Yanbao Yu (Presenter)
J. Craig Venter Institute
Authorship: Yanbao Yu, Shiferaw B. Negesso, Rembert Pieper
J. Craig Venter Institute, Rockville MD 20850
| Short Abstract We analyzed the proteomes of Klebsiella pneumoniae (Kp) strains derived from UTIs by LC-MS/MS. Over 45% of the in silico proteome was identified from analyses of strain KPC27. This strain produces several b-lactamases and a carbapenemase resulting in only a few last-resort antibiotic drugs for therapy of infections. We observed that KPC27 responded to antibacterial effectors with increased expression of iron uptake and transport systems. In addition, we found differences in the use of iron acquisition systems and MFA pathways in the in vivo environment when comparing cultured and in vivo KPC27 proteomes. We hypothesize that iron starvation induces a regulatory shift towards energy metabolism via MFA pathways. Strategies interfering with the regulatory circuit of iron starvation and energy generation could potentially lead to inhibitors that form a new class of antibiotic drugs. |
Long Abstract
Introduction
Carbapenem-resistant Klebsiella pneumoniae (Kp) have emerged as a cause of life-threatening infections worldwide (1). One underlying resistance mechanism is the production of carbapenemases, a class of β-lactamase which can break down a broad variety of β-lactams such as carbapenems and penicillins (2). As these are common antibacterial agents against many microorganisms, the prevalence of Kp carbapenemase (KPC)-producing pathogens leave few available therapeutic options and create an increasing fear for public health (3). KPC is the most frequently observed class A carbapenemases (3). It has many variants emerged recently, including KPC-1 through KPC-11 which usually are distinguished by one or two amino acid substitutions. Mass spectrometry-based approach, such as matrix-assisted laser desorption ionization–time of flight MS (MALDI-TOF MS) has been applied to identify carbapenem hydrolysis products, thus confirming carbapenemase activity in Gram-negative isolates (4). However, experience with this methodology is still limited. To gain a comprehensive view of the molecules potentially involved in the success of carbapenem resistance, we used a quantitative proteomics approach to discover the global proteome of KPC-27 strain, which was isolated from a patient with urinary tract infection (UTI). These data contribute several new findings of the mechanistic study of drug-resistance and pathogenesis by Carbapenem-resistant Klebsiella pneumoniae.
Methods
The Kp cells stocks frozen in 30% glycerol/Luria broth (LB) were cultured overnight at 37˚C in LB medium, diluted 100-fold into fresh LB, and grown to an OD600 of 0.35-0.5 or 0.9-1.2 in a shaker at 880 rpm. Cells were collected, washed and lysed with SDS buffer (2% SDS, 5mM EDTA, 50mM DTT). Around 50 µg proteins were aliquoted and subjected to gel-based and FASP digestion, respectively. Two types of 96-well plates with roughly 30K MWCO were used (Millipore MultiScreen-PCR96 Filter Plate, MSNU03050; Pall AcroPrep Plate 30K Omega, 8035). Peptide digests were desalted afterwards using the spinnable StageTip protocol. LC-MS/MS analysis was performed on an Ultimate 3000 nano LC and Q Exactive mass spectrometer system (Thermo Scientific). The LCMS raw data were processed using the Proteome Discoverer-Sequest-HT platform (version 1.4, Thermo Scientific) and the MaxQuant-Andromeda software suite (version 1.5.4.0). Science) algorithm was employed. Hierarchical clustering and other quantitative analyses were performed in Perseus software (version 1.5) using normalized label free quantitation (LFQ values generated by MaxQuant.
Results
We identified ~3,000 non-redundant KPC27 proteins, which covered over 50% of the sequenced genome. To achieve maximum proteome coverage while maintaining a reasonable measurement time, in this study, we employed multiple sample processing approaches, including SDS PAGE separation/in-gel digestion and filter-based processing. Notably, we improved our 96FASP approach and developed a quicker version (q96FASP), which significantly speeds up sample processing and scales up the throughput. In this method, a 30k MWCO filter based 96-well plate was used. The centrifugation time reduced from 1.5 hour for original 96FASP method to 15~20 min, and up to 96 samples can be processed simultaneously. We systematically investigate the efficiency, reproducibility and throughput of the q96FASP method. It worked as quick as conventional individual filter-based FASP. The correlation between them in terms of protein identification and quantification was high (R2 > 0.9). Processing the same sample in five wells suggested that the multi-well format FASP can generate highly reproducible protein digests with minimal variations in the context of protein identification and quantitation. The filters with different membrane chemistries also showed complementary effects for proteome analysis.
Quantitative proteome analysis of Kp cells growing in neutrophil rich and healthy urine medium revealed that KPC27 responded to antibacterial effectors, some of which sequester iron, with increased expression of iron uptake and transport systems (SitA, FepA, FhuA) and enzymes responsible for energy generation via mixed acid fermentation (MFA) pathways. We hypothesize that iron starvation induces a regulatory shift towards energy metabolism via MFA pathways due to the high number of enzymes in the TCA cycle and the NADH-quinone oxidoreductase complex with iron cofactors. We compared the KPC proteome with proteomes of four other Kp strains derived from UTIs and urethral catheter biofilms and found that there are differences in the use of iron acquisition systems and MFA pathways in the in vivo environment.
References & Acknowledgements:
1. Nordmann, P., Cuzon, G. and Naas, T. (2009) The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. The Lancet Infectious Diseases 9, 228-236
2. Lee, C.-R., Lee, J. H., Park, K. S., Kim, Y. B., Jeong, B. C. and Lee, S. H. (2016) Global Dissemination of Carbapenemase-Producing Klebsiella pneumoniae: Epidemiology, Genetic Context, Treatment Options, and Detection Methods. Frontiers in Microbiology 7,
3. Queenan, A. M. and Bush, K. (2007) Carbapenemases: the Versatile β-Lactamases. Clinical Microbiology Reviews 20, 440-458
4. Burckhardt, I. and Zimmermann, S. (2011) Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry To Detect Carbapenem Resistance within 1 to 2.5 Hours. J. Clin. Microbiol. 49, 3321-3324
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