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In Vivo and In Vitro Studies of Cytosolic Phospholipase A2 Expression in Helicobacter pylori Infection

Nardone, Gerardo; Holicky, Eileen L.; Uhl, James R.; Sabatino, Lina; Staibano, Stefania; Rocco, Alba; Colantuoni, Vittorio; Manzo, Barbara A.; Romano, Marco; Budillon, Gabriele; Cockerill, Franklin R.; Miller, Laurence J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/2001 EN
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Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2 activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H. pylori infection. In gastric biopsies from 10 H. pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H. pylori-negative gastritis patients. To clarify whether H. pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with different H. pylori strains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover...

An Essential Role of the Enhancer for Murine Cytomegalovirus In Vivo Growth and Pathogenesis

Ghazal, Peter; Messerle, Martin; Osborn, Kent; Angulo, Ana
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2003 EN
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The transcription of cytomegalovirus (CMV) immediate-early (IE) genes is regulated by a large and complex enhancer containing an array of binding sites for a variety of cellular transcription factors. Previously, using bacterial artificial chromosome recombinants of the virus genome, it was reported that the enhancer region of murine CMV (MCMV) is dispensable but performs a key function for viral multiplication (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502-8509, 1998). In the present study, we defined, through the reconstitution of infectious enhancerless MCMVs, the growth requirement for the enhancer in tissue culture and explored its significance for steering a productive infection in vivo. A comparison of cis and trans complementation systems for infection of enhancerless virus in permissive fibroblasts revealed a multiplicity-dependent growth phenotype that is severely compromised in the rate of infectious-virus multiplication. The in vivo impact of viruses that have an amputated enhancer was investigated in an extremely sensitive model of MCMV infection, the SCID mouse. Histological examination of spleens, livers, lungs, and salivary glands from animals infected with enhancer-deficient MCMV demonstrated an absence of tissue damage associated with CMV infection. The lack of pathogenic lesions correlated with a defect in replication competence. Enhancerless viruses were not detectable in major target organs harvested from SCID mice. The pathogenesis and growth defect reverted upon restoration of the enhancer. Markedly...

Interleukin-10 and Pathogenesis of Murine Ocular Toxoplasmosis

Lu, Fangli; Huang, Shiguang; Kasper, Lloyd H.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2003 EN
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To understand the role of interleukin-10 (IL-10) in ocular toxoplasmosis, we compared C57BL/6 (B6) and BALB/c background mice lacking a functional IL-10 gene (IL-10−/−) and B6 transgenic mice expressing IL-10 under the control of the IL-2 promoter. Increased cellular infiltration and necrosis were observed in the eye tissue of IL-10−/− mice of both the B6 and BALB/c backgrounds with associated changes in the levels of cytokines in serum. In contrast, there was no evidence of necrosis in the eye tissue from IL-10 transgenic mice following parasite exposure. Our results demonstrate that IL-10 is important in the regulation of inflammation during acute ocular toxoplasmosis.

Differences in Chlamydia trachomatis Serovar E Growth Rate in Polarized Endometrial and Endocervical Epithelial Cells Grown in Three-Dimensional Culture▿

Guseva, Natalia V.; Dessus-Babus, Sophie; Moore, Cheryl G.; Whittimore, Judy D.; Wyrick, Priscilla B.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
EN
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In vitro studies of obligate intracellular chlamydia biology and pathogenesis are highly dependent on the use of experimental models and growth conditions that mimic the mucosal architecture and environment these pathogens encounter during natural infections. In this study, the growth of Chlamydia trachomatis genital serovar E was monitored in mouse fibroblast McCoy cells and compared to more relevant host human epithelial endometrium-derived HEC-1B and cervix-derived HeLa cells, seeded and polarized on collagen-coated microcarrier beads, using a three-dimensional culture system. Microscopy analysis of these cell lines prior to infection revealed morphological differences reminiscent of their in vivo architecture. Upon infection, early chlamydial inclusion distribution was uniform in McCoy cells but patchy in both epithelial cell lines. Although no difference in chlamydial attachment to or entry into the two genital epithelial cell lines was noted, active bacterial genome replication and transcription, as well as initial transformation of elementary bodies to reticulate bodies, were detected earlier in HEC-1B than in HeLa cells, suggesting a faster growth, which led to higher progeny counts and titers in HEC-1B cells upon completion of the developmental cycle. Chlamydial development in the less relevant McCoy cells was very similar to that in HeLa cells...

Genome-Wide Analysis of Cellular Response to Bacterial Genotoxin CdtB in Yeast▿ †

Kitagawa, Takao; Hoshida, Hisashi; Akada, Rinji
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
EN
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The cytolethal distending toxins (CDTs) are secreted virulence proteins produced by several bacterial pathogens, and the subunit CdtB has the ability to create DNA lesions, primarily DNA single-strand breaks (SSBs) in vitro, and cause cell cycle arrest, cellular distension, and cell death in both mammalian and yeast cells. To elucidate the components of the mechanisms underlying the response to CdtB-induced DNA lesions, a CdtB expression plasmid was transformed into a series of diploid yeast strains harboring deletions in 4,708 nonessential genes. A total of 4,706 of these clones were successfully transformed, which we have now designated as a systematic transformation array (STA), and were subsequently screened. We identified 61 sensitive strains from the STA whose deleted genes can be categorized into a number of groups, including DNA metabolism, chromosome segregation, vesicular traffic, RNA catabolism, protein translation, morphogenesis, and nuclear transport, as well as one unknown open reading frame. However, only 28 of these strains were found to be sensitive to HO endonuclease, which is known to create a DNA double-strand break (DSB), suggesting that CdtB-induced DNA lesion is not similar to the direct DSB. Amazingly, CdtB expression elicits severe growth defects in haploid yeast cells...

Histophilus somni IbpA DR2/Fic in Virulence and Immunoprotection at the Natural Host Alveolar Epithelial Barrier▿

Zekarias, Bereket; Mattoo, Seema; Worby, Carolyn; Lehmann, Jason; Rosenbusch, Ricardo F.; Corbeil, Lynette B.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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Newly recognized Fic family virulence proteins may be important in many bacterial pathogens. To relate cellular mechanisms to pathogenesis and immune protection, we studied the cytotoxicity of the Histophilus somni immunoglobulin-binding protein A (IbpA) direct repeat 2 Fic domain (DR2/Fic) for natural host target cells. Live virulent IbpA-producing H. somni strain 2336, a cell-free culture supernatant (CCS) of this strain, or recombinant DR2/Fic (rDR2/Fic) caused dramatic retraction and rounding of bovine alveolar type 2 (BAT2) epithelial cells. IbpA-deficient H. somni strain 129Pt and a Fic motif His298Ala mutant rDR2/Fic protein were not cytotoxic. The cellular mechanism of DR2/Fic cytotoxicity was demonstrated by incubation of BAT2 cell lysates with strain 2336 CCS or rDR2/Fic in the presence of [α-32P]ATP, which resulted in adenylylation of Rho GTPases and cytoskeletal disruption. Since IbpA is not secreted by type III or type IV secretion systems, we determined whether DR2/Fic entered the host cytoplasm to access its Rho GTPase targets. Although H. somni did not invade BAT2 cells, DR2/Fic was internalized by cells treated with H. somni, CCS, or the rDR2/Fic protein, as shown by confocal immunomicroscopy. Transwell bacterial migration assays showed that large numbers of strain 2336 bacteria migrated between retracted BAT2 cells...

ArcA-Regulated Glycosyltransferase Lic2B Promotes Complement Evasion and Pathogenesis of Nontypeable Haemophilus influenzae ▿

Wong, Sandy M. S.; St. Michael, Frank; Cox, Andrew; Ram, Sanjay; Akerley, Brian J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2011 EN
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Signaling mechanisms used by Haemophilus influenzae to adapt to conditions it encounters during stages of infection and pathogenesis are not well understood. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and contributes to resistance to bactericidal effects of serum and to bloodstream infection by H. influenzae. We show that ArcA of nontypeable H. influenzae (NTHI) activates expression of a glycosyltransferase gene, lic2B. Structural comparison of the lipooligosaccharide (LOS) of a lic2B mutant to that of the wild-type strain NT127 revealed that lic2B is required for addition of a galactose residue to the LOS outer core. The lic2B gene was crucial for optimal survival of NTHI in a mouse model of bacteremia and for evasion of serum complement. The results demonstrate that ArcA, which controls cellular metabolism in response to environmental reduction and oxidation (redox) conditions, also coordinately controls genes that are critical for immune evasion, providing evidence that NTHI integrates redox signals to regulate specific countermeasures against host defense.

Defining the Roles of Human Carcinoembryonic Antigen-Related Cellular Adhesion Molecules during Neutrophil Responses to Neisseria gonorrhoeae

Sarantis, Helen; Gray-Owen, Scott D.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2012 EN
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580.77137%
Symptomatic infection of humans with Neisseria gonorrhoeae is characterized by a neutrophil-rich cervical or urethral exudate, suggesting that neutrophils are important both for the clearance of these bacteria and for the pathogenesis of gonorrhea. Neisseria interacts with neutrophils through ligation of human carcinoembryonic antigen related-cellular adhesion molecules (CEACAMs) by their surface-expressed Opa proteins, resulting in bacterial binding, engulfment, and neutrophil activation. Multiple CEACAMs are expressed by human neutrophils, and yet their coexpression has precluded understanding of the relative contribution of each CEACAM to functional responses of neutrophils during neisserial infection. In this work, we directly address the role of each CEACAM during infection by introducing individual human CEACAMs into a differentiated murine MPRO cell line-derived neutrophil model. Murine neutrophils cannot bind the human-restricted Neisseria; however, we show that introducing any of the Opa-binding CEACAMs of human neutrophils (CEACAM1, CEACAM3, and CEACAM6) allows binding and entry of Neisseria into murine neutrophils. While CEACAM1- and CEACAM6-expressing neutrophils bind more bacteria, neisserial uptake via these two receptors unexpectedly proceeds without appreciable neutrophil activation. In stark contrast...

Receptor-Dependent and -Independent Immunomodulatory Effects of Phenol-Soluble Modulin Peptides from Staphylococcus aureus on Human Neutrophils Are Abrogated through Peptide Inactivation by Reactive Oxygen Species

Forsman, Huamei; Christenson, Karin; Bylund, Johan; Dahlgren, Claes
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2012 EN
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670.1197%
The virulence and pathogenesis mechanisms of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains depend on a newly described group of phenol-soluble modulin (PSM) peptides (the PSMα peptides) with cytolytic activity. These toxins are α-helical peptides with a formyl group at the N terminus, and they activate neutrophils through formyl peptide receptor 2 (FPR2), a function closely correlated to the capacity of staphylococcal species to cause invasive infections. The effects of two synthetic PSMα peptides were investigated, and we show that they utilize FPR2 and promote neutrophils to produce reactive oxygen species (ROS) which in turn trigger inactivation of the peptides. Independently of FPR2, the PSMα peptides also downregulate the neutrophil response to other stimuli and exert a cytolytic effect to which apoptotic neutrophils are more sensitive than viable cells. The novel immunomodulatory functions of the PSMα peptides were sensitive to ROS generated by the neutrophil myeloperoxidase (MPO)-H2O2 system, suggesting a role for this enzyme system in counteracting bacterial virulence.

Vaccine-Elicited CD8+ T Cells Protect against Respiratory Syncytial Virus Strain A2-Line19F-Induced Pathogenesis in BALB/c Mice

Lee, Sujin; Stokes, Kate L.; Currier, Michael G.; Sakamoto, Kaori; Lukacs, Nicholas W.; Celis, Esteban; Moore, Martin L.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2012 EN
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580.63418%
CD8+ T cells may contribute to vaccines for respiratory syncytial virus (RSV). Compared to CD8+ T cells responding to RSV infection, vaccine-elicited anti-RSV CD8+ T cells are less well defined. We used a peptide vaccine to test the hypothesis that vaccine-elicited RSV-specific CD8+ T cells are protective against RSV pathogenesis. BALB/c mice were treated with a mixture (previously termed TriVax) of an M282-90 peptide representing an immunodominant CD8 epitope, the Toll-like receptor (TLR) agonist poly(I·C), and a costimulatory anti-CD40 antibody. TriVax vaccination induced potent effector anti-RSV CD8+ cytotoxic T lymphocytes (CTL). Mice were challenged with RSV strain A2-line19F, a model of RSV pathogenesis leading to airway mucin expression. Mice were protected against RSV infection and against RSV-induced airway mucin expression and cellular lung inflammation when challenged 6 days after vaccination. Compared to A2-line19F infection alone, TriVax vaccination followed by challenge resulted in effector CD8+ T cells with greater cytokine expression and the more rapid appearance of RSV-specific CD8+ T cells in the lung. When challenged 42 days after TriVax vaccination, memory CD8+ T cells were elicited with RSV-specific tetramer responses equivalent to TriVax-induced effector CD8+ T cells. These memory CD8+ T cells had lower cytokine expression than effector CD8+ T cells...

Congenic Strains of the Filamentous Form of Cryptococcus neoformans for Studies of Fungal Morphogenesis and Virulence

Zhai, Bing; Zhu, Pinkuan; Foyle, Dylan; Upadhyay, Srijana; Idnurm, Alexander; Lin, Xiaorong
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2013 EN
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663.53805%
Cryptococcus neoformans is an unconventional dimorphic fungus that can grow either as a yeast or in a filamentous form. To facilitate investigation of genetic factors important for its morphogenesis and pathogenicity, congenic a and α strains for a filamentous form were constructed. XL280 (α) was selected as the background strain because of its robust ability to undergo the morphological transition from yeast to the filamentous form. The MATa allele from a sequenced strain JEC20 was introgressed into the XL280 background to generate the congenic a and α pair strains. The resulting congenic strains were then used to test the impact of mating type on virulence. In both the inhalation and the intravenous infection models of murine cryptococcosis, the congenic a and α strains displayed comparable levels of high virulence. The a-α coinfections displayed equivalent virulence to the individual a or α infections in both animal models. Further analyses of the mating type distribution in a-α coinfected mice suggested no influence of a-α interactions on cryptococcal neurotropism, irrespective of the route of inoculation. Furthermore, deletion or overexpression of a known transcription factor, Znf2, in XL280 abolished or enhanced filamentation and biofilm formation...

Differential Roles of ASK1 and TAK1 in Helicobacter pylori-Induced Cellular Responses

Hayakawa, Yoku; Hirata, Yoshihiro; Kinoshita, Hiroto; Sakitani, Kosuke; Nakagawa, Hayato; Nakata, Wachiko; Takahashi, Ryota; Sakamoto, Kei; Maeda, Shin; Koike, Kazuhiko
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2013 EN
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583.11113%
The mitogen-activated protein kinase (MAPK) signaling pathway regulates various cellular functions, including those induced by Helicobacter pylori. TAK1 is an upstream MAPK kinase kinase (MAP3K) required for H. pylori-induced MAPK and NF-κB activation, but it remains unclear whether other MAP3Ks are involved in H. pylori-induced cellular responses. In this study, we focused on the MAP3K ASK1, which plays a critical role in gastric tumorigenesis. In gastric epithelial cells, H. pylori activates ASK1 in a reactive oxygen species (ROS)- and cag pathogenicity island-dependent manner, and ASK1 regulates sustained JNK activation and apoptosis induced by H. pylori. In contrast, TAK1 regulates H. pylori-mediated early JNK activation and cytokine production. We also found reciprocal regulation between ASK1 and TAK1 in H. pylori-related responses, whereby inhibition of TAK1 or downstream p38 MAPK activates ASK1 through ROS production, and ASK1 suppresses TAK1 and downstream NF-κB activation. We identified ROS/ASK1/JNK as a new signaling pathway induced by H. pylori, which regulates apoptotic cell death. The balance of ASK1-induced apoptosis and TAK1-induced antiapoptotic or inflammatory responses may determine the fate of epithelial cells infected with H. pylori and thus be involved in the pathogenesis of gastritis and gastric cancer.

NOD2, RIP2 and IRF5 Play a Critical Role in the Type I Interferon Response to Mycobacterium tuberculosis

Pandey, Amit K.; Yang, Yibin; Jiang, Zhaozhao; Coulombe, Francois; Behr, Marcel A.; Fitzgerald, Katherine A.; Sassetti, Christopher M.; Kelliher, Michelle A.; Fortune, Sarah Merritt
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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670.3112%
While the recognition of microbial infection often occurs at the cell surface via Toll-like receptors, the cytosol of the cell is also under surveillance for microbial products that breach the cell membrane. An important outcome of cytosolic recognition is the induction of IFNα and IFNβ, which are critical mediators of immunity against both bacteria and viruses. Like many intracellular pathogens, a significant fraction of the transcriptional response to Mycobacterium tuberculosis infection depends on these type I interferons, but the recognition pathways responsible remain elusive. In this work, we demonstrate that intraphagosomal M. tuberculosis stimulates the cytosolic Nod2 pathway that responds to bacterial peptidoglycan, and this event requires membrane damage that is actively inflicted by the bacterium. Unexpectedly, this recognition triggers the expression of type I interferons in a Tbk1- and Irf5-dependent manner. This response is only partially impaired by the loss of Irf3 and therefore, differs fundamentally from those stimulated by bacterial DNA, which depend entirely on this transcription factor. This difference appears to result from the unusual peptidoglycan produced by mycobacteria, which we show is a uniquely potent agonist of the Nod2/Rip2/Irf5 pathway. Thus...

The Pathogenic Properties of a Novel and Conserved Gene Product, KerV, in Proteobacteria

Apidianakis, Yiorgos; Boechat, Ana Laura; Baldini, Regina L.; Goumnerov, Boyan C.; An, Dingding; Rahme, Laurence G.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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683.2295%
Identification of novel virulence factors is essential for understanding bacterial pathogenesis and designing antibacterial strategies. In this study, we uncover such a factor, termed KerV, in Proteobacteria. Experiments carried out in a variety of eukaryotic host infection models revealed that the virulence of a Pseudomonas aeruginosa kerV null mutant was compromised when it interacted with amoebae, plants, flies, and mice. Bioinformatics analyses indicated that KerV is a hypothetical methyltransferase and is well-conserved across numerous Proteobacteria, including both well-known and emerging pathogens (e.g., virulent Burkholderia, Escherichia, Shigella, Vibrio, Salmonella, Yersinia and Brucella species). Furthermore, among the 197 kerV orthologs analyzed in this study, about 89% reside in a defined genomic neighborhood, which also possesses essential DNA replication and repair genes and detoxification gene. Finally, infection of Drosophila melanogaster with null mutants demonstrated that KerV orthologs are also crucial in Vibrio cholerae and Yersinia pseudotuberculosis pathogenesis. Our findings suggested that KerV has a novel and broad significance as a virulence factor in pathogenic Proteobacteria and it might serve as a new target for antibiotic drug design.

PerR Confers Phagocytic Killing Resistance and Allows Pharyngeal Colonization by Group A Streptococcus

Gryllos, Ioannis; Kalish, Leslie A.; Wessels, Michael Robert; Grifantini, Renata; Colaprico, Annalisa; Cary, Max E.; Hakansson, Anders; Carey, David W.; Suarez-Chavez, Maria; Kalish, Leslie A.; Mitchell, Paul D.; White, Gary L.; Wessels, Michael Robert
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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670.4343%
The peroxide response transcriptional regulator, PerR, is thought to contribute to virulence of group A Streptococcus (GAS); however, the specific mechanism through which it enhances adaptation for survival in the human host remains unknown. Here, we identify a critical role of PerR-regulated gene expression in GAS phagocytosis resistance and in virulence during pharyngeal infection. Deletion of perR in M-type 3 strain 003Sm was associated with reduced resistance to phagocytic killing in human blood and by murine macrophages in vitro. The increased phagocytic killing of the perR mutant was abrogated in the presence of the general oxidative burst inhibitor diphenyleneiodonium chloride (DPI), a result that suggests PerR-dependent gene expression counteracts the phagocyte oxidative burst. Moreover, an isogenic perR mutant was severely attenuated in a baboon model of GAS pharyngitis. In competitive infection experiments, the perR mutant was cleared from two animals at 24 h and from four of five animals by day 14, in sharp contrast to wild-type bacteria that persisted in the same five animals for 28 to 42 d. GAS genomic microarrays were used to compare wild-type and perR mutant transcriptomes in order to characterize the PerR regulon of GAS. These studies identified 42 PerR-dependent loci...

Plasma Gelsolin Depletion and Circulating Actin in Sepsis—A Pilot Study

Lee, Po-Shun; Patel, Sanjay R; Christiani, David C.; Bajwa, Ednan Khalid; Stossel, Thomas Peter; Waxman, Aaron Bradley
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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663.4149%
Background: Depletion of the circulating actin-binding protein, plasma gelsolin (pGSN) has been described in septic patients and animals. We hypothesized that the extent of pGSN reduction correlates with outcomes of septic patients and that circulating actin is a manifestation of sepsis. Methodology/Principal Findings: We assayed pGSN in plasma samples from non-surgical septic patients identified from a pre-existing database which prospectively enrolled patients admitted to adult intensive care units at an academic hospital. We identified 21 non-surgical septic patients for the study. Actinemia was detected in 17 of the 21 patients, suggesting actin released into circulation from injured tissues is a manifestation of sepsis. Furthermore, we documented the depletion of pGSN in human clinical sepsis, and that the survivors had significantly higher pGSN levels than the non-survivors (163±47 mg/L vs. 89±48 mg/L, p = 0.01). pGSN levels were more strongly predictive of 28-day mortality than APACHE III scores. For every quartile reduction in pGSN, the odds of death increased 3.4-fold. Conclusion: We conclude that circulating actin and pGSN deficiency are associated with early sepsis. The degree of pGSN deficiency correlates with sepsis mortality. Reversing pGSN deficiency may be an effective treatment for sepsis.

Bacterial and Host Determinants of MAL Activation upon EPEC Infection: The Roles of Tir, ABRA, and FLRT3

Leong, John M.; Machesky, Laura M.; Heath, Robert John William; Xavier, Ramnik; Visegrády, Balázs
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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Infection of host cells by pathogenic microbes triggers signal transduction pathways leading to a multitude of host cell responses including actin cytoskeletal re-arrangements and transcriptional programs. The diarrheagenic pathogens Enteropathogenic E. coli (EPEC) and the related Enterohemorrhagic E. coli (EHEC) subvert the host-cell actin cytoskeleton to form attaching and effacing lesions on the surface of intestinal epithelial cells by injecting effector proteins via a type III secretion system. Here we use a MAL translocation assay to establish the effect of bacterial pathogens on host cell signaling to transcription factor activation. MAL is a cofactor of Serum response factor (SRF), a transcription factor with important roles in the regulation of the actin cytoskeleton. We show that EPEC induces nuclear accumulation of MAL-GFP. The translocated intimin receptor is essential for this process and phosphorylation of Tyrosine residues 454 and 474 is important. Using an expression screen we identify FLRT3, C22orf28 and TESK1 as novel activators of SRF. Importantly we demonstrate that ABRA (actin-binding Rho-activating protein, also known as STARS) is necessary for EPEC-induced nuclear accumulation of MAL and the novel SRF activator FLRT3...

DNA Damage and Reactive Nitrogen Species are Barriers to Vibrio cholerae Colonization of the Infant Mouse Intestine

Dupes, Nicole M.; Simmons, Lyle A.; Davies, Bryan William; Bogard, Ryan; Gerstenfeld, Tyler A. I.; Mekalanos, John J.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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670.2271%
Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover...

Roles of Cellular Activation and Sulfated Glycans in Haemophilus somnus Adherence to Bovine Brain Microvascular Endothelial Cells

Behling-Kelly, E.; Vonderheid, H.; Kim, Kwang Sik; Corbeil, L. B.; Czuprynski, C. J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/2006 EN
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Haemophilus somnus can cause a devastating fibrinopurulent meningitis with thrombotic vasculitis and encephalitis in cattle. The mechanisms used by H. somnus to migrate from the bloodstream into the central nervous system (CNS) are unknown. In this study, we demonstrate that H. somnus adheres to, but does not invade, bovine brain endothelial cells (BBEC) in vitro. The number of adherent H. somnus was significantly increased by prior activation of the BBEC with tumor necrosis factor alpha (TNF-α). Addition of exogenous glycosaminoglycans significantly reduced H. somnus adherence to resting and TNF-α-activated BBEC. Heparinase digestion of the endothelial cell's glycocalyx or sodium chlorate inhibition of endothelial cell sulfated glycan synthesis significantly reduced the number of adherent H. somnus. In contrast, addition of hyaluronic acid, a nonsulfated glycosaminoglycan, had no inhibitory effect. These findings suggest a critical role for both cellular activation and sulfated glycosaminoglycans in adherence of H. somnus to BBEC. Using heparin-labeled agarose beads, we demonstrated a high-molecular-weight heparin-binding protein expressed by H. somnus. Heparin was also shown to bind H. somnus in a 4°C binding assay. These data suggest that heparin-binding proteins on H. somnus could serve as initial adhesins to sulfated proteoglycans on the endothelial cell surface...

Streptococcus pyogenes Arginine and Citrulline Catabolism Promotes Infection and Modulates Innate Immunity

Cusumano, Zachary T.; Watson, Michael E.; Caparon, Michael G.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2014 EN
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A bacterium's ability to acquire nutrients from its host during infection is an essential component of pathogenesis. For the Gram-positive pathogen Streptococcus pyogenes, catabolism of the amino acid arginine via the arginine deiminase (ADI) pathway supplements energy production and provides protection against acid stress in vitro. Its expression is enhanced in murine models of infection, suggesting an important role in vivo. To gain insight into the function of the ADI pathway in pathogenesis, the virulence of mutants defective in each of its enzymes was examined. Mutants unable to use arginine (ΔArcA) or citrulline (ΔArcB) were attenuated for carriage in a murine model of asymptomatic mucosal colonization. However, in a murine model of inflammatory infection of cutaneous tissue, the ΔArcA mutant was attenuated but the ΔArcB mutant was hyperattenuated, revealing an unexpected tissue-specific role for citrulline metabolism in pathogenesis. When mice defective for the arginine-dependent production of nitric oxide (iNOS−/−) were infected with the ΔArcA mutant, cutaneous virulence was rescued, demonstrating that the ability of S. pyogenes to utilize arginine was dispensable in the absence of nitric oxide-mediated innate immunity. This work demonstrates the importance of arginine and citrulline catabolism and suggests a novel mechanism of virulence by which S. pyogenes uses its metabolism to modulate innate immunity through depletion of an essential host nutrient.