Natural and experimental feline coronavirus (FCoV) infection leads to systemic viral spread via monocyte-associated viraemia and induces systemic proliferation of monocytes/macrophages. In the majority of naturally infected animals, FCoV infection remains subclinical and is associated with generalised B and T cell hyperplasia, but no other pathological findings. A minority of cats, however, develop feline infectious peritonitis (FIP), a fatal systemic granulomatous disease. This is generally accompanied by B and T cell depletion. The obvious functional differences of lymphatic tissues in FCoV-infected cats with and without FIP suggest that they contribute to the outcome of FCoV infection. This study attempted to evaluate the functional changes in haemolymphatic tissues after natural FCoV infection, with special emphasis on the magnitude, phenotype and function of the monocyte/macrophage population. The spleen, mesenteric lymph nodes and bone marrow from naturally FCoV-infected cats with and without FIP and specific pathogen-free (SPF) control cats were examined for the quantity and activation state of monocytes/macrophages both by immunohistology and by quantitative real time PCR for the transcription of interleukin (IL)-1beta, IL-6, IL-10, IL-12 p40, tumour necrosis factor (TNF), granulocyte colony stimulating factor (G-CSF), macrophage-CSF (M-CSF) and GM-CSF. Compared to cats with FIP, FCoV-infected cats without FIP exhibited significantly higher IL-10 levels in the spleen and significantly lower levels of IL-6, G- and M-CSF in mesenteric lymph nodes. In cats with FIP, however, IL-12 p40 levels were significantly lower in lymphatic tissues in comparison to both SPF cats and FCoV-infected cats without FIP. In comparison to SPF cats, FIP cats had significantly higher IL-1beta levels and lower TNF levels in mesenteric lymph nodes and lower M-CSF levels in the spleen. Findings indicate that FCoV-infected cats which do not develop FIP are able to mount an effective FCoV-specific immune response and can avoid excessive macrophage activation and FIP, possibly by upregulation of IL-10 production. Development of FIP, however, might be due to a lack of IL-12 which inhibits an effective cellular immune response and allows for monocyte/macrophage activation and the development of FIP.

Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin, which is a cyclic peptide with one exocyclic amino acid linked to a branched-chain fatty acid acyl residue. The structural relationship to daptomycin and the excellent antibacterial performance of friulimicin make the antibiotic an attractive drug candidate. The complete friulimicin biosynthetic gene cluster of 24 open reading frames from A. friuliensis was sequenced and analyzed. In addition to genes for regulation, self-resistance, and transport, the cluster contains genes encoding peptide synthetases, proteins involved in the synthesis and linkage of the fatty acid component of the antibiotic, and proteins involved in the synthesis of the nonproteinogenic amino acids pipecolinic acid, methylaspartic acid, and 2,3-diaminobutyric acid. By using heterologous gene expression in Escherichia coli, we provide biochemical evidence for the stereoselective synthesis of L-pipecolinic acid by the deduced protein of the lysine cyclodeaminase gene pip. Furthermore, we show the involvement of the dabA and dabB genes in the biosynthesis of 2,3-diaminobutyric acid by gene inactivation and subsequent feeding experiments.In recent years, the sequences of numerous gene clusters involved in the synthesis of many secondary metabolites have become available for comparison and exploitation. The programmed manipulation of genes encoding enzymes in the biosynthetic pathways offers promise for redesigning existing antibiotic structures to create antibiotics with new activities and the ability to overcome bacterial resistance (50). Therefore, each newly analyzed gene cluster represents a new tool for combinatorial biosynthesis and can provide information about the synthesis of unusual building blocks, such as nonproteinogenic amino acids, acyl residues, and sugar moieties. An interesting group of secondary metabolites for such experiments seems to be bioactive lipopeptides isolated from streptomycetes. So far, only a few biosynthetic gene clusters corresponding to these lipopeptides have been isolated and characterized, such as the clusters for calcium-dependent antibiotic (CDA) from Streptomyces coelicolor (17), daptomycin from Streptomyces roseosporus (26), and A54145 from Streptomyces fradiae (24). By targeted modification and gene exchange, it is possible to generate new lipopeptide structures (12,25).Another member of this group of secondary metabolites is the antibiotic friulimicin that is produced by the actinomycete Actinoplanes friuliensis. This compound is highly active against multidrug-resistant gram-positive bacteria, such as methicillinresistant Staphylococcus aureus strains (4). The biosynthesis of this lipopeptide is catalyzed by nonribosomal peptide synthetases (NRPS) (15).The eight bioactive lipopeptides isolated from A. friuliensis (4) consist of 10 amino acids that form a ring structure with one exocyclic amino acid linked to an acyl residue of various chain lengths. The acyl residue is a branched-chain fatty acid with a ⌬cis3 double bond. The ...

The antibiotic phosphinothricin tripeptide (PTT) consists of two molecules of L-alanine and one molecule of the unusual amino acid phosphinothricin (PT) which are nonribosomally combined. The bioactive compound PT has bactericidal, fungicidal, and herbicidal properties and possesses a COPOC bond, which is very rare in natural compounds. Previously uncharacterized flanking and middle regions of the PTT biosynthetic gene cluster from Streptomyces viridochromogenes Tü494 were isolated and sequenced. The boundaries of the gene cluster were identified by gene inactivation studies. Sequence analysis and homology searches led to the completion of the gene cluster, which consists of 24 genes. Four of these were identified as undescribed genes coding for proteins that are probably involved in uncharacterized early steps of antibiotic biosynthesis or in providing precursors of PTT biosynthesis (phosphoenolpyruvate, acetyl-coenzyme A, or L-alanine). The involvement of the genes orfM and trs and of the regulatory gene prpA in PTT biosynthesis was analyzed by gene inactivation and overexpression, respectively. Insight into the regulation of PTT was gained by determining the transcriptional start sites of the pmi and prpA genes. A previously undescribed regulatory gene involved in morphological differentiation in streptomycetes was identified outside of the left boundary of the PTT biosynthetic gene cluster.

Phosphinothricyl-alanyl-alanine (Pt tripeptide (Ptt), bialaphos) is a metabolite produced by Streptomyces viridochromogenes and Streptomyces hygroscopicus. It contains the unique phosphinoamino acid phosphinothricin (Pt), which after cleavage from Ptt is active as an inhibitor of glutamine synthetase. We have isolated three enzymes that assemble the building block of the Ptt peptide backbone in a nonribosomal mechanism. The first enzyme, named Ptt-synthetase I (PTTS I), activates N-acetyldemethylphosphinothricin (AcDMPt) as adenylate and thioester. Pt is not activated. PTTS I can also activate N-acetylphosphinothricin (AcPt) or N-acetylglutamate as structural analogues of AcDMPT. Native PTTS I has an estimated size of 62 kDa whereas the denatured form displays a size of 76 kDa. Immunoblot analysis and determination of its N-terminal protein sequence revealed that PTTS I is identical with the gene product of phsA. The phsA gene was previously identified near the Pt-resistance gene pat in the Ptt biosynthesis gene cluster in S. viridochromogenes. Besides PTTS I, two alanine-activating enzymes (PTTS II/III) were partially purified from S. viridochromogenes with estimated native sizes of ca. 120 kDa (enzyme 1) and ca. 140 kDa (enzyme 2). Both enzymes bind alanine as a thioester via the corresponding adenylate. Level of PTTS II/III and product formation were correlated with each other in several different strains of S. viridochromogenes. These results indicate that Ptt is synthesized by three peptide synthetases, each activating one single amino acid. The data also confirm previous genetic data, which suggest that AcDMPt-Ala-Ala is the precursor of Ptt.