A penicillin-binding protein inhibits selection of colistin-resistant, lipooligosaccharide-deficient Acinetobacter baumannii
ABSTRACT: We report the transcriptional expression from wild type, a ponA mutant, and lipooligosaccharide-deficient A. baumannii in order to understand the cellular changes after inactivation of lipid A biosynthesis. Among all strains, genes in the Localization Of Lipoprotein (Lol) transport pathway were upregulated. This study provides a framework to understand how some Acinetobacter baumannii strains can survive without lipid A and lipopolysaccharide/lipooligosaccharide. Overall design: Examination of the global transcriptome in four different A. baumannii strains.
Proceedings of the National Academy of Sciences of the United States of America 20160928 41
The Gram-negative bacterial outer membrane fortifies the cell against environmental toxins including antibiotics. Unique glycolipids called lipopolysaccharide/lipooligosaccharide (LPS/LOS) are enriched in the cell-surface monolayer of the outer membrane and promote antimicrobial resistance. Colistin, which targets the lipid A domain of LPS/LOS to lyse the cell, is the last-line treatment for multidrug-resistant Gram-negative infections. Lipid A is essential for the survival of most Gram-negative ...[more]
Project description:Colistin is a crucial last-line drug used for the treatment of life-threatening infections caused by multi-drug resistant strains of the Gram-negative bacteria, Acinetobacter baumannii. However, colistin resistant A. baumannii isolates can be isolated following failed colistin therapy. Resistance is most often mediated by the addition of phosphoethanolamine (pEtN) to lipid A by PmrC, following missense mutations in the pmrCAB operon encoding PmrC and the two-component signal transduction system PmrA/PmrB. We recovered an isogenic pair of A. baumannii isolates from a single patient before (6009-1) and after (6009-2) failed colistin treatment that displayed low/intermediate and high levels of colistin resistance, respectively. To understand how increased colistin-resistance arose, we genome sequenced each isolate which revealed that 6009-2 had an extra copy of the insertion sequence element ISAba125 within a gene encoding an H-NS-family transcriptional regulator. Consequently, transcriptomic analysis of the clinical isolates identified was performed and more than 150 genes as differentially expressed in the colistin-resistant, hns mutant, 6009-2. Importantly, the expression of eptA, encoding a second lipid A-specific pEtN transferase, but not pmrC, was significantly increased in the hns mutant. This is the first time an H-NS-family transcriptional regulator has been associated with a pEtN transferase and colistin resistance. Overall design: mRNA profiles of two isolates taken from a single patient, one before and one after colistin treatment, cDNA libraries were sequenced on an Illumina HiSeq 2000
Project description:Objectives: Colistin remains a last-line treatment for multidrug-resistant Acinetobacter baumannii and combined use of colistin and carbapenems has shown synergistic effects against multidrug-resistant strains. In order to understand the bacterial responses to these antibiotics we analysed the transcriptome of A. baumannii following exposure to each. Overall design: Methods: RNA sequencing was employed to determine changes in the transcriptome following treatment with colistin and doripenem, both alone and in combination, using an in vitro pharmacokinetics/pharmacodynamics (PK/PD) model to mimic the pharmacokinetics of both antibiotics in patients.
Project description:We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis. Consequently, strains harboring these mutations are unable to produce the major Gram negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC19606, to that of an isogenic, LPS-deficient, lpxA mutant strain. Analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, up-regulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-beta-1,6-N-acetylglucosamine (PNAG) were also up-regulated and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein, AssC, in culture supernatants of the A. baumannii wild-type strain, but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface. Comparison of a gene expression in biological duplicate samples derived from parent bacterial strain to an isogenic mutant strain.
Project description:Total RNA isolated from mid-log-grown cultures of A. baumannii and mutant strain in three independent times. Expression profile of A. baumannii and its protein kinase muatant was compared. Overall design: Agilent one-color experiment, Organism: Acinetobacter baumannii, Agilent Custom Acinetobacter baumannii 8x15k Microarray designed by Genotypic Technology Private Limited (AMADID: 079361).
Project description:Acinetobacter baumannii AB042, a triclosan-resistant mutant, was examined for modulated gene expression using whole genome sequencing, transcriptomics, and proteomics in order to understand the mechanism of triclosan-resistance as well as its impact on A. Baumannii. Overall design: A triclosan mutant (AB042) was isolated by culturing A. baumannii ATCC 17978 in increasing concentrations of triclosan.
Project description:We analyzed the extracellular proteome of colistin-resistant Korean Acinetobacter baumannii (KAB) strains to identify proteome profiles that can be used to characterize extensively drug-resistant KAB strains.
Project description:Total RNA isolated from mid-log-grown cultures of A. baumannii and mutant strain in three independent times. Expression profile of A. baumannii and its byk mutant was compared. Overall design: Agilent one-color experiment, Organism: Acinetobacter baumannii, Agilent Custom Acinetobacter baumannii 8x15k Microarray designed by Genotypic Technology Private Limited (AMADID: 079361).
Project description:In recent years, the Gram-negative bacterium Acinetobacter baumannii has garnered considerable attention for its unprecedented capacity to rapidly develop resistance to antibacterial therapeutics. This is coupled with the seemingly epidemic emergence of new hyper-virulent strains. Although strain-specific differences for A. baumannii isolates have been well described, these studies have primarily focused on proteinaceous factors. At present, only limited publications have investigated the presence and role of small regulatory RNA (sRNA) transcripts. Herein, we perform such an analysis, describing the RNA-seq-based identification of 78 A. baumannii sRNAs in the AB5075 background. Together with six previously identified elements, we include each of these in a new genome annotation file, which will serve as a tool to investigate regulatory events in this organism. Our work reveals that the sRNAs display high expression, accounting for >50 % of the 20 most strongly expressed genes. Through conservation analysis we identified six classes of similar sRNAs, with one found to be particularly abundant and homologous to regulatory, C4 antisense RNAs found in bacteriophages. These elements appear to be processed from larger transcripts in an analogous manner to the phage C4 molecule and are putatively controlled by two further sRNAs that are strongly antisense to them. Collectively, this study offers a detailed view of the sRNA content of A. baumannii, exposing sequence and structural conservation amongst these elements, and provides novel insight into the potential evolution, and role, of these understudied regulatory molecules. This study is based on the annotation of novel sRNAs on basis of an Acinetobacter baumannii RNA sequencing dataset. Each sample was generated by pooling three independent biological replicate RNA preps
Project description:RNA sequencing was carried out by ARK genomics, Edinburgh on an Illumina HiSeq platform to compare gene expression in Acinetobacter baumannii strain AYE and an adeRS deletion mutant in this strain.