Identification of uropathogenic Escherichia coli surface proteins by shotgun proteomics
Introduction
Urinary tract infections (UTIs) are among the most frequent bacterial infections affecting humans. It is estimated that there are 11.3 million community-acquired UTIs in the United States annually, which result in projected public health costs of over two billion dollars (Foxman et al., 2000, Litwin et al., 2005). Forty percent of all women will experience at least one UTI in their lifetime and one in four will have a recurrent UTI within six months (Foxman, 1990). Up to 90% of uncomplicated UTI cases in adults are caused by the extraintestinal pathogen uropathogenic Escherichia coli (UPEC) (Zhang and Foxman, 2003). Oftentimes, the UPEC bacteria originate from the patients' own intestinal flora. Infection begins when the bacteria ascend the urethra from the periurethral area and colonize the bladder leading to cystitis. If left untreated, the infection can ascend the ureters, leading to the development of pyelonephritis and potentially bacteremia.
Bacteria sense and interact with their environment using proteins expressed on their surface. Proteins on the bacterial surface are also likely to be readily accessible to host immune responses, making them attractive vaccine targets to neutralize or eradicate infecting pathogens (Grandi, 2001). The UPEC genome contains numerous surface-expressed virulence factors that aid in the colonization of the urinary tract. UPEC utilizes several surface appendages termed fimbriae or adhesins, such as P, type 1, F1C, S, M, and Dr fimbriae, to colonize the mucosal epithelium and endothelial cells of the urinary tract (Johnson, 1991, Nowicki et al., 1989). Other virulence factors include secreted toxins, such as cytotoxic necrotizing factor 1 (Caprioli et al., 1987), secreted autotransporter toxin (Maroncle et al., 2006) and hemolysin (Smith, 1963, Welch, 1991), as well as numerous iron acquisition systems that aid in survival within the urinary tract by scavenging iron molecules from the host (Opal et al., 1990, Russo et al., 2001, Russo et al., 2002, Torres et al., 2001).
Many proteins associated with the phospholipid lipid membrane layers are hydrophobic and contain numerous transmembrane domains, making them more challenging to study than readily soluble proteins. These technical limitations have hindered the ability to define many membrane proteins at the structural and functional level and have led to an underrepresentation of particular classes of proteins. Recent advances in proteomics technology have led to a better understanding of the microbial outer membrane proteome. The use of carbonate extraction and the strong zwitterionic detergent amido sulfobetaine-14 (ASB-14) allowed for identification of 21 of the 26 predicted outer membrane proteins present in E. coli K-12 (Molloy et al., 2000). Two-dimensional gel electrophoresis (2-DE) has improved the solubilization and separation of proteins and become a method of choice for studying membrane proteins. However, intrinsic properties of 2-DE lead to an underrepresentation of proteins that are highly hydrophobic or highly basic and proteins of low abundance, high molecular weight, or extreme isoelectric points.
Recently, gel-free methods have been developed that overcome many of the problems associated with 2-DE and provide a comprehensive analysis of bacterial membrane proteins (Wu et al., 2003, Wu and Yates, 2003). Gel-free methods utilize two-dimensional liquid chromatography (2-DLC), frequently strong cation exchange in the first dimension followed by reverse-phase chromatography in the second dimension, and tandem mass spectrometry (MS/MS) to separate mixtures of proteins that have been subjected to protease digestion and individually identify them. The 2-DLC-MS/MS approach does not show the bias against membrane proteins which are highly hydrophobic or highly basic, proteins with low abundance, high molecular weight, or extreme isoelectric points typically observed with 2-DE analysis (Cordwell, 2006).
In this report, we examine two methods aimed at identifying the outer membrane proteome of UPEC cultured in pooled human urine. Our initial method attempted to isolate surface-exposed peptides from UPEC through the use of a protease to shave the extracellular domains of outer membrane proteins from the bacterial surface, similar to an approach taken with group A Streptococcus by Rodriguez-Ortega et al. (2006). Bacterial cell lysis proved problematic with this approach, so we utilized a more direct approach and isolated outer membrane proteins by differential centrifugation and identified them with 2-DLC-MS/MS. Sulfo-NHS-SS-Biotin was used to label lysines on extracellular loops of outer membrane proteins to identify domains that may be surface-exposed and available to interact with the host immune system. This technique allowed us to identify 25 predicted outer membrane proteins expressed during growth in human urine, one of which is currently being applied in the development of an UPEC UTI vaccine.
Section snippets
Bacterial strains, media, and growth conditions
CFT073 is a representative UPEC strain that was isolated from the urine and blood of a patient with acute pyelonephritis (Mobley et al., 1990); the genome has been sequenced and fully annotated (Welch et al., 2002). Overnight bacterial cultures were inoculated from isolated colonies and cultured in Luria-Bertani (LB) aerobically at 37 °C. Bacteria from the overnight cultures were centrifuged (3500 ×g, 10 min, 25 °C) to collect the cells, washed with sterile phosphate-buffered saline (PBS), and
Buffer selection for surface-exposed protein trypsin digestion
Initially, buffers used during the trypsin digestion of surface-exposed proteins were tested for the amount of cell lysis occurring during the 30 min incubation at 37 °C by plating samples pre- and post-digestion on Luria agar to determine CFUs (Table 1). The first buffer tested was PBS, pH 7.4 containing 40% sucrose as used by Rodriguez-Ortega et al. in their identification of vaccine candidates from protease digestion of group A Streptococcus surface proteins (Rodriguez-Ortega et al., 2006).
Discussion
Several new technologies for studying bacterial surface proteomics have been recently developed that surmount a number of limitations presented by the traditional method of choice, 2-DE. Identification of surface-exposed portions of the outer membrane proteome may significantly improve the efficacy of a vaccine by focusing on portions of the protein that are exposed to and recognized by the host immune system. The first method attempted to determine surface-exposed domains of outer membrane
Acknowledgements
Proteomics data were provided by the Michigan Proteome Consortium (http://www.proteomeconsortium.org), which is supported in part by funds from the Michigan Life Sciences Corridor (State of Michigan MEDC grant no. GR239). Funding was provided by NIH grant AI-043363 to H.L.T. Mobley.
References (27)
Technologies for bacterial surface proteomics
Curr. Opin. Microbiol.
(2006)- et al.
Urinary tract infection: self-reported incidence and associated costs
Ann. Epidemiol.
(2000) Antibacterial vaccine design using genomics and proteomics
Trends Biotechnol.
(2001)- et al.
Urologic diseases in America project: analytical methods and principal findings
J. Urol.
(2005) - et al.
Cytotoxic necrotizing factor production by hemolytic strains of Escherichia coli causing extraintestinal infections
J. Clin. Microbiol.
(1987) Recurring urinary tract infection: incidence and risk factors
Am. J. Public Health
(1990)- et al.
Uropathogenic Escherichia coli outer membrane antigens expressed during urinary tract infection
Infect. Immun.
(2007) - et al.
Haem acquisition is facilitated by a novel receptor Hma and required by uropathogenic Escherichia coli for kidney infection
Mol. Microbiol.
(2008) Virulence factors in Escherichia coli urinary tract infection
Clin. Microbiol. Rev.
(1991)- et al.
Defining genomic islands and uropathogen-specific genes in uropathogenic Escherichia coli
J. Bacteriol.
(2007)
Protease activity, secretion, cell entry, cytotoxicity, and cellular targets of secreted autotransporter toxin of uropathogenic Escherichia coli
Infect. Immun.
Pyelonephritogenic Escherichia coli and killing of cultured human renal proximal tubular epithelial cells: role of hemolysin in some strains
Infect. Immun.
Proteomic analysis of the Escherichia coli outer membrane
Eur. J. Biochem.
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