QcrC is a potential target for antibody therapy and vaccination to control Campylobacter jejuni infection by suppressing its energy metabolism.

Introduction: Campylobacter spp. are a public health concern, yet there is still no effective vaccine or medicine available. Methods: Here, we developed a Campylobacter jejuni-specific antibody and found that it targeted a menaquinol cytochrome c reductase complex QcrC. Results: The antibody was specifically reactive to multiple C. jejuni strains including clinical isolates from patients with acute enteritis and was found to inhibit the energy metabolism and growth of C. jejuni. Different culture conditions produced different expression levels of QcrC in C. jejuni, and these levels were closely related not only to the energy metabolism of C. jejuni but also its pathogenicity. Furthermore, immunization of mice with recombinant QcrC induced protective immunity against C. jejuni infection. Discussion: Taken together, our present findings highlight a possible antibody- or vaccination-based strategy to prevent or control Campylobacter infection by targeting the QcrC-mediated metabolic pathway.

The omega-3 postbiotic trans-10-cis-15-octadecadienoic acid attenuates contact hypersensitivity in mice through downregulation of vascular endothelial growth factor A.

Intestinal bacteria metabolize dietary substances to produce bioactive postbiotics, among which some are recognized for their role in promoting host health. We here explored the postbiotic potential of two omega-3 α-linolenic acid-derived metabolites: trans-10-cis-15-octadecadienoic acid (t10,c15-18:2) and cis-9-cis-15-octadecadienoic acid (c9,c15-18:2). Dietary intake of lipids rich in omega-3 α-linolenic acid elevated levels of t10,c15-18:2 and c9,c15-18:2 in the serum and feces of mice, an effect dependent on the presence of intestinal bacteria. Notably, t10,c15-18:2 mitigated skin inflammation in mice that became hypersensitive after exposure to 2,4-dinitrofluorobenzene, an experimental model for allergic contact dermatitis. In particular, t10,c15-18:2-but not c9,c15-18:2-attenuated ear swelling and edema, characteristic symptoms of contact hypersensitivity. The anti-inflammatory effects of t10,c15-18:2 were due to its ability to suppress the release of vascular endothelial growth factor A from keratinocytes, thereby mitigating the enhanced vascular permeability induced by hapten stimulation. Our study identified retinoid X receptor as a functional receptor that mediates the downregulation of skin inflammation upon treatment with t10,c15-18:2. Our results suggest that t10,c15-18:2 holds promise as an omega-3 fatty acid-derived postbiotic with potential therapeutic implications for alleviating the skin edema seen in allergic contact dermatitis-induced inflammation.

Induction of unique macrophage subset by simultaneous stimulation with LPS and IL-4.

Macrophages manifest as various subtypes that play diverse and important roles in immunosurveillance and the maintenance of immunological homeostasis in various tissues. Many in vitro studies divide macrophages into two broad groups: M1 macrophages induced by lipopolysaccharide (LPS), and M2 macrophages induced by interleukin 4 (IL-4). However, considering the complex and diverse microenvironment in vivo, the concept of M1 and M2 is not enough to explain diversity of macrophages. In this study, we analyzed the functions of macrophages induced by simultaneous stimulation with LPS and IL-4 (termed LPS/IL-4-induced macrophages). LPS/IL-4-induced macrophages were a homogeneous population showing a mixture of the characteristics of M1 and M2 macrophages. In LPS/IL-4-induced macrophages, expression of cell-surface M1 markers (I-Ab) was higher than in M1 macrophages, but lower expression of iNOS, and expression of M1-associated genes (Tnfα and Il12p40) were decreased in comparison to expression in M1 macrophages. Conversely, expression of the cell-surface M2 marker CD206 was lower on LPS/IL-4-induced macrophages than on M2 macrophages and expression of M2-associated genes (Arg1, Chi3l3, and Fizz1) varied, with Arg1 being greater than, Fizz1 being lower than, and Chi3l3 being comparable to that in M2 macrophages. Glycolysis-dependent phagocytic activity of LPS/IL-4-induced macrophages was strongly enhanced as was that of M1 macrophages; however, the energy metabolism of LPS/IL-4-induced macrophages, such as activation state of glycolytic and oxidative phosphorylation, was quite different from that of M1 or M2 macrophages. These results indicate that the macrophages induced by LPS and IL-4 had unique properties.

Capsular polysaccharide from a fish-gut bacterium induces/promotes apoptosis of colon cancer cells in vitro through Caspases' pathway activation.

Among the widespread malignancies colorectal cancer is the most lethal. Treatments of this malignant tumor include surgery for lesions and metastases, radiotherapy, immunotherapy, and chemotherapy. Nevertheless, novel therapies to reduce morbidity and mortality are demanding. Natural products, such as polysaccharides, can be a valuable alternative to sometimes very toxic chemotherapeutical agents, also because they are biocompatible and biodegradable biomaterials. Microbial polysaccharides have been demonstrated to fulfill this requirement. In this paper, the results about the structure and the activity of a capsular polysaccharide isolated from the psychrotroph Pseudoalteromonas nigrifaciens Sq02-Rifr, newly isolated from the fish intestine, have been described. The characterization has been obtained by spectroscopic and chemical methods, and it is supported by the bioinformatic analysis. The polymer activates Caspases 3 and 9 on colon cancer cells CaCo-2 and HCT-116, indicating a promising antitumor effect, and suggesting a potential capacity of CPS to induce apoptosis.

Complete Lipooligosaccharide Structure from Pseudoalteromonas nigrifaciens Sq02-Rifr and Study of Its Immunomodulatory Activity.

Lipopolysaccharides (LPS) are surface glycoconjugates embedded in the external leaflet of the outer membrane (OM) of the Gram-negative bacteria. They consist of three regions: lipid A, core oligosaccharide (OS), and O-specific polysaccharide or O-antigen. Lipid A is the glycolipid endotoxin domain that anchors the LPS molecule to the OM, and therefore, its chemical structure is crucial in the maintenance of membrane integrity in the Gram-negative bacteria. In this paper, we reported the characterization of the lipid A and OS structures from Pseudoalteromonas nigrifaciens Sq02-Rifr, which is a psychrotrophic Gram-negative bacterium isolated from the intestine of Seriola quinqueradiata. The immunomodulatory activity of both LPS and lipid A was also examined.

Development of a regulatable low-temperature protein expression system using the psychrotrophic bacterium, Shewanella livingstonensis Ac10, as the host.

A low-temperature protein expression system is useful for the production of thermolabile proteins. We previously developed a system that enables constitutive protein production at low temperatures, using the psychrotrophic bacterium Shewanella livingstonensis Ac10 as the host. To increase the utility of this system, in the present study, we introduced a repressible promoter of the trp operon of this bacterium into the system. When ß-lactamase was produced under the control of this promoter at 18°C and 4°C, the yields were 75 and 33 mg/L-culture, respectively, in the absence of L-Trp, and the yields were decreased by 72% and 77%, respectively, in the presence of L-Trp. We also found that 3-indoleacrylic acid, a competitive inhibitor of the Escherichia coli trp repressor, increased the expression of the reporter gene. This repressible gene expression system would be useful for regulatable recombinant protein production at low temperatures.

Isolation of a Novel Bacterial Strain Capable of Producing Abundant Extracellular Membrane Vesicles Carrying a Single Major Cargo Protein and Analysis of Its Transport Mechanism.

Extracellular membrane vesicles (EMVs) play an important role in various bacterial activities. EMVs have potential for use as vaccines, drug-delivery vehicles, platforms for extracellular production of recombinant proteins, and so on. In this study, we newly isolated a cold-adapted bacterium, Shewanella vesiculosa HM13, which abundantly produces EMVs, characterized them, and analyzed their cargo transport mechanism. S. vesiculosa HM13, isolated from the intestine of a horse mackerel as a prospective host for a low-temperature secretory protein expression system, produced a single major secretory protein, P49, of unknown function in the culture supernatant. Analysis using sucrose density gradient ultracentrifugation indicated that P49 is a cargo protein carried by EMVs. S. vesiculosa HM13 displayed extensive blebbing on the surface of the outer membrane, and the size of blebs was comparable to that of EMVs. These blebs are thought to be precursors of the EMVs. Disruption of the P49 gene resulted in only a marginal decrease in the EMV production, indicating that the EMVs are produced even in the absence of the major cargo protein. Whole genome sequencing of S. vesiculosa HM13 revealed that this bacterium has a gene cluster coding for a non-canonical type II protein secretion system (T2SS) homolog in addition to a gene cluster coding for canonical T2SS. The P49 gene was located downstream of the former gene cluster. To examine the role of the putative non-canonical T2SS-like translocon, we disrupted the gene coding for a putative outer membrane channel of the translocon, named GspD2. The gspD2 disruption lead to disappearance of P49 in the EMV fraction, whereas the production of EMVs was not significantly affected by this mutation. These results are indicative that the T2SS-like machinery functions as a novel type of protein translocon responsible for selective cargo loading to the EMVs. We also found that GFP fused to the C-terminus of P49 expressed in S. vesiculosa HM13 was transported to EMVs, indicating that P49 is useful as a carrier to deliver the fusion partner to EMVs. These findings deepen our understanding of the mechanism of biogenesis of EMVs and facilitate their applications.