The entomopathogenic fungi were an important natural pathogenic of insects that has been developed as potential biological control agents for many important agricultural, forest and medical pests. Several these fungi produce a wide range of secondary metabolites with high therapeutic value as antibiotics, cytotoxic substances, insecticides, compounds that promote or inhibit growth, attractor and repellent. Therefore, this study was performed to select the antibacterial activity of liquid culture filtrates of 347 entomopathogenic fungi form Korea soils against two pathogenic bacteria including Ralstonia solanacearum and Escherichia coli using novel method which represents a quick and easily applicable tool obtaining large number of samples. As results, eight-five strains (24%) and seventy-six strains (22%) of these fungal metabolites produced anti-R. solanacearum and anti-E. coli compounds, respectively. The preferential antibacterial activity against R. solanacearum and E. coli gives evidence that these entomopathogenic fungal metabolites might be useful as an agent for bacteria control and the technique was simple to operate and allowed a large number of samples to be handled concurrently.

느타리버섯은 가장 중요한 식용버섯 중 하나이다. 느타리버섯은 Pseudomonas tolaasii에 의한 세균성 갈변병에 매우 감수성이므로, 저항성 품종을 만들기 위한 노력의 하나로 누에에서 분리된 항 세균성 단백질인 누에신을 느타리버섯에서 과발현시키고자 하였다. 누에신 cDNA는 여름 느타리버섯의 β-tubulin 프로모터에 결합되어 pTRura3-2 vector와 함께 우라실 영양요구성 돌연변이 균주에 형질전환되었다. 누에신 cDNA가 형질전환된 느타리버섯을 genomic PCR과 Southern blot을 통하여 분리할 수가 있었으며, 이들 중 3개의 형질전환체가 누에신 유전자를 발현시킴을 확인하였다. 그러나 이들 형질전환체들에서 누에신 단백질을 검출할 수 없었으며, 또한 항 세균 효과도 확인할 수 없었다. 이들 결과는 형질전환기술을 이용한 병 저항성 개발 가능성을 보여주고 있다.

Salmonellosis is a major bacterial zoonosis that causes self-limited enteritis to fatal infection in animals and food-borne infection and typhoid fever in humans. Multidrug-resistant strains of Salmonella spp. has increased over the last several decades and recently causes more serious problems in public health. The present study was investigated bacteriocidal effects of sodium chlorate, sodium azide, sodium cyanide, and sodium salts mixture containing sodium chlorate, sodium azide, and sodium cyanide on infection with S. typhimurium in macrophage RAW 264.7 cells, and antibacterial effects of sodium salts mixture for murine salmonellosis. In infection assay of S. typhimurium in RAW 264.7 cells, bacterial survival rates within macrophage in all treated groups was significantly reduced comparing to that of the control group with the passage of incubation time. Administration of sodium salts mixture showed a therapeutic effect for S. typhimurium infected ICR mice. The mortality of mice treated with sodium salts mixture was 70% until 12 days, while that of control mice was 100% until 9 days after S. typhimurium infection. The results of this study strongly indicate that sodium salts mixture has a potency treatment for murine salmonellosis.

The present study was investigated the antibacterial effect of several sodium salts and the sodium salt mixture composed with sodium chlorate, sodium azide and sodium cyanide on Salmonella gallinarum (S. gallinarum) infection in murine derived macrophage RAW 264.7 cells. In the infection assay of S. gallinarum in macrophage cells pretreated with 15 mM sodium chlorate, 0.3 mM sodium cyanide, 0.3 mM sodium azide and the sodium salt mixture (15 mM sodium chlorate, 0.3 mM sodium cyanide, 0.3 mM sodium azide), respectively, the numbers of S. gallinarum in all treated-groups tended to decrease in the process of time after treatment, but the group treated with sodium cyanide was no significant difference compared with control. After 24 hours of treatment, the number of S. gallinarum in sodium azide (p<0.05), sodium chlorate (p<0.001) and the sodium salt mixture (p<0.001) treated-group was significantly decreased compared with control, and that in the sodium salt mixture treated-group was decreased the higher than all groups. The results of this study demonstrated that the sodium salt mixture composed with sodium chlorate, sodium azide and sodium cyanide, has the antimicrobial activity for S. gallinarum and may be beneficial on the control of intracellular pathogens.

본 시험은 무궁화과에 속하는 초본 식물인 Kenaf (Hibiscus cannabinus L.)를 경상대학교 시험농장 에서 2010년 6월 1일 파종하여 같은 해 11월 18일 수확하여 건조 한 후, 꽃 (HCME-F)과 잎 (HCME-L)에서 추출한 메탄올 추출물을 이용하여, 세포 독성 시험 및 주요 병원균에 대한 항균효과를 규명하였다. 항균효과에 사용된 균주는 가축에서 피부 질환, 유방염 및 소화기질병을 유발하는 그람양성 균인 St. aureus 와 Str. epidermidis, 그람음성균인 S. typhimurium 과 E. coli 균을 공시균주로 사용하였다. 추출물의 안전성을 평가하기 위해 두 추출물 HCME-F과 HCME-L을 0, 25, 50, 100 및 200 ㎍/ml 농도로 첨가한 배지에서 RAW 264.7 세포와 24시간 반응 후 세포 독성을 측정해 본 결과, 세포 독성이 인정되지 않았으며, 항균효과 시험결과 그람양성균인 St. aureus와 Str. epidermidis 균에 대하여 1, 50 및 100 ㎍/ml의 추출물 농도 및 반응시간 경과에 따라 항균 효과가 증가되었으나, 그람음 성균인 S. typhimurium 과 E. coli 에서는 항균효과가 인정되지 않았다. 종합적으로 Kenaf의 꽃과 잎 에서 추출한 메탄올 추출물이 세포에 대한 안전성이 입증되었고, 가축과 사람의 피부 질환 및 유방염의 대표적 그람양성균인 St. aureus와 Str. epidermidis 균에 대한 항균효과를 보여, Kenaf의 꽃과 잎을 이용한 선택적 그람양성균 치료제 및 사료첨가제 개발이 가능할 것으로 판단된다.

Salmonellosis is the commonest zoonosis worldwide that generally causes enterocolitis and foodborne poisoning which represents a considerable public health burden. Salmonella spp. are potential enteric pathogens and intracellularly replicates in host cells resulting in chronic infections. The medical treatments for salmonellosis have been difficult yet and had a serious problem including the increasing emergence of antibiotic resistance. The present report was designated to investigate the antibacterial effects of Saururus chinensis Baill ethanol extract (SCEE) on pure culture and infection with Salmonella enterica serovar Typhimurium (S. typhimurium) in murine derived macrophage RAW 264.7 cells. In determination of antibacterial activity of SCEE against S. typhimurium, bacterial viability was markedly decreased compared to the control. Also, SCEE significantly induced morphological change (p<0.05) of RAW 264.7 cells. In infection assay of S. typhimurium in RAW 264.7 cells pretreated with 100㎍/㎖ of SCEE, which is a non-cytotoxic concentration, bacterial uptake ability of macrophage was increased corresponding with morphological change, whereas bacterial survival rates within macrophage were markedly reduced compared with untreated control. Furthermore, nitric oxide (NO) production in SCEE-treated cells was slightly increased until 2 h but showed a tendency of decrease after 4 h until 24 h post infection compared with untreated control with S. typhimurium infection. Taken together, these findings demonstrated that SCEE has the antibacterial activity for S. typhimurium and the protective effects against S. typhimurium infection through activating murine macrophage independent on NO, suggesting that SCEE may be beneficial on the disease caused by intracellularly replicating pathogens as a safe alternatives of conventional chemotherapies.

This study was carried out to investigate the effects of Hericium erinaceus extract on the Anti- inflammatory and Anti-bacterial. To investigate in vitro Anti-bacterial activity assay, hot water extracts of Hericium erinaceus carried out using a broth micro-dillution method following the Clinical and Laboratory Standards Institute guidlines(Citron and Hecht, 2003; Jorgensen and Turnidge, 2003). In the next experiment, to investigate anti-inflammatory test, the RAW 264.7 macrophage, cells was cultured using DMEM including the 10% FBS. Hericium erinaceus extract has the effects of anti-inflammation and, which may be due to its inhibitory potential on the macrophage activation. Furthermore, Hericium erinaceus extract has the anti-bacterial effects through the inhibitory Propionibacterium acnes(P. acnes) and periodontopathic bacteria, Porphyromonas gingivalis , Fusobacterium nucleatum and Tannerella forsythia. The above results suggest that Hericium erinaceus hot water extract could be applicable for improvement of several inflammatory disease.

살모넬라증은 대표적인 인수공통전염병의 하나로서 세포내 기생하며 질병을 유발하며 장염과 식중독 등을 유발하여 공중보건학적으로 심각한 문제를 야기하고 있다. 본 연구는 삼백초의 수용성 추출물을 (SCWE) 이용하여 숙주세포에 대한 안전성, S. typhimurium 균에 대한 항균효과 및 대식세포 내 균 증 식억제 기능을 규명하였다. 본 실험을 통하여 SCWE 1, 10 및 100 μg/ml 농도로 첨가한 배지에서 RAW 264.7 체포와 24 시간 반응 후 평가해본 결과 세포독성이 인정되지 않았으며, S. typhimurium 균에 대하여 시간 경과에 따라 항균효과가 증가되는 것을 확인하였고, SCWE 처리에 의해 대식세포의 형태적 변화가 증가되는 것으로 나타났으며 (p<0.05), 살모넬라균의 탐식능력 및 대식세포 내 균 증식 억제 능 력이 비 처리군에 비해 현저히 증가되는 것이 확인되었다. 또한 SCWE 처리 한 대식세포에 살모넬라균 감염을 수행하였을 때 대식세포의 nitric oxide (NO) 산생능력이 비 처리 군에 비해 저하되는 것으로 나타나, 살모넬라균에 의한 대식세포의 세포독성을 억제하는 것으로 나타났다. 종합적으로 SCWE의 숙 주세포에 대한 안전성, 살모넬라균에 대한 항균효과 및 대식세포 내 균 증식 억제 효과가 있는 것으로 나타나 SCWE를 이용한 세포내 기생세균의 치료제 개발이 가능할 것으로 판단된다.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of the innate immune system that recognize peptidoglycan, a unique cell wall component of bacteria. Here we cloned and characterized PGRP-S from the bumblebee Bombus ignitus (BiPGRP-S). The BiPGRP-S gene consists of four exons encoding 194 amino acid residues. Comparative analysis indicates that the predicted amino acid sequence of BiPGRP-S shares high identity with enzymatically active PGRP-S proteins and contains the amino acids required for amidase activity. BiPGRP-S in B. ignitus worker bees is constitutively expressed in boththe fat body and epidermis, and it is secreted into the hemolymph. Quantitative real-time PCR assays revealed that in both the fat body and epidermis, the BiPGRP-S gene is highly induced by an injection of Bacillus thuringiensis. In addition, recombinant BiPGRP-S expressed as a 19-kDa protein in baculovirus-infected insect cells can bind to B. megaterium and B. thuringiensis but not to Staphylococcus aureus, Escherichia coli or Beauveria bassiana. Consistent with these data, BiPGRP-S shows antibacterial activity against B. megaterium and B. thuringiensis. These results indicate that BiPGRP-S is an inducible protein that may be involved in the immune response against bacterial infection of the genus Bacillus as an amidase-type PGRP-S.

Attacin is a well-studied glycine-rich antibacterial protein in insect immune response, which has limitary antibacterial effect to some Gram-negative bacteria. A cDNA encoding the attacin gene was screened and isolated from the immunized larvae of the swallowtail butterfly, Papilio xuthus. The complete P. xuthus attacin cDNA is 949 nucleotides encoding a 250 amino acid precursor that contains a putative 18-residue signal peptide, a common 42-residue propeptide sequence and a presumed 190-residue mature protein with a theoretical mass of 19904.01 and a pI of 9.13. The putative mature protein of P. xuthus attacin showed 48%~52% and 24%~30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. The attacin transcript was induced at significant level after injection with bacterial lipopolysaccharide (LPS). Recombinant attacin was highly expressed in E. coli BL21 (DE3) cells by fusing with an N-terminal S-tag/thrombin cleavage site configuration protein to avoid the cell death during induction. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC). After desalting and cleavage with thrombin, the recombinant attacin was released and showed considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35. Our results proved that this protein family with a potent antibacterial activity may play a role in the immune response of butterflies.

The present study was undertaken to estimate the antibacterial effect of a combination of C. rhizoma,L. Flos, and P. japonica (1:1:1) extracts (CLP1000) and a combination of the herbal extract mixture and dioctahedral smectite (CLPS1000) against murine salmonellosis. At the concentration of CLP1000 and CLPS10000.5 mg/ml, the antibacterial effect was not showed on Salmonella typhimurium (S. typhimurium). On the other hand,the antibacterial effect against S. typhimurium was observed at the concentration of CLP1000 and CLPS1000 1.0 mg/ml. Oral administration of Smectite, CLP1000, and CLPS1000 at the dose of 10 mg/ml showed a therapeutic effect for S. typhimurium infected BALB/c mice. The mortality of Smectite, CLP1000 and CLPS1000-treated mice was 90%,90%, and 70% at 12 days, respectively, while that of untreated mice was 100% at 9 days after a lethal dose of S. typhimurium infection. The results of our study strongly indicate that CLPS1000 has potential as an effective of salmonellosis.

A novel beetle antimicrobial protein from stimulated Copris tripartitus and the corresponding gene were isolated in parallel through differential display-PCR and expression in Escherichia coli. To find cDNA clones responsible for bacteria resistance, the suppression subtractive hybridization and GeneFishing differentially expressed genes system were employed in the dung beetle, Copris tripartitus immunized with lipopolysaccaride. One cDNA clone from eight subtracted clones was selected through dot blot analysis and confirmed by northern blot analysis. The 516-bp, selected cDNA clone was determined by 5' and 3'rapid amplication of cDNA ends and cloned into the GST fusion expression vector pGEX-4T-1 for expression of the protein. The expressed protein was predicted 14.7 kDa and inhibited the growth of gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. These results implied that the expressed protein is related to immune defense mechanism against microorganism.

Thrombin-induced platelet microbicidal protein (tPMP) is a small cationic peptide that exerts potent in vitro microbicidal activity against a broad spectrum of human pathogens, including Staphylococcus aureus and Streptococcus rattus BHT. Earlier evidence has suggested that tPMP targets and disrupts the bacterial membrane. However, it is not yet clear whether membrane disruption itself is sufficient to kill the bacteria or whether subsequent, presumably intracellular, events are also involved in this process. In this study, we investigated the microbicidal activity of rabbit tPMP toward S. rattus BHT cells in the presence or absence of a pretreatment with antibiotics that differ in their mechanisms of action. The streptocidal effects of tPMP on control cells (no antibiotic pretreatment) were rapid and concentration-dependent. Pretreatment of S. rattus BHT cells with either penicillin or amoxicillin (inhibitors of bacterial cell wall synthesis) significantly enhanced the anti-S. rattus BHT effects of tPMP compared with the effects against the respective control cells over most tPMP concentration ranges tested. On the other hand, pretreatment of S. rattus BHT cells with tetracycline or doxycycline (30S ribosomal subunit inhibitors) significantly decreased the streptocidal effects of tPMP over a wide peptide concentration range. Furthermore, pretreatment with rifampin (an inhibitor of DNA-dependent RNA polymerase) essentially blocked the killing of S. rattus BHT by tPMP at most concentrations compared with the respective control cells. These results suggest that tPMP exerts anti-S. rattus BHT activity through mechanisms involving both the cell membrane and intracellular targets.

The cultural characteristics and antibacterial activities of Cordyceps militaris and Paecilomyces tenuipes were compared. The mycelial growth was the highest on MCM (Mushroom Complete Medium) for C. militaris and on YMA (Yeast Malt Agar) for P. tenuipes. But the mycelial density on MMM (Mushroom Minimal Medium) was lower than other on media. The optimum mycelial growth was observed at 25℃. C. militaris was low mycelial growth when it was transferred over 5 times generation. The carbon source for the optimum mycelial growth was fructose of monosaccharide, maltose of disaccharide and dextrin of polysaccharide. The calcium nitrate of organonitrogen was found the best mycelial growth on C. militaris, while the sodium nitrate observed to be well for mycelial growth on P. tenuipes. The ammonium tartrate was observed to be the best among the inorganonitrogen used for mycelial growth. Antibacterial activities were found out just C. militaris against Bacillus cereus of Gram (+).