title: Identification of Outer Membrane and Exoproteins of Carbapenem-Resistant Multilocus Sequence Type 258 Klebsiella pneumoniae.
abstract: Carbapenem-resistant Klebsiella pneumoniae strains have emerged as a cause of life-threatening infections in susceptible individuals (e.g., transplant recipients and critically ill patients). Strains classified as multilocus sequence type (ST) 258 are among the most prominent causes of carbapenem-resistant K. pneumoniae infections worldwide, but the basis for the success of this lineage remains incompletely determined. To gain a more comprehensive view of the molecules potentially involved in the success of ST258, we used a proteomics approach to identify surface-associated and culture supernatant proteins produced by ST258. Protein samples were prepared from varied culture conditions in vitro, and were analyzed by a combination of two-dimensional electrophoresis and liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). We identified a total of 193 proteins in outer membrane preparations from bacteria cultured in Luria-Bertani broth (LB) or RPMI 1640 tissue culture media (RPMI). Compared with LB, several iron-acquisition proteins, including IutA, HmuR, HmuS, CirA, FepA, FitA, FoxA, FhuD, and YfeX, were more highly expressed in RPMI. Of the 177 proteins identified in spent media, only the fimbrial subunit, MrkA, was predicted to be extracellular, a finding that suggests few proteins (or a limited quantity) are freely secreted by ST258. Notably, we discovered 203 proteins not reported in previous K. pneumoniae proteome studies. In silico modeling of proteins with unknown function revealed several proteins with beta-barrel transmembrane structures typical of porins, as well as possible host-interacting proteins. Taken together, these findings contribute several new targets for the mechanistic study of drug-resistance and pathogenesis by ST258 K. pneumoniae isolates.
authors.affiliation: Laboratory of Human Bacterial Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, National Institute of Health, Hamilton, MT, United States of America.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Brinkworth
authors.affiliation: Research Technologies Branch, National Institute of Allergy and Infectious Disease, National Institute of Health, Bethesda, MD, United States of America.
authors.forename: Carl H
authors.initials: CH
authors.lastname: Hammer
authors.affiliation: Research Technologies Branch, National Institute of Allergy and Infectious Disease, National Institute of Health, Bethesda, MD, United States of America.
authors.forename: L Renee
authors.initials: LR
authors.lastname: Olano
authors.affiliation: Laboratory of Human Bacterial Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, National Institute of Health, Hamilton, MT, United States of America.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Kobayashi
authors.affiliation: Public Health Research Institute Tuberculosis Center, New Jersey Medical School, Rutgers the State University of New Jersey, Newark, NJ, United States of America.
authors.forename: Liang
authors.initials: L
authors.lastname: Chen
authors.affiliation: Public Health Research Institute Tuberculosis Center, New Jersey Medical School, Rutgers the State University of New Jersey, Newark, NJ, United States of America.
authors.forename: Barry N
authors.initials: BN
authors.lastname: Kreiswirth
authors.affiliation: Laboratory of Human Bacterial Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, National Institute of Health, Hamilton, MT, United States of America.
authors.forename: Frank R
authors.initials: FR
authors.lastname: DeLeo
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D015373:Bacterial Typing Techniques; D015780:Carbapenems / Q000494:pharmacology*; D003470:Culture Media; D024881:Drug Resistance, Bacterial / Q000187:drug effects*; D015180:Electrophoresis, Gel, Two-Dimensional; D007711:Klebsiella pneumoniae / Q000145:classification* / Q000187:drug effects / Q000378:metabolism*; D013058:Mass Spectrometry; D058885:Multilocus Sequence Typing
chemical_list: D001425:Bacterial Outer Membrane Proteins; D015780:Carbapenems; D003470:Culture Media