Visualizing the invisible: novel approaches to visualizing bacterial proteins and host-pathogen interactions.

Host-pathogen interactions play a critical role in infectious diseases, and understanding the underlying mechanisms is vital for developing effective therapeutic strategies. The visualization and characterization of bacterial proteins within host cells is key to unraveling the dynamics of these interactions. Various protein labeling strategies have emerged as powerful tools for studying host-pathogen interactions, enabling the tracking, localization, and functional analysis of bacterial proteins in real-time. However, the labeling and localization of Salmonella secreted type III secretion system (T3SS) effectors in host cells poses technical challenges. Conventional methods disrupt effector stoichiometry and often result in non-specific staining. Bulky fluorescent protein fusions interfere with effector secretion, while other tagging systems such as 4Cys-FLaSH/Split-GFP suffer from low labeling specificity and a poor signal-to-noise ratio. Recent advances in state-of-the-art techniques have augmented the existing toolkit for monitoring the translocation and dynamics of bacterial effectors. This comprehensive review delves into the bacterial protein labeling strategies and their application in imaging host-pathogen interactions. Lastly, we explore the obstacles faced and potential pathways forward in the realm of protein labeling strategies for visualizing interactions between hosts and pathogens.

The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity.

Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We next show that salivary sfRNA is protected in detergent-sensitive compartments, likely extracellular vesicles. In support of this hypothesis, we visualized viral RNAs in vesicles in mosquito saliva and noted a marked enrichment of signal from 3'UTR sequences, which is consistent with the presence of sfRNA. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in a human hepatoma cell line and human primary dermal fibroblasts. Transfection of 3'UTR RNA prior to DENV2 infection inhibited type I and III interferon induction and signaling, and enhanced viral replication. Therefore, we posit that sfRNA present in salivary extracellular vesicles is delivered to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.

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 cells. Upon entry, the injected effector proteins cooperatively modulate eukaryotic signaling pathways and reprogram cellular functions, enabling bacterial entry and survival. Monitoring and localizing these secreted effector proteins in the context of infections provides a footprint for defining the dynamic interface of host-pathogen interactions. However, labeling and imaging bacterial proteins in host cells without disrupting their structure/function is technically challenging. Constructing fluorescent fusion proteins does not resolve this problem, because the fusion proteins jam the secretory apparatus and thus are not secreted. To overcome these obstacles, we recently employed a method for site-specific fluorescent labeling of bacterial secreted effectors, as well as other difficult-to-label proteins, using genetic code expansion (GCE). This paper provides a complete step-by-step protocol to label Salmonella secreted effectors using GCE site-specifically, followed by directions for imaging the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM) Recent findings suggest that the incorporation of non-canonical amino acids (ncAAs) via GCE, followed by bio-orthogonal labeling with tetrazine-containing dyes, is a viable technique for selective labeling and visualization of bacterial secreted proteins and subsequent image analysis in the host. The goal of this article is to provide a straightforward and clear protocol that can be employed by investigators interested in conducting super-resolution imaging using GCE to study various biological processes in bacteria and viruses, as well as host-pathogen interactions.

INTS13 variants causing a recessive developmental ciliopathy disrupt assembly of the Integrator complex.

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.

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

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

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.

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

After Salmonella 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 Salmonella; modifying acid survival pathways represents a target for inhibiting Salmonella.

Role of Ser65, His148 and Thr203 in the Organic Solvent-dependent Spectral Shift in Green Fluorescent Protein.

The photophysics of green fluorescent protein (GFP) is remarkable because of its exceptional property of excited state proton transfer (ESPT) and the presence of a functional proton wire. Another interesting property of wild-type GFP is that its absorption and fluorescence excitation spectra are sensitive to the presence of polar organic solvents even at very low concentrations. Here, we use a combination of methodologies including site-specific mutagenesis, absorption spectroscopy, steady-state and time-resolved fluorescence measurements and all-atom molecular dynamics simulations in explicit solvent, to uncover the mechanism behind the unique spectral sensitivity of GFP toward organic solvents. Based on the evidences provided herein, we suggest that organic solvent-induced changes in the proton wire prevent ground state movement of a proton through the wire and thus bring about the spectral changes observed. The present study can not only help to understand the mechanism of proton transfer by further dissecting the intricate steps in GFP photophysics but also encourages to develop GFP-based organic solvent biosensors.

Effect of T·T Mismatch on DNA Dynamics Probed by Minor Groove Binders: Comparison of Dynamic Stokes Shifts of Hoechst and DAPI.

Recognition of DNA base mismatches and their subsequent repair by enzymes is vital for genomic stability. However, it is difficult to comprehend such a process in which enzymes sense and repair different types of mismatches with different ability. It has been suggested that the differential structural changes of mismatched bases act as cues to the repair enzymes, although the effect of such DNA structural changes on surrounding water and ion dynamics is inevitable due to strong electrostatic coupling among them. Thus, collective dynamics of DNA, water, and ions near the mismatch site is believed to be important for mismatch recognition and repair mechanism. Here we show that introduction of a T·T mismatch in the minor groove of DNA induces dispersed (collective) power-law solvation dynamics (of exponent ∼0.24), measured by monitoring the time-resolved fluorescence Stokes shifts (TRFSS) of two popular minor groove binders (Hoechst 33258 and DAPI) over five decades of time from 100 fs to 10 ns. The same ligands however sense different dynamics (power-law of exponent ∼0.15 or power-law multiplied with biexponential relaxation) in the minor groove of normal-DNA. The similar fluorescence anisotropy decays of ligands measured in normal- and T·T-DNA suggest that Stokes shift dynamics and their changes in T·T-DNA purely originate from the solvation process, and not from any internal rotational motion of probe-ligands. The dispersed power-law solvation dynamics seen in T·T-DNA indicate that the ligands do not sense any particular (exponential) relaxation specific to T·T wobbling and/or other conformational changes. This could be the reason why T·T mismatch is recognized by enzymes with lower efficiency compared to purine-pyrimidine and purine-purine mismatches.

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

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 (Lß') 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 (Lα) 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.

New insight into probe-location dependent polarity and hydration at lipid/water interfaces: comparison between gel- and fluid-phases of lipid bilayers.

Environment polarity and hydration at lipid/water interfaces play important roles in membrane biology, which are investigated here using a new homologous series of 4-aminophthalimide-based fluorescent molecules (4AP-Cn; n = 2-10, 12) having different lipophilicities (octanol/water partition coefficient - log P). We show that 4AP-Cn molecules probe a peculiar stepwise polarity (E) profile at the lipid/water interface of the gel-phase (Lß') DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) bilayer at room temperature, which was not anticipated in earlier studies. However, the same molecules probe only a subtle but continuous polarity change at the interface of water and the fluid-phase (Lα) DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) bilayer at room temperature. Fluorescence quenching experiments indicate that solutes with different log P values adsorb at different depths across DPPC/water and DOPC/water interfaces, which correlate with the polarity profiles observed at the interfaces. Molecular dynamics simulations performed on eight probe-lipid systems (four in each of the DPPC and DOPC bilayers - a total run of 2.6 µs) support experimental results, providing further information on the relative position and angle distributions as well as hydration of probes at the interfaces. Simulation results indicate that besides positions, probe orientations also play an important role in defining the local dielectric environment by controlling the probes' exposure to water at the interfaces especially of the gel-phase DPPC bilayer. The results suggest that 4AP-Cn probes are well suited for studying solvation properties at lipid/water interfaces of gel- and fluid-phases simultaneously.

Measuring Size, Size Distribution, and Polydispersity of Water-in-Oil Microemulsion Droplets using Fluorescence Correlation Spectroscopy: Comparison to Dynamic Light Scattering.

Water-in-oil microemulsion droplets (MEDs) are thermodynamically stable supramolecular structures formed in a mixture of water and oil, stabilized by surfactant layer. Here we use fluorescence correlation spectroscopy (FCS) to measure the diffusion, and the size, size distribution, and polydispersity of MEDs prepared in ternary mixtures of water/oil/sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in heptane, isooctane, and nonane at (near) single droplet level. We compare FCS data directly to dynamic light scattering (DLS) data, which shows that the optical matching point (OMP) conditions of MEDs in different oils (where excess optical polarizability of droplets vanish) severely influence DLS data, while FCS extracts the accurate size, size distribution, and polydispersity of AOT-MEDs in all three oils. This suggests that extreme precaution must be taken in acquiring and explaining DLS data of MEDs in solution. FCS data show nearly identical W0-dependent (peak) size variations of AOT-MEDs in all three oils, though a subtle increase in (average) polydispersity of droplets is observed with increase in carbon chain length of oils. Establishing the accuracy of FCS data for AOT-MEDs, we further apply FCS to measure the size parameters of MEDs prepared in a quaternary mixture of water/oil/cetyltrimethylammonium bromide (CTAB)/1-butanol in hexane, heptane, and isooctane. Unlike AOT-MEDs, FCS data show substantial effect of added cosurfactant (1-butanol) and external oil on size, size distribution and polydispersity of quaternary CTAB-MEDs. Analysis of size distributions reveals large variation of polydispersity which possibly indicates the existence of larger shape heterogeneity, together with size heterogeneity, of CTAB-MEDs compared to AOT-MEDs in solution.

Dispersed dynamics of solvation in G-quadruplex DNA: comparison of dynamic Stokes shifts of probes in parallel and antiparallel quadruplex structures.

G-quadruplex DNA (GqDNA) structures play an important role in many specific cellular functions and are promising anti-tumor targets for small molecules (ligands). Here, we measured the dynamic Stokes shift of a ligand (Hoechst) bound to parallel c-Myc (mPu22) GqDNA over five decades of time from 100 fs to 10 ns, and compared it with the previously reported dynamics of DAPI bound to antiparallel human telomeric (hTelo22) GqDNA (Pal et al 2015 J. Phys. Chem. Lett. 6 1754). Stokes shift data from fluorescence up-conversion and time-correlated single photon counting experiments was combined to cover the broad dynamic range. The results show that the solvation dynamics of Hoechst in parallel mPu22 GqDNA follow a power law relaxation, added to fast 2 ps exponential relaxation, from 100 fs to 10 ns, with only a subtle difference of power law exponents in the two ligand-GqDNA systems (0.06 in Hoechst-mPu22 compared to 0.16 in DAPI-hTelo22). We measured steady-state fluorescence spectra and time-resolved anisotropy decays which confirm the tight binding of Hoechst to parallel mPu22 with a binding constant of ~1 × 105 M-1. The molecular docking of Hoechst in parallel GqDNA followed by a 50 ns molecular dynamics (MD) simulation on a Hoechst-GqDNA complex reveals that Hoechst binds to one of the outer G-tetrads by end-stacking near G13 and G4, which is different from the binding site of DAPI inside a groove of antiparallel hTelo22 GqDNA. Reconciling previous experimental and simulation results, we assign the 2 ps component to the hydration dynamics of only weakly perturbed water near mPu22 and the power law relaxation to the coupled motion of water and DNA (i.e. DNA backbone, unpaired bases and loops connecting G-tetrads) which come near the Hoechst inside parallel GqDNA.

Power-Law Solvation Dynamics in G-Quadruplex DNA: Role of Hydration Dynamics on Ligand Solvation inside DNA.

G-quadruplex DNA (GqDNA) structures act as promising anticancer targets for small-molecules (ligands). Solvation dynamics of a ligand (DAPI: 4',6-diamidino-2-phenylindole) inside antiparallel-GqDNA is studied through direct comparison of time-resolved experiments to molecular dynamics (MD) simulation. Dynamic Stokes shifts of DAPI in GqDNA prepared in H2O buffer and D2O are compared to find the effect of water on ligand solvation. Experimental dynamics (in H2O) is then directly compared with the dynamics computed from 65 ns simulation on the same DAPI-GqDNA complex. Ligand solvation follows power-law relaxation (summed with fast exponential relaxation) from ~100 fs to 10 ns. Simulation results show relaxation below ~5 ps is dominated by water motion, while both water and DNA contribute comparably to dictate long-time power-law dynamics. Ion contribution is, however, found to be negligible. Simulation results also suggest that anomalous solvation dynamics may have origin in subdiffusive motion of perturbed water near GqDNA.

Sequence-Dependent Solvation Dynamics of Minor-Groove Bound Ligand Inside Duplex-DNA.

Ligand binding to minor-grooves of DNA depends on DNA-base sequence near its binding-site. However, it is not known how base-sequences affect the local solvation of ligand inside minor-grooves of DNA. Here we present a comprehensive study on sequence-dependent solvation dynamics of ligand inside duplex-DNA by measuring the static and dynamic fluorescence Stokes shifts of a popular groove-binder, DAPI, inside DNA minor-grooves created by four different sequences; d(5'-CGCGAATTCGCG-3')2, d(5'-CGCGTTAACGCG-3')2, d(5'-CGCGCAATTGCGCG-3')2, and d(5'-CGCGCTTAAGCGCG-3')2, having different sequences near DAPI-binding site. Fluorescence up-conversion and time-correlated single photon counting techniques are employed to capture the dynamic Stokes shifts of DAPI over five decades in time from 100 fs to 10 ns. We show that the ligands sense different static and dynamic solvation inside minor-grooves created by different sequences: Only subtle change in the dynamics is seen in DNA containing -AATTG-, -TTAAG-, and -AATTC- sequences, which show power-law relaxation in initial time-decades, followed by biexponential decay in nanosecond time-scales. However, changing a single base (and the complementary base) near ligand-binding site from -TTAAG- to -TTAAC- drastically induces the dynamics to follow a single power-law relaxation over the entire five decades. The observed variation of dynamics possibly relate to the local DNA motions, coupled to the hydration dynamics near the ligand-binding site.

Understanding ligand interaction with different structures of G-quadruplex DNA: evidence of kinetically controlled ligand binding and binding-mode assisted quadruplex structure alteration.

The study of ligand interaction with G-quadruplex DNA is an active research area, because many ligands are shown to bind G-quadruplex structures, showing anticancer effects. Here, we show, for the first time, how fluorescence correlation spectroscopy (FCS) can be used to study binding kinetics of ligands with G-quadruplex DNA at the single molecule level. As an example, we study interaction of a benzo-phenoxazine ligand (Cresyl Violet, CV) with antiparallel and (3 + 1) hybrid G-quadruplex structures formed by human telomeric sequence. By using simple modifications in FCS setup, we describe how one can extract the reaction kinetics from diffusion-coupled correlation curves. It is found that the ligand (CV) binds stronger, by an order of magnitude, to a (3 + 1) hybrid structure, compared to an antiparallel one. Ensemble-averaged time-resolved fluorescence experiments are also carried out to obtain the binding equilibrium constants (K) of ligand-quadruplex interactions in bulk solution for the first time, which are found to match very well with FCS results. Global analysis of FCS data provides association (k(+)) and dissociation (k(-)) rates of the ligand in the two structures. Results indicate that stronger ligand binding to the (3 + 1) hybrid structure is controlled by the dissociation rate, rather than the association rate of ligand in the quadruplexes. Circular dichroism (CD) and induced-CD spectra show that the ligand not only binds at different conformations in the quadruplexes, but also induces antiparallel structure to form a mixed-type hybrid structure in Na(+) solution. However, in K(+) solution, the ligand stabilizes the (3 + 1) hybrid structure. Molecular docking studies predict the possible differences in binding sites of the ligand inside two quadruplexes, which strongly support the experimental observations. Results suggest that different binding modes of the ligand to the quadruplex structures actually assist the alteration of structures differently.

Probe Position-Dependent Counterion Dynamics in DNA: Comparison of Time-Resolved Stokes Shift of Groove-Bound to Base-Stacked Probes in the Presence of Different Monovalent Counterions.

Time-resolved fluorescence Stokes shifts (TRFSS) of 4',6-diamidino-2-phenylindole (DAPI) inside the minor groove of DNA are measured in the presence of three different monovalent counterions: sodium (Na(+)), rubidium (Rb(+)), and tetrabutylammonium (TBA(+)). Fluorescence up-conversion and time-correlated single photon counting are combined to obtain the time-resolved emission spectra (TRES) of DAPI in DNA from 100 fs to 10 ns. Time-resolved Stokes shift data suggest that groove-bound DAPI can not sense the counterion dynamics because the ions are displaced by DAPI far from the probe-site. However, when these results are compared to the earlier base-stacked coumarin data, the same ions are found to affect the nanosecond dynamics significantly. This suggests that the ions come close to the probe-site, such that they can affect the dynamics when measured by base-stacked coumarin. These results support previous molecular dynamics (MD) simulation data of groove-bound and base-stacked probes inside DNA.

Fluorescence correlation spectroscopy: an efficient tool for measuring size, size-distribution and polydispersity of microemulsion droplets in solution.

Fluorescence correlation spectroscopy (FCS) is an ideal tool for measuring molecular diffusion and size under extremely dilute conditions. However, the power of FCS has not been utilized to its best to measure diffusion and size parameters of complex chemical systems. Here, we apply FCS to measure the size, and, most importantly, the size distribution and polydispersity of a supramolecular nanostructure (i.e., microemulsion droplets, MEDs) in dilute solution. It is shown how the refractive index mismatch of a solution can be corrected in FCS to obtain accurate size parameters of particles, bypassing the optical matching problem of light scattering techniques that are used often for particle-size measurements. We studied the MEDs of 13 different W(0) values from 2 to 50 prepared in a ternary mixture of water, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), and isooctane, with sulforhodamine-B as a fluorescent marker. We find that, near the optical matching point of MEDs, the dynamic light scattering (DLS) measurements underestimate the droplet sizes while FCS estimates the accurate ones. A Gaussian distribution model (GDM) and a maximum-entropy-based FCS data fitting model (MEMFCS) are used to analyze the fluorescence correlation curves that unfold Gaussian-type size distributions of MEDs in solution. We find the droplet size varies linearly with W(0) up to ~20, but beyond this W(0) value, the size variation deviates from this linearity. To explain nonlinear variation of droplet size for W(0) values beyond ~20, we invoke a model (the coated-droplet model) that incorporates the size polydispersity of the droplets.