Cellular Microbiology via MedWorm.com

An insider's guide to the microtubule cytoskeleton of Giardia

Cellular Microbiology — Wed, 17 Mar 2010 00:00:00 +0100

Giardia intestinalis is a zoonotic, parasitic protist with a complex microtubule cytoskeleton critical for motility, attachment, intracellular transport, cell division and transitioning between its two life cycle stages [ndash] the cyst and the trophozoite. This review focuses on the structures of the primary elements of the microtubule cytoskeleton and cytoskeletal dynamics throughout this complex giardial life cycle. The giardial cytoskeleton has both highly dynamic elements and more stable MT structures, including several novel structures like the ventral disc that change conformation via unknown mechanisms. While our knowledge of the giardial cytoskeleton is primarily cytological, the completed Giardia genome and recently developed reverse genetic tools affords an opportunity to uncove...

Mycobacterium tuberculosis protein ESAT-6 is a potent activator of the NLRP3/ASC inflammasome

Cellular Microbiology — Tue, 09 Mar 2010 00:00:00 +0100

Interleukin-1[beta] (IL-1[beta]) represents one of the most important mediators of inflammation and host responses to infection. Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, induces IL-1[beta] secretion at the site of infection, but the underlying mechanism(s) are poorly understood. In this work we show that Mtb infection of macrophages stimulates caspase-1 activity and promotes the secretion of IL-1[beta]. This stimulation requires live intracellular bacteria expressing a functional ESX-1 secretion system. ESAT-6, an ESX-1 substrate implicated in membrane damage, is both necessary and sufficient for caspase-1 activation and IL-1[beta] secretion. ESAT-6 promotes the access of other immunostimulatory agents such as AG85 into the macrophage cytosol, indicating...

Microtubule-dependent retrograde transport of bovine immunodeficiency virus

Cellular Microbiology — Fri, 05 Mar 2010 00:00:00 +0100

In this study, we found that pharmacological disruption of microtubules remarkably blocked bovine immunodeficiency virus (BIV) movement from the cell periphery to the perinuclear region, a process known as retrograde transport. A similar effect was observed by inhibiting function of the microtubule-associated motor protein dynein. By yeast two-hybrid assay, we found that the capsid protein (CA) of BIV interacted with the dynein light-chain component LC8. Immunoprecipitation and GST-pulldown assays further demonstrated an interaction between CA and LC8 in mammalian cells. In addition, our data revealed LC8 as a linker between BIV particles and microtubules. Retrograde transport of BIV was significantly inhibited by knockdown of LC8 expression. Our findings present the first evidence that in...

Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

Cellular Microbiology — Thu, 04 Mar 2010 00:00:00 +0100

The immunity-related GTPases (IRGs) constitute an interferon-induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion-driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild-type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and...

H. pylori-induced promoter hypermethylation downregulates USF1 and USF2 transcription factor gene expression

Cellular Microbiology — Tue, 23 Feb 2010 00:00:00 +0100

Helicobacter pylori infection is associated with the development of gastric adenocarcinoma. Upstream stimulatory factors USF1 and USF2 regulate the transcription of genes related to immune response, cell cycle and cell proliferation. A decrease in their expression is observed in human gastric epithelial cells infected with H. pylori, associated to a lower binding to their DNA E-box recognition site as shown by electrophoretic mobility shift assay. DNA methylation leads to gene silencing. The treatment of cells with 5'-azacytidine, an inhibitor of DNA methylation, restored the USF1 and USF2 gene expression in the presence of infection. Using promoter PCR methylation assay, a DNA hypermethylation was shown in the promoter region of USF1 and USF2 genes, in infected cells. The inhibition of US...

Subpatent infection with nucleoside transporter 1-deficient Plasmodium blood stage parasites confers sterile protection against lethal malaria in mice

Cellular Microbiology — Mon, 22 Feb 2010 00:00:00 +0100

Repeated immunizations with whole Plasmodium blood stage parasites and concomitant drug cure of infection confer protective immunity against parasite challenge in mice, monkeys and humans. Moreover, it was recently shown that infections with genetically modified rodent malaria blood stage parasites conferred sterile protection against lethal blood stage challenge. However, in these models vaccination resulted in high parasitemias and, in consequence, carries risk of vaccine-induced pathology and death. Herein, we generated a novel, completely blood stage-attenuated P. yoelii rodent malaria strain by targeted deletion of parasite nucleoside transporter 1 (NT1). Immunization of inbred and outbred mouse strains with a single low dose of Pynt1- blood stages did not induce any patent infections...

New insights into protein export in malaria parasites

Cellular Microbiology — Fri, 19 Feb 2010 00:00:00 +0100

In order to survive and promote its virulence the malaria parasite must export hundreds of its proteins beyond an encasing vacuole and membrane into the host red blood cell. In the last few years, several major advances have been made that have significantly contributed to our understanding of this export process. These include: (i) the identification of sequences that direct protein export (a signal sequence and a motif termed PEXEL), which have allowed predictions of the exportomes of Plasmodium species that are the cause of malaria, (ii) the recognition that the fate of proteins destined for export is already decided within the parasite's endoplasmic reticulum and involves the PEXEL motif being recognized and cleaved by the aspartic protease plasmepsin V and (iii) the discovery of the P...

Pupylation versus ubiquitylation: tagging for proteasome-dependent degradation

Cellular Microbiology — Thu, 18 Feb 2010 00:00:00 +0100

Prokaryotic ubiquitin-like protein (Pup) is the first identified prokaryotic protein that is functionally analogous to ubiquitin. Despite using the proteasome as the end-point for proteolysis, Pup differs from ubiquitin both biochemically and structurally. We will discuss these differences that have been highlighted by several recent studies. Finally, we will speculate on the possible interactions between the two analogous pathways in pathogen and host. (Source: Cellular Microbiology)

Cryptococcus neoformans responds to mannitol by increasing capsule size in vitro and in vivo

Cellular Microbiology — Fri, 12 Feb 2010 00:00:00 +0100

In this study, we determined the effects of mannitol and glucose on the capsule and exopolysaccharide production. Growth in mannitol significantly increased capsular volume compared with cultivation in glucose. However, cells grown in glucose concentrations higher than 62.5 mM produced more exopolysaccharide than cells grown in mannitol. The fibre lengths and glycosyl composition of capsular polysaccharide from yeast grown in mannitol was structurally different from that of yeast grown in glucose. Furthermore, mannitol treatment of mice infected intratracheally with C. neoformans resulted in fungal cells with significantly larger capsules and the mice had reduced fungal dissemination to the brain. Our results demonstrate the capacity of carbohydrate source and concentration to modify the e...

The chemokine-binding protein encoded by the poxvirus orf virus inhibits recruitment of dendritic cells to sites of skin inflammation and migration to peripheral lymph nodes

Cellular Microbiology — Thu, 11 Feb 2010 00:00:00 +0100

Orf virus (ORFV) is a zoonotic parapoxvirus that induces acute pustular skin lesions in sheep and humans. ORFV can reinfect its host and the discovery of several secreted immune modulatory factors that include a chemokine-binding protein (CBP) may explain this phenomenon. Dendritic cells (DC) are professional antigen presenting cells that induce adaptive immunity and their recruitment to sites of infection in skin and migration to peripheral lymph nodes is critically dependent on inflammatory and constitutive chemokine gradients respectively. Here we examined whether ORFV-CBP could disable these gradients using mouse models. Previously we established that ORFV-CBP bound murine inflammatory chemokines with high affinity and here we show that this binding spectrum extends to constitutive che...

HIF-1α mediates the induction of IL-8 and VEGF expression on infection with Afa/Dr diffusely adhering E. coli and promotes EMT-like behaviour

Cellular Microbiology — Wed, 10 Feb 2010 00:00:00 +0100

Microbes regulate a large panel of intracellular signalling events that can promote inflammation and/or enhance tumour progression. Indeed, it has been shown that infection of human intestinal cells with the Afa/Dr diffusely adhering E. coli C1845 strain induces expression of pro-angiogenic and pro-inflammatory genes. Here, we demonstrate that exposure of cryptic-like intestinal epithelial cells to C1845 bacteria induces HIF-1[alpha] protein levels. This effect depends on the binding of F1845 adhesin to the membrane-associated DAF receptor that initiates signalling cascades promoting translational mechanisms. Indeed, inhibition of MAPK and PI-3K decreases HIF-1[alpha] protein levels and blocks C1845-induced phosphorylation of the ribosomal S6 protein. Using RNA interference we show that ba...

Endoplasmic reticulum stress is induced and modulated by enterovirus 71

Cellular Microbiology — Fri, 05 Feb 2010 00:00:00 +0100

Picornavirus infection alters the endoplasmic reticulum (ER) membrane but it is unclear whether this induces ER stress. Infection of rhabdomyosarcoma cells with enterovirus 71 (EV71), a picornavirus, caused overexpression of the ER-resident chaperone proteins, BiP and calreticulin, and phosphorylation of eIF2[alpha], but infection with UV-inactivated virus did not, indicating that ER stress was induced by viral replication and not by viral attachment or entry. Silencing (si)RNA knockdown demonstrated that phosphorylation of eIF2[alpha] was dependent on PKR: eIF2[alpha] phosphorylation was reduced by siPKR but not by siPERK. We provided evidence showing that PERK is upstream of PKR and is thus able to negatively regulate the PKR-eIF2[alpha] pathway. Pulse-chase experiments revealed that EV7...

Secreted cysteine proteases of the carcinogenic liver fluke, Opisthorchis viverrini: regulation of cathepsin F activation by autocatalysis and trans-processing by cathepsin B

Cellular Microbiology — Tue, 02 Feb 2010 00:00:00 +0100

Opisthorchis viverrini is an important helminth pathogen of humans that is endemic in Thailand and Laos. Adult flukes reside within host bile ducts and feed on epithelial tissue and blood cells. Chronic opisthorchiasis is associated with severe hepatobiliary diseases such as cholangiocarcinoma. Here we report that adult O. viverrini secrete two major cysteine proteases: cathepsin F (Ov-CF-1) and cathepsin B1 (Ov-CB-1). Ov-CF-1 is secreted as an inactive zymogen that autocatalytically processes and activates to a mature enzyme at pH 4.5 via an intermolecular cleavage at the prosegment[ndash]mature domain junction. Ov-CB-1 is also secreted as a zymogen but, in contrast to Ov-CF-1, is fully active against peptide and macromolecular substrates despite retaining the N-terminal prosegment. The a...

N-terminal region of Mannheimia haemolytica leukotoxin serves as a mitochondrial targeting signal in mammalian cells

Cellular Microbiology — Tue, 26 Jan 2010 00:00:00 +0100

In this study, using the bioinformatics tool MitoProt II we identified an N-terminal amino acid sequence of LktA that represents a mitochondrial targeting signal (MTS). We show that expression of this sequence, as a GFP fusion protein within mammalian cells, directs GFP to mitochondria. By immunoprecipitation we demonstrate that LktA interacts with the Tom22 and Tom40 components of the translocase of the outer mitochondrial membrane (TOM), which suggests that import of this toxin into mitochondria involves a classical import pathway for endogenous proteins. We also analysed the amino acid sequences of other RTX toxins and found a MTS in the N-terminal region of Actinobacillus pleuropneumoniae ApxII and enterohaemorrhagicEscherichia coli EhxA, but not in A. pleuropneumoniae ApxI, ApxIII, Ag...

Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation

Cellular Microbiology — Tue, 26 Jan 2010 00:00:00 +0100

In this study we show that capsids from various sources encapsidate active protein kinase C[alpha], irrespective of hepatitis B virus genotype and host cell. Treatment of a virion expressing cell line with a pseudosubstrate inhibitor showed that inhibition of protein kinase C phosphorylation did not affect genome maturation but resulted in capsid accumulation and inhibited virion release to the medium. Our results imply that different protein kinases have distinct functions within the hepadnaviral life cycle. (Source: Cellular Microbiology)

Chromatin modifications: implications in the regulation of gene expression in Toxoplasma gondii

Cellular Microbiology — Tue, 26 Jan 2010 00:00:00 +0100

The apicomplexan Toxoplasma gondii completes its life cycle by successive processes of parasite differentiation that rely on a tight control of gene expression to ensure appropriate protein profiles on time. During the last 5 years, several groups have pioneered this field of investigation, suggesting that epigenetics could play an important role in the control of parasite gene expression. Histone modifications serve as an effective way to regulate gene transcription but they do not operate alone; rather, they act in concert with other putative epigenetic information carriers (histone variants, small RNAs) and DNA sequence-specific transcription factors to modulate the higher-order structure of the chromatin fibre and govern the on-time recruitment of the transcriptional machinery to speci...

CRK adaptor protein expression is required for efficient replication of avian influenza A viruses and controls JNK-mediated apoptotic responses

Cellular Microbiology — Wed, 20 Jan 2010 00:00:00 +0100

The non-structural protein 1 (A/NS1) of influenza A viruses (IAV) harbours several src-homology domain (SH) binding motifs that are required for interaction with cellular proteins. The SH3 binding motif at aa212-217 [PPLPPK] of A/NS1 was shown to be essential for binding to the cellular adaptor proteins CRK and CRKL. Both regulate diverse cellular effector pathways, including activation of the MAP-kinase JNK that in turn mediates antiviral responses to IAV infection. By studying functional consequences of A/NS1[ndash]CRK interaction we show here that A/NS1 binding to CRK contributes to suppression of the antiviral-acting JNK[ndash]ATF2 pathway. However, only IAV that encode an A/NS1-protein harbouring the CRK/CRKL SH3 binding motif PPLPPK were attenuated upon downregulation of CRKI/II and ...

Transcriptome dysregulation by anthrax lethal toxin plays a key role in induction of human endothelial cell cytotoxicity

Cellular Microbiology — Wed, 20 Jan 2010 00:00:00 +0100

We have investigated how Bacillus anthracis lethal toxin (LT) triggers caspase-3 activation and the formation of thick actin cables in human endothelial cells. By DNA array analysis we show that LT has a major impact on the cell transcriptome and we identify key host genes involved in LT cytotoxic effects. Indeed, upregulation of TRAIL and downregulation of XIAP both participate in LT-induced caspase-3 activation. LT induces a downregulation of the immediate early gene and master regulator of transcription egr1. Importantly, its re-expression in LT-intoxicated cells blocks caspase-3 activation. In parallel, we found that the formation of actin cables induced by LT occurs in the absence of direct activation of RhoA/ROCK signalling. We show that knock-down of cortactin and rhophilin-2 under ...

Proteophosphoglycan confers resistance of Leishmania major to midgut digestive enzymes induced by blood feeding in vector sand flies

Cellular Microbiology — Wed, 20 Jan 2010 00:00:00 +0100

Leishmania synthesize abundant phosphoglycan-containing molecules made up of [Gal-Man-PO4] repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis sand flies was tested using L. major knockout mutants deficient in either total phosphoglycans (lpg2- or lpg5A-/5B-) or LPG alone (lpg1-) along with their respective gene add-back controls. Our results confirm that LPG, the major cell surface molecule of Leishmania promastigotes known to mediate attachment to the vector midgut, is necessary to prevent the loss of infection during excretion of the blood meal remnants from a natural vector, P. duboscqi, but not an unnatural vector, L. longipalpis. Midgut digestive...

E. coli secreted protein F promotes EPEC invasion of intestinal epithelial cells via an SNX9-dependent mechanism

Cellular Microbiology — Wed, 20 Jan 2010 00:00:00 +0100

Enteropathogenic Escherichia coli (EPEC) infection requires the injection of effector proteins into intestinal epithelial cells (IECs) via type 3 secretion. Type 3-secreted effectors can interfere with IEC signalling pathways via specific protein[ndash]protein interactions. For example, E. coli secreted protein F (EspF) binds sorting nexin 9 (SNX9), an endocytic regulator, resulting in tubulation of the plasma membrane. Our aim was to determine the mechanism of EspF/SNX9-induced membrane tubulation. Point mutation of the SNX9 lipid binding domains or truncation of the EspF SNX9 binding domains significantly inhibited tubulation, as did inhibition of clathrin coated pit (CCP) assembly. Although characterized as non-invasive, EPEC are known to invade IECs in vitro and in vivo. Indeed, we fou...

Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus

Cellular Microbiology — Wed, 20 Jan 2010 00:00:00 +0100

Coronaviruses (CoV) are enveloped positive-strand RNA viruses that induce different membrane rearrangements in infected cells in order to efficiently replicate and assemble. The origin, the protein composition and the function of these structures are not well established. To shed further light on these structures, we have performed a time-course experiment in which the mouse hepatitis virus (MHV)-induced membrane rearrangements were examined qualitatively and quantitatively by (immuno)-electron microscopy. With our approach we were able to confirm the appearance of 6, previously reported, membranous structures during the course of a complete infection cycle. These structures include the well-characterized double-membrane vesicles (DMVs), convoluted membranes (CMs) and virions but also the ...

The innate immune molecule, NOD1, regulates direct killing of Helicobacter pylori by antimicrobial peptides

Cellular Microbiology — Wed, 20 Jan 2010 00:00:00 +0100

This study demonstrates, for the first time, the involvement of NOD1 and hBD-2 in direct killing of H. pylori bacteria by epithelial cells and confirms the importance of NOD1 in host defence mechanisms against cagPAI+H. pylori infection. (Source: Cellular Microbiology)

Malaria hemozoin modulates susceptibility of immature monocyte-derived dendritic cells to HIV-1 infection by inducing a mature-like phenotype

Cellular Microbiology — Wed, 13 Jan 2010 00:00:00 +0100

We report here that HZ promotes transmission of HIV-1 by immature monocyte-derived DCs (iMDDCs). Moreover, we noted that in the presence of HZ, iMDDCs were less permissive to productive HIV-1 infection. The HZ-dependent modulation of the interaction between iMDDCs and HIV-1 seems to be partly due to a decreased expression of CCR5 and also to the induction of a more mature phenotype as proven by microscopy and flow cytometry analyses. Therefore, exposure of iMDDCs to malaria pigments provokes their maturation rendering them more potent to trans-infect CD4+ T cells with HIV-1. (Source: Cellular Microbiology)

Reactive oxygen species induced by Streptococcus pyogenes invasion trigger apoptotic cell death in infected epithelial cells

Cellular Microbiology — Mon, 11 Jan 2010 00:00:00 +0100

Streptococcus pyogenes (group A streptococcus, GAS), one of the most common pathogens of humans, attaches and invades into human pharyngeal or skin epithelial cells. We have previously reported that induction of apoptosis is associated with GAS invasion, which induces mitochondrial dysfunction and apoptotic cell death. We demonstrate here that GAS-induced apoptosis is mediated by reactive oxygen species (ROS) production. Both the induction of apoptosis and ROS production markedly increased upon invasion of wild-type GAS strain JRS4 into HeLa cells; however, the apoptotic response was not observed in fibronectin-binding protein F1-disrupted mutant SAM1-infected cells. In Bcl-2-overexpressing HeLa cells (HBD98-2-4), the induction of apoptosis, ROS production and mitochondrial dysfunction wer...

Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuole rupture in real time

Cellular Microbiology — Mon, 11 Jan 2010 00:00:00 +0100

Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen-induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram-negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We ...

Interleukin-1 receptor phosphorylation activates Rho kinase to disrupt human gastric tight junctional claudin-4 during Helicobacter pylori infection

Cellular Microbiology — Mon, 11 Jan 2010 00:00:00 +0100

Helicobacter pylori infects more than half of the human population worldwide. In the absence of treatment, this persistent infection leads to asymptomatic gastritis, which in some cases can progress into gastric ulcers and adenocarcinomas. The host[ndash]microbial interactions that govern the clinical outcome of infection remain incompletely understood. H. pylori is known to disrupt gastric epithelial tight junctions, which may represent a significant component of disease pathogenesis. The present study demonstrates that H. pylori disrupt epithelial tight junctional claudin-4 in a Rho kinase (ROCK)-dependent manner in human gastric epithelial (HGE-20) cell monolayers, independently of the virulence factors CagA and VacA, and without altering claudin-4 transcription. In the same epithelial ...

Secretion of an acid phosphatase provides a possible mechanism to acquire host nutrients by Plasmodium falciparum

Cellular Microbiology — Mon, 11 Jan 2010 00:00:00 +0100

As an intracellular proliferating parasite, Plasmodium falciparum exploits the human host to acquire nutrients. However, nutrients such as nucleotides and cofactors are mostly phosphorylated in the host cell cytosol and thus have to be dephosphorylated in order to be taken up by the parasite. Here we report the functional characterization of a unique secreted phosphatase in P. falciparum, which is expressed throughout the developmental stages in the red blood cell. We show that this enzyme, formerly described as anchoring glideosome-associated protein 50 (GAP50), reveals a broad substrate profile with preference for di- and triphosphates at pH 5[ndash]7. Bioinformatic studies of the protein sequence identified an N-terminal signal anchor (SA) as well as a C-terminal transmembrane domain. B...

Helicobacter pylori-derived neutrophil-activating protein increases the lifespan of monocytes and neutrophils

Cellular Microbiology — Mon, 11 Jan 2010 00:00:00 +0100

In conclusion, our data reinforce the notion that HP-NAP has a pivotal role in sustaining a prolonged activation of myeloid cells. (Source: Cellular Microbiology)

Loss of Dictyostelium ATG9 results in a pleiotropic phenotype affecting growth, development, phagocytosis and clearance and replication of Legionella pneumophila

Cellular Microbiology — Mon, 11 Jan 2010 00:00:00 +0100

Infection of Dictyostelium discoideum with Legionella pneumophila resulted in a large number of differentially regulated genes among them three core autophagy genes, ATG8, ATG9 and ATG16. Macroautophagy contributes to many physiological and pathological processes and might also constitute an important mechanism in cell-autonomous immunity. For further studies we selected the highly conserved ATG9. In colocalization studies with GFP-tagged ATG9 and different organelle marker proteins we neither observed colocalization with mitochondria, the ER nor lysosomes. However, there was partial colocalization with the Golgi apparatus and many ATG9-GFP-containing vesicles localized along microtubules and accumulated around the microtubule organizing centre. ATG9-deficient cells had pleiotropic defects...

Rac1 inactivation by lethal toxin from Clostridium sordellii modifies focal adhesions upstream of actin depolymerization

Cellular Microbiology — Tue, 05 Jan 2010 00:00:00 +0100

Inactivation of different small GTPases upon their glucosylation by lethal toxin from Clostridium sordellii strain IP82 (LT-82) is already known to lead to cell rounding, adherens junction (AJ) disorganization and actin depolymerization. In the present work, we observed that LT-82 induces a rapid dephosphorylation of paxillin, a protein regulating focal adhesion (FA), independently of inactivation of paxillin kinases such as Src, Fak and Pyk2. Among the small GTPases inactivated by this toxin, including Rac, Ras, Rap and Ral, we identified Rac1, as responsible for paxillin dephosphorylation using cells overexpressing Rac1V12. Rac1 inactivation by LT-82 modifies interactions between proteins from AJ and FA complexes as shown by pull-down assays. We showed that in Triton X-100-insoluble memb...

Anthrax lethal toxin promotes dephosphorylation of TTP and formation of processing bodies

Cellular Microbiology — Thu, 31 Dec 2009 00:00:00 +0100

Anthrax lethal toxin (LeTx) is composed of protective antigen (PA) and lethal factor (LF) [ndash] PA is the receptor-binding moiety and LF is a protease that cleaves mitogen-activated protein kinase kinases (MAPKKs). LeTx subverts the immune response to Bacillus anthracis in several ways, such as downregulating interleukin-8 (IL-8) by increasing the rate of IL-8 mRNA degradation. Many transcripts are regulated through cis-acting elements that bind proteins that either impede or promote degradation. Some of these RNA-binding proteins are regulated by MAPKs and previous work has demonstrated that interfering with MAPK signalling decreases the half-life of IL-8 mRNA. Here, we have localized a segment within the IL-8 3' untranslated region responsible for LeTx-induced transcript destabilizatio...

EspM2 is a RhoA guanine nucleotide exchange factor

Cellular Microbiology — Mon, 21 Dec 2009 00:00:00 +0100

We investigated how the type III secretion system WxxxE effectors EspM2 of enterohaemorrhagic Escherichia coli, which triggers stress fibre formation, and SifA of Salmonella enterica serovar Typhimurium, which is involved in intracellular survival, modulate Rho GTPases. We identified a direct interaction between EspM2 or SifA and nucleotide-free RhoA. Nuclear Magnetic Resonance Spectroscopy revealed that EspM2 has a similar fold to SifA and the guanine nucleotide exchange factor (GEF) effector SopE. EspM2 induced nucleotide exchange in RhoA but not in Rac1 or H-Ras, while SifA induced nucleotide exchange in none of them. Mutating W70 of the WxxxE motif or L118 and I127 residues, which surround the catalytic loop, affected the stability of EspM2. Substitution of Q124, located within the cat...

Penitentiary or penthouse condo: the tuberculous granuloma from the microbe's point of view

Cellular Microbiology — Mon, 21 Dec 2009 00:00:00 +0100

Granuloma formation represents a pivotal point during human infection with Mycobacterium tuberculosis, for this structure may limit mycobacterial spread and prevent active disease, while at the same time allow for the survival and persistence of viable mycobacteria within the host. The current therapeutic regimens for treating tuberculosis disease have proven effective in developing countries. However, in countries with large populations, limited access to health care, and high incidence of HIV co-infection, tuberculosis disease continues to represent a major global health emergency. Particularly, the emergence of extensively and multi-drug-resistant forms of tuberculosis underscores the need develop new treatment strategies. Recent mechanistic studies have identified bacterial virulence m...

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite

Cellular Microbiology — Mon, 21 Dec 2009 00:00:00 +0100

In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop ...

EspM inhibits pedestal formation by enterohaemorrhagic Escherichia coli and enteropathogenic E. coli and disrupts the architecture of a polarized epithelial monolayer

Cellular Microbiology — Tue, 15 Dec 2009 00:00:00 +0100

Enterohaemorrhagic Escherichia coli and enteropathogenic E. coli are enteropathogens characterized by their ability to induce the host cell to form actin-rich structures, termed pedestals. A type III secretion system, through which the pathogens deliver effector proteins into infected host cells, is essential for their virulence and pedestal formation. Enterohaemorrhagic E. coli encodes two similar effectors, EspM1 and EspM2, which activate the RhoA signalling pathway and induce the formation of stress fibres upon infection of host cells. We confirm these observations and in addition show that EspM inhibits the formation of actin pedestals. Moreover, we show that translocation of EspM into polarized epithelial cells induces dramatic changes in the tight junction localization and in the mor...

Human Sec3 protein is a novel transcriptional and translational repressor of flavivirus

Cellular Microbiology — Thu, 10 Dec 2009 00:00:00 +0100

This study identified human Sec3 exocyst protein (hSec3p) as a novel interacting partner of WNV and DENV C protein. Mutagenesis studies showed that the SH2 domain-binding motif of hSec3p binds to the first 15 amino acids of C protein. We report for the first time that hSec3p can modulate virus production by affecting viral RNA transcription and translation through the sequestration of elongation factor 1[alpha] (EF1[alpha]). This molecular discovery shed light on the protective role of hSec3p during flavivirus infection. This study also highlighted the antagonistic mechanism adopted by flavivirus C protein that can negatively regulate the formation of hSec3p[ndash]EF1[alpha] complex by sequestering hSec3p to establish successful infection. (Source: Cellular Microbiology)

Modelling parasite dissemination: host cell subversion and immune evasion by Toxoplasma gondii

Cellular Microbiology — Tue, 08 Dec 2009 00:00:00 +0100

Protozoan parasites belong to the most widespread and devastating human pathogens. Their ability to manipulate host responses and establish infection in their hosts continues to puzzle researchers. Recent developments of experimental model systems are contributing to the discovery of new aspects of the biology of parasite dissemination. Here, we review current knowledge on strategies utilized by the apicomplexan parasite Toxoplasma gondii to disseminate and establish infection in its host. Recent findings have revealed intricate mechanisms by which this obligate intracellular protozoan sequesters cellular functions of the immune system to assure propagation. These mechanisms include the hijacking of migratory leucocytes, modulation of migratory properties of infected cells and rapid transf...

Positioning of large organelles by a membrane- associated cytoskeleton in Plasmodium sporozoites

Cellular Microbiology — Fri, 04 Dec 2009 00:00:00 +0100

Cellular organelles are usually linked to the cytoskeleton, which often provides a scaffold for organelle function. In malaria parasites, no link between the cytoskeleton and the major organelles is known. Here we show that during fast, stop-and-go motion of Plasmodium sporozoites, all organelles stay largely fixed in respect to the moving parasite. Cryogenic electron tomography reveals that the nucleus, mitochondrion, apicoplast and the microtubules of Plasmodium sporozoites are linked to the parasite pellicle via long tethering proteins. These tethers originate from the inner membrane complex and are arranged in a periodic fashion following a 32 nm repeat. The tethers pass through a subpellicular structure that encompasses the entire parasite, probably as a network of membrane-associated...

Galectin-3, a marker for vacuole lysis by invasive pathogens

Cellular Microbiology — Fri, 27 Nov 2009 00:00:00 +0100

Shigella bacteria invade macrophages and epithelial cells and following internalization lyse the phagosome and escape to the cytoplasm. Galectin-3, an abundant protein in macrophages and epithelial cells, belongs to a family of beta-galactoside-binding proteins, the galectins, with many proposed functions in immune response, development, differentiation, cancer and infection. Galectins are synthesized as cytosolic proteins and following non-classical secretion bind extracellular beta-galactosides. Here we analysed the localization of galectin-3 following entry of Shigella into the cytosol and detected a striking phenomenon. Very shortly after bacterial invasion, intracellular galectin-3 accumulated in structures in vicinity to internalized bacteria. By using immuno-electron microscopy anal...

The MAP kinase-activated protein kinase 2 (MK2) contributes to the Shiga toxin-induced inflammatory response

Cellular Microbiology — Fri, 27 Nov 2009 00:00:00 +0100

Infection with Shiga toxin (STx)-producing bacteria can progress to a toxemic, extraintestinal injury cascade known as haemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children. Mounting evidence suggests that STx activates stress response pathways in susceptible cells and has implicated the p38 mitogen-activated protein kinase (MAPK) pathway. More importantly, some of the pathology associated with HUS is believed to be a result of a STx-induced inflammatory response. From a siRNA screen of the human kinome adapted to a high-throughput format, we found that knock-down of the MAPK-activated protein kinase 2 (MK2), a downstream target of the p38 MAPK, protected against Shiga toxicity. Further characterization of the in vitro role of MK2 revealed that STx activate...

Pro-apoptotic activity of mBNIP-21 depends on its BNIP-2 and Cdc42GAP homology (BCH) domain and is enhanced by coxsackievirus B3 infection

Cellular Microbiology — Fri, 27 Nov 2009 00:00:00 +0100

Our previous study reported that mouse BNIP-21 (mBNIP-21) induces apoptosis through a mitochondria-dependent pathway. To map the functional domains of mBNIP-21, we performed mutational analyses and demonstrated that the BNIP-2 and Cdc42GAP homology (BCH) domain is required for apoptosis induction by mBNIP-21 targeting the mitochondria and inducing cytochrome c release. This pro-apoptotic activity was enhanced by coxsackievirus infection. However, deletion of the Bcl-2 homology 3 (BH3)-like domain, a well-known cell 'death domain' in proapoptotic Bcl-2 family proteins, did not affect the activity of mBNIP-21. These data were further supported by transfection of a mouse Bax (mBax) mutant, whose BH3 was replaced by the mBNIP-21 BH3-like domain. This replacement significantly reduced the pro-a...

Modulation of caspases and their non-apoptotic functions by Legionella pneumophila

Cellular Microbiology — Mon, 23 Nov 2009 00:00:00 +0100

Legionella pneumophila has become a model system to decipher the non-apoptotic functions of caspases and their role in immunity. In permissive cells, the L. pneumophila-containing vacuole evades endosomal traffic and is remodelled by the endoplasmic reticulum. Evasion of the endosomes is mediated by the Dot/Icm type IV secretion system. Upon L. pneumophila infection of genetically restrictive cells such as wild-type (WT) C57Bl/6J murine macrophages, flagellin is sensed by the NOD-like receptor Nlrc4 leading to caspase-1 activation by the inflammasome complex. Then, caspase-7 is activated downstream of the Nlrc4 inflammasome, promoting non-apoptotic functions such as L. pneumophila-containing phagosome maturation and bacterial degradation. Interestingly, caspase-3 is activated in permissive...

Interactions of Candida albicans with epithelial cells

Cellular Microbiology — Mon, 16 Nov 2009 00:00:00 +0100

The fungus, Candida albicans, interacts with epithelial cells in the human host both as a normal commensal and as an invasive pathogen. It has evolved multiple complementary mechanisms to adhere to epithelial cells. Adherent C. albicans cells can invade epithelial surfaces both by penetrating into individual epithelial cells, and by degrading interepithelial cell junctions and passing between epithelial cells. Invasion into epithelial cells is mediated by both induced endocytosis and active penetration, whereas degradation of epithelial cell junction proteins, such as E-cadherin, occurs mainly via proteolysis by secreted aspartyl proteinases. C. albicans invasion of epithelial cells results in significant epithelial cell damage, which is probably induced by lytic enzymes, such as proteases...

Recent insights into host–pathogen interactions from Dictyostelium

Cellular Microbiology — Mon, 16 Nov 2009 00:00:00 +0100

This report reviews recent insights gained for several bacterial pathogens using Dictyostelium as host. (Source: Cellular Microbiology)

Phosphothioate oligodeoxynucleotides inhibit Plasmodium sporozoite gliding motility

Cellular Microbiology — Thu, 12 Nov 2009 00:00:00 +0100

Plasmodium sporozoites, transmitted to the mammalian host through a mosquito bite, travel to the liver, where they invade hepatocytes, and develop into a form that is then able to infect red blood cells. In spite of the importance of innate immunity in controlling microbial infections, almost nothing is known about its role during the liver stage of a malaria infection. Here, we tested whether synthetic CpG phosphothioate (PS) oligodeoxynucleotides (ODNs), which bind to Toll-like receptor 9 (Tlr9), could have a protective effect on Plasmodium berghei infection in hepatocytes. Surprisingly, CpG PS-ODNs potently impair P. berghei infection in hepatoma cell lines independently of Tlr9 activation. Indeed, not only CpG but also non-CpG PS-ODNs, which do not activate Tlr9, decreased parasite inf...

Aspergillus fumigatus MedA governs adherence, host cell interactions and virulence

Cellular Microbiology — Wed, 04 Nov 2009 00:00:00 +0100

In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared with A. nidulans. The A. fumigatus[Delta]medA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, end...

In vivo biofilm composition of Aspergillus fumigatus

Cellular Microbiology — Wed, 04 Nov 2009 00:00:00 +0100

The in vivo composition of the mycelial extracellular matrix (ECM) of Aspergillus fumigatus during host invasion is reported here for the first time. A new galactosaminogalactan and the galactomannan were the major polysaccharides of the in vivo ECM. The composition of the ECM in vivo varied with the aspergillosis pathologies. (Source: Cellular Microbiology)

Bacterial-type oxygen detoxification and iron-sulfur cluster assembly in amoebal relict mitochondria

Cellular Microbiology — Tue, 03 Nov 2009 00:00:00 +0100

The assembly of vital reactive iron-sulfur (Fe-S) cofactors in eukaryotes is mediated by proteins inherited from the original mitochondrial endosymbiont. Uniquely among eukaryotes, however, Entamoeba and Mastigamoeba lack such mitochondrial-type Fe-S cluster assembly proteins and possess instead an analogous bacterial-type system acquired by lateral gene transfer. Here we demonstrate, using immunomicroscopy and biochemical methods, that beyond their predicted cytosolic distribution the bacterial-type Fe-S cluster assembly proteins NifS and NifU have been recruited to function within the relict mitochondrial organelles (mitosomes) of Entamoeba histolytica. Both Nif proteins are 10-fold more concentrated within mitosomes compared with their cytosolic distribution suggesting that active Fe-S ...

Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells

Cellular Microbiology — Mon, 02 Nov 2009 00:00:00 +0100

Gram-negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1-dependent manner to Gram-negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram-negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram-negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-[kappa]B and...

Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers

Cellular Microbiology — Mon, 02 Nov 2009 00:00:00 +0100

This study reconciles the apparent controversy regarding the nature of the HCMV assembly site and suggests that HCMV has the ability to generate a novel membrane compartment containing markers for both TGN and endosomes, or that the membranes that HCMV uses for its envelope may be vesicles in transit between the TGN and endosomes. (Source: Cellular Microbiology)

Leishmania cell surface prohibitin: role in host–parasite interaction

Cellular Microbiology — Mon, 02 Nov 2009 00:00:00 +0100

Proteins selectively upregulated in infective parasitic forms could be critical for disease pathogenesis. A mammalian prohibitin orthologue is upregulated in infective metacyclic promastigotes of Leishmania donovani, a parasite that causes visceral leishmaniasis. Leishmania donovani prohibitin shares 41% similarity with mammalian prohibitin and 95[ndash]100% within the genus. Prohibitin is concentrated at the surface of the flagellar and the aflagellar pole, the aflagellar pole being a region through which host[ndash]parasite interactions occur. Prohibitin is attached to the membrane through a GPI anchor. Overexpression of wild-type prohibitin increases protein surface density resulting in parasites with higher infectivity. However, parasites overexpressing a mutant prohibitin with an amin...

Bacterial pathogens and the autophagic response

Cellular Microbiology — Mon, 02 Nov 2009 00:00:00 +0100

The host cell recognition and removal of invading pathogens are crucial for the control of microbial infections. However, several microorganisms have developed mechanisms that allow them to survive and replicate intracellularly. Autophagy is an ubiquitous physiological pathway in eukaryotic cells, which maintains the cellular homeostasis and acts as a cell quality control mechanism to eliminate aged organelles and unnecessary structures. In addition, autophagy has an important role as a housekeeper since cells that have to get rid of invading pathogens use this pathway to assist this eradication. In this review we will summarize some strategies employed by bacterial pathogens to modulate autophagy to their own benefit and, on the other hand, the role of autophagy as a protective process of...

Selective and specific internalization of clostridial C3 ADP-ribosyltransferases into macrophages and monocytes

Cellular Microbiology — Thu, 29 Oct 2009 00:00:00 +0100

In conclusion, we identified macrophages/monocytes as target cells for clostridial C3 transferases and shed light on their selective uptake mechanism, which might contribute to understand the role of C3 transferases in pathogenesis. (Source: Cellular Microbiology)

Variable expression of surface-exposed polymorphic membrane proteins in in vitro-grown Chlamydia trachomatis

Cellular Microbiology — Wed, 28 Oct 2009 00:00:00 +0100

The hypothesized variable expression of polymorphic membrane proteins (PmpA[ndash]PmpI) in Chlamydia trachomatis-infected patients was tested by examination of the expression of each Pmp subtype in in vitro-grown C. trachomatis. A panel of monospecific polyclonal and monoclonal antibodies was used to demonstrate surface exposure of Pmps of each subtype by differential immunofluorescence (IF) with and without prior detergent permeabilization of paraformaldehyde-fixed inclusions and for selected Pmps by immunogold labelling. Although specific transcript was detected for each pmp gene late in development, IF experiments with Pmp subtype-specific antibodies reveal that a number of inclusions in a single infection do not express Pmps of a given subtype. Coexpression experiments suggest that pmp...

Caenorhabditis is a metazoan host for Legionella