super-resolution-imaging-bacterial-secreted-proteins-using-genetic

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host ...

University of Texas Medical Branch

university of texas medical branch

Despite significant interest in understanding the role of the local dielectric environment and lipid-bilayer fluidity/rigidity in resonance energy transfer between chromophores at lipid/water interfaces, a comprehensive approach to quantify such environmental dependence on energy transfer is missing - primarily because of the scarcity of suitable probes. Here we present the results on multi-chromophoric Förster resonance energy transfer (FRET) from a series of 4-aminophthalimide-based molecules (4AP-Cn; n = 2-10, 12) of different lipophilicity (donors), which reside at different depths across the lipid/water interfaces, to rhodamine-6G (Rh6G; acceptor) molecules that stay in a water-rich region near the lipid headgroups. We apply steady-state and time-resolved fluorescence spectroscopy, and find that multi-chromophoric FRET from the series of 4AP-Cn donors to the Rh6G acceptor occurs in a peculiar stepwise fashion at the lipid/water interface of a gel-phase (L) DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) bilayer at room temperature. However, the same donor-acceptor pairs show only subtle but continuous donor-depth-dependent FRET at the lipid/water interface of a fluid-phase (L) DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) bilayer. These features were found to correlate with the lipid-phase dependent local environmental polarity sensed by 4AP-Cn donors at the interfaces. Molecular dynamics (MD) simulations, combined with experimental results, show that relative depth (and angle) variation of the 4AP-Cn donors and Rh6G acceptor directly controls the FRET efficiencies through fine tuning of the emission and absorption spectra of the donors and acceptor, respectively. The results indicate that the 4AP-Cn probes are well-suited as donors for FRET studies, which allow the FRET parameters at lipid/water interfaces of gel- and fluid-phases of lipid-bilayers to be quantified and compared simultaneously.

Probe-location dependent resonance energy transfer at lipid/water interfaces: comparison between the gel- and fluid-phase of lipid bilayer.

After  is phagocytosed, it resides in an acidic vacuole. Its cytoplasm acidifies to pH 5.6; acidification activates pathogenicity island 2 (SPI-2). SPI-2 encodes a type three secretion system whose effectors modify the vacuole, driving endosomal tubulation. Using super-resolution imaging in single bacterial cells, we show that low pH induces expression of the SPI-2 SsrA/B signaling system. Single particle tracking, atomic force microscopy, and single molecule unzipping assays identified pH-dependent stimulation of DNA binding by SsrB. A so-called phosphomimetic form (D56E) was unable to bind to DNA in live cells. Acid-dependent DNA binding was not intrinsic to regulators, as PhoP and OmpR binding was not pH-sensitive. The low level of SPI-2 injectisomes observed in single cells is not due to fluctuating SsrB levels. This work highlights the surprising role that acid pH plays in virulence and intracellular lifestyles of ; modifying acid survival pathways represents a target for inhibiting .

Single cell, super-resolution imaging reveals an acid pH-dependent conformational switch in SsrB regulates SPI-2.

Recent advances in super-resolution imaging techniques, together with new fluorescent probes have enhanced our understanding of bacterial pathogenesis and their interplay within the host. In this review, we provide an overview of what these techniques have taught us about the bacterial lifestyle, the nucleoid organization, its complex protein secretion systems, as well as the secreted virulence factors.

Super-resolution imaging of bacterial pathogens and visualization of their secreted effectors.

Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA, a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.

Genetic code expansion enables visualization of  type three secretion system components and secreted effectors.

Oral-facial-digital (OFD) syndromes are a heterogeneous group of congenital disorders characterized by malformations of the face and oral cavity, and digit anomalies. Mutations within 12 cilia-related genes have been identified that cause several types of OFD, suggesting that OFDs constitute a subgroup of developmental ciliopathies. Through homozygosity mapping and exome sequencing of two families with variable OFD type 2, we identified distinct germline variants in INTS13, a subunit of the Integrator complex. This multiprotein complex associates with RNA Polymerase II and cleaves nascent RNA to modulate gene expression. We determined that INTS13 utilizes its C-terminus to bind the Integrator cleavage module, which is disrupted by the identified germline variants p.S652L and p.K668Nfs*9. Depletion of INTS13 disrupts ciliogenesis in human cultured cells and causes dysregulation of a broad collection of ciliary genes. Accordingly, its knockdown in Xenopus embryos leads to motile cilia anomalies. Altogether, we show that mutations in INTS13 cause an autosomal recessive ciliopathy, which reveals key interactions between components of the Integrator complex.

Article	Year
Probe-location dependent resonance energy transfer at lipid/water interfaces: comparison between the gel- and fluid-phase of lipid bilayer. Physical chemistry chemical physics : PCCP\| PubMed ID: 28726898	2017
Single cell, super-resolution imaging reveals an acid pH-dependent conformational switch in SsrB regulates SPI-2. eLife\| PubMed ID: 31033442	2019
Super-resolution imaging of bacterial pathogens and visualization of their secreted effectors. FEMS microbiology reviews\| PubMed ID: 32970796	2021
Genetic code expansion enables visualization of type three secretion system components and secreted effectors. eLife\| PubMed ID: 34061032	2021
INTS13 variants causing a recessive developmental ciliopathy disrupt assembly of the Integrator complex. Nature communications\| PubMed ID: 36229431	2022

Moirangthem Kiran Singh

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