Immune response mechanisms against Pseudomonas aeruginosa associated with mucosal immunization with protein antigens in a rat model of acute lung infection.

Pseudomonas aeruginosa is a major cause of nosocomal and community acquired chronic infections in subjects with compromised respiratory function. The microbe is environmentally ubiquitious and has a high level of innate antimicrobial resistance. This has led researchers to investigate vaccine and immunotherapeutic approaches to prevent and treat P. aeruginosa infections. Seven cytosolic non-integral proteins were studied as vaccine candidates in an acute lung infection model in the rat. Five of these (amidase, amidopeptidase, KatE, KatE and Pa13 a novel 13kDa protein) enhanced bacterial clearance from the lung compared to control animals following challenge and are worthy of further study. Immune mechanisms stimulated by these proteins in response to both immunization and infection varied. The most pronounced degree of bacterial clearance from the lung was associated with antigens, which demonstrated greater surface exposure and induced an increase in phagocyte recruitment, in particular, an increased proportion of polymorphonuclear leukocytes. Lymphocytic proliferation and specific antibody responses in the absence of enhanced clearance were less informative as immune correlates.

SMb20651 is another acyl carrier protein from Sinorhizobium meliloti.

Acyl carrier proteins (ACPs) are small acidic proteins that carry growing acyl chains during fatty acid or polyketide synthesis. In rhizobia, there are four different and well-characterized ACPs: AcpP, NodF, AcpXL and RkpF. The genome sequence of Sinorhizobium meliloti 1021 reveals two additional ORFs that possibly encode additional ACPs. One of these, smb20651, is located on the plasmid pSymB as part of an operon. The genes of the operon encode a putative asparagine synthetase (AsnB), the predicted ACP (SMb20651), a putative long-chain fatty acyl-CoA ligase (SMb20650) and a putative ammonium-dependent NAD+ synthetase (NadE1). When SMb20651 was overexpressed in Escherichia coli, [3H]beta-alanine, a biosynthetic building block of 4'-phosphopantetheine, was incorporated into the protein in vivo. The purified SMb20651 was modified with 4'-phosphopantetheine in the presence of S. meliloti holo-ACP synthase (AcpS). Also, holo-SMb20651 was modified in vitro with a malonyl group by malonyl CoA-ACP transacylase. In E. coli, coexpression of SMb20651 together with other proteins such as AcpS and SMb20650 led to the formation of additional forms of SMb20651. In this bacterium, acylation of SMb20651 with C12 : 0 or C18 : 0 fatty acids was detected, demonstrating that this protein is involved in fatty acid biosynthesis or transfer. Expression of SMb20651 was detected in S. meliloti as holo-SMb20651 and acyl-SMb20651.

Purification and partial characterization of acyl carrier protein from Euglena gracilis variety bacillaris.

Acyl carrier protein (ACP) was purified from Euglena gracilis variety bacillaris in yields of about 1 mg/100 g (wet wt) of cells. Antibodies against the purified protein were raised in hens and isolated from eggs. Antibodies raised against Euglena ACP inhibited the Euglena chloroplast nonaggregated fatty acid synthetase using either Euglena or Escherichia coli ACP as a substrate. Comparisons with other ACPs included the following items: biologic activity, acidic pI, size, behavior in size exclusion media, and amino acid sequence of the N-terminal portion of the molecule.

In vivo pools of free and acylated acyl carrier proteins in spinach. Evidence for sites of regulation of fatty acid biosynthesis.

In order to examine potential regulatory steps in plant fatty acid biosynthesis, we have developed procedures for the analysis of the major acyl-acyl carrier protein (ACP) intermediates of this pathway. These techniques have been used to separate and identify acyl-ACPs with chain configurations ranging from 2:0 to 18:1 and to determine the relative in vivo concentrations of acyl-ACPs in spinach leaf and developing seed. In both leaf and seed as much as 60% of the total ACPs were nonesterified (free), with the remaining proportion consisting of acyl-ACP intermediates leading to the formation of palmitate, stearate, and oleate. In spinach leaf the proportions of the various acyl groups esterified to each ACP isoform were indistinguishable, indicating that these isoforms are utilized similarly in de novo fatty acid biosynthesis in vivo. However, the acyl group distribution pattern of seed ACP-II differed significantly from that of leaf ACP-II. The malonyl-ACP levels were less than the 4:0-ACP and 6:0-ACP levels in leaf, and in contrast, the malonyl-ACP-II levels in seed were approximately 3-fold higher than the 4:0-ACP-II and 6:0-ACP-II levels. In addition, the ratio of oleoyl-ACP-II (18:1) to stearoyl-ACP-II (18:0) was higher in seed than in leaf. These data suggest that the differences in acyl-ACP patterns reflect a tissue/organ-specific difference rather than an isoform-specific difference. In extracts prepared from leaf samples collected in the dark, the levels of acetyl-ACPs were approximately 5-fold higher compared to samples collected in the light. The levels of free ACPs showed an inverse response, increasing in the light and decreasing in the dark. Notably there was no concomitant increase in the malonyl-ACP levels. The most likely explanation for the major increase in acetyl-ACP levels in the dark is that light/dark control over the rate of fatty acid biosynthesis occurs at the reaction catalyzed by acetyl-CoA carboxylase.

Acyl carrier protein is present in the mitochondria of plants and eucaryotic micro-organisms.

Proteins antigenically similar to the acyl carrier protein (ACP) found in the mitochondria of Neurospora crassa were detected by immunoblotting and radioimmunoassay techniques in mitochondria isolated from yeast, potatoes, and pea leaves. These mitochondrial proteins were similar to Neurospora ACP both in their electrophoretic mobility and in their unusual decrease in mobility upon reduction. Authentic ACP(s) show this type of change upon conversion of the acylated to the unacylated form. Purified ACP from both spinach chloroplasts and Escherichia coli cells cross-reacted with antibodies raised against Neurospora ACP. Purified ACP from Neurospora cross-reacted with antibodies raised against spinach chloroplast ACP and E. coli ACP. Mitochondria isolated from beef heart and rat brain were tested extensively and exhibited no cross-reaction with any of the three anti-ACP preparations. The discovery of ACP in the mitochondria of other organisms raises questions concerning the possible relationship between ACP and beta-oxidation in mitochondria, the involvement of ACP in de novo biosynthesis of some of the acyl chains in mitochondria and the subcellular locations of fatty acid biosynthesis in plants and eucaryotic micro-organisms.