Bacterial Uptak

The study of host immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) is critical to the identification and de­velopment of tools to control and manage the disease.

The primary route of transmission is through ingestion of faecal matter, or milk and colos­trum containing MAP. It is generally thought that neonates are most susceptible to infection due to their undeveloped immune system com­bined with the high level of exposure to MAP, especially if their dams are clinically infected (Larsen et al., 19 75; Smith et al., 2015). By nature, MAP is an enteric pathogen, preferen­tially homing to regions of the small intestine to initiate the infectious process. Lymphoid ag­gregates such as gut-associated lymphoid tis­sue (GALT) line the mucosa of the intestines and are key components of the infectious pro­cess. The most critical GALT lymphoid aggre­gates involved in MAP infection are the Peyer's patches.

‘Corresponding author: judy.stabel@ars.usda.gov

© CAB International 2020. Paratuberculosis: Organism, Disease, Control, 2nd Edition

(eds M.A. Behr et al.)

allowing endocytosis of antigens (Featherstone, 199 7). Transcytosis of antigens through the cell then occurs, engaging with macrophages or dendritic cells (DCs) on the basolateral side. The process of MAP uptake in the small intestine was first defined in a pioneering study using ileal loops of neonatal calves, in which it was found that MAP was preferentially taken up by M cells and then by subepithelial and intraepithelial macrophages within the domes of the Peyer's patches (Momotani et al., 1988). Additional studies using the everted sleeve method demon­strated that uptake of MAP by intestinal mucosa of goat kids was not restricted to M cells but also included regional enterocytes (SigurSardottir et al., 2004, 2005). This was further corrobo­rated more recently wherein the presence of M cells was evaluated in a temporal study of MAP uptake within loops of Peyer's patch and non­Peyer's patch ileum and jejunum from lambs (Ponnusamy et al., 2013). Results demonstrated that MAP was able to invade both M cells and enterocytes, but translocation was greater for M cells in both ileum and jejunum. Furthermore, uptake of MAP was more pronounced for ileal loops compared with the jejunal regions of the small intestine. Uptake of MAP has experimen­tally been shown to occur as rapidly as 30 min post-inoculation (SigurSardottir et al., 2001), with colonization of distant organs such as liver and lymph nodes occurring within 1 h.

A high density of B1 integrin receptors is noted on the apical membrane of the M cell, whereas integrin receptors are located more exclusively on the basolateral side of entero- cytes, suggesting a probable mechanism for preferential uptake of MAP by M cells. The role of integrin receptors in the uptake of MAP was demonstrated after pre-opsonization of MAP with fibronectin increased uptake by M cells 2.6-fold compared with non-opsonized MAP (Secott et al., 2001). Further, uptake of MAP by M cells was inhibited 52-73% following treatment of fibronectin-opsonized MAP with monoclonal antibodies directed against inte- grin subunits a5, aV, b1 and B3 (Secott et al., 2004). In addition to integrins, other pathogen recognition receptors (PRRs) such as Toll-like receptors (TLRs) and platelet-activating factor receptor are involved in the recognition of bacte­rial pathogens (Tyrer et al., 2006). Cell-surface PRRs such as TLR2 and TLR4 recognize micro­bial components such as lipoproteins (TLR2) and lipopolysaccharide (LPS) (TLR4) upon infec­tion and act as ‘gatekeepers' to the intestinal epi­thelium, including M cells (Chabot et al., 2006). In MAP infection, gene expression of TLR2 and TLR4 was upregulated in the ileum and jejunum of naturally infected sheep compared with non­infected animals, with increasing TLR expres­sion correlating with bacterial load in the tissue (Taylor et al., 2008). Interestingly, divergent expression of TLR2 and TLR4 was observed on peripheral blood mononuclear cells (PBMCs) in early and late stages of infection, respectively, in sheep that ultimately progressed to multibacil- lary disease (Thirunavukkarasu et al., 2013). In addition, blocking of TLR2 expression in RAW

264.7 macrophages upon exposure to MAP pre­cluded the expression of nitric oxide, suggesting that TLR2 may be a critical factor in controlling early infection in the host.

2009). Using an in vitro model of infection, greater than 6-fold upregulation of MAP3464 gene was observed in MDBK cells within 30 min of exposure to live MAP (Alonso-Hearn et al., 2008). The MAP3464 gene is purported to be part of the ABC transporter family, a series of genes acting as membrane pumps in biological membranes. The composition of the cell wall of MAP has been implicated in the efficiency of invasion of epithelial cells (Alonso-Hearn et al.,

2010). A mycobacterial LuxR transcriptional regulator controlling the expression of genes such as FadE8, MAP1420, LprL and MAP1203 demonstrated that envelope genes could control the entry of MAP into cells. Further studies with MAP modified with a pJAM:MAP1203 plasmid showed that invasion of bovine MDBK cells was enhanced compared with pJAM MAP controls (Everman et al., 2018). MAP1203 contains an arginine-glycine-aspartic acid (RGD) peptide motif that facilitates the binding and invasion of microorganisms (Everman et al., 2018). The role of bacterial factors and host receptors in the initial uptake of MAP by epithelial cells re­quires further study, as it conceptually repre­sents a point in the infection process amenable to intervention.

17.3