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Intracellular Streptococcus pyogenes in human macrophages display an altered gene expression profile.

ABSTRACT: Streptococcus pyogenes is an important human pathogen, which has recently gained recognition as an intracellular microorganism during the course of severe invasive infections such as necrotizing fasciitis. Although the surface anchored M protein has been identified as a pivotal factor affecting phagosomal maturation and S. pyogenes survival within macrophages, the overall transcriptional profile required for the pathogen to adapt and persist intracellularly is as of yet unknown. To address this, the gene expression profile of S. pyogenes within human macrophages was determined and compared to that of extracellular bacteria using customized microarrays and real-time qRT-PCR. In order to model the early phase of infection involving adaptation to the intracellular compartment, samples were collected 2h post-infection. Microarray analysis revealed that the expression of 145 streptococcal genes was significantly altered in the intracellular environment. The majority of differentially regulated genes were associated with metabolic and energy-dependent processes. Key up-regulated genes in early phase intracellular bacteria were ihk and irr, encoding a two-component gene regulatory system (TCS). Comparison of gene expression of selected genes at 2h and 6h post-infection revealed a dramatic shift in response regulators over time with a down-regulation of ihk/irr genes concurring with an up-regulation of the covR/S TCS. In re-infection assays, intracellular bacteria from the 6h time point exhibited significantly greater survival within macrophages than did bacteria collected at the 2h time point. An isogenic S. pyogenes mutant deficient in ihk/irr displayed significantly reduced bacterial counts when compared to wild-type bacteria following infection of macrophages. The findings illustrate how gene expression of S. pyogenes during the intracellular life cycle is fine-tuned by temporal expression of specific two-component systems.

SUBMITTER: Hertzen E

PROVIDER: S-EPMC3325220 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Intracellular Streptococcus pyogenes in human macrophages display an altered gene expression profile.

Hertzén Erika E Johansson Linda L Kansal Rita R Hecht Alexander A Dahesh Samira S Janos Marton M Nizet Victor V Kotb Malak M Norrby-Teglund Anna A

PloS one 20120412 4

Streptococcus pyogenes is an important human pathogen, which has recently gained recognition as an intracellular microorganism during the course of severe invasive infections such as necrotizing fasciitis. Although the surface anchored M protein has been identified as a pivotal factor affecting phagosomal maturation and S. pyogenes survival within macrophages, the overall transcriptional profile required for the pathogen to adapt and persist intracellularly is as of yet unknown. To address this, ...[more]

PMID: 22511985

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Project description:The objective of this study was to investigate which genes are important for Streptococcus pyogenes during intracellular survival in human macrophages. Streptococcus pyogenes is an important human pathogen, which has recently gained recognition as an intracellular microorganism during the course of severe invasive infections such as necrotizing fasciitis. Although the surface anchored M protein has been identified as a pivotal factor affecting phagosomal maturation and S. pyogenes survival within macrophages, the overall transcriptional profile required for the pathogen to adapt and persist intracellularly is yet unknown. To address this, gene expression profiles of S. pyogenes within human macrophages were determined and compared to those of extracellular bacteria using customized microarrays and real-time qRT-PCR. In order to model the early phase of infection involving adaptation to the intracellular compartment, samples were collected 2h post-infection and within 2 h post infection, the expression of 145 streptococcal genes was significantly altered in the intracellular environment. The majority of differentially regulated genes were associated with metabolic and energy-dependent processes. Key upregulated genes in early phase intracellular bacteria were ihk and irr, encoding a two-component gene regulatory system (TCS). We observed that an isogenic S. pyogenes mutant deficient in ihk/irr displayed significantly reduced bacterial counts as compared to wild-type bacteria following infection of macrophages. Comparison of gene expression of selected genes at 2h and 6h post-infection revealed a dramatic shift in response regulators over time with a down-regulation of ihk/irr genes concurrent with an upreguation of the well-studied covR/S two component regulator. In reinfection assays, intracellular bacteria from the 6h time point exhibited significantly greater survival within macrophages than did bacteria collected at the 2h time point. The findings illustrate how gene expression of S. pyogenes during the intracellular life cycle is fine-tuned by temporal expression of specific two-component systems.

Project description:Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]), this system is encoded by the copYAZ operon. This study demonstrates that although the site of GAS infection represents a Cu-rich environment, inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde-3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of noncognate metal-binding sites by Cu. Surprisingly, the onset of these effects is delayed by ?4 h even though expression of copZ is upregulated immediately upon exposure to Cu. Further biochemical investigations show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. These results indicate that GSH buffers excess intracellular Cu when the transcriptionally responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance and allows bacteria to maintain metabolism even in the presence of an excess of this metal ion.IMPORTANCE The control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it has been established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and has long been assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this physiological role of glutathione beyond measuring growth of glutathione-deficient mutants in the presence of Cu. Our work with group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.

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Intracellular Streptococcus pyogenes in human macrophages display an altered gene expression profile.

Publications

Intracellular Streptococcus pyogenes in human macrophages display an altered gene expression profile.

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