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| Status |
Public on Mar 25, 2010 |
| Title |
Comparison of Clinical hVISA Parent and Laboratory-Derived VISA Mutants |
| Organism |
Staphylococcus aureus |
| Experiment type |
Expression profiling by array
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| Summary |
The mechanisms leading to the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype are incompletely understood. In an effort to identify VISA determinants, clinical hVISA strain MM66 and its 1st-step VISA mutants were compared. S. aureus microarrays, array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center), as well as comparative genomic sequencing (CGS) were utilized to determine VISA transcriptome alterations and mutations that define the MM66 VISA mechanism. In addition, whole cell autolysis, antimicrobial susceptibility tests, growth in the presence of salt, coagulase and hemolysis assays were performed to confirm VISA transcriptional alterations. Overall, 2 MM66 VISA demonstrated 85 up-regulated and 8 down-regulated genes in common. SAMMD (Staphylococcus aureus Microarray Metadatabase) analysis identified a significant differential gene expression overlap between the global stress response regulator sigB-controlled genes in MM66-3 and MM66-4. CGS analysis on a MM66 VISA revealed two mutations in: a sensory box histidine kinase (yycG) and a putative adenine phosphoribosyltransferase (apt). The yycFG two-component system has been reported to regulate whole cell autolysis and MM66 VISA demonstrated reduced whole cell autolysis. Five ssaA homologues (putative amidases) and a gene that expresses a bifunctional autolysin (atlA) controlled by YycFG were downregulated in MM66 VISA. One MM66 VISA mutant demonstrated drastic downregulation of a gene cluster (betA, gbsA, betB, and cudT) required for the osmoprotective response of S. aureus and MM66 VISA mutants demonstrated reduced growth in high salt media compared to MM66. Virulence genes were also downregulated (coa, hla, hlb, hlgA, hlgB, and hlgC) in 2 MM66 VISA mutants which also demonstrated reduced coagulase and hemolytic activities compared to MM66. pur operon genes were also upregulated in MM66 VISA and it is interesting that we have also found an apt mutation, since Apt activity can be inhibited by high AMP levels and reduces the conversion of AMP into adenine. Conclusion: Mutations in yycG and apt allow hVISA to acquire the VISA phenotype and yycFG is involved with altered autolytic activity in MM66 VISA.
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| Overall design |
S. aureus pangenome microarrays were utilized to determine transcriptome alterations occurring in VISA mutants MM66-3 and MM66-4 isolated from a clinical hVISA parent strain MM66. Total bacterial RNA was isolated as previously described from S. aureus strains MM66, MM66-3 and MM66-4 grown in Luria broth to an OD580nm of 0.7 (37°C, 200 rpm). The RNA samples were then converted to fluorescently-labeled cDNA and hybridized to S. aureus microarrays version 4 (NIAID's Pathogen Functional Genomics Resource Center). The Staphylococcus aureus microarray meta-database (SAMMD) was used to compare and analyze the microarray data obtained from this experiment to the microarray data obtained from different published protocols and experiments. Real-time PCR and physiological experiments were performed to validate the microarray data.
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| Contributor(s) |
Lamichhane-Khadka R, Dupre J, Cuaron J, Muthaiyan A, Delgado A, Zaman S, Reynolds T, Nagarajan V, Elasri MO, Wilkinson BJ, Yu W, Li Z, Gustafson JE |
| Citation(s) |
34069103 |
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| Submission date |
Jun 07, 2009 |
| Last update date |
Sep 15, 2021 |
| Contact name |
John E Gustafson, Arunachalam Muthaiyan |
| E-mail(s) |
jgustafs@nmsu.edu, amuthai@gmail.com
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| Phone |
575-646-5660
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| Fax |
575-646-5665
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| Organization name |
New Mexico State University
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| Department |
Department of Biology and Molecular Biology Program
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| Lab |
Staph lab
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| Street address |
PO Box 30001 Dept. 3AF
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| City |
Las Cruces |
| State/province |
NM |
| ZIP/Postal code |
88003-8001 |
| Country |
USA |
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| Platforms (1) |
| GPL7072 |
JCVI PFGRC Staphylococcus aureus version 4 array |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA116317 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE16479_RAW.tar |
4.5 Mb |
(http)(custom) |
TAR (of TAV) |
| Processed data included within Sample table |
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