Adh Promotes Actinobacillus pleuropneumoniae Survival in Porcine Alveolar Macrophages by Inhibiting CHAC2-Mediated Respiratory Burst and Inflammatory Cytokine Expression.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes porcine pleuropneumonia that seriously endangers pig's health. Adh, located in the head region of trimeric autotransporter adhesion of A. pleuropneumoniae, affects bacterial adhesion and pathogenicity. However, how Adh mediates A. pleuropneumoniae immune invasion is still unclear. Here, we established the A. pleuropneumoniae strain L20 or L20 ΔAdh-infected porcine alveolar macrophages (PAM) model, and applied protein overexpression, RNA interference, qRT-PCR, Western blot and immunoflourescence techniques to dissect the effects of Adh on PAM during A. pleuropneumoniae infection. We found that Adh could increase the A. pleuropneumoniae adhesion and intracellular survival in PAM. Gene chip analysis of piglet lungs further showed that Adh significantly induced cation transport regulatory-like protein 2 (CHAC2) expression, whose overexpression suppressed the phagocytic capacity of PAM. Furthermore, CHAC2 overexpression dramatically increased glutathione (GSH) expression, decreased reactive oxygen species (ROS), and promoted A. pleuropneumoniae survival in PAM, while the knockdown of CHAC2 reversed these phenomena. Meanwhile, CHAC2 silence activated the NOD1/NF-κB pathway, resulting in an increase in IL-1ß, IL-6, and TNF-α expression, whereas this effect was weakened by CHAC2 overexpression and addition of NOD1/NF-κB inhibitor ML130. Moreover, Adh enhanced the secretion of LPS of A. pleuropneumoniae, which regulated the expression of CHAC2 via TLR4. In conclusion, through a LPS-TLR4-CHAC2 pathway, Adh inhibits respiratory burst and inflammatory cytokines expression to promote A. pleuropneumoniae survival in PAM. This finding may provide a novel target for the prevention and treatment of A. pleuropneumoniae.

IL-5 enhances the resistance of Actinobacillus pleuropneumoniae infection in mice through maintaining appropriate levels of lung M2, PMN-II and highly effective neutrophil extracellular traps.

Interleukin 5 (IL-5) regulates the maturation, activation, proliferation and function of immune cells, and plays an important role in the inflammatory response induced by an allergy. However, its anti-pathogen effect is poorly understood currently, especially on pneumonia. Here, this study was designed to elucidate the immunological role of IL-5 in the infection of mice with Actinobacillus pleuropneumoniae (APP). We established an acute lung infection model of APP in IL-5 knockout mice (IL-5-/-) and wild-type mice (WT) through nasal infusion or intraperitoneal injection, compared the survival rate, clinical symptoms, lung bacterial load, proportion of various immune cells, immune molecular expression, and neutrophil germicidal ability through flow cytometry, RT-qPCR, ELISA and immunofluorescence. Compared to WT mice, the IL-5-/- mice had a lower survival rate, more severe clinical symptoms, significantly increased bacterial load, and inflammatory cell infiltration in the lung after APP infection. In an uninfected state, IL-5 deficiency decreased the number of M1 interstitial macrophages and CD14- monocytes, while after infection, IL-5 deficiency significantly reduced the M2 alveolar macrophages, and increased PMN-II cells in the lung. Furthermore, the expression of IL-10, IL-4, IL-33, TNF-α, iNOS in the lung was lower in IL-5-/- mice under an uninfected condition, and the secretion of IL-18 was significantly increased after infection. In addition, IL-5 deficiency decreased bactericidal ability by inhibiting the formation of neutrophil extracellular traps (NETs). Collectively, these results provide evidence that IL-5 can enhance the resistance of APP infection, and its anti-infection mechanism, implying new targets and ideas for APP or similar respiratory agents' prevention and treatment.

Dynamic immune response characteristics of piglets infected with Actinobacillus pleuropneumoniae through omic.

Porcine infectious pleuropneumonia is characterized by a high-rate of carriage and mixed infection with other pathogens. The host immune response induced by Actinobacillus pleuropneumoniae (APP) is the basis for elucidating pathogenesis and controlling disease. However, there is currently no comprehensive and dynamic data characterising the host immune response. In this study, piglets were infected with APP and differentially expressed proteins of bronchoalveolar lavage fluid (BALF) and peripheral serum were identified by iTRAQ-LC-MS/MS, and differentially expressed genes of peripheral blood mononuclear cells (PBMC) by RNA-seq. The results of the integrated analysis of serum, BALF and PBMC showed significant metabolism and local immune responses in BALF, the general immune response in PBMC mainly involves cytokines, while that in serum mainly involves biosynthesis, phagosome, and complement and coagulation cascades. Furthermore, immune responses in PBMCs and serum were rapid and maintained compared to the lung where metabolism and cell adhesion activities were enriched. Some innate immunity pathways of the cellular response to ROS, neutrophil mediated immunity, granulocyte activation and leukocyte cell-cell adhesion were identified as central points, connecting multiple signaling pathways to form an integrated large network. At 24 h post-infection, 14 molecules were up regulated in BALF, 10 of which were shared with PBMC, but at 120 h, 20 down-regulated molecules were identified in BALF, 11 of them still up- regulated in PBMC. We conclude that, the immune response in the lung is different from that in blood, but there is a similarity in response in PBMC and serum.

IFN-γ-/- Mice Resist Actinobacillus pleuropneumoniae Infection by Promoting Early Lung IL-18 Release and PMN-I Accumulation.

Porcine pleuropneumonia is a common infectious disease of pigs caused by Actinobacillus pleuropneumoniae Interferon gamma (IFN-γ) expression increases in the lung of pigs after A. pleuropneumoniae infection, but the role of IFN-γ during the infection is still obscure. In this study, an IFN-γ-/- mouse infection model was established, and bacterial load, levels of inflammatory cytokines, and types of neutrophils in the lungs were studied at different times post-A. pleuropneumoniae infection. We found that wild-type (WT) mice were more susceptible to A. pleuropneumoniae than IFN-γ-/- mice. At 6 h postinfection (hpi), the expression of interleukin 18 (IL-18) and IL-1ß in the lungs of IFN-γ-/- mice was significantly increased compared to WT mice. The bacterial load and levels of inflammatory cytokines (IL-1ß and IL-6) of IFN-γ-/- mice were significantly reduced at 12 hpi compared to WT mice. After an initial loss, the numbers of lung polymorphonuclear (PMN)-I cells dramatically increased in the lungs of IFN-γ-/- but not WT mice, whereas PMN-II cells continually decreased. Finally, in vivo administration of IL-18 significantly reduced clinical scores and bacterial load in the lungs of A. pleuropneumoniae-infected mice. This study identifies IFN-γ as a target for regulating the inflammatory response in the lung and provides a basis for understanding the course of clinical bacterial pneumonia and for the formulation of treatment protocols.

Streptococcus suis serotype 2 enolase interaction with host brain microvascular endothelial cells and RPSA-induced apoptosis lead to loss of BBB integrity.

Host proteins interacting with pathogens are receiving more attention as potential therapeutic targets in molecular medicine. Streptococcus suis serotype 2 (SS2) is an important cause of meningitis in both humans and pigs worldwide. SS2 Enolase (Eno) has previously been identified as a virulence factor with a role in altering blood brain barrier (BBB) integrity, but the host cell membrane receptor of Eno and The mechanism(s) involved are unclear. This study identified that SS2 Eno binds to 40S ribosomal protein SA (RPSA) on the surface of porcine brain microvascular endothelial cells leading to activation of intracellular p38/ERK-eIF4E signalling, which promotes intracellular expression of HSPD1 (heat-shock protein family D member 1), and initiation of host-cell apoptosis, and increased BBB permeability facilitating bacterial invasion. This study reveals novel functions for the host-interactional molecules RPSA and HSPD1 in BBB integrity, and provides insight for new therapeutic strategies in meningitis.

RPSA distribution and expression in tissues and immune cells of pathogen-infected mice.

AIMS: 40S ribosomal protein SA (RPSA), a component of the small ribosomal subunit, is a high-affinity receptor of laminin that is widely expressed in cells and involves in many biological processes. However, it hasn't been reported which tissues and cells may be targeted by RPSA-mediated pathogen regulation. Therefore, in this study, a gram-positive bacterium Streptococcus suis Type 2 (SS2), gram-negative bacterium Actinobacillus pleuropneumoniae (A.pleuropneumoniae), and porcine circovirus Type 2 (PCV2) were used to infect ICR mice. METHODS AND RESULTS: The effects of infection with the three pathogens on expression levels of RPSA in mouse tissues and peripheral blood immune cells were analysed by immunohistochemistry and flow cytometry. The results suggested that the pathological changes in mice infected with SS2 were mainly manifested as congestion and inflammatory infiltration in the meninges, lungs, hearts and livers. The mice infected with A.pleuropneumoniae or PCV2 showed lung lesions and mild hepatocyte degeneration, respectively. In uninfected mice, RPSA protein was expressed to various degrees in all tissues except the spleen. After SS2 infection for 3 d, the expression of RPSA in the liver and brain increased, while decreased significantly in the heart and duodenum. These results were corroborated on examining the correlation between RPSA expression and the process of SS2 infection, except that there was no significant difference between the expression levels in the heart at 1 d and 3 d. After A.pleuropneumoniae and PCV2 infection for 3 d, the expression of RPSA decreased in the heart, and brain, respectively. Additionally, under physiological conditions, RPSA expression in CD4+ T cells, CD8+ T cells, neutrophils, and macrophages in the peripheral blood of mice was higher than that in B cells and NK cells. After SS2 infection for 3 d, RPSA expression increased significantly in CD4+ T cells and CD8+ T cells but decreased significantly in macrophages. The expression of RPSA after A.pleuropneumoniae and PCV2 infection were similar, and RPSA expression decreased only in macrophages. CONCLUSIONS: The results revealed that RPSA showed different expression levels in tissues and immune cells due to different pathogens causing disease courses, suggesting different target tissues and target cells in RPSA-mediated pathogenesis after infection, which supports the systematic study of the pathogenesis of RPSA in infectious diseases.

Recombinant tandem epitope vaccination provides cross protection against Actinobacillus pleuropneumoniae challenge in mice.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae/APP) is the pathogen that causes porcine contagious pleuropneumonia. Actinobacillus pleuropneumoniae is divided into 18 serovars, and the cross protection efficacy of epitopes is debatable, which has resulted in the slow development of a vaccine. Consequently, epitope-based vaccines conferring Actinobacillus pleuropneumoniae cross protection have rarely been reported. In this study, B cell epitopes in the head domain of trimeric autotransporter adhesin were predicted, and 6 epitopes were selected. Then, the predicted epitopes (Ba1, Bb5, C1, PH1 and PH2) were connected by linkers to construct a recombinant tandem antigen (rta) gene. The RTA protein encoded by the recombinant rta gene was expressed, and finally the ICR mice were immunized with the RTA protein with or without inactivated Actinobacillus pleuropneumoniae (serovars 1 and 5b) and challenged with Actinobacillus pleuropneumoniae to evaluate the protective effect of the epitope-based vaccine and combined vaccine. The mice in the RTA-immunized group and RTA plus inactivated Actinobacillus pleuropneumoniae vaccine group had a significant improvement in clinical symptoms and a higher level of antibody in the serum than those in the control group. The RTA immune group had a 40% survival rate after Actinobacillus pleuropneumoniae infection, whereas the combination of RTA and inactivated Actinobacillus pleuropneumoniae produced very strong cross immune protection in mice, at least 50% (RTA IB1 + C5) and at most 100% (RTA IB5 + C1), whereas no cross immunoprotection was found in the solo Actinobacillus pleuropneumoniae immune group. Overall, the combination of the RTA protein and inactivated bacteria significantly enhanced the cross protection effects. This implies that RTA protein in combination with a suitable inactivated Actinobacillus pleuropneumoniae strain could be a candidate vaccine for porcine contagious pleuropneumonia.

Differences in pig respiratory tract and peripheral blood immune responses to Actinobacillus pleuropneumoniae.

Excessive cytokine production is an important component of the acute respiratory distress syndrome and multiple organ failure. Pneumonia can lead to an overexpression of cytokines, although comparatively little is known about the relevance and differences in cytokines between blood and lung. In this study, piglets were experimentally infected intranasally with Actinobacillus pleuropneumoniae (APP), and transcriptomes of lung tissue and peripheral blood mononuclear cells determined. In addition, the levels of 30 cytokines in broncheoalveolar lavage fluid (BALF) and sera were determined by ELISA. Post infection, there was an early increase in lung monocytes, and a later rise in inflammatory cytokines in BALF. Blood lymphocytes increased early in infection and there was a rise in inflammatory cytokines in the peripheral blood of infected piglets. Genes involved in cytokine production, leukocyte migration and differentiation, lymphocyte activation, and cytokine-mediated signaling pathways in the transcriptomes of lung tissue were significantly down-regulated early in infection. At this early phase of APP infection (0-6 h), the cytokines IL-1ß, MCP-1, and IL-5 in sera increased rapidly and significantly, while many cytokines in BALF decreased. At 48 h post-infection, cytokines in sera were no longer significantly increased, although some were up-regulated in BALF, and there was aggravated pathological damage in the lungs at this time. The data indicate there are substantial differences between immune cells and cytokines in the lung and peripheral blood of APP infected piglets at equivalent time points. The results increase our understanding of pig-APP host interactive biology, and will be important in formulating future therapeutic and preventative strategies to prevent disease caused by APP.

iTRAQ-based quantitative proteomic analysis of peripheral blood serum in piglets infected with Actinobacillus pleuropneumoniae.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae (APP) is a swine respiratory disease with an important impact around the world either as a single infection or part of the porcine respiratory disease complex. The data of interaction between hosts and pathogens has becoming more crucial for exploration of the mechanism. However, up to now, comparatively little information is available on the systemic and dynamic changes that occur in pig serum in response to APP infection. This study used iTRAQ to identify differentially expressed proteins (DEPs) in pig serum in response to APP infection. Compared with the APP un-infected group (S0),there were 137 up-regulated and 68 down-regulated proteins at 24 h (S24), and 81 up-regulated and 107 down-regulated proteins at 120 h (S120). At 24 h, the immune response was not significantly enriched, but cell adhesion, cytosol, Golgi apparatus, GTP and ATP binding and regulation of cell cycle were extremely active, implying host preparation of immune response starting. Subsequently, innate immune response, negative regulation of apoptotic process, immunological synapse, adaptive immune response, the regulation of inflammatory response, positive regulation of T cell proliferation were more enhanced at 120 h then that of 24 h, representing innate immunity transferring to the adaptive, while endocytosis, cell adhesion and platelet aggregation showed obvious decline. The pathways of T cell receptor signaling pathway, cytokine-cytokine receptor interaction, complement and coagulation cascades, leukocyte transendothelial migration were active remarkably during all infection period, and more pathways could connect to form innate immune defense networks. Surprisingly, the pathways like amoebiasis, rheumatoid arthritis and malaria had been found up-regulated. As a conclusion, APP could delay host inflammatory response to the infection at early stage, and induced innate immunity to convert from adhesion, interaction into complement activation, proteasome digestion, bacterial invasion at later stage. This would increase our understanding of the porcine distinct response to APP infection.

Proteomics analysis of important molecules in serum from meningitic piglets caused by Streptococcus suis serotype 2.

INTRODUCTION: Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes meningitis in China. This study's aim was comparative analysis of serum proteomics from meningitis and non-meningitis piglets. METHODOLOGY: SS2 meningitis and non-meningitis piglet models were established. The serum samples were collected and analyzed by label-free LC-MS/MS proteomics technology. Differentially expressed proteins (DEPs) from serum were screened out by comparing the meningitis group and non-meningitis group to the healthy group (M/C; N/C), respectively. And then, globally and comparative analysis of DEPs in "M/C" and "N/C" in serum were performed using bioinformatics method. Finally, we comparatively analyzed the serum and cerebrospinal fluid proteomics in piglets that lived with meningitis. RESULTS: We obtained 316 and 191 DEPs from "M/C" and "N/C" which classification visualizations were established. 157 DEPs were common in both groups and 159 DEPs were unique to the "M/C". These DEPs and the signaling pathways which they participated in were visualized. Moreover, some DEPs which participated in multiple pathways were discovered and the interaction between 159 DEPs was also mapped. 39 common DEPs were also screened out in serum and cerebrospinal fluid during meningitis, and signaling pathways associated with these DEPs were further visualized. CONCLUSIONS: DEPs in "M/C" and "N/C" were comparatively analyzed and the similarities and differences of these DEPS which were involved in signal pathways were summarized. Moreover, several important molecules were screened out.

Porcine circovirus type 2 promotes Actinobacillus pleuropneumoniae survival during coinfection of porcine alveolar macrophages by inhibiting ROS production.

Actinobacillus pleuropneumoniae (APP) and porcine circovirus type 2 (PCV2) are both important pathogens of the porcine respiratory disease complex (PRDC), which results in significant worldwide economic losses. Recently, PCV2 and APP coinfection has been described in the worldwide pork industry, and represents an extremely complex situation in veterinary medicine. However, the mechanism of their coinfection has not been investigated. In this study, we found that PCV2 promoted APP adhesion to and invasion of porcine alveolar macrophages (PAMs) during coinfection. Additionally, PCV2 suppressed reactive oxygen species (ROS) production by inhibiting cytomembrane NADPH oxidase activity, which was beneficial for APP survival in PAMs in vitro. During coinfection, PCV2 weakened the inflammatory response and macrophage antigen presentation by decreasing TNF-α, IFN-γ and IL-4 expression, and reduced clearance of the invading bacteria. The host-cell experimental results were verified in a mouse model. The findings provide a deeper and novel understanding of porcine coinfection, and will be extremely helpful for the design of strategies for PRDC control.

Establishment and comparison of Actinobacillus pleuropneumoniae experimental infection model in mice and piglets.

Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia, a disease responsible for substantial losses in the worldwide pig industry. In this study, outbred Kunming (KM) and Institute of Cancer Research (ICR) mice were evaluated as alternative mice models for APP research. After intranasal infection of serotype 5 reference strain L20, there was less lung damage and a lower clinical sign score in ICR compared to KM mice. However, ICR mice showed more obvious changes in body weight loss, the amount of immune cells (such as neutrophils and lymphocytes) and cytokines (such as IL-6, IL-1ß and TNF-α) in blood and bronchoalveolar lavage fluid (BALF). The immunological changes observed in ICR mice closely mimicked those found in piglets infected with L20. While both ICR and KM mice are susceptible to APP and induce pathological lesions, we suggest that ICR and KM mice are more suitable for immunological and pathogenesis studies, respectively. The research lays the theoretical basis for determine that mice could replace pigs as the APP infection model and it is of significance for the study of APP infection in the laboratory.

Transcriptomic analysis of porcine PBMCs in response to Actinobacillus pleuropneumoniae reveals the dynamic changes of differentially expressed genes related to immuno-inflammatory responses.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, for which the mortality rate is high. Host peripheral blood is a body site for the immune clearance of pathogens mediated by release of inflammatory factors. However, "out of control" inflammatory factor release can contribute to host death. To further understand the changes in the transcription level of immune-related effectors, samples of peripheral blood mononuclear cells (PBMCs) collected from piglets at different stages of infection (0, 24 and 120 h) were sequenced on an Illumina HiSeq™ 4000 platform. We found 3818 differentially expressed genes (DEGs) in the 24 h-infection group compared to the 0 h-infection group (Pb24-Vs-Pb0). DEGs mainly involved in the Gene ontology and KEGG pathways that included nucleic acid metabolism regulation, cell growth, cell differentiation, and organ morphological maintenance were not significantly enriched (P > 0.05). However, DEGs associated with protein kinase activity, receptor activation, metabolism, local adhesion and immune inflammatory responses were significantly enriched in Pb120-Vs-Pb24 (P < 0.05), as were those related to the T cell receptor signalling pathway, with most being down-regulated compared to the preceding stage (Pb24-Vs-Pb0). In PBMCs there were some changes in glucose metabolism, local adhesion and the immune inflammatory response (Pb120-Vs-Pb0). In addition, up-regulated DEGs, such as IL8, IL1ß, and CCL2, and were significantly enriched in immune-inflammatory related pathways compared to the uninfected stage, although they began to decline after 24 h.

Differential Protein Profiling of Cerebrospinal Fluid in Piglets with Severe Meningoencephalitis Caused by Streptococcus suis Type 2 Compared to Controls.

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen that can cause meningitis both in pigs and in human beings. However, the pathogenesis of central nervous system (CNS) infection caused by SS2 have not yet been elucidated. To find the key molecules in cerebrospinal fluid (CSF) needed for the pathogenesis, a SS2 meningoencephalitic pig model and a SS2 non-meningoencephalitic pig model were established in this study. CSF was collected from infected piglets, and protein profiling was performed with label-free proteomics technology. A total of 813 differential proteins, including 52 up-regulated proteins and 761 down-regulated proteins, were found in the CSF of meningoencephalitic pigs compared with both non-meningoencephalitic pigs and healthy pigs. These 813 differential proteins were clustered into three main categories, namely, cellular component, biological process, and molecular function by gene ontology (GO) analysis. The most enriched subclasses of differential proteins in each category were exosome (44.3%), energy pathway (25.0%) and catalytic activity (11.3%), respectively. The most enriched subclasses of upregulated proteins were extracellular (62.1%), protein metabolism (34.5%) and cysteine-type peptidase activity (6.9%), and of downregulated proteins were exosomes (45.0%), energy pathway (24.0%) and catalytic activity (9.4%). Then, the differential proteins were further investigated by using the KEGG database and were found to participate in 16 KEGGs. The most enriched KEGG was citrate cycle (56.6%), and some of these differential proteins are associated with brain diseases such as Huntington's disease (18.6%), Parkinson's disease (23.8%) and Alzheimer's disease (17.6%). Sixteen of the 813 differential proteins, chosen randomly as examples, were further confirmed by enzyme-linked immunosorbent assay (ELISA) to support the proteomic data. To our knowledge, this is the first study to analyze the differential protein profiling of CSF between SS2 meningoencephalitic piglets and non-meningoencephalitic piglets by employing proteomic technology. The discovery and bioinformatics analysis of these differential proteins provides reference data not only for research on pathogenesis of SS2 CNS infection but also for diagnosis and drug therapy research.

Characteristic Comparison of Meningitis and Non-meningitis of Streptococcus suis in an Experimentally Infected Porcine Model.

This study tested the differences of meningitis and non-meningitis of Streptococcus suis (SS). In this study, an infected pig model of streptococcal meningitis was established. Compared with the non-meningitis Streptococcus suis group (JZLQ001 group), the meningitis Streptococcus suis group (JZLQ022) exhibited neurological symptoms, such as ataxia and foaming at the mouth, and the brain showed a large area of congestion at 5 days post-infection (p.i.). Moreover, bacterial counts, white blood cells (WBCs), neutrophils, and blood glucose in the blood reached a peak and were significantly higher than those of the JZLQ001 group at 3 days p.i. These values then decreased at 5 days p.i. However, the content of total protein in the blood was lower in the JZLQ022 group than that in the JZLQ001 group, and the difference was most significant at 5 days p.i. When neurological symptoms appeared on 5 days p.i., the bacterial counts in the brain in the JZLQ022 group were significantly higher than those in the JZLQ001 group. The levels of cytokines in the peripheral blood and cerebrospinal fluid (CSF) were an important indicator of inflammation. By ELISA detection, the secretion levels of IL-6, IL-8, and IL-17 in the peripheral blood in the JZLQ022 group were significantly higher than those in the JZLQ001 group at 12 and 24 h and 3 days p.i.; however, TNF-α showed no difference. At 5 days p.i., the secretion levels of IL-6, IL-8, and IL-17 in the JZLQ022 group were significantly lower than those in the JZLQ001 group. The results were similar in CSF. HE staining revealed that the JZLQ022 group exhibited neuronophagia and hyperemia in the brain, but no change was found in the JZLQ001 group. A further study investigating the impact of meningitis Streptococcus suis on blood-brain barrier (BBB) integrity found that the brain tissue content of endogenous IgG in the JZLQ022 group was significantly higher than that in the JZLQ001 group. The present study demonstrated that pigs infected with meningitis and non-meningitis Streptococcus suis exhibit significant differences in immunological aspects such as bacterial counts, WBCs, neutrophils, blood glucose and total protein in the peripheral blood, the secretion levels of IL-6, IL-8, and IL-17, and BBB integrity. These data provide the necessary evidence to better understand SS meningitis.

An anti-Propionibacterium acnes antibody shows heterologous resistance to an Actinobacillus pleuropneumoniae infection independent of neutrophils in mice.

Porcine contagious pleuropneumonia is a highly fatal respiratory disease that is caused by Actinobacillus pleuropneumoniae (APP) and results in tremendous economic losses for the pig breeding industry worldwide. Previous studies have demonstrated that Propionibacterium acnes (PA) could effectively prevent APP infection in mice and pigs. The humoral immune response played a primary role during this process and anti-PA antibody could mediate macrophages to kill the bacteria. However, the role of neutrophils in this process is currently unknown. In this study, mice were injected with cyclophosphamide to deplete neutrophils and then passively immunized with anti-PA serum or negative serum. Mice were subsequently challenged with APP serotype 1. The results showed that the mice exhibited less bacterial colonization, less lung damage, and a high survival rate, which were immunized with the anti-PA antibody whether neutrophils were depleted or not. Worse still, the presence of neutrophils increased the damage to the mice after challenge. These results suggest that the activity of the anti-PA antibody against APP infection was independent of neutrophils. These findings have important significance for understanding the mechanisms of humoral immunity conferred by heterologous immunization and lay a good foundation for preventing APP infection.

The N-terminal and central domain of colicin A enables phage lysin to lyse Escherichia coli extracellularly.

Multidrug-resistant Escherichia coli has seriously threatened antibiotic resources and international public health. Bacteriophage lysin preparations have been widely considered as valid agents for solving multidrug resistances. Many lysins have been derived to treat diseases caused by Gram-positive bacteria, but only a few lysin preparations have been found that successively treat diseases caused by Gram-negative bacteria. The outer membrane of Gram-negative bacteria effectively blocks the interactions between peptidoglycan in the periplasmic space and bacteriophage lysins, which therefore hampers the antimicrobial effects of bacteriophage lysins. In this study, a new fusion protein (Colicin-Lysep3) was constructed by fusing the translocation and receptor binding domains of colicin A with an E. coli phage lysin, which endows Colicin-Lysep3 bactericidal activity against E. coli from outside of Gram-negative bacteria. These results show that Colicin-Lysep3 could lyse the E. coli broadly in vitro and significantly reduce the number of E. coli in an intestinal infection mouse model. Overall, our findings first demonstrated that a colicin A fragment could enable a bacteriophage lysin to lyse E. coli from the outside, promoting the application of phage lysin preparations in control of Gram-negative bacteria.

B cell cross-epitope of Propionibacterium acnes and Actinobacillus pleuropneumonia selected by phage display library can efficiently protect from Actinobacillus pleuropneumonia infection.

Contagious porcine pleuropneumonia (CPP), caused by Actinobacillus pleuropneumoniae (APP), is a highly transmissible and fatal respiratory illness that causes tremendous economic losses for the pig breeding industry worldwide. Propionibacterium acnes (PA) has a strong cross-reaction with anti-APP1 and anti-APP5 serum and can efficiently prevent APP infection, which was fortuitously found in researching the differential gene between the different APP serotypes. There seems to be some natural cross-protection between PA and APP. To identify the common epitope, the phage display library of a PA whole genome was constructed, whose size is 105. The DNA sequence of the positive clone was determined after three rounds of biopanning, and ten common protein types were identified and the epitope was predicted by computer software. Six peptide epitopes were selected and synthesized for further analysis. Among these epitopes, Ba1, Bb5 and C1 could bind to anti-PA serum and anti-APP1 serum and vice versa. Furthermore, the IgG and IL-4 levels and CD4+/CD8+ T cell ratios in the Ba1, Bb5 and C1 groups were significantly higher than that in the control group, indicating that the epitopes could trigger an immune response, which was mainly humoral immunity. Moreover, Ba1 and Bb5 equally protected 80% of mice from a fatal dose of APP1 infection compared with the control group. Mice could resist APP1 and APP5 challenge after being treated with the combination of Ba1 and Bb5, with survival rates of 80% and 90%, respectively. These findings suggest that the PA epitope confers antigenicity and can heterologously resist to the APP infection. This finding provides a novel strategy for preventing APP infection.

Enhancement of the direct antimicrobial activity of Lysep3 against Escherichia coli by inserting cationic peptides into its C terminus.

Phage lysins are considered promising antimicrobials against resistant bacterial infections. Some lysins have been reported for the prevention and treatment of Gram-positive bacterial infection. Gram-negative bacterial phage lysins, however, can only destroy the bacterial cell wall from inside because of the obstruction of the bacterial outer membrane that prevents direct hydrolysis of the bacterial wall peptidoglycan from the outside, severely restricting the development of lysins against Gram-negative bacteria. In this study, genetic engineering techniques were used to fuse a 5 cationic amino acid polypeptide (KRKRK), a 10 cationic amino acid polypeptide (KRKRKRKRKR), a 15 cationic amino acid polypeptide (KRKRKRKRKRKRKRK), and a polypeptide including both cationic and hydrophobic amino acids (KRKRKFFVAIIP) to the C-terminus of the Escherichia coli phage lysin Lysep3 to obtain four fusion lysins (5aa, 10aa, 15aa, Mix). The bactericidal effects of those four lysins on E. coli were then compared in vitro. Our results showed that the fusion of hydrophobic and positively charged amino acids, Mix, can kill E. coli effectively; the fusion of positively charged amino acids alone at the C-terminus (5aa, 10aa, 15aa) also showed bactericidal activity against E. coli from the outside, with the bactericidal activity gradually increasing with the positive charge at the C-terminus of the lysin. Collectively, improving the positive charge at the C-terminus of E. coli bacteriophage lysin Lysep3 increases its bactericidal ability from outside E. coli, providing a new practical method for the development of anti-Gram-negative bacterial lysins.

Case Report Associated with Aspergillosis and Hepatitis E Virus Coinfection in Himalayan Griffons.

This study involved a death which occurred in four Himalayan griffons housed in Beijing zoo, China. Based on pathogen identification and the pathological changes observed, we did characterize the fungi and Hepatitis E virus (HEV) in four dead Himalayan griffons. Pathological changes were severe. Membranous-like material was observed on the surface of the internal organs. Spleen was necrotic. Focal lymphocyte infiltration in the liver and many sunflower-like fungi nodules were evident in the tissues, especially in the kidney. PCR was used to identify the pathogen. Based on the 18SrRNA genomic sequence of known fungi, the results confirmed that all four dead Himalayan griffons were infected with Aspergillus. At the same time the detection of HEV also showed positive results. To the best of our knowledge, this work appears to be the first report of concurrent presence of Aspergillosis and Hepatitis E virus in rare avian species.