Biology subjects

Thanassi, D. G.

Publications and source records attributed to Thanassi, D. G..

3 recordsLinked to original sources

bioRxiv2026

Protein secretion by the type IV pilus machinery in Francisella tularensis

Francisella tularensis is a highly virulent, Gram-negative bacterial pathogen that causes the zoonotic disease tularemia. F. tularensis infects a variety of host cells and replicates intracellularly while evading and interfering with host immune responses. The molecular mechanisms that facilitate the intracellular replication and virulence of F. tularensis are poorly understood. The Francisella genome contains a set of pil genes that code for the assembly of surface fibers termed type IV pili (T4P). T4P are major bacterial virulence determinants but the function of the pil system during F. tularensis infection and intracellular growth is unclear. T4P are closely related to the type II secretion pathway and the pil system of a related Francisella species, F. novicida, was shown to function in protein secretion as well as pilus assembly. To identify proteins secreted by F. tularensis, we analyzed the F. tularensis Live Vaccine Strain (LVS) using bio-orthogonal non-canonical amino acid tagging (BONCAT). Using BONCAT in conjunction with proteomics, we identified candidate proteins secreted by the wild-type LVS, as well as candidate proteins whose extracellular abundance decreased in the absence of the PilF ATPase or the PilE4 pilus subunit. Using epitope tagging of selected candidates, we validated T4P-mediated secretion of the ChiA and ChiD chitinases and the KatG catalase by the LVS. These results further our understanding of the pil system and protein secretion pathways in F. tularensis. IMPORTANCEFrancisella tularensis is a highly virulent Gram-negative bacterial pathogen and the causative agent of tularemia. F. tularensis lacks secretion systems utilized by other intracellular bacterial pathogens but contains pil genes that encode for type IV pili (T4P) and may also function in protein secretion. T4P are observed on the surface of all Francisella spp. but pil-mediated protein secretion has only been reported for F. novicida, which is not normally pathogenic in humans. In this study, we used bio-orthogonal non-canonical amino acid tagging to identify proteins secreted by F. tularensis, for which there is limited information. We demonstrate that the F. tularensis pil system is capable of protein secretion and validate T4P-medeated secretion of the ChiA and ChiD chitinases and the KatG catalase. These results will facilitate investigation of Francisella virulence mechanisms and may provide targets for therapeutic intervention.

microbiology↗

bioRxiv2024

Outer membrane tube formation by Francisella novicida involves extensive envelope modifications and is linked with type VI secretion and alterations to the host phagosomal membrane

Francisella tularensis is a Gram-negative, intracellular pathogen that causes the zoonotic disease tularemia. Due to its ease of dissemination and high lethality, F. tularensis is classified as a Tier 1 Select Agent with potential for misuse as a bioweapon. The mechanisms by which Francisella replicates intracellularly and interacts with the host during infection are not well understood. Francisella produces spherical outer membrane vesicles (OMV) and novel tubular extensions of its cell surface that are also released extracellularly. These OMV and outer membrane tubes (OMT) contain Francisella virulence factors and are produced in response to amino acid starvation and during infection of macrophages. To investigate how the OMT are formed, we used cryogenic electron tomography to examine the model Francisella species, F. novicida, during in vitro culture and within the macrophage phagosome. OMT formation involved progressive alterations of the bacterial envelope, resulting in extensions of both the inner and outer membranes. A dynamic cytoplasmic structure was present at the base of the OMT that extended into the tubes during elongation, together with cytoplasmic material. OMT produced within the macrophage phagosome were associated with changes to the phagosomal membrane, suggesting a role in phagosomal escape. Consistent with this, using confocal microscopy, we observed colocalization of the Francisella type VI secretion system with the OMT, both within bacteria and in released tubular vesicles. These findings reveal the cellular transformations that occur during membrane tubulation by Francisella and provide insights into the function of membrane-derived structures during host-pathogen interactions.

microbiology↗

bioRxiv2020

Processive Dynamics of the Usher Assembly Platform During Uropathogenic Escherichia coli P Pilus Biogenesis

Uropathogenic Escherichia coli (UPEC) assemble hair-like surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili mediate the adherence of UPEC to the kidney epithelium, facilitating bacterial colonization and pyelonephritis1. P pili are assembled through the conserved chaperone-usher (CU) pathway2-4. In this pathway, a dedicated chaperone facilitates the folding of nascent pilus subunits in the periplasm and an integral outer membrane (OM) protein termed the usher provides the assembly platform and secretion channel for the pilus fiber. Much of the structural and functional understanding of the CU pathway has been gained through investigations of type 1 pili, which promote UPEC binding to the bladder epithelium and the development of cystitis5. In contrast, the structural basis for P pilus biogenesis at the usher has remained elusive. This is in part due to the flexible and variable-length P pilus tip fiber, creating structural heterogeneity, as well as difficulties in isolating stable P pilus assembly intermediates from bacteria. Here, we have devised a method to circumvent these hindrances and determined cryo-EM structures of the activated PapC usher in the process of secreting two- and three-subunit P pilus assembly intermediates. These structures show processive steps in P pilus biogenesis, reveal differences between P and type 1 pili, and capture new conformational dynamics of the usher assembly machine.

microbiology↗