Immunotherapy Of Multidrug-resistant Mycobacterium Tuberculosis Infection And Study Of Clostridium Difficile Toxins’ Domain Functions And Their Role In Pathogenesis

Mycobacterium tuberculosis and Clostridium difficile are both infectious bacteria threatening public health. In this study, we are focusing on the immunotherapy of M. tubercosis infection and the domain functions and pathogenesis of C. difficile toxins.Tuberculosis (TB) is prevalent globally. In China, it is one of the most threatening epidemic diseases. The emergence of multi-drug resistant TB (MDR-TB) makes it difficult to treat TB because the current regimens failed to clear up MDR-TB in patients. New therapeutic regimens to treat TB are urgently needed due to the emergence of multidrug resistant tuberculosis (MDR-TB). Cytokine-based regulation of host immune response is likely to be one of the available methods for clinical treatment of tuberculosis. In this study, we investigated the therapeutic effects of IL-2and GM-CSF co-administrated with INH (isoniazid) and RIP (rifampin) to treat TB in mouse model of TB infection. We first evaluated the effective regimens in drug susceptible M. tuberculosis H37Rv infected mice based on bacterial counts in lungs and spleens and the degree of histopathology in the lungs. Compared with untreated group, both IL-2and GM-CSF monotherapy reduced the bacterial number in the lungs by0.82(P<0.01) and0.58(P<0.05) log10respectively while in spleens by1.42(P<0.01) and1.22(P<0.01) log10respectively. Although the combination of chemical drugs and single cytokine regimens in mice did not show any advantage in terms of reducing the CFU, the lungs had much less lesions compared with the chemotherapy alone group. Next, the effects of immunotherapy regimens were assessed for MDR-TB strain OB35infected mice by detecting mean body weight and survival, bacterial counts in lungs and spleens, and histopathological changes in the lungs. Compared with untreated control and chemotherapy alone groups, immunotherapy with either cytokine had a significant reduction of CFU in lungs and spleens. The pathological changes of lungs from immunotherapy groups were also less severe and the lesions were limited compared with that of the control mice. Notably, the chemotherapy in combination with both cytokines group (IR-IL-2-GM-CSF) reduced the lung and spleen bacterial loads by1.02,1.34log10, respectively compared with chemotherapy alone. Additionally, the average body weights, survival rates, and histopathological results also revealed the synergistic effect of IL-2and GM-CSF when co-administrated with INH-RIF (HR).Clostridium difficile is one of the leading causes of nosocomial diarrhea associated with antibiotic treatment in developed countries. The increasing morbidity and mortality of C. difficile infection (CDI) have attracted more attentions globally. The emergence of hypervirulent antibiotic-resistant strains contributes the recently sharp increase of incidence and severity of CDI. The pathogenesis of CDI is not well-understood. Two exotoxins, TcdA and TcdB, which belong to large clostridia toxin family, are thought to be the main causes of disease. Both toxins are multi-domain proteins and intoxicate target cells by glucosylating Rho GTPases. The toxins consist of at least4functional domains including a glucosyltransferase (GT) domain, a cystine protease domain (CPD), a transmembrane domain (TD), and a receptor binding domain (RBD). The functions of these molecular domains and their roles in pathogenesis of disease are not fully understood; especially the TD, which is the largest segment of toxins and comprises nearly50%of the protein, is rarely investigated. There are two research objectives in this study:to investigate domain function and to determine toxins’role in the pathogenesis of the disease. As for the first objective, we investigated the functions of toxin domains in their molucular action and disease manifestation. We identified that a97-amino-acid fragment (D97), located in the C-terminus of the TD, is essential for the cellular activity of TcdB. We deleted D97and expressed a truncated protein (designated TxB-D97), and found that the deletion did not appear to affect the functions of the other domains. The cell binding and uptake were similar between the mutant and wild type TcdB. Both wild type and mutant toxins released their GT domains similarly in the presence of inositol hexakisphosphate (Insp6), and showed a similar intrinsic glucosyltransferase activity in a cell-free glucosylating assay. Despite these, the cytopathic and cytotoxic activities of TxB-D97were reduced by more than5logs compared to wild type toxin and the mutant toxin failed to glucosylate Rho GTPase Racl of intact cells. Unlike wild type TcdB, the mutant toxin failed to induce macrophages to produce tumor necrosis factor alpha (TNF-a), an outcome dependent on the GT activity of the toxin. Cellular fractionation demonstrated that the TxB-D97was unable to liberate its GT domain efficiently into cytosol. Our data demonstrated that the D97fragment, located in the C-terminus of the TD, adjacent to the RBD, is essential for the delivery of the GT domain into the cell cytosol. In order to define the enterotoxicity of the toxins within their molecular domain(s), we generated several chimeric toxins by switching domains between the two toxins. We examined the enterotoxicity of those chimeric toxins in a well-established mouse ileal loop model. The enterotoxicity of these chimeric toxins was defined by their abilities to cause fluid accumulation, induce the production of proinflammatory cytokines and influx of neutrophils, and destruct intestinal epithelia. Chimera TxdA-Br with the RBD of TcdB induced mouse intestinal tissue damage, numerous neutrophil infiltration, inflammatory cytokine production and fluid accumulation in ileum loops whereas chimera TxdB-Ar with the RBD of TcdA failed to induced any enterotoxic response. Meanwhile, glucosyltransferase-deficient mutant TcdA was essentially lost their enterotoxicity. These data indicate that the enterotoxicity of TcdA is not determined by its RBD. For the second research objective, we explored the differential role of TcdA and TcdB in CDI pathogenesis. We established a Bacillus megaterium oral challenged mouse model. B. megaterium was used as a surrogate host to express individual toxins, allowing their delivery into mouse gastrointestinal tracts. Utilizing this model, we found that B. megaterium expressing TcdB cause disease and death independent of TcdA or any other cofactors from C. difficile. TcdB caused severe lesions not only in intestinal tissues but also in lung, which is likely directly caused by systemic TcdB after its liberation into circulation. Compared to TcdB, TcdA appeared to be a more potent enterotoxin by inducing severe lesions and inflammatory response in intestine, although it can also liberate into circulation. Pro-inflammatory cytokines IL-1, IL-6, KC and TNF-a were significantly elevated in the serum and pleural fluid of mouse challenged with B. megaterium expressing TcdA, suggesting this toxin is highly pro-inflammatory.Conclusions:For the study of immunotherapy of Mycobacterium tuberculosis infection, we conclude that IL-2and GM-CSF are potential immunomodulators to enhance the effects of chemical drugs to treat MDR-TB, which suggests that the immunotherapy with combination cytokines is likely to be a promising strategy for tuberculosis treatment in the future. For the study of Clostridium difficile toxin study, we found that:(1) D97is essential for delievery of GT domain of TcdB into cytosol of target cells;(2) RBD was not responsible for enterotoxicity and GT was required for enterotoxicity;(3) TcdA and TcdB alone caused epithelial barrier dysfunction both in vivo and in vitro, subsequently increased paracellular flux and toxin translocation;(4) Although TcdB was not an exactly defined enterotoxin, independent of TcdA or other factors from C. difficile, TcdB caused disease in vivo;(5) Both toxins liberated to circulation, resulting in toxemia and severe and fatal systemic disease;(6) TcdB is less pro-inflammatory in comparison to TcdA;(7) TcdB have potent systemic toxicity and is able to cause both intestinal and systemic destructions. These results are useful to further understand the pathogenesis of these toxins in vivo and supply new evidences for study of pathological model.