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Determination of transcriptome of SptR in group A Streptococcus during growth in human saliva

ABSTRACT: The molecular genetic mechanisms used by bacteria to persist in humans are poorly understood. Group A Streptococcus (GAS) causes the majority of bacterial pharyngitis cases in humans and is prone to persistently inhabit the upper respiratory tract. To gain information about how GAS survives in and infects the oropharynx, we analyzed the transcriptome of a serotype M1 strain grown in saliva. The dynamic pattern of changes in transcripts of genes [spy0874/0875, herein named sptR and sptS (sptR/S), for saliva persistence] encoding a two-component gene regulatory system of unknown function suggested that SptR/S contributed to persistence of GAS in saliva. Consistent with this idea, an isogenic nonpolar mutant strain (DeltasptR) was dramatically less able to survive in saliva compared with the parental strain. Iterative expression microarray analysis of bacteria grown in saliva revealed that transcripts of several known and putative GAS virulence factor genes were decreased significantly in the DeltasptR mutant strain. Compared with the parental strain, the isogenic mutant strain also had altered transcripts of multiple genes encoding proteins involved in complex carbohydrate acquisition and utilization pathways. Western immunoblot analysis and real-time PCR analysis of GAS in throat swabs taken from humans with pharyngitis confirmed the findings. We conclude that SptR/S optimizes persistence of GAS in human saliva, apparently by strategically influencing metabolic pathways and virulence factor production. The discovery of a genetic program that significantly increased persistence of a major human pathogen in saliva enhances understanding of how bacteria survive in the host and suggests new therapeutic strategies.

ORGANISM(S): Yersinia pestis Chlamydia trachomatis Streptococcus pyogenes Staphylococcus aureus Streptococcus sp. 'group A' Borreliella burgdorferi Coxiella burnetii

PROVIDER: GSE16124 | GEO | 2009/05/16

SECONDARY ACCESSION(S): PRJNA115013

REPOSITORIES: GEO

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Project description:The molecular genetic mechanisms used by bacteria to persist in humans are poorly understood. Group A Streptococcus (GAS) causes the majority of bacterial pharyngitis cases in humans and is prone to persistently inhabit the upper respiratory tract. To gain information about how GAS survives in and infects the oropharynx, we analyzed the transcriptome of a serotype M1 strain grown in saliva. The dynamic pattern of changes in transcripts of genes [spy0874/0875, herein named sptR and sptS (sptR/S), for saliva persistence] encoding a two-component gene regulatory system of unknown function suggested that SptR/S contributed to persistence of GAS in saliva. Consistent with this idea, an isogenic nonpolar mutant strain (DeltasptR) was dramatically less able to survive in saliva compared with the parental strain. Iterative expression microarray analysis of bacteria grown in saliva revealed that transcripts of several known and putative GAS virulence factor genes were decreased significantly in the DeltasptR mutant strain. Compared with the parental strain, the isogenic mutant strain also had altered transcripts of multiple genes encoding proteins involved in complex carbohydrate acquisition and utilization pathways. Western immunoblot analysis and real-time PCR analysis of GAS in throat swabs taken from humans with pharyngitis confirmed the findings. We conclude that SptR/S optimizes persistence of GAS in human saliva, apparently by strategically influencing metabolic pathways and virulence factor production. The discovery of a genetic program that significantly increased persistence of a major human pathogen in saliva enhances understanding of how bacteria survive in the host and suggests new therapeutic strategies. We determined the transcriptome of SptR (spy0874) in human saliva by comparing expression microarray date for parental wild-type strain MGAS5005 and its delta-spy0874 isogenic mutant strain. 4 replicates of each strain were grown to mid-exponential and stationary growth phases in human saliva. RNA was isolated, converted to cDNA, labeled and hybridized to a custom-made Affymetrix GeneChip. Data was analyzed using GCOS.

Project description:Relatively little is understood about the dynamics of global hostâpathogen transcriptome changes that occur during bacterial infection of mucosal surfaces. To test the hypothesis that group A Streptococcus (GAS) infection of the oropharynx provokes a host transcriptome response, we performed genome-wide transcriptome analysis using a nonhuman primate model of experimental pharyngitis. We also identified host and pathogen biological processes and individual host and pathogen gene pairs with correlated patterns of expression, suggesting interaction. For this study, 509 host genes and seven biological pathways were differentially expressed throughout the entire 32-day infection cycle. GAS infection produced an initial widespread significant decrease in expression of many host genes, including those involved in cytokine production, vesicle formation, metabolism, and signal transduction. This repression lasted until day 4, at which time a large increase in expression of host genes was observed, including those involved in protein translation, antigen presentation, and GTP-mediated signaling. The interactome analysis identified 73 host and pathogen gene pairs with correlated expression levels. We discovered significant correlations between transcripts of GAS genes involved in hyaluronic capsule production and host endocytic vesicle formation, GAS GTPases and host fibrinolytic genes, and GAS response to interaction with neutrophils. We also identified a strong signal, suggesting interaction between host Î³Î´ T cells and genes in the GAS mevalonic acid synthesis pathway responsible for production of isopentenyl-pyrophosphate, a short-chain phospholipid that stimulates these T cells. Taken together, our Q:2 results are unique in providing a comprehensive understanding of the hostâpathogen interactome during mucosal infection by a bacterial pathogen. Longitudinal pharyngeal infection of cynomolgus macaques by group A Streptococcus Briefly, animals were GAS-culture negative and had negligible antistreptolysin O titers, indicating no recent history of GAS exposure. Twenty animals were subjected to a mock-inoculation protocol (PBS only) for 5 weeks, rested for 4 weeks, and inoculated in the upper respiratory tract with 107 CFUsMGAS5005. Blood, saliva, and throat swabs were collected on days 0, 1, 2, 4, 7, 9, 16, 23, 32, 45, 58, 72, and 86. Only the first nine time-points were studied because specimens collected during days 0 to 32 had matching comparator specimens from the mock-infection protocol. Thirty-two clinical and laboratory parameters were measured by the same veterinarian during mock and infection periods. Array data was only for the tonsil swabs, while blood and saliva used for other tests.

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Determination of transcriptome of SptR in group A Streptococcus during growth in human saliva

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