Patrick Munro

Activation and Proteasomal Degradation of Rho GTPases by Cytotoxic Necrotizing Factor-1 Elicit a Controlled Inflammatory Response

The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 penetrates autonomously into cells and confers phagocytic properties to epithelial and endothelial cells. CNF1 acts at the molecular level by constitutively activating Rho GTPases attenuated by their cellular ubiquitin-mediated proteasomal degradation. Here we report the relationship between the ubiquitin-mediated proteasomal degradation of activated Rho and the endothelial cell response to the toxin. The type of cellular response to CNF1 intoxication, first screened by DNA microarray analysis, revealed the launching of a program oriented toward an inflammatory response. Parallel to Rho protein activation by CNF1, we also established the kinetics of production of monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), IL-6, monocyte inflammatory protein-3α (MIP-3α) and E-selectin. Both the mutation of the catalytic domain of the toxin (CNF1-C866S) and the inhibition of Rho proteins abrogate the CNF1-induced production of the immunomodulators MIP-3α, MCP-1, and IL-8. These immunomodulators are also produced upon activation of Cdc42 and Rac preferentially. Our results indicate that, in addition to pathogen molecular pattern recognition by host-receptors, a direct activation of Rho proteins by the CNF1 virulence factor efficiently triggers a cellular reaction of host alert. Consistently, we assume that the CNF1-induced ubiquitin-mediated proteasomal degradation of activated Rho proteins may limit the amplitude of the host cell immune responses.

Toxins from anaerobic bacteria: specificity and molecular mechanisms of action

Major advances have been made in the past five years in the identification of cellular targets of toxins produced by anaerobic bacteria. These targets include the vesicular membrane docking and fusion apparatus, the actin cytoskeleton, the signal transduction machinery and the cell membrane. The recent discovery that large clostridial toxins (Clostridium difficile A and B toxins, C. sordellii lethal and hemorrhagic toxins, and alpha C. novyi toxin) are monoglucosyltransferases, together with the establishment of the perfringolysin crystal structure, has led to new insights in the field of toxins from anaerobic bacteria.

Injection of Staphylococcus aureus EDIN by the Bacillus anthracis protective antigen machinery induces vascular permeability

ABSTRACT Systemic injection of Bacillus anthracis lethal toxin (LT) produces vascular leakage and animal death. Recent studies suggest that LT triggers direct endothelial cell cytotoxicity that is responsible for the vascular leakage. LT is composed of heptamers of protective antigen (PA), which drives the endocytosis and translocation into host cells of the lethal factor (LF), a mitogen-activated protein kinase kinase protease. Here we investigated the consequences of injection of an endothelium-permeabilizing factor using LT as a “molecular syringe.” To this end, we generated the chimeric factor LE, corresponding to the PA-binding domain of LF (LF1-254) fused to EDIN exoenzyme. EDIN ADP ribosylates RhoA, leading to actin cable disruption and formation of transcellular tunnels in endothelial cells. We report that systemic injection of LET (LE plus PA) triggers a PA-dependent increase in the pulmonary endothelium permeability. We also report that native LT induces a progressive loss of endothelium barrier function. We established that there is a direct correlation between the extent of endothelium permeability induced by LT and the cytotoxic activity of LT. This suggests new ways to design therapeutic drugs against anthrax directed toward vascular permeability.

Luciferase-expressing Leishmania infantum allows the monitoring of amastigote population size, in vivo, ex vivo and in vitro.

Here we engineered transgenic Leishmania infantum that express luciferase, the objectives being to more easily monitor in real time their establishment either in BALB/c mice—the liver and spleen being mainly studied—or in vitro. Whatever stationary phase L. infantum promastigotes population—wild type or engineered to express luciferase—the parasite burden was similar in the liver and the spleen at day 30 post the intravenous inoculation of BALB/c mice. Imaging of L. infantum hosting BALB/C mice provided sensitivity in the range of 20,000 to 40,000 amastigotes/mg tissue, two tissues—liver and spleen—being monitored. Once sampled and processed ex vivo for their luciferin-dependent bioluminescence the threshold sensitivity was shown to range from 1,000 to 6,000 amastigotes/mg tissue. This model further proved to be valuable for in vivo measurement of the efficiency of drugs such as miltefosine and may, therefore, additionally be used to evaluate vaccine-induced protection.

The Staphylococcus aureus Epidermal Cell Differentiation Inhibitor Toxin Promotes Formation of Infection Foci in a Mouse Model of Bacteremia

ABSTRACT Inactivation of the host GTPase RhoA by staphylococcal epidermal cell differentiation inhibitor (EDIN) exotoxins triggers the formation of large transcellular tunnels, named macroapertures, in endothelial cells. We used bioluminescent strains of Staphylococcus aureus to monitor the formation of infection foci during the first 24 h of hematogenous bacterial dissemination. Clinically derived EDIN-expressing S. aureus strains S25 and Xen36 produced many disseminated foci. EDIN had no detectable impact on infection foci in terms of histopathology or the intensity of emitted light. Moreover, EDIN did not modify the course of bacterial clearance from the bloodstream. In contrast, we show that EDIN expression promotes a 5-fold increase in the number of infection foci produced by Xen36. This virulence activity of EDIN requires RhoA ADP-ribosyltranferase activity. These results suggest that EDIN is a risk factor for S. aureus dissemination through the vasculature by virtue of its ability to promote the formation of infection foci in deep-seated tissues.

Effect of thermal, oxidative, acidic, osmotic, or nutritional stresses on subsequent culturability of Escherichia coli in seawater.

Survival of stressed Escherichia coli with or without the rpoS gene was assessed after 2 and 6 days in sterile seawater. Cells were submitted to thermal (48°C), acidic (pH 5.1), oxidative (H2O2 1mm), nutritional (C, N, P starvation), or osmotic (NaCl 0.5m) stresses for periods ranging from 0 to 4 h. We found a stress-mediated cross protection against seawater relative to controls. Viability was higher when cells were acid, oxidatively, nutritionally or osmotically stressed. Survival increased in cells stressed at 37°C as compared with 20°C. With the exception of osmotic stress, we found that this stress-induced cross protection was rpoS dependent.

Cherubism allele heterozygosity amplifies microbe-induced inflammatory responses in murine macrophages.

Cherubism is a rare autoinflammatory bone disorder that is associated with point mutations in the SH3-domain binding protein 2 (SH3BP2) gene, which encodes the adapter protein 3BP2. Individuals with cherubism present with symmetrical fibro-osseous lesions of the jaw, which are attributed to exacerbated osteoclast activation and defective osteoblast differentiation. Although it is a dominant trait in humans, cherubism appears to be recessively transmitted in mice, suggesting the existence of additional factors in the pathogenesis of cherubism. Here, we report that macrophages from 3BP2-deficient mice exhibited dramatically reduced inflammatory responses to microbial challenge and reduced phagocytosis. 3BP2 was necessary for LPS-induced activation of signaling pathways involved in macrophage function, including SRC, VAV1, p38MAPK, IKKα/β, RAC, and actin polymerization pathways. Conversely, we demonstrated that the presence of a single Sh3bp2 cherubic allele and pathogen-associated molecular pattern (PAMP) stimulation had a strong cooperative effect on macrophage activation and inflammatory responses in mice. Together, the results from our study in murine genetic models support the notion that infection may represent a driver event in the etiology of cherubism in humans and suggest limiting inflammation in affected individuals may reduce manifestation of cherubic lesions.

The loss of culturability by Escherichia coli cells in seawater depends on availability of phosphate ions and phosphate transport systems

Using strains with or without the PhoE porin or different components of the phosphate regulon, we determined that maintenance of the culturability of Escherichia coli in seawater depended significantly on the presence of structures allowing access of phosphate ions to the periplasm, then to the cytoplasm of cells. Cells totally deprived of the two main phosphate transport systems (Pit, Pst) exhibited the highest loss of culturability. Most of this effect resulted from the loss of the high-affinity Pst system, and more specifically that of the periplasmic phosphate-binding protein PhoS. Survival was enhanced in seawater supplemented with phosphate (0.5 mm), whether or not these structures were present. From an ecological point of view, it is assumed that the presence of phosphate ions, even at low concentrations, can influence the behavior of E. coli cells in seawater.

Immunoadjuvant Properties of the Rho Activating Factor CNF1 in Prophylactic and Curative Vaccination against Leishmania infantum.

There is a need to develop new effective immunoadjuvants for prophylactic or therapeutic vaccines against intracellular pathogens. The activation of Rho GTPases by bacterial cytotoxic necrotizing factor 1 (CNF1) elicits humoral protective responses against protein antigens. Here, we set out to investigate whether CNF1 activity initiates humoral immunity against co-administered parasite antigens and anti-microbial immune signaling. We report that co-administration of wild-type (WT) CNF1 with Leishmania (L.) promastigote antigens at the nasal mucosa triggered prophylactic and curative vaccine responses against this parasite. Vaccination of the mucosa with promastigote lysate antigens combined with WT CNF1 conferred protection against high inoculum L. infantum infection, which reached 82% in the spleen. Immune parameter analysis by antigen recall indicated robust T-helper (Th)1 polarization of immune memory cells, with high IL-2 and IFN-γ production combined with decreased IL-4 production. Additionally, we explored the curative effect of WT CNF1 on previously infected animals. We observed that PL combined with WT CNF1, but not the inactive C866S mutant CNF1 (mCNF1), induced a 58% decrease in the parasite burden in the spleen.

EDIN-B Promotes the Translocation of Staphylococcus aureus to the Bloodstream in the Course of Pneumonia

It is crucial to define risk factors that contribute to host invasion by Staphylococcus aureus. Here, we demonstrate that the chromosomally encoded EDIN-B isoform from S. aureus contributes to the onset of bacteremia during the course of pneumonia. Deletion of edinB in a European lineage community-acquired methicillin resistant S. aureus (CA-MRSA) strain (ST80-MRSA-IV) dramatically decreased the frequency and magnitude of bacteremia in mice suffering from pneumonia. This deletion had no effect on the bacterial burden in both blood circulation and lung tissues. Re-expression of wild-type EDIN-B, unlike the catalytically inactive mutant EDIN-R185E, restored the invasive characteristics of ST80-MRSA-IV.