Comparison of the genome sequence of the poultry pathogen Bordetella avium with those of B. bronchiseptica, B. pertussis, and B. parapertussis reveals extensive diversity in surface structures associated with host interaction

Bordetella avium is a pathogen of poultry and is phylogenetically distinct from Bordetella bronchiseptica, Bordetella pertussis, and Bordetella parapertussis, which are other species in the Bordetella genus that infect mammals. In order to understand the evolutionary relatedness of Bordetella species and further the understanding of pathogenesis, we obtained the complete genome sequence of B. avium strain 197N, a pathogenic strain that has been extensively studied. With 3,732,255 base pairs of DNA and 3,417 predicted coding sequences, it has the smallest genome and gene complement of the sequenced bordetellae. In this study, the presence or absence of previously reported virulence factors from B. avium was confirmed, and the genetic bases for growth characteristics were elucidated. Over 1,100 genes present in B. avium but not in B. bronchiseptica were identified, and most were predicted to encode surface or secreted proteins that are likely to define an organism adapted to the avian rather than the mammalian respiratory tracts. These include genes coding for the synthesis of a polysaccharide capsule, hemagglutinins, a type I secretion system adjacent to two very large genes for secreted proteins, and unique genes for both lipopolysaccharide and fimbrial biogenesis. Three apparently complete prophages are also present. The BvgAS virulence regulatory system appears to have polymorphisms at a poly(C) tract that is involved in phase variation in other bordetellae. A number of putative iron-regulated outer membrane proteins were predicted from the sequence, and this regulation was confirmed experimentally for five of these.     

Structural characteristics and plant-beneficial effects of bacteria colonizing the shoots of field grown conventional and genetically modified T4-lysozyme producing potatoes

Genetically modified potatoes expressing antibacterial protein T4 lysozyme may offer effective control strategies for bacterial pathogens causing severe potato diseases. Apart from this beneficial effect, it is very important to investigate such engineered potatoes carefully for potential adverse effects on potato-associated bacteria which frequently exhibit plant beneficial functions such as plant growth promotion and antagonism towards pathogens invading the plant. Two field experiments were carried out in Spain to analyze the potential effects of conventional and genetically modified T4-lysozyme producing potatoes on shoot-associated bacteria. The first baseline field trial 2002 was performed in Meliana in which three conventional potato lines, Achirana Inta, Desirée, and Merkur, were cultivated and sampled at flowering. The second field trial was conducted in Cella in 2003 in order to compare the effects of a senescent transgenic, T4 lysozyme expressing potato trait, Desirée DL 12, with its isogenic, non-transformed parental line Desirée. Structural characteristics of potato shoot-associated bacteria was assayed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and dominant community members within T-RFLP profiles were identified by sequence analysis of generated 16S rRNA gene libraries. Cultivable bacteria isolated from shoots of potatoes grown in the Meliana field trial were monitored for antibiosis against Ralstonia solanacearum, whereas isolates derived from shoots of potatoes cultivated in the Cella trial were screened for antagonism against Ralstonia solanacearum and Rhizoctonia solani, and for 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production. Determined antagonists were identified by 16S rRNA gene analysis. All potato traits hosted a cultivar-specific community of bacteria with antagonism against the pathogens and/or potential to produce ACC deaminase. Several antagonists obtained from the Cella field potatoes were also observed as ACC deaminase producers. Community profiling revealed a greater diversity differentiation between the senescent T4 lysozyme expressing and parental Desirée lines grown in the Cella field as compared to the variations between the three flowering conventional lines cultivated in the Meliana field trial. Effects of the two varying field sites and different vegetation stages were greater than those of T4 lysozyme when investigating the community composition of bacteria colonizing the shoots of the Desirée line cultivated in both field trials.     

Correlated fragile site expression allows the identification of candidate fragile genes involved in immunity and associated with carcinogenesis

Background  

Common fragile sites (cfs) are specific regions in the human genome that are particularly prone to genomic instability under conditions of replicative stress. Several investigations support the view that common fragile sites play a role in carcinogenesis. We discuss a genome-wide approach based on graph theory and Gene Ontology vocabulary for the functional characterization of common fragile sites and for the identification of genes that contribute to tumour cell biology.     

Novel scabies mite serpins inhibit the three pathways of the human complement system

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.     

VEGF signalling enhances lesion burden in KRIT1 deficient mice

The exact molecular mechanisms underlying CCM pathogenesis remain a complicated and controversial topic. Our previous work illustrated an important VEGF signalling loop in KRIT1 depleted endothelial cells. As VEGF is a major mediator of many vascular pathologies, we asked whether the increased VEGF signalling downstream of KRIT1 depletion was involved in CCM formation. Using an inducible KRIT1 endothelial‐specific knockout mouse that models CCM, we show that VEGFR2 activation plays a role in CCM pathogenesis in mice. Inhibition of VEGFR2 using a specific inhibitor, SU5416, significantly decreased the number of lesions formed and slightly lowered the average lesion size. Notably, VEGFR2 inhibition also decreased the appearance of lesion haemorrhage as denoted by the presence of free iron in adjacent tissues. The presence of free iron correlated with increased microvessel permeability in both skeletal muscle and brain, which was completely reversed by SU5416 treatment. Finally, we show that VEGFR2 activation is a common downstream consequence of KRIT1, CCM2 and CCM3 loss of function, though the mechanism by which VEGFR2 activation occurs likely varies. Thus, our study clearly shows that VEGFR2 activation downstream of KRIT1 depletion enhances the severity of CCM formation in mice, and suggests that targeting VEGF signalling may be a potential future therapy for CCM.     

Differential gene expression of pathogens inside infected hosts

DNA microarray is a useful technology for studying differential gene expression in the context of microbe-host interactions. This review concentrates on recent findings of the survival strategies of three intracellular pathogens: Shigella flexneri, Salmonella enterica serovar Typhimurium and Mycobacterium tuberculosis.     

Protein Kinase C and Extracellular Signal-Regulated Kinase Regulate Movement,Attachment, Pairing and Egg Release in Schistosoma mansoni

Protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using ‘smart’ antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated) throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis'' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.     

Reduced prevalence of Epstein-Barr virus-related lymphocryptovirus infection in sera from a new world primate

The recent discovery of an Epstein-Barr virus (EBV)-related lymphocryptovirus (LCV) naturally infecting common marmosets demonstrated that gamma-1 herpesviruses are not limited to human and Old World nonhuman primate hosts. We developed serologic assays to detect serum antibodies against lytic- and latent-infection marmoset LCV antigens in order to perform the first seroepidemiologic study of LCV infection in New World primates. In three different domestic colonies and in animals recently captured from the wild, we found that the seroprevalence of marmoset LCV infection was not as ubiquitous as with EBV or Old World LCV. These biologic differences in LCV infection of New World versus human and Old World primate hosts correlate with the evolution of the LCV viral gene repertoire.