Different strains of Streptococcus suis serotypes 1 and 2 isolated from pigs either contained a restrictionmodification (R-M) system or lacked it. The R-M system was an isoschizomer of Streptococcus pneumoniae DpnII, which recognizes nucleotide sequence 5-GATC-3. The nucleotide sequencing of the genes encoding the R-M system in S. suis DAT1, designated SsuDAT1I, showed that the SsuDAT1I gene region contained two methyltransferase genes, designated ssuMA and ssuMB, as does the DpnII system. The deduced amino acid sequences of M.SsuMA and M.SsuMB showed 70 and 90% identity to M.DpnII and M.DpnA, respectively. However, the SsuDAT1I system contained two isoschizomeric restriction endonuclease genes, designated ssuRA and ssuRB. The deduced amino acid sequence of R.SsuRA was 49% identical to that of R.DpnII, and R.SsuRB was 72% identical to R.LlaDCHI of Lactococcus lactis subsp. cremoris DCH-4. The four SsuDAT1I genes overlapped and were bounded by purine biosynthetic gene clusters in the following gene order: purF-purMpurN-purH-ssuMA-ssuMB-ssuRA-ssuRB-purD-purE. The G؉C content of the SsuDAT1I gene region (34.1%) was lower than that of the pur region (48.9%), suggesting horizontal transfer of the SsuDAT1I system. No transposable element or long-repeat sequence was found in the flanking regions. The SsuDAT1I genes were functional by themselves, as they were individually expressed in Escherichia coli. Comparison of the sequences between strains with and without the R-M system showed that only the region from 53 bp upstream of ssuMA to 5 bp downstream of ssuRB was inserted in the intergenic sequence between purH and purD and that the insertion target site was not the recognition site of SsuDAT1I. No notable substitutions or insertions could be found, and the structures were conserved among all the strains. These results suggest that the SsuDAT1I system could have been integrated into the S. suis chromosome by an illegitimate recombination mechanism.More than 3,000 restriction-modification (R-M) systems have been identified in a wide variety of microorganisms, where they are thought to protect the host from invasion by foreign DNA. Only a minority of R-M systems have been sequenced (6,37,56). Among them, some type II R-M systems, which recognize 4-bp palindromic sequence 5Ј-GATC-3Ј, involve a variety of isoschizomers. Three classes can be distinguished by their manners of DNA cleavage and susceptibilities to DNA methylation. The first class of isoschizomers, represented by Sau3AI, which was described for Staphylococcus aureus, is prevented from digesting host DNA by a cognate 5-methylcytosine methyltransferase and is not influenced by the modification of N 6 -methyladenine (48, 56). The second class, represented by DpnI, which was described for Streptococcus pneumoniae, is unique among restriction endonucleases in that it cleaves only at methylated DNA sequence 5Ј-GmeATC-3Ј, and thus the cells producing DpnI do not carry the corresponding methyltransferase gene (22,23). The third class, including MboI, DpnII, and LlaDCHI...

Tissue-resident memory T cells (TRMs) are a novel nonvascular memory T cell subset. Although CD8 + TRMs are well-characterized, CD4 + TRMs-especially lung-resident memory Th17 cells-are still being defined. In this study, we characterized lung-resident memory Th17 cells (lung TRM17) and their role in protection against the highly virulent fungus Cryptococcus gattii. We found that intravenously transferred DCs preferentially migrated to lungs and attracted recipient DCs and led to the induction of long-lived Th17 cells expressing characteristic markers. This population could be clearly discriminated from circulating T cells by intravascular staining and was not depleted by the immunosuppressive agent FTY720. The C. gattii antigen re-stimulation assay revealed that vaccine-induced lung Th17 cells produced IL-17A but not IFNγ. The DC vaccine significantly increased IL-17A production and suppressed fungal burden in the lungs and improved the survival of mice infected with C. gattii. This protective effect was significantly reduced in the IL-17A knockout (KO) mice, but not in the FTY720-treated mice. The protective effect also coincided with the activation of neutrophils and multinucleated giant cells, and these inflammatory responses were suppressed in the vaccinated IL-17A KO mice. Overall, these data demonstrated that the systemic DC vaccine induced lung TRM17, which played a substantial role in anti-fungal immunity.Mucosal Immunology (2019) 12:265-276; https://doi.

Cryptococcus gattii is a capsular fungal pathogen, which causes life-threatening cryptococcosis in immunocompetent individuals. This emerging pathogen is less likely to be recognized by innate immunity compared to traditional Cryptococcus neoformans strains. Previous studies indicate that C-type lectin receptors (CLRs), including dectin-1 and dectin-2, play a role in recognizing cryptococcal cells; however, it remains to be elucidated whether the receptors physically associate with C . gattii yeast cell surfaces. Based on the previous findings, we hypothesized that culture conditions influence the expression or exposure of CLR ligands on C . gattii . Therefore, in the present study, we first investigated the culture conditions that induce exposure of CLR ligands on C . gattii yeast cells via the binding assay using recombinant fusion proteins of mouse CLR and IgG Fc, Fc dectin-1 and Fc dectin-2. Common fungal culture media, such as yeast extract–peptone–dextrose (YPD) broth, Sabouraud broth, and potato dextrose agar, did not induce the exposure of dectin-1 ligands, including β-1,3-glucan, on both capsular and acapsular C . gattii strains, in contrast to Fc dectin-1 and Fc dectin-2 bound to C . gattii cells growing in the conventional synthetic dextrose (SD) medium [may also be referred to as a yeast nitrogen base with glucose medium]. The medium also induced the exposure of dectin-1 ligands on C . neoformans , whereas all tested media induced dectin-1 and dectin-2 ligands in a control fungus Candida albicans . Notably, C . gattii did not expose dectin-1 ligands in SD medium supplemented with yeast extract or neutral buffer. In addition, compared to YPD medium-induced C . gattii , SD medium-induced C . gattii more efficiently induced the phosphorylation of Syk, Akt, and Erk1/2 in murine dendritic cells (DCs). Afterwards, the cells were considerably engulfed by DCs and remarkably induced DCs to secrete the inflammatory cytokines. Overall, the findings suggest that C . gattii alters its immunostimulatory potential in response to the environment.