Biology subjects

Galan, J. E.

Publications and source records attributed to Galan, J. E..

7 recordsLinked to original sources

bioRxiv2025

Interplay between SpaO variants shapes the architecture of the Salmonella type III secretion sorting platform

Salmonella enterica utilizes a virulence-associated type III secretion system (T3SS) to inject bacterial effectors directly into host cells. Central to this machinery is the sorting platform (SP), a cytosolic assembly whose core scaffolding protein, SpaO, is produced in two isoforms: a full-length (SpaOL) and a shorter variant (SpaOshort) comprising the C-terminal 101 residues of SpaOL. Although SpaOshort is evolutionarily conserved across type III secretion systems, its precise function has remained elusive. Here, we combined a sensitive, real-time translocation assay with site-directed photo-crosslinking to elucidate the role of SpaOshort in Salmonella SPI-1 T3SS. We found that while SpaOshort is not absolutely required for effector secretion, its absence significantly dampens T3SS-mediated protein delivery. Further biochemical and structural probing revealed that SpaOshort is a structural component of the sorting platform, arranged as a homodimer associated to SpaOL via an N-terminal "docking motif." This interaction occurs while SpaOL is associated with other SP components, supporting a model in which SpaOshort is integrated into the SP pods alongside SpaOL, OrgA, and OrgB. Collectively, these findings show that SpaOshort, while not strictly essential, functions as a critical structural component of the sorting platform, providing new insights into how Salmonella and related bacteria assemble and maintain these specialized protein-injection systems.

microbiology↗

bioRxiv2024

Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

Salmonella enterica serovar Typhi and Paratyphi A are the cause of typhoid and paratyphoid fever in humans, which are systemic life-threatening illnesses. Both serovars are exclusively adapted to the human host, where they can cause life-long persistent infection. A distinct feature of these serovars is the presence of a relatively high number of degraded coding sequences coding for metabolic pathways, most likely a consequence of their adaptation to a single host. As a result of convergent evolution, these serovars shared many of the degraded coding sequences although often affecting different genes in the same metabolic pathway. However, there are several coding sequences that appear intact in one serovar while clearly degraded in the other, suggesting differences in their metabolic capabilities. Here, we examined the functionality of metabolic pathways that appear intact in S. Typhi but that show clear signs of degradation in S. Paratyphi A. We found that, in all cases, the existence of single amino acid substitutions in S. Typhi metabolic enzymes, transporters, or transcription regulators resulted in the inactivation of these metabolic pathways. Thus, the inability of S. Typhi to metabolize Glucose-6-Phosphate or 3-phosphoglyceric acid is due to the silencing of the expression of the genes encoding the transporters for these compounds due to point mutations in the transcriptional regulatory proteins. In contrast, its inability to utilize glucarate or galactarate is due to the presence of point mutations in the transporter and enzymes necessary for the metabolism of these sugars. These studies provide additional support for the concept of adaptive convergent evolution of these two human-adapted Salmonella enterica serovars and highlight a limitation of bioinformatic approaches to predict metabolic capabilities.

microbiology↗

bioRxiv2023

Cryo-EM structure of the bacterial effector protein SipA bound to F-actin reveals a unique mechanism for filament stabilization

The bacterial pathogen Salmonella spp. modulates cellular processes by delivering effector proteins through its type III secretion systems. Among these effectors, SipA facilitates bacterial invasion and promotes intestinal inflammation. The mechanisms by which this effector carries out these functions are incompletely understood, although SipAs ability to modulate actin dynamics is central to some of these activities. Here, we report the cryo-EM structure of SipA bound to filamentous actin. The structure shows that this effector stabilizes actin filaments through unique interactions of its carboxy-terminal domain with four actin subunits. Furthermore, our structure-function studies revealed that SipAs actin-binding activity is independent of its ability to stimulate intestinal inflammation. Overall, these studies illuminate critical aspects of Salmonella pathogenesis and provide unique insight into the mechanisms by which a bacterial effector modulates actin dynamics.

microbiology↗

bioRxiv2022

Assembly and architecture of the type III secretion sorting platform

Type III secretion systems are bacterial nanomachines specialized in protein delivery into target eukaryotic cells. The structural and functional complexity of these machines demand highly coordinated mechanisms for their assembly and operation. The sorting platform is a critical component of type III secretion machines that ensures the timely engagement and secretion of proteins destined to travel this export pathway. However, the mechanisms that lead to the assembly of this multi-component structure have not been elucidated. Herein, employing structure modeling and an extensive in vivo crosslinking strategy, we provide a detailed inter-subunit-contact survey of the entire sorting platform complex. Using the identified crosslinks as signatures for pairwise inter-subunit interactions in combination with systematic genetic deletions, we mapped the assembly process of this unique bacterial structure. Insights generated by this study could serve as the bases for the development of anti-virulence strategies to combat several medically important bacterial pathogens.

microbiology↗

bioRxiv2021

Typhoid toxin sorting and exocytic transport from Salmonella Typhi infected cells

Typhoid toxin is an essential virulence factor for Salmonella Typhi, the cause of typhoid fever in humans. This toxin has an unusual biology in that it is produced by Salmonella Typhi only when located within host cells. Once synthesized, the toxin is secreted to the lumen of the Salmonella-containing vacuole from where it is transported to the extracellular space by vesicle carrier intermediates. Here we report the identification of the typhoid toxin sorting receptor and the cellular machinery that packages the toxin into vesicle carriers, and exports it to the extracellular space. We found that the cation- independent mannose-6-phosphate receptor serves as typhoid toxin sorting receptor and that the coat protein COPII and the GTPase Sar1 mediate its packaging into vesicle carriers. Formation of the typhoid toxin carriers requires the specific environment of the Salmonella Typhi-containing vacuole, which is determined by the activities of specific effectors of its type III protein secretion systems. We also found that Rab11B and its interacting protein Rip11 control the intracellular transport of the typhoid toxin carriers, and the SNARE proteins VAMP7, SNAP23, and Syntaxin 4 their fusion to the plasma membrane. Typhoid toxins cooption of specific cellular machinery for its transport to the extracellular space illustrates the remarkable adaptation of an exotoxin to exert its function in the context of an intracellular pathogen.

microbiology↗

bioRxiv2019

Alternate subunit assembly diversifies the function of a bacterial toxin

Bacterial toxins with an AB5 architecture are central to bacterial pathogenesis. Functionally diverse and evolutionarily distant AB5 toxins adopt synonymous structures in which a discrete domain of the toxins active (A) subunit is inserted into a ring-like platform comprised of five delivery (B) subunits. Salmonella Typhi, the cause of typhoid fever, produces an unusual A2B5 toxin known as typhoid toxin, a major virulence factor. Here, we report that upon infection of human cells, S. Typhi produces two forms of typhoid toxin that have distinct delivery components but share common active subunits. We demonstrate that the two typhoid toxins exhibit substantially different trafficking properties, elicit markedly different effects when administered to laboratory animals, and are expressed in response to different regulatory mechanisms and distinct metabolic cues. Collectively, these results indicate that the evolution of two typhoid toxin variants has conferred functional versatility to this virulence factor. More broadly, this study reveals a new paradigm in toxin biology and suggests that the evolutionary expansion of AB5 toxins was likely fueled by the remarkable plasticity inherent to their structural design coupled to the functional versatility afforded by the combination of homologous toxin components.

microbiology↗

bioRxiv2018

Visualization of the type III secretion mediated Salmonella-host cell interface using cryo-electron tomography

Many important gram-negative bacterial pathogens use highly sophisticated type III secretion systems (T3SSs) to establish complex host-pathogen interactions. Bacterial-host cell contact triggers the activation of the T3SS and the subsequent insertion of a translocon pore into the target cell membrane, which serves as a conduit for the passage of effector proteins. Therefore the initial interaction between T3SS-bearing bacteria and host cells is the critical step in the deployment of the protein secretion machine, yet this process remains poorly understood. Here, we use high-throughput cryo-electron tomography (cryo-ET) to visualize the T3SS-mediated Salmonella-host cell interface. Our analysis reveals the intact translocon at an unprecedented level of resolution, its deployment in the host cell membrane, and the establishment of an intimate association between the bacteria and the target cells, which is essential for effector translocation. Our studies provide critical data supporting the long postulated direct injection model for effector translocation.

microbiology↗