Restrictions on the Importation of Zebrafish into Canada Associated with Spring Viremia of Carp Virus.

The zebrafish model system is helping researchers improve the health and welfare of people and animals and has become indispensable for advancing biomedical research. As genetic engineering is both resource intensive and time-consuming, sharing successfully developed genetically modified zebrafish lines throughout the international community is critical to research efficiency and to maximizing the millions of dollars in research funding. New restrictions on importation of zebrafish into Canada based on putative susceptibility to infection by the spring viremia of carp virus (SVCV) have been imposed on the scientific community. In this commentary, we review the disease profile of SVCV in fish, discuss the findings of the Canadian government's scientific assessment, how the interpretations of their assessment differ from that of the Canadian research community, and describe the negative impact of these regulations on the Canadian research community and public as it pertains to protecting the health of Canadians.

The Burkholderia cenocepacia sensor kinase hybrid AtsR is a global regulator modulating quorum-sensing signalling.

Burkholderia cenocepacia is commonly found in the environment and also as an important opportunistic pathogen infecting patients with cystic fibrosis. Successful infection by this bacterium requires coordinated expression of virulence factors, which is achieved through different quorum sensing (QS) regulatory systems. Biofilm formation and Type 6 secretion system (T6SS) expression in B. cenocepacia K56-2 are positively regulated by QS and negatively regulated by the sensor kinase hybrid AtsR. This study reveals that in addition to affecting biofilm and T6SS activity, the deletion of atsR in B. cenocepacia leads to overproduction of other QS-regulated virulence determinants including proteases and swarming motility. Expression of the QS genes, cepIR and cciIR, was upregulated in the ΔatsR mutant and resulted in early and increased N-acylhomoserine lactone (AHL) production, suggesting that AtsR plays a role in controlling the timing and fine-tuning of virulence gene expression by modulating QS signalling. Furthermore, a ΔatsRΔcepIΔcciI mutant could partially upregulate the same virulence determinants indicating that AtsR also modulates the expression of virulence genes by a second mechanism, independently of any AHL production. Together, our results strongly suggest that AtsR is a global virulence regulator in B. cenocepacia.

A unique regulator contributes to quorum sensing and virulence in Burkholderia cenocepacia.

Burkholderia cenocepacia causes chronic and life-threatening respiratory infections in immunocompromized people. The B. cenocepacia N-acyl-homoserine lactone (AHL)-dependent quorum sensing system relies on the production of AHLs by the synthases CepI and CciI while CepR, CciR and CepR2 control expression of many genes important for pathogenesis. Downstream from, and co-transcribed with cepI, lies BCAM1871 encoding a hypothetical protein that was uncharacterized prior to this study. Orthologs of B. cenocepacia BCAM1871 are uniquely found in Burkholderia spp and are conserved in their genomic locations in pathogenic Burkholderia. We observed significant effects on AHL activity upon mutation or overexpression of BCAM1871, although these effects were more subtle than those observed for CepI indicating BCAM1871 acts as an enhancer of AHL activity. Transcription of cepI, cepR and cciIR was significantly reduced in the BCAM1871 mutant. Swimming and swarming motilities as well as transcription of fliC, encoding flagellin, were significantly reduced in the BCAM1871 mutant. Protease activity and transcription of zmpA and zmpB, encoding extracellular zinc metalloproteases, were undetectable in the BCAM1871 mutant indicating a more significant effect of mutating BCAM1871 than cepI. Exogenous addition of OHL restored cepI, cepR and fliC transcription but had no effect on motility, protease activity or zmpA or zmpB transcription suggesting AHL-independent effects. The BCAM1871 mutant exhibited significantly reduced virulence in rat chronic respiratory and nematode infection models. Gene expression and phenotypic assays as well as vertebrate and invertebrate infection models showed that BCAM1871 significantly contributes to pathogenesis in B. cenocepacia.

The Burkholderia cenocepacia LysR-type transcriptional regulator ShvR influences expression of quorum-sensing, protease, type II secretion, and afc genes.

Burkholderia cenocepacia is a significant opportunistic pathogen in individuals with cystic fibrosis. ShvR, a LysR-type transcriptional regulator, has previously been shown to influence colony morphology, biofilm formation, virulence in plant and animal infection models, and some quorum-sensing-dependent phenotypes. In the present study, it was shown that ShvR negatively regulates its own expression, as is typical for LysR-type regulators. The production of quorum-sensing signal molecules was detected earlier in growth in the shvR mutant than in the wild type, and ShvR repressed expression of the quorum-sensing regulatory genes cepIR and cciIR. Microarray analysis and transcriptional fusions revealed that ShvR regulated over 1,000 genes, including the zinc metalloproteases zmpA and zmpB. The shvR mutant displayed increased gene expression of the type II secretion system and significantly increased protease and lipase activities. Both ShvR and CepR influence expression of a 24-kb genomic region adjacent to shvR that includes the afcA and afcC operons, required for the production of an antifungal agent; however, the reduction in expression was substantially greater in the shvR mutant than in the cepR mutant. Only the shvR mutation resulted in reduced antifungal activity against Rhizoctonia solani. ShvR, but not CepR, was shown to directly regulate expression of the afcA and afcC promoters. In summary, ShvR was determined to have a significant influence on the expression of quorum-sensing, protease, lipase, type II secretion, and afc genes.

Burkholderia cenocepacia differential gene expression during host-pathogen interactions and adaptation to the host environment.

Members of the Burkholderia cepacia complex (Bcc) are important in medical, biotechnological, and agricultural disciplines. These bacteria naturally occur in soil and water environments and have adapted to survive in association with plants and animals including humans. All Bcc species are opportunistic pathogens including Burkholderia cenocepacia that causes infections in cystic fibrosis and chronic granulomatous disease patients. The adaptation of B. cenocepacia to the host environment was assessed in a rat chronic respiratory infection model and compared to that of high cell-density in vitro grown cultures using transcriptomics. The distribution of genes differentially expressed on chromosomes 1, 2, and 3 was relatively proportional to the size of each genomic element, whereas the proportion of plasmid-encoded genes differentially expressed was much higher relative to its size and most genes were induced in vivo. The majority of genes encoding known virulence factors, components of types II and III secretion systems and chromosome 2-encoded type IV secretion system were similarly expressed between in vitro and in vivo environments. Lower expression in vivo was detected for genes encoding N-acyl-homoserine lactone synthase CepI, orphan LuxR homolog CepR2, zinc metalloproteases ZmpA and ZmpB, LysR-type transcriptional regulator ShvR, nematocidal protein AidA, and genes associated with flagellar motility, Flp type pilus formation, and type VI secretion. Plasmid-encoded type IV secretion genes were markedly induced in vivo. Additional genes induced in vivo included genes predicted to be involved in osmotic stress adaptation or intracellular survival, metal ion, and nutrient transport, as well as those encoding outer membrane proteins. Genes identified in this study are potentially important for virulence during host-pathogen interactions and may be associated with survival and adaptation to the host environment during chronic lung infections.

Reciprocal regulation by the CepIR and CciIR quorum sensing systems in Burkholderia cenocepacia.

BACKGROUND: Burkholderia cenocepacia belongs to a group of closely related organisms called the B. cepacia complex (Bcc) which are important opportunistic human pathogens. B. cenocepacia utilizes a mechanism of cell-cell communication called quorum sensing to control gene expression including genes involved in virulence. The B. cenocepacia quorum sensing network includes the CepIR and CciIR regulatory systems. RESULTS: Global gene expression profiles during growth in stationary phase were generated using microarrays of B. cenocepacia cepR, cciR and cepRcciIR mutants. This is the first time CciR was shown to be a global regulator of quorum sensing gene expression. CepR was primarily responsible for positive regulation of gene expression while CciR generally exerted negative gene regulation. Many of the genes that were regulated by both quorum sensing systems were reciprocally regulated by CepR and CciR. Microarray analysis of the cepRcciIR mutant suggested that CepR is positioned upstream of CciR in the quorum sensing hierarchy in B. cenocepacia. A comparison of CepIR-regulated genes identified in previous studies and in the current study showed a substantial amount of overlap validating the microarray approach. Several novel quorum sensing-controlled genes were confirmed using qRT-PCR or promoter::lux fusions. CepR and CciR inversely regulated flagellar-associated genes, the nematocidal protein AidA and a large gene cluster on Chromosome 3. CepR and CciR also regulated genes required for iron transport, synthesis of extracellular enzymes and surface appendages, resistance to oxidative stress, and phage-related genes. CONCLUSION: For the first time, the influence of CciIR on global gene regulation in B. cenocepacia has been elucidated. Novel genes under the control of the CepIR and CciIR quorum sensing systems in B. cenocepacia have been identified. The two quorum sensing systems exert reciprocal regulation of many genes likely enabling fine-tuned control of quorum sensing gene expression in B. cenocepacia strains carrying the cenocepacia island.

A Burkholderia cenocepacia orphan LuxR homolog is involved in quorum-sensing regulation.

Burkholderia cenocepacia utilizes quorum sensing to control gene expression, including the expression of genes involved in virulence. In addition to CepR and CciR, a third LuxR homolog, CepR2, was found to regulate gene expression and virulence factor production. All B. cenocepacia strains examined contained this orphan LuxR homolog, which was not associated with an adjacent N-acyl-homoserine lactone synthase gene. Expression of cepR2 was negatively autoregulated and was negatively regulated by CciR in strain K56-2. Microarray analysis and quantitative reverse transcription-PCR determined that CepR2 did not influence expression of cepIR or cciIR. However, in strain K56-2, CepR2 negatively regulated expression of several known quorum-sensing-controlled genes, including genes encoding zinc metalloproteases. CepR2 exerted positive and negative regulation on genes on three chromosomes, including strong negative regulation of a gene cluster located adjacent to cepR2. In strain H111, which lacks the CciIR quorum-sensing system, CepR2 positively regulated pyochelin production by controlling transcription of one of the operons required for the biosynthesis of the siderophore in an N-acyl-homoserine lactone-independent manner. CepR2 activation of a luxI promoter was demonstrated in a heterologous Escherichia coli host, providing further evidence that CepR2 can function in the absence of signaling molecules. This study demonstrates that the orphan LuxR homolog CepR2 contributes to the quorum-sensing regulatory network in two distinct strains of B. cenocepacia.

Pseudomonas aeruginosa RsmA plays an important role during murine infection by influencing colonization, virulence, persistence, and pulmonary inflammation.

The ability of Pseudomonas aeruginosa to cause a broad range of infections in humans is due, at least in part, to its adaptability and its capacity to regulate the expression of key virulence genes in response to specific environmental conditions. Multiple two-component response regulators have been shown to facilitate rapid responses to these environmental conditions, including the coordinated expression of specific virulence determinants. RsmA is a posttranscriptional regulatory protein which controls the expression of a number of virulence-related genes with relevance for acute and chronic infections. Many membrane-bound sensors, including RetS, LadS, and GacS, are responsible for the reciprocal regulation of genes associated with acute infection and chronic persistence. In P. aeruginosa this is due to sensors influencing the expression of the regulatory RNA RsmZ, with subsequent effects on the level of free RsmA. While interactions between an rsmA mutant and human airway epithelial cells have been examined in vitro, the role of RsmA during infection in vivo has not been determined yet. Here the function of RsmA in both acute and chronic models of infection was examined. The results demonstrate that RsmA is involved in initial colonization and dissemination in a mouse model of acute pneumonia. Furthermore, while loss of RsmA results in reduced colonization during the initial stages of acute infection, the data show that mutation of rsmA ultimately favors chronic persistence and results in increased inflammation in the lungs of infected mice.

Manipulation of host Kruppel-like factor (KLF) function by exotoxins from diverse bacterial pathogens.

Diverse pathogenic bacteria have developed similar mechanisms to subvert host cell responses. In this Progress article, we focus on bacterial virulence factors with different enzymatic activities that can increase the expression of the Kruppel-like factor (KLF) family of mammalian transcriptional regulators through their ability to modify the activity of a common host-cell target - the Rho protein family. By using a common virulence strategy, both Gram-negative and Gram-positive pathogens exploit the KLF regulatory cascade to modulate nuclear factor kappaB activation, pro-inflammatory cytokine expression, actin cytoskeletal dynamics and phagocytosis.

Pseudomonas aeruginosa infection of airway epithelial cells modulates expression of Kruppel-like factors 2 and 6 via RsmA-mediated regulation of type III exoenzymes S and Y.

Pseudomonas aeruginosa is an important opportunistic pathogen which is capable of causing both acute and chronic infections in immunocompromised patients. Successful adaptation of the bacterium to its host environment relies on the ability of the organism to tightly regulate gene expression. RsmA, a small RNA-binding protein, controls the expression of a large number of virulence-related genes in P. aeruginosa, including those encoding the type III secretion system and associated effector proteins, with important consequences for epithelial cell morphology and cytotoxicity. In order to examine the influence of RsmA-regulated functions in the pathogen on gene expression in the host, we compared global expression profiles of airway epithelial cells in response to infection with P. aeruginosa PAO1 and an rsmA mutant. The RsmA-dependent response of host cells was characterized by significant changes in the global transcriptional pattern, including the increased expression of two Kruppel-like factors, KLF2 and KLF6. This increased expression was mediated by specific type III effector proteins. ExoS was required for the enhanced expression of KLF2, whereas both ExoS and ExoY were required for the enhanced expression of KLF6. Neither ExoT nor ExoU influenced the expression of the transcription factors. Additionally, the increased gene expression of KLF2 and KLF6 was associated with ExoS-mediated cytotoxicity. Therefore, this study identifies for the first time the human transcription factors KLF2 and KLF6 as targets of the P. aeruginosa type III exoenzymes S and Y, with potential importance in host cell death.

The posttranscriptional regulator RsmA plays a role in the interaction between Pseudomonas aeruginosa and human airway epithelial cells by positively regulating the type III secretion system.

Posttranscriptional regulation of certain virulence-related genes in Pseudomonas aeruginosa is brought about by RsmA, a small RNA-binding protein. During interaction with airway epithelial cells, RsmA promoted actin depolymerization, cytotoxicity, and anti-internalization of P. aeruginosa by positively regulating the virulence-associated type III secretion system.