Lanlan Bai

Antimicrobial activity of a bovine myeloid antimicrobial peptide (BMAP-28) against methicillin-susceptible and methicillin-resistant Staphylococcus aureus.

A bovine myeloid antimicrobial peptide (BMAP-28) is a member of the cathelicidin family which is included in the innate immune system of mammals. Recently, there have been many studies about antimicrobial peptides. This study aims to clarify whether BMAP-28 has bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) and compares its activity against methicillin-susceptible S. aureus (MSSA) and MRSA. We found that the peptide was effective in killing MRSA (minimal inhibitory concentration (MIC) range; 5-20 µg/mL). It was also revealed that MSSA (MIC range; 1.25-20 µg/mL) had two levels of susceptibility to BMAP-28. We also examined the effect of BMAP-28 on bacterial shape to visually show its activity. After exposure to the peptide, both MSSA and MRSA cells showed the morphological changes on their surfaces. Our results indicate that BMAP-28 is a promising candidate for medicine against drug-resistant bacteria.

Novel CD8+ cytotoxic T cell epitopes in bovine leukemia virus with cattle

Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T cell leukemia virus (HTLV). The cytotoxic T lymphocyte (CTL) plays a key role in suppressing the progression of disease caused by BLV. T and B cell epitopes in BLV have been studied, but CD8(+) CTL epitopes remain poorly understood. We used a library of 115 synthetic peptides covering the entirety of the Env proteins (gp51 and gp30), the Gag proteins (p15, p24, and p12), and the Tax protein of BLV to identify 11 novel CD8(+) T cell epitopes (gp51N5, gp51N11, gp51N12, gp30N5, gp30N6, gp30N8, gp30N16, tax16, tax18, tax19, and tax20) in four calves experimentally infected with BLV. The number of CD8(+) T cell epitopes that could be identified in each calf correlated with the BLV proviral load. Interestingly, among the 11 epitopes identified, only gp51N11 was capable of inducing CD8(+) T cell-mediated cytotoxicity in all four calves, but it is not a suitable vaccine target because it shows a high degree of polymorphism according to the Wu-Kabat variability index. By contrast, no CTL epitopes were identified from the Gag structural protein. In addition, several epitopes were obtained from gp30 and Tax, indicating that cellular immunity against BLV is strongly targeted to these proteins. CD8(+) CTL epitopes from gp30 and Tax were less polymorphic than epitopes from. Indeed, peptides tax16, tax18, tax19, and tax20 include a leucine-rich activation domain that encompasses a transcriptional activation domain, and the gp30N16 peptide contains a proline-rich region that interacts with a protein tyrosine phosphatase SHP1 to regulate B cell activation. Moreover, at least one CD8(+) CTL epitope derived from gp30 was identified in each of the four calves. These results indicate that BLV gp30 may be the best candidate for the development of a BLV vaccine.

Interaction between Leptospiral Lipopolysaccharide and Toll-like Receptor 2 in Pig Fibroblast Cell Line, and Inhibitory Effect of Antibody against Leptospiral Lipopolysaccharide on Interaction

Leptospiral lipopolysaccharide (L-LPS) has shown potency in activating toll-like receptor 2 (TLR2) in pig fibroblasts (PEFs_NCC1), and causes the expression of proinflammatory cytokines. However, the stimulation by L-LPS was weak eliciting the function of TLR2 sufficiently in pig innate immunity responses during Leptospira infection. In this study, the immune response of pig embryonic fibroblast cell line (PEFs_SV40) was investigated and was found to be the high immune response, thus TLR2 is the predominate receptor of L-LPS in pig cells. Further, we found a strategy using the antibody against L-LPS, to prevent L-LPS interaction with TLR2 in pig cells which could impact on immune activation.

A bovine myeloid antimicrobial peptide (BMAP-28) kills methicillin-resistant Staphylococcus aureus but promotes adherence of the bacteria.

The cathelicidin family is one of the several families of antimicrobial peptides (AMPs). A bovine myeloid antimicrobial peptide (BMAP-28) belongs to this family. Recently, the emergence of drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) has become a big problem. AMPs are expected to be leading compounds of new antibiotics against drug-resistant bacteria. In this study, we focused on the activity of BMAP-28 against bacterial cell surfaces. First, we observed morphological change of MRSA caused by BMAP-28 using a scanning probe microscope. We also studied activities of BMAP-28 against adherence of S. aureus to fibronectin, collagen type I, collagen type IV. We confirmed whether BMAP-28 can bind to lipoteichoic acid (LTA) of S. aureus. BMAP-28 was indicated as damaging the cell surface of MRSA. In a particular range of concentrations, BMAP-28 promoted adherence of S. aureus against fibronectin and collagens. It was revealed that BMAP-28 and LTA of S. aureus bound with each other. Our study showed the potential of BMAP-28 which can damage MRSA and interact with LTA of S. aureus but promote its adherence in some concentrations. This study provides new points of which to take notice when we use AMPs as medicines.

Antimicrobial activity of tea catechin against canine oral bacteria and the functional mechanisms

Epigallocatechin gallate (EGCG) is the major polyphenolic compound of green tea. Polyphenolic compounds were extracted from the leaf of Camellia sinensis (Japanese green tea), and the minimum inhibitory concentration against canine oral bacteria was measured. Subsequently, we investigated the inhibitory effects of polyphenolic compounds and EGCG on the growth of canine oral bacteria. EGCG showed antimicrobial activity against a model bacterium, Streptococcus mutans. Our results indicate that EGCG can inhibit the growth and biofilm formation of S. mutans and that EGCG does not interact with streptococcal lipoteichoic acid (LTA). Furthermore, our findings suggest that EGCG interacts with other component(s) of the bacterial membrane aside from streptococcal LTA to inhibit biofilm formation and damage biofilms.

Susceptibility difference between methicillin-susceptible and methicillin-resistant Staphylococcus aureus to a bovine myeloid antimicrobial peptide (BMAP-28)

A bovine myeloid antimicrobial peptide antimicrobial peptide (BMAP-28) is a member of the cathelicidin family and acts as a component of innate immunity. There are few reports of susceptibility difference of methicillin-resistant Staphylococcus aureus (MRSA) and susceptible strains (MSSA) against BMAP-28. This study aims to clarify how a few amino acid substitutions of BMAP-28 are related to its antimicrobial activity using four analog peptides of BMAP-28. We also compared cellular fatty acid components of MSSA and MRSA using gas chromatography. We found that a few amino acid substitutions of BMAP-28 do not change antimicrobial activity. It was also revealed that the percentage of cis-11-eicosenoic acid in total detected fatty acids of MRSA was significantly higher than that of MSSA. In addition, the percentage of palmitic acid in total detected fatty acids of MRSA tended to be lower than that of MSSA. Our results will provide new information to deal with the question of differences in bacterial susceptibility against BMAP-28.

Epitope mapping of CD8+ T cells on bovine leukemia virus Gag, Env and Tax protein in cattle with different bovine MHC DRB3 alleles

The bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leucosis (EBL) which is the most common neoplastic disease of cattle. Recent studies show bovine leukocyte antigen (BoLA)-DRB3 gene plays a direct role in controlling the number of proviral load in cattle and the homozygote BoLA-DRB3*1601 is associated with high proviral load. The other hand, the CD8+ cytotoxic T cell (CTL) response is an important defense against viral invasion. To find the candidate of vaccine for BLV infection we performed CD8+ T cell epitopes mapping in BLV-infected cattle with different BoLA-DRB3 alleles. In this study, 20 amino acids in length of 115 synthetic peptides were made from the sequence of Gag, Env and Tax protein of BLV, overlapping by 10 amino acids. Then total 11 CD8+ T cell epitopes were found out in BLV-infected four Japanese Black cattle with different BoLA-DRB3 alleles that are both of DRB3 *1601/1601, and DRB3 *1501/2703 and DRB3 *1501/0503 allele by WST assay. Different CD8+ T cell epitopes were recognized by cattle with different BoLA-DRB3 alleles and then dose-dependent cytotoxicity activity were measured by non-radioactive cytotoxicity assay. All of four cattle responded to gp51N11, but it has higher variability values by Wu-Kabat variability index analysis. By contrast, CD8+ T cell epitopes were determined from all of four cattle on gp30 region and they have high conservatives. gp30N16 among 4 epitopes was detected in each low proviral load cattle from susceptibility and neutrality groups. Those results were suggested that gp30 region maybe the best candidate for vaccine that can induce cell-mediated immunity against this disease.

Development of a new recombinant p24 ELISA system for diagnosis of bovine leukemia virus in serum and milk

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leucosis. Here, we designed a p24 enzyme-linked immunosorbent assay (ELISA) to detect antibodies specific for BLV capsid protein p24 (encoded by the gag gene) in bovine serum samples. The p24 gene was inserted into an Escherichia coli expression system, and recombinant proteins (GST-p24, p24, and His-p24) were purified. His-p24 was the most suitable antigen for using in the ELISA. The cut-off point was calculated from a receiver operating characteristic curve derived from a set of 582 field samples that previously tested positive or negative by BLV-CoCoMo-qPCR-2, which detects BLV provirus. The new p24 ELISA showed almost the same specificity and sensitivity as a commercial gp51 ELISA kit when used to test field serum samples, and allowed monitoring of p24 antibodies in raw milk and whey. Comparing the results for the p24 ELISA and gp51 ELISA revealed that p24 antibodies were detected earlier than gp51 antibodies in three out of eight calves experimentally infected with BLV, indicating improved detection without diminishing BLV serodiagnosis. Thus, the p24 ELISA is a robust and reliable assay for detecting BLV antibodies in serum or milk, making it is a useful tool for large-scale BLV screening.

Peptide microarray mapping of B cell epitopes on bovine leukemia virus and peptide ELISA analysis of conservation of epitopes in BLV infected Japanese cattle

The bovine leukemia virus (BLV), a retrovirus structurally and functionally related to the human T-lymphotropic viruses HTLV-1 and HTLV-2, is the etiological agent of bovine leucosis. B cell immune mechanisms may play a major role in protection against BLV infection. A battery of 157 synthetic peptides, 15-mer length, 4 amino acids overlapped, was used to mapping B cell epitopes on BLV envelope glycoprotein gp5l and capsid protein by peptide microarrays. Two susceptible cattle with the homozygote BoLA-DRB3 *1601 allele and two non-susceptible cattle with DRB3 *1501/*2703 alleles and DRB3 *1501/*0503 alleles were infected with BLV, and serums were collected from those cattle each of having challenged BLV before and after. Only one epitope A out of 157 synthetic peptides responds with all of four BLV positive serums not their negative serums. To demonstrate whether epitope A is common B cell epitope or not by our established peptide ELISA system that uses maleimide activated mariculture keyhole limpet hemocyanin (mcKLH) carrier protein. To found, there are 7 kinds of BLV positive serum no response with epitope A among 232 kinds of positive serum. Furthermore, we searched other peptides respond with BLV positive serum that contain 7 kinds of serum no responded with epitope A and found epitope B among peptides of non-specifically responded on peptide microarray. Epitope B strongly responded with all of 232 kinds of serum as estimated by peptide ELISA system. Our results shows that epitope B has a tendency to react some BLV negative serums, thus we will perform further experiments to determine the common B cell epitope just responses with positive serum by peptides that are 3 alanine substitutions as shifting one amino acid on epitope sequence.

A novel bovine leukemia virus peptide vaccine targeting susceptible cattle-Estimating vaccine effectiveness using susceptible cattle constructed by fertilized ovum transplantation

Bovine leukemia virus (BLV), the aetiological agent of the enzootic bovine leukosis, was widely spread in the worldwide, and it is desired to develop vaccine. However, the disease progression is greatly affected by host factors, and the difference of disease susceptibility is caused the reason to make hard to estimate the BLV vaccine effect. In this study, we confirmed the association of the genotype of cattle major histocompatibility complex (BoLA) class II DRB3 gene and disease susceptibility by experimentally infection study using the cattle made by fertilized ovum transplantation technique. Susceptibility cattle were also used for estimating the effect of our developed new p12-4R1/gp51R1 peptide-conjugated Carbonate apatite (CO3Ap) vaccine and succeeded to detect the novel vaccines effects such as A) pathologic suppression, B) Transmission interference, and C) infection inhibition. We produced six heads of disease susceptibility cattle (BoLA-DRB3*1601/*1601) which made by fertilized ovum transplantation technique, and also prepared four normal cattle (BoLA-DRB3*1501/*2703, DRB3*1501/*0503 and DRB3*1501/*1201). These cattle were attacked by BLV (copy number: 3-4 x 107), and measured the proviral load, lymphocyte count, and antibody titer for 161 days (susceptibility cattle) and 119 days (normal cattle) for estimating the disease susceptibility and also the vaccine effect. Within the three kind of test, A) pathologic suppression effect was most strong. In six BLV-infected susceptibility cattle, three vaccinated group were significantly suppressed the proliferation of proviral load, lymphocyte count, and CD5+B cell count, compared with unvaccinated group. On the contrary, in four normal cattle test, there is no significantly difference between the vaccinated group and control group because of high host factors effect. This result clearly showed that the advantage of using susceptibility cattle for estimating the effect of anti-BLV vaccine, drugs or any treatments, and the BoLA genotyping is one of the good markers for using these strategies.