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Proteomic Analysis of the Ehrlichia chaffeensis Phagosome in Cultured DH82 Cells  [PDF]
Yan Cheng, Yan Liu, Bin Wu, Jian-zhi Zhang, Jiang Gu, Ya-ling Liao, Fu-kun Wang, Xu-hu Mao, Xue-jie Yu
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0088461
Abstract: Ehrlichia chaffeensis is an obligately intracellular bacterium that resides and multiplies within cytoplasmic vacuoles of phagocytes. The Ehrlichia-containing vacuole (ECV) does not fuse with lysosomes, an essential condition for Ehrlichia to survive inside phagocytes, but the mechanism of inhibiting the fusion of the phagosome with lysosomes is not clear. Understanding the ECV molecular composition may decipher the mechanism by which Ehrlichia inhibits phagosome-lysosome fusion. In this study, we obtained highly purified ECVs from E. chaffeensis-infected DH82 cells by sucrose density gradient centrifugation and analyzed their composition by mass spectrometry-based proteomics. The ECV composition was compared with that of phagolysosomes containing latex beads. Lysosomal proteins such as cathepsin D, cathepsin S, and lysosomal acid phosphatase were not detected in E. chaffeensis phagosome preparations. Some small GTPases, involved in membrane dynamics and phagocytic trafficking, were detected in ECVs. A notable finding was that Rab7, a late endosomal marker, was consistently detected in E. chaffeensis phagosomes by mass spectrometry. Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment. Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway. Ehrlichia chaffeensis did not inhibit phagosomes containing latex beads from fusing with lysosomes in infected cells. We concluded that the E. chaffeensis vacuole was a late endosome and E. chaffeensis might inhibit phagosome-lysosome fusion by modifying its vacuolar membrane composition, rather than by regulating the expression of host genes involved in trafficking.
Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages
Takeshi Haneda, Mariko Sugimoto, Yukie Yoshida-Ohta, Yoshio Kodera, Masamichi Oh-Ishi, Tadakazu Maeda, Satomi Shimizu-Izumi, Tsuyoshi Miki, Yoshinori Kumagai, Hirofumi Danbara, Nobuhiko Okada
BMC Microbiology , 2010, DOI: 10.1186/1471-2180-10-324
Abstract: Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2.A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.The facultative intracellular bacterium Salmonella enterica causes a broad spectrum of diseases, such as gastroenteritis and bacteremia, which are typically acquired by oral ingestion of contaminated food or water. S. enterica serovar Typhimurium (S. Typhimurium) causes enterocolitis in humans and a typhoid-like systemic infection in mice.Several virulence genes associated with Salmonella pathogenicity islands (SPIs) and the virulence plasmid have been characterized in S. Typhimurium. Two type III secretion systems (T3SS) encoded by SPI-1 and SPI-2 play central roles in Salmonella pathogenesis. SPI-1 is essential for the invasion of host cells and the induction of apoptosis in infected macrophages [1,2]. SPI-2 T3SS primarily confers survival and replication on macrophages and is required for systemic infection in the mouse infection model [3,4]. Expression of SPI-2 genes is induced within a modified phagosome, called the Salmonella-containing vacuole (SCV), in infected macrophages [5]. Induction of SPI-2 genes dep
Subcellular Localization of Extracytoplasmic Proteins in Monoderm Bacteria: Rational Secretomics-Based Strategy for Genomic and Proteomic Analyses  [PDF]
Sandra Renier, Pierre Micheau, Régine Talon, Michel Hébraud, Micka?l Desvaux
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0042982
Abstract: Genome-scale prediction of subcellular localization (SCL) is not only useful for inferring protein function but also for supporting proteomic data. In line with the secretome concept, a rational and original analytical strategy mimicking the secretion steps that determine ultimate SCL was developed for Gram-positive (monoderm) bacteria. Based on the biology of protein secretion, a flowchart and decision trees were designed considering (i) membrane targeting, (ii) protein secretion systems, (iii) membrane retention, and (iv) cell-wall retention by domains or post-translocational modifications, as well as (v) incorporation to cell-surface supramolecular structures. Using Listeria monocytogenes as a case study, results were compared with known data set from SCL predictors and experimental proteomics. While in good agreement with experimental extracytoplasmic fractions, the secretomics-based method outperforms other genomic analyses, which were simply not intended to be as inclusive. Compared to all other localization predictors, this method does not only supply a static snapshot of protein SCL but also offers the full picture of the secretion process dynamics: (i) the protein routing is detailed, (ii) the number of distinct SCL and protein categories is comprehensive, (iii) the description of protein type and topology is provided, (iv) the SCL is unambiguously differentiated from the protein category, and (v) the multiple SCL and protein category are fully considered. In that sense, the secretomics-based method is much more than a SCL predictor. Besides a major step forward in genomics and proteomics of protein secretion, the secretomics-based method appears as a strategy of choice to generate in silico hypotheses for experimental testing.
Myocarditis Mimicking an Acute Coronary Syndrome: A Case Related to Salmonella enteritis  [PDF]
B. Rossetti,G. Nguisseu,A. Buracci,L. Migliorini,G. Zanelli
Gastroenterology Research and Practice , 2009, DOI: 10.1155/2009/931853
Abstract: Infective myocarditis is most commonly due to a viral infection; occasionally it has been related to bacteria. Gastrointestinal infections associated with myocarditis have only rarely been described in young people, and the pathogenesis is unclear. We report a case of myocarditis mimicking an acute coronary syndrome (ACS) in a patient hospitalized for fever and diarrhoea. Salmonella enteritidis was isolated from stool, and no other pathogens were found. The coronary angiography was normal, and there were not other coronary artery risk factors, other than hypertension. The patient was treated with ciprofloxacin, acetylsalicylate acid, and ramipril with rapid clinical improvement and normalization of cardiac abnormalities. Final diagnosis of Salmonella enteritis and related myocarditis was made based on clinical, laboratory, ECG and echocardiographical findings.
Salmonella enterica serotype dublin bacteraemia mimicking enteric fever  [cached]
Dias M,Antony B,Pinto H,Rekha B
Indian Journal of Medical Microbiology , 2009,
Abstract: Salmonella enterica serotype Dublin, a bovine adapted serotype, is the commonest cause of salmonellosis in cattle. Salmonellosis in animals always presents a potential zoonotic threat. Infected cattles serves as a source of infection to humans. We present here Salmonella Dublin Bacteraemia in an elderly patient, with all the clinical details, due to the rarity of its occurrence. He was treated successfully with ciprofloxacin and his follow up period was uneventful.
Proteomic analysis of Salmonella enterica serovar Enteritidis following propionate adaptation
Leona N Calhoun, Rohana Liyanage, Jackson O Lay, Young Kwon
BMC Microbiology , 2010, DOI: 10.1186/1471-2180-10-249
Abstract: In this study, we used 2 D gel electrophoresis to examine the proteomes of PA adapted and unadapted S. Enteritidis and have identified five proteins that are upregulated in PA adapted cultures using standard peptide mass fingerprinting by MALDI-TOF-MS and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of these five, two significant stress-related proteins (Dps and CpxR) were shown (via qRT-PCR analysis) to be upregulated at the transcriptional level as well. Unlike the wild type when adapted to PA (which demonstrates significant acid resistance), PA adapted S. Enteritidis ?dps and S. Enteritidis ?cpxR were at a clear disadvantage when challenged to a highly acidic environment. However, we found the acid resistance to be fully restorable after genetic complementation.This work reveals a significant difference in the proteomes of PA adapted and unadapted S. Enteritidis and affirms the contribution of Dps and CpxR in PA induced acid resistance.Salmonella enterica Serovar Enteritidis (S. Enteritidis) is a facultative intracellular pathogen responsible for acute gastroenteritis and is currently the second most frequently isolated serovar in the United States - accounting for nearly 15% of total cases of human salmonellosis [1]. S. Enteritidis maintains its status as a leading cause of foodborne infections mainly due to its prevalence in poultry products and its environmental persistence despite the harsh conditions it encounters. The survival of this pathogen under intense conditions has been linked to its remarkable ability to quickly respond to environmental signals and adapt to its surroundings, as well as the induction of specific stress responses during environmental adaptation [2-6].Throughout its infection cycle, S. Enteritidis encounters several distinctive environments including those rich in the short chain fatty acids (SCFAs) acetate, propionate (PA), and butyrate. PA is one of many SCFAs deemed acceptable for use in food preservation and is freque
Mass spectrometry-based quantitative proteomic analysis of Salmonella enterica serovar Enteritidis protein expression upon exposure to hydrogen peroxide
Kihoon Kim, Edward Yang, Gia-Phong Vu, Hao Gong, Jing Su, Fenyong Liu, Sangwei Lu
BMC Microbiology , 2010, DOI: 10.1186/1471-2180-10-166
Abstract: Using stable isotope labeling coupled with mass spectrometry, we performed quantitative proteomic analysis of Salmonella enterica serovar Enteritidis and identified 76 proteins whose expression is modulated upon exposure to H2O2. SPI-1 effector SipC was expressed about 3-fold higher and SopB was expressed approximately 2-fold lower in the presence of H2O2, while no significant change in the expression of another SPI-1 protein SipA was observed. The relative abundance of SipA, SipC, and SopB was confirmed by Western analyses, validating the accuracy and reproducibility of our approach for quantitative analysis of protein expression. Furthermore, immuno-detection showed substantial expression of SipA and SipC but not SopB in the late phase of infection in macrophages and in the spleen of infected mice.We have identified Salmonella proteins whose expression is modulated in the presence of H2O2. Our results also provide the first direct evidence that SipC is highly expressed in the spleen at late stage of salmonellosis in vivo. These results suggest a possible role of SipC and other regulated proteins in supporting survival and replication of Salmonella under oxidative stress and during its systemic infection in vivo.Salmonella enterica is one of the leading causes of food-borne illnesses around the world [1,2]. There are two major serotypes of Salmonella enterica, namely Salmonella enterica serovar Enteritidis (S. Enteritidis) and Typhimurium (S. Typhimurium). In recent years, S. Enteritidis represents one of the most commonly reported serotypes associated with food poisoning illness in the United States [3]. Two hallmarks of Salmonella pathogenesis are the invasion of non-phagocytic cells such as the epithelial cells of the intestinal mucosa, and the survival inside macrophages during systemic infection. The mechanisms of both processes are linked to the functions of two type III secretion systems (T3SS) of Salmonella that are encoded and regulated by a cluster of gen
Phagosome proteomes open the way to a better understanding of phagosome function
Gareth Griffiths, Luis Mayorga
Genome Biology , 2007, DOI: 10.1186/gb-2007-8-3-207
Abstract: Early in their evolution, eukaryotic cells acquired the capacity to take up microbes by phagocytosis as a source of food. Since then, phagocytosis has evolved into a highly complex and regulated process, and is one of the main ways in which multicellular animals clear the body of pathogenic microbes and cellular debris. Microbes and other particles are taken up by phagocytosis into an intracellular membrane-bounded organelle called a phagosome (Figure 1). This eventually fuses with other organelles, notably endosomes and lysosomes, resulting in a gradual alteration of the composition and function of the phagosome, a process referred to as phagosome maturation. After full maturation, the phagosome will contain a battery of hydrolytic enzymes and have an internal pH as low as 4-4.5 (Figure 1).In the vast majority of cases, the microbe inside the phagosome is killed and digested, but a number of important pathogens, including the bacterium Mycobacterium tuberculosis, which kills around two million people each year, have acquired the ability to survive, and even replicate, in this hostile environment. Each type of pathogen that exploits intracellular vesicles seems to have evolved a different survival strategy. Phagosome maturation follows a defined biochemical program, and different pathogens probably redirect this program in a unique fashion. Pathogen proteins and/or lipids released inside phagosomes alter signaling pathways in the phagosomal membrane or in the cytoplasm.A pathogen-containing phagosome in, for example, a macrophage, has three distinct 'compartments'. These are the pathogen itself; the luminal contents, which are enriched in hydrolases, protons, and ions such as Ca2+, and have a still poorly defined redox state; and the phagosomal membrane, the boundary between the pathogen and the cytoplasm. This last controls most phagosome functions, including their fusion, recycling, and interactions with the cytoskeleton. Determining the molecular composition of t
Comparative Proteomic Analysis of the PhoP Regulon in Salmonella enterica Serovar Typhi Versus Typhimurium  [PDF]
Richelle C. Charles, Jason B. Harris, Michael R. Chase, Lauren M. Lebrun, Alaullah Sheikh, Regina C. LaRocque, Tanya Logvinenko, Sean M. Rollins, Abdullah Tarique, Elizabeth L. Hohmann, Ian Rosenberg, Bryan Krastins, David A. Sarracino, Firdausi Qadri, Stephen B. Calderwood, Edward T. Ryan
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0006994
Abstract: Background S. Typhi, a human-restricted Salmonella enterica serovar, causes a systemic intracellular infection in humans (typhoid fever). In comparison, S. Typhimurium causes gastroenteritis in humans, but causes a systemic typhoidal illness in mice. The PhoP regulon is a well studied two component (PhoP/Q) coordinately regulated network of genes whose expression is required for intracellular survival of S. enterica. Methodology/Principal Findings Using high performance liquid chromatography mass spectrometry (HPLC-MS/MS), we examined the protein expression profiles of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro and compared these results to profiles of phoP?/Q? mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Our analysis identified 53 proteins in S. Typhimurium LT2 and 56 proteins in S. Typhi that were regulated in a PhoP-dependent manner. As expected, many proteins identified in S. Typhi demonstrated concordant differential expression with a homologous protein in S. Typhimurium. However, three proteins (HlyE, STY1499, and CdtB) had no homolog in S. Typhimurium. HlyE is a pore-forming toxin. STY1499 encodes a stably expressed protein of unknown function transcribed in the same operon as HlyE. CdtB is a cytolethal distending toxin associated with DNA damage, cell cycle arrest, and cellular distension. Gene expression studies confirmed up-regulation of mRNA of HlyE, STY1499, and CdtB in S. Typhi in PhoP-inducing conditions. Conclusions/Significance This study is the first protein expression study of the PhoP virulence associated regulon using strains of Salmonella mutant in PhoP, has identified three Typhi-unique proteins (CdtB, HlyE and STY1499) that are not present in the genome of the wide host-range Typhimurium, and includes the first protein expression profiling of a live attenuated bacterial vaccine studied in humans (Ty800).
Actin-interacting and flagellar proteins in Leishmania spp.: bioinformatics predictions to functional assignments in phagosome formation
Diniz, Michely C.;Costa, Marcília P.;Pacheco, Ana C.L.;Kamimura, Michel T.;Silva, Samara C.;Carneiro, Laura D.G.;Sousa, Ana P.L.;Soares, Carlos E.A.;Souza, Celeste S.F.;Oliveira, Diana Magalh?es de;
Genetics and Molecular Biology , 2009, DOI: 10.1590/S1415-47572009000300033
Abstract: several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (aips) during both processes of motility and host cell entry might be key events for successful infection. for a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. in a search for aips involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. results presented here provide the first report of leishmania coronin and arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. this is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented.
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