A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Abstract

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level, but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy, and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses, and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

The Accessory Gene saeP of the SaeR/S Two-Component Gene Regulatory System Impacts Staphylococcus aureus Virulence During Neutrophil Interaction

“…results, USA300 formed clearly-defined abscesses containing a nidus of staphylococci also referred to as a staphylococcal abscess community (SAC). These bacteria exhibit spatial regulation of nuc-gfp as we previously reported (Behera et al, 2019). That is, we detected strong expression of nuc-gfp in the core of the SAC and muted expression on the periphery of the SAC.…”

“…To investigate if the increased mortality in mice challenged with the USA300 saePQ mutant was due to increased expression of SaeR/S-dependent virulence factors, we infected mice with either USA300 or USA300 saePQ mutant carrying an integrated nuc-gfp translational fusion (Behera et al, 2019). Three days post-infection, groups of three-eight C57BL/6 mice were euthanized.…”

“…Next, we transduced the strain to chloramphenicol resistance, moving in the P saeP3 -saeRS construct (saeRS under the control of their native promoter, cloned into pCL55 and integrated into the geh locus, Cm R ). Then, we integrated the nuc-gfp reporter as described by allelic exchange (Behera et al, 2019). Briefly, DNA fragments upstream and downstream of the gene or gene fragment of interest were amplified using primers listed in Table 1, purified by agarose gel electrophoresis, then combined in a two-step overlap PCR reaction and cloned into pJB38 .…”

“…Infections were allowed to progress for 72 h or until humane endpoints were reached. To analyze nuc-gfp expression in tissues, infections were performed as described for USA300 LAC and the saePQ double mutant essentially described above (and specifically in Behera et al, 2019); notably, animals were fed AIN-93 purified diet (Reeves et al, 1993). Briefly, mice were euthanized 72 h post-infection and kidneys were harvested, fixed with 10% (v/v) buffered formalin, embedded in Sub Xero clear tissue freezing medium (Mercedes Medical), and sectioned into 10 µm slices.…”