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.

Re-burying Artificially Exposed Surface of Viral Subunit Vaccines Through Oligomerization Enhances Vaccine Efficacy.

Viral subunit vaccines often suffer low efficacy. We recently showed that when taken out of the context of whole virus particles, recombinant subunit vaccines contain artificially exposed surface regions that are non-neutralizing and reduce their efficacy, and thus these regions need to be re-buried in vaccine design. Here we used the envelope protein domain III (EDIII) of Japanese encephalitis virus (JEV), a subunit vaccine candidate, to further validate this important concept for subunit vaccine designs. We constructed monomeric EDIII, dimeric EDIII via a linear space, dimeric EDIII via an Fc tag, and trimeric EDIII via a foldon tag. Compared to monomeric EDIII or linearly linked dimeric EDIII, tightly packed EDIII oligomers via the Fc or foldon tag induce higher neutralizing antibody titers in mice and also protect mice more effectively from lethal JEV challenge. Structural analyses demonstrate that part of the artificially exposed surface areas on recombinant EDIII becomes re-buried in Fc or foldon-mediated oligomers. This study further establishes the artificially exposed surfaces as an intrinsic limitation of subunit vaccines, and suggests that re-burying these surfaces through tightly packed oligomerization is a convenient and effective approach to overcome this limitation.

Anti-Influenza Effect and Mechanisms of Lentinan in an ICR Mouse Model.

Influenza virus is a serious threat to global human health and public health security. There is an urgent need to develop new anti-influenza drugs. Lentinan (LNT) has attracted increasing attention in recent years. As potential protective agent, LNT has been shown to have anti-tumor, anti-inflammatory, and antiviral properties. However, there has been no further research into the anti-influenza action of lentinan in vivo, and the mechanism is still not fully understood. In this study, the anti-influenza effect and mechanism of Lentinan were studied in the Institute of Cancer Research (ICR) mouse model. The results showed that Lentinan had a high degree of protection in mice against infection with influenza A virus, delayed the emergence of clinical manifestations, improved the survival rate of mice, significantly prolonged the middle survival days, attenuated the weight loss, and reduced the lung coefficient of mice. It alleviated the pathological damage of mice infected with the influenza virus and improved blood indices. Lentinan treatment considerably inhibited inflammatory cytokine (TNF-α, IL-1ß, IL-4, IL-5, IL-6) levels in the serum and lung and improved IFN-γ cytokine levels, which reduced cytokine storms caused by influenza virus infection. The underlying mechanisms of action involved Lentinan inhibiting the inflammatory response by regulating the TLR4/MyD88 signaling pathway. This study provides a foundation for the clinical application of Lentinan, and provides new insight into the development of novel immunomodulators.