Specific Degradation of the Mucus Adhesion-Promoting Protein (MapA) of Lactobacillus reuteri to an Antimicrobial Peptide

The intestinal flora of mammals contains lactic acid bacteria (LAB) that may provide positive health effects for the host. Such bacteria are referred to as probiotic bacteria. From a pig, we have isolated a Lactobacillus reuteri strain that produces an antimicrobial peptide (AMP). The peptide was purified and characterized, and it was unequivocally shown that the AMP was a well-defined degradation product obtained from the mucus adhesion-promoting protein (MapA); it was therefore termed AP48-MapA. This finding demonstrates how large proteins might inherit unexpected pleiotropic functions by conferring antimicrobial capacities on the producer. The MapA/AP48-MapA system is the first example where a large protein of an intestinal LAB is shown to give rise to such an AMP. It is also of particular interest that the protein that provides this AMP is associated with the binding of the bacterium producing it to the surface/lining of the gut. This finding gives us new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota.Mammals have a microbiota in their digestive tract that contains lactic acid bacteria (LAB). It has been increasingly evident that some of these lactic acid bacteria produce antimicrobial peptides that may contribute to the positive effect on their host. Such bacteria are often referred to as probiotics, and one of their important beneficial effects is their ability to produce antimicrobial compounds that prevent or interfere with the growth of pathogenic bacteria in the host.It is known that the fecal microflora of pigs/piglets is large and diverse and develops rapidly after birth. Lactobacillus reuteri is among the very first lactic acid bacteria that colonize the intestine of new-born piglets, and their numbers gradually increase until they become the most dominant LAB in pigs (5, 17, 28). Other lactobacilli that are also part of the gut microbiota of pigs include L. amylovorus, L. acidophilus, L. salivarius, and L. casei (4, 8). Probiotic isolates have been identified within all these species, and many of them are today used as food/feed supplements to support good health (4, 11, 27). An important part of the antimicrobial arsenal produced by lactic acid bacteria (LAB) is a group of peptides called bacteriocins, which are ribosomally synthesized antibiotic-like peptides (antimicrobial peptides [AMPs]) (3, 7, 19). The bacteriocins constitute a wide range of structurally different peptides that are divided into different classes and subclasses. Some are modified (the lantibiotics, or class I), while others are basically unmodified (class II) (3, 6, 19).Most bacteriocins are derived from prepeptides, each containing a short leader sequence (14 to 30 amino acids [aa]) which is cleaved off during the secretion of the mature peptide (19). In recent years, a new group of AMPs have been recognized (18); these are different from regular bacteriocins in that they are derived from larger proteins through specific degradations, leading to a defined peptide possessing antimicrobial activity. Such antimicrobial peptides have been known for a long time in mammalian systems. For instance, lactoferrin, a protein in milk, is readily degraded to a specific antimicrobial peptide through heat, acid treatment, or pepsin digestion (14, 24, 26). Defined histone fragments with antimicrobial properties have been isolated from different eukaryotic species (1, 2, 15, 21, 23), and a few antimicrobial peptides derived from larger proteins have been isolated in bacteria, including Helicobacter pylori (22), propionic acid bacteria (9, 10), and Clostridium beijerinckii (13). Such antimicrobial peptides are most likely formed by proteolytic degradation during cell proliferation or death.     

Formation of phosphatidic acid, ceramide, and diglyceride on radiolysis of lipids: identification by MALDI-TOF mass spectrometry

By use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, phosphatidic acid was found to be the main product of γ radiolysis of cardiolipin, phosphatidylinositol, and phosphatidylglycerol. It has been shown that γ irradiation of such glycolipids as cerebroside and galactosyl diglyceride leads to formation of ceramide and diglyceride, respectively. These findings, combined with those obtained earlier, allowed an assumption to be made that, owing to radiation-induced free radical fragmentation of lipids in their polar moiety, formation of signaling molecules can occur.     

Transgenic mouse models of angiotensin receptor subtype function in the cardiovascular system

Angiotensin II mediates is biological actions via different subtypes of G protein-coupled receptors, termed AT(1) and AT(2) receptors. In rodents, two AT(1) receptors have been identified, AT(1A) and AT(1B), whereas in humans a single AT(1) receptor exists. Recently, a number of transgenic animal models have been generated which overexpress or lack functional angiotensin II receptor subtypes. This review focuses on the physiological significance of angiotensin II receptor subtype diversity in the cardiovascular system. In the mouse, AT(1A) receptors are the major regulators of cardiovascular homeostasis by determining vascular tone and natriuresis. In addition, AT(1A) receptors mediate growth-stimulating signals in vascular and cardiac myocytes. AT(1B) receptors participate in blood pressure regulation, and their functions become apparent when the AT(1A) receptor gene is deleted. Deletion of the mouse gene for the AT(2) receptor subtype led to hypersensitivity to pressor and antinatriuretic effects of angiotensin II in vivo, suggesting that the AT(2) receptor subtype counteracts some of the biological effects of AT(1) receptor signalling.     

Molecular and genetic characterization of propionicin F, a bacteriocin from Propionibacterium freudenreichii

This work describes the purification and characterization of propionicin F, the first bacteriocin isolated from Propionibacterium freudenreichii. The bacteriocin has a bactericidal activity and is only active against strains of P. freudenreichii. Propionicin F appears to be formed through a processing pathway new to bacteriocins. The mass of the purified bacteriocin was determined by mass spectrometry, and the N-terminal amino acid sequence was determined by Edman degradation. Sequencing of pcfA, the bacteriocin structural gene, revealed that propionicin F corresponds to a 43-amino-acid peptide in the central part of a 255-amino-acid open reading frame, suggesting that mature propionicin F is excised from the probacteriocin by N- and C-terminal proteolytic modifications. DNA sequencing and Northern blot hybridizations revealed that pcfA is cotranscribed with genes encoding a putative proline peptidase and a protein from the radical S-adenosylmethionine family. A gene encoding an ABC transporter was also identified in close proximity to the bacteriocin structural gene. The potential role of these genes in propionicin F maturation and secretion is discussed.     

Expression and characterization of human glycosylated interleukin-1 receptor antagonist in Pichia pastoris

Interleukin-1 receptor antagonist is an inhibitor of the pro-inflammatory action of interleukin-1. The gene encoding for interleukin-1 receptor antagonist (IL-1ra) was cloned into a Pichia pastoris expression vector pPICzalphaA (Invitrogen, USA) and transformed into P. pastoris strain SMD1168H. Multi-copy selection of the gene produced a high expressing strain of IL-1ra that produced 17mg/L of total secreted purified protein. The IL-1ra produced in P. pastoris was a mixture of glycosylated and non-glycosylated IL-1ra where 70% of the total protein was glycosylated. SP-Sepharose purification allowed for separation of the two expressed forms of IL-1ra, which permits biochemical investigation of glycosylated and non-glycosylated IL-1ra using one expression system. Mass spectrometric analysis revealed the expression of the full-length protein and that the glycosylated IL-1ra contained high mannose glycoforms that ranged from Man(9)GlcNAc(2) to Man(14)GlcNAc(2).     

Heterologous production of antimicrobial peptides in Propionibacterium freudenreichii

Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P4) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P4 and Ppamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.     

Trans lipid formation induced by thiols in human monocytic leukemia cells

Trans lipids in humans originate exogenously from the ingestion of isomerized fats. An endogenous path comprising a thiyl radical-catalyzed cis-trans isomerization of cis-unsaturated phospholipids was proposed. However, whether an isomerization process might be feasible in eukaryotic cells remained to be established. Here we report the presence of trans lipids in human monocytic leukemia cell membranes (THP-1) before and after treatment with a 10 mM series of thiols. Oleic, linoleic, and arachidonic acid residues of membrane phospholipids were analyzed and, unexpectedly, an initial trans lipid content was found in control cells. Then, incubation for 24 h with thiols under physiological conditions slightly increased trans lipid content. Formation of trans isomers was also evaluated in the presence of thiol and under free radical stress induced by gamma-irradiation or by thermal decomposition of azo-compounds. The similarity of isomer trends formed under incubation and stress conditions, together with the reactivity order of fatty acid residues (arachidonic > linoleic approximately oleic), indicated a common radical path and some mechanistic considerations are advanced. These results offer the first evidence that trans lipids are formed in eukaryotic cells and confirm that thiyl radicals are harmful to the integrity of cis lipid geometry. This work motivates further studies into the relationship between lipid isomerization outcome and thiyl radicals in cellular systems, as well as the formation of trans lipids and the metabolic response to such a perturbation introduced into biological membranes.