Global changes in cellular gene expression during bacteriophage PRD1 infection

Virus-induced changes in cellular gene expression and host physiology have been studied extensively. Still, there are only a few analyses covering the entire viral replication cycle and whole-host gene pool expression at the resolution of a single gene. Here we report changes in Escherichia coli gene expression during bacteriophage PRD1 infection using microarray technology. Relative mRNA levels were systematically measured for over 99% of the host open reading frames throughout the infection cycle. Although drastic modifications could be detected in the expression of individual genes, global changes at the whole-genome level were moderate. Notably, the majority of virus-induced changes took place only after the synthesis of virion components, indicating that there is no major reprogramming of the host during early infection. The most highly induced genes encoded chaparones and other stress-inducible proteins.     

A mutation in the latency-related gene of bovine herpesvirus 1 inhibits protein expression from open reading frame 2 and an adjacent reading frame during productive infection

The latency-related (LR) gene of bovine herpesvirus 1 (BHV-1) is abundantly expressed during latency. A mutant BHV-1 strain that contains three stop codons at the 5′ terminus of the LR gene (LR mutant) does not reactivate from latency. This study demonstrates that the LR mutant does not express open reading frame 2 or an adjacent reading frame that lacks an initiating ATG (reading frame C). Since the LR mutant and wild-type BHV-1 express similar levels of LR RNA, we conclude that LR protein expression plays an important role in regulating the latency reactivation cycle in cattle.     

Analysis of host responses to microbial infection using gene expression profiling

Gene expression profiling offers new opportunities for understanding host-cell responses to microbial pathogens and their products. Current strategies involve either first identifying mRNAs that differ in their expression status under different experimental conditions and later defining the identity of the respective genes (for example, differential display or serial analysis of gene expression), or alternatively assessing changes in the expression of already defined genes (for example, cDNA or oligonucleotide microarrays). Early studies indicate the power of gene expression profiling for providing new insights into groups of genes whose expression is altered during the course of host-microbe interactions, and for the discovery of cellular genes that were not previously recognized to be regulated by infection.     

Evaluation of protein expression in bovine bronchoalveolar fluid following challenge with Mannheimia haemolytica

Proteomics analysis of bovine bronchoalveolar fluid (BAF) following induction of pneumonia with Mannheimia haemolytica using nanoflow liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) resulted in the identification of 88 unique proteins. Proteins detected in BAF included antimicrobial peptides (AMPs), complement factors, acute-phase proteins, protease inhibitors, and proteins involved in oxidation-reduction. Notwithstanding biological variation, differences in relative protein abundance, determined using normalized peptide counts, were detected for select proteins in BAF from genuinely infected versus sham-infected animals. To demonstrate the applicability of using normalized peptide counts to assess protein expression trends, LC-MS/MS data for the acute-phase protein haptoglobin (HPT) were compared with ELISA data, and statistical evaluation of the relationship between the data revealed a strong measure of association. Differences were detected between sham- and genuinely infected animals for haptoglobin, as well as the AMPs cathelicidin-1 and cathelicidin-4, and inter-α-trypsin inhibitor heavy chain-4, a fairly novel protein involved in the acute phase response. Though the small sample size limited the scope of the inferences, the results indicate the likely importance of AMPs and acute-phase proteins during respiratory infection, and provide additional information regarding potential mechanisms involved in the bovine mucosal barrier defense.     

Gene expression analysis of host innate immune responses during Lethal H5N1 infection in ferrets

How viral and host factors contribute to the severe pathogenicity of the H5N1 subtype of avian influenza virus infection in humans is poorly understood. We identified three clusters of differentially expressed innate immune response genes in lungs from H5N1 (A/Vietnam/1203/04) influenza virus-infected ferrets by oligonucleotide microarray analysis. Interferon response genes were more strongly expressed in H5N1-infected ferret lungs than in lungs from ferrets infected with the less pathogenic H3N2 subtype. In particular, robust CXCL10 gene expression in H5N1-infected ferrets led us to test the pathogenic role of signaling via CXCL10's cognate receptor, CXCR3, during H5N1 influenza virus infection. Treatment of H5N1-infected ferrets with the drug AMG487, a CXCR3 antagonist, resulted in a reduction of symptom severity and delayed mortality compared to vehicle treatment. We contend that unregulated host interferon responses are at least partially responsible for the severity of H5N1 infection and provide evidence that attenuating the CXCR3 signaling pathway improves the clinical course of H5N1 infection in ferrets.     

Fungal gene expression during pathogenesis-related development and host plant colonization

To successfully infect plants, pathogenic fungi must recognize and communicate with their host during different stages of the disease cycle. In past years, techniques such as insertional mutagenesis, sensitive GFP-based reporter systems and microarray techniques have been developed to analyze these processes at the molecular level, and now novel insights into this fascinating aspect of pathogen-plant communication are beginning to emerge. This is exemplified by a number of pathogenicity genes functioning in distinct stages of pathogenic development in Magnaporthe grisea.     

Microarray analysis of gene expression profiles in the bovine mammary gland during lactation

Mammary glands undergo functional and metabolic changes during virgin, lactation and dry periods. A total of 122 genes were identified as differentially expressed, including 79 up-regulated and 43 down-regulated genes during lactation compared with virgin and dry periods. Gene ontology analysis showed the functional classification of the up-regulated genes in lactation, including transport, biosynthetic process, signal transduction, catalytic activity, immune system process, cell death, and positive regulation of the developmental process. Microarray data clarified molecular events in bovine mammary gland lactation.     

Roles of genes 44, 50, and 51 in regulating gene expression and host takeover during infection of Bacillus subtilis by bacteriophage SPO1

We show that the products of SPO1 genes 44, 50, and 51 are required for the normal transition from early to middle gene expression during infection of Bacillus subtilis by bacteriophage SPO1; that they are also required for control of the shutoff of host DNA, RNA, and protein synthesis; and that their effects on host shutoff could be accounted for by their effects on the regulation of gene expression. These three gene products had four distinguishable effects in regulating SPO1 gene expression: (i) gp44-50-51 acted to restrain expression of all SPO1 genes tested, (ii) gp44 and/or gp50-51 caused additional specific repression of immediate-early genes, (iii) gp44 and/or gp50-51 stimulated expression of middle genes, and (iv) gp44 and/or gp50-51 stimulated expression of some delayed-early genes. Shutoff of immediate-early gene expression also required the activity of gp28, the middle-gene-specific sigma factor. Shutoff of host RNA and protein synthesis was accelerated by either the 44- single mutant or the 50(-)51(-) double mutant and more so by the 44(-)50(-)51(-) triple mutant. Shutoff of host DNA synthesis was accelerated by the mutants early in infection but delayed by the 44(-)50(-)51(-) triple mutant at later times. Although gp50 is a very small protein, consisting almost entirely of an apparent membrane-spanning domain, it contributed significantly to each activity tested. We identify SPO1 genes 41 to 51 and 53 to 60 as immediate-early genes; genes 27, 28, and 37 to 40 as delayed-early genes; and gene 52 as a middle gene.     

Mutation in the Sp1 motif of the bovine leptin gene affects its expression

Leptin is expressed mainly by adipocytes and plays a crucial role in the regulation of energy expenditure, food intake, and adiposity. Using PCR-heteroduplex analysis and sequencing, we investigated a C/G substitution in the promoter region of the bovine leptin gene. Application of the electrophoretic mobility shift assay showed that the C→G transversion decreased the leptin gene promoter binding capacity for nuclear proteins. With real-time PCR and Western blotting, we showed that the leptin expression level was higher in cattle with the CC than with the GG genotype.