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Novel algorithms reveal streptococcal transcriptomes and clues about undefined genes.

Ryan PA, Kirk BW, Euler CW, Schuch R, Fischetti VA - PLoS Comput. Biol. (2007)

Bottom Line: We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application.We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data.Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacterial Pathogenesis and Immunology, Rockefeller University, New York, New York, USA. ryanp@mail.rockfeller.edu

ABSTRACT
Bacteria-host interactions are dynamic processes, and understanding transcriptional responses that directly or indirectly regulate the expression of genes involved in initial infection stages would illuminate the molecular events that result in host colonization. We used oligonucleotide microarrays to monitor (in vitro) differential gene expression in group A streptococci during pharyngeal cell adherence, the first overt infection stage. We present neighbor clustering, a new computational method for further analyzing bacterial microarray data that combines two informative characteristics of bacterial genes that share common function or regulation: (1) similar gene expression profiles (i.e., co-expression); and (2) physical proximity of genes on the chromosome. This method identifies statistically significant clusters of co-expressed gene neighbors that potentially share common function or regulation by coupling statistically analyzed gene expression profiles with the chromosomal position of genes. We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application. We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data. Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence.

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Confirmation of speH Deletion Mutant and Pharyngeal Cell Adherence Assay(A) Results of RT-PCR and PCR analyses of total RNA preparations isolated from mid-log (OD = 0.4) and stationary phase (OD = 1) cultures of the ΔspeH deletion mutant (ΔL and ΔS, respectively), and stationary phase cultures of the SF370 parental strain (P). RNA was reverse-transcribed as described in Methods. To assess genomic DNA contamination, control reactions containing Taq DNA polymerase instead of reverse transcriptase were included. cDNA products were separated on a 1% agarose gel and visualized by ethidium bromide staining. Lanes containing products from either the RT-PCR or PCR analysis are designated at the bottom of the panel. Lanes labeled MW contain 1 kb Plus DNA ladder (1 μg; Invitrogen).(B) Results of the pharyngeal cell adherence assay (detailed in Methods), comparing parental strain SF370 with the deletion mutant SF370ΔspeH (abbreviated ΔspeH). Adherent streptococci are reported as the percentage of total number of streptococci added as inoculum to pharyngeal cell monolayers. Statistical significance (reported as p value) was determined by Student's t-test.
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pcbi-0030132-g001: Confirmation of speH Deletion Mutant and Pharyngeal Cell Adherence Assay(A) Results of RT-PCR and PCR analyses of total RNA preparations isolated from mid-log (OD = 0.4) and stationary phase (OD = 1) cultures of the ΔspeH deletion mutant (ΔL and ΔS, respectively), and stationary phase cultures of the SF370 parental strain (P). RNA was reverse-transcribed as described in Methods. To assess genomic DNA contamination, control reactions containing Taq DNA polymerase instead of reverse transcriptase were included. cDNA products were separated on a 1% agarose gel and visualized by ethidium bromide staining. Lanes containing products from either the RT-PCR or PCR analysis are designated at the bottom of the panel. Lanes labeled MW contain 1 kb Plus DNA ladder (1 μg; Invitrogen).(B) Results of the pharyngeal cell adherence assay (detailed in Methods), comparing parental strain SF370 with the deletion mutant SF370ΔspeH (abbreviated ΔspeH). Adherent streptococci are reported as the percentage of total number of streptococci added as inoculum to pharyngeal cell monolayers. Statistical significance (reported as p value) was determined by Student's t-test.

Mentions: Increased expression of speH during pharyngeal cell adherence suggests that the SpeH exotoxin is either necessary for adherence, or is a component of a downstream infection process. Adherence-mediated upregulation of speH is likely not the result of phage induction, as the remaining phage 370.2 genes identified in our analysis were downregulated. To determine if SpeH plays a direct role in the adherence process, we created a deletion mutant in strain SF370 (SF370ΔspeH), which was confirmed by PCR (unpublished data) and RT-PCR (Figure 1A) and tested in vitro for adherence to human pharyngeal cells. We observed no significant difference in adherence between the wild-type (SF370) and mutant strains (Figure 1B), indicating that SpeH is not involved directly in attachment to the pharyngeal cell. The significant upregulation of the speH gene during adherence suggests that the gene product may function instead during a subsequent stage of infection.


Novel algorithms reveal streptococcal transcriptomes and clues about undefined genes.

Ryan PA, Kirk BW, Euler CW, Schuch R, Fischetti VA - PLoS Comput. Biol. (2007)

Confirmation of speH Deletion Mutant and Pharyngeal Cell Adherence Assay(A) Results of RT-PCR and PCR analyses of total RNA preparations isolated from mid-log (OD = 0.4) and stationary phase (OD = 1) cultures of the ΔspeH deletion mutant (ΔL and ΔS, respectively), and stationary phase cultures of the SF370 parental strain (P). RNA was reverse-transcribed as described in Methods. To assess genomic DNA contamination, control reactions containing Taq DNA polymerase instead of reverse transcriptase were included. cDNA products were separated on a 1% agarose gel and visualized by ethidium bromide staining. Lanes containing products from either the RT-PCR or PCR analysis are designated at the bottom of the panel. Lanes labeled MW contain 1 kb Plus DNA ladder (1 μg; Invitrogen).(B) Results of the pharyngeal cell adherence assay (detailed in Methods), comparing parental strain SF370 with the deletion mutant SF370ΔspeH (abbreviated ΔspeH). Adherent streptococci are reported as the percentage of total number of streptococci added as inoculum to pharyngeal cell monolayers. Statistical significance (reported as p value) was determined by Student's t-test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1913099&req=5

pcbi-0030132-g001: Confirmation of speH Deletion Mutant and Pharyngeal Cell Adherence Assay(A) Results of RT-PCR and PCR analyses of total RNA preparations isolated from mid-log (OD = 0.4) and stationary phase (OD = 1) cultures of the ΔspeH deletion mutant (ΔL and ΔS, respectively), and stationary phase cultures of the SF370 parental strain (P). RNA was reverse-transcribed as described in Methods. To assess genomic DNA contamination, control reactions containing Taq DNA polymerase instead of reverse transcriptase were included. cDNA products were separated on a 1% agarose gel and visualized by ethidium bromide staining. Lanes containing products from either the RT-PCR or PCR analysis are designated at the bottom of the panel. Lanes labeled MW contain 1 kb Plus DNA ladder (1 μg; Invitrogen).(B) Results of the pharyngeal cell adherence assay (detailed in Methods), comparing parental strain SF370 with the deletion mutant SF370ΔspeH (abbreviated ΔspeH). Adherent streptococci are reported as the percentage of total number of streptococci added as inoculum to pharyngeal cell monolayers. Statistical significance (reported as p value) was determined by Student's t-test.
Mentions: Increased expression of speH during pharyngeal cell adherence suggests that the SpeH exotoxin is either necessary for adherence, or is a component of a downstream infection process. Adherence-mediated upregulation of speH is likely not the result of phage induction, as the remaining phage 370.2 genes identified in our analysis were downregulated. To determine if SpeH plays a direct role in the adherence process, we created a deletion mutant in strain SF370 (SF370ΔspeH), which was confirmed by PCR (unpublished data) and RT-PCR (Figure 1A) and tested in vitro for adherence to human pharyngeal cells. We observed no significant difference in adherence between the wild-type (SF370) and mutant strains (Figure 1B), indicating that SpeH is not involved directly in attachment to the pharyngeal cell. The significant upregulation of the speH gene during adherence suggests that the gene product may function instead during a subsequent stage of infection.

Bottom Line: We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application.We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data.Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacterial Pathogenesis and Immunology, Rockefeller University, New York, New York, USA. ryanp@mail.rockfeller.edu

ABSTRACT
Bacteria-host interactions are dynamic processes, and understanding transcriptional responses that directly or indirectly regulate the expression of genes involved in initial infection stages would illuminate the molecular events that result in host colonization. We used oligonucleotide microarrays to monitor (in vitro) differential gene expression in group A streptococci during pharyngeal cell adherence, the first overt infection stage. We present neighbor clustering, a new computational method for further analyzing bacterial microarray data that combines two informative characteristics of bacterial genes that share common function or regulation: (1) similar gene expression profiles (i.e., co-expression); and (2) physical proximity of genes on the chromosome. This method identifies statistically significant clusters of co-expressed gene neighbors that potentially share common function or regulation by coupling statistically analyzed gene expression profiles with the chromosomal position of genes. We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application. We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data. Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence.

Show MeSH
Related in: MedlinePlus