In the intricate battlefield of host-pathogen interactions, the ability of pathogens to adapt swiftly to fluctuating environments often determines their survival and virulence. Salmonella enterica serovar Typhimurium (S. Typhimurium), a leading cause of gastrointestinal infections worldwide, exemplifies this adaptive ingenuity, especially as it invades and proliferates within the hostile confines of macrophages. These immune cells represent formidable barriers, deploying a barrage of antimicrobial mechanisms designed to neutralize invading microbes. However, S. Typhimurium has evolved dynamic transcriptional responses enabling it to not only survive but thrive inside these cellular fortresses. Despite significant advances leveraging RNA sequencing technologies, the nuances governing the timing, magnitude, and variability of these bacterial gene expression programs have remained incompletely understood. Addressing these gaps, a pioneering study has now constructed a comprehensive library of fluorescent promoter reporters that systematically maps out the transcriptional landscape of S. Typhimurium during macrophage infection, revealing hitherto uncharted regulatory dynamics with profound implications.
Central to this breakthrough is the development of an ambitious GFP-reporter strain library encompassing nearly 2,901 promoter regions computationally predicted across the S. Typhimurium genome. Each promoter element was fused to a green fluorescent protein gene, allowing real-time visualization and quantification of promoter activity under diverse conditions. This expansive reporter platform was leveraged to monitor transcriptional shifts during standard in vitro growth phases as well as across the complex intracellular milieu of RAW 264.7 macrophages, a widely employed murine macrophage cell line. Importantly, the study deployed both bulk fluorescence measurements and cutting-edge single-cell imaging modalities, enabling deconvolution of population-level responses into discrete subpopulations and individual-cell heterogeneity. This dual approach uncovered not just averaged promoter activities but also revealed the stochasticity and condition-specific regulation often masked in ensemble assays.
Within the intracellular environment of macrophages, bacterial pathogens encounter oxidative stress, nutrient limitation, and metal ion fluctuations. The transcriptional reprogramming of S. Typhimurium in response to these challenges is orchestrated by a suite of virulence factors, notably the Salmonella Pathogenicity Island 2 (SPI-2) locus. The reporter library confirmed intense activity within SPI-2-related promoters upon macrophage infection, consistent with prior findings that SPI-2 is critical for intracellular survival and pathogenesis. However, beyond these expected regulators, the study uncovered startling heterogeneity in promoter activity at the single-cell level. This heterogeneity suggests that within a clonal bacterial population, subpopulations may adopt divergent transcriptional states, potentially facilitating phenotypic diversification as a bet-hedging strategy in the face of fluctuating host defenses.
A particularly compelling revelation emerged in the form of 234 previously uncharacterized promoters exhibiting transcriptional activity under either in vitro or intracellular conditions. This finding significantly expands the catalog of potentially functional regulatory sequences in S. Typhimurium and points to a more complex transcriptional network than previously appreciated. Many of these novel promoters might drive expression of genes involved in metabolic adaptation and stress response that have so far eluded detection via conventional RNA-seq due to their transient or low-level expression.
Key among the metabolic adaptations uncovered was the induction of genes related to manganese homeostasis, spotlighting the small RNA mntS as an essential component in regulating intracellular manganese availability. Manganese acts as a cofactor for numerous enzymes and also mediates resistance to oxidative stress, a common weapon employed by macrophages. The insights from this study underscore the critical balance S. Typhimurium must maintain in trace metal acquisition and detoxification within the phagosomal niche, pivoting on finely tuned transcriptional circuits.
Moreover, the research illuminated the activation of the Entner–Doudoroff (ED) pathway genes within macrophages, a less commonly utilized glycolytic route in many bacteria. This pathway provides metabolic flexibility, enabling S. Typhimurium to efficiently catabolize sugars and generate energy under nutrient-restricted intracellular conditions. The data suggest that reliance on the ED pathway may represent a strategic metabolic rewiring to circumvent bottlenecks encountered during infection, highlighting the metabolic plasticity of this pathogen.
The integrative dataset generated from this extensive screening effort was made accessible through SalComKinetics, an innovative online platform for visualizing and interrogating transcriptional dynamics across conditions and time points. This resource empowers researchers worldwide to probe gene expression patterns at unprecedented resolution, facilitating systems-level analyses that could accelerate discovery of novel therapeutic targets and illuminate bacterial adaptive strategies.
Methodologically, the study represents a tour de force in combining computational prediction, genetic engineering, quantitative fluorescence assays, and live-cell microscopy. The generation of nearly three thousand individual promoter-GFP fusions required precise cloning and validation workflows, underscored by rigorous quantitative measurements to ensure reproducibility and sensitivity. The fusion of bulk and single-cell data streams allowed insights into both overarching trends and microscopic nuances of bacterial gene regulation, bridging a critical gap in current understanding of infection dynamics.
From a pathogenesis perspective, this work provides an invaluable window into how S. Typhimurium modulates its transcriptional landscape in real time during host cell invasion and residence. The finding that promoter activity is not only temporally regulated but also subject to population heterogeneity challenges simplistic models of uniform bacterial behavior inside host cells. Such heterogeneity may underlie differential survival strategies, resistance to immune clearance, or emergence of persister cells contributing to chronic infections.
Moreover, the discovery of numerous transcriptionally active yet previously unannotated promoters raises fundamental questions about the evolution and organization of bacterial genomes. It suggests that extensive layers of regulatory complexity remain to be deciphered, with potential consequences for understanding bacterial physiology, virulence, and adaptation. Such elements could encode small RNAs, alternative sigma factor-dependent transcripts, or uncharacterized open reading frames that modulate bacterial fitness under infection-related stresses.
The implications of manganese regulation through mntS also extend beyond Salmonella biology. Trace metal homeostasis is a central battlefield in host–pathogen interactions, and elucidating regulatory RNAs controlling metal acquisition could unveil novel antimicrobial intervention points. Targeting metal scavenging and detoxification pathways offers an exciting frontier in combating intracellular pathogens that have evolved sophisticated strategies to circumvent host nutritional immunity.
On the metabolic front, the elucidation of ED pathway activation shifts the conventional paradigm dominated by glycolysis toward appreciating alternative biochemical routes exploited by pathogens during infection. Understanding these shifts can inform metabolic modeling efforts aimed at predicting vulnerabilities and optimizing antimicrobial therapies that disrupt pathogen energy metabolism within host environments.
By openly sharing the promoter library and the comprehensive transcriptional datasets via SalComKinetics, this study embodies the ethos of open science and collaborative discovery. It provides a foundational toolkit not only for microbiologists investigating Salmonella but also for researchers exploring gene regulation, bacterial pathogenesis, and host–microbe interactions across diverse systems.
In summary, this landmark study redefines our understanding of Salmonella’s transcriptional responsiveness to the intracellular niche of macrophages, revealing complex, time-dependent, and heterogeneous gene expression patterns. The integration of high-throughput promoter screening with live-cell imaging charts a path forward for dissecting bacterial adaptation at an unprecedented level of detail. Such insights are poised to catalyze new avenues in infection biology, translational research, and the development of innovative antimicrobial strategies.
As infectious diseases continue to pose global health challenges, especially with rising antibiotic resistance, comprehensively mapping bacterial transcriptional dynamics in relevant host environments becomes ever more critical. The approach and findings presented herein illuminate fundamental principles of pathogen survival and open new doors for targeted interventions aimed at disarming stealthy intracellular invaders like Salmonella enterica serovar Typhimurium.
Subject of Research: Salmonella enterica serovar Typhimurium transcriptional dynamics during macrophage infection
Article Title: Profiling Salmonella transcriptional dynamics during macrophage infection using a comprehensive reporter library
Article References:
Nguyen, T.H., Wang, B.X., Diaz, O.R. et al. Profiling Salmonella transcriptional dynamics during macrophage infection using a comprehensive reporter library. Nat Microbiol 10, 1006–1023 (2025). https://doi.org/10.1038/s41564-025-01953-5
Image Credits: AI Generated
