Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Melanin as a virulence factor of Paracoccidioides brasiliensis and other dimorphic pathogenic fungi: a minireview

Melanin pigments are substances produced by a broad variety of pathogenic microorganisms, including bacteria, fungi, and helminths. Microbes predominantly produce melanin pigment via tyrosinases, laccases, catecholases, and the polyketide synthase pathway. In fungi, melanin is deposited in the cell wall and cytoplasm, and melanin particles (""ghosts"") can be isolated from these fungi that have the same size and shape of the original cells. Melanin has been reported in several human pathogenic dimorphic fungi including Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides posadasii. Melanization appears to contribute to virulence by reducing the susceptibility of melanized fungi to host defense mechanisms and antifungal drugs.

Immune-Deficient Drosophila melanogaster: A Model for the Innate Immune Response to Human Fungal Pathogens

We explored the host-pathogen interactions of the human opportunistic fungus Candida albicans using Drosophila melanogaster. We established that a Drosophila strain devoid of functional Toll receptor is highly susceptible to the human pathogen C. albicans. Using this sensitive strain, we have been able to show that a set of specific C. albicans mutants of different virulence in mammalian infection models are also impaired in virulence in Drosophila and remarkably display the same rank order of virulence. This immunodeficient insect model also revealed virulence properties undetected in an immunocompetent murine model of infection. The genetic systems available in both host and pathogen will enable the identification of host-specific components and C. albicans genes involved in the host-fungal interplay.

Identification and characterization of virulence determinants in the Lyme disease spirochete Borrelia burgdorferi

Borrelia burgdorferi is the etiological agent of Lyme disease, the most common tick-borne disease in the United States. Although the most frequently reported symptom is arthritis, patients can also experience severe cardiac, neurologic, and dermatologic abnormalities. The identification of virulence determinants in infectious B. burgdorferi strains has been limited by their slow growth rate, poor transformability, and general lack of genetic tools. The present study demonstrates the use of transposon mutagenesis for the identification of infectivity-related factors in infectious B. burgdorferi, examines the potential role for chemotaxis in mammalian infection, and describes the development of a novel method for the analysis of recombination events at the Ids antigenic variation locus. A pool of Himar1 mutants was isolated using an infectious B. burgdorferi clone and the transposon vector pMarGent. Clones exhibiting reduced infectivity in mice possessed insertions in virulence determinants putatively involved in host survival and dissemination. These results demonstrated the feasibility of extensive transposon mutagenesis studies for the identification of additional infectivity-related factors. mcp-5 mutants were chosen for further study to determine the role of chemotaxis during infection. Animal studies indicated that mcp-5 mutants exhibited a reduced infectivity potential, and suggested a role for mcp-5 during the early stages of infection. An in vitro phenotype for an mcp-5 mutant was not detected. Genetic complementation of an mcp-5 mutant resulted in restoration of Mcp-5 expression in the complemented clone, as demonstrated by western blotting, but the organisms were not infectious in mice. We believe this result is a consequence of differences in expression between genes located on the linear chromosome and genes present on the circular plasmid used for trans-complementation. Overall, this work implicates mcp-5 as an important determinant of mammalian infectivity. Finally, the development of a computer-assisted method for the analysis of recombination events occurring at the B. burgdorferi vls antigenic variation locus has proven highly valuable for the detailed examination of vls gene conversion. The studies described here provide evidence for the importance of chemotaxis during infection in mice and demonstrate advances in both genetic and computational approaches for the further characterization of the Lyme disease spirochete. ^

Molecular pathogenesis of H5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif

The emergence of H5N1 highly pathogenic avian influenza has caused a heavy socio-economic burden through culling of poultry to minimise human and livestock infection. Although human infections with H5N1 have to date been limited, concerns for the pandemic potential of this zoonotic virus have been greatly intensified following experimental evidence of aerosol transmission of H5N1 viruses in a mammalian infection model. In this review, we discuss the dominance of the haemagglutinin cleavage site motif as a pathogenicity determinant, the host-pathogen molecular interactions driving cleavage activation, reverse genetics manipulations and identification of residues key to haemagglutinin cleavage site functionality and the mechanisms of cell and tissue damage during H5N1 infection. We specifically focus on the disease in chickens, as it is in this species that high pathogenicity frequently evolves and from which transmission to the human population occurs. With >75% of emerging infectious diseases being of zoonotic origin, it is necessary to understand pathogenesis in the primary host to explain spillover events into the human population.

An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: Proteins associated with invasion and infection of the mammalian host

To infect their mammalian hosts, Fasciola hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum, and penetrate the liver. After migrating through and feeding on the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and produce eggs. Here we integrated a transcriptomics and proteomics approach to profile Fasciola secretory proteins that are involved in host-pathogen interactions and to correlate changes in their expression with the migration of the parasite. Prediction of F. hepatica secretory proteins from 14,031 expressed sequence tags (ESTs) available from the Wellcome Trust Sanger Centre using the semiautomated EST2Secretome pipeline showed that the major components of adult parasite secretions are proteolytic enzymes including cathepsin L, cathepsin B, and asparaginyl endopeptidase cysteine proteases as well as novel trypsin-like serine proteases and carboxypeptidases. Proteomics analysis of proteins secreted by infective larvae, immature flukes, and adult F. hepatica showed that these proteases are developmentally regulated and correlate with the passage of the parasite through host tissues and its encounters with different host macromolecules. Proteases such as FhCL3 and cathepsin B have specific functions in larvae activation and intestinal wall penetration, whereas FhCL1, FhCL2, and FhCL5 are required for liver penetration and tissue and blood feeding. Besides proteases, the parasites secrete an array of antioxidants that are also highly regulated according to their migration through host tissues. However, whereas the proteases of F. hepatica are secreted into the parasite gut via a classical endoplasmic reticulum/Golgi pathway, we speculate that the antioxidants, which all lack a signal sequence, are released via a non-classical trans-tegumental pathway.

UNDERSTANDING THE RELATIONSHIP BETWEEN ONCOLYTIC AD5 DELETED E1b55KDA LYTIC INFECTION AND P53 IN MAMMALIAN CELLS

Adenoviruses are the most commonly used in the development of oncolytic therapy. Oncolytic adenoviruses are genetically modified to selectivity replicate in and kill tumor cells. The p53 molecule is a tumor suppressor protein that responds to viral infection through the activation of apoptosis, which is inhibited by adenovirus E1B55kDa protein leading to progressive viral lytic cycle. The non-specificity of replication has limited the use of wild type adenovirus in cancer therapy. This issue was resolved by using an E1b deleted Ad that can only replicate in cells with a deficiency in the p53 protein, a common feature of most cancer cells. Although demonstrating a moderate success rate, E1b55kDa deleted Ad has not been approved as a standard therapy for all cancer types. Several studies have revealed that E1b deleted Ad replication was independent of p53 status in the cell, as the virus replicated better in some p53 deficient cancers more than others. However, this mechanism has not been investigated deeply. Therefore, the objective of this study is to understand the relationship between p53 status, levels and functional activity, and oncolytic Ad5dlE1b55kDa replication efficiency. Firstly, five transient p53 expression vectors that contain different regulatory elements were engineered and then evaluated in H1299, HEK293 and HeLa cell lines. Data indicated that vector that contains the MARs and HPRE regulatory elements achieved the highest stability of p53 expression. Secondly, we used these vectors to examine the effect of various p53 expression levels on the replication efficiency of oncolytic Ad5dlE1b55kDa. We found that the level of p53 in the cell had an insignificant effect on the oncolytic viruses’ replication. However, the functional activity of p53 had a significant effect on its replication, as Ad5dlE1b55kDa was shown to have selective activity in H1299 cells (p53-null). In contrast, a decrease in viral replication was found in HeLa cells (p53-positive). Finally, the effect of p53’s functional activity on the replication efficiency of oncolytic Ad5dlE1b55kDa was examined. Viral growth was evaluated in H1299 cells expressing number of p53 mutants. P53-R175H mutant successfully rescued viral growth by allowing the virus to exert its mechanism of selectivity. The mechanism entailed deregulating the expression of specific genes, cell cycle and apoptosis, in the p53 pathway to promote its production leading to efficient oncolytic effect. These results confirmed that oncolytic Ad5dlE1b55kDa sensitivity is mutation-type specific. Therefore, before it is applied clinically as cancer therapy for p53 deficient tumors, the type of p53 mutation must be determined for efficient antitumor effect.

Caspase-3-dependent phagocyte death during systemic Salmonella enterica serovar Typhimurium infection of mice.

Growth of Salmonella enterica in mammalian tissues results from continuous spread of bacteria to new host cells. Our previous work indicated that infective S. enterica are liberated from host cells via stochastic necrotic burst independently of intracellular bacterial numbers. Here we report that liver phagocytes can undergo apoptotic caspase-3-mediated cell death in vivo, with apoptosis being a rare event, more prevalent in heavily infected cells. The density-dependent apoptotic cell death is likely to constitute an alternative mechanism of bacterial spread as part of a bet-hedging strategy, ensuring an ongoing protective intracellular environment in which some bacteria can grow and persist.

Characterization and expression analysis of Paralichthys olivaceus voltage-dependent anion channel (VDAC) gene in response to virus infection

Voltage-dependent anion channel (VDAC, also known as mitochondrial porin) is acknowledged to play an important role in stress-induced mammalian apoptosis. In this study, Paralichthys olivaceus VDAC (PoVDAC) gene was identified as a virally induced gene from Scophthalmus Maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). The full length of PoVDAC cDNA is 1380 bp with an open reading frame of 852 bp encoding a 283 amino acid protein. The deduced PoVDAC contains one alpha-helix, 13 transmembrane beta-strands and one eukaryotic mitochondrial porin signature motif. Constitutive expression of PoVDAC was confirmed in all tested tissues by real-time PCR. Further expression analysis revealed PoVDAC mRNA was upregulated by viral infection. We prepared fish antiserum against recombinant VDAC proteins and detected the PoVDAC in heart lysates from flounder as a 32 kDa band on western blot. Overexpression of PoVDAC in fish cells induced apoptosis. Immunofluoresence localization indicated that the significant distribution changes of PoVDAC have occurred in virus-induced apoptotic cells. This is the first report on the inductive expression of VDAC by viral infection, suggesting that PoVDAC might be mediated flounder antiviral immune response through induction of apoptosis. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular characterization and IFN signal pathway analysis of Carassius auratus CaSTAT1 identified from the cultured cells in response to virus infection

Type I interferon (IFN) exerts its pleiotropic effects mainly through the JAK-STAT signaling pathway, which is presently best described in mammals. By subtractive suppression hybridization, two fish signaling factors, JAK1 and STAT1, had been identified in the IFN-induced crucian carp Carassius auratus L. blastulae embryonic (CAB) cells after treatment with UV-inactivated grass carp hemorrhagic virus (GCHV). Further, the full-length cDNA of STAT1, termed CaSTAT1, was obtained. It contains 2926 bp and encodes a protein of 718 aa. CaSTAT1 is most similar to rat STAT1 with 59% identity overall and displays all highly conserved domains that the STAT family possesses. Like human STAT1beta, it lacks the C-terminus acting as transcriptional activation domain in mammals. By contrast, only a single transcript was detected in virus-induced CAB cells. Expression analysis showed that CaSTAT1 could be activated by stimulation of CAB cells with poly I:C, active GCHV, UV-inactivated GCHV or CAB IFN, and displayed diverse expression patterns similar to that of mammalian STATI. Additionally, the expression of an antiviral gene CaMx1 was also induced under the same conditions, and expression difference between CaSTAT1 and CaMx1 was revealed by induction of CAB IFN. These results provide molecular evidence supporting the notion that the fish IFN signaling transduction pathway is similar to that in mammals. Fish IFN exerts its multiple functions, at least antiviral action, through a JAK-STAT pathway. (C) 2004 Elsevier Ltd. All rights reserved.

Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved

Two genes on A/J chromosome 18 are associated with susceptibility to Staphylococcus aureus infection by combined microarray and QTL analyses.

Although it has recently been shown that A/J mice are highly susceptible to Staphylococcus aureus sepsis as compared to C57BL/6J, the specific genes responsible for this differential phenotype are unknown. Using chromosome substitution strains (CSS), we found that loci on chromosomes 8, 11, and 18 influence susceptibility to S. aureus sepsis in A/J mice. We then used two candidate gene selection strategies to identify genes on these three chromosomes associated with S. aureus susceptibility, and targeted genes identified by both gene selection strategies. First, we used whole genome transcription profiling to identify 191 (56 on chr. 8, 100 on chr. 11, and 35 on chr. 18) genes on our three chromosomes of interest that are differentially expressed between S. aureus-infected A/J and C57BL/6J. Second, we identified two significant quantitative trait loci (QTL) for survival post-infection on chr. 18 using N(2) backcross mice (F(1) [C18A]xC57BL/6J). Ten genes on chr. 18 (March3, Cep120, Chmp1b, Dcp2, Dtwd2, Isoc1, Lman1, Spire1, Tnfaip8, and Seh1l) mapped to the two significant QTL regions and were also identified by the expression array selection strategy. Using real-time PCR, 6 of these 10 genes (Chmp1b, Dtwd2, Isoc1, Lman1, Tnfaip8, and Seh1l) showed significantly different expression levels between S. aureus-infected A/J and C57BL/6J. For two (Tnfaip8 and Seh1l) of these 6 genes, siRNA-mediated knockdown of gene expression in S. aureus-challenged RAW264.7 macrophages induced significant changes in the cytokine response (IL-1 beta and GM-CSF) compared to negative controls. These cytokine response changes were consistent with those seen in S. aureus-challenged peritoneal macrophages from CSS 18 mice (which contain A/J chromosome 18 but are otherwise C57BL/6J), but not C57BL/6J mice. These findings suggest that two genes, Tnfaip8 and Seh1l, may contribute to susceptibility to S. aureus in A/J mice, and represent promising candidates for human genetic susceptibility studies.

Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.

UNLABELLED: The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. IMPORTANCE: Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.