Quantitative Profiling and Identification of Outer Membrane Proteins of β-Lactam Resistant Strain of Acinetobacter baumannii Jitendra Vashist,† Vishvanath Tiwari,† Arti Kapil,‡ and Moganty R. Rajeswari*,† Department of Biochemistry and Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, 110029, India Received December 5, 2009
In the recent past, Acinetobacter baumannii in hospitals has become increasingly resistant to a number of antibiotics including the latest carbapenems and this is of great concern. Here, for the first time, we report 2D-Differential In-Gel Electrophoresis (DIGE) analysis of outer membrane proteome of ATCC 19606 and carbapenem-resistant strain of A. baumannii. Using biological variance analysis (BVA), we identified 25 differentially expressed proteins with a fold difference g2; confidence >90% and statistical significance p e 0.05. By applying MALDI TOF/TOF and ESI MS/MS proteomic approach to carbapenemresistant A. baumannii, we identified four porins whose expression was significantly downregulated and a large number of upregulated proteins with specific transport and enzymatic properties. All the following synergistically give advantage to the resistant A. baumannii for survival: (i) high expression of altered CarO isoforms with possible low influx efficiency, (ii) elevated levels of CsuA/B with high adhesive properties, (iii) overexpression of pivotal nutrient transporters, (iv) continued expression of the major OmpAb, and (v) induced production of proteins of hypothetical origin. This proteomic study provides a platform to understand the complex nature of β-lactam resistance in A. baumannii. Keywords: β-lactams • Acinetobacter baumannii • DIGE • Outer membrane proteins
1. Introduction Acinetobacter baumannii is a Gram-negative, coccobacilli aerobic pathogen responsible for nosocomial infections in hospitals worldwide. A regular microbiological screening of clinical samples at the All India Institute of Medical Sciences (AIIMS), India, identified 458 bacterial samples as Acinetobacter strains (total 1460 Gram-negative bacterial strains). Within the population of Acinetobacter spp., A. baumannii accounts for more than 90% (data unpublished). A. baumannii can be cultured from sputum/respiratory secretion, wound or urine of hospitalized patients. A. baumannii exhibits an exceptional catabolic capability which gives high competence for natural transformation associated with a genetic flexibility and versatility to the bacterium.1 The ability to grow at wide range of temperature (28-44 °C), humidity and pH range makes the A. baumannii an adaptive pathogen in the hospital environment. A. baumannii has emerged during the past decade as an important nosocomial pathogen involved in pneumonia, septicemia, urinary tract infections and meningitis.2,3 Such infections are often treated by usual drug families including β-lactams, aminoglycosides and quinolones.1 β-Lactams are the armamentarium of our antibiotic source and are routinely given to treat a variety of bacterial infections. Carbapenems are the last resort of β-lactams which have the highest efficacy and broad spectrum against wide verity of Gram-negative bacteria. * To whom correspondence should be addressed. E-mail: mrraji@ hotmail.com. Phone: 91-11-26593416. Fax: 91-11-26588663. † Department of Biochemistry, All India Institute of Medical Sciences. ‡ Department of Microbiology, All India Institute of Medical Sciences. 10.1021/pr9011188
2010 American Chemical Society
Emergence of resistant strains even to carbapenems like imipenem is a failure in combating the infections caused by A. baumannii and is a threat to mankind.4-8 Further, the intrinsic resistance to a number of antimicrobial agents allows the A. baumannii to spread in the hospital settings. Therefore, A. baumannii combined with resistance is thus very difficult to treat and challenges the development of newer drugs. In such a scenario, the understanding of the resistance mechanism is of utmost importance. Antimicrobial resistance is a global health problem. Generally, a bacterium exploits all the stratagems via multigenic phenomenon to escape from mortal effects of antibiotics. Coevolution of β-lactam antibiotics and the resistance strategy developed by bacteria have become a major bottleneck and opened up new avenues such as proteomics in understanding the antimicrobial resistance.9-12 The high-throughput identification and characterization of specific proteins are generally associated with newly evolved proteomic technology. In the context of host-pathogen interactions, outer membrane proteins (Omps) of Gram-negative bacteria play a key role in cellular adhesion, signal transduction and pathogenesis. In general, higher antibiotic resistance of Gram-negative bacteria as compared to Gram-positive bacteria, has been attributed to the presence of Omps that regulate antibiotic entry into cells by the reduction of outer membrane permeability and the activation of multidrug efflux pumps.13-16 However, efforts toward characterization of the membrane proteome have lagged behind because of the hydrophobic nature and low solubility of membrane proteins as compared to soluble Journal of Proteome Research 2010, 9, 1121–1128 1121 Published on Web 12/30/2009
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Vashist et al.
Table 1. Minimal inhibitory concentrations (µg/mL) of various β-lactams for clinical strains of A. baumannii strain
ceftazidime piperacillin cefotaxime meropenem imipenem
ATCC RS 307
0.1 128
0.1 128
