Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone

ABSTRACT It is well appreciated that oxygen- and other nutrient-limiting gradients characterize microenvironments within chronic infections that foster bacterial tolerance to treatment and the immune response. However, determining how bacteria respond to these microenvironments has been limited by a...

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Autores principales:	Peter Jorth, Melanie A. Spero, J. Livingston, Dianne K. Newman
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	HCR Pseudomonas aeruginosa aggregate biofilms gene expression in situ hybridization Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/bc92388167244d53b79778dac5035526
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spelling	oai:doaj.org-article:bc92388167244d53b79778dac50355262021-11-15T15:54:47ZQuantitative Visualization of Gene Expression in Mucoid and Nonmucoid <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone10.1128/mBio.02622-192150-7511https://doaj.org/article/bc92388167244d53b79778dac50355262019-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02622-19https://doaj.org/toc/2150-7511ABSTRACT It is well appreciated that oxygen- and other nutrient-limiting gradients characterize microenvironments within chronic infections that foster bacterial tolerance to treatment and the immune response. However, determining how bacteria respond to these microenvironments has been limited by a lack of tools to study bacterial functions at the relevant spatial scales in situ. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to provide analog mRNA relative quantitation of Pseudomonas aeruginosa single cells as a step toward this end. To assess the potential for this method to be applied to bacterial populations, we visualized the expression of genes needed for the production of alginate (algD) and the dissimilatory nitrate reductase (narG) at single-cell resolution within laboratory-grown aggregates. After validating new HCR probes, we quantified algD and narG expression across microenvironmental gradients within both single aggregates and aggregate populations using the agar block biofilm assay (ABBA). For mucoid and nonmucoid ABBA populations, narG was expressed in hypoxic and anoxic regions, while alginate expression was restricted to the hypoxic zone (∼40 to 200 μM O2). Within individual aggregates, surface-adjacent cells expressed alginate genes at higher levels than interior cells, revealing that alginate expression is not constitutive in mucoid P. aeruginosa but instead varies with oxygen availability. These results establish HCR v3.0 as a versatile and robust tool to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. This advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen activities during infections. IMPORTANCE A goal for microbial ecophysiological research is to reveal microbial activities in natural environments, including sediments, soils, or infected human tissues. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to quantitatively measure microbial gene expression in situ at single-cell resolution in bacterial aggregates. Using quantitative image analysis of thousands of Pseudomonas aeruginosa cells, we validated new P. aeruginosa HCR probes. Within in vitro P. aeruginosa aggregates, we found that bacteria just below the aggregate surface are the primary cells expressing genes that protect the population against antibiotics and the immune system. This observation suggests that therapies targeting bacteria growing with small amounts of oxygen may be most effective against these hard-to-treat infections. More generally, this proof-of-concept study demonstrates that HCR v3.0 has the potential to identify microbial activities in situ at small spatial scales in diverse contexts.Peter JorthMelanie A. SperoJ. LivingstonDianne K. NewmanAmerican Society for MicrobiologyarticleHCRPseudomonas aeruginosaaggregatebiofilmsgene expressionin situ hybridizationMicrobiologyQR1-502ENmBio, Vol 10, Iss 6 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	HCR Pseudomonas aeruginosa aggregate biofilms gene expression in situ hybridization Microbiology QR1-502
spellingShingle	HCR Pseudomonas aeruginosa aggregate biofilms gene expression in situ hybridization Microbiology QR1-502 Peter Jorth Melanie A. Spero J. Livingston Dianne K. Newman Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone
description	ABSTRACT It is well appreciated that oxygen- and other nutrient-limiting gradients characterize microenvironments within chronic infections that foster bacterial tolerance to treatment and the immune response. However, determining how bacteria respond to these microenvironments has been limited by a lack of tools to study bacterial functions at the relevant spatial scales in situ. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to provide analog mRNA relative quantitation of Pseudomonas aeruginosa single cells as a step toward this end. To assess the potential for this method to be applied to bacterial populations, we visualized the expression of genes needed for the production of alginate (algD) and the dissimilatory nitrate reductase (narG) at single-cell resolution within laboratory-grown aggregates. After validating new HCR probes, we quantified algD and narG expression across microenvironmental gradients within both single aggregates and aggregate populations using the agar block biofilm assay (ABBA). For mucoid and nonmucoid ABBA populations, narG was expressed in hypoxic and anoxic regions, while alginate expression was restricted to the hypoxic zone (∼40 to 200 μM O2). Within individual aggregates, surface-adjacent cells expressed alginate genes at higher levels than interior cells, revealing that alginate expression is not constitutive in mucoid P. aeruginosa but instead varies with oxygen availability. These results establish HCR v3.0 as a versatile and robust tool to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. This advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen activities during infections. IMPORTANCE A goal for microbial ecophysiological research is to reveal microbial activities in natural environments, including sediments, soils, or infected human tissues. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to quantitatively measure microbial gene expression in situ at single-cell resolution in bacterial aggregates. Using quantitative image analysis of thousands of Pseudomonas aeruginosa cells, we validated new P. aeruginosa HCR probes. Within in vitro P. aeruginosa aggregates, we found that bacteria just below the aggregate surface are the primary cells expressing genes that protect the population against antibiotics and the immune system. This observation suggests that therapies targeting bacteria growing with small amounts of oxygen may be most effective against these hard-to-treat infections. More generally, this proof-of-concept study demonstrates that HCR v3.0 has the potential to identify microbial activities in situ at small spatial scales in diverse contexts.
format	article
author	Peter Jorth Melanie A. Spero J. Livingston Dianne K. Newman
author_facet	Peter Jorth Melanie A. Spero J. Livingston Dianne K. Newman
author_sort	Peter Jorth
title	Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone
title_short	Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone
title_full	Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone
title_fullStr	Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone
title_full_unstemmed	Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone
title_sort	quantitative visualization of gene expression in mucoid and nonmucoid <named-content content-type="genus-species">pseudomonas aeruginosa</named-content> aggregates reveals localized peak expression of alginate in the hypoxic zone
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/bc92388167244d53b79778dac5035526
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Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone

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