The biological reducing agent Oxyrase improves the resuscitation of dormant Mycobacterium smegmatis and Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease, a chronic intestinal disease of cattle and other ruminants. Diagnostic culture for MAP is typically unrewarding until latter stages of the disease. This may be a consequence of oxidative damage to dormant organisms, which results from culture. The purpose of this investigation was to determine the effect of the commercial reducing agent Oxyrase® for Broth (OB) on the recovery of dormant Mycobacterium smegmatis (MS) and MAP. Dormant organisms were inoculated into medium only or that supplemented with media containing serial dilutions of OB. Growth was monitored by optical density for up to 21 days. Treatment of MAP and MS with OB led to significant increases in recovery and growth yield. However, the concentration of OB necessary to promote recovery was dependent on the number of viable organisms present in seed cultures. Mitigating oxidative damage by using OB can facilitate the recovery of dormant mycobacteria. Whether this is a direct or indirect effect has yet to be be established

Tactics of Mycobacterium avium subsp. paratuberculosis for intracellular survival in mononuclear phagocytes

Johne's disease is a condition that refers to chronic granulomatous enteritis in ruminants. It is believed that survival and replication of Mycobacterium (M.) paratuberculosis in mononuclear phagocytes plays an important role in the pathogenesis of Johne's disease. However, it is not clear how M. paratuberculosis survives for long time periods in mononuclear phagocytes, nor is it clear which factors trigger multiplication of these bacilli and result in the development of Johne's disease. Investigating the intracellular fate of M. paratuberculosis is challenging because of its very slow growth (more than two months to form visible colonies on media). Existing animal models also have limitations. Despite those obstacles, there has been progress in understanding the intracellular survival tactics of M. paratuberculosis and the host response against them. In this review, we compare known aspects of the intracellular survival tactics of M. paratuberculosis with those of other mycobacterial species, and consider possible mycobactericidal mechanisms of mononuclear phagocytes

Conversion of Commensal Escherichia coli K-12 to an Invasive Form via Expression of a Mutant Histone-Like Protein

The HUαE38K, V42L mutant of the bacterial histone-like protein HU causes a major change in the transcription profile of the commensal organism Escherichia coli K-12 (Kar S, Edgar R, Adhya S, Proc. Natl. Acad. Sci. U. S. A. 102:16397–16402, 2005). Among the upregulated genes are several related to pathogenic interactions with mammalian cells, as evidenced by the expression of curli fibers, Ivy, and hemolysin E. When E. coli K-12/ HUαE38K, V42L was added to Int-407 cells, there was host cell invasion, phagosomal disruption, and intracellular replication. The invasive trait was also retained in a murine ileal loop model and intestinal explant assays. In addition to invasion, the internalized bacteria caused a novel subversion of host cell apoptosis through modification and regulation of the BH3-only proteins BimEL and Puma. Changes in the transcription profile were attributed to positive supercoiling of DNA leading to the altered availability of relevant promoters. Using the E. coli K-12/HUαE38K, V42L variant as a model, we propose that traditional commensal E. coli can adopt an invasive lifestyle through reprogramming its cellular transcription, without gross genetic changes

The Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein, a toll-like receptor 4 agonist, enhances dendritic cell-based cancer vaccine potency

In this study, we showed the direct interaction between Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein (FAP) and toll-like receptor4 (TLR4) via co-localization and binding by using confocal microscopy and co-immunoprecipitation assays. FAP triggered the expression of pro- and anti-inflammatory cytokines in a TLR4-dependent manner. In addition, FAP-induced cytokine expression in bone marrow-derived dendritic cells (BMDCs) was modulated in part by glycogen synthase kinase-3 (GSK-3). FAP-induced expression of CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II in TLR4+/+ BMDCs was not observed in TLR4-/- BMDCs. Furthermore, FAP induced DC-mediated CD8+ T cell proliferation and cytotoxic T lymphocyte (CTL) activity, and suppressed tumor growth with DC-based tumor vaccination in EG7 thymoma murine model. Taken together, these results indicate that the TLR4 agonist, FAP, a potential immunoadjuvant for DC-based cancer vaccination, improves the DC-based immune response via the TLR4 signaling pathway

TGF-β-Mediated Sustained ERK1/2 Activity Promotes the Inhibition of Intracellular Growth of Mycobacterium avium in Epithelioid Cells Surrogates

Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth

The role of the fibronectin -attachment protein of Mycobacterium avium subsp. paratuberculosis in the pathogenesis of Johne\u27s disease

Fibronectin (FN)-attachment proteins (FAPs) are a family of mycobacterial proteins that have been shown to facilitate FN-mediated attachment and internalization of mycobacteria in vitro. The purpose of the present study was to investigate the role that FAP plays in the attachment and invasion of M cells in the dome epithelium covering Peyer\u27s patches by Mycobacterium avium subsp. paratuberculosis (Mpt). Fibronectin binding by Mpt was activated by acid treatment, demonstrating the physiologic relevance of FN binding as a potential virulence factor of Mpt. Mycobacterium avium subsp. paratuberculosis expresses a FAP, designated FAP-P, that is necessary for FN binding by this organism. However, this protein is not present on the surface of the organism, suggesting that one or more proteins cooperate with FAP-P to enable FN binding by Mpt in addition to FAP-P. Fibronectin enhanced attachment and ingestion of Mpt by Caco-2 and T-24 cells. Blocking with the FN binding peptide from M. avium subsp. avium FAP did not impede Mpt attachment to these cells, but virtually eliminated bacterial ingestion. Attachment and invasion of epithelial cells in vitro were significantly reduced in Mpt strains carrying antisense FAP-P mutations, indicating that these phenomena were FN-dependent processes that were mediated by FAP-P. Mycobacterium avium subsp. paratuberculosis selectively invaded M cells in vivo, and this process was enhanced in the presence of FN. Preferential invasion of M cells in vivo was eliminated in Mpt carrying antisense FAP-P mutations. Co-injection of the RGD cell-binding or synergy site peptides from FN or α5, αV, β1 or β3 integrin subunit blocking antibodies with Mpt into murine gut loops significantly impaired the preferential invasion of M cells by Mpt. This demonstrated that FN-opsonized Mpt specifically bound to integrins displayed on the lumenal surface of M cells. The results indicate that FAP-P dictates M cell targeting and invasion by Mpt by using FN to form a bridge with M cell surface integrins

Mycobacterium avium subsp. paratuberculosis Fibronectin Attachment Protein Facilitates M-Cell Targeting and Invasion through a Fibronectin Bridge with Host Integrins

Efficient attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by cultured epithelial cells requires the expression of a fibronectin (FN) attachment protein homologue (FAP-P) which mediates FN binding by M. avium subsp. paratuberculosis. Invasion of Peyer's patches by M. avium subsp. paratuberculosis occurs through M cells, which, unlike other intestinal epithelial cells, express integrins on their luminal faces. We sought to determine if the interaction between FAP-P of M. avium subsp. paratuberculosis and soluble FN enabled targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo via these surface integrins. Wild-type and antisense FAP-P mutant M. avium subsp. paratuberculosis strains were injected alone or coinjected with blocking peptides or antibodies into murine gut loops, and immunofluorescence microscopy was performed to assess targeting and invasion of M cells by M. avium subsp. paratuberculosis. Nonopsonized M. avium subsp. paratuberculosis preferentially invaded M cells in murine gut loops. M-cell invasion was enhanced 2.6-fold when M. avium subsp. paratuberculosis was pretreated with FN. Invasion of M cells by the antisense FAP-P mutant of M. avium subsp. paratuberculosis was reduced by 77 to 90% relative to that observed for the control strains. Peptides corresponding to the RGD and synergy site integrin recognition regions of FN blocked M. avium subsp. paratuberculosis invasion of M cells by 75 and 45%, respectively, whereas the connecting segment 1 peptide was noninhibitory. Antibodies against the α5, αV, β1, and β3 integrin subunits inhibited M-cell invasion by 52 to 73%. The results indicate that targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo is mediated primarily by the formation of an FN bridge formed between FAP-P of M. avium subsp. paratuberculosis and integrins on M cells