A candidate subunit vaccine induces protective immunity against Mycobacterium avium subspecies paratuberculosis in mice.

Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis (PTB), which is a granulomatous enteritis in ruminants that threatens the dairy industry's healthy development and public health safety worldwide. Because the commercial inactivated vaccines are not completely protective and interfere with bovine tuberculosis diagnostics, we tested four fusion proteins, namely 66NC, 66CN, 90NC, and 90CN, which were constructed with MAP3527, Ag85B, and Hsp70 of MAP in different tandem combinations. Notably, 66NC, which encodes a 66 kDa fusion protein that combines in linear order MAP3527N40-232, Ag85B41-330, and MAP3527C231-361, induced a powerful and specific IFN-γ response. Immunization of C57BL/6 mice with the 66NC fusion protein formulated in Montanide ISA 61 VG adjuvant generated robust Th1, Th2, and Th17 type immune responses and strong antibody responses. The 66NC vaccine protected C57BL/6 mice against virulent MAP K-10 infection. This resulted in a reduction of bacterial load and improvement of pathological damage in the liver and intestine, in addition to a reduction of body weight loss; significantly better protection than the reported 74 F vaccine was also induced. Furthermore, vaccine efficacy correlated with the levels of IFN-γ-, TNF-α-, and IL-17A-secreting antigen-specific CD4+ and CD8+ T lymphocytes as well as with serum IFN-γ and TNF-α levels after vaccination. These results demonstrate that recombinant protein 66NC is an efficient candidate for further development into a protective vaccine in terms of inducing specific protection against MAP.

Mycobacterium tuberculosis protease Rv3090 is associated with late cell apoptosis and participates in organ injuries and mycobacterial dissemination in mice.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). Mtb can overcome macrophage intracellular killing and lead to persistent infections. The proteases of Mtb are critical virulence factors that participate in immune responses. We determined that Rv3090 is a cell wall-associated protease and a potential pathogenic factor. To characterize the role of Rv3090 in Mtb, recombinant Msg_Rv3090 and Msg_pAIN strains were constructed to infect macrophages and mice. Lactate dehydrogenase assays and flow cytometry results showed that Rv3090 induces late macrophage apoptosis. In vivo infection experiments indicated that Rv3090 could induce hepatocyte and lung cell apoptosis and cause pathological damage to the spleen, livers and lungs. Msg_Rv3090 specifically stimulated the secretion of inflammatory cytokines including TNF-α, IL-6 and IL-1ß. Overexpression of Rv3090 significantly promoted the survival of Msg in livers and lungs. Thus, Rv3090 protease triggered late cell apoptosis and contributed to the pathogenicity and dissemination of Mtb.

Extracellular DNase MAP3916c attacks the neutrophil extracellular traps and is needed for Mycobacterium avium subsp. paratuberculosis virulence.

Extracellular DNases/nucleases are important virulence factors in many bacteria. However, no DNase/nucleases have been reported in Mycobacterium avium subsp. paratuberculosis (MAP), which is a pathogen of paratuberculosis. Genome analyses of MAP K-10 revealed that the map3916c gene putatively encodes a nuclease. In this study, we show that MAP3916c is an extracellular nonspecific DNase requiring a divalent cation, especially Mg2+. The optimum DNase activity of MAP3916c was exhibited at 41 °C and pH 9.0. Site-directed mutagenesis studies indicated that 125-Histidine is necessary for MAP3916c DNase activity. In addition, MAP3916c DNase could destroy the neutrophil extracellular traps (NETs) induced by Phorbol 12-myristate 13-acetate in vitro and degrade the NETs induced by MAP K-10 upon infection. Furthermore, MAP3916c DNase promoted the colonization of MAP K-10, induced the formation of granulomas in the liver and small intestine and promoted the release of IL-1ß, IL-6 and TNF-α inflammatory cytokines during the infection of mice. These results indicated that MAP3916c is relevant to NETs escape and the pathogenicity of MAP. It also provides a basis for further study of the function of nuclease activity on the MAP immune evasion.

NanoFe3O4 accelerates anoxic biodegradation of 3, 5, 6-trichloro-2-pyridinol.

3, 5, 6-trichloro-2-pyridinol (TCP) is a widespread organic pollutant with persistent, mobile and high antimicrobial effects. Here, nanoFe3O4 was firstly introduced into the anoxic biodegradation of TCP. It was found that nanoFe3O4 significantly accelerated TCP biodegradation. The removal rate of TCP (100 mg L-1) increased from 83.03% to 98.74% within 12 h in the presence of nanoFe3O4, and the addition of nanoFe3O4 also promoted the accumulation of CO2. Reductive dechlorination mechanism was involved in anoxic biodegradation of TCP. Molecular approaches further revealed that nanoFe3O4 distinctly induced the shifts of bacterial community. The dominant genus Ochrobactrum was converted to genus Delftia in nanoFe3O4 treatment, and the relative abundance of Delftia increased from 10.26% to 44.62%. Meanwhile, the total relative abundance of bacteria related to TCP dechlorination and degradation significantly increased in the presence of nanoFe3O4. These results indicated that nanoFe3O4 induced the enrichment of TCP-degrading bacteria to promote the anoxic biodegradation of TCP.

Phospholipid transfer protein (PLTP) deficiency attenuates high fat diet induced obesity and insulin resistance.

Increased phospholipid transfer protein (PLTP) activity has been found to be associated with obesity, and metabolic syndrome in humans. However, whether or not PLTP has a direct effect on insulin sensitivity and obesity is largely unknown. Here we analyzed the effect by using PLTP knockout (PLTP-/-) mouse model. Although, PLTP-/- mice have normal body-weight-gain under chow diet, these mice were protected from high-fat-diet-induced obesity and insulin resistance, compared with wild type mice. In order to understand the mechanism, we evaluated insulin receptor and Akt activation and found that PLTP deficiency significantly enhanced phosphorylated insulin receptor and Akt levels in high-fat-diet fed mouse livers, adipose tissues, and muscles after insulin stimulation, while total Akt and insulin receptor levels were unchanged. Moreover, we found that the PLTP deficiency induced significantly more GLUT4 protein in the plasma membranes of adipocytes and muscle cells after insulin stimulation. Finally, we found that PLTP-deficient hepatocytes had less sphingomyelins and free cholesterols in the lipid rafts and plasma membranes than that of controls and this may provide a molecular basis for PLTP deficiency-mediated increase in insulin sensitivity. We have concluded that PLTP deficiency leads to an improvement in tissue and whole-body insulin sensitivity through modulating lipid levels in the plasma membrane, especially in the lipid rafts.

Degradation of 3,5,6-trichloro-2-pyridinol by a microbial consortium in dryland soil with anaerobic incubation.

Biodegradation of 3,5,6-trichloro-2-pyridinol (TCP) in drylands is an important biological process of detoxification. Flooding in drylands can result in the formation of anaerobic habitats. However, little is known about the microbial metabolism of TCP in dryland soil under anaerobic conditions. Here, chlorpyrifos-contaminated dryland soil was incubated to enrich the TCP-degrading microbial consortium under anaerobic conditions. Chloridion and CO2 were released with TCP degradation, and the enrichment cultures of dryland soil could metabolize 97% of TCP (100 mg/L) within 20 h. Both reductive and hydrolysis dechlorination mechanisms were involved in TCP biodegradation under anaerobic conditions. Bacterial taxonomic analysis revealed that the aerobic TCP-degrading bacteria Ochrobactrum and dechlorination bacteria Delftia were the dominant genera. Anaerobic and facultative bacteria; i.e., Bacteroides, Bacillus, and Cupriavidus had lower relative abundances, but they were significantly enriched following treatment with TCP. These results indicate that the enrichment cultures of dryland soil dominated by aerobic bacteria could dechlorinate and degrade TCP under anaerobic conditions.