Dr. Secott's Research Projects
Johne's disease, or paratuberculosis, is an intestinal disease of ruminants that has continued to plague the US dairy industry. This condition is a result of chronic infection by the acid-fast bacillus known as Mycobacterium avium subsp paratuberculosis (Mpt). The progression of disease is subtle: although animals are usually infected within the few weeks of life, the chronic wasting and profuse diarrhea that indicate the development of clinical paratuberculosis usually don't appear until 3 to 5 years after infection. The relatively high national prevalence of paratuberculous cattle may have public health consequences, as Mpt has been implicated as one of the etiologic agents of Crohn's disease in humans.
We have suggested that one of the keys to controlling and preventing Johne's disease involves an understanding of the very early events in pathogenesis. Specifically, if the mechanisms by which Mpt attaches to intestinal epithelial cells and is translocated to underlying tissues can be identified, one can begin to develop strategies that may prevent or limit Mpt infection.
Mpt has been shown to traverse the intestinal epithelium through M cells, which are present in the follicle associated epithelium covering intestinal lymphoid tissue known as Peyer's patches, wherein the organism replicates. M cells are unique among intestinal mucosa cells in that they express FN-binding integrins on their apical surfaces. The ability of microorganisms to bind host fibronectin (FN). We have found that Mpt expresses a protein that belongs to the fibronectin-attachment protein family. This protein, designated FAP-P, is necessary for FN binding. Interestingly, experimental evidence indicated that FAP-P is not present on the surface of the organism. We have also found that invasion of cultured epithelial cells by Mpt is a FN-dependent process that requires the expression of FAP-P. Further, the targeting and selective invasion of M cells by Mpt in murine gut loops in vivo occurs as a result of a soluble FN bridge that links Mpt (through FAP-P) to M cells (through FN-binding integrins).
The fact that FAP-P is not exposed on the surface of Mpt may indicate that other bacterial surface components make the initial contact with FN and either propagate a signal that leads to the translocation of FAP-P to the surface of Mpt, or shuttle FN inward through the bacterial cell envelope to FAP-P. Which of these scenarios - if either - explains how FAP-P engages FN, and how are these proteins and processes regulated? The possibility that multiple proteins act in concert to bind FN would suggest that there may be several points at which the FN-Mpt interaction can be interrupted, thereby greatly reducing the likelihood of infection. Identification of FN motifs recognized by the FN binding proteins of Mpt may lead to the design of inhibitors that can be used can prevent or limit the spread of Johne's disease.
We observed that Mpt invaded non-M intestinal epithelial cells significantly less often than M cells, but in a manner that was largely opsonin-independent. By what means does Mpt recognize and invade these cells? What other gene products contribute to the invasive capacity of Mpt?
Fecal culture is a very reliable means of detecting Mpt infection in cattle with clinical paratuberculosis. However, detection of asymptomatically infected cattle by fecal culture is considerably less accurate. It may simply be due to the fact that these animals are shedding organisms below the threshold of detection. On the other hand, it may be that the organisms shed cannot survive the decontamination process or grow in the presence of the inhibitors used in selective culture media. This phenomenon - viable but not culturable, or VBNC - was observed in studies of tissues from patients with latent tuberculosis in the 1950s. Research by several groups of investigators has shown that mycobacteria, including Mpt can enter a dormant state when oxygen or nutrients become limiting. Some strains of Micrococcus luteus synthesize a protein known as resuscitation protection factor (RPF) when grown in a nutrient-poor medium. This protein has been shown to promote the growth of low numbers of several species of Gram-positive organisms, including Mycobacterium bovis BCG. The genomes of pathogenic mycobacteria, including Mpt, contain open reading frames that share varying degrees of homology with RPF. Does Mpt synthesize a protein such as RPF that enables dormant organisms to resume active growth and contribute to the progression from asymptomatic carriage to clinical disease? Could this factor be exploited to improve the accuracy of fecal culture?