Biology subjects

Carabeo, R. A.

Publications and source records attributed to Carabeo, R. A..

5 recordsLinked to original sources

bioRxiv2021

Regulation of invasion-associated actin dynamics by the Chlamydia trachomatis effectors TarP and TmeA

The obligate intracellular pathogen Chlamydia trachomatis manipulates the host actin cytoskeleton to assemble actin-rich structures that drive pathogen entry. The recent discovery of TmeA, which like TarP is an invasion-associated type III effector implicated in actin remodeling, raised questions regarding the nature of their functional interaction. Quantitative live-cell imaging of actin remodeling at invasion sites revealed differences in recruitment and turnover kinetics associated with TarP and TmeA pathways, with the former accounting for most of the robust actin dynamics at invasion sites. TarP-mediated recruitment of the actin nucleators formin and the Arp2/3 complex were crucial for rapid actin kinetics, generating a collaborative positive feedback loop that enhanced their respective actin-nucleating activities within invasion sites. In contrast, Fmn1 is neither recruited to invasion sites nor collaborates with Arp2/3 within the context of TmeA-associated actin recruitment. While the TarP-Fmn1-Arp2/3 signaling axis is responsible for the majority of actin dynamics, its inhibition had similar effects as deletion of TmeA on invasion efficiency, consistent with the proposed model that TarP and TmeA acting on different stages of the same invasion pathway. Summary StatementKinetic analysis of actin recruitment during C. trachomatis invasion reveals TarP as the major contributor relative to TmeA, via its ability to facilitate collaboration between actin nucleators Formin 1 and Arp2/3.

cell biology↗

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↗

bioRxiv2018

The Chlamydia type III effector TarP alters the dynamics and organization of host cell focal adhesions

The human pathogen Chlamydia trachomatis targets epithelial cells lining the genital mucosa. We observed that infection of various cell types, including fibroblasts and epithelial cells resulted in the formation of unusually stable focal adhesions that resisted disassembly induced by the myosin II inhibitor, blebbistatin. Super-resolution microscopy revealed in infected cells the vertical displacement of paxillin and FAK from the signaling layer of focal adhesions; while vinculin remained in its normal position within the force transduction layer. The candidate type III effector TarP which localized to focal adhesions during infection and when expressed ectopically, was sufficient to mimic both the reorganization and blebbistatin-resistant phenotypes. These effects of TarP, including its localization to focal adhesions required interaction with the host protein vinculin through a specific domain at the C-terminus of TarP. The consequence of Chlamydia-stabilized focal adhesions was restricted cell motility and enhanced attachment to the extracellular matrix. Thus, via a novel mechanism, Chlamydia inserts TarP within focal adhesions to alter their organization and dynamics.

cell biology↗

bioRxiv2017

Genome-wide transcriptional responses of iron-starved Chlamydia trachomatis reveal prioritization of metabolic precursor synthesis over protein translation

Iron is essential for growth and development of Chlamydia. Its long-term starvation in cultured mammalian cells leads to production of aberrant non-infectious chlamydial forms, also known as persistence. Immediate transcriptional responses to iron limitation have not been characterized, leaving a knowledge gap of how Chlamydia regulates its response to changes in iron availability. We used the fast-chelating agent 2,2-Bipyridyl (BPDL) to homogeneously starve Chlamydia trachomatis serovar L2 of iron, starting at 6 or 12h post-infection. Immediate transcriptional responses were monitored after only 3 or 6h of BPDL-treatment, well before formation of aberrant Chlamydia. The first genome-wide transcriptional response of C. trachomatis to iron-starvation was subsequently determined utilizing RNA-sequencing. Only 7% and 8% of the genome was differentially expressed in response to iron-starvation at early and mid-stages of development, respectively. Biological pathway analysis revealed an overarching theme. Synthesis of macromolecular precursors (deoxynucleotides, amino acids, charged tRNAs, and acetyl-coA) was up-regulated, while energy-expensive processes (ABC transport and translation) were down-regulated. A large fraction of differentially down-regulated genes are involved in translation, including ribosome assembly, initiation and termination factors, which could be analogous to the translation down-regulation triggered by stress in other prokaryotes during stringent responses. Additionally, transcriptional up-regulation of DNA repair, oxidative stress, and tryptophan salvage genes reveals a possible coordination of responses to multiple antimicrobial and immunological insults. These responses of replicative-phase Chlamydia to iron-starvation indicate a prioritization of survival over replication, enabling the pathogen to \"stock the pantry\" with ingredients needed for rapid growth once optimal iron levels are restored.\n\nIMPORTANCEBy utilizing an experimental approach that monitors the immediate global response of Chlamydia trachomatis to iron-starvation, clues to long-standing questions in Chlamydia biology are revealed, including how Chlamydia adapts to this stress. We determined that this pathogen initiates a transcriptional program that prioritizes replenishment of nutrient stores over replication, possibly in preparation for rapid growth once optimal iron levels are restored. Transcription of genes for biosynthesis of metabolic precursors was generally up-regulated, while those involved in multiple steps of translation were down-regulated. We also observed an increase in transcription of genes involved in DNA repair and neutralizing oxidative stress, indicating that Chlamydia employs an \"all-or-nothing\" strategy. Its small genome limits its ability to tailor a specific response to a particular stress. Therefore, the \"all-or-nothing\" strategy may be the most efficient way of surviving within the host, where the pathogen likely encounters multiple simultaneous immunological and nutritional insults.

microbiology↗