Microarrays for microbiologists

Lucchini, Sacha; Thompson, Arthur R.; Hinton, Jay C. D.

doi:10.1099/00221287-147-6-1403

Cited by 115 publications

(64 citation statements)

References 109 publications

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“…We defined the genes controlled by PNPase activity by doing gene expression profiling on MC1 and MC2 (34). In short, bacteria were grown in LB, and RNA was prepared from MC1 and MC2, reversetranscribed to cDNA, and fluorescently labeled.…”

Section: Pnpase Acts As a Global Regulator Of Virulence Gene Expressionmentioning

confidence: 99%

Polynucleotide phosphorylase is a global regulator of virulence and persistency in Salmonella enterica

Clements

Eriksson

Thompson

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

154

133

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For many pathogens, the ability to regulate their replication in host cells is a key element in establishing persistency. Here, we identified a single point mutation in the gene for polynucleotide phosphorylase (PNPase) as a factor affecting bacterial invasion and intracellular replication, and which determines the alternation between acute or persistent infection in a mouse model for Salmonella enterica infection. In parallel, with microarray analysis, PNPase was found to affect the mRNA levels of a subset of virulence genes, in particular those contained in Salmonella pathogenicity islands 1 and 2. The results demonstrate a connection between PNPase and Salmonella virulence and show that alterations in PNPase activity could represent a strategy for the establishment of persistency.T he species Salmonella enterica is a classic example of the facultative intracellular pathogen that can cause acute and persistent infections (1-4). Typhoid fever is a serious manifestation of systemic salmonellosis in humans and involves an estimated 16.6 million new cases per year (2) with between 2% and 5% of the cases developing into the carrier state (3, 4).Much of the knowledge of salmonellosis pathogenesis comes from work on the murine salmonellosis model (1, 5), and this model has been used to define bacterial virulence factors (1, 6, 7). An important virulence trait of Salmonella is its ability to invade and grow within host cells, and many of the genetic determinants essential for these processes have been well characterized (8-11). Invasion of epithelial cells requires the expression of a type III secretion apparatus, which translocates effector proteins from the bacterium to the host cell, inducing the cell to internalize the bacteria by bacterial-mediated endocytosis (8,11). Proteins involved in this process are encoded by a large cluster of genes collectively termed Salmonella pathogenicity island 1 (SPI 1). The ability to grow intracellularly, once internalized within the host cell, requires the coordinate expression of additional sets of virulence factors (6), such as SPI 2 (1, 10).Although chronic carriage of Salmonella is recognized as a significant complication that facilitates spread of the disease (2) and predisposes victims to additional clinical conditions (12, 13) the mechanism underlying persistency has remained poorly understood. We discovered the link between polynucleotide phosphorylase (PNPase) and chronic infection while characterizing a mutant of Salmonella typhimurium that was defective in its expression of virulence-associated AgfA fimbriae and known to establish persistent infection in BALB͞c mice (14). Materials and Methods Mapping and Sequencing of MC2 Mutation.A random Tn10camd insertion library from S. typhimurium 14028 (22) was transduced into MC1 by P22 transduction selecting for chloramphenicolresistant colonies. Transductants were pooled and used to propagate phage KB1, which was then used to transduce MC2. Chloramphenicol-resistant colonies were screened for agf expression on Congo red pla...

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Section: Pnpase Acts As a Global Regulator Of Virulence Gene Expressionmentioning

confidence: 99%

Polynucleotide phosphorylase is a global regulator of virulence and persistency in Salmonella enterica

Clements

Eriksson

Thompson

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

154

133

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“…Microarray approaches have been discussed by Lucchini et al (2001) and Thompson et al (2001). RNA (10 mg) was fluorescently labelled during reverse transcription into cDNA.…”

Section: Methodsmentioning

confidence: 99%

A global role for Fis in the transcriptional control of metabolism and type III secretion in Salmonella enterica serovar Typhimurium

et al. 2004

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Fis is a key DNA-binding protein involved in nucleoid organization and modulation of many DNA transactions, including transcription in enteric bacteria. The regulon of genes whose expression is influenced by Fis in Salmonella enterica serovar Typhimurium (S. typhimurium) has been defined by DNA microarray analysis. These data suggest that Fis plays a central role in coordinating the expression of both metabolic and type III secretion factors. The genes that were most strongly up-regulated by Fis were those involved in virulence and located in the pathogenicity islands SPI-1, SPI-2, SPI-3 and SPI-5. Similarly, motility and flagellar genes required Fis for full expression. This was shown to be a direct effect as purified Fis protein bound to the promoter regions of representative flagella and SPI-2 genes. Genes contributing to aspects of metabolism known to assist the bacterium during survival in the mammalian gut were also Fis-regulated, usually negatively. This category included components of metabolic pathways for propanediol utilization, biotin synthesis, vitamin B 12 transport, fatty acids and acetate metabolism, as well as genes for the glyoxylate bypass of the tricarboxylic acid cycle. Genes found to be positively regulated by Fis included those for ethanolamine utilization. The data reported reveal the central role played by Fis in coordinating the expression of both housekeeping and virulence factors required by S. typhimurium during life in the gut lumen or during systemic infection of host cells. INTRODUCTIONSalmonella enterica serovar Typhimurium (S. typhimurium) is the most common and best studied of the S. enterica serovars that infect humans (Finlay & Brumell, 2000). It is able to infect a range of animal species, including chicken, cattle and mice, and intensive study of this organism is providing important insights into key processes involved in bacterial pathogenesis. In the mouse, S. typhimurium is a facultative intracellular pathogen capable of invading epithelial cells and it has the ability to survive and proliferate within macrophages. The bacterium can be manipulated genetically with relative ease and the complete genome sequence is available (McClelland et al., 2001), allowing a combination of genetic analysis, cell biology and animal infection studies. This multidisciplinary approach has provided a picture of the major events involved when S. typhimurium infects the murine host. Following ingestion and passage through the stomach, the bacteria cross the lining of the intestine by invading the intestinal epithelium, predominantly via M cells. The Salmonellae are subsequently phagocytosed by macrophages before entering the blood stream and establishing a systemic infection (Finlay & Brumell, 2000; Galán, 2001;Groisman & Mouslim, 2000;Holden, 2002;Scherer & Miller, 2001).S. typhimurium is dependent upon the products of a large number of genes (up to 200) to cause infection (Finlay & Brumell, 2000). Some of the virulence genes are located on a 90 kb pathogenicity plasmid, of which the spv gen...

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“…Briefly, for each reaction, 500 ng enriched mRNA obtained in our experiments or 1 µl control RNA offered by the SuperScript TM FirstStrand Synthesis System for RT-PCR (Life Technologies) was reverse transcribed using the SuperScript II system. For the reaction, 1 µl (2 µM) gene-specific primers that hybridize to the 3' terminus of the mRNA listed in Table 2 for enriched mRNA and 0.5 µg Oligo (dT) [12][13][14][15][16][17][18] for control RNA were mixed together in a total volume of 10 µl respectively and heated to 65 C for 10 min. After cooling on ice for at least 1 min, the reaction buffer was added according to the manufacturer's recommendations.…”

Section: Determination Of the Binding Capacity Of The Magnetic Beadsmentioning

confidence: 99%

Bacterial mRNA Purification by Magnetic Capture‐Hybridization Method

Pang

Zhou

Song

et al. 2004

Microbiology and Immunology

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A magnetic capture‐hybridization method was developed for purification of bacterial messenger RNA (mRNA) from total RNA by removing 5S, 16S and 23S ribosomal RNAs (rRNA). The quality of mRNA was evaluated by A260nm/A280nm value, denatured gel electrophoresis and RT‐PCR. The results showed that highly purified and intact mRNA was obtained by this method. The magnetic capture‐hybridization is a rapid and simple method for bacterial mRNA purification and has promising potential for improving studies using bacterial mRNA.

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Microarrays for microbiologists

Cited by 115 publications

References 109 publications

Polynucleotide phosphorylase is a global regulator of virulence and persistency in Salmonella enterica

Polynucleotide phosphorylase is a global regulator of virulence and persistency in Salmonella enterica

A global role for Fis in the transcriptional control of metabolism and type III secretion in Salmonella enterica serovar Typhimurium

Bacterial mRNA Purification by Magnetic Capture‐Hybridization Method

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