Yongming Sang

Antimicrobial peptides and bacteriocins: alternatives to traditional antibiotics.

Abstract Antimicrobial peptides (AMPs) are ubiquitous, gene-encoded natural antibiotics that have gained recent attention in the search for new antimicrobials to combat infectious disease. In multicellular organisms, AMPs, such as defensins and cathelicidins, provide a coordinated protective response against infection and are a principal component of innate immunity in vertebrates. In unicellular organisms, AMPs, such as bacteriocins, function to suppress competitor species. Because many AMPs kill bacteria by disruption of membrane integrity and are thus thought to be less likely to induce resistance, AMPs are being extensively evaluated as novel antimicrobial drugs. This review summarizes and discusses the antibiotic properties of AMPs highlighting their potential as alternatives to conventional antibiotics.

Porcine host defense peptides: Expanding repertoire and functions

Host defense peptides (HDPs) are a large group of innate immune effectors that are also termed antimicrobial peptides. Because of the rapid progress that has been made in completing several animal genomes, many HDPs have been systemically defined using bioinformatic analysis and partially characterized using reverse genomic approaches. In pigs, about 30 HDPs have been identified and partially characterized relative to structure and function. Antimicrobial activity of porcine HDPs has been extensively evaluated against a broad spectrum of microorganisms in vitro and evaluated for their protective role in vivo. Increasing evidence indicates that HDPs are functionally differentiated during posttranslational and postsecretory processing, and that the structural units for antimicrobial and immunoregulatory functions are separate. These findings suggest promising new avenues for therapeutic drug design based on HDPs, including porcine HDPs. This review summarizes and discusses advances in porcine HDPs research during the last decade with an emphasis on the rapidly expanding profiles and biological functions.

Structural and functional annotation of the porcine immunome

BackgroundThe domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems.ResultsThe Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome.ConclusionsThis extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

Bioinformatic and expression analysis of novel porcine β-defensins

Abstractβ-Defensins are a major group of mammalian antimicrobial peptides. Although more than 30 β-defensins have been identified in humans, only one porcine β-defensin has been reported. In this article we report the identification and initial characterization of 11 novel porcine β-defensins (pBD). Using bioinformatic approaches, we screened 287,821 porcine expressed sequence tags for similarity of their predicted peptides to known human β-defensins and identified full-length or partial sequences for the 11 novel pBDs. Similar to the previously identified pBD1, all of these peptides have a consensus β-defensin motif. A differential expression pattern for these newly identified genes was found. For example, unlike most β-defensins, pBD2 and pBD3 were expressed in bone marrow and in other lymphoid tissues including thymus, spleen, lymph nodes, duodenum, and liver. Including pBD2 and pBD3, six porcine β-defensins were expressed in lung and skin. Several newly identified porcine β-defensins, including pBD123, pBD125, and pBD129, were expressed in male reproductive tissues, including lobuli testis and some segments of the epididymis. Phylogenetic analysis indicates that in most cases the evolutionary relationship between individual porcine β-defensins and their human orthologs is closer than the relationship among β-defensins in the same species. These findings establish the existence of multiple porcine β-defensins and suggest that the pig may be an ideal model for the characterization of β-defensin diversity and function.

Differential expression and activity of the porcine type I interferon family

Type I interferons (IFNs) are central to innate and adaptive immunity, and many have unique developmental and physiological functions. However, in most species, only two subtypes, IFN-alpha and IFN-beta, have been well studied. Because of the increasing importance of zoonotic viral diseases and the use of pigs to address human research questions, it is important to know the complete repertoire and activity of porcine type I IFNs. Here we show that porcine type I IFNs comprise at least 39 functional genes distributed along draft genomic sequences of chromosomes 1 and 10. These functional IFN genes are classified into 17 IFN-alpha subtypes, 11 IFN-delta subtypes, 7 IFN-omega subtypes, and single-subtype subclasses of IFN-alphaomega, IFN-beta, IFN-epsilon, and IFN-kappa. We found that porcine type I IFNs have diverse expression profiles and antiviral activities against porcine reproductive and respiratory syndrome virus (PRRSV) and vesicular stomatitis virus (VSV), with activity ranging from 0 to >10(5) U.ng(-1).ml(-1). Whereas most IFN-alpha subtypes retained the greatest antiviral activity against both PRRSV and VSV in porcine and MARC-145 cells, some IFN-delta and IFN-omega subtypes, IFN-beta, and IFN-alphaomega differed in their antiviral activity based on target cells and viruses. Several IFNs, including IFN-alpha7/11, IFN-delta2/7, and IFN-omega4, exhibited minimal or no antiviral activity in the tested target cell-virus systems. Thus comparative studies showed that antiviral activity of porcine type I IFNs is virus- and cell-dependent, and IFN-alphas are positively correlated with induction of MxA, an IFN-stimulated gene. Collectively, these data provide fundamental genomic information for porcine type I IFNs, information that is necessary for understanding porcine physiological and antiviral responses.

Gene Silencing and Overexpression of Porcine Peptidoglycan Recognition Protein Long Isoforms: Involvement in β-Defensin-1 Expression

ABSTRACT Peptidoglycan recognition proteins (PGRPs) are a group of newly identified proteins with emerging functions in mammalian innate immunity. Here we report the identification and characterization of two long isoforms of porcine PGRP. Their complete cDNA sequences encode predicted peptides of 252 and 598 residues and are named pPGRP-L1 and pPGRP-L2, respectively. These porcine isoforms share identical PGRP domains at their C terminus, which are highly conserved with human and mouse orthologs. pPGRP-L1 is expressed constitutively in several tissues, including bone marrow, intestine, liver, spleen, kidney, and skin. pPGRP-L2 is highly expressed in the duodenum and liver, and expression in intestinal tissues is increased by Salmonella infection. In intestinal cells, expression of both pPGRP-L1 and pPGRP-L2 is increased by bacterial infection. Recombinant pPGRP-L1 and pPGRP-L2 have N-acetylmuramoyl-L-alanine amidase activity. Loss-of-function and gain-of-function experiments indicate that these two pPGRPs are involved in expression of the antimicrobial peptide β-defensin-1. Silencing of pPGRP-L2 in intestinal cells challenged with Listeria monocytogenes results in downregulation of β-defensin-1. Conversely, overexpression of pPGRP-L1 or pPGRP-L2 dramatically upregulates expression of β-defensin-1. Collectively, these findings suggest that porcine PGRPs are involved in antimicrobial peptide expression.

Molecular Cloning and Characterization of Three β-Defensins from Canine Testes

ABSTRACT Mammalian β-defensins are small cationic peptides possessing broad antimicrobial and physiological activities. Because dogs are particularly resilient to sexually transmitted diseases, it has been proposed that their antimicrobial peptide repertoire might provide insight into novel antimicrobial therapeutics and treatment regimens. To investigate this proposal, we cloned the full-length cDNA of three canine β-defensin isoforms (cBD-1, -2, and -3) from canine testicular tissues. Their predicted peptides share identical N-terminal 65-amino-acid residues, including the β-defensin consensus six-cysteine motif. The two longer isoforms, cBD-2 and -3, possess 4 and 34 additional amino acids, respectively, at the C terminus. To evaluate the antimicrobial activity of cBD, a 34-amino-acid peptide derived from the shared mature peptide region was synthesized. Canine β-defensin displayed broad antimicrobial activity against gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus; MICs of 6 and 100 μg/ml, respectively), gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, and Neisseria gonorrhoeae; MICs of 20 to 50, 20, and 50 μg/ml, respectively), and yeast (Candida albicans; MIC of 5 to 50 μg/ml) and lower activity against Ureaplasma urealyticum and U. canigenitalium (MIC of 200 μg/ml). Antimicrobial potency was significantly reduced at salt concentrations higher than 140 mM. All three canine β-defensins were highly expressed in testis. In situ hybridization indicated that cBD-1 was expressed primarily in Sertoli cells within the seminiferous tubules. In contrast, cBD-2 was located primarily within Leydig cells. The longest isoform, cBD-3, was detected in Sertoli cells and to a lesser extent in the interstitium. The tissue-specific expression and broad antimicrobial activity suggest that canine β-defensins play an important role in host defense and other physiological functions of the male reproductive system.

Toll-like Receptor 3 Activation Decreases Porcine Arterivirus Infection

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that initiates infection in pulmonary alveolar macrophages (PAMs), elicits weak immune responses, and establishes a persistent infection. To understand the role of dsRNA intermediates in eliciting host immunity, we sought to determine if toll-like receptor-3 (TLR3), a well-known dsRNA sensor, is involved in the regulation of PRRSV infection. TLR3 gene expression was increased in PAMs of congenitally infected 2-wk-old pigs. Stimulation of PAMs with dsRNA increased gene expression for TLR3 and interferon-beta and suppressed PRRSV infectivity. To investigate activation and signaling parameters, expression constructs of wild-type and functional-domain-truncated porcine TLR3 were used in cell transfection studies. When cells that overexpressed porcine TLR3 were stimulated with dsRNA a rapid and robust calcium influx was induced. Moreover, ligand activation of porcine TLR3 expressed in MARC-145 cells elicited an antiviral response to PRRSV. Conversely, transfection of PAMs with small-interfering RNA targeting porcine TLR3 resulted in up to 80% suppression of TLR3 mRNA expression and an increase in PRRSV infectivity. These data provide fundamental genetic and molecular information for porcine TLR3, and implicate its involvement in PRRSV infection, findings that may suggest new strategies to limit this costly pandemic disease.

Molecular identification and functional expression of porcine Toll-like receptor (TLR) 3 and TLR7.

To investigate porcine Toll-like receptors (TLR) responding to viral pathogen associated molecular patterns, the full-length cDNA of porcine TLR3 and TLR7 were identified and characterized. Porcine TLR3 and TLR7 cDNA encode 904- and 1050-amnio-acid polypeptides, respectively. Both porcine TLR3 and TLR7 contain typical functional TLR domains and share about 80% sequence identity to other mammalian orthologues. Tissue expression profiles showed that TLR3 was highly expressed in kidney, duodenum, spleen and liver, and moderately expressed in bone marrow, lung, and skin. Conversely, TLR7 was moderately and constitutively expressed in all tissues evaluated. Stimulation of mammalian cells transfected with porcine TLR3 and TLR7 constructs with TLR3 and TLR7 agonists [poly (I:C) and imiquimod (R837), respectively], and adenovirus elicited activation of interferon regulatory factors (IRFs). These data provide molecular and functional information for porcine TLR3 and TLR7, and implicate their role in mediating immune protection against porcine viral diseases.

Interaction between innate immunity and porcine reproductive and respiratory syndrome virus

Abstract Innate immunity provides frontline antiviral protection and bridges adaptive immunity against virus infections. However, viruses can evade innate immune surveillance potentially causing chronic infections that may lead to pandemic diseases. Porcine reproductive and respiratory syndrome virus (PRRSV) is an example of an animal virus that has developed diverse mechanisms to evade porcine antiviral immune responses. Two decades after its discovery, PRRSV is still one of the most globally devastating viruses threatening the swine industry. In this review, we discuss the molecular and cellular composition of the mammalian innate antiviral immune system with emphasis on the porcine system. In particular, we focus on the interaction between PRRSV and porcine innate immunity at cellular and molecular levels. Strategies for targeting innate immune components and other host metabolic factors to induce ideal anti-PRRSV protection are also discussed.