Biology subjects

Grin, I.

Publications and source records attributed to Grin, I..

4 recordsLinked to original sources

bioRxiv2026

Gene synteny and translational coupling of sctS and sctT facilitate assembly of the unique helical T3SS export apparatus in Salmonella Typhimurium

Virulence-associated type III secretion systems (T3SS) are employed by many gram-negative pathogens to translocate effector proteins into hosts. These nanomachines are composed of cytoplasmic components, a needle complex base, housing the export apparatus and anchoring the machine in the bacterial cell envelope, a needle filament, and a translocon that penetrates the host membrane. Assembly of the T3SS is a hierarchical process, which is initiated by coordinated association of the five subunits comprising the export apparatus, SctR, SctS, SctT, SctU, and SctV, in the bacterial inner membrane. In this study, we report that proper assembly of this uniquely helical export apparatus is fine-tuned by the genetic organization of the export apparatus genes in Salmonella Typhimurium. The sctS mRNA harbors a stem-loop structure which conceals the Shine-Dalgarno sequence of sctT from recognition by a ribosome. sctT translation is only possible upon melting of the stem-loop by a sctS-translating ribosome. This mechanism translationally couples sctS and sctT. We show that this strict regulation prevents uncontrolled overexpression of SctT and its assembly into futile multimers that disrupt export apparatus assembly and reduce pathogen fitness. Based on the strong synteny of T3SS export apparatus genes and their highly conserved unique structure, also among closely related bacterial flagella, similar mechanisms are likely needed to enable a tightly regulated and stoichiometric assembly process of these molecular machines in general.

microbiology↗

bioRxiv2024

Discovery of a synthetic small molecule targeting the central regulator of Salmonella pathogenicity

The enteric pathogen Salmonella enterica serovar Typhimurium relies on the activity of effector proteins to invade, replicate, and disseminate into host epithelial cells and other tissues, thereby causing disease. Secretion and injection of effector proteins into host cells is mediated by dedicated secretion systems, which hence represent major virulence determinants. Here, we report the identification of a synthetic small molecule with drug-like properties, C26, which suppresses the secretion of effector proteins, and consequently hinders bacterial invasion of eukaryotic cells. C26 binds to and inhibits HilD, the transcriptional regulator of the major secretion systems. While sharing the same binding pocket as the previously described long-chain fatty acid ligands, C26 inhibits HilD with a unique binding mode and a distinct mechanism. We provide evidence for target engagement within infected eukaryotic cells and present analogs with improved potency and suitability as scaffolds to develop anti-virulence agents against Salmonella infections in humans and animals.

microbiology↗

bioRxiv2024

The T4bSS of Legionella features a two-step secretion pathway with an inner membrane intermediate for secretion of transmembrane effectors

To promote intracellular survival and infection, Legionella spp. translocate hundreds of effector proteins into eukaryotic host cells using a type IV b protein secretion system (T4bSS). T4bSS are well known to translocate soluble as well as transmembrane domain-containing effector proteins (TMD-effectors) but the mechanisms of secretion are still poorly understood. Herein we investigated the secretion of hydrophobic TMD-effectors, of which about 80 were previously reported to be encoded by L. pneumophila. A proteomic analysis of fractionated membranes revealed that TMD-effectors are targeted to and inserted into the bacterial inner membranes of L. pneumophila independent of the presence of a functional T4bSS. While the T4bSS chaperones IcmS and IcmW were critical for secretion of all tested TMD-effectors, they did not influence inner membrane targeting of these proteins. As for soluble effector proteins, translocation of TMD-effectors into host cells depended on a C-terminal secretion signal and this signal needed to be presented towards the cytoplasmic side of the inner membrane. A different secretion behavior of TMD- and soluble effectors and the need for small periplasmic loops within TMD-effectors provided strong evidence that TMD-effectors are secreted in a two-step secretion process: Initially, an inner membrane intermediate is formed, that is extracted towards the cytoplasmic side, possibly by the help of the type IV coupling protein complex and subsequently secreted into eukaryotic host cells by the T4bSS core complex. Overall, our study highlights the amazing versatility of T4bSS to secrete soluble and TMD-effectors from different subcellular locations of the bacterial cell.

microbiology↗

bioRxiv2019

A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a lack of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess secretion and injection through the type III secretion system encoded by Salmonella pathogenicity island 1. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed NanoLuc and split-NanoLuc-based assays that enable the monitoring of type III secretion-dependent injection of effector proteins into host cells.\n\nImportanceThe ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or even into target cells is one of the foundations of interbacterial as well as pathogen-host interaction. While great progress has been made in elucidating assembly and structure of secretion systems, our understanding of their secretion mechanism often lags behind, not last because of the challenge to quantitatively assess secretion function. Here, we developed a luciferase-based assay to enable the simple, quick, quantitative, and high throughput-compatible assessment of secretion and injection through virulence-associated type III secretion systems. The assay allows detection of minute amounts of secreted substrate proteins either in the supernatant of the bacterial culture or within eukaryotic host cells. It thus provides an enabling technology to elucidate the mechanisms of secretion and injection of type III secretion systems and is likely adaptable to assay secretion through other bacterial secretion systems.

microbiology↗