A novel Lawsonia intracellularis autotransporter protein is a prominent antigen

Abstract

Investigation of antigenic determinants of the microaerophilic obligate intracellular bacterium Lawsonia intracellularis using a mass spectrometry approach identified a novel bacterial protein present in an extract of cell culture medium aspirated from heavily infected in vitro cell cultures. Western immunoblotting analysis of SDS-PAGE-resolved proteins using immune sera pooled from L. intracellularis-infected pigs revealed the presence of a strongly immunoreactive band of ∼ 72 kDa. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of this component and database mining using a fully annotated L. intracellularis genome sequence and the comprehensive GenBank prokaryotic genomic database highlighted the presence of a protein that shares little sequence similarity with other prokaryotic proteins and appears to be highly species specific. Detailed bioinformatic analyses identified the protein as member of the autotransporter protein family of surface-exposed proteins, and the designation LatA (Lawsonia autotransporter protein A) is suggested. Recognition of recombinant LatA on Western blots by a panel of sera from infected and control pigs corresponded 100% with a commercial serodiagnostic that relies on in vitro culture of this fastidious organism. LatA therefore represents a potential candidate for the development of a rapid and species-specific serodiagnostic reagent.

Fig. 1.

Recognition of L. intracellularis proteins by sera from infected pigs. Shown is a Western blot of L. intracellularis protein extract probed with sera pooled from 10 pigs naturally infected with L. intracellularis (lane A) and the corresponding SDS-PAGE gel (lane B). A protein of approximately 72 kDa was strongly recognized by sera, and the corresponding region of the SDS-PAGE gel was excised and analyzed by LC-ESI-MS/MS.

Fig. 2.

Schematic diagram showing the predicted features of LatA, including the signal sequence (amino acids 1 to 31), the passenger domain (amino acids 32 to 546), the ArgE/DapE/ACY1/CPG2/YscS-like metallopeptidase motif (amino acids 227 to 236; IPR001261), the α-helical linker domain (amino acids 556 to 583), and the β-barrel domain (amino acids 587 to 851). A putative structural model of the LatA β-barrel domain was generated using the Phyre program.

Fig. 3.

Recognition of purified rLatA by pig sera. Western blots were probed with sera from infected animals (numbers 1 to 9) or uninfected animals (numbers 10 to 16). Sera from infected animals reacted specifically with rLatA, whereas reactivity of sera from uninfected animals was negligible. Induction of specific antibodies indicates that LatA is expressed during in vivo infection with L. intracellularis.