Growth phase-dependent production of the adhesive nanofiber protein AtaA in Acinetobacter sp. Tol 5.

AtaA, the sticky, long, and peritrichate nanofiber protein from Acinetobacter sp. Tol 5, mediates autoagglutination and is highly adhesive to various material surfaces, resulting in a biofilm. Although the production of the adhesive nanofiber protein is likely to require a large amount of energy and material sources, the relationship between AtaA fiber production and cell growth remains unknown. Here, we report the growth phase-dependent AtaA fiber production in Tol 5. We examined the ataA gene expression in different growth phases using a reporter gene assay with an originally developed reporter plasmid and using reverse transcription-quantitative polymerase chain reaction. Bacterial cells with surface-displayed AtaA at different growth phases were immunostained and analyzed using fluorescence flow cytometry and confocal laser scanning microscopy. The results indicate that Tol 5 modulated the amount of surface-displayed AtaA at the transcriptional level. AtaA production was low in the early growth phase but remarkably increased in the late growth phase, covering the whole bacterial cell with AtaA fibers in the stationary phase. Tol 5 displayed AtaA fibers poorly in the early growth phase and showed less autoagglutination and adhesiveness than those in the stationary phase. Although Tol 5 grew as fast as its ataA-deficient mutant in the early growth phase, the optical density of Tol 5 culture was slightly lower than that of the ataA-deficient mutant in the late growth phase. Based on these experimental results, we propose the growth-phase-dependent production of AtaA fiber for efficient and fast cell growth.

Perturbation by Antimicrobial Bacteria of the Epidermal Bacterial Flora of Rainbow Trout in Flow-Through Aquaculture.

The bacterial flora of the epidermal mucus of fish is closely associated with the host's health and susceptibility to pathogenic infections. In this study, we analyzed the epidermal mucus bacteria of rainbow trout (Oncorhynchus mykiss) reared in flow-through aquaculture under environmental perturbations. Over ~2 years, the bacteria present in the skin mucus and water were analyzed based on the 16S rDNA sequences. The composition of the mucus bacterial community showed significant monthly fluctuations, with frequent changes in the dominant bacterial species. Analysis of the beta- and alpha-diversity of the mucus bacterial flora showed the fluctuations of the composition of the flora were caused by the genera Pseudomonas, Yersinia, and Flavobacterium, and some species of Pseudomonas and Yersinia in the mucus were identified as antimicrobial bacteria. Examination of the antimicrobial bacteria in the lab aquarium showed that the natural presence of antimicrobial bacteria in the mucus and water, or the purposeful addition of them to the rearing water, caused a transition in the mucus bacteria community composition. These results demonstrate that specific antimicrobial bacteria in the water or in epidermal mucus comprise one of the causes of changes in fish epidermal mucus microflora.

Establishing a Percutaneous Infection Model Using Zebrafish and a Salmon Pathogen.

To uncover the relationship between skin bacterial flora and pathogen infection, we developed a percutaneous infection model using zebrafish and Yersinia ruckeri, a pathogen causing enteric redmouth disease in salmon and in trout. Pathogen challenge, either alone or together with pricking by a small needle, did not cause infection of the fish. However, cold stress given by water temperature shift from the optimum 28 °C for zebrafish to 20 °C caused fatal infection of injured fish following pathogen challenge. We investigated the effects of cold stress, injury, and pathogen challenge, alone and in combination, on fish skin bacterial flora using 16S rDNA metagenomics. We found that cold stress drastically altered the skin bacterial flora, which was dominated by Y. ruckeri on infected fish. In addition, fish whose intrinsic skin bacterial flora was disrupted by antibiotics had their skin occupied by Y. ruckeri following a challenge with this pathogen, although the fish survived without injury to create a route for invasion into the fish body. Our results suggest that the intrinsic skin bacterial flora of fish protects them from pathogen colonization, and that its disruption by stress allows pathogens to colonize and dominate their skin.

On-fiber display of a functional peptide at sites distant from the cell surface using a long bacterionanofiber of a trimeric autotransporter adhesin.

In the cell surface display system, the distance of a surface-displayed molecule from the cell surface should influence its functionality due to the interference by other surface structures. For the purpose of developing this distance-variable surface display system, we utilized a long fibrous adhesin, Acinetobacter trimeric autotransporter adhesin (AtaA) of the strain Tol 5. We constructed His-tagged full-length and shorter AtaA fibers designed by N-terminal deletion and expressed them in the ΔataA mutant. Immunoelectron microscopy clearly showed that they formed fibers on the cell surface and the His-tag was displayed on the fiber tip located at fixed distances from the cell surface. N-terminal deletion of AtaA shortened the distance between the His-tag and the cell surface, as designed. Time-course analyses of the cell-to-Ni-Sepharose beads binding revealed that cells producing the longer fibers bound more rapidly to the beads. The His-tagged AtaA derivatives were also displayed on Escherichia coli cells, and a similar tendency was shown; the His-tag on the longer fiber was more functional than that on the shorter one. Thus, we developed an on-fiber display system of a functional peptide using a long trimeric autotransporter adhesin (TAA) fiber, which can vary the distance between the displayed molecule and the cell surface.

Reversible bacterial immobilization based on the salt-dependent adhesion of the bacterionanofiber protein AtaA.

BACKGROUND: Immobilization of microbial cells is an important strategy for the efficient use of whole-cell catalysts because it simplifies product separation, enables the cell concentration to be increased, stabilizes enzymatic activity, and permits repeated or continuous biocatalyst use. However, conventional immobilization methods have practical limitations, such as limited mass transfer in the inner part of a gel, gel fragility, cell leakage from the support matrix, and adverse effects on cell viability and catalytic activity. We previously showed a new method for bacterial cell immobilization using AtaA, a member of the trimeric autotransporter adhesin family found in Acinetobacter sp. Tol 5. This approach is expected to solve the drawbacks of conventional immobilization methods. However, similar to all other immobilization methods, the use of support materials increases the cost of bioprocesses and subsequent waste materials. RESULTS: We found that the stickiness of the AtaA molecule isolated from Tol 5 cells is drastically diminished at ionic strengths lower than 10 mM and that it cannot adhere in deionized water, which also inhibits cell adhesion mediated by AtaA. Cells immobilized on well plates and polyurethane foam in a salt solution were detached in deionized water by rinsing and shaking, respectively. The detached cells regained their adhesiveness in a salt solution and could rapidly be re-immobilized. The cells expressing the ataA gene maintained their adhesiveness throughout four repeated immobilization and detachment cycles and could be repeatedly immobilized to polyurethane foam by a 10-min shake in a flask. We also demonstrated that both bacterial cells and a support used in a reaction could be reused for a different type of reaction after detachment of the initially immobilized cells from the support and a subsequent immobilization step. CONCLUSIONS: We invented a unique reversible immobilization method based on the salt-dependent adhesion of the AtaA molecule that allows us to reuse bacterial cells and supports by a simple manipulation involving a deionized water wash. This mitigates problems caused by the use of support materials and greatly helps to enhance the efficiency and productivity of microbial production processes.

An Acinetobacter trimeric autotransporter adhesin reaped from cells exhibits its nonspecific stickiness via a highly stable 3D structure.

Trimeric autotransporter adhesins (TAAs), cell surface proteins of Gram-negative bacteria, mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific, high adhesiveness to abiotic material surfaces as well as to biotic surfaces. AtaA is a homotrimer of polypeptides comprising 3,630 amino acids and forms long nanofibers; therefore, it is too large and structurally complex to be produced as a recombinant protein. In this study, we isolated AtaA's passenger domain (AtaA PSD), which is translocated to the cell surface through the C-terminal transmembrane domain and exhibits biological functions, using a new method. We introduced a protease recognition site and reaped AtaA nanofibers 225 nm in length from the cell surface through proteolytic cleavage with a specific protease. Biochemical and biophysical analyses of the purified native AtaA PSD revealed that it has a stable structure under alkaline and acidic conditions. Temperatures above 80 °C, which disrupted AtaA's higher-order structure but maintained the full-length AtaA polypeptide, inactivated AtaA's nonspecific adhesiveness, suggesting that the stickiness of AtaA requires its 3D structure. This finding refutes the widespread but vague speculation that large unfolded polypeptides readily stick to various surfaces.

Effectiveness of direct immobilization of bacterial cells onto material surfaces using the bacterionanofiber protein AtaA.

The bacterionanofiber protein AtaA, a member of the trimeric autotransporter adhesin family found in Acinetobacter sp. Tol 5, is responsible for the nonspecific, high adhesiveness and autoagglutination of this strain. Previously, we introduced the ataA gene into the nonadhesive Acinetobacter strain ST-550, which conferred high adhesiveness to this strain, immobilized its cells, and improved indigo productivity due to enhanced tolerance to the toxic substrate. In this study, we again demonstrated the effectiveness of this new microbial immobilization method using AtaA in a number of conditions. AtaA enabled the effective immobilization of growing, resting, and lyophilized cells of a type strain of Acinetobacter, ADP1, which is also intrinsically nonadhesive, onto the surface of several kinds of support ranging from artificial to natural materials and from hydrophobic polyurethane to hydrophilic glass. Immobilization with AtaA enabled exclusive cell growth in the support space and only a few cells existed in the bulk medium. Immobilization of resting cells drastically increased cell concentration, depending on the support material; dry cells of approximately 110 g/L could be immobilized onto glass wool. Finally, we demonstrated that ADP1 cells immobilized on polyurethane foam can undergo at least 10 repetitive reactions without inactivation during a 5-h period. Even after drying and storing for 3 days, the immobilized cells showed enzymatic activity and an ester hydrolysis reaction was repeated by simply transferring the support with the cells into a fresh reaction buffer.

A protective effect of milk fat globule EGF factor VIII (MFG-E8) on the spontaneous fusion of milk fat globules in breast milk.

Lipid droplets synthesized in mammary epithelial cells are secreted into breast milk by the budding-off mechanism. The milk lipids, termed mik fat globules (MFGs), are surrounded with the cell plasma membrane and contain various membrane proteins, including milk fat globule epidermal growth factor (EGF)-factor VIII (MFG-E8), on their surface. We report here that the MFGs in the milk of MFG-E8-deficient mice fused each other and turned into abnormally large size of lipid droplets within ∼48 h after being secreted into mammary alveolar lumen in situ or being incubated at 37°C in vitro. This biophysical degeneration of MFGs in the MFG-E8-deficient milk was efficiently rescued in vitro by adding the milk serum of wild-type mice, isolated MFG-E8 or annexin V. Moreover, addition of ethylenediaminetetraacetic acid (30 mM) also protected the MFG fusion remarkably in vitro. In addition, bovine MFGs also fused each other when isolated from milk serum, and the fusion was inhibited by adding isolated MFG-E8 or mouse milk serum, but not the milk serum of MFG-E8-deficient mice. MFG-E8 in breast milk may mask the phosphatidylserine exposed on the surface of MFGs with time after secretion and thereby suppress the membrane fusion among MFGs resulting in the enlargement of MFGs in the breast milk.

Repression of mammary adipogenesis by genistein limits mammosphere formation of human MCF-7 cells.

Mammary adipose tissue may contribute to breast cancer development and progression by altering neighboring epithelial cell behavior and phenotype through paracrine signaling. Dietary exposure to soy foods is associated with lower mammary tumor risk and reduced body weight and adiposity in humans and in rodent breast cancer models. Despite the suggested linkage between obesity and breast cancer, the local influence of bioactive dietary components on mammary adiposity for antitumor effects remains unknown. Herein, we report that post-weaning dietary exposure to soy protein isolate and its bioactive isoflavone genistein (GEN) lowered mammary adiposity and increased mammary tumor suppressor PTEN and E-cadherin expression in female mice, relative to control casein diet. To ascertain GEN's role in mammary adipose deposition that may affect underlying epithelial cell phenotype, we evaluated GEN's effects on SV40-immortalized mouse mammary stromal fibroblast-like (MSF) cells during differentiation into adipocytes. MSF cells cultured in a differentiation medium with 40 nM GEN showed reductions in mature adipocyte numbers, triglyceride accumulation, and Pparγ (Pparg) and fatty acid synthase transcript levels. GEN inhibition of adipose differentiation was accompanied by increased estrogen receptor ß (Erß (Esr2)) gene expression and was modestly recapitulated by ERß-selective agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN). Reduction of Erß expression by siRNA targeting increased Pparγ transcript levels and stromal fibroblast differentiation into mature adipocytes; the latter was reversed by GEN but not by DPN. Conditioned medium from GEN-treated adipocytes diminished anchorage-independent mammosphere formation of human MCF-7 breast cancer cells. Our results suggest a mechanistic pathway to support direct regulation of mammary adiposity by GEN for breast cancer prevention.

Post-weaning increases in the milk-fat globule EGF-factor VIII on fat globules in mouse milk and in the uptake of the fat globules by HC11 mammary epithelial cells.

Milk fat globules (MFGs) secreted by lactating mammary gland are unique lipid surrounded by a phospholipid bi-layer. We report here post-weaning changes in MFG EGF factor VIII (MFG-E8) and annexin V-accessible phosphatidyl-l-serine on the surface of MFGs. The MFG content in milk markedly decreased to about one-half within 2 days after forced weaning, despite a slight increase in milk protein content. Immunofluorescence-staining of MFGs using anti-MFG-E8 and annexin V indicated that MFG-E8 was present on some, but not all, MFGs before weaning, whereas most of MFGs were MFG-E8-positive and annexin V-negative after weaning. Free MFG-E8 with binding activity to phosphatidyl-l-serine was present abundantly in the post-weaning milk, and indeed exhibited binding to MFGs in pre-weaning milk. MFGs were taken up by HC11 mouse mammary epithelial cells in vitro, and those from post-weaning milk were remarkable for such cellular uptake. Moreover, the uptake of MFGs by the cells was inhibited by an anti-MFG-E8 antibody. Taken together, these findings suggest that MFG-E8 plays a critical role in regulation of MFG dynamics after weaning or during the suckling interval through the control of MFG-epithelial cell interaction in lactating mammary glands.

AtaA, a new member of the trimeric autotransporter adhesins from Acinetobacter sp. Tol 5 mediating high adhesiveness to various abiotic surfaces.

Acinetobacter sp. Tol 5 exhibits an autoagglutinating nature and noteworthy adhesiveness to various abiotic surfaces from hydrophobic plastics to hydrophilic glass and stainless steel. Although previous studies have suggested that bacterionanofibers on Tol 5 cells are involved in the adhesive phenotype of Tol 5, the fiber that directly mediates Tol 5 adhesion has remained unknown. Here, we present a new member of trimeric autotransporter adhesins designated AtaA, which we discovered by analyzing a less adhesive mutant of Tol 5, T1, obtained by transposon mutagenesis. AtaA forms thinner and shorter nanofibers than fimbriae on Tol 5 cells. We performed target disruption of ataA by allelic marker exchange, and the resulting ΔataA strain was complemented with ataA on the Escherichia coli-Acinetobacter shuttle vector, which was newly constructed. These results proved that AtaA is essential for Tol 5's autoagglutinating nature and high adhesiveness to surfaces of various materials. In addition, the adhesiveness to solid surfaces mediated by AtaA is notably higher than that mediated by YadA of Yersinia enterocolitica WA-314. Moreover, and importantly, these characteristics can be conferred to the non-adhesive, non-agglutinating bacterium Acinetobacter sp. ADP1 in trans by transformation with ataA, with expected applications to microbial immobilization.

Re-evaluation of milk-fat globule EGF-factor VIII (MFG-E8) as an intrinsic component of the mouse milk-fat globule membrane.

In lactating mammary glands, milk fat is secreted as fat globules surrounded by a cell plasma membrane containing characteristic membrane-associated proteins. Among these, butyrophilin has been shown to be specific and intrinsic to the fat globule membrane, whereas milk-fat globule EGF-factor VIII (MFG-E8) is uncertain. We characterized in the present study MFG-E8 in milk fat globules and in the culture medium of HC11 mammary epithelial cells. MFG-E8 was immunologically detected in the mammary tissues of both pregnant and lactating mice. Double-immunofluorescence staining for MFG-E8 and butyrophilin showed diversity in the MFG-E8-staining intensity among different fat globules in milk. HC11 cells secreted monomeric MFG-E8 with phosphatidylserine-binding activity, despite no fat globules being detected in the cells. This secretion was upregulated by not only prolactin but also by insulin or EGF. These results suggest that milk MFG-E8 was not equally present among fat globules and not necessarily intrinsic to the fat globules.

High serum levels of both carcinoembryonic antigen and carbohydrate antigen 19-9 in a patient with sigmoid colon cancer without metastasis.

Carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9 are well known as the most common tumor markers of colon cancer, and levels are used not only for preoperative assessment of extent and outcome of cancer, but also postoperative monitoring of recurrence. We encountered a patient with sigmoid colon cancer showing abnormally high serum levels of CEA (311.1 ng/ml) and CA19-9 (5731.2 U/ml) preoperatively. We could not detect any metastases on computed tomography (CT) or (18)F-fluorodeoxyglucose positron emission tomography/CT. Sigmoidectomy and lymph node dissection were performed. Pathological analysis revealed well-differentiated tubular adenocarcinoma of the sigmoid colon with cancer cells infiltrating to the subserosa, but no lymph node metastases. As of postoperative day 60, serum levels of CEA and CA19-9 were 3.4 ng/ml and 9.2 U/ml, respectively, without any further anti-tumor treatment. This represents a rare case of sigmoid colon cancer with high levels of tumor markers in sera that improved following sigmoidectomy without further anti-cancer treatment.

Control of dry weight and tube feeding improved the general condition of a hemodialysis patient: report of a case.

Patients on long-term hemodialysis are at risk of developing malnutrition,and poor nutrient intake is an important factor in this. In the present case, we encountered a 55-year-old Japanese woman with end-stage renal failure and a past history of schizophrenia. Severe systemic edema was observed. Hemodialysis was started, but after one year she suddenly became unable to consume food orally, despite provision of a dietary plan by the nutrition support team (NST). Tube feeding was eventually implemented. Because the systemic edema did not improve, we decided to remove body fluid by intense hemodialysis. Hypotension was often observed during this hemodialysis, requiring dopamine. Over approximately 2 months, the patient's dry weight fell from 73 kg to 62 kg, the patient's activity improved and she became able to eat orally again, allowing tube feeding to be stopped. Although the reason for the sudden anorexia has not been clarified, tube feeding and dry weight control was successful in the treatment of this malnourished hemodialysis patient.

Acute abdomen caused by both acute appendicitis and epididymitis.

Acute appendicitis often presents as right lower quadrant (RLQ) pain, severe tenderness at the point of McBurny or Lanz, and Blumberg's sign. Scrotal events with appendicitis are very rare. In our case, a 63-year-old Japanese man presented with severe RLQ pain and high fever. Physical examination revealed severe tenderness (including both points of McBurny and Lanz) and Blumberg's sign. The scrotum was slightly swollen and showed local heat with severe testicular pain. Abdominal computed tomography revealed ascites in a pelvic space and the right side of the spermatic cord was swollen. Emergency operation was performed and the final diagnosis was catarrhal appendicitis and acute epididymitis. This is the first report of acute appendicitis concomitant with acute epididymitis.

Fournier's gangrene in elderly patient: report of a case.

Fournier's gangrene (FG) is rapidly progressing acute gangrenous infection of the anorectal and urogenital area. FG needs precocious diagnosis and aggressive treatment with the use of wide spectrum antibioticus and surgical debridement. In our case, a 91-year-old Japanese female who had rehabilitation after treatment of pneumonia and her past history was rheumatoid arthritis treated with steroid and chronic heart failure. Her activities of daily living was bedridden with dementia. Necrotic skin was observed in urogenital and anorectal area and skin redness enlarged to the hip with high fever. Surgical debridement was performed. Both Peptostreptococcus Sp. and Fusobacterium Sp. was cultured from resected necrotic tissue. We used antibioticus, PAPM and PIPC, which had sensitivity for them. But unfortunately, disseminated intravascular coagulation occurred after 4th day of operation, and finally she died after 10th day of operation. We discussed the treatment for FG in patient with complication.

Transplantation of a mammary stromal cell line into a mammary fat pad: development of the site-specific in vivo analysis system for mammary stromal cells.

The interaction between mammary epithelial and stromal tissue is considered to be important in breast tissue development. In this study, we developed a transplantation procedure for the mammary stromal fibroblastic cell line (MSF) to examine its life in vivo. First we established MSF cells which stably expressed lacZ (lacZ/MSF) and had characteristics of mammary stromal cells. The lacZ/MSF cells were then transplanted into a cleared mammary fat pad of syngenic mice with and without mammary primary epithelial organoids. Whole mount X-gal and carmine staining of the transplants revealed that a number of undifferentiated lacZ/MSF cells survived around the mammary epithelial tissue when transplanted with organoids. These results indicate that transplantation of MSF cells into mammary fat pad was accomplished by co-transplantation with primary mammary organoids. Finally, we discuss the application of transplantation procedure for in vivo studies of the mammary stromal tissue development and stromal-epithelial interactions.

Vascular endothelial growth factors and their receptors in the novel human cell line, HN-Eso-1, established from esophageal spindle cell carcinoma.

Human carcinosarcomas of esophagus are uncommon malignant neoplasms that are composed both carcinomatous and sarcomatous components. We established a novel cell line, HN-Eso-1, from the metastatic esophageal spindle cell carcinoma (so-called carcinosarcoma). In this study, we estimated the vascular endothelial growth factors (VEGFs) and VEGF receptors (VEGFRs). Reverse transcription polymerase chain reaction (RT-PCR) studies revealed that VEGF-A, -C, -D and VEGFR-1, -2 were upregulated. Cisplatin reduced the cell viability of HN-Eso-1 cells and VEGF attenuated its effect. These results suggest that expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-1, and VEGFR-2 are involved in the cell's autocrine system and that VEGF protected these cells from the anti-tumor agent.

Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs.

By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approximately 100 nm in diameter from bovine milk. We also found that approximately 1700 and 1000 ng of total RNA, in which small RNAs were major components, was contained inside the microvesicles isolated from 6 ml of colostrum and mature milk, respectively, despite high RNase activity in the milk. Polyadenylated gene transcripts for major milk proteins and translation elongation factor-1alpha (EF-1alpha) were present in the microvesicles, and integrity of some transcripts was confirmed by real-time PCR targeting 5'- and 3'-ends of mRNA and by in vitro translation analysis. Moreover, a considerable amount of mammary gland and immune-related microRNAs were present in the milk-derived microvesicles. Acidification of milk to mimic gastrointestinal tract did not mostly affected RNA yield and quality. The milk related gene transcripts were detected in cultured cells when incubated with milk-derived microvesicles, suggesting cellular uptake of the microvesicle contents including RNA. Our findings suggest that bovine breast milk contains RNAs capable for being transferred to living cells and involved in the development of calf's gastrointestinal and immune systems.

Establishment of a mammary stromal fibroblastic cell line for in vitro studies in mice of mammary adipocyte differentiation.

Mammary stromal adipose tissue remodeling is important for appropriate mammary gland development during pregnancy, lactation, and involution. However, the precise mechanisms underlying mammary stromal adipose tissue remodeling remain unclear. We have established a mammary stromal, fibroblastlike cell line (MSF) from primary mouse mammary culture by introducing a temperature-sensitive simian virus-40 large tumor antigen. Among several hormones related to mammary gland development, hydrocortisone was found to commit MSF cells to a preadipocyte lineage, whereas insulin was found to induce extracellular matrix-dependent adipogenic differentiation of the cells, as assessed by lipid accumulation and marker gene expression. Interestingly, such hormone-induced adipogenic differentiation of MSF cells, but not 3T3-L1 cells, was suppressed by prolactin through its receptor and downstream STAT5. Furthermore, coculture of MSF cells with mammary epithelial HC11 cells and culture in HC11-conditioned medium also suppressed adipogenic differentiation of MSF cells. We have demonstrated that adipogenic differentiation of at least some populations of mammary stromal cells is modulated by lactogenic hormones and humoral factors from epithelial cells, suggesting that the response of these mammary cells may differ from adipocytes at other sites. We believe that the MSF cell line will prove a useful model to elucidate mammary stromal adipose development in vitro as well as represent an important first step toward developing stable adipocyte cell lines that faithfully represent their site of origin.