Biology subjects

Clemens, D. L.

Publications and source records attributed to Clemens, D. L..

4 recordsLinked to original sources

bioRxiv2025

Discovery and CryoEM Structure of FPM13, a Periplasmic Metalloprotein Unique to Francisella

We report the cryoEM structure of the Francisella protein FTN_1118, identifying it as a novel 13 kDa periplasmic protein unique to the Francisella genus, which we now designate FPM13 (Francisella Periplasmic Metalloprotein, 13 kDa) based on its structural and biochemical properties. FPM13 was serendipitously identified during purification of Francisella type VI secretion system (T6SS) effector proteins, co-purifying with them. Its identity, initially unknown, was established using the novel cryoID method. The structure reveals a symmetrical, donut-shaped 18-mer with 9-fold dihedral symmetry, formed by two stacked nonamers head-to-head. It measures [~]8 nm both in height and in outer diameter, and has a 3.5 nm central channel. The complex features a double-layered wall with an inner {beta}-sheet core and an outer -helical shell. Each monomer adopts a compact fold comprising an N-terminus {beta}-strand, an -helix and two additional {beta} strands at the C-terminus. The assembly is stabilized by inter-ring loop interactions and hydrophobic and electrostatic contacts between neighboring subunits. Biochemical analyses, as shown by APEX-biotinylation and Triton X-114 phase partitioning, confirmed that FPM13 is a soluble periplasmic protein. ICP-MS demonstrated that FPM13 binds iron and copper. Deletion of FPM13 in Francisella novicida strains caused no growth defects in macrophages or mice but show increased copper sensitivity under iron-depleted conditions, suggesting a role for FPM13 in metal transport or detoxification.

microbiology↗

bioRxiv2025

Atomic Structure and Phospholipid Binding Properties of the Francisella Type VI Secretion System Effector Protein PdpC

Francisella tularensis, a gram-negative facultative intracellular pathogen, causes tularemia, a potentially fatal infection. The Francisella pathogenicity island (FPI) encodes a type VI secretion system (T6SS), critical for the bacteriums ability to replicate within host cells and cause disease. PdpC, an FPI-encoded T6SS effector protein, is essential for phagosomal escape, intracellular replication, and virulence in animals, yet its structure and function remain poorly understood. Here, we expressed PdpC recombinantly and determined the cryoEM structure of PdpC at 3.4 [A] resolution, revealing a monomeric 156 kDa protein with a seahorse-shaped architecture (80 [A] x 75 [A] x 120 [A]). PdpC comprises five domains: an N-terminal domain (NTD) forming a prominent lateral head lobe, a central body domain, a C-terminal tail domain, a small wedge domain at the groove between the body and tail, and an unmodeled "mouth" domain that completes the seahorse-like shape. Functional studies demonstrate that PdpC binds specific host cell phospholipids, including phosphatidylinositol-3-phosphate. These findings provide a foundation for elucidating PdpCs mechanism of action and developing strategies to prevent or treat tularemia.

microbiology↗

bioRxiv2025

Structure, identification and characterization of the RibD-enolase complex in Francisella

Francisella tularensis is a highly infectious bacterium, a Tier 1-select agent, and the causative agent of tularemia, a potentially fatal zoonotic disease. In this study originally aiming to identify anti-tularemia drug targets, we serendipitously determined the atomic structures and identified their components of the native RibD-enolase protein complex in Francisella novicida; and subsequently systematically characterized the catalytic functions of the RibD-enolase complex. Originally discovered as individually protein in Escherichia coli and yeast, respectively, RibD and enolase are two essential enzymes involved in distinct metabolic pathways, both of which could serve as potential therapeutic targets for tularemia treatment and prevention. Our biochemical validation using pull-down assays confirmed the formation of this complex in vivo, revealing that all eluted RibD is bound to enolase, while the majority of enolase remained uncomplexed. Structural analysis reveals unique features of the Francisella complex, including key RibD-enolase interactions that mediate complex assembly and {beta}-strand swapping between RibD subunits. Furthermore, molecular dynamics simulations of the ligand-bound RibD-enolase complex highlight localized conformational changes within the substrate-binding sites and suggest a gating mechanism between RibDs substrate and cofactor-binding sites to ensure efficient uptake and turnover. Despite the physical association between RibD and enolase, enzymatic assays indicated their catalytic activities are independent of each other, thus the complex may have alternative functional roles that warrant further exploration. Our study provides the first structural and biochemical characterization of the RibD-enolase complex, establishing a foundation for further investigations into its functional significance in Francisella and potential antibacterial development. SignificanceFrancisella tularensis, a dangerous bacterium classified as a high-priority bioterrorism threat, causes tularemia, a severe and sometimes deadly disease spread from animals to humans. In a study originally aimed at finding new treatments, we accidentally uncovered a unique partnership between two enzymatic proteins--RibD and enolase--in Francisella. These proteins, previously known to work alone in other non-pathologic organisms, were found to form a heterodimer of RibD and enolase dimers in Francisella. Biochemical experiments confirmed that enolase works both alone and together with RibD in living cells. In addition to the novel heterodimer-of-homodimer configuration, the RibD subunits feature swapped {beta} strands for dimerization as compared to E. coli RibD homodimer. The results offer clues for designing future antibiotics against tularemia and beyond.

microbiology↗

bioRxiv2021

Human alveolar lining fluid from the elderly promotes Mycobacterium tuberculosis growth in alveolar epithelial cells and bacterial translocation into the cytosol

The elderly population is at significant risk of developing respiratory diseases, including tuberculosis (TB) caused by the airborne Mycobacterium tuberculosis (M.tb). Once M.tb reaches the alveolar space, it contacts alveolar lining fluid (ALF) which dictates host cell interactions. We previously determined that age-associated dysfunctionality in human ALF soluble innate components lead to accelerated M.tb growth within human alveolar macrophages. Here we determined the impact of human ALF on M.tb infection of alveolar epithelial cells (ATs), another critical cellular determinant of infection. We observed that E-ALF-exposed M.tb had significantly increased intracellular growth in ATs compared to adult ALF (A-ALF)-exposed bacteria. Despite this, there were no alterations in AT inflammatory mediators or cell activation. However, exposure to E-ALF altered endosomal trafficking of M.tb, driving bacterial translocation to both endosomal and cytosolic compartments in ATs. Our results indicate that exposure of M.tb to E-ALF promotes translocation of bacteria into the AT cytosol as a potential favorable niche for rapid bacterial growth and at the same time dampens ATs immune responses. Thus, our findings highlight the influence of the elderly lung mucosa on M.tb infection of ATs, an unexplored contributing factor to the elderly populations increased susceptibility of developing active TB disease.

immunology↗