-negative opportunistic pathogens and the host at an important infection site, and demonstrates the utility of purified lung surfactant preparations for dissecting host-lung pathogen interactions in vitro. The goal of this study was to compare the transcriptional responses of Pseudomonas aeruginosa PA14, Burkholderia thailandensis E264, Klebsiella pneumoniae MGH 78578, and Stenotrophomonas maltophilia K279A exposed to pulmonary surfactant using a custom affymetrix chip designed for their genomes. The goal of this study was to compare the transcriptional responses of Pseudomonas aeruginosa PA14, Burkholderia thailandensis E264, Klebsiella pneumoniae MGH 78578, and Stenotrophomonas maltophilia K279A exposed to pulmonary surfactant using a custom affymetrix chip designed for their genomes. Overall design: Microarrays were performed with total RNA collected from two independent gene induction experiments, where each strain was cultured in MOPS minimal media in the presence and absence of Survanta (purified pulmonary surfactant). Total RNA collected from these experiments were then mixed together 1:1 (P. aeruginosa & B. thailandensis, or K. pneumoniae & S. maltophilia) prior to hybridization to custom Affymetrix microarray chips (PaKpSmBta521148F). The resulting eight chips were then processed via RMA using Affymetrix's Expression Console and Transcriptome Analysis Console.
Project description:Genome-wide identification of RNA polymerase (RNAP) binding sites were performed in Klebsiella pneumoniae MGH 78578 (KP). Anti-RNAP is used to capture the RNAP in KP. ChIP-chip was performed on tiling array specifically made for KP. Comparison ChIP by anti-RNAP antibody vs ChIP by normal mouse IgG (control, mock IP)
Project description:Genome-wide gene expression analysis was performed with the cells in exponential and stationary growth phases. Through these two growth status, 89.6% of currently annotated genes were expressed. High-density oligonucleotide tiling arrays consisting of 379,528 50-mer probes spaced 30 bp apart across the whole Klebsiella pneumoniae MGH 78578 genome was used (Roche NimbleGen).
Project description:Liao2011 - Genome-scale metabolic
reconstruction of Klebsiella pneumoniae (iYL1228)
This model is described in the article:
An experimentally validated
genome-scale metabolic reconstruction of Klebsiella pneumoniae
MGH 78578, iYL1228.
Liao YC, Huang TW, Chen FC,
Charusanti P, Hong JS, Chang HY, Tsai SF, Palsson BO, Hsiung
J. Bacteriol. 2011 Apr; 193(7):
Klebsiella pneumoniae is a Gram-negative bacterium of the
family Enterobacteriaceae that possesses diverse metabolic
capabilities: many strains are leading causes of
hospital-acquired infections that are often refractory to
multiple antibiotics, yet other strains are metabolically
engineered and used for production of commercially valuable
chemicals. To study its metabolism, we constructed a
genome-scale metabolic model (iYL1228) for strain MGH 78578,
experimentally determined its biomass composition,
experimentally determined its ability to grow on a broad range
of carbon, nitrogen, phosphorus and sulfur sources, and
assessed the ability of the model to accurately simulate growth
versus no growth on these substrates. The model contains 1,228
genes encoding 1,188 enzymes that catalyze 1,970 reactions and
accurately simulates growth on 84% of the substrates tested.
Furthermore, quantitative comparison of growth rates between
the model and experimental data for nine of the substrates also
showed good agreement. The genome-scale metabolic
reconstruction for K. pneumoniae presented here thus provides
an experimentally validated in silico platform for further
studies of this important industrial and biomedical
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Project description:Screening of 14 novel proteins derived from Klebsiella pneumoniae MGH 78578 identified prior via screening of cDNA libraries. The full-length proteins were attached using a specific HaloTag to their corresponding ligand surface, HaloLink. Screening was performed using two different polyclonal antibodies to Klebsiella pneumoniae (Acris AP00792PU-N and Abcam ab20947) and detection achieved by Goat polyclonal to rabbit IgG conjugated with Chromeo-546 (Abcam ab60317). In order to assess their potential immungenic nature and rank the proteins investigated, comparative analysis using already described antigens from K. pneumoniae were used in the assay. Each microarray was seperated into different incubation chambers using the 16-well ProPlate (Grace Biolabs) multi-well gaskets. As positive references ompA and mdh were used. For negative control gapA was used and the crude lysates of the expression host (Acella E.coli) and buffer were spotted as well.Samples and controls were spotted with five replicates each. Incubation was performed using different antibodies reactive to K. pneumoniae.
Project description:Elucidating the RamA Regulon in Klebsiella pneumoniae and the transcriptome profiles of multidrug resistant Klebsiella pneumoniae Overall design: various strains of Klebsiella pneumoniae
Project description:Sequences of 11 amino acids belonging to the KPN_00363 protein and KPN_00459 protein from Klebsiella pneumoniae MGH 78578 which was previously identified as potentially immunogenic was analyzed via alanine scanning to narrow down the significant amino acid residues within the sequence. Overall design: 26 peptides, two representing the original sequences, 22 peptides with each residue replaced by alanine (or glycin, if the original sequence contained alanine) as well as two related peptides were synthesized on microarrays by JPTs Pepstar Technology. For each microarray, nine replicates for each peptide were spotted. The microarray was seperated into three incubation chambers by the ProPlate 3-well module (Grace Biolabs) to allow for incubation with different antibodies in parallel. For specific interaction rabbit polyclonal IgG to K. pneumoniae (Abcam ab20947) was used, while non-specific binding to the epitope sequences was checked using rabbit polyclonal IgG to E. coli (Abcam ab137967) and S. enterica (Abcam ab35156).
Project description:Bacteria can circumvent the effect of antibiotics by transitioning to a poorly understood physiological state that does not involve conventional genetic elements of resistance. Here we examine antibiotic susceptibility with a Class A β-lactamase+ invasive strain of Klebsiella pneumoniae that was isolated from a lethal outbreak within laboratory colonies of Chlorocebus aethiops sabaeus monkeys. Bacterial responses to the ribosomal synthesis inhibitors streptomycin and doxycycline resulted in distinct proteomic adjustments that facilitated decreased susceptibility to each antibiotic. Drug-specific changes to proteomes included proteins for receptor-mediated membrane transport and sugar utilization, central metabolism, and capsule production, while mechanisms common to both antibiotics included elevated scavenging of reactive oxygen species and turnover of misfolded proteins. Resistance to combined antibiotics presented integrated adjustments to protein levels as well as unique drug-specific proteomic features. Our results demonstrate that dampening of Klebsiella pneumoniae susceptibility involves global remodeling of the bacterial proteome to counter the effects of antibiotics and stabilize growth.
Project description:Total RNA isolated from mid log grown cultures of K.pneumoniae and mutant strain in three independent times.Expression profile of K.pneumoniae and its pk muatant was compared. Overall design: Agilent one-color experiment,Organism: Klebsiella pneumoniae ,Agilent Custom Klebsiella pneumoniae 8x15k Microarray designed by Genotypic Technology Private Limited (AMADID: 079362)