Single Host Cell Research Articles

Metabolic changes in Toxoplasma gondii-infected host cells measured by autofluorescence imaging.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite that infects warm-blooded vertebrates across the world. In humans, seropositivity rates of T. gondii range from 10% to 90% across communities. Despite its prevalence, few studies address how T. gondii infection changes the metabolism of host cells. In this study, we investigate how T. gondii manipulates the host cell metabolic environment by monitoring the metabolic response over time using noninvasive autofluorescence lifetime imaging of single cells, metabolite analysis, extracellular flux analysis, and reactive oxygen species (ROS) production. Autofluorescence lifetime imaging indicates that infected host cells become more oxidized and have an increased proportion of bound NAD(P)H compared to uninfected controls. Over time, infected cells also show decreases in levels of intracellular glucose and lactate, increases in oxygen consumption, and variability in ROS production. We further examined changes associated with the pre-invasion "kiss and spit" process using autofluorescence lifetime imaging, which also showed a more oxidized host cell with an increased proportion of bound NAD(P)H over 48 hours compared to uninfected controls, suggesting that metabolic changes in host cells are induced by T. gondii kiss and spit even without invasion.IMPORTANCEThis study sheds light on previously unexplored changes in host cell metabolism induced by T. gondii infection using noninvasive, label-free autofluorescence imaging. In this study, we use optical metabolic imaging (OMI) to measure the optical redox ratio (ORR) in conjunction with fluorescence lifetime imaging microscopy (FLIM) to noninvasively monitor single host cell response to T. gondii infection over 48 hours. Collectively, our results affirm the value of using autofluorescence lifetime imaging to noninvasively monitor metabolic changes in host cells over the time course of a microbial infection. Understanding this metabolic relationship between the host cell and the parasite could uncover new treatment and prevention options for T. gondii infections worldwide.

Experimental Evolution Studies in Φ6 Cystovirus.

Experimental evolution studies, in which biological populations are evolved in a specific environment over time, can address questions about the nature of spontaneous mutations, responses to selection, and the origins and maintenance of novel traits. Here, we review more than 30 years of experimental evolution studies using the bacteriophage (phage) Φ6 cystovirus. Similar to many lab-studied bacteriophages, Φ6 has a high mutation rate, large population size, fast generation time, and can be genetically engineered or cryogenically frozen, which facilitates its rapid evolution in the laboratory and the subsequent characterization of the effects of its mutations. Moreover, its segmented RNA genome, outer membrane, and capacity for multiple phages to coinfect a single host cell make Φ6 a good non-pathogenic model for investigating the evolution of RNA viruses that infect humans. We describe experiments that used Φ6 to address the fitness effects of spontaneous mutations, the consequences of evolution in the presence of coinfection, the evolution of host ranges, and mechanisms and consequences of the evolution of thermostability. We highlight open areas of inquiry where further experimentation on Φ6 could inform predictions for pathogenic viruses.

Facultative endosymbiosis between cellulolytic protists and methanogenic archaea in the gut of the Formosan termite Coptotermes formosanus.

Anaerobic protists frequently harbour methanogenic archaea, which apparently contribute to the hosts' fermentative metabolism by consuming excess H2. However, the ecological properties of endosymbiotic methanogens remain elusive in many cases. Here we investigated the ecology and genome of the endosymbiotic methanogen of the Cononympha protists in the hindgut of the termite Coptotermes formosanus. Microscopic and 16S rRNA amplicon sequencing analyses revealed that a single species, designated here "Candidatus Methanobrevibacter cononymphae", is associated with both Cononympha leidyi and Cononympha koidzumii and that its infection rate in Cononympha cells varied from 0.0% to 99.8% among termite colonies. Fine-scale network analysis indicated that multiple 16S rRNA sequence variants coexisted within a single host cell and that identical variants were present in both Cononympha species and also on the gut wall. Thus, "Ca. Methanobrevibacter cononymphae" is a facultative endosymbiont, transmitted vertically with frequent exchanges with the gut environment. Indeed, transmission electron microscopy showed escape or uptake of methanogens from/by a Cononympha cell. The genome of "Ca. Methanobrevibacter cononymphae" showed features consistent with its facultative lifestyle: i.e., the genome size (2.7 Mbp) comparable to those of free-living relatives; the pseudogenization of the formate dehydrogenase gene fdhA, unnecessary within the non-formate-producing host cell; the dependence on abundant acetate in the host cell as an essential carbon source; and the presence of a catalase gene, required for colonization on the microoxic gut wall. Our study revealed a versatile endosymbiosis between the methanogen and protists, which may be a strategy responding to changing conditions in the termite gut.

Abstract 5658: MG-Ig (molecularly grafted immunoglobulin): An alternative platform for production of bispecific antibodies

Abstract The generation of bispecific molecules, in which two target-specific moieties are engineered into a single entity, offers an attractive approach to optimize biological activity which may improve clinical outcomes. Among various formats of bispecific molecules, the heterodimeric Fc currently is a favorable scaffold for the design of bispecific antibodies because of its structural similarity to the natural antibody. However, development of molecules based on this concept is challenged by the presence of potential homodimer contamination, reduced yield, and stability loss relative to the natural Fc. Here, we report the development of the MG-Ig platform by grafting a single chain fragment variable (scFv) onto the hinge region of a full-size immunoglobulin with a different specificity to produce a trivalent IgG-like bispecific antibody that retains natural homodimer Fc. Whereas the MG-Ig platform is evolved from the Dock-and-Lock (DNL) technology which exploits the interaction between the anchoring domain (AD) of A kinase anchor proteins and the dimerization and docking domain (DDD) of regulatory subunit of cAMP-dependent protein kinase, the main distinctions include 1) the AD-fused scFv monomer is judiciously designed to graft onto the DDD dimer inserted into the hinge region of IgG, forming a bispecific antibody with three (1+2) binding arms and 2) all modular subunits are expressed in a single host cell and self-assemble to generate the bioactive conjugate in situ. To validate the method, bispecific T-cell engagers, designated 3scFv × hL0125C (CD3 × Trop2) and 3scFv × hACD20C (CD3 × CD20), which redirect T cells to Trop2 and CD20, respectively, have been produced with high yield and purity. Flow cytometry analysis demonstrated the bispecific binding of 3scFv × hL0125C to both CD3 on Jurkat and Trop2 on ME-180 with unaltered affinities. These T-cell redirecting bispecific antibodies mediated dose-dependent killing of target cancer cells when co-incubated with human PBMCs. Based on the 60-h viability assays using MTS, the IC50 of 3scFv × hL0125C was about 6 pM for MDA-MB-468, 13 pM for MDA-MB-231, 20 pM for BT-474, and 18 pM for HCT116. In 3D culture of HCT116 cells, the formation of multicellular tumor spheroids was impeded by 3scFv × hL0125C at 19.5 pM or higher, which is consistent with the cytotoxicity assays for tumor cells grown in monolayer. In addition, 3scFv × hL0125C was stable in human plasma for 7 days at 37°C, retaining bispecific integrity and binding activity. These data are promising and merit further development of MG-Ig bispecific antibodies for preclinical and clinical studies. Citation Format: Donglin Liu, Li Zeng, Berenice E. Liu, Chien-Hsing Chang. MG-Ig (molecularly grafted immunoglobulin): An alternative platform for production of bispecific antibodies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5658.

Enhanced recombination among Omicron subvariants of SARS‐CoV‐2 contributes to viral immune escape

Genetic recombination is an important driver of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) evolution, which requires the coinfection of a single host cell with different SARS-CoV-2 strains. To understand the emergence and prevalence of recombinant SARS-CoV-2 lineages through time and space, we analyzed SARS-CoV-2 genome sequences collected from November 2019 to July 2022. We observed an extraordinary increase in the emergence of SARS-CoV-2 recombinant lineages during the Omicron wave, particularly in Northern America and Europe. This phenomenon was independent of the sequencing frequency or genetic diversity of circulating SARS-CoV-2 strains. The recombination breakpoints were more prevalent in the 3'-untranslated region ofthe viral genome. Importantly, we noted the enrichment of certain amino acids in the Spike protein of recombinant lineages, which have been reported to confer immune escape from neutralizing antibodies and increase angiotensin-converting enzyme 2 receptorbinding in some cases. We also observed I42V amino acid change genetically fixated in the NSP14 of the Omicron lineage, which needs further characterization for its potential role in enhanced recombination. Overall, we report the important and timely observation of accelerated recombination in the currently circulating SARS-CoV-2 Omicron variants and explore their potential contribution to viral fitness, particularly immune escape.

On the formation of a super stop-band in finite mono-coupled periodic structures using an array of vibration absorbers: Controlling parameters and physical insight

There are two fundamental strategies in the design of metamaterials used for noise and vibration control. The first uses structural periodicity and explores the Bragg scattering to produce frequency ranges where waves do not propagate or are severely attenuated (stop-bands). This condition is usually limited by the constitutive size of the structural elements and tends to occur at higher frequencies. A second strategy consists of adding vibration absorbers to produce a resonance(s), and this can occur at much lower frequencies. The addition of periodically attached vibration absorbers alone creates a periodic medium, and Bragg stop-bands also occurs in this situation. There is a particular condition where the Bragg and local resonance stop-bands can be combined to produce an ultra-wide or a super stop-band. This paper investigates the conditions required to produce a super stop-band in a finite mono-coupled periodic system composed of symmetric cells, each with a vibration absorber attached. Using a lumped parameter system, the dynamic features and the transmissibility of a single cell are determined, and the physical mechanisms behind the super stop-band formation are investigated, including the absorber tuning frequency and the influence of damping. Two experimental setups to support the theoretical findings are described. One involves a lumped-parameter system and the other involves a rod, both with a vibration absorber attached. The results show that a smooth super stop-band only occurs when the vibration absorber is tuned to the free-free natural frequency of the single host cell, creating a union between the Bragg stop-bands due to mass and stiffness of the absorber, and that the damping in the absorber should be equal to the damping in the host cell.

Looking Inside Non-Destructively: Label-Free, Raman-Based Visualization of Intracellular Coxiella burnetii.

The life cycle of intracellular pathogens is often complex and can include different morphoforms. Treatment of intracellular infections and unperturbed studying of the pathogen inside the host cell are frequently challenging. Here, we present a Raman-based, label-free, non-invasive, and non-destructive method to localize, visualize, and even quantify intracellular bacteria in 3D within intact host cells in a Coxiella burnetii infection model. C. burnetii is a zoonotic obligate intracellular pathogen that causes infections in ruminant livestock and humans with an acute disease known as Q fever. Using statistical data analysis, no isolation is necessary to gain detailed information on the intracellular pathogen's metabolic state. High-quality false color image stacks with diffraction-limited spatial resolution enable a 3D spatially resolved single host cell analysis that shows excellent agreement with results from transmission electron microscopy. Quantitative analysis at different time points post infection allows to follow the infection cycle with the transition from the large cell variant (LCV) to the small cell variant (SCV) at around day 6 and a gradual change in the lipid composition during vacuole maturation. Spectral characteristics of intracellular LCV and SCV reveal a higher lipid content of the metabolically active LCV.

Bacterial filamentation as a mechanism for cell-to-cell spread within an animal host

Intracellular pathogens are challenged with limited space and resources while replicating in a single host cell. Mechanisms for direct invasion of neighboring host cells have been discovered in cell culture, but we lack an understanding of how bacteria directly spread between host cells in vivo. Here, we describe the discovery of intracellular bacteria that use filamentation for spreading between the intestinal epithelial cells of a natural host, the rhabditid nematode Oscheius tipulae. The bacteria, which belong to the new species Bordetella atropi, can infect the nematodes following a fecal-oral route, and reduce host life span and fecundity. Filamentation requires UDP-glucose biosynthesis and sensing, a highly conserved pathway that is used by other bacteria to detect rich conditions and inhibit cell division. Our results indicate that B. atropi uses a pathway that normally regulates bacterial cell size to trigger filamentation inside host cells, thus facilitating cell-to-cell dissemination.

High-Definition DIC Imaging Uncovers Transient Stages of Pathogen Infection Cycles on the Surface of Human Adult Stem Cell-Derived Intestinal Epithelium.

ABSTRACTInteractions between individual pathogenic microbes and host tissues involve fast and dynamic processes that ultimately impact the outcome of infection. Using live-cell microscopy, these dynamics can be visualized to study, e.g., microbe motility, binding and invasion of host cells, and intrahost-cell survival. Such methodology typically employs confocal imaging of fluorescent tags in tumor-derived cell line infections on glass. This allows high-definition imaging but poorly reflects the host tissue’s physiological architecture and may result in artifacts. We developed a method for live-cell imaging of microbial infection dynamics on human adult stem cell-derived intestinal epithelial cell (IEC) layers. These IEC layers are grown in apical imaging chambers, optimized for physiological cell arrangement and fast, but gentle, differential interference contrast (DIC) imaging. This allows subsecond visualization of both microbial and epithelial surface ultrastructure at high resolution without using fluorescent reporters. We employed this technology to probe the behavior of two model pathogens, Salmonella enterica serovar Typhimurium and Giardia intestinalis, at the intestinal epithelial surface. Our results reveal pathogen-specific swimming patterns on the epithelium and show that Salmonella lingers on the IEC surface for prolonged periods before host cell invasion, while Giardia uses circular swimming with intermittent attachments to scout for stable adhesion sites. The method even permits tracking of individual Giardia flagella, demonstrating that active flagellar beating and attachment to the IEC surface are not mutually exclusive. This work describes a generalizable and relatively inexpensive approach to resolving dynamic pathogen-IEC layer interactions, applicable even to genetically nontractable microorganisms.

Systems analysis shows that thermodynamic physiological and pharmacological fundamentals drive COVID-19 and response to treatment.

Why a systems analysis view of this pandemic? The current pandemic has inflicted almost unimaginable grief, sorrow, loss, and terror at a global scale. One of the great ironies with the COVID‐19 pandemic, particularly early on, is counter intuitive. The speed at which specialized basic and clinical sciences described the details of the damage to humans in COVID‐19 disease has been impressive. Equally, the development of vaccines in an amazingly short time interval has been extraordinary. However, what has been less well understood has been the fundamental elements that underpin the progression of COVID‐19 in an individual and in populations. We have used systems analysis approaches with human physiology and pharmacology to explore the fundamental underpinnings of COVID‐19 disease. Pharmacology powerfully captures the thermodynamic characteristics of molecular binding with an exogenous entity such as a virus and its consequences on the living processes well described by human physiology. Thus, we have documented the passage of SARS‐CoV‐2 from infection of a single cell to species jump, to tropism, variant emergence and widespread population infection. During the course of this review, the recurrent observation was the efficiency and simplicity of one critical function of this virus. The lethality of SARS‐CoV‐2 is due primarily to its ability to possess and use a variable surface for binding to a specific human target with high affinity. This binding liberates Gibbs free energy (GFE) such that it satisfies the criteria for thermodynamic spontaneity. Its binding is the prelude to human host cellular entry and replication by the appropriation of host cell constituent molecules that have been produced with a prior energy investment by the host cell. It is also a binding that permits viral tropism to lead to high levels of distribution across populations with newly formed virions. This thermodynamic spontaneity is repeated endlessly as infection of a single host cell spreads to bystander cells, to tissues, to humans in close proximity and then to global populations. The principal antagonism of this process comes from SARS‐CoV‐2 itself, with its relentless changing of its viral surface configuration, associated with the inevitable emergence of variants better configured to resist immune sequestration and importantly with a greater affinity for the host target and higher infectivity. The great value of this physiological and pharmacological perspective is that it reveals the fundamental thermodynamic underpinnings of SARS‐CoV‐2 infection.

Indigo derivatives-incorporated functional polyhydroxybutyrate polymer with controlled biodegradability

Indigo derivatives incorporating Poly(3-hydroxybutyrate) (IDPs) were produced in a single recombinant Escherichia coli strain by a one-pot process. The E. coli cells expressed enzymes involved in two different synthesis pathways: genes for Poly(3-hydroxybutyrate) (PHB) synthesis (phaB, phaC, and bktb), and the gene for synthesizing indigo derivatives (CYP102G4). PHB is a well-known biodegradable polymer, the monomers of which are synthesized and polymerized in a single host cell, while indigo is a well-known indole-derived colorant that has been used for denim dyeing. Depending on the indole derivatives used in the feed, indigo derivatives with different colors were synthesized through the action of the CYP102G4 enzyme. The indole derivatives included 5-Cl, 6-Cl, 7-Cl, 5-Br, 5-OH, 5-OCH3, 4-NO2, and 5-NO2 indole. Engineered Escherichia coli cells fed with each indole derivative simultaneously generated IDPs. Several biological and physical properties, such as the antioxidant activity, biodegradability, melting temperature (Tm), glass transition temperature (Tg), average molecular weight, polydispersity index, hardness, and elasticity of the IDPs were analyzed. Among the IDPs, 5-OH IDP showed a higher Tm of 181.1 °C and thermostability, whereas 4-NO2 IDP showed the lowest Tm of 154.7 °C. And the biodegradability varied depending on the incorporated indigo derivatives. 5-OH IDP was the most resistant to biodegradation, with the lowest weight loss of 2.6%, while 4-NO2 IDP showed the highest weight loss of 18.9%. Moreover, significant changes in the hardness, analyzed by nanoindentation, were observed with 4-NO2 incorporated IDP. Finally, the produced IDPs were used as a cellulose coating, which resulted in differently packed surface structures without loss of coloration. The results of this study can be further extended to developing functional biodegradable polymers with characteristic coloration.

Cyanobiont genetic diversity and host specificity of cyanobiont-bearing dinoflagellate Ornithocercus in temperate coastal waters

Cyanobacteria are ubiquitous in marine environments and play an important role as primary producers. Some cyanobacteria, the so-called cyanobionts (cyanobacterial symbionts), have a symbiotic relationship with unicellular organisms. Among these relationships, in particular, the nature (e.g., genetic diversity, host or cyanobiont specificity, and cyanobiont seasonality) of the cyanobiont-dinoflagellate host consortia remains poorly understood. In this study, 16S rDNA of cyanobionts in 138 single host cells isolated over four seasons from temperate waters were sequenced using the MiSeq platform. Genetic analysis of cyanobionts from the dinoflagellate host Ornithocercus revealed that three genetic types of Synechococcales cyanobionts occurred in a wide range of water temperatures (11–24 °C), and their distribution seemed to be closely associated with variations in salinity. Furthermore, a certain degree of host (or cyanobiont) specificity in cyanobionts (or the host) among Ornithocercus species as well as among other dinophysoid species (i.e. Amphisolenia, Citharistes, and Histioneis), was observed. In addition to the Synechococcales cyanobionts, this study identified OTU sequences affiliated with Vampirovibrionales and Chroococcidiopsidales in some Ornithocercus cells, suggesting that Ornithocercus species are an additional habitat for these bacterial groups.

Model-based approach for predicting the impact of genetic modifications on product yield in biopharmaceutical manufacturing-Application to influenza vaccine production.

A large group of biopharmaceuticals is produced in cell lines. The yield of such products can be increased by genetic engineering of the corresponding cell lines. The prediction of promising genetic modifications by mathematical modeling is a valuable tool to facilitate experimental screening. Besides information on the intracellular kinetics and genetic modifications the mathematical model has to account for ubiquitous cell-to-cell variability. In this contribution, we establish a novel model-based methodology for influenza vaccine production in cell lines with overexpressed genes. The manipulation of the expression level of genes coding for host cell factors relevant for virus replication is achieved by lentiviral transduction. Since lentiviral transduction causes increased cell-to-cell variability due to different copy numbers and integration sites of the gene constructs we use a population balance modeling approach to account for this heterogeneity in terms of intracellular viral components and distributed kinetic parameters. The latter are estimated from experimental data of intracellular viral RNA levels and virus titers of infection experiments using cells overexpressing a single host cell gene. For experiments with cells overexpressing multiple host cell genes, only final virus titers were measured and thus, no direct estimation of the parameter distributions was possible. Instead, we evaluate four different computational strategies to infer these from single gene parameter sets. Finally, the best computational strategy is used to predict the most promising candidates for future modifications that show the highest potential for an increased virus yield in a combinatorial study. As expected, there is a trend to higher yields the more modifications are included.

Parallel reductive genome evolution in Desulfovibrio ectosymbionts independently acquired by Trichonympha protists in the termite gut

Several Trichonympha protist species in the termite gut have independently acquired Desulfovibrio ectosymbionts in apparently different stages of symbiosis. Here, we obtained the near-complete genome sequence of Desulfovibrio phylotype ZnDsv-02, which attaches to the surface of Trichonympha collaris cells, and compared it with a previously obtained genome sequence of ‘Candidatus Desulfovibrio trichonymphae’ phylotype Rs-N31, which is almost completely embedded in the cytoplasm of Trichonympha agilis. Single-nucleotide polymorphism analysis indicated that although Rs-N31 is almost clonal, the ZnDsv-02 population on a single host cell is heterogeneous. Despite these differences, the genome of ZnDsv-02 has been reduced to 1.6 Mb, which is comparable to that of Rs-N31 (1.4 Mb), but unlike other known ectosymbionts of protists with a genome similar in size to their free-living relatives. Except for the presence of a lactate utilization pathway, cell-adhesion components and anti-phage defense systems in ZnDsv-02, the overall gene-loss pattern between the two genomes is very similar, including the loss of genes responsive to environmental changes. Our study suggests that genome reduction can occur in ectosymbionts, even when they can be transmitted horizontally and obtain genes via lateral transfer, and that the symbiont genome size depends heavily on their role in the symbiotic system.

Bioinformatics design and experimental validation of influenza A virus multi-epitopes that induce neutralizing antibodies.

Pandemics caused by influenza A virus (IAV) are responsible for the deaths of millions of humans around the world. One of these pandemics occurred in Mexico in 2009. Despite the impact of IAV on human health, there is no effective vaccine. Gene mutations and translocation of genome segments of different IAV subtypes infecting a single host cell make the development of a universal vaccine difficult. The design of immunogenic peptides using bioinformatics tools could be an interesting strategy to increase the success of vaccines. In this work, we used the predicted amino acid sequences of the neuraminidase (NA) and hemagglutinin (HA) proteins of different IAV subtypes to perform multiple alignments, epitope predictions, molecular dynamics simulations, and experimental validation. Peptide selection was based on the following criteria: promiscuity, protein surface exposure, and the degree of conservation among different medically relevant IAV strains. These peptides were tested using immunological assays to test their ability to induce production of antibodies against IAV. We immunized rabbits and mice and measured the levels of IgG and IgA antibodies in serum samples and nasal washes. Rabbit antibodies against the peptides P11 and P14 (both of which are hybrids of NA and HA) recognized HA from both group 1 (H1, H2, and H5) and group 2 (H3 and H7) IAV and also recognized the purified NA protein from the viral stock (influenza A Puerto Rico/916/34). IgG antibodies from rabbits immunized with P11 and P14 were capable of recognizing viral particles and inhibited virus hemagglutination. Additionally, intranasal immunization of mice with P11 and P14 induced specific IgG and IgA antibodies in serum and nasal mucosa, respectively. Interestingly, the IgG antibodies were found to have neutralizing capability. In conclusion, the peptides designed through in silico studies were validated in experimental assays.Electronic supplementary materialThe online version of this article (10.1007/s00705-020-04537-2) contains supplementary material, which is available to authorized users.

Pediludiella daitoensis gen. et sp. nov. (Scenedesmaceae, Chlorophyceae), a large coccoid green alga isolated from a Loxodes ciliate

Freshwater protists often harbor unicellular green algae within their cells. In ciliates, possibly because of large host cell sizes and the small size of algal coccoids, a single host cell typically contains more than a hundred algal cells. While surveying such algae-bearing protists on Minami Daito Jima Island in Japan, we found a green Loxodes ciliate (Loxodida, Karyorelictea) that contained one or two dozens of very large coccoid algae. We isolated one of these algae and analyzed its characteristics in detail. A small subunit (SSU) rDNA phylogeny indicated Pseudodidymocystis species (Scenedesmaceae, Chlorophyceae) to be the taxon closest to the alga, although it was clearly separated from this by 39 or more different sites (inclusive of gaps). SSU rRNA structure analyses indicated that these displacements included eight compensatory base changes (CBCs) and seven hemi-CBCs. We therefore concluded that this alga belongs to a separate genus, and described it as Pediludiella daitoensis gen. et sp. nov. The shape of the isolated and cultured P. daitoensis was nearly spherical and reached up to 30 µm in diameter. Chloroplasts were arranged peripherally and often split and elongated. Cells were often vacuolated and possessed a net-like cytoplasm that resembled a football (soccer ball) in appearance, which was reflected in the genus name.

RTX Toxins Ambush Immunity's First Cellular Responders.

The repeats-in-toxin (RTX) family represents a unique class of bacterial exoproteins. The first family members described were toxins from Gram-negative bacterial pathogens; however, additional members included exoproteins with diverse functions. Our review focuses on well-characterized RTX family toxins from Aggregatibacter actinomycetemcomitans (LtxA), Mannheimia haemolytica (LktA), Bordetella pertussis (CyaA), uropathogenic Escherichia coli (HlyA), and Actinobacillus pleuropneumoniae (ApxIIIA), as well as the studies that have honed in on a single host cell receptor for RTX toxin interactions, the β2 integrins. The β2 integrin family is composed of heterodimeric members with four unique alpha subunits and a single beta subunit. β2 integrins are only found on leukocytes, including neutrophils and monocytes, the first responders to inflammation following bacterial infection. The LtxA, LktA, HlyA, and ApxIIIA toxins target the shared beta subunit, thereby targeting all types of leukocytes. Specific β2 integrin family domains are required for the RTX toxin’s cytotoxic activity and are summarized here. Research examining the domains of the RTX toxins required for cytotoxic and hemolytic activity is also summarized. RTX toxins attack and kill phagocytic immune cells expressing a single integrin family, providing an obvious advantage to the pathogen. The critical question that remains, can the specificity of the RTX-β2 integrin interaction be therapeutically targeted?

Escalating Threat Levels of Bacterial Infection Can Be Discriminated by Distinct MAPK and NF-κB Signaling Dynamics in Single Host Cells.

During an infection, immune cells must identify the specific level of threat posed by a given bacterial input in order to generate an appropriate response. Given that they use a general non-self-recognition system, known as Toll-like receptors (TLRs), to detect bacteria, it remains unclear how they transmit information about a particular threat. To determine whether host cells can use signaling dynamics to transmit contextual information about a bacterial stimulus, we use live-cell imaging to make simultaneous quantitative measurements of host MAPK and NF-κB signaling, two key pathways downstream of TLRs, and bacterial infection and load. This combined, single-cell approach reveals that NF-κB and MAPK signaling dynamics are sufficient to discriminate between (1) pathogen-associated molecular patterns (PAMPs) versus bacteria, (2) extracellular versus intracellular bacteria, (3) pathogenic versus non-pathogenic bacteria, and (4) the presence or absence of features indicating an active intracellular bacterial infection, such as replication and effector secretion.

Single-cell genomics of co-sorted Nanoarchaeota suggests novel putative host associations and diversification of proteins involved in symbiosis

BackgroundNanoarchaeota are obligate symbionts of other Archaea first discovered 16 years ago, yet little is known about this largely uncultivated taxon. While Nanoarchaeota diversity has been detected in a variety of habitats using 16S rRNA gene surveys, genome sequences have been available for only three Nanoarchaeota and their hosts. The host range and adaptation of Nanoarchaeota to a wide range of environmental conditions has thus largely remained elusive. Single-cell genomics is an ideal approach to address these questions as Nanoarchaeota can be isolated while still attached to putative hosts, enabling the exploration of cell-cell interactions and fine-scale genomic diversity.ResultsFrom 22 single amplified genomes (SAGs) from three hot springs in Yellowstone National Park, we derived a genome-based phylogeny of the phylum Nanoarchaeota, linking it to global 16S rRNA gene diversity. By exploiting sequencing of co-sorted tightly attached cells, we associated Nanoarchaeota with 6 novel putative hosts, 2 of which were found in multiple SAGs, and showed that the same host species may associate with multiple species of Nanoarchaeota. Comparison of single nucleotide polymorphisms (SNPs) within a population of Nanoarchaeota SAGs indicated that Nanoarchaeota attached to a single host cell in situ are likely clonal. In addition to an overall pattern of purifying selection, we found significantly higher densities of non-synonymous SNPs in hypothetical cell surface proteins, as compared to other functional categories. Genes implicated in interactions in other obligate microbe-microbe symbioses, including those encoding a cytochrome bd-I ubiquinol oxidase and a FlaJ/TadC homologue possibly involved in type IV pili production, also had relatively high densities of non-synonymous SNPs.ConclusionsThis population genetics study of Nanoarchaeota greatly expands the known potential host range of the phylum and hints at what genes may be involved in adaptation to diverse environments or different hosts. We provide the first evidence that Nanoarchaeota cells attached to the same host cell are clonal and propose a hypothesis for how clonality may occur despite diverse symbiont populations.

Viral Anti-CRISPR Tactics: No Success without Sacrifice

Co-evolution with their bacterial hosts has led to viral countermeasures against CRISPR-mediated immunity. In a recent issue of Cell, Landsberger etal. (2018) and Borges etal. (2018) report that cooperation among bacteriophages and multiple infection events are necessary to overcome CRISPR immune responses.