Autoimmune susceptibility gene PTPN2 is required for clearance of adherent-invasive Escherichia coli by integrating bacterial uptake and lysosomal defence

ObjectivesAlterations in the intestinal microbiota are linked with a wide range of autoimmune and inflammatory conditions, including inflammatory bowel diseases (IBD), where pathobionts penetrate the intestinal barrier and promote inflammatory reactions. In patients with IBD, the ability of intestinal macrophages to efficiently clear invading pathogens is compromised resulting in increased bacterial translocation and excessive immune reactions. Here, we investigated how an IBD-associated loss-of-function variant in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene, or loss of PTPN2 expression affected the ability of macrophages to respond to invading bacteria.DesignIBD patient-derived macrophages with wild-type (WT) PTPN2 or carrying the IBD-associated PTPN2 SNP, peritoneal macrophages from WT and constitutive PTPN2-knockout mice, as well as mice specifically lacking PTPN2 in macrophages were infected with non-invasive K12 Escherichia coli, the human adherent-invasive E. coli (AIEC) LF82, or a novel mouse AIEC (mAIEC) strain.ResultsLoss of PTPN2 severely compromises the ability of macrophages to clear invading bacteria. Specifically, loss of functional PTPN2 promoted pathobiont invasion/uptake into macrophages and intracellular survival/proliferation by three distinct mechanisms: Increased bacterial uptake was mediated by enhanced expression of carcinoembryonic antigen cellular adhesion molecule (CEACAM)1 and CEACAM6 in PTPN2-deficient cells, while reduced bacterial clearance resulted from defects in autophagy coupled with compromised lysosomal acidification. In vivo, mice lacking PTPN2 in macrophages were more susceptible to mAIEC infection and mAIEC-induced disease.ConclusionsOur findings reveal a tripartite regulatory mechanism by which PTPN2 preserves macrophage antibacterial function, thus crucially contributing to host defence against invading bacteria.

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Bactericidal Activities of Meropenem and Ertapenem against Extended-Spectrum-β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae in a Neutropenic Mouse Thigh Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00752-06 ◽

2007 ◽

Vol 51 (4) ◽

pp. 1481-1486 ◽

Cited By ~ 34

Author(s):

C. Andrew DeRyke ◽

Mary Anne Banevicius ◽

Hong Wei Fan ◽

David P. Nicolau

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Infection Model ◽

Bacterial Reduction ◽

Efficacy Analysis ◽

Percent Time ◽

Extended Spectrum ◽

Wide Range

ABSTRACT The purpose of this study was to examine the in vivo efficacies of meropenem and ertapenem against extended-spectrum-β-lactamase (ESBL)-producing isolates with a wide range of MICs. Human-simulated dosing regimens in mice were designed to approximate the free drug percent time above the MIC (fT>MIC) observed for humans following meropenem at 1 g every 8 h and ertapenem at 1 g every 24 h. An in vivo neutropenic mouse thigh infection model was used to examine the bactericidal effects against 31 clinical ESBL Escherichia coli and Klebsiella pneumoniae isolates and 2 non-ESBL isolates included for comparison at a standard 105 inoculum. Three isolates were examined at a high 107 inoculum as well. Meropenem displayed greater in vitro potency, with a median MIC (range) (μg/ml) of 0.125 (0.03 to 32), than did ertapenem, with 0.5 (0.012 to 128). Seven of the 31 ESBL isolates were removed from the efficacy analysis due to their inability to establish infection in the mouse model. When MICs were ≤1.5 μg/ml for ertapenem (≤0.5 μg/ml for meropenem), similar reductions in CFU (≈ 2-log kill) were observed for both ertapenem (fT>MIC ≥ 23%) and meropenem (fT>MIC ≥ 75%). Ertapenem showed bacterial regrowth for seven of eight isolates, with MICs of ≥2 μg/ml (fT>MIC ≤ 20%), while meropenem displayed antibacterial potency that varied from a static effect to a 1-log bacterial reduction in these isolates (fT>MIC = 30 to 65%). At a 107 inoculum, both agents eradicated bacteria due to adequate exposures (fT>MIC = 20 to 45%). Due to low MICs, no difference in bacterial kill was noted for the majority of ESBL isolates tested. However, for isolates with raised ertapenem MICs of ≥2 μg/ml, meropenem displayed sustained efficacy due to its greater in vitro potency and higher resultant fT>MIC.

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A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression

Applied and Environmental Microbiology ◽

10.1128/aem.00509-20 ◽

2020 ◽

Vol 86 (24) ◽

Author(s):

Erin M. Nawrocki ◽

Hillary M. Mosso ◽

Edward G. Dudley

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Microbial Interactions ◽

Severe Illness ◽

Content Type ◽

E Coli ◽

Wide Range ◽

Lambdoid Prophage

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo. Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

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The PDZ Binding Motif of Human Papillomavirus Type 16 E6 Induces PTPN13 Loss, Which Allows Anchorage-Independent Growth and Synergizes with Ras for Invasive Growth

Journal of Virology ◽

10.1128/jvi.02188-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2493-2500 ◽

Cited By ~ 85

Author(s):

William C. Spanos ◽

Andrew Hoover ◽

George F. Harris ◽

Shu Wu ◽

Guinevere L. Strand ◽

...

Keyword(s):

Human Papillomavirus ◽

Tyrosine Phosphatase ◽

Binding Motif ◽

Ras Signaling ◽

Invasive Growth ◽

Dependent Manner ◽

Loss Of Function ◽

Hpv16 E6 ◽

Anchorage Independent Growth

ABSTRACT The human papillomavirus (HPV) oncogene E6 has been shown to perform multiple functions (p53 degradation, telomerase activation, etc.) that play a role in oncogenic transformation. Beyond known E6 functions, an undefined mechanism that allows cellular invasion requires the E6 PDZ binding motif (PDZBM). Here, we show that HPV type 16 (HPV16) E6 interacts with and induces loss of a protein tyrosine phosphatase (PTPN13) in a PDZBM-dependent manner. PTPN13 loss induced either by the presence of E6 or by a short hairpin RNA strategy allows for anchorage-independent growth (AIG) and synergy with a known oncogene, Rasv12, resulting in invasive growth in vivo. Restoring PTPN13 expression reverses AIG in cells lacking PTPN13. A genomic analysis of colorectal carcinoma has identified an association between PTPN13 loss-of-function mutations and aberrant Ras signaling. Our findings support this correlation and provide methods for further evaluation of the mechanisms by which PTPN13 loss/Ras expression leads to invasive growth, the results of which will be important for treatment of HPV-related and non-HPV cancer.

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An in vivo genetic screen in Drosophila identifies the orthologue of human cancer/testis gene SPO11 among a network of targets to inhibit lethal(3)malignant brain tumour growth

Open Biology ◽

10.1098/rsob.170156 ◽

2017 ◽

Vol 7 (8) ◽

pp. 170156 ◽

Cited By ~ 5

Author(s):

Fabrizio Rossi ◽

Cristina Molnar ◽

Kazuya Hashiyama ◽

Jan P. Heinen ◽

Judit Pampalona ◽

...

Keyword(s):

Brain Tumour ◽

Target Genes ◽

Human Cancer ◽

Loss Of Function ◽

Malignant Growth ◽

Malignant Brain Tumour ◽

Rnai Technology ◽

Wide Range ◽

Cancer Testis

Using transgenic RNAi technology, we have screened over 4000 genes to identify targets to inhibit malignant growth caused by the loss of function of lethal(3)malignant brain tumour in Drosophila in vivo . We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt tumours hence showing that, rather counterintuitively, tumour-linked overexpression is not a good predictor of functional requirement. Moreover, we have found that most of the genes upregulated in mbt tumours remain overexpressed in tumour-suppressed double-mutant conditions, hence revealing that most of the tumour transcriptome signature is not necessarily correlated with malignant growth. One of the identified target genes is meiotic W68 ( mei-W68 ), the Drosophila orthologue of the human cancer/testis gene Sporulation-specific protein 11 ( SPO11 ), the enzyme that catalyses the formation of meiotic double-strand breaks. We show that Drosophila mei-W68/SPO11 drives oncogenesis by causing DNA damage in a somatic tissue, hence providing the first instance in which a SPO11 orthologue is unequivocally shown to have a pro-tumoural role. Altogether, the results from this screen point to the possibility of investigating the function of human cancer relevant genes in a tractable experimental model organism like Drosophila.

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Functions of the Ectodomain and Cytoplasmic Tyrosine Phosphatase Domains of Receptor Protein Tyrosine Phosphatase Dlar In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.23.19.6909-6921.2003 ◽

2003 ◽

Vol 23 (19) ◽

pp. 6909-6921 ◽

Cited By ~ 23

Author(s):

Neil X. Krueger ◽

R. Sreekantha Reddy ◽

Karl Johnson ◽

Jack Bateman ◽

Nancy Kaufmann ◽

...

Keyword(s):

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

P Element ◽

Receptor Protein ◽

Loss Of Function ◽

Protein Tyrosine ◽

Receptor Protein Tyrosine Phosphatase ◽

Catalytically Active

ABSTRACT The receptor protein tyrosine phosphatase (PTPase) Dlar has an ectodomain consisting of three immunoglobulin (Ig)-like domains and nine fibronectin type III (FnIII) repeats and a cytoplasmic domain consisting of two PTPase domains, membrane-proximal PTP-D1 and C-terminal PTP-D2. A series of mutant Dlar transgenes were introduced into the Drosophila genome via P-element transformation and were then assayed for their capacity to rescue phenotypes caused by homozygous loss-of-function genotypes. The Ig-like domains, but not the FnIII domains, are essential for survival. Conversely, the FnIII domains, but not the Ig-like domains, are required during oogenesis, suggesting that different domains of the Dlar ectodomain are involved in distinct functions during Drosophila development. All detectable PTPase activity maps to PTP-D1 in vitro. The catalytically inactive mutants of Dlar were able to rescue Dlar −/− lethality nearly as efficiently as wild-type Dlar transgenes, while this ability was impaired in the PTP-D2 deletion mutants DlarΔPTP-D2 and Dlarbypass . Dlar-C1929S, in which PTP-D2 has been inactivated, increases the frequency of bypass phenotype observed in Dlar −/− genotypes, but only if PTP-D1 is catalytically active in the transgene. These results indicate multiple roles for PTP-D2, perhaps by acting as a docking domain for downstream elements and as a regulator of PTP-D1.

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Genome-Wide Transposon Mutagenesis Reveals a Role for pO157 Genes in Biofilm Development in Escherichia coli O157:H7 EDL933

Infection and Immunity ◽

10.1128/iai.00156-10 ◽

2010 ◽

Vol 78 (6) ◽

pp. 2377-2384 ◽

Cited By ~ 49

Author(s):

Supraja Puttamreddy ◽

Nancy A. Cornick ◽

F. Chris Minion

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Microtiter Plate ◽

Virulence Plasmid ◽

Microtiter Plate Assay ◽

Severe Diarrhea ◽

Wide Range ◽

Intergenic Regions ◽

Biofilm Development

ABSTRACT Enterohemorrhagic Escherichia coli O157:H7, a world-wide human food-borne pathogen, causes mild to severe diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The ability of this pathogen to persist in the environment contributes to its dissemination to a wide range of foods and food processing surfaces. Biofilms are thought to be involved in persistence, but the process of biofilm formation is complex and poorly understood in E. coli O157:H7. To better understand the genetics of this process, a mini-Tn5 transposon insertion library was constructed in strain EDL933 and screened for biofilm-negative mutants using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm negative, and transposon insertions were located in 51 distinct genes/intergenic regions that must be involved either directly or indirectly in biofilm formation. All of the 51 biofilm-negative mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Thirty-six genes were unique to this study, including genes on the virulence plasmid pO157. The type V secreted autotransporter serine protease EspP and the enterohemolysin translocator EhxD were found to be directly involved in biofilm formation. In addition, EhxD and EspP were also important for adherence to T84 intestinal epithelial cells, suggesting a role for these genes in tissue interactions in vivo.

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Dendritic cell expression of the transcription factor T-bet regulates mast cell progenitor homing to mucosal tissue

Journal of Experimental Medicine ◽

10.1084/jem.20060626 ◽

2007 ◽

Vol 204 (2) ◽

pp. 431-439 ◽

Cited By ~ 34

Author(s):

Pilar Alcaide ◽

Tatiana G. Jones ◽

Graham M. Lord ◽

Laurie H. Glimcher ◽

Jenny Hallgren ◽

...

Keyword(s):

Transcription Factor ◽

Mast Cell ◽

Adhesion Molecule ◽

Trichinella Spiralis ◽

Cellular Adhesion ◽

Cell Trafficking ◽

Peripheral Tissues ◽

Cellular Adhesion Molecule

The transcription factor T-bet was identified in CD4+ T cells, and it controls interferon γ production and T helper type 1 cell differentiation. T-bet is expressed in certain other leukocytes, and we recently showed (Lord, G.M., R.M. Rao, H. Choe, B.M. Sullivan, A.H. Lichtman, F.W. Luscinskas, and L.H. Glimcher. 2005. Blood. 106:3432–3439) that it regulates T cell trafficking. We examined whether T-bet influences homing of mast cell progenitors (MCp) to peripheral tissues. Surprisingly, we found that MCp homing to the lung or small intestine in T-bet−/− mice is reduced. This is reproduced in adhesion studies using bone marrow–derived MCs (BMMCs) from T-bet−/− mice, which showed diminished adhesion to mucosal addresin cellular adhesion molecule–1 (MAdCAM-1) and vascular cell adhesion molecule–1 (VCAM-1), endothelial ligands required for MCp intestinal homing. MCp, their precursors, and BMMCs do not express T-bet, suggesting that T-bet plays an indirect role in homing. However, adoptive transfer experiments revealed that T-bet expression by BM cells is required for MCp homing to the intestine. Furthermore, transfer of WT BM-derived dendritic cells (DCs) to T-bet−/− mice restores normal MCp intestinal homing in vivo and MCp adhesion to MAdCAM-1 and VCAM-1 in vitro. Nonetheless, T-bet−/− mice respond vigorously to intestinal infection with Trichinella spiralis, eliminating a role for T-bet in MC recruitment to sites of infection and their activation and function. Therefore, remarkably, T-bet expression by DCs indirectly controls MCp homing to mucosal tissues.

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ICAM-directed vascular immunotargeting of antithrombotic agents to the endothelial luminal surface

Blood ◽

10.1182/blood-2002-09-2853 ◽

2003 ◽

Vol 101 (10) ◽

pp. 3977-3984 ◽

Cited By ~ 69

Author(s):

Juan-Carlos Murciano ◽

Silvia Muro ◽

Lauren Koniaris ◽

Melpo Christofidou-Solomidou ◽

David W. Harshaw ◽

...

Keyword(s):

Drug Delivery ◽

Plasminogen Activator ◽

Cellular Adhesion ◽

Tissue Type ◽

Luminal Surface ◽

Pulmonary Endothelium ◽

Type Plasminogen Activator ◽

Cellular Adhesion Molecule ◽

Rats And Mice

Abstract Drug targeting to a highly expressed, noninternalizable determinant up-regulated on the perturbed endothelium may help to manage inflammation and thrombosis. We tested whether inter-cellular adhesion molecule-1 (ICAM-1) targeting is suitable to deliver antithrombotic drugs to the pulmonary vascular lumen. ICAM-1 antibodies bind to the surface of endothelial cells in culture, in perfused lungs, and in vivo. Proinflammatory cytokines enhance anti-ICAM binding to the endothelium without inducing internalization. 125I-labeled anti-ICAM and a reporter enzyme (β-Gal) conjugated to anti-ICAM bind to endothelium and accumulate in the lungs after intravenous administration in rats and mice. Anti-ICAM is seen to localize predominantly on the luminal surface of the pulmonary endothelium by electron microscopy. We studied the pharmacological effect of ICAM-directed targeting of tissue-type plasminogen activator (tPA). Anti-ICAM/tPA, but not control IgG/tPA, conjugate accumulates in the rat lungs, where it exerts plasminogen activator activity and dissolves fibrin microemboli. Therefore, ICAM may serve as a target for drug delivery to endothelium, for example, for pulmonary thromboprophylaxis. Enhanced drug delivery to sites of inflammation and the potential anti-inflammatory effect of blocking ICAM-1 may enhance the benefit of this targeting strategy.

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Proximal binding pocket Arg717 substitutions in Escherichia coli AcrB cause clinically relevant divergencies in resistance profiles

10.1101/2021.12.16.473095 ◽

2021 ◽

Author(s):

Martijn Zwama ◽

Kunihiko Nishino

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Fold Increase ◽

Inhibition Zone ◽

Binding Pocket ◽

Loss Of Function ◽

Gain Of Function ◽

Multidrug Efflux Pumps ◽

Wide Range ◽

Resistance Nodulation Division

Multidrug resistance (MDR) in bacteria can be caused by the over-expression of multidrug efflux pumps belonging to the Resistance-Nodulation-Division (RND) superfamily of proteins. These intrinsic or acquired pumps can export a wide range of antibiotics. Recently, amino acid substitutions within these pumps have been observed in resistant clinical strains. Among others, two of these worrying gain-of-function mutations are R717L and R717Q in the proximal binding pocket of efflux pump AcrB (AcrB-Sa) found in azithromycin-resistant Salmonella enterica spp. We investigated the ramifications of these (and other) mutations in phylogenetically closely related AcrB from Escherichia coli (AcrB-Ec). We found that AcrB-Ec harboring Arg717 substitutions were significantly more effective in exporting all tested macrolides, with an up to 8-fold increase in the minimum inhibitory concentration (MIC) values (from 16 to 128 µg/mL for azithromycin). Interestingly, gain-of-function was also seen for fluoroquinolones (2-fold higher MICs), while there was a consistent loss-of-function for the export of novobiocin and β-lactam cloxacillin (2-fold lower MICs), pointing to a protein adaptation, which simultaneously partly compromised the efflux ability of other compounds with different molecular properties. Disk diffusion susceptibility testing corroborated the findings, as the R717Q and R717L mutant strains had significantly smaller inhibition zones for macrolides and fluoroquinolones and a larger inhibition zone for novobiocin, compared to the wild type. The spread and independent emergences of these potent efflux pump mutations highlight the necessity of control of, and adjustments to, treatments with antibiotics and the need for novel antibiotics and efflux pump inhibitors.

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NFAT Signaling in Osteoblasts Regulates the Hematopoietic Niche in the Bone Microenvironment

Clinical and Developmental Immunology ◽

10.1155/2013/107321 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 8

Author(s):

Cheryl L. Sesler ◽

Majd Zayzafoon

Keyword(s):

B Cells ◽

Cellular Adhesion ◽

Dominant Negative ◽

Mineral Apposition Rate ◽

B Lymphopoiesis ◽

Bone Microenvironment ◽

Activated T Cells ◽

Trabecular Thickness ◽

Cellular Adhesion Molecule

Osteoblasts support hematopoietic cell development, including B lymphopoiesis. We have previously shown that the nuclear factor of activated T cells (NFAT) negatively regulates osteoblast differentiation and bone formation. Interestingly, in smooth muscle, NFAT has been shown to regulate the expression of vascular cellular adhesion molecule-1 (VCAM-1), a mediator of cell adhesion and signaling during leukocyte development. To examine whether NFAT signaling in osteoblasts regulates hematopoietic developmentin vivo, we generated a mouse model expressing dominant-negative NFAT driven by the 2.3 kb fragment of the collagen-αI promoter to disrupt NFAT activity in osteoblasts (dnNFATOB). Bone histomorphometry showed that dnNFATOBmice have significant increases in bone volume (44%) and mineral apposition rate (131%) and decreased trabecular thickness (18%). In the bone microenvironment, dnNFATOBmice displayed a significant increase (87%) in Lineage−cKit+Sca-1+(LSK) cells and significant decreases in B220+CD19−IgM−pre-pro-B cells (41%) and B220+CD19+IgM+immature B cells (40%). Concurrent with these findings, LSK cell differentiation into B220+cells was inhibited when cocultured on differentiated primary osteoblasts harvested from dnNFATOBmice. Gene expression and protein levels of VCAM-1 in osteoblasts decreased in dnNFATOBmice compared to controls. These data suggest that osteoblast-specific NFAT activity mediates early B lymphopoiesis, possibly by regulating VCAM-1 expression on osteoblasts.

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