Iron Acquisition Proteins Research Articles

Possible role of Escherichia coli in propagation and perpetuation of chronic inflammation in ulcerative colitis

BackgroundThis study investigated a possible role of Escherichia coli in propagation and perpetuation of the chronic inflammation in ulcerative colitis (UC). The lesions of UC are located superficially on the rectal and/or colonic mucosa. It is suggested that the commensal bacteria of the digestive tract may play a role in the pathogenesis of UC. Several studies have demonstrated proliferation of E. coli in the gut of UC patients. An increase in the number of E. coli in the inflamed tissue is most probably related to the abundance of iron ions produced by the bacteria.MethodsColon mucosal biopsies were collected from 30 patients with acute-phase UC, both from tissues with inflammatory changes (n = 30) and unchanged tissue with no inflammatory changes (n = 30) from the same patient. Biopsies were also taken from 16 patients with irritable bowel syndrome diarrhea who comprised the control group. Quantitative and qualitative analysis of the biopsy specimens was performed using culture methods and real-time polymerase chain reaction (PCR). Genotyping of the E. coli isolates was done using pulsed-field gel electrophoresis. Multiplex PCR was used to compare the E. coli strains for the presence of genes responsible for synthesis of iron acquisition proteins: iroN, iutA, iha, ireA, chuA, and hlyA.ResultsWe demonstrated that there was a significant increase in the number of E. coli at the sites of inflammation in patients with UC compared to the control group (P = 0.031). Comparative analysis of the restriction patterns of E. coli isolated from inflammatory and unchanged tissues showed that the local inflammatory changes did not promote specific E. coli strains. There was a significant difference in the frequency of the iroN gene in E. coli isolated from patients with UC as compared to the control group.ConclusionsThe increase in the numbers of E. coli in the inflammatory tissues is related to the presence of chuA and iutA genes, which facilitate iron acquisition during chronic intestinal inflammatory processes.

Flexible Survival Strategies of Pseudomonas aeruginosa in Biofilms Result in Increased Fitness Compared with Candida albicans

The majority of microorganisms persist in nature as surface-attached communities often surrounded by an extracellular matrix, called biofilms. Most natural biofilms are not formed by a single species but by multiple species. Microorganisms not only cooperate as in some multispecies biofilms but also compete for available nutrients. The Gram-negative bacterium Pseudomonas aeruginosa and the polymorphic fungus Candida albicans are two opportunistic pathogens that are often found coexisting in a human host. Several models of mixed biofilms have been reported for these organisms showing antagonistic behavior. To investigate the interaction of P. aeruginosa and C. albicans in more detail, we analyzed the secretome of single and mixed biofilms of both organisms using MALDI-TOF MS/MS at several time points. Overall 247 individual proteins were identified, 170 originated from P. aeruginosa and 77 from C. albicans. Only 39 of the 131 in mixed biofilms identified proteins were assigned to the fungus whereby the remaining 92 proteins belonged to P. aeruginosa. In single-species biofilms, both organisms showed a higher diversity of proteins with 73 being assigned to C. albicans and 154 to P. aeruginosa. Most interestingly, P. aeruginosa in the presence of C. albicans secreted 16 proteins in significantly higher amounts or exclusively among other virulence factors such as exotoxin A and iron acquisition systems. In addition, the high affinity iron-binding siderophore pyoverdine was identified in mixed biofilms but not in bacterial biofilms, indicating that P. aeruginosa increases its capability to sequester iron in competition with C. albicans. In contrast, C. albicans metabolism was significantly reduced, including a reduction in detectable iron acquisition proteins. The results obtained in this study show that microorganisms not only compete with the host for essential nutrients but also strongly with the present microflora in order to gain a competitive advantage.

Abstract 236: Heart Failure Remodeling: Local Endogenous Erythropoietin and Erythropoietin Receptor Expression

Erythropoietin (EPO) via erythropoietin receptor (EPOR) maintains myeloid erythropoiesis, however in a failing heart (FH) it seems to exert cytoprotective, antiapoptotic, proangiogenic effects. Aim: to assess (myocardial) M-EPOR expression and to find out if erythropoietin (M-EPO) is produced in human heart; secondary to elucidate the mechanisms related to M-EPO/M-EPOR expression. Methods: We used 33 explanted FH, compared to 11 non-failing hearts (NFH). Patients characteristics: left ventricle (LV - EF 22±11%), NTproBNP (5464±4825 pg/ml). Serum variables: iron (62±32ug/dl), ferritin (156±122ng/ml), transferrin receptor (3.8±2.6mg/l), EPO (29.5±44.4mIU/ml), HT (39.8±5.3%), Hb (13.2±1.7g/dl), creatinine clearance (CrCl 68±23 ml/min), Na+ (138±5.4 mmol/l). Myocardial assesment: M-EPO, M-EPOR, iron storage protein - ferritin (M-FR), iron acquisition protein transferrin receptor (M-TfR), Iron Regulatory Protein 1 (M-IRP1) - by ELISA - ng/mg protein - expression; myocardial iron load - by Instrumental Neutron Activation Analysis, µg/g. Results: In FH and NFH M-EPO/M-EPOR expression were detected; M-EPO was elevated in failing LV (in FH vs NFH 4.7±1.6 vs 3.7±1.4, p=0.0346; ng/mg protein), no changes in M-EPOR was found. M-EPO correlated with M-EPOR from both ventricles (LV- left r=0.63, p=0.0002; RV - right r=0.42, p=0.0161). With regard to anemia (by WHO), patients were divided into two subgroups: A+ (n=11) and A- (n=22). In A+ vs A- serum EPO (47±23 vs 28±30 mIU/l, p=0.0473) was higher. In myocardium A+ M-TfR was lower than in A- both LV (136±61 vs 218±134; p=0.0446) and RV (130±92 vs 199±143; p=0.0688), however no differences in M-EPO/M-EPOR/M-FR/M-IRP1 and iron load were found. In LV, A+ subgroup, IRP1 correlation with M-EPO (r=0.79, p=0.0107) and M-EPOR (r=0.76, p=0.0184). Conclusions: M-EPO/M-EPOR are synthesized locally in human hearts. In failing LV M-EPO synthesis is significantly increased. M-EPO production correlated with M-EPOR expression. M-EPO/EPOR expression is associated with regulatory protein IRP1. The presence of anemia accompanied by high plasma EPO is not connected with modification of myocardial expression: M-EPO/M-EPOR/M-FR/M-IRP1, iron load, except for M-TfR.

Luminal Iron Levels Govern Intestinal Tumorigenesis after Apc Loss In Vivo

It is clear from epidemiological studies that excess iron is associated with increased risk of colorectal cancer; however, questions regarding the mechanism of how iron increases cancer risk, the source of the excess iron (circulating or luminal), and whether iron reduction represents a potential therapeutic option remain unanswered. In this study, we show that after Apc deletion, the cellular iron acquisition proteins TfR1 and DMT1 are rapidly induced. Conversely, restoration of APC reduces cellular iron due to repression of these proteins. To test the functional importance of these findings, we performed in vivo investigations of the impact of iron levels on intestinal tumorigenesis. Strikingly, depletion of luminal (but not systemic) iron strongly suppressed murine intestinal tumorigenesis, whereas increased luminal iron strongly promoted tumorigenesis. Taken together, our data definitively delineate iron as a potent modifier of intestinal tumorigenesis and have important implications for dietary iron supplementation in patients at high risk of colorectal cancer.

1088 ESCHERICHIA COLI STRAINS FROM CHRONIC PROSTATITIS PATIENTS INVADE PROSTATE EPITHELIAL CELLS AND INDUCE SECRETION OF INFLAMMATORY MEDIATORS

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Prostate & Genitalia1 Apr 20121088 ESCHERICHIA COLI STRAINS FROM CHRONIC PROSTATITIS PATIENTS INVADE PROSTATE EPITHELIAL CELLS AND INDUCE SECRETION OF INFLAMMATORY MEDIATORS Li-Lin Cheng, Laura E. Goeser, Alexandra E. Burleigh, Justin J. Lemke, Rodney A. Welch, and Walter J. Hopkins Li-Lin ChengLi-Lin Cheng Madison, WI More articles by this author , Laura E. GoeserLaura E. Goeser Madison, WI More articles by this author , Alexandra E. BurleighAlexandra E. Burleigh Madison, WI More articles by this author , Justin J. LemkeJustin J. Lemke Madison, WI More articles by this author , Rodney A. WelchRodney A. Welch Madison, WI More articles by this author , and Walter J. HopkinsWalter J. Hopkins Madison, WI More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1195AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Category IIIA chronic prostatitis is characterized by the presence of leukocytes in prostatic secretions without culturable bacteria. One possible explanation is that infecting bacteria are harbored within prostate epithelial cells and their intracellular presence induces local secretion of inflammatory mediators. We examined the ability of uropathogenic E. coli to invade mouse prostate epithelial cells in vitro and to induce production of cytokines and chemokines by these cells. METHODS Twenty-four E. coli isolates from chronic prostatitis patients were genotyped for urovirulence factor genes associated with adherence, toxin production, iron acquisition, secretion, and outer membrane proteins. Motility was measured by a swimming assay. Isolates were tested for their invasion of primary epithelial cells derived from the dorsal and lateral prostate lobes of C3H/HeOuJ mice using a gentamicin protection assay. A multiplex assay was used to quantify cytokines/chemokines secreted by cells exposed to E. coli for two hours before addition of gentamicin. RESULTS Among the 24 strains, 10 were intermediate to highly invasive and 14 were low to non-invasive. There was no significant link between any individual gene and invasion intensity; however, a significant positive correlation between motility and epithelial cell invasion was noted. An initial study with eight isolates showed strong responses of IL-6, monocyte chemotactic protein (MCP-1) and granulocyte-macrophage colony stimulating factor (GM-CSF) at 24 hours post exposure, while IL-1β and TNF-α were undetectable. The mediators were induced by seven invasive strains but not by a non-invasive isolate. In addition, strains with cnf-1, cdt, and hlyA toxin genes appeared to induce the least amounts of mediators in prostate epithelial cells. CONCLUSIONS E. coli isolates from chronic prostatitis patients are able to invade prostate epithelial cells with a wide range of intensities. Inflammatory mediators are produced by prostate epithelial cells when exposed to invasive, chronic prostatitis E. coli strains. The secretion of MCP-1, GM-CSF, and IL-6 is associated with recruitment and production of mononuclear inflammatory cells, implying their roles in promoting chronic inflammation. These results thus support a model of chronic prostatic inflammation in the absence of extracellular bacteria, which is the primary characteristic of Category IIIA prostatitis. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e441 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Li-Lin Cheng Madison, WI More articles by this author Laura E. Goeser Madison, WI More articles by this author Alexandra E. Burleigh Madison, WI More articles by this author Justin J. Lemke Madison, WI More articles by this author Rodney A. Welch Madison, WI More articles by this author Walter J. Hopkins Madison, WI More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Sodium nitroprusside may modulate Escherichia coli antioxidant enzyme expression by interacting with the ferric uptake regulator

Efforts to explore possible relationships between nitric oxide (NO) and antioxidant enzymes in an Escherichia coli model have uncovered a possible interaction between sodium nitroprusside (SNP), a potent, NO-donating drug, and the ferric uptake regulator (Fur), an iron(II) – dependent regulator of antioxidant and iron acquisition proteins present in Gram-negative bacteria. The enzymatic profiles of superoxide dismutase and hydroperoxidase during logarithmic phase of growth were studied via non-denaturing polyacrylamide gel electrophoresis and activity staining specific to each enzyme. Though NO is known to induce transcription of the manganese-bearing isozyme of SOD (MnSOD), treatment with SNP paradoxically suppressed MnSOD expression and greatly enhanced the activity of the iron-containing equivalent (FeSOD). Fur, one of six global regulators of MnSOD transcription, is uniquely capable of suppressing MnSOD while enhancing FeSOD expression through distinct mechanisms. We thus hypothesize that Fur is complacent in causing this behaviour and that the iron(II) component of SNP is activating Fur. E. coli was also treated with the SNP structural analogues, potassium ferricyanide (PFi) and potassium ferrocyanide (PFo). Remarkably, the ferrous PFo was capable of mimicking the SNP-related pattern, whereas the ferric PFi was not. As Fur depends upon ferrous iron for activation, we submit this observation of redox-specificity as preliminary supporting evidence for the hypothesized Fur–SNP interaction.Iron is an essential metal that the human innate immune system sequesters to prevent its use by invading pathogens. As NO is known to inhibit iron-bound Fur, and as activated Fur regulates iron uptake through feedback inhibition, we speculate that the administration of this drug may disrupt this strategic management of iron in favour of residing Gram-negative species by providing a source of iron in an otherwise iron-scarce environment capable of encouraging its own uptake. However, these gains may be counteracted by the oxidative consequences of iron and NO, as the former can catalyse the formation of toxic free radical species while the latter can inhibit enzymes and contribute to the formation of other toxic compounds. The potential consequences of SNP on microbial growth warrant future investigation.

Comparative Genomics and Transcriptomics of Propionibacterium acnes

The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that is occasionally associated with inflammatory diseases. Recent work has indicated that evolutionary distinct lineages of P. acnes play etiologic roles in disease while others are associated with maintenance of skin homeostasis. To shed light on the molecular basis for differential strain properties, we carried out genomic and transcriptomic analysis of distinct P. acnes strains. We sequenced the genome of the P. acnes strain 266, a type I-1a strain. Comparative genome analysis of strain 266 and four other P. acnes strains revealed that overall genome plasticity is relatively low; however, a number of island-like genomic regions, encoding a variety of putative virulence-associated and fitness traits differ between phylotypes, as judged from PCR analysis of a collection of P. acnes strains. Comparative transcriptome analysis of strains KPA171202 (type I-2) and 266 during exponential growth revealed inter-strain differences in gene expression of transport systems and metabolic pathways. In addition, transcript levels of genes encoding possible virulence factors such as dermatan-sulphate adhesin, polyunsaturated fatty acid isomerase, iron acquisition protein HtaA and lipase GehA were upregulated in strain 266. We investigated differential gene expression during exponential and stationary growth phases. Genes encoding components of the energy-conserving respiratory chain as well as secreted and virulence-associated factors were transcribed during the exponential phase, while the stationary growth phase was characterized by upregulation of genes involved in stress responses and amino acid metabolism. Our data highlight the genomic basis for strain diversity and identify, for the first time, the actively transcribed part of the genome, underlining the important role growth status plays in the inflammation-inducing activity of P. acnes. We argue that the disease-causing potential of different P. acnes strains is not only determined by the phylotype-specific genome content but also by variable gene expression.

Population coverage analysis of T-Cell epitopes of Neisseria meningitidis serogroup B from Iron acquisition proteins for vaccine design.

Although the concept of Reverse Vaccinology was first pioneered for sepsis and meningococcal meningitidis causing bacterium, Neisseria meningitides, no broadly effective vaccine against serogroup B meningococcal disease is yet available. In the present investigation, HLA distribution analysis was undertaken to select three most promiscuous T-cell epitopes out of ten computationally validated epitopes of Iron acquisition proteins from Neisseria MC58 by using the population coverage tool of Immune Epitope Database (IEDB). These epitopes have been determined on the basis of their binding ability with maximum number of HLA alleles along with highest population coverage rate values for all the geographical areas studied. The comparative population coverage analysis of moderately immunogenic and high immunogenic peptides suggests that the former may activate T-cell response in a fairly large proportion of people in most geographical areas, thus indicating their potential for development of epitope-based vaccine.

Transcriptional and Translational Regulatory Responses to Iron Limitation in the Globally Distributed Marine Bacterium Candidatus Pelagibacter ubique

Iron is recognized as an important micronutrient that limits microbial plankton productivity over vast regions of the oceans. We investigated the gene expression responses of Candidatus Pelagibacter ubique cultures to iron limitation in natural seawater media supplemented with a siderophore to chelate iron. Microarray data indicated transcription of the periplasmic iron binding protein sfuC increased by 16-fold, and iron transporter subunits, iron-sulfur center assembly genes, and the putative ferroxidase rubrerythrin transcripts increased to a lesser extent. Quantitative peptide mass spectrometry revealed that sfuC protein abundance increased 27-fold, despite an average decrease of 59% across the global proteome. Thus, we propose sfuC as a marker gene for indicating iron limitation in marine metatranscriptomic and metaproteomic ecological surveys. The marked proteome reduction was not directly correlated to changes in the transcriptome, implicating post-transcriptional regulatory mechanisms as modulators of protein expression. Two RNA-binding proteins, CspE and CspL, correlated well with iron availability, suggesting that they may contribute to the observed differences between the transcriptome and proteome. We propose a model in which the RNA-binding activity of CspE and CspL selectively enables protein synthesis of the iron acquisition protein SfuC during transient growth-limiting episodes of iron scarcity.

Covalently linked cell wall proteins ofCandida albicans and their role in fitness and virulence

The cell wall of Candida albicans consists of an internal skeletal layer and an external protein coat. This coat has a mosaic-like nature, containing c. 20 different protein species covalently linked to the skeletal layer. Most of them are GPI proteins. Coat proteins vary widely in function. Many of them are involved in the primary interactions between C. albicans and the host and mediate adhesive steps or invasion of host cells. Others are involved in biofilm formation and cell-cell aggregation. They further include iron acquisition proteins, superoxide dismutases, and yapsin-like aspartic proteases. In addition, several covalently linked carbohydrate-active enzymes are present, whose precise functions remain hitherto largely elusive. The expression levels of the genes that encode covalently linked cell wall proteins (CWPs) can vary enormously. They depend on the mode of growth and the combined inputs of several signaling pathways that sense environmental conditions. This is reflected in the unusually long intergenic regions of most of these genes. Finally, the precise location of several covalently linked CWPs is temporally and spatially regulated. We conclude that covalently linked CWPs of C. albicans play a crucial role in fitness and virulence and that their expression is tightly controlled.

Identification of iron-acquisition proteins of Avibacterium paragallinarum

When Avibacterium paragallinarum reference strain 0083 (serovar A) was grown in an iron-restricted culture medium, the expression of the 60, 68 and 93 kDa outer membrane proteins increased as compared with normal media. Sera of chickens experimentally infected with Av. paragallinarum recognized these iron-restriction induced proteins, suggesting their expression in vivo. The three outer membrane proteins were identified as transferrin receptor and iron transport proteins by mass spectroscopy and a search in sequence databases. As these proteins have been reported to be regulated by the Fur protein in many bacteria, we investigated, through molecular methods, the presence of the fur gene in Av. paragallinarum. A candidate fur gene of Av. paragallinarum was amplified by polymerase chain reaction using complementary primers to conserved regions of fur gene sequences from members of the Pasteurellaceae family. The nucleotide sequence of the cloned gene, from ATG to TAA stop codon, was 453 base pairs in length and the deduced amino acid sequence showed 94% identity with Fur sequences of Actinobacillus pleuropneumoniae and Haemophilus ducreyi. The Av. paragallinarum deduced Fur protein (17.8 kDa) amino acid sequence contains the N-terminal helix–turn–helix DNA-binding domain and the two iron-binding sites in the C-terminal end, typical of other described Fur proteins. The study of iron-restriction-induced proteins and the mechanism regulating their expression could lead to an understanding of the responses of Av. paragallinarum to survive in an iron-restricted environment on host mucosal surfaces.

Outer membrane proteomics of Pasteurella multocida isolates to identify putative host-specificity determinants

Pasteurella multocida is a Gram-negative bacterium responsible for diseases affecting a broad range of farm-reared animals. Although there is an association between capsular serotype and disease, the molecular basis of host specificity is poorly understood. Outer membrane proteins (OMPs) are at the interface of bacterium and host and are likely to play important roles in host specificity and disease. Two classes are of particular importance—adhesins that are adapted for colonization of specific host niches and iron-acquisition proteins that allow pathogens to acquire iron from host-specific iron complexes. A comparative analysis of the outer membrane (OM) proteome of eight P. multocida isolates associated with disease of avian, bovine, ovine and porcine species was performed to identify putative host-specificity determinants. Isolates were cultured in iron-replete media, and also in iron-limited conditions to mimic the iron-limited host environment, and induce expression of iron-regulated OMPs expressed in vivo. The OMP-rich sarcosyl-insoluble cell fraction was isolated and the OMPs were separated by SDS-PAGE and identified by matrix-assisted light desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The expressed proteome was compared with the in silico predicted proteome from the genome sequence of P. multocida strain Pm70, using PSORTb and Proteome Analyst subcellular localization software. In iron-rich conditions isolates were clustered into three groups based on high molecular weight (HMW) OMP similarity. Isolates responsible for invasive disease were clustered into a single group. Putative colonization OMPs were present in isolates recovered from different host species, but showed molecular weight heterogeneity. Such proteins are good candidates for further study as disease or host-specificity determinants, as variation between these proteins may be a consequence of adaptation to different host niches. HMW OMPs were identified as being involved in iron-uptake. However, isolates associated with different diseases and host species expressed different iron-uptake proteins, or regulated expression differently, suggesting adaptation to specific host niches.

Outer Membrane Antigens of the UropathogenProteus mirabilisRecognized by the Humoral Response during Experimental Murine Urinary Tract Infection

Proteus mirabilis, a gram-negative bacterium, is a frequent cause of complicated urinary tract infections in those with functional or anatomical abnormalities or those subject to long-term catheterization. To systematically identify surface-exposed antigens as potential vaccine candidates, proteins in the outer membrane fraction of bacteria were separated by two-dimensional gel electrophoresis and subjected to Western blotting with sera from mice experimentally infected with P. mirabilis. Protein spots reactive with sera were identified by mass spectrometry, which in conjunction with the newly completed genome sequence of P. mirabilis HI4320, was used to identify surface-exposed antigens. Culture conditions that may mimic in vivo conditions more closely than Luria broth (growth in human urine and under iron limitation and osmotic stress) were also used. Thirty-seven antigens to which a humoral response had been mounted, including 23 outer membrane proteins, were identified. These antigens are presumably expressed during urinary tract infection. Protein targets that are both actively required for virulence and antigenic may serve as protective antigens for vaccination; thus, five representative antigens were selected for use in virulence studies. Strains of P. mirabilis with mutations in three of the corresponding genes (the PMI0047 gene, rafY, and fadL) were not attenuated in the murine model of urinary tract infection. Putative iron acquisition proteins PMI0842 and PMI2596, however, both contribute to fitness in the urinary tract and thus emerge as vaccine candidates.

Limited Role for Iron Regulation in Coxiella burnetii Pathogenesis

In gram-negative bacteria, iron acquisition proteins are commonly regulated by Fur (ferric uptake regulator), which binds iron-regulated promoters (the Fur box). We hypothesized that Coxiella burnetii requires iron and employs an iron-regulatory system and used various approaches to define a Fur regulon. Cloned C. burnetii fur complemented an Escherichia coli fur deletion mutant. A ferrous iron transporter gene (CBU1766), a putative iron binding protein-encoding gene (CBU0970), and a cation efflux pump gene (CBU1362) were identified by genome annotation and using a Fur titration assay. Bioinformatically predicted Fur box-containing promoters were tested for transcriptional control by iron. Five genes demonstrated at least a twofold induction with minimal iron. Putatively regulated genes were evaluated in a two-plasmid regulator/promoter heterologous expression system. These data suggested a very limited Fur-regulated system in C. burnetii. In an in vitro tissue culture model, a significant increase in bacterial growth was observed with infected cells treated with deferoxamine in comparison to growth under iron-replete conditions. In an iron-overloaded animal model in vivo, the level of bacterial growth detected in the iron-injected animals was significantly decreased in comparison to growth in control animals. In a low-iron-diet animal model, a significant increase in splenomegaly was observed, but no significant change in bacterial growth was identified. The small number of predicted iron acquisition systems, few Fur-regulated genes, and enhanced replication under a decreased iron level predict a requirement of a low level of iron for survival, perhaps to avoid creation of additional reactive oxygen radicals.

Predicting the Interactome of Xanthomonas oryzae pathovar oryzae for target selection and DB service

BackgroundProtein-protein interactions (PPIs) play key roles in various cellular functions. In addition, some critical inter-species interactions such as host-pathogen interactions and pathogenicity occur through PPIs. Phytopathogenic bacteria infect hosts through attachment to host tissue, enzyme secretion, exopolysaccharides production, toxins release, iron acquisition, and effector proteins secretion. Many such mechanisms involve some kind of protein-protein interaction in hosts. Our first aim was to predict the whole protein interaction pairs (interactome) of Xanthomonas oryzae pathovar oryzae (Xoo) that is an important pathogenic bacterium that causes bacterial blight (BB) in rice. We developed a detection protocol to find possibly interacting proteins in its host using whole genome PPI prediction algorithms. The second aim was to build a DB server and a bioinformatic procedure for finding target proteins in Xoo for developing pesticides that block host-pathogen protein interactions within critical biochemical pathways.DescriptionA PPI network in Xoo proteome was predicted by bioinformatics algorithms: PSIMAP, PEIMAP, and iPfam. We present the resultant species specific interaction network and host-pathogen interaction, XooNET. It is a comprehensive predicted initial PPI data for Xoo. XooNET can be used by experimentalists to pick up protein targets for blocking pathological interactions. XooNET uses most of the major types of PPI algorithms. They are: 1) Protein Structural Interactome MAP (PSIMAP), a method using structural domain of SCOP, 2) Protein Experimental Interactome MAP (PEIMAP), a common method using public resources of experimental protein interaction information such as HPRD, BIND, DIP, MINT, IntAct, and BioGrid, and 3) Domain-domain interactions, a method using Pfam domains such as iPfam. Additionally, XooNET provides information on network properties of the Xoo interactome.ConclusionXooNET is an open and free public database server for protein interaction information for Xoo. It contains 4,538 proteins and 26,932 possible interactions consisting of 18,503 (PSIMAP), 3,118 (PEIMAP), and 8,938 (iPfam) pairs. In addition, XooNET provides 3,407 possible interaction pairs between two sets of proteins; 141 Xoo proteins that are predicted as membrane proteins and rice proteomes. The resultant interacting partners of a query protein can be easily retrieved by users as well as the interaction networks in graphical web interfaces. XooNET is freely available from .

Pasteurella multocida pathogenesis: 125 years after Pasteur

Pasteurella multocida was first shown to be the causative agent of fowl cholera by Louis Pasteur in 1881. Since then, this Gram-negative bacterium has been identified as the causative agent of many other economically important diseases in a wide range of hosts. The mechanisms by which these bacteria can invade the mucosa, evade innate immunity and cause systemic disease are slowly being elucidated. Key virulence factors identified to date include capsule and lipopolysaccharide. The capsule is clearly involved in bacterial avoidance of phagocytosis and resistance to complement, while complete lipopolysaccharide is critical for bacterial survival in the host. A number of other virulence factors have been identified by both directed and random mutagenesis, including Pasteurella multocida toxin (PMT), putative surface adhesins and iron acquisition proteins. However, it is likely that many key virulence factors are yet to be identified, including those required for initial attachment and invasion of host cells and for persistence in a relatively nutrient poor and hostile environment.

Molecular genetic anatomy of inter- and intraserotype variation in the human bacterial pathogen group A Streptococcus

In recent years we have studied the relationship between strain genotypes and patient phenotypes in group A Streptococcus (GAS), a model human bacterial pathogen that causes extensive morbidity and mortality worldwide. We have concentrated our efforts on serotype M3 organisms because these strains are common causes of pharyngeal and invasive infections, produce unusually severe invasive infections, and can exhibit epidemic behavior. Our studies have been hindered by the lack of genome-scale phylogenies of multiple GAS strains and whole-genome sequences of multiple serotype M3 strains recovered from individuals with defined clinical phenotypes. To remove some of these impediments, we sequenced to closure the genome of four additional GAS strains and conducted comparative genomic resequencing of 12 contemporary serotype M3 strains representing distinct genotypes and phenotypes. Serotype M3 strains are a single phylogenetic lineage. Strains from asymptomatic throat carriers were significantly less virulent for mice than sterile-site isolates and evolved to a less virulent phenotype by multiple genetic pathways. Strain persistence or extinction between epidemics was strongly associated with presence or absence, respectively, of the prophage encoding streptococcal pyrogenic exotoxin A. A serotype M3 clone significantly underrepresented among necrotizing fasciitis cases has a unique frameshift mutation that truncates MtsR, a transcriptional regulator controlling expression of genes encoding iron-acquisition proteins. Expression microarray analysis of this clone confirmed significant alteration in expression of genes encoding iron metabolism proteins. Our analysis provided unprecedented detail about the molecular anatomy of bacterial strain genotype-patient phenotype relationships.

Iron-activated iron uptake: a positive feedback loop mediated by iron regulatory protein 1.

The love-hate relationship between iron and living matter has generated mechanisms to maintain iron concentration in a narrow range, above and below which deleterious effects occur. At the cellular level, iron homeostasis is accomplished by the activity of the IRP proteins, which, under conditions of iron depletion, up-regulate the expression of the iron acquisition proteins TfR and DMT1. It has been shown that hydrogen peroxide activates IRP1 and that this activation mediates a potentially harmful increase in cell iron uptake. Here we show that IRP1 activity is also induced by iron-mediated oxidative stress. When cells were incubated with up to 20 microM of iron, a typical decrease in IRP1 and IRP2 activity was observed. Interestingly, when iron was further increased to 40 or 80 microM. IRP1 was reactivated in three of the four different cell lines tested, i.e., Caco-2 cells, N2A cells and HepG2 cells. In the fourth cell line (K562) IRP1 activity did not increase, but neither did it decrease. This response to iron was largely abrogated when the antioxidant N-acetyl cysteine was added along with iron to the culture medium. Thus, the effect of iron was mediated by oxidative stress. Increases in IRP1 activity were accompanied by increases in cell iron uptake, an indication that the activated IRP1 was functional in the activation of iron uptake. Hence, this iron-induced iron uptake feedback loop results in the increase of intracellular iron and increased oxidative stress.

Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins.

A collection of large virulence exoproteins, including Ca2+-independent cytolysins, an iron acquisition protein and several adhesins, are secreted by the two-partner secretion (TPS) pathway in various Gram-negative bacteria. The hallmarks of the TPS pathway are the presence of an N-proximal module called the 'secretion domain' in the exoproteins that we have named the TpsA family, and the channel-forming beta-barrel transporter proteins we refer to as the TpsB family. The genes for cognate exoprotein and transporter protein are usually organized in an operon. Specific secretion signals are present in a highly conserved region of the secretion domain of TpsAs. TpsBs probably serve as specific receptors of the TpsA secretion signals and as channels for the translocation of the exoproteins across the outer membrane. A subfamily of transporters also mediates activation of their cognate cytolysins upon secretion. The exoproteins are synthesized as precursors with an N-terminal cleavable signal peptide, and a subset of them carries an extended signal peptide of unknown function. According to our current model, the exoproteins are probably translocated across the cytoplasmic membrane in a Sec-dependent fashion, and their signal peptide is probably processed by a LepB-type signal peptidase. The N-proximal secretion domain directs the exoproteins towards their transporters early, so that translocation across both membranes is coupled. The exoproteins transit through the periplasm in an extended conformation and fold progressively at the cell surface before eventually being released into the extracellular milieu. Several adhesins also undergo extensive proteolytic processing upon secretion. The genes of many new TpsAs and TpsBs are found in recently sequenced genomes, suggesting that the TPS pathway is widespread.

Helicobacter pylori Factors Associated With Disease Development

Although certain factors appear to predispose the host to infection by Helicobacter pylori, clearly the bacterium possesses a well-defined battery of virulence factors that allow the organism to: (1) colonize the gastric mucosa (urease, flagella, adhesins, acidinhibitory protein, iron acquisition proteins, and heat shock proteins); (2) evade host defense (shedding of surface proteins, catalase, superoxide dismutase, and poorly reactive Iipopolysaccharide); and (3) damage host tissue (vacuolating cytotoxin, protease, CagArelated factors, inducers of cytokines, and chemotaxins). Together these factors allow H. pylori to persist in the host, establishing a chronic infection. Although many of these virulence factors are produced by all strains of H. pylori, there are also well-defined pathogenicity islands (contiguous stretches of chromosomal DNA) present in some strains that encode additional proteins including CagA that potentiate virulence. Strains possessing these "virulence cassettes" are isolated more frequently from patients with the more serious clinical manifestations associated with duodenal ulcer than from patients with gastritis alone or nonulcer dyspepsia.