Biology subjects

Pokorzynski, N. D.

Publications and source records attributed to Pokorzynski, N. D..

5 recordsLinked to original sources

bioRxiv2025

Chlamydia iron starvation links nutritional immunity to pathogen recognition

Nutritional immunity is an antimicrobial strategy that evolved to starve pathogens of essential nutrients, with death as the desired outcome. Here, we report that transient iron starvation of the obligate intracellular pathogen Chlamydia trachomatis, growing in endocervical epithelial cells, enhances pathogen recognition by the host cell through the dysregulation of a peptidoglycan (PG) remodeling enzyme, resulting in the activation of the nucleotide-binding oligomerization domain 2 (NOD2) pathway that recognizes PG fragments, increased production of tumor necrosis factor alpha (TNF) via increased activation of NF-{kappa}B, which correlated with death of infected cells. Activation of the NOD2/ NF-{kappa}B signaling axis is linked to the dysregulated overexpression of the PG remodeling enzyme AmiA and the subsequent cleavage and mislocalization of D-Ala-D-Ala analog. Inhibiting amiA transcriptional upregulation by CRISPR interference reduced pathogen recognition. We propose that nutritional immunity in general mediate abnormal expression of bacterial genes linked to pathogen-associated molecular patterns. ImportanceLimiting pathogen access to essential nutrients is the central tenet of nutritional immunity, with the outcome being severe starvation and eventual death of the pathogen. However, pathogen starvation induces several physiological changes prior to its death. They include errors in several biological processes, including metabolism and gene expression, which could lead to pathogen death. Here, we demonstrate that iron starvation of the clinically relevant human pathogen Chlamydia trachomatis significantly dysregulates the expression of a peptidoglycan remodeling amidase, AmiA to enhance chlamydial recognition by the host cell and the subsequent increased production of tumor necrosis factor and death of infected cells to the detriment of Chlamydia.

microbiology↗

bioRxiv2023

Machine Learning and Metabolic Model Guided CRISPRi Reveals a Central Role for Phosphoglycerate Mutase in Chlamydia trachomatis Persistence

Upon nutrient starvation, Chlamydia trachomatis serovar L2 (CTL) shifts from its normal growth to a non-replicating form, termed persistence. It is unclear if persistence is an adaptive response or lack of it. To understand that transcriptomics data were collected for nutrient-sufficient and nutrient-starved CTL. Applying machine learning approaches on transcriptomics data revealed a global transcriptomic rewiring of CTL under stress conditions without having any global stress regulator. This indicated that CTLs stress response is due to lack of an adaptive response mechanism. To investigate the impact of this on CTL metabolism, we reconstructed a genome-scale metabolic model of CTL (iCTL278) and contextualized it with the collected transcriptomics data. Using the metabolic bottleneck analysis on contextualized iCTL278, we observed phosphoglycerate mutase (pgm) regulates the entry of CTL to the persistence. Later, pgm was found to have the highest thermodynamics driving force and lowest enzymatic cost. Furthermore, CRISPRi-driven knockdown of pgm and tryptophan starvation experiments revealed the importance of this gene in inducing persistence. Hence, this work, for the first time, introduced thermodynamics and enzyme-cost as tools to gain deeper understanding on CTL persistence.

systems biology↗

bioRxiv2021

Host cell amplification of nutritional stress contributes to persistence in Chlamydia trachomatis

Persistence, a viable but non-replicating growth state, has been implicated in diseases caused by Chlamydia trachomatis. Starvation of distinct nutrients produce a superficially similar persistent state, implying convergence on a common intracellular environment. We employed host-pathogen dual RNA-sequencing under both iron- and tryptophan-starved conditions to systematically characterize the persistent chlamydial transcriptome and to define common contributions of the host cell transcriptional stress response in shaping the intracellular environment. The transcriptome of the infected host cells was highly specific to each nutritional stress, despite comparable effects on chlamydial growth and development in each condition. In contrast, the chlamydial transcriptomes between nutritional conditions were highly similar, suggesting some overlap in host cell responses to iron limitation and tryptophan starvation that contribute to a common persistent phenotype. We demonstrate that a commonality in the host cell responses is the suppression of guanosine triphosphate (GTP) biosynthesis, a nucleotide for which Chlamydia are auxotrophic. Pharmacological inhibition of host inosine monophosphate dehydrogenase (IMPDH1), which catalyzes the rate-limiting step in de novo guanine nucleotide synthesis, resulted in comparable GTP depletion to both iron and tryptophan starvation and induced chlamydial persistence. Moreover, IMPDH1 inhibition and iron starvation acted synergistically to control chlamydial growth. Thus, host cell reduction in GTP levels amplifies the nutritional stress to intracellular chlamydiae in infection-relevant models of persistence, illustrating the determinative role the infected host cell plays in bacterial stress responses. IMPORTANCEBacteria respond to nutritional stress through universal and unique mechanisms. Genome reduction in the Chlamydiaceae, a consequence of coevolution with their obligate eukaryotic hosts, has reduced their repertoire of stress response mechanisms. Here we demonstrate that the infected host cell may provide the context within which universal stress responses emerge for Chlamydia trachomatis. We report that during starvation of the essential nutrients iron or tryptophan, a common response of the infected epithelial cell is the suppression of GTP biosynthesis, which induces a persistent developmental state in the pathogen. Thus, chlamydial persistence results from the combined effects of primary stresses on the pathogen and the host, with the latter eliciting a secondary host cell response that intensifies the inhospitable intracellular environment.

microbiology↗

bioRxiv2020

Regulation of an iron-dependent repressor by tryptophan availability attenuates transcription of the tryptophan salvage genes in Chlamydia trachomatis

The trp operon of Chlamydia trachomatis has a genetic organization that is distinct from other model bacteria. The operon contains the trpR open-reading frame (ORF), an intergenic region (IGR), and the trpB and trpA ORFs. TrpR mediates tryptophan-dependent regulation of the operon from the major promoter upstream of trpR(PtrpR). We recently reported that trpBA is additionally regulated by the iron-dependent repressor YtgR via an operator sequence within the IGR upstream of an alternative promoter for TrpR-independent trpBA expression (PtrpBA). Here we report that YtgR repression of PtrpBA is also dependent on tryptophan levels via a rare triple-tryptophan motif (WWW) in the N-terminal permease domain of the YtgCR precursor. Tryptophan limitation inhibits translation at the WWW motif and subsequently promotes transcription termination of ytgCR in a Rho-independent manner. This regulatory schematic resembles mechanisms of transcriptional attenuation for trp operons described in model bacteria, such as cis-attenuation by TrpL in E. coli or trans-attenuation by TRAP in B. subtilis. YtgR performs an analogous function by sensing both iron and tryptophan levels, with the latter highlighting the unique strategy of Chlamydia to retain a trans-attenuator mechanism for regulating expression of the trpRBA operon.

microbiology↗

bioRxiv2018

The tryptophan salvage pathway is dynamically regulated by the iron-dependent repressor YtgR in Chlamydia trachomatis

During infection, pathogens are starved of essential nutrients such as iron and tryptophan by host immune effectors. Without conserved global stress response regulators, how the obligate intracellular bacterium Chlamydia trachomatis arrives at a physiologically similar \"persistent\" state in response to starvation of either nutrient remains unclear. Here, we report on the iron-dependent regulation of the trpRBA tryptophan salvage pathway in C. trachomatis. Iron starvation specifically induces trpBA expression from a novel promoter element within an intergenic region flanked by trpR and trpB. YtgR, the only known iron-dependent regulator in Chlamydia, can bind to the trpRBA intergenic region upstream of the alternative trpBA promoter to repress transcription. Simultaneously, YtgR binding promotes the termination of transcripts from the primary promoter upstream of trpR. This is the first description of an iron-dependent mechanism regulating prokaryotic tryptophan biosynthesis that may indicate the existence of novel approaches to gene regulation and stress response in Chlamydia.

microbiology↗