Expression by Lawsonia intracellularis of type III secretion system components during infection.

Contact-dependent secretion systems, such as the type III secretion system (T3SS), have been shown to play significant roles in the pathogenicity of many gram-negative bacterial pathogens. Lawsonia intracellularis is a novel, obligate intracellular gram-negative bacterium, which has been identified as the etiological agent of proliferative enteropathies in numerous animal species. Analysis of the genome sequence of the L. intracellularis strain PHE/MN1-00 has revealed the presence of a T3SS secretion system in this bacterium. In this study we aimed to determine whether this important virulence mechanism is also present in L. intracellularis strain LR189/5/83. Using a PCR-based approach, we verified the presence of a genomic region encoding a T3SS. Specifically, a gene highly homologous to the yscN energiser component of the prototypic T3SS of Yersinia spp. was identified and termed lscN. Two further open reading frames (ORFs) contiguous with lscN were also identified: lscO and lscQ, which are also homologues of ORFs within the T3SS of Yersinia spp. To establish whether this T3SS may be functional, expression was monitored directly by RT-PCR and indirectly by detection of serological responses in vaccinated and infected animals. Transcripts for lscN and lscQ were detected and purified rLscQ was recognized by antiserum from infected pigs, indicating expression in vivo during infection. By analogy to other bacteria, this T3SS may be crucial for intracellular development and is likely to play a significant role in the virulence of this unusual pathogen.

LsaA, an antigen involved in cell attachment and invasion, is expressed by Lawsonia intracellularis during infection in vitro and in vivo.

Lawsonia intracellularis has been identified recently as the etiological agent of proliferative enteropathies, which are characterized by intestinal epithelial hyperplasia and associated moderate immune responses. This disease complex has been reported in a broad range of animals, prevalently in pigs, and L. intracellularis has been linked with ulcerative colitis in humans. L. intracellularis is an obligate intracellular bacterium, and the pathogenic mechanisms used to cause disease are unknown. Using in vitro-grown organisms as a source of genomic DNA, we identified a Lawsonia gene which encodes a surface antigen, LsaA (for Lawsonia surface antigen), associated with attachment to and entry into cells. The deduced amino acid sequence of this protein showed some similarity to members of a novel protein family identified in a number of other bacterial pathogens but for which roles are not fully defined. Transcription of this gene was detected by reverse transcription-PCR in L. intracellularis grown in vitro in IEC18 cells and in bacteria present in ileal tissue from infected animals. Immunohistochemistry with specific monoclonal antibody and immunoblotting with sera from infected animals demonstrated that LsaA protein is synthesized by L. intracellularis during infection. Expression of this gene during infection in vitro and in vivo suggests that this surface antigen is involved during infection, and phenotypic analysis indicated a role during L. intracellularis attachment to and entry into intestinal epithelial cells