Role of the N-Acetylmuramoyl-l-Alanyl Amidase, AmiA, of Helicobacter pylori in Peptidoglycan Metabolism, Daughter Cell Separation, and Virulence.

The human gastric pathogen, Helicobacter pylori, is becoming increasingly resistant to most available antibiotics. Peptidoglycan (PG) metabolism is essential to eubacteria, hence, an excellent target for the development of new therapeutic strategies. However, our knowledge on PG metabolism in H. pylori remains poor. We have further characterized an isogenic mutant of the amiA gene encoding a N-acetylmuramoyl-l-alanyl amidase. The amiA mutant displayed long chains of unseparated cells, an impaired motility despite the presence of intact flagella and a tolerance to amoxicillin. Interestingly, the amiA mutant was impaired in colonizing the mouse stomach suggesting that AmiA is a valid target in H. pylori for the development of new antibiotics. Using reverse phase high-pressure liquid chromatography, we analyzed the PG muropeptide composition and glycan chain length distribution of strain 26695 and its amiA mutant. The analysis showed that H. pylori lacked muropeptides with a degree of cross-linking higher than dimeric muropeptides. The amiA mutant was also characterized by a decrease of muropeptides carrying 1,6-anhydro-N-acetylmuramic acid residues, which represent the ends of the glycan chains. This correlated with an increase of very long glycan strands in the amiA mutant. It is suggested that these longer glycan strands are trademarks of the division site. Taken together, we show that the low redundancy on genes involved in PG maturation supports H. pylori as an actractive alternative model to study PG metabolism and cell shape regulation.

Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics.

Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.

Differential expression of Ixodes ricinus salivary gland proteins in the presence of the Borrelia burgdorferi sensu lato complex.

In Europe, Ixodes ricinus is the main vector of Lyme borreliosis. Their salivary glands play a critical role in the biological success of ticks. To better understand the cross-talk between Borrelia burgdorferi and tick salivary glands, we analyzed protein expression in the salivary glands of I. ricinus adult ticks that were infected by various strains of the B. burgdorferi sl complex. iTRAQ allowed the identification of more than 120 proteins, providing the first proteomic data pertaining to I. ricinus salivary glands. Among these proteins, only 12 were modulated in the presence of various Borrelia strains. Most of them are up-regulated and are involved in cell defense and protein synthesis and processing. Down-regulated proteins are mostly implicated in the cytoskeleton. The DIGE analysis allowed us to identify 35 proteins and showed the down-regulation of 4 proteins. All 15 proteins were not modulated by all strains. Overall, these observations showed that the presence of Borrelia in tick salivary glands is a factor of stress for the protein machinery, and also that some Borrelia strains produce a dysregulation of cytoskeletal proteins. Interestingly, a protein from Borrelia, OspA, was found in infected salivary glands. The consequence of its presence in salivary glands is discussed. BIOLOGICAL SIGNIFICANCE: Lyme borreliosis is still the most prevalent arthropod-borne disease in the temperate regions of the northern hemisphere. The geographical distribution of Lyme borreliosis is expanding, especially towards higher altitudes and latitudes. Human pathogenic spirochetes causing Lyme borreliosis belong to the B. burgdorferi sensu lato complex. They are extracellular pathogens transmitted to humans through the bite of Ixodes spp. ticks. The bioactive molecules present in tick saliva not only promote tick feeding, but also create an advantageous microenvironment at the tick bite site for survival and replication of Borrelia bacteria. Investigation of the tick-host-pathogen interface would provide new strategies to control tick-borne infections. We chose to analyze the interaction of several strains of the B. burgdorferi sensu lato complex with I. ricinus salivary glands. We also investigated the presence of bacterial proteins in salivary glands. For these purposes, we undertook a proteomic study implying the complementary approaches of iTRAQ and DIGE. Our study allowed identifying several salivary markers of infection that were shown to vary according to the strain. Moreover, OspA, a bacterial protein was shown to be expressed in salivary glands and may be implied in the pathogenicity of some Borrelia strains.

Characterization of the enzymatic activity of Clostridium perfringens TpeL.

TpeL is a toxin produced by Clostridium perfringens which belongs to the large clostridial glucosylating toxin family. It was shown that TpeL modifies Ras using UDP-glucose or UDP-N-acetylglucosamine as cosubstrates (Guttenberg et al., 2012; Nagahama et al., 2011). We confirmed that TpeL preferentially glucosaminates the three isoforms of Ras (cH-Ras, N-Ras, and K-Ras) from UDP-N-acetylglucosamine and to a lower extent Rap1a and R-Ras3, and very weakly Rac1. In contrast to previous report, we observed that Ral was not a substrate of TpeL. In addition, we confirmed by in vitro glucosylation and mass spectrometry that TpeL modifies cH-Ras at Thr35.

Infection by chikungunya virus modulates the expression of several proteins in Aedes aegypti salivary glands.

BACKGROUND: Arthropod-borne viral infections cause several emerging and resurging infectious diseases. Among the diseases caused by arboviruses, chikungunya is responsible for a high level of severe human disease worldwide. The salivary glands of mosquitoes are the last barrier before pathogen transmission. METHODS: We undertook a proteomic approach to characterize the key virus/vector interactions and host protein modifications that occur in the salivary glands that could be responsible for viral transmission by using quantitative two-dimensional electrophoresis. RESULTS: We defined the protein modulations in the salivary glands of Aedes aegypti that were triggered 3 and 5 days after an oral infection (3 and 5 DPI) with chikungunya virus (CHIKV). Gel profile comparisons showed that CHIKV at 3 DPI modulated the level of 13 proteins, and at 5 DPI 20 proteins. The amount of 10 putatively secreted proteins was regulated at both time points. These proteins were implicated in blood-feeding or in immunity, but many have no known function. CHIKV also modulated the quantity of proteins involved in several metabolic pathways and in cell signalling. CONCLUSION: Our study constitutes the first analysis of the protein response of Aedes aegypti salivary glands infected with CHIKV. We found that the differentially regulated proteins in response to viral infection include structural proteins and enzymes for several metabolic pathways. Some may favour virus survival, replication and transmission, suggesting a subversion of the insect cell metabolism by arboviruses. For example, proteins involved in blood-feeding such as the short D7, an adenosine deaminase and inosine-uridine preferring nucleoside hydrolase, may favour virus transmission by exerting an increased anti-inflammatory effect. This would allow the vector to bite without the bite being detected. Other proteins, like the anti-freeze protein, may support vector protection.

Identification of Outer Membrane Proteins Altered in Response to UVC-Radiation in Vibrio parahaemolyticus and Vibrio alginolyticus.

Vibrio parahaemolyticus and V. alginolyticus, marine foodborne pathogens, were treated with UVC-radiation (240 J/m(2)) to evaluate alterations in their outer membrane protein profiles. Outer membrane protein patterns of UVC-irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Altered proteins were identified by mass spectrometry (MS and MS/MS) and analysis revealed that OmpW, OmpA, Long-chain fatty acid transport protein, Outer membrane receptor protein, Putative uncharacterized protein VP0167, Maltoporin (lamB), Polar flagellin B/D, Agglutination protein Peptidoglycan-associated lipoprotein and MltA-interacting protein MipA were appeared, thereby they can be considered as UVC-stress proteins in some vibrios. In addition, expression of OmpK decreased to non-detectable level. Furthermore, we observed a decrease or an increase in the expression level of other outer membrane proteins.

Identification of outer membrane proteins of Vibrio parahaemolyticus and Vibrio alginolyticus altered in response to γ-irradiation or long-term starvation.

Vibrio parahaemolyticus and Vibrio alginolyticus were subjected to γ-irradiation (0.5 kGy) or starvation by incubation for 8 months in seawater to study modifications in their outer membrane protein patterns. After treatment, outer membrane protein profiles of starved or γ-irradiated bacteria were found to be altered when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Altered proteins were identified by mass spectrometry (MS and MS/MS) and analyses revealed that OmpU can be considered a starvation stress-induced protein. In addition, expression of OtnA, OmpW, OmpA and peptidoglycan-associated lipoprotein decreased to non-detectable levels in starved cells. Furthermore, MltA-interacting protein MipA appeared under γ-irradiation or starvation conditions. Thus, it can be considered to be a γ-irradiation, long-term starvation stress protein in some vibrios.

CCSV-MPase, a novel procoagulant metalloproteinase from Cerastes cerastes venom: purification, biochemical characterization and protein identification.

A procoagulant metalloproteinase called CCSV-MPase was purified from C. cerastes venom by successive chromatographic methods starting with gel-filtration through Sephadex G-75; ion-exchange DEAE-Cellulose A-50; affinity chromatography on Benzamidine Sepharose 6B and RP-HPLC on a C8 column. CCSV-MPase has been isolated to an extent of about tenfolds and its molecular mass was evaluated at 70 kDa by SDS-PAGE. CCSV-MPase hydrolyzes casein and fibrinogene as natural substrates. Its proteolytic activity was inhibited by EDTA and 1.10-phenanthroline, a chelators of bivalent cation metals and Zn(2+) respectively. CCSV-MPase is therefore a Zn(2+)-metalloproteinase with fibrinogenolytic but not hemorrhagic activity. It greatly decreased levels of plasmatic fibrinogen when administered to rats for 24 h. This fibrinogenase hydrolyzes the Bß chain of human fibrinogen in vitro releasing fibrinopeptide B only. LC MS/MS analysis of tryptic fragments of CCSV-MPase demonstrated that it showed some sequence similarities with four other venom metalloproteinases. CCSV-MPase could be considered as a potential therapeutic agent as it is a non-hemorrhagic enzyme and could be useful in thrombotic diseases because of its defibrinogenation of blood.

AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX.

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage.

Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1(-/-) null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases.

Interleukin-7 compartmentalizes its receptor signaling complex to initiate CD4 T lymphocyte response.

Interleukin (IL)-7 is a central cytokine that controls homeostasis of the CD4 T lymphocyte pool. Here we show on human primary cells that IL-7 binds to preassembled receptors made up of proprietary chain IL-7Ralpha and the common chain gammac shared with IL-2, -4, -9, -15, and -21 receptors. Upon IL-7 binding, both chains are driven in cholesterol- and sphingomyelin-rich rafts where associated signaling proteins Jak1, Jak3, STAT1, -3, and -5 are found to be phosphorylated. Meanwhile the IL-7.IL-7R complex interacts with the cytoskeleton that halts its diffusion as measured by single molecule fluorescence autocorrelated spectroscopy monitored by microimaging. Comparative immunoprecipitations of IL-7Ralpha signaling complex from non-stimulated and IL-7-stimulated cells confirmed recruitment of proteins such as STATs, but many others were also identified by mass spectrometry from two-dimensional gels. Among recruited proteins, two-thirds are involved in cytoskeleton and raft formation. Thus, early events leading to IL-7 signal transduction involve its receptor compartmentalization into membrane nanodomains and cytoskeleton recruitment.

IgG autoantibody to brain beta tubulin III associated with cytokine cluster-II discriminate cerebral malaria in central India.

BACKGROUND: The main processes in the pathogenesis of cerebral malaria caused by Plasmodium falciparum involved sequestration of parasitized red blood cells and immunopathological responses. Among immune factors, IgG autoantibodies to brain antigens are increased in P. falciparum infected patients and correlate with disease severity in African children. Nevertheless, their role in the pathophysiology of cerebral malaria (CM) is not fully defined. We extended our analysis to an Indian population with genetic backgrounds and endemic and environmental status different from Africa to determine if these autoantibodies could be either a biomarker or a risk factor of developing CM. METHODS/PRINCIPAL FINDINGS: We investigated the significance of these self-reactive antibodies in clinically well-defined groups of P. falciparum infected patients manifesting mild malaria (MM), severe non-cerebral malaria (SM), or cerebral malaria (CM) and in control subjects from Gondia, a malaria epidemic site in central India using quantitative immunoprinting and multivariate statistical analyses. A two-fold complete-linkage hierarchical clustering allows classifying the different patient groups and to distinguish the CM from the others on the basis of their profile of IgG reactivity to brain proteins defined by PANAMA Blot. We identified beta tubulin III (TBB3) as a novel discriminant brain antigen in the prevalence of CM. In addition, circulating IgG from CM patients highly react with recombinant TBB3. Overall, correspondence analyses based on singular value decomposition show a strong correlation between IgG anti-TBB3 and elevated concentration of cluster-II cytokine (IFNgamma, IL1beta, TNFalpha, TGFbeta) previously demonstrated to be a predictor of CM in the same population. CONCLUSIONS/SIGNIFICANCE: Collectively, these findings validate the relationship between antibody response to brain induced by P. falciparum infection and plasma cytokine patterns with clinical outcome of malaria. They also provide significant insight into the immune mechanisms associated to CM by the identification of TBB3 as a new disease-specific marker and potential therapeutic target.

In vivo interactome of Helicobacter pylori urease revealed by tandem affinity purification.

In the human gastric bacterium Helicobacter pylori, two metalloenzymes, hydrogenase and urease, are essential for in vivo colonization, the latter being a major virulence factor. The UreA and UreB structural subunits of urease and UreG, one of the accessory proteins for Ni(2+) incorporation into apourease, were taken as baits for tandem affinity purification. The method allows the purification of protein complexes under native conditions and physiological expression levels of the bait protein. Furthermore the tandem affinity purification technology was combined with in vivo cross-link to capture transient interactions. The results revealed different populations of urease complexes: (i) urease captured during activation by Ni(2+) ions comprising all the accessory proteins and (ii) urease in association with metabolic proteins involved e.g. in ammonium incorporation and the cytoskeleton. Using UreG as a bait protein, we copurified HypB, the accessory protein for Ni(2+) incorporation into hydrogenase, that is reported to play a role in urease activation. The interactome of HypB partially overlapped with that of urease and revealed interactions with SlyD, which is known to be involved in hydrogenase maturation as well as with proteins implicated in the formation of [Fe-S] clusters present in the small subunit of hydrogenase. In conclusion, this study provides new insight into coupling of ammonium production and assimilation in the gastric pathogen and the intimate link between urease and hydrogenase maturation.

Comprehensive proteome analysis of Mycobacterium ulcerans and quantitative comparison of mycolactone biosynthesis.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rapidly emerging human disease in which mycolactone, a cytotoxic and immunosuppressive macrocyclic polyketide, is responsible for massive skin destruction. The genome sequencing of M. ulcerans has recently been accomplished (http://genolist.pasteur.fr/BuruList/) enabling the first proteome study of this important human pathogen. Here, we present a comprehensive proteome analysis of different subcellular fractions and culture supernatant of in vitro grown M. ulcerans. By a combination of gel-based and gel-free techniques for protein and peptide separation with subsequent analysis by MS, we identified 1074 different proteins, corresponding to 25% of the protein-coding DNA sequence. Interestingly, new information was obtained about central metabolism and lipid biosynthesis, and as many as 192 conserved hypothetical proteins were found. Comparative analysis of the wild-type strain and an isogenic mycolactone-deficient mutant, by 2-DE and iTRAQ labeling of the cytoplasmic fraction, revealed differences in the expression profiles of proteins involved in lipid metabolism and information pathways, as well as stress responses.

60S ribosomal subunit assembly dynamics defined by semi-quantitative mass spectrometry of purified complexes.

During the highly conserved process of eukaryotic ribosome formation, RNA follows a maturation path with well-defined, successive intermediates that dynamically associate with many pre-ribosomal proteins. A comprehensive description of the assembly process is still lacking. To obtain data on the timing and order of association of the different pre-ribosomal factors, a strategy consists in the use of pre-ribsomal particles isolated from mutants that block ribosome formation at different steps. Immunoblots, inherently limited to only a few factors, have been applied to evaluate the accumulation or decrease of pre-ribosomal intermediates under mutant conditions. For a global protein-level description of different 60S ribosomal subunit maturation intermediates in yeast, we have adapted a method of in vivo isotopic labelling and mass spectrometry to study pre-60S complexes isolated from strains in which rRNA processing was affected by individual depletion of five factors: Ebp2, Nog1, Nsa2, Nog2 or Pop3. We obtained quantitative data for 45 distinct pre-60S proteins and detected coordinated changes for over 30 pre-60S factors in the analysed mutants. These results led to the characterisation of the composition of early, intermediate and late pre-ribosomal complexes, specific for crucial maturation steps during 60S assembly in eukaryotes.

Linking functionally related genes by sensitive and quantitative characterization of genetic interaction profiles.

Describing at a genomic scale how mutations in different genes influence one another is essential to the understanding of how genotype correlates with phenotype and remains a major challenge in biology. Previous studies pointed out the need for accurate measurements of not only synthetic but also buffering interactions in the characterization of genetic networks and functional modules. We developed a sensitive and efficient method that allows such measurements at a genomic scale in yeast. In a pilot experiment (41 genome-wide screens), we quantified the fitness of 140,000 double deletion strains relative to the corresponding single mutants and identified many genetic interactions. In addition to synthetic growth defects (validated experimentally with factors newly identified as genetically interfering with mRNA degradation), most of the identified genetic interactions measured weak epistatic effects. These weak effects, rarely meaningful when considered individually, were crucial to defining specific signatures for many gene deletions and had a major contribution in defining clusters of functionally related genes.

Phosphoproteomic analysis of Leishmania donovani pro- and amastigote stages.

Following transmission to the vertebrate host, the protozoan parasite Leishmania donovani differentiates into the pathogenic amastigote stage that is adapted for intracellular survival. This developmental transition is induced by environmental factors including elevated temperature and acidic pH and is likely transduced by signaling cascades involving protein kinases and their downstream phosphoprotein substrates. These signaling networks are highly adapted to the specific nutritional and physiological requirements of the organism and thus studying Leishmania phosphorylation may allow important insight into the parasite-specific biology. We used a gel-based approach to investigate qualitative and quantitative changes of the phosphoproteome of the major L. donovani life cycle stages. Phosphoproteins were purified by immobilized metal affinity chromatography (IMAC), separated by IEF and SDS-PAGE using pH 4-7 IPG immobiline strips, revealed by fluorescent multiplex staining, and identified by MALDI-MS and MS/MS. Our analysis allowed us to establish a first repertoire of the Leishmania phosphoproteome and to identify phosphoproteins implicated in stress- and heat shock response, RNA/protein turnover, metabolism, and signaling.

The salivary glands and saliva of Anopheles gambiae as an essential step in the Plasmodium life cycle: a global proteomic study.

Proteins synthesized in the salivary glands of the Anopheles gambiae mosquito are thought to be important in the life cycle of the malaria parasite Plasmodium. To describe A. gambiae salivary gland and saliva contents, we combined several techniques: 1-DE, 2-DE and LC MS/MS. This study has identified five saliva proteins and 122 more proteins from the salivary glands, including the first proteomic description for 89 of these salivary gland proteins. Since the invasion and sporozoite maturation take place during the process of salivary glands ageing, the effect of salivary gland age on salivary component composition was examined. LC MS/MS profiling of young versus old salivary gland proteomes suggests that there is an over-representation of proteins involved in signaling and proteins related to the immune response in the proteins from older mosquitoes. The iTRAQ labeling was used for a comparative proteomic analysis of salivary gland samples from infected or Plasmodium berghei-free mosquitoes. The expression levels of five secreted proteins were altered when the parasite was present. These observations will serve as a basis for future work concerning the possible role of these proteins in the interaction between A. gambiae, Plasmodium and the mammalian host.

The Pseudomonas aeruginosa LasB metalloproteinase regulates the human urokinase-type plasminogen activator receptor through domain-specific endoproteolysis.

Pseudomonas aeruginosa is an opportunistic pathogen in human lungs, where its secretable LasB metalloproteinase can be a virulence factor. The urokinase-type plasminogen activator receptor (uPAR) participates in pericellular proteolysis and the adherence/migration of epithelial cells and leukocytes recruited during infection and shows functional regulation by various proteinases via limited endoproteolysis occurring within its three domains (D1 to D3). We thus examined the proteolytic activity of LasB on uPAR by using recombinant uPAR as well as uPAR-expressing, human monocytic, and bronchial epithelial cell lines. Protein immunoblotting and flow immunocytometry using a panel of domain-specific anti-uPAR antibodies showed that LasB is able to cleave uPAR both within the sequence linking D1 to D2 and at the carboxy terminus of D3. Comparison of LasB-producing and LasB-deficient bacterial strains indicated that LasB is entirely responsible for the uPAR cleavage ability of P. aeruginosa. Based on amino-terminal protein microsequencing and mass spectrometry analysis of the cleavage of peptides mimicking the uPAR sequences targeted by LasB, cleavage sites were determined to be Ala(84)-Val(85) and Thr(86)-Tyr(87) (D1-D2) and Gln(279)-Tyr(280) (D3). Such a dual cleavage of uPAR led to the removal of amino-terminal D1, the generation of a truncated D2D3 species, and the shedding of D2D3 from cells. This proteolytic processing of uPAR was found to (i) drastically reduce the capacity of cells to bind urokinase and (ii) abrogate the interaction between uPAR and the matrix adhesive protein vitronectin. The LasB proteinase is thus endowed with a high potential for the alteration of uPAR expression and functioning on inflammatory cells during infections by P. aeruginosa.