What's next after the clot? Residual pulmonary vascular obstruction after pulmonary embolism: From imaging finding to clinical consequences.

Surviving an embolism exposes patients to potential long-term complications, such as altered quality of life, persistent dyspnea, impaired exercise capacity or pulmonary hypertension. The common objective factor in most of these situations is the presence of residual pulmonary vascular obstruction (RPVO). Planar ventilation/perfusion scintigraphy (V/Q lung scan) is the gold standard for assessing RPVO, which occurs in 46 to 66% of patients at 3 months and persists in 25 to 29% of patients a year after acute PE. Assessed early (i.e. before discharge), RPVO could predict acute PE development with a high negative predictive value. Evaluated after anticoagulation therapy, RPVO could help to manage anticoagulation treatment and predict the risk of PE recurrence and patients identified at risk of developing chronic thromboembolic pulmonary hypertension. In this comprehensive review, we provide an overview of the current knowledge of RPVO after PE from imaging diagnosis to clinical consequences. In the first part, we mainly focus on the imaging modalities capable of detecting and quantifying RPVO. We then focus on the symptoms and syndromes linked with this residual obstruction after PE. Although the occurrence of RPVO and long-term complications varies greatly from one patient to another, we finally aim to identify the patients and diseases at risk of developing residual obstruction.

The AprX protein of Pseudomonas aeruginosa: a new substrate for the Apr type I secretion system.

Protein secretion in Pseudomonas aeruginosa involves different mechanisms. The type II and type III secretory pathways control the extracellular release of a wide range of substrates. The type I secretion process, or ABC transporter, was believed to be exclusively involved in alkaline protease secretion. Recently, it was discovered that a P. aeruginosa heme binding protein, HasAp, is also secreted by a type I process. We present here the identification of a third putative type I-dependent protein of P. aeruginosa, AprX. The function of this protein has not yet been elucidated but very interestingly it appears to be linked to the apr cluster, and organized in one single operon together with the aprD, -E and -F genes.

Protein secretion by heterologous bacterial ABC-transporters: the C-terminus secretion signal of the secreted protein confers high recognition specificity.

Pseudomonas aeruginosa releases several extracellular proteins which are secreted via two independent secretion pathways. Alkaline protease (AprA) Is released by its own specific secretion machinery which is an ABC-transporter. Despite sequence similarities between components of ABC-transporters in different bacteria, each transporter is dedicated to the secretion of a particular protein or a family of closely related proteins. Heterologous complementation between ABC-transporters for unrelated polypeptides can occur, but only at a very low level. We show that the 50 C-terminal amino acids of AprA constitute an autonomous secretion signal. By heterologous complementation experiments between the unrelated alpha-haemolysin (HlyA) and Apr secretion systems we demonstrated that it is only the recognition of the secretion signal by the translocator which confers specificity to the secretion process. Secretion was size-dependent. However inclusion of glycine-rich repeats from HlyA in AprA seems to overcome the size limitation exerted by the Apr secretion apparatus such that the machinery secreted a hybrid protein 20 kDa larger than the normal maximal size.

The Pseudomonas fluorescens lipase has a C-terminal secretion signal and is secreted by a three-component bacterial ABC-exporter system.

Both Pseudomonas aeruginosa and Pseudomonas fluorescens secrete a lipase into the extracellular medium. Unlike the lipase of P. aeruginosa, the lipase produced by P. fluorescens does not contain any N-terminal signal sequence. We show that the P. fluorescens lipase is secreted through the signal peptide-independent pathway of the alkaline protease that we previously identified in P. aeruginosa. Secretion of this protease (AprA) is dependent on the presence of three genes located adjacent to the aprA gene, aprD, aprE and aprF. The three secretion functions permit an efficient secretion of P. fluorescens lipase. Inactivation of one of them (AprE) prevented this secretion. In Escherichia coli, the three proteins AprD, AprE, AprF are necessary and sufficient for efficient secretion of lipase to the extracellular medium. The secretion signal is located within the C-terminal part of the lipase sequence and can promote efficient secretion of a passenger protein. Thus the P. fluorescens lipase secretion system belongs to the group of the three-component bacterial ABC-exporter systems.

Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa: relationships to other secretory pathways.

A genetic locus implicated in the synthesis and secretion of alkaline protease (APR) in Pseudomonas aeruginosa has been previously described [Guzzo et al., J. Bacteriol. 172 (1990) 942-948]. The nucleotide sequence of the DNA fragment encoding these functions was determined and revealed the existence of five open reading frames: aprA, the structural gene encoding APR; aprI, which encodes a protease inhibitor; and aprD, aprE, aprF whose products are involved in protease secretion. The AprD, AprE and AprF proteins share significant homology with proteins implicated in secretion of Erwinia chrysanthemi proteases and Escherichia coli alpha-haemolysin. These results provide further evidence for the existence of a specialized secretory system widespread among Gram- bacteria.

Protein secretion in Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into the extracellular medium. At least two distinct secretion pathways can be discerned. The majority of the exoproteins are secreted via a two-step mechanism. These proteins are first translocated across the inner membrane in a signal sequence-dependent fashion. The subsequent translocation across the outer membrane requires the products of at least 12 distinct xcp genes. The exact role of one of these proteins, the XcpA protein, has been resolved. It is a peptidase that is required for the processing of the precursors of four other Xcp proteins, thus allowing their assembly into the secretion apparatus. This peptidase is also required for the processing of the precursors of type IV pili subunits. Two other Xcp proteins, XcpR and XcpS, display extensive homology to proteins involved in pili biogenesis, which suggests that the assembly of the secretion apparatus and the biogenesis of type IV pili are related processes. The secretion of alkaline protease does not require the xcp gene products. This enzyme, which is encoded by the aprA gene, is not synthesized in a precursor form with an N-terminal signal sequence. Secretion across the two membranes probably takes place in one step at adhesion zones that may be constituted by three accessory proteins, designated AprD, AprE and AprF. The two secretion pathways found in P. aeruginosa appear to have disseminated widely among Gram-negative bacteria.

Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism.

A 6.5-kb DNA fragment carrying the functions required for specific secretion of the extracellular alkaline protease produced by Pseudomonas aeruginosa was cloned. The whole 6.5-kb DNA fragment was transcribed in one direction and probably carried three genes involved in secretion. The expression in trans of these genes, together with the apr gene, in Escherichia coli allowed synthesis and secretion of the alkaline protease, which was extensively investigated by performing pulse-chase experiments under various conditions. We demonstrated the absence of a precursor form, as well as the independence of alkaline protease translocation from SecA. The absence of secretion genes impaired alkaline protease secretion; the protein then remained intracellular and was partially degraded.

The secretion genes of Pseudomonas aeruginosa alkaline protease are functionally related to those of Erwinia chrysanthemi proteases and Escherichia coli alpha-haemolysin.

The extracellular alkaline protease produced by Pseudomonas aeruginosa is secreted by a specific pathway, independent of the pathway used by most of the other extracellular proteins of this organism. Secretion of this protease is dependent on the presence of several genes located adjacent to the apr gene. Complementation studies have shown that PrtD, E, and F, the three secretion functions for Erwinia chrysanthemi proteases B and C (Létoffé et al., 1990), can mediate the secretion of the alkaline protease by Escherichia coli. The secretion functions involved in alpha-haemolysin secretion in E. coli (hlyB, hlyD, tolC) can also be used to complement alkaline protease secretion by E. coli, although less efficiently. These data indicate that protease secretion mechanisms in Pseudomonas and Erwinia are very similar and are homologous to that of E. coli alpha-haemolysin.

Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli.

Pseudomonas virulence is thought to depend on multiple characteristics, including the production of an extracellular alkaline protease. We report the isolation, from a PAO1 DNA genomic bank, of a cosmid carrying the structural gene coding for alkaline protease. By in vivo mutagenesis using transposon Tn1735, which functions as a transposable promoter, the expression of an 8.8-kilobase DNA fragment under control the tac promoter was obtained. When expressed in Escherichia coli, active alkaline protease was synthesized and secreted to the extracellular medium in the absence of cell lysis.

Secretion of extracellular proteins by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a bacterial species of commercial value secreting numerous extracellular proteins, involved in pathogenesis. Most strains produce at least a lipase, a phospholipase, an alkaline phosphatase, an exotoxin and 2 proteases (elastase and alkaline protease). Various mechanisms for secretion of exoproteins appear to exist in P aeruginosa. Genetic analysis has led to the identification of 2 secretion pathways: i) a "general" secretion pathway, defined by the xcp mutations, which mediates secretion of most extracellular proteins, and; ii) an independent secretion pathway specific for alkaline protease. Our present knowledge on the pathways and components of the secretion machinery in P aeruginosa is reviewed in this article.

Cloning of xcp genes located at the 55 min region of the chromosome and involved in protein secretion in Pseudomonas aeruginosa.

Pleiotropic mutations (xcp) affecting secretion of proteins in Pseudomonas aeruginosa have been previously characterized and mapped at 0 min, 55 min and 65 min. Genomic libraries of this organism have been constructed and the genes xcp-5 and xcp-54, located at the 55 min region, were cloned using the adjacent met allele as a marker, and complementation of xcp strains. From our linkage and cloning analysis, the most probable gene order in this region appears to be pyrD... xcp-5/xcp-54/met-9011/oru-314/trpF/leu-10. Restriction mapping and transposon (Tn1725) insertion mutagenesis demonstrated that: (i) the overall size of DNA necessary for xcp expression was 9kb, (ii) the two loci are not adjacent on the chromosome, and (iii) the two loci are expressed independently. The xcp-5 gene has been subcloned on a 4kb EcoRI fragment.

Phosphate regulation in Pseudomonas aeruginosa: cloning of the alkaline phosphatase gene and identification of phoB- and phoR-like genes.

In Pseudomonas aeruginosa, phosphate limitation results in the synthesis of several protein species. We report the cloning of the P. aeruginosa alkaline phosphatase structural gene, phoA, and we show that this gene is regulated normally in Escherichia coli. We have also identified and cloned two P. aeruginosa genes which can complement phoB and phoR mutations in E. coli. This suggests that a pho regulon system similar to that in E. coli may exist in P. aeruginosa, using at least two similar regulatory factors.

Mutation that affects pepN translation initiation in Escherichia coli.

Mutants altered in their expression of the hybrid pepN-lacZ gene have been selected for resistance to p-nitrophenyl-beta-D-thiogalactopyranoside (a bacteriostatic compound that enters the cells via lac permease). A unique mutation decreasing the level of pepN expression to 9% of that of the wild type has been studied in detail. This mutation controls in cis the expression of the pepN gene. The pepN region from a pepN-lacZ gene fusion has been cloned and sequenced. Comparison of the mutant and wild type sequences indicates that the mutation lies between the Shine-Dalgarno sequence (AGGT) and the initiation codon (AUG). This mutation is a T----C transition which might allow the formation of a stable secondary structure in the region of translation initiation thus decreasing the level of pepN expression.

Nucleotide sequence of the promoter and amino-terminal encoding region of the Escherichia coli pepN gene.

The nucleotide sequence of the region probably responsible for regulation of pepN expression and of the region encoding the amino-terminal part of aminopeptidase N, has been determined. The transcription start site was identified by S1 nuclease mapping. All features of the promoter are those of a weak promoter and no obvious structure responsible for regulation was identified, although a possible Pho box is located 63 base pairs upstream from the Pribnow box. The reading frame was unambiguously determined by purifying the protein and by sequencing the first 21 NH2-terminal residues. The NH2-terminal region of aminopeptidase N does not contain any fragment resembling signal sequence and the protein is not produced in a precursor form. A divergent promoter, which might be that of pncB, encoding the nicotinic acid phosphoribosyltransferase (P. Terpstra, personal communication), has also been identified, which allows the assignment of the gene organization in this chromosomal region as ompF asnS pncB pepN.

Multiple controls exerted on in vivo expression of the pepN gene in Escherichia coli: studies with pepN-lacZ operon and protein fusion strains.

Three physiological conditions were shown to promote transcriptional regulation of pepN expression: phosphate limitation, the nature of the source of carbon and energy, and anaerobiosis. The transcriptional level of regulation can be deduced from the observation of these effects in strains carrying operon fusion pepN-lacZ. Mutations in the various genes phoB, phoM, phoR, crp, and fnr (oxrA) did not affect pepN expression.

Cloning and orientation of the gene encoding aminopeptidase N in Escherichia coli.

The pepN gene, that encodes aminopeptidase N in Escherichia coli, has been cloned into the multicopy plasmid pBR322. Expression of the cloned pepN gene results in overproduction of the enzyme. The restriction map of the 6.7 Kb insert was established and the gene was further localized by analysis of the in vitro constructed delection plasmid and mutant plasmids generated by Tn5 insertions. Chromosome mobilization experiments, using pep-N-lac fusion strains allowed us to infer a clockwise direction of transcription for the pepN gene.

Physical mapping of the gene for aminopeptidase N in Escherichia coli K12.

The pepN gene has been cloned into the multicopy plasmid pBR322. The restriction map of the insert was established and the gene was localized. By comparison with the restriction map of the plasmid pJP30 bearing the ompF region, it has been possible to order the ompF, asnS, and pepN genes. The ompF and asnS genes are contiguous and pepN is separated from asnS by a DNA fragment of about 1.6 kb.

Fusion of the lac genes to the promoter for the aminopeptidase N gene of Escherichia coli.

Strains of Escherichia coli K12 were isolated in which the lac structural genes were fused to the promoter for the aminopeptidasee N structural gene (pepN). Although this enzyme is constitutively produced, the differential rate of synthesis is increased about 4-fold upon phosphate starvation. The pepN-lac fusions were shown to respond to phosphate specific regulatory signals. A plaque forming lambda transducing phage bearing the pepN-lac fusion was isolated. This phage was used to prove genetically the fusion of lac genes to the promoter for the aminopeptidase. These results demonstrate a control at the transcriptional level of aminopeptidase synthesis.