Quantitative proteomic analysis of type III secretome of enteropathogenic Escherichia coli reveals an expanded effector repertoire for attaching/effacing bacterial pathogens.
Type III secretion systems are central to the pathogenesis and virulence of many important Gram-negative bacterial pathogens, and elucidation of the secretion mechanism and identification of the secreted substrates are critical to our understanding of their pathogenic mechanisms and developing potential therapeutics. Stable isotope labeling with amino acids in cell culture-based mass spectrometry is a quantitative and highly sensitive proteomics tool that we have previously used to successfully analyze the type III secretomes of Citrobacter rodentium and Salmonella enterica serovar Typhimurium. In this report, stable isotope labeling with amino acids in cell culture was used to analyze the type III secretome of enteropathogenic Escherichia coli (EPEC), an important human pathogen, which, together with enterohemorrhagic E. coli and C. rodentium, represents the family of attaching and effacing bacterial pathogens. We not only confirmed all 25 known EPEC type III-secreted proteins and effectors previously identified by conventional molecular and bioinformatical techniques but also identified several new type III-secreted proteins, including two novel effectors, C_0814/NleJ and LifA, that were shown to be translocated into host cells. LifA is a known virulence factor believed to act as a toxin as well as an adhesin, but its mechanism of secretion and function is not understood. With a predicted molecular mass of 366 kDa, LifA is the largest type III effector identified thus far in any pathogen. We further demonstrated that Efa1, ToxB, and Z4332 (homologs of LifA in enterohemorrhagic E. coli) are also type III effectors. This study has comprehensively characterized the type III secretome of EPEC, expanded the repertoire of type III-secreted effectors for the attaching and effacing pathogens, and provided new insights into the mode of function for LifA/Efa1/ToxB/Z4332, an important family of virulence factors.
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
Wanyin
W
Deng
Hong B
HB
Yu
Carmen L
CL
de Hoog
Nikolay
N
Stoynov
Yuling
Y
Li
Leonard J
LJ
Foster
B Brett
BB
Finlay
D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D058947:Bacterial Secretion Systems; D001427:Bacterial Toxins / Q000302:isolation & purification / Q000378:metabolism; D054308:Enteropathogenic Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D007553:Isotope Labeling; D020543:Proteome
D058947:Bacterial Secretion Systems; D001427:Bacterial Toxins; C404907:Efa1 protein, E coli; D029968:Escherichia coli Proteins; D020543:Proteome; C406587:lymphostatin protein, E coli; C439629:toxB protein, E coli