Biology subjects

Chetrit, D.

Publications and source records attributed to Chetrit, D..

4 recordsLinked to original sources

bioRxiv2026

Type IV Secretion System Drives Lipid Mixing

Type IV secretion systems (T4SSs) are versatile molecular machines used by bacteria to secrete protein effectors into host cells, promoting pathogenesis, and to transfer DNA between bacteria through conjugation, driving horizontal gene transfer. Most, like Dot/Icm of the pathogen Legionella pneumophila (L. pneumophila) or Escherichia coli (E. coli) RK2, are primed for substrate delivery only upon contact with a target membrane, but mechanisms are unknown. A pilus could bind a receptor to initiate priming, but many T4SSs, especially those that deliver effectors, lack a pilus. Here, we present evidence that T4SSs are primed by direct contact with target membrane lipids. Combining fluorescence assays with genetics and biochemistry, we found that Dot/Icm drives lipid exchange between bacterial cells and between bacteria and synthetic membranes containing only lipids. Lipid exchange requires membrane contact but does not require ATP hydrolysis or even full complex assembly. Minimally, the outer membrane core complex protein DotG needs to be present in at least one of the apposed membranes. We similarly observed lipid mixing with the simpler E. coli RK2 T4SS, where we could follow lipid mixing and plasmid transfer simultaneously. We found that lipid mixing always preceded or accompanied plasmid transfer, suggesting it may be part of the contact-dependent priming mechanism. Lipid mixing was inhibited or promoted by lipids that inhibit or promote membrane fusion, respectively. Lipids inhibiting lipid mixing also inhibited substrate transfer. Together, our results suggest that initial contact between DotG outer segments and target membrane lipids promotes lipid mixing as part of the mechanism that primes T4SS for substrate translocation. HighlightsO_LIThe Legionella pneumophila Dot/Icm secretion system requires contact with a target membrane for effector translocation, but how membrane contact primes the machinery for this is unknown. C_LIO_LIWe found that Dot/Icm drives contact-dependent lipid mixing between bacteria or between bacteria and liposomes, showing that Dot/Icm priming does not require a protein receptor. C_LIO_LILipid mixing also occurs with the simpler Type IVA system (Escherichia coli RK2) during conjugation, suggesting lipid mixing is a general feature of Type IV Secretion System function. C_LIO_LIThe core complex protein DotG is sufficient to drive lipid mixing, suggesting DotG-target membrane interactions may destabilize the target membrane. C_LI

microbiology↗

bioRxiv2025

In-situ structures of the Legionella Dot/Icm T4SS identify the DotA-IcmX complex as the gatekeeper for effector translocation

The Dot/Icm machine in Legionella pneumophila is one of the most versatile type IV secretion systems (T4SSs), with a remarkable capacity to translocate over 330 different effector proteins across the bacterial envelope into host cells. At least 27 Dot and Icm proteins are required for assembly and function of the system, yet the architecture and activation mechanism remain poorly understood at the molecular level. Here, we deploy cryo-electron microscopy to reveal in-situ structures of the Dot/Icm machine at near-atomic resolution. Importantly, two proteins essential for effector translocation, DotA and IcmX, form a pentameric protochannel at an inactive state. Upon activation, the DotA-IcmX protochannel undergoes extensive rearrangements to form an extended transenvelope passage capable of transporting effector proteins from the bacterial cytoplasm into host cells as revealed by cryo-electron tomography. Collectively, our findings suggest that the DotA-IcmX complex functions as the gatekeeper for effector translocation of the Dot/Icm T4SS.

molecular biology↗

bioRxiv2024

Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1

Synaptotagmin-1 (Syt1) is a major calcium sensor for rapid neurotransmitter release in neurons and hormone release in many neuroendocrine cells. It possesses two tandem cytosolic C2 domains that bind calcium, negatively charged phospholipids, and the neuronal SNARE complex. Calcium binding to Syt1 triggers exocytosis, but how this occurs is not well understood. Syt1 has additional roles in docking dense core vesicles (DCV) and synaptic vesicles (SV) to the plasma membrane (PM) and in regulating fusion pore dynamics. Thus, Syt1 perturbations could affect release through vesicle docking, fusion triggering, fusion pore regulation, or a combination of these. Here, using a human neuroendocrine cell line, we show that neutralization of highly conserved polybasic patches in either C2 domain of Syt1 impairs both DCV docking and efficient release of serotonin from DCVs. Interestingly, the same mutations resulted in larger fusion pores and faster release of serotonin during individual fusion events. Thus, Syt1s roles in vesicle docking, fusion triggering, and fusion pore control may be functionally related.

biophysics↗

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↗