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.

Peeling the onion: additional layers of regulation in the acid stress response.

Bacteria are capable of withstanding large changes in osmolality and cytoplasmic pH, unlike eukaryotes that tightly regulate their pH and cellular composition. Previous studies on the bacterial acid stress response described a rapid, brief acidification, followed by immediate recovery. More recent experiments with better pH probes have imaged single living cells, and we now appreciate that following acid stress, bacteria maintain an acidic cytoplasm for as long as the stress remains. This acidification enables pathogens to sense a host environment and turn on their virulence programs, for example, enabling survival and replication within acidic vacuoles. Single-cell analysis identified an intracellular pH threshold of ~6.5. Acid stress reduces the internal pH below this threshold, triggering the assembly of a type III secretion system in Salmonella and the secretion of virulence factors in the host. These pathways are significant because preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. Although we refer to the acid stress response as singular, it is actually a complex response that involves numerous two-component signaling systems, several amino acid decarboxylation systems, as well as cellular buffering systems and electron transport chain components, among others. In a recent paper in the Journal of Bacteriology, M. G. Gorelik, H. Yakhnin, A. Pannuri, A. C. Walker, C. Pourciau, D. Czyz, T. Romeo, and P. Babitzke (J Bacteriol 206:e00354-23, 2024, https://doi.org/10.1128/jb.00354-23) describe a new connection linking the carbon storage regulator CsrA to the acid stress response, highlighting new additional layers of complexity.

Effects of pleiotropic ompR and envZ alleles of Escherichia coli on envelope stress and antibiotic sensitivity.

The EnvZ-OmpR two-component system of Escherichia coli regulates the expression of the ompF and ompC porin genes in response to medium osmolarity. However, certain mutations in envZ confer pleiotropy by affecting the expression of genes of the iron and maltose regulons not normally controlled by EnvZ-OmpR. In this study, we obtained two novel envZ and ompR pleiotropic alleles, envZT15P and ompRL19Q, among revertants of a mutant with heightened envelope stress and an outer membrane (OM) permeability defect. Unlike envZ, pleiotropic mutations in ompR have not been described previously. The mutant alleles reduced the expression of several outer membrane proteins (OMPs), overcame the temperature-sensitive growth defect of a protease-deficient (ΔdegP) strain, and lowered envelope stress and OM permeability defects in a background lacking the BamB protein of an essential ß-barrel assembly machinery complex. Biochemical analysis showed OmpRL19Q, like wild-type OmpR, is readily phosphorylated by EnvZ, but the EnvZ-dependent dephosphorylation of OmpRL19Q~P was drastically impaired compared to wild-type OmpR. This defect would lead to a prolonged half-life for OmpRL19Q~P, an outcome remarkably similar to what we had previously described for EnvZR397L, resulting in pleiotropy. By employing null alleles of the OMP genes, it was determined that the three pleiotropic alleles lowered envelope stress by reducing OmpF and LamB levels. The absence of LamB was principally responsible for lowering the OM permeability defect, as assessed by the reduced sensitivity of a ΔbamB mutant to vancomycin and rifampin. Possible mechanisms by which novel EnvZ and OmpR mutants influence EnvZ-OmpR interactions and activities are discussed.IMPORTANCEMaintenance of the outer membrane (OM) integrity is critical for the survival of Gram-negative bacteria. Several envelope homeostasis systems are activated when OM integrity is perturbed. Through the isolation and characterization of novel pleiotropic ompR/envZ alleles, this study highlights the involvement of the EnvZ-OmpR two-component system in lowering envelope stress and the OM permeability defect caused by the loss of proteins that are involved in OM biogenesis, envelope homeostasis, and structural integrity.

Small, Dynamic Clusters of Tir-Intimin Seed Actin Polymerization.

The understanding of actin pedestal formation by enteropathogenic Escherichia coli (EPEC) relies mainly on static ensemble information obtained from cell lysates. Here, the dynamic nature of signaling components on the subsecond timescale, which resemble phase condensates, is demonstrated. Unlike in vitro phase condensates, transfected intimin receptor (Tir) and downstream component form clusters 200 nm in diameter that are spaced ≈500 nm on average, indicating cellular regulation. On supported lipid bilayers with diffusive intimin, Tir-expressing fibroblasts formed Tir-intimin clusters even without Tir tyrosines, although Tir tyrosine phosphorylation is necessary for actin polymerization from clusters. Single-molecule tracking showed that Tir is diffusive in the clusters and exchanges with Tir in the plasma membrane. Further, Nck and N-WASP bind to the clusters and exchange with cytoplasmic molecules. Tir has a similar cluster lifetime to Nck, but longer than that of N-WASP. Actin polymerization from the clusters requires N-WASP binding, involved Arp2/3 activation, and stabilized N-WASP clusters. These dynamic properties are distinct from larger in vitro systems and do not depend significantly upon crosslinking. Thus, Tir-intimin clusters in the plasma membrane are limited in size by exchange and enhance signaling needed for actin polymerization that enables strong and stable bacterial attachment to host cells.

Distinct Interaction Mechanism of RNA Polymerase and ResD at Proximal and Distal Subsites for Transcription Activation of Nitrite Reductase in Bacillus subtilis.

The ResD-ResE signal transduction system plays a pivotal role in anaerobic nitrate respiration in Bacillus subtilis. The nasD operon encoding nitrite reductase is essential for nitrate respiration and is tightly controlled by the ResD response regulator. To understand the mechanism of ResD-dependent transcription activation of the nasD operon, we explored ResD-RNA polymerase (RNAP), ResD-DNA, and RNAP-DNA interactions required for nasD transcription. Full transcriptional activation requires the upstream promoter region where five molecules of ResD bind. The distal ResD-binding subsite at -87 to -84 partially overlaps a sequence similar to the consensus distal subsite of the upstream (UP) element with which the Escherichia coli C-terminal domain of the α subunit (αCTD) of RNAP interacts to stimulate transcription. We propose that interaction between αCTD and ResD at the promoter-distal site is essential for stimulating nasD transcription. Although nasD has an extended -10 promoter, it lacks a reasonable -35 element. Genetic analysis and structural simulations predicted that the absence of the -35 element might be compensated by interactions between σA and αCTD and between αCTD and ResD at the promoter-proximal ResD-binding subsite. Thus, our work suggested that ResD participates in nasD transcription activation by binding to two αCTD subunits at the proximal and distal promoter sites, representing a unique configuration for transcription activation. IMPORTANCE A significant number of ResD-controlled genes have been identified, and transcription regulatory pathways in which ResD participates have emerged. Nevertheless, the mechanism of how ResD activates transcription of different genes in a nucleotide sequence-specific manner has been less explored. This study suggested that among the five ResD-binding subsites in the promoter of the nasD operon, the promoter-proximal and -distal ResD-binding subsites play important roles in nasD activation by adapting different modes of protein-protein and protein-DNA interactions. The finding of a new type of protein-promoter architecture provides insight into the understanding of transcription activation mechanisms controlled by transcription factors, including the ubiquitous response regulators of two-component regulatory systems, particularly in Gram-positive bacteria.

Polyimidazolium Protects against an Invasive Clinical Isolate of Salmonella Typhimurium.

Frequent outbreaks of Salmonella Typhimurium infection, in both animal and human populations and with the potential for zoonotic transmission, pose a significant threat to the public health sector. The rapid emergence and spread of more invasive multidrug-resistant clinical isolates of Salmonella further highlight the need for the development of new drugs with effective broad-spectrum bactericidal activities. The synthesis and evaluation of main-chain cationic polyimidazolium 1 (PIM1) against several Gram-positive and Gram-negative bacteria have previously demonstrated the efficacy profile of PIM1. The present study focuses on the antibacterial and anti-biofilm activities of PIM1 against Salmonella in both in vitro and in ovo settings. In vitro, PIM1 exhibited bactericidal activity against three strains of Salmonella at a low dosage of 8 µg/mL. The anti-biofilm activity of PIM1 was evident by its elimination of planktonic cells within preformed biofilms in a dose-dependent manner. During the host cell infection process, PIM1 reduces the extracellular bacterial load, which reduces adhesion and invasion to limit the establishment of infection. Once intracellular, Salmonella strains were tolerant and protected from PIM1 treatment. In a chicken egg infection model, PIM1 exhibited therapeutic activity for both Salmonella strains, using stationary-phase and exponential-phase inocula. Moreover, PIM1 showed a remarkable efficacy against the stationary-phase inocula of drug-resistant Salmonella by eliminating the bacterial burden in >50% of the infected chicken egg embryos. Collectively, our results highlight the potential for PIM1 as a replacement therapy for existing antibiotic applications on the poultry farm, given the efficiency and low toxicity profile demonstrated in our agriculturally relevant chicken embryo model.

Salmonella biofilms program innate immunity for persistence in Caenorhabditis elegans.

The adaptive in vivo mechanisms underlying the switch in Salmonella enterica lifestyles from the infectious form to a dormant form remain unknown. We employed Caenorhabditis elegans as a heterologous host to understand the temporal dynamics of Salmonella pathogenesis and to identify its lifestyle form in vivo. We discovered that Salmonella exists as sessile aggregates, or in vivo biofilms, in the persistently infected C. elegans gut. In the absence of in vivo biofilms, Salmonella killed the host more rapidly by actively inhibiting innate immune pathways. Regulatory cross-talk between two major Salmonella pathogenicity islands, SPI-1 and SPI-2, was responsible for biofilm-induced changes in host physiology during persistent infection. Thus, biofilm formation is a survival strategy in long-term infections, as prolonging host survival is beneficial for the parasitic lifestyle.

A single molecule analysis of H-NS uncouples DNA binding affinity from DNA specificity.

Heat-stable nucleoid structuring protein (H-NS) plays a crucial role in gene silencing within prokaryotic cells and is important in pathogenesis. It was reported that H-NS silences nearly 5% of the genome, yet the molecular mechanism of silencing is not well understood. Here, we employed a highly-sensitive single-molecule counting approach, and measured the dissociation constant (KD) of H-NS binding to single DNA binding sites. Charged residues in the linker domain of H-NS provided the most significant contribution to DNA binding affinity. Although H-NS was reported to prefer A/T-rich DNA (a feature of pathogenicity islands) over G/C-rich DNA, the dissociation constants obtained from such sites were nearly identical. Using a hairpin unzipping assay, we were able to uncouple non-specific DNA binding steps from nucleation site binding and subsequent polymerization. We propose a model in which H-NS initially engages with non-specific DNA via reasonably high affinity (∼60 nM KD) electrostatic interactions with basic residues in the linker domain. This initial contact enables H-NS to search along the DNA for specific nucleation sites that drive subsequent polymerization and gene silencing.

Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells.

Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.

To ∼P or Not to ∼P? Non-canonical activation by two-component response regulators.

Bacteria sense and respond to their environment through the use of two-component regulatory systems. The ability to adapt to a wide range of environmental stresses is directly related to the number of two-component systems an organism possesses. Recent advances in this area have identified numerous variations on the archetype systems that employ a sensor kinase and a response regulator. It is now evident that many orphan regulators that lack cognate kinases do not rely on phosphorylation for activation and new roles for unphosphorylated response regulators have been identified. The significance of recent findings and suggestions for further research are discussed.

A FRET-based DNA biosensor tracks OmpR-dependent acidification of Salmonella during macrophage infection.

In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of a sensor kinase (usually a membrane protein) and a response regulator (usually a DNA binding protein). The EnvZ/OmpR two-component system responds to osmotic stress and regulates expression of outer membrane proteins. In Salmonella, EnvZ/OmpR also controls expression of another two-component system SsrA/B, which is located on Salmonella Pathogenicity Island (SPI) 2. SPI-2 encodes a type III secretion system, which functions as a nanomachine to inject bacterial effector proteins into eukaryotic cells. During the intracellular phase of infection, Salmonella switches from assembling type III secretion system structural components to secreting effectors into the macrophage cytoplasm, enabling Salmonella to replicate in the phagocytic vacuole. Major questions remain regarding how bacteria survive the acidified vacuole and how acidification affects bacterial secretion. We previously reported that EnvZ sensed cytoplasmic signals rather than extracellular ones, as intracellular osmolytes altered the dynamics of a 17-amino-acid region flanking the phosphorylated histidine. We reasoned that the Salmonella cytoplasm might acidify in the macrophage vacuole to activate OmpR-dependent transcription of SPI-2 genes. To address these questions, we employed a DNA-based FRET biosensor ("I-switch") to measure bacterial cytoplasmic pH and immunofluorescence to monitor effector secretion during infection. Surprisingly, we observed a rapid drop in bacterial cytoplasmic pH upon phagocytosis that was not predicted by current models. Cytoplasmic acidification was completely dependent on the OmpR response regulator, but did not require known OmpR-regulated genes such as ompC, ompF, or ssaC (SPI-2). Microarray analysis highlighted the cadC/BA operon, and additional experiments confirmed that it was repressed by OmpR. Acidification was blocked in the ompR null background in a Cad-dependent manner. Acid-dependent activation of OmpR stimulated type III secretion; blocking acidification resulted in a neutralized cytoplasm that was defective for SPI-2 secretion. Based upon these findings, we propose that Salmonella infection involves an acid-dependent secretion process in which the translocon SseB moves away from the bacterial cell surface as it associates with the vacuolar membrane, driving the secretion of SPI-2 effectors such as SseJ. New steps in the SPI-2 secretion process are proposed.

A divalent switch drives H-NS/DNA-binding conformations between stiffening and bridging modes.

Heat-stable nucleoid structuring protein (H-NS) is an abundant prokaryotic protein that plays important roles in organizing chromosomal DNA and gene silencing. Two controversial binding modes were identified. H-NS binding stimulating DNA bridging has become the accepted mechanism, whereas H-NS binding causing DNA stiffening has been largely ignored. Here, we report that both modes exist, and that changes in divalent cations drive a switch between them. The stiffening form is present under physiological conditions, and directly responds to pH and temperature in vitro. Our findings have broad implications and require a reinterpretation of the mechanism by which H-NS regulates genes.

Lipid-Mediated Regulation of Embedded Receptor Kinases via Parallel Allosteric Relays.

Membrane-anchored receptors are essential cellular signaling elements for stimulus sensing, propagation, and transmission inside cells. However, the contributions of lipid interactions to the function and dynamics of embedded receptor kinases have not been described in detail. In this study, we used amide hydrogen/deuterium exchange mass spectrometry, a sensitive biophysical approach, to probe the dynamics of a membrane-embedded receptor kinase, EnvZ, together with functional assays to describe the role of lipids in receptor kinase function. Our results reveal that lipids play an important role in regulating receptor function through interactions with transmembrane segments, as well as through peripheral interactions with nonembedded domains. Specifically, the lipid membrane allosterically modulates the activity of the embedded kinase by altering the dynamics of a glycine-rich motif that is critical for phosphotransfer from ATP. This allostery in EnvZ is independent of membrane composition and involves direct interactions with transmembrane and periplasmic segments, as well as peripheral interactions with nonembedded domains of the protein. In the absence of the membrane-spanning regions, lipid allostery is propagated entirely through peripheral interactions. Whereas lipid allostery impacts the phosphotransferase function of the kinase, extracellular stimulus recognition is mediated via a four-helix bundle subdomain located in the cytoplasm, which functions as the osmosensing core through osmolality-dependent helical stabilization. Our findings emphasize the functional modularity in a membrane-embedded kinase, separated into membrane association, phosphotransferase function, and stimulus recognition. These components are integrated through long-range communication relays, with lipids playing an essential role in regulation.

Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif.

ß-Barrelmembrane proteins (ßMPs) form barrel-shaped pores in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Because of the robustness of their barrel structures, ßMPs have great potential as nanosensors for single-molecule detection. However, natural ßMPs currently employed have inflexible biophysical properties and are limited in their pore geometry, hindering their applications in sensing molecules of different sizes and properties. Computational engineering has the promise to generate ßMPs with desired properties. Here we report a method for engineering novel ßMPs based on the discovery of sequence motifs that predominantly interact with the cell membrane and appear in more than 75% of transmembrane strands. By replacing ß1-ß6 strands of the protein OmpF that lack these motifs with ß1-ß6 strands of OmpG enriched with these motifs and computational verification of increased stability of its transmembrane section, we engineered a novel ßMP called OmpGF. OmpGF is predicted to form a monomer with a stable transmembrane region. Experimental validations showed that OmpGF could refold in vitro with a predominant ß-sheet structure, as confirmed by circular dichroism. Evidence of OmpGF membrane insertion was provided by intrinsic tryptophan fluorescence spectroscopy, and its pore-forming property was determined by a dye-leakage assay. Furthermore, single-channel conductance measurements confirmed that OmpGF function as a monomer and exhibits increased conductance than OmpG and OmpF. These results demonstrated that a novel and functional ßMP can be successfully engineered through strand replacement based on sequence motif analysis and stability calculation.

The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm.

Two-component systems mediate bacterial signal transduction, employing a membrane sensor kinase and a cytoplasmic response regulator (RR). Environmental sensing is typically coupled to gene regulation. Understanding how input stimuli activate kinase autophosphorylation remains obscure. The EnvZ/OmpR system regulates expression of outer membrane proteins in response to osmotic stress. To identify EnvZ conformational changes associated with osmosensing, we used HDXMS to probe the effects of osmolytes (NaCl, sucrose) on the cytoplasmic domain of EnvZ (EnvZ(c)). Increasing osmolality decreased deuterium exchange localized to the four-helix bundle containing the autophosphorylation site (His(243)). EnvZ(c) exists as an ensemble of multiple conformations and osmolytes favoured increased helicity. High osmolality increased autophosphorylation of His(243), suggesting that these two events are linked. In-vivo analysis showed that the cytoplasmic domain of EnvZ was sufficient for osmosensing, transmembrane domains were not required. Our results challenge existing claims of robustness in EnvZ/OmpR and support a model where osmolytes promote intrahelical H-bonding enhancing helix stabilization, increasing autophosphorylation and downstream signalling. The model provides a conserved mechanism for signalling proteins that respond to diverse physical and mechanical stimuli.

Single-molecule studies on the mechanical interplay between DNA supercoiling and H-NS DNA architectural properties.

The Escherichia coli H-NS protein is a major nucleoid-associated protein that is involved in chromosomal DNA packaging and gene regulatory functions. These biological processes are intimately related to the DNA supercoiling state and thus suggest a direct relationship between H-NS binding and DNA supercoiling. Here, we show that H-NS, which has two distinct DNA-binding modes, is able to differentially regulate DNA supercoiling. H-NS DNA-stiffening mode caused by nucleoprotein filament formation is able to suppress DNA plectoneme formation during DNA supercoiling. In contrast, when H-NS is in its DNA-bridging mode, it is able to promote DNA plectoneme formation during DNA supercoiling. In addition, the DNA-bridging mode is able to block twists diffusion thus trapping DNA in supercoiled domains. Overall, this work reveals the mechanical interplay between H-NS and DNA supercoiling which provides insights to H-NS organization of chromosomal DNA based on its two distinct DNA architectural properties.

H-NS Regulates Gene Expression and Compacts the Nucleoid: Insights from Single-Molecule Experiments.

A set of abundant nucleoid-associated proteins (NAPs) play key functions in organizing the bacterial chromosome and regulating gene transcription globally. Histone-like nucleoid structuring protein (H-NS) is representative of a family of NAPs that are widespread across bacterial species. They have drawn extensive attention due to their crucial function in gene silencing in bacterial pathogens. Recent rapid progress in single-molecule manipulation and imaging technologies has made it possible to directly probe DNA binding by H-NS, its impact on DNA conformation and topology, and its competition with other DNA-binding proteins at the single-DNA-molecule level. Here, we review recent findings from such studies, and provide our views on how these findings yield new insights into the understanding of the roles of H-NS family members in DNA organization and gene silencing.

Rapid, high-throughput tracking of bacterial motility in 3D via phase-contrast holographic video microscopy.

Tracking fast-swimming bacteria in three dimensions can be extremely challenging with current optical techniques and a microscopic approach that can rapidly acquire volumetric information is required. Here, we introduce phase-contrast holographic video microscopy as a solution for the simultaneous tracking of multiple fast moving cells in three dimensions. This technique uses interference patterns formed between the scattered and the incident field to infer the three-dimensional (3D) position and size of bacteria. Using this optical approach, motility dynamics of multiple bacteria in three dimensions, such as speed and turn angles, can be obtained within minutes. We demonstrated the feasibility of this method by effectively tracking multiple bacteria species, including Escherichia coli, Agrobacterium tumefaciens, and Pseudomonas aeruginosa. In addition, we combined our fast 3D imaging technique with a microfluidic device to present an example of a drug/chemical assay to study effects on bacterial motility.

An Escherichia coli Nissle 1917 missense mutant colonizes the streptomycin-treated mouse intestine better than the wild type but is not a better probiotic.

Previously we reported that the streptomycin-treated mouse intestine selected for two different Escherichia coli MG1655 mutants with improved colonizing ability: nonmotile E. coli MG1655 flhDC deletion mutants that grew 15% faster in vitro in mouse cecal mucus and motile E. coli MG1655 envZ missense mutants that grew slower in vitro in mouse cecal mucus yet were able to cocolonize with the faster-growing flhDC mutants. The E. coli MG1655 envZ gene encodes a histidine kinase that is a member of the envZ-ompR two-component signal transduction system, which regulates outer membrane protein profiles. In the present investigation, the envZP41L gene was transferred from the intestinally selected E. coli MG1655 mutant to E. coli Nissle 1917, a human probiotic strain used to treat gastrointestinal infections. Both the E. coli MG1655 and E. coli Nissle 1917 strains containing envZP41L produced more phosphorylated OmpR than their parents. The E. coli Nissle 1917 strain containing envZP41L also became more resistant to bile salts and colicin V and grew 50% slower in vitro in mucus and 15% to 30% slower on several sugars present in mucus, yet it was a 10-fold better colonizer than E. coli Nissle 1917. However, E. coli Nissle 1917 envZP41L was not better at preventing colonization by enterohemorrhagic E. coli EDL933. The data can be explained according to our "restaurant" hypothesis for commensal E. coli strains, i.e., that they colonize the intestine as sessile members of mixed biofilms, obtaining the sugars they need for growth locally, but compete for sugars with invading E. coli pathogens planktonically.