Quantitative visualization of gene expression inPseudomonas aeruginosaaggregates reveals peak expression of alginate in the hypoxic zone

AbstractIt is well appreciated that oxygen- and 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 scalesin situ. Here we report the application of the hybridization chain reaction (HCR) v3.0 toPseudomonas aeruginosaaggregates as a step towards this end. As proof-of-principle, we visualize the expression of genes needed for the production of alginate (algD) and the dissimilatory nitrate reductase (narG). Using an inducible bacterial gene expression construct to calibrate the HCR signal, we were able to quantifyalgDandnarGgene expression across microenvironmental gradients both within single aggregates and within aggregate populations using the Agar Block Biofilm Assay (ABBA). For the ABBA population, alginate gene expression was restricted to hypoxic regions within the environment (~40-200 μM O2), as measured by an oxygen microelectrode. Within individual biofilm aggregates, cells proximal to the surface expressed alginate genes to a greater extent than interior cells. Lastly, mucoid biofilms consumed more oxygen than nonmucoid biofilms. These results establish that HCR has a sensitive dynamic range and can be used to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. Because HCR v3.0 can be performed on diverse cell types, this methodological advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen behavior in human chronic infections.ImportanceThe visualization of microbial activities in natural environments is an important goal for numerous studies in microbial ecology, be the environment a sediment, soil, or infected human tissue. Here we report the application of the hybridization chain reaction (HCR) v3.0 to measure microbial gene expressionin situat single-cell resolution in aggregate biofilms. UsingPseudomonas aeruginosawith a tunable gene expression system, we show that this methodology is quantitative. Leveraging HCR v3.0 to measure gene expression within aP. aeruginosaaggregate, we find 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, HCR v3.0 has potential for broad application into microbial activitiesin situat small spatial scales.

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Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid Pseudomonas aeruginosa Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone

mBio ◽

10.1128/mbio.02622-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 2

Author(s):

Peter Jorth ◽

Melanie A. Spero ◽

J. Livingston ◽

Dianne K. Newman

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Single Cell ◽

Spatial Scales ◽

Hybridization Chain Reaction ◽

Microbial Activities ◽

Chain Reaction ◽

Content Type ◽

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 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.

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Combined in situ hybridization chain reaction and immunostaining to visualize gene expression in whole-mount Drosophila central nervous systems

10.1101/2021.08.02.454831 ◽

2021 ◽

Author(s):

Julia C Duckhorn ◽

Ian P Junker ◽

Yun Ding ◽

Troy R Shirangi

Keyword(s):

Gene Expression ◽

Nervous System ◽

In Situ Hybridization ◽

Hybridization Chain Reaction ◽

Chain Reaction ◽

Nervous Systems ◽

Whole Mount ◽

Central Nervous Systems ◽

Mrna And Protein Expression

Methods to visualize gene expression in the Drosophila central nervous system are important in fly neurogenetic studies. In this chapter, we describe a detailed protocol that sequentially combines in situ hybridization chain reaction (HCR) and immunostaining to detect mRNA and protein expression in whole-mount Drosophila larval and adult central nervous systems. We demonstrate the application of in situ HCR in comparisons of nervous system gene expression between Drosophila species, and in the validation of single-cell RNA-Seq results in the fly nervous system. Our protocol provides a simple, robust, multiplexable, and relatively affordable means to quantitatively visualize gene expression in the nervous system of flies, facilitating its general use in fly neurogenetic studies.

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Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

10.1101/214619 ◽

2017 ◽

Author(s):

Vikas Trivedi ◽

Harry M.T. Choi ◽

Scott E. Fraser ◽

Niles A. Pierce

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Mrna Expression ◽

Hybridization Chain Reaction ◽

Chain Reaction ◽

Developmental Gene Expression ◽

Vertebrate Embryos ◽

Anatomical Space ◽

Subcellular Resolution

ABSTRACTFor decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting, and/or homogenization. Here, we eliminate the tradeoff between quantitation and anatomical context, using multiplexed in situ hybridization chain reaction (HCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable iterative bi-directional queries that open a new era for in situ hybridization.SUMMARYMultiplexed in situ hybridization chain reaction (HCR) enables quantitative multidimensional analyses of developmental gene expression with subcellular resolution in an anatomical context.

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A new method for the in situ assay of α-glucosidase activity and inhibitor screening through an enzyme substrate-mediated DNA hybridization chain reaction

New Journal of Chemistry ◽

10.1039/c9nj01868a ◽

2019 ◽

Vol 43 (24) ◽

pp. 9458-9465

Author(s):

Xiquan Yue ◽

Lihong Su ◽

Xu Chen ◽

Junfeng Liu ◽

Longpo Zheng ◽

...

Keyword(s):

Small Molecule ◽

Dna Hybridization ◽

New Method ◽

Hybridization Chain Reaction ◽

Chain Reaction ◽

Inhibitor Screening ◽

Glucosidase Activity ◽

Enzyme Substrate ◽

In Situ Assay

The strategy is based on small molecule-mediated hybridization chain reaction.

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Optimization and evaluation of fluorescence in situ hybridization chain reaction in cleared fresh-frozen brain tissues

Brain Structure and Function ◽

10.1007/s00429-020-02194-4 ◽

2021 ◽

Author(s):

Vivek Kumar ◽

David M. Krolewski ◽

Elaine K. Hebda-Bauer ◽

Aram Parsegian ◽

Brian Martin ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Hybridization Chain Reaction ◽

Chain Reaction ◽

Fresh Frozen ◽

Brain Tissues

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Detection of MBL-2 gene expression in intestinal biopsies of celiac patients by in situ reverse transcription polymerase chain reaction

European Journal of Histochemistry ◽

10.4081/825 ◽

2009 ◽

Vol 47 (2) ◽

pp. 177 ◽

Cited By ~ 13

Author(s):

M Boniotto ◽

O Radillo ◽

L Braida ◽

D Pirulli ◽

A Città

Keyword(s):

Gene Expression ◽

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Chain Reaction ◽

Polymerase Chain

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The contribution of increased collagen synthesis to human glomerulosclerosis: a quantitative analysis of alpha 2IV collagen mRNA expression by competitive polymerase chain reaction.

Journal of Experimental Medicine ◽

10.1084/jem.176.6.1571 ◽

1992 ◽

Vol 176 (6) ◽

pp. 1571-1576 ◽

Cited By ~ 49

Author(s):

E P Peten ◽

L J Striker ◽

M A Carome ◽

S J Elliott ◽

C W Yang ◽

...

Keyword(s):

Gene Expression ◽

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Collagen Synthesis ◽

Type Iv Collagen ◽

Chain Reaction ◽

Collagen Mrna ◽

Type Iv ◽

Polymerase Chain

We previously reported that one of the main components of the sclerotic material in human glomerular diseases was type IV collagen. In this study we examined the contribution of increased synthesis to this process at the gene expression level. Sufficient material has not been available to study type IV collagen synthesis by normal or sclerotic glomeruli in humans. We took advantage of the availability of nephrectomy specimens from patients with renal carcinoma, and of the observation that approximately 50% of these patients develop varying degrees of glomerulosclerosis. We microdissected glomeruli from 10 patients and analyzed them using in situ reverse transcription coupled with polymerase chain reaction (PCR) analyses (in situ RT-PCR). alpha 2IV collagen mRNA, after reverse transcription into cDNA, was detected in all patients and appeared to be increased in those with glomerulosclerosis (n = 5). A competitive PCR assay was developed to quantitate this change. There was an average 3.7-fold increase in glomerular type IV collagen cDNA in patients with significant sclerosis. This change was not due to an increased number of glomerular cells. Thus, glomerulosclerosis in humans is associated with an elevation of glomerular type IV collagen gene expression, suggesting that increased synthesis of type IV collagen may represent one component of this process.

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In situ amplified electrochemical aptasensing for sensitive detection of adenosine triphosphate by coupling target-induced hybridization chain reaction with the assembly of silver nanotags

Talanta ◽

10.1016/j.talanta.2015.08.035 ◽

2016 ◽

Vol 146 ◽

pp. 23-28 ◽

Cited By ~ 20

Author(s):

Qian Zhou ◽

Youxiu Lin ◽

Yuping Lin ◽

Qiaohua Wei ◽

Guonan Chen ◽

...

Keyword(s):

Adenosine Triphosphate ◽

Sensitive Detection ◽

Hybridization Chain Reaction ◽

Chain Reaction

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PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

Infection and Immunity ◽

10.1128/iai.01388-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1638-1647 ◽

Cited By ~ 28

Author(s):

Ziyu Sun ◽

Jing Shi ◽

Chang Liu ◽

Yongxin Jin ◽

Kewei Li ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Bacterial Colonization ◽

Mrna Level ◽

Sos Response ◽

Opportunistic Pathogen ◽

Host Cells ◽

Chronic Infections ◽

Mobility Shift ◽

Content Type

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced byP. aeruginosathat are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component ofP. aeruginosa. In this study, we demonstrate that mutation inprtRresults in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by atacpromoter repressed the endogenousprtRpromoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

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