Jianhe Hu

Potential of Novel Antimicrobial Peptide P3 from Bovine Erythrocytes and Its Analogs To Disrupt Bacterial Membranes In Vitro and Display Activity against Drug-Resistant Bacteria in a Mouse Model

ABSTRACT With the emergence of many antibiotic-resistant strains worldwide, antimicrobial peptides (AMPs) are being evaluated as promising alternatives to conventional antibiotics. P3, a novel hemoglobin peptide derived from bovine erythrocytes, exhibited modest antimicrobial activity in vitro. We evaluated the antimicrobial activities of P3 and an analog, JH-3, both in vitro and in vivo. The MICs of P3 and JH-3 ranged from 3.125 μg/ml to 50 μg/ml when a wide spectrum of bacteria was tested, including multidrug-resistant strains. P3 killed bacteria within 30 min by disrupting the bacterial cytoplasmic membrane and disturbing the intracellular calcium balance. Circular dichroism (CD) spectrometry showed that P3 assumed an α-helical conformation in bacterial lipid membranes, which was indispensable for antimicrobial activity. Importantly, the 50% lethal dose (LD50) of JH-3 was 180 mg/kg of mouse body weight after intraperitoneal (i.p.) injection, and no death was observed at any dose up to 240 mg/kg body weight following subcutaneous (s.c.) injection. Furthermore, JH-3 significantly decreased the bacterial count and rescued infected mice in a model of mouse bacteremia. In conclusion, P3 and an analog exhibited potent antimicrobial activities and relatively low toxicities in a mouse model, indicating that they may be useful for treating infections caused by drug-resistant bacteria.

Thymol kills bacteria, reduces biofilm formation, and protects mice against a fatal infection of Actinobacillus pleuropneumoniae strain L20

Actinobacillus pleuropneumoniae is the causative agent of the highly contagious and deadly respiratory infection porcine pleuropneumonia, resulting in serious losses to the pig industry worldwide. Alternative to antibiotics are urgently needed due to the serious increase in antimicrobial resistance. Thymol is a monoterpene phenol and efficiently kills a variety of bacteria. This study found that thymol has strong bactericidal effects on the A. pleuropneumoniae 5b serotype strain, an epidemic strain in China. Sterilization occurred rapidly, and the minimum inhibitory concentration (MIC) is 31.25μg/mL; the A. pleuropneumoniae density was reduced 1000 times within 10min following treatment with 1 MIC. Transmission electron microscopy (TEM) analysis revealed that thymol could rapidly disrupt the cell walls and cell membranes of A. pleuropneumoniae, causing leakage of cell contents and cell death. In addition, treatment with thymol at 0.5 MIC significantly reduced the biofilm formation of A. pleuropneumoniae. Quantitative RT-PCR results indicated that thymol treatment significantly increased the expression of the virulence genes purC, tbpB1 and clpP and down-regulated ApxI, ApxII and Apa1 expression in A. pleuropneumoniae. Therapeutic analysis of a murine model showed that thymol (20mg/kg) protected mice from a lethal dose of A. pleuropneumoniae, attenuated lung pathological lesions. This study is the first to report the use of thymol to treat A. pleuropneumoniae infection, establishing a foundation for the development of new antimicrobials.

The role of natural antimicrobial peptides during infection and chronic inflammation

Natural antimicrobial peptides (AMPs), a family of small polypeptides that are produced by constitutive or inducible expression in organisms, are integral components of the host innate immune system. In addition to their broad-spectrum antibacterial activity, natural AMPs also have many biological activities against fungi, viruses and parasites. Natural AMPs exert multiple immunomodulatory roles that may predominate under physiological conditions where they lose their microbicidal properties in serum and tissue environments. Increased drug resistance among microorganisms is occurring far more quickly than the discovery of new antibiotics. Natural AMPs have shown promise as ‘next generation antibiotics’ due to their broad-spectrum curative effects, low toxicity, the fact that they are not residual in animals, and the low rates of resistance exhibited by many pathogens. Many types of synthetic AMPs are currently being tested in clinical trials for the prevention and treatment of various diseases such as chemotherapy-associated infections, diabetic foot ulcers, catheter-related infections, and other conditions. Here, we provide an overview of the types and functions of natural AMPs and their role in combating microorganisms and different infectious and inflammatory diseases.

Development of an Indirect Dot-PPA-ELISA using glutamate dehydrogenase as a diagnostic antigen for the rapid and specific detection of Streptococcus suis and its application to clinical specimens

Streptococcus suis is an important zoonotic pathogen causing infections in pigs and humans. Bacterial surface-related proteins are often explored as potential vaccine candidates and diagnostic antigens. In the present study, glutamate dehydrogenase, a highly conserved immunogenic extracellular protein, was used to establish a dot horseradish peroxidase enzyme-linked staphylococcal protein A immunosorbent assay (Dot-PPA-ELISA) for diagnosis of S. suis infection. The antigen–antibody reaction was optimised through checkerboard titration involving serial dilutions, followed by selective blocking tests and evaluations of cross-reaction, repeatability, and stability. Comparative analysis by using a conventional plate ELISA kit showed that the specificity and sensitivity of the Dot-PPA-ELISA were 97.5 and 96.6%, respectively. Furthermore, dynamic changes in the levels of antibody in rabbits immunised with a propolis inactivated vaccine were monitored by Dot-PPA-ELISA. A total seroprevalence of 73.1% in 305 pig serum samples indicated the method’s applicability to detect S. suis infection. Cumulatively, the results suggested that Dot-PAA-ELISA is a convenient, rapid, sensitive, and specific diagnostic method suitable for studying large numbers of samples obtained from clinical and epidemiological studies, thereby helping reduce important economic losses.

Halofuginone-induced autophagy suppresses the migration and invasion of MCF-7 cells via regulation of STMN1 and p53

Traditional Chinese medicines have been recognized as especially promising anticancer agents in modern anticancer research. Halofuginone (HF), an analog of quinazolinone alkaloid extracted from Dichroa febrifuga, is widely used in traditional medicine. However, whether HF inhibits the growth of breast cancer cells and/or reduces the migration and invasion of MCF‐7 human breast cancer cells, as well as the underlying mechanisms in vitro, remains unclear. In this study, we report that an HF extract inhibits the growth of MCF‐7 cells and reduces their migration and invasion, an important feature of potential anticancer agents. In addition, HF significantly increases the activation of autophagy, which is closely associated with tumor metastasis. As STMN1 and p53 have been closely implicated in breast cancer progression, we analyzed their expression in the context of HF extract treatment. Western blot analysis showed that HF suppresses STMN1 and p53 expression and activity in an autophagy‐dependent manner. Collectively, these data indicate that activation of autophagy reduces expression of STMN1 and p53, and the migration and invasion of cancer cells contributes to the anti‐cancer effects of the HF. These findings may provide new insight into breast cancer prevention and therapy.

HJH-1, a Broad-Spectrum Antimicrobial Activity and Low Cytotoxicity Antimicrobial Peptide

With the overuse of antibiotics, multidrug-resistant bacteria pose a significant threat to human health. Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. This study examines the antimicrobial and membrane activity of HJH-1, a cationic peptide derived from the hemoglobin α-subunit of bovine erythrocytes P3. HJH-1 shows potent antimicrobial activity against different bacterial species associated with infection and causes weaker hemolysis of erythrocytes, at least five times the minimum inhibitory concentration (MIC). HJH-1 has good stability to tolerance temperature, pH value, and ionic strength. The anionic membrane potential probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] and propidium iodide are used as indicators of membrane integrity. In the presence of HJH-1 (1× MIC), Escherichia coli membranes rapidly depolarise, whereas red blood cells show gradual hyperpolarisation. Scanning electron microscopy and transmission electron micrographs show that HJH-1 (1× MIC) damaged the membranes of Escherichia coli, Staphylococcus aureus, and Candida albicans. In conclusion, HJH-1 damages the integrity of the bacterial membrane, preventing the growth of bacteria. HJH-1 has broad-spectrum antibacterial activity, and these activities are performed by changing the normal cell transmembrane potential and disrupting the integrity of the bacterial membrane.

GCN2 controls the cellular checkpoint: potential target for regulating inflammation

Accumulating evidence suggests that cellular stress signals, including those induced by nutrient availability, strongly influence the function of the immune system. The integrated stress response (ISR) is a cytoprotective response mediated by eukaryotic translation initiation factor 2α (eIF2α) that enables cells to sense and respond to diverse cellular signals, including endoplasmic reticulum (ER) stress, the unfolded protein response and nutrient deprivation. GCN2, one of four known sensors of the ISR, can be activated by single amino acid deprivation. In the context of low levels of an amino acid, uncharged tRNA molecules accumulate and bind to the HisRS domain of GCN2 (Fig. 1). Subsequently, the phosphorylation of eIF2αin Ser-51 occurs, which inhibits its function, leading to reduced mRNA translation and protein synthesis, thereby reducing the amino acid supply. Concurrently, eIF2α phosphorylation enhances the translation of specific mRNA molecules that contain 5ʹ-terminal leader sequences. These molecules include the transcription factor ATF4, which controls the transcription and expression of hundreds of genes, maintains cell homeostasis, and participates in protein metabolism, host responses to infection, responses to immunization, inflammation and other physiological and pathological processes. As nutrients are metabolized, dynamic changes in nutrient bioavailability occur in the intestine, which can trigger metabolic sensors such as GCN2 and potentially modulate gut immune responses. In a recent report published in Nature, Ravindran et al. demonstrated that GCN2-mediated amino acid starvation-sensing mechanisms can shape intestinal inflammation in the dextran sodium sulfate (DSS) mouse model of colitis, indicating that GCN2 has the ability to modulate immune responses, especially inflammation (Fig. 1). In our previous study, we showed another setting in which GCN2 is activated and IFN-gamma (IFN-γ) accelerates bovine mammary epithelial cell (BMEC) arginine consumption, resulting in activation of the amino acid starvation response that further drives GCN2 activity in a lactating Holstein cow model, suggesting that GCN2 can be activated by non-infectious (i.e., sterile) inflammation (Fig. 1). During infection, bacterial or viral growth may cause a nutrient shortage in cells or tissues, which might activate GCN2 signaling (Fig. 1). Damage of the plasma membrane or inhibition of amino acid uptake by poreforming toxins (PFT), such as α-toxin, also causes amino acid starvation and energy loss, resulting in GCN2 activation (Fig. 1). Thus, nutrient and energy sensors such as GCN2 may serve as sentinels for the initiation of immune responses. In the experimental autoimmune encephalomyelitis mouse model, central nervous system inflammation was enhanced after GCN2 deletion, which was characterized by increased expression of IL-17 and IFN-γ and decreased expression of IL-10 during the remission phase, resulting in enhanced nervous tissue inflammation and demyelinating lesions. Activation of GCN2 in macrophages promotes the expression of anti-inflammatory cytokine IL-10 in vitro. In vivo, apoptotic cells can stimulate the production of anti-inflammatory cytokines IL-10 and TGF-β in macrophage in a GCN2-dependent manner, whereas myeloid cell-specific deletion of GCN2 abrogated regulatory cytokine production that resulted in increased immune cell activation, humoural autoimmunity, renal

Methods for the detection and characterization of Streptococcus suis: from conventional bacterial culture methods to immunosensors

One of the most important zoonotic pathogens worldwide, Streptococcus suis is a swine pathogen that is responsible for meningitis, toxic shock and even death in humans. S. suis infection develops rapidly with nonspecific clinical symptoms in the early stages and a high fatality rate. Recently, much attention has been paid to the high prevalence of S. suis as well as the increasing incidence and its epidemic characteristics. As laboratory-acquired infections of S. suis can occur and it is dangerous to public health security, timely and early diagnosis has become key to controlling S. suis prevalence. Here, the techniques that have been used for the detection, typing and characterization of S. suis are reviewed and the prospects for future detection methods for this bacterium are also discussed.

M protein is sufficient for assembly and release of Peste des petits ruminants virus-like particles

Peste des petits ruminants virus (PPRV), belonging to paramyxoviruses, has six structure proteins (such as matrix protein (M), nucleocapsid proteins (N), fusion protein (F) and hemagglutinin protein (H)) and could cause high morbidity and mortality in sheep and goats. Although a vaccine strain of PPRV has been rescued and co-expression of M and N could yield PPRV-like particles, the roles of structure proteins in virion assembly and release have not been investigated in detail. In this study, plasmids carrying PPRV cDNA sequences encoding the N, M, H, and F proteins were expressed in Vero cells. The co-expression of all four proteins resulted in the release of virus-like particles (VLPs) with similar release efficiency to that of authentic virions. Moreover, the co-expression of M together with F also resulted in efficient VLPs release. In the absence of M protein, the expression of no combination of the other proteins resulted in particle release. In summary, a VLPs production system for PPRV has been established and M protein is necessary for promoting the assembly and release of VLPs, of which the predominant protein is M protein. Further study will be focused on the immunogenicity of the VLPs.

Serial expression and activity analysis of LNK-16: a bovine antimicrobial peptide analogue.

Abstract Indolicidin is a broad-spectrum antimicrobial peptide (AMP) with great therapeutic potential; however, high manufacturing costs associated with industrial-scale chemical synthesis have limited its delivery. Therefore, the use of recombinant DNA technology to produce this peptide is urgently needed. In this study, a new methodology for the large-scale production of a novel bovine AMP was developed. LNK-16 is an analogue of indolicidin that contains a kallikrein protease site at its C-terminus. The amino acid sequence of LNK-16 was synthesized using Escherichia coli-preferred codons. Three copies of the target gene were assembled in series by overlapping PCR and cloned into pET-30a(+) for the expression of His-(LNK-16)3 in E. coli BL21 (DE3) cells. The expressed fusion protein His-(LNK-16)3 was purified by Ni2+-chelating chromatography and then cleaved by kallikrein to release LNK-16. The recombinant LNK-16 peptide showed antimicrobial activity similar to that of chemically synthesized LNK-16 and indolicidin. Together, these data indicate that the use of serial expression can improve the large-scale production of AMPs for clinical and research applications.