Distinct Upstream Role of Type I IFN Signaling in Hematopoietic Stem Cell-Derived and Epithelial Resident Cells for Concerted Recruitment of Ly-6Chi Monocytes and NK Cells via CCL2-CCL3 Cascade

Type I interferon (IFN-I)-dependent orchestrated mobilization of innate cells in inflamed tissues is believed to play a critical role in controlling replication and CNS-invasion of herpes simplex virus (HSV). However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown. Here, we found that IFN-I signaling promoted the differentiation of CCL2-producing Ly-6C^hi monocytes and IFN-γ/granzyme B-producing NK cells, whereas deficiency of IFN-I signaling induced Ly-6C^lo monocytes producing CXCL1 and CXCL2. More interestingly, recruitment of Ly-6C^hi monocytes preceded that of NK cells with the levels peaked at 24 h post-infection in IFN-I–dependent manner, which was kinetically associated with the CCL2-CCL3 cascade response. Early Ly-6C^hi monocyte recruitment was governed by CCL2 produced from hematopoietic stem cell (HSC)-derived leukocytes, whereas NK cell recruitment predominantly depended on CC chemokines produced by resident epithelial cells. Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6G^hi neutrophil recruitment to ameliorate immunopathology. Finally, tissue resident CD11b^hiF4/80^hi macrophages and CD11c^hiEpCAM⁺ dendritic cells appeared to produce initial CCL2 for migration-based self-amplification of early infiltrated Ly-6C^hi monocytes upon stimulation by IFN-I produced from infected epithelial cells. Ultimately, these results decipher a detailed IFN-I–dependent pathway that establishes orchestrated mobilization of Ly-6C^hi monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells. Therefore, this cascade response of resident–to-hematopoietic–to-resident cells that drives cytokine–to-chemokine–to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues.     

Co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing swine interleukin-18 and interferon-α provides enhanced Th1-biased protective immunity against inactivated vaccine of pseudorabies virus

The co-administration of two or more cytokines may generate additive or synergistic effects for controlling infectious diseases. However, the practical use of cytokine combinations for the modulation of immune responses against inactivated vaccine has not been demonstrated in livestock yet, primarily due to protein stability, production, and costs associated with mass administration. In light of the current situation, we evaluated the immunomodulatory functions of the combined administration of swine interleukin-18 (swIL-18) and interferon-α (swIFN-α) against an inactivated PrV vaccine using attenuated Salmonella enterica serovar Typhimurium as a cytokine delivery system. Co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α produced enhanced Th1-biased humoral and cellular immune responses against the inactivated PrV vaccine, when compared to single administration of S. enterica serovar Typhimurium expressing either swIL-18 or swIFN-α. Also, enhanced immune responses in co-administered piglets occurred rapidly after virulent PrV challenge, and piglets that received co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α displayed a greater alleviation of clinical severity following the virulent PrV challenge, as determined by clinical scores and cumulative daily weight gain. Furthermore, this enhancement was confirmed by reduced nasal shedding of PrV following viral challenge. Therefore, these results suggest that oral co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α provide enhanced Th1-biased immunity against inactivated PrV vaccine to alleviate clinical signs caused by PrV challenge.     

Identification and characterization of a xyloglucan-specific family 74 glycosyl hydrolase from Streptomyces coelicolor A3(2)

The sco6545 gene of Streptomyces coelicolor A3(2) was nominated as a putative cellulase with 863 mature-form amino acids (90.58 kDa). We overexpressed and purified Sco6545 and demonstrated that the protein is not a cellulase but a xyloglucan-specific glycosyl hydrolase which cleaves xyloglucan at unbranched glucose residues.     

Identification and characterization of a novel β-galactosidase from Victivallis vadensis ATCC BAA-548, an anaerobic fecal bacterium

Victivallis vadensis ATCC BAA-548 is a Gram-negative, anaerobic bacterium that was isolated from a human fecal sample. From the genomic sequence of V. vadensis, one gene was found to encode agarase; however, its enzymatic properties have never been characterized. The gene encoding the putative agarase (NCBI reference number ZP_01923925) was cloned by PCR and expressed in E. coli Rosetta-gami by using the inducible T₇ promoter of pET28a(+). The expressed protein with a 6×His tag at the N-terminus was named His6-VadG925 and purified as a soluble protein by Ni²⁺-NTA agarose affinity column chromatography. The purification of the enzyme was 26.8-fold, with a yield of 73.2% and a specific activity of 1.02 U/mg of protein. The purified His6-VadG925 produced a single band with an approximate MW of 155 kDa, which is consistent with the calculated value (154,660 Da) including the 6×His tag. Although VadG925 and many of its homologs were annotated as agarases, it did not hydrolyze agarose. Instead, purified His₆-VadG925 hydrolyzed an artificial chromogenic substrate, p-nitrophenyl-β-d-galactopyranoside, but not p-nitrophenyl-α-d-galactopyranoside. The optimum pH and temperature for this β-galactosidase activity were pH 7.0 and 40°C, respectively. The K _m and V _max of His6-VadG925 towards p-nitrophenyl-β-d-galactopyranoside were 1.69 mg/ml (0.0056 M) and 30.3 U/mg, respectively. His6-VadG925 efficiently hydrolyzed lactose into glucose and galactose, which was demonstrated by TLC and mass spectroscopy. These results clearly demonstrated that VadG925 is a novel β-galactosidase that can hydrolyze lactose, which is unusual because of its low homology to validated β-galactosidases.     

Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2), an exo- and endo-type β-agarase-producing neoagarobiose

Streptomyces coelicolor can degrade agar, the main cell wall component of red macroalgae, for growth. To constitute a crucial carbon source for bacterial growth, the alternating α-(1,3) and β-(1,4) linkages between the 3,6-anhydro-L-galactoses and D-galactoses of agar must be hydrolyzed by α/β-agarases. In S. coelicolor, DagA was confirmed to be an endo-type β-agarase that degrades agar into neoagarotetraose and neoagarohexaose. Genomic sequencing data of S. coelicolor revealed that Sco3487, annotated as a putative hydrolase, has high similarity to the glycoside hydrolase (GH) GH50 β-agarases. Sco3487 encodes a primary translation product (88.5 kDa) of 798 amino acids, including a 45-amino-acid signal peptide. The sco3487 gene was cloned and expressed under the control of the ermE promoter in Streptomyces lividans TK24. β-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-β-D-galactopyranoside. Mature Sco3487 (83.9 kDa) was purified 52-fold with a yield of 66% from the culture broth. The optimum pH and temperature for Sco3487 activity were 7.0 and 40°C, respectively. The K(m) and V(max) for agarose were 4.87 mg/ml (4 × 10(-5) M) and 10.75 U/mg, respectively. Sco3487 did not require metal ions for its activity, but severe inhibition by Mn(2+) and Cu(2+) was observed. Thin-layer chromatography analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and Fourier transform-nuclear magnetic resonance spectrometry of the Sco3487 hydrolysis products revealed that Sco3487 is both an exo- and endo-type β-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose.     

Overexpression and biochemical characterization of DagA from <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose

The DagA product of Streptomyces coelicolor is an agarase with a primary translation product (35 kDa) of 309 amino acids, including a 30-amino acid signal peptide. Although dagA expression in Streptomyces lividans under the control of its own set of promoters was previously reported, its enzymatic properties have never been elucidated. To develop an improved expression system for dagA, three types of strong promoters for the Streptomyces host were linked to dagA, and their efficiencies in DagA production were compared in S. lividans TK24. All of the transformants with dagA grew at improved rates and produced larger amounts of DagA in the modified R2YE medium containing 0.5% agar as the sole carbon source. Of the three transformants, the S. lividans TK24/pUWL201-DagA (ermE promoter) produced the highest agarase activity (A ₅₄₀ = 4.24), and even the S. lividans TK24/pHSEV1-DagA (tipA promoter) and S. lividans TK24/pWHM3-DagA (sprT promoter) produced higher agarase activity (A ₅₄₀ = 0.24 and 0.12, respectively) than the control (A ₅₄₀ = 0.01) in the modified R2YE medium. The mature form of DagA protein (32 kDa) was successfully purified by one-step affinity column chromatography by using agarose beads with excellent yield. The purified DagA was found to exhibit maximal agarase activity at 40°C and pH 7.0. The K _m, V _max, and K _cat values for agarose were 2.18 mg/ml (approximately 1.82 × 10⁻⁵ M), 39.06 U/mg of protein, and 9.5 × 10³/s, respectively. Thin layer chromatography (TLC) analysis, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, and Fourier transform nuclear magnetic resonance (FT-NMR) spectrometry of the hydrolyzed products of agarose by DagA revealed that DagA is an endo-type β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose.