Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth

Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.Infectious diseases typically arise when pathogens grow to high tissue loads, causing extensive damage and immunopathology. An outstanding example is Shigella flexneri, which rapidly grow from a small infectious dose of 10–100 bacteria (1) to intestinal loads causing life-threatening bloody diarrhea (bacillary dysentery) within a few hours (2, 3). This vigorous Shigella growth occurs inside human colon epithelial cells and requires an integrated Shigella pathogenesis program, including a type three secretion system encoded on the Shigella virulence plasmid. Using this system, Shigella translocates enzymes into the host cell cytosol, where they target key cellular functions, allowing Shigella to enter the host cell and escape bacterial killing by innate immune responses (4). After Shigella reaches the host cell cytosol, many virulence factors are down-regulated (5), and Shigella starts rapid proliferation.Biomass generation at such high rates depends on extensive exploitation of intracellular host nutrients (6). The host cell cytoplasm contains hundreds of metabolites, but it is unclear which of these potential nutrients Shigella uses, how the host cell can supply them at sufficiently high rates to support rapid Shigella growth, and why host cells can sustain viability while being vigorously exploited by intracellular Shigella. For related enteroinvasive Escherichia coli, previous research has shown that glucose and other host metabolites, such as diverse amino acids, can be incorporated into the biomass of these closely related pathogens (7). However, quantitative data are still lacking, and energy production, which is usually a major part of nutrient use (8), could not be analyzed because of technical limitations.In general, pathogen metabolism has been recognized as a fundamentally important aspect of infectious diseases, but available data are mostly restricted to qualitative presence/absence of enzymes in pathogen genomes and metabolite or gene expression profiles in various infection models (9–13). Comprehensive quantitative studies on pathogen nutrition, metabolism, and growth are largely lacking. This limited knowledge reflects, in part, the fact that suitable methodologies are just becoming available. In this study, we combined various metabolomics approaches, proteomics, and microbial genetics to elucidate the metabolic basis of Shigella rapid growth in infected human host cells.     

Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus

Atypical chemokine receptor 1 (ACKR1) is a G protein–coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein–binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1−Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.

The emergence of various multidrug-resistant strains of Staphylococcus aureus (SA) makes it a major concern for public health (1, 2). SA produces a large arsenal of virulence factors, among which bicomponent leukocidins—also referred to as leukotoxins—stand out as interesting targets for developing novel antivirulence strategies (3, 4). Leukotoxins belong to the family of β-barrel pore-forming toxins that assemble into heteromeric pores to lyse specific cells (5–8). Five different types of leukotoxins are expressed by SA infecting humans: Panton–Valentine leukocidin (PVL), LukED, HlgAB, HlgCB, and LukAB. Each one is formed by two subunits, the host cell–targeting S component (S for slow elution during biochemical purification) and the polymerization F (fast elution) component. In the current view, with the exception of LukAB, all the subunits are believed to be secreted as monomers that will heterodimerize upon specific interaction with host myeloid and erythroid cells. Dimerization will lead to a subsequent toxin oligomerization and pore formation in cell membranes (9–11).Recent identification of leukotoxin receptors in targeted host cells increased our knowledge of the mechanism behind cellular specificity of these toxins and their role in pathogenesis (12–17). Based on these findings, the predicted mechanism is that only the monomeric S component specifically interacts with various complement and chemokine receptors present on the surface of leukocytes, all related to the family of G protein–coupled receptors (GPCRs). The S component later recruits the F component to trigger oligomerization and pore formation. Though the F components HlgB and LukD were shown to bind to the surface of erythrocytes independently from their S component partners (18, 19), it was considered a receptor-independent binding. Recently, one of the F components, LukF-PV, was shown to specifically require a receptor in order to recognize targeted cells (20), therefore challenging the proposed initial steps of receptor recognition and pore formation (21).Out of all targeted receptors, the atypical chemokine receptor 1 (ACKR1, previously called DARC) (22), recognized by both HlgA and LukE, is a key player. Indeed, in addition to being expressed in myeloid cells, ACKR1 is expressed in erythrocytes and endothelial cells, making it necessary for SA to escape the immune system, to grow, and to cause cell death (12, 13). Unlike canonical chemokine receptors, ACKR1 lacks the conserved DRYLAIV motif and is thus structurally unable to activate G proteins by dissociating the subunits upon chemokine engagement (23, 24). Rather, it internalizes and transports chemokines to the degradative compartment, acting as a chemokine buffer by modulating chemokine concentration and bioavailability (25–28). The high-resolution structure of ACKR1 is still unknown; however, its homologs of known structures share the highly conserved GPCR structure consisting of a single polypeptide chain with three intracellular and extracellular loops, an external N-terminal region essential for the specificity of ligand binding, and an intracellular carboxyl-terminal region that is involved in receptor signaling. Although increasing amounts of structural and molecular data of chemokine receptors are being discovered (29, 30), the structural immunology and pharmacology related to ACKR1 is still in its infancy.Binding of leukotoxins to GPCRs is poorly understood at the molecular and structural level. Various residues in the loops of the rim domain of HlgA and LukE, as well as a four-residue region in the cap domain of HlgA, were shown to be necessary for hemolytic activity and/or binding to erythrocytes (31–33). From the receptor side, LukE and HlgA seem to target different regions of the ACKR1 N-terminal part, a highly flexible region, whereas both require sulfation of tyrosine residues in this same part of the receptor (13). In addition, little is known of the effects of leukotoxins on ACKR1 binding to its natural ligands and downstream molecular signaling. Although biochemical and cell biology work has been done since the discovery of receptors targeted by leukotoxins, direct evidence capturing purified leukotoxin−receptor complexes has only been provided for the LukE−CCR5 pair (16).In this study, we used an integrative molecular pharmacology and structural mass spectrometry (MS) approach in order to characterize the effect of HlgAB binding on ACKR1 structure and function. We demonstrate that both leukotoxins, HlgA and HlgB, form independent complexes with purified ACKR1 in vitro using native MS (nMS). In living cells, time-resolved fluorescence resonance energy transfer (TR-FRET) experiments revealed that both HlgA and HlgB binding to ACKR1 compete with CCL5, an endogenous ligand. We also monitored the effect of leukotoxin binding on ACKR1 conformation using a combination of hydrogen/deuterium exchange MS (HDX-MS) and cell-based resonance energy transfer. Surprisingly, in addition to the expected accessibility changes in the extracellular domain of the receptor, binding of leukotoxins induced long-range allosteric conformational changes in the intracellular domain of ACKR1 that leads to the dissociation of and/or changes in the architecture of preassembled ACKR1−Gαi1 protein complexes. Altogether, our study brings insights into the initial steps of leukotoxin biology through GPCR, namely, the toxins’ effect on GPCR structure and function.     

The bacterial signal indole increases epithelial-cell tight-junction resistance and attenuates indicators of inflammation

Interkingdom signaling is established in the gastrointestinal tract in that human hormones trigger responses in bacteria; here, we show that the corollary is true, that a specific bacterial signal, indole, is recognized as a beneficial signal in intestinal epithelial cells. Our prior work has shown that indole, secreted by commensal Escherichia coli and detected in human feces, reduces pathogenic E. coli chemotaxis, motility, and attachment to epithelial cells. However, the effect of indole on intestinal epithelial cells is not known. Because intestinal epithelial cells are likely to be exposed continuously to indole, we hypothesized that indole may be beneficial for these cells, and investigated changes in gene expression with the human enterocyte cell line HCT-8 upon exposure to indole. Exposure to physiologically relevant amounts of indole increased expression of genes involved in strengthening the mucosal barrier and mucin production, which were consistent with an increase in the transepithelial resistance of HCT-8 cells. Indole also decreased TNF-α-mediated activation of NF-κB, expression of the proinflammatory chemokine IL-8, and the attachment of pathogenic E. coli to HCT-8 cells, as well as increased expression of the antiinflammatory cytokine IL-10. The changes in transepithelial resistance and NF-κB activation were specific to indole: other indole-like molecules did not elicit a similar response. Our results are similar to those observed with probiotic strains and suggest that indole could be important in the intestinal epithelial cells response to gastrointestinal tract pathogens.     

Characteristics of the MicroRNA Expression Profile of Exosomes Released by Vero Cells Infected with Porcine Epidemic Diarrhea Virus

Exosomes are nanoscale vesicles actively secreted by a variety of cells. They contain regulated microRNA (miRNA), allowing them to function in intercellular communication. In the present study, the role of exosomal miRNAs in porcine epidemic diarrhea virus (PEDV) infection was investigated using exosomes isolated from Vero cells infected with PEDV. The results of transmission electron microscopy observation showed that the exosomes are spherical in shape, uniform in size, and negatively stained in the membrane. Nanoparticle tracking analysis showed that the average exosome particle size is 130.5 nm. The results of miRNA sequencing showed that, compared with the control group, a total of 115 miRNAs are abnormally expressed in the exosomes of infected cells. Of these, 80 miRNAs are significantly upregulated and 35 miRNAs are significantly downregulated. Functional annotation analysis showed that the differentially expressed miRNAs are associated with PEDV infection through interaction with the cAMP, Hippo, TGF-beta, HIF-1, FoxO, MAPK, and Ras signaling pathways. Thus, our findings provide important information about the effects of PEDV infection on exosomal miRNA expression and will aid the search for potential anti-PEDV drug candidates.     

Structural basis for the removal of ubiquitin and interferon-stimulated gene 15 by a viral ovarian tumor domain-containing protease

The attachment of ubiquitin (Ub) and the Ub-like (Ubl) molecule interferon-stimulated gene 15 (ISG15) to cellular proteins mediates important innate antiviral responses. Ovarian tumor (OTU) domain proteases from nairoviruses and arteriviruses were recently found to remove these molecules from host proteins, which inhibits Ub and ISG15-dependent antiviral pathways. This contrasts with the Ub-specific activity of known eukaryotic OTU-domain proteases. Here we describe crystal structures of a viral OTU domain from the highly pathogenic Crimean–Congo haemorrhagic fever virus (CCHFV) bound to Ub and to ISG15 at 2.5-Å and 2.3-Å resolution, respectively. The complexes provide a unique structural example of ISG15 bound to another protein and reveal the molecular mechanism of an ISG15 cross-reactive deubiquitinase. To accommodate structural differences between Ub and ISG15, the viral protease binds the β-grasp folds of Ub and C-terminal Ub-like domain of ISG15 in an orientation that is rotated nearly 75° with respect to that observed for Ub bound to a representative eukaryotic OTU domain from yeast. Distinct structural determinants necessary for binding either substrate were identified and allowed the reengineering of the viral OTU protease into enzymes with increased substrate specificity, either for Ub or for ISG15. Our findings now provide the basis to determine in vivo the relative contributions of deubiquitination and deISGylation to viral immune evasion tactics, and a structural template of a promiscuous deubiquitinase from a haemorrhagic fever virus that can be targeted for inhibition using small-molecule-based strategies.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Shigella impairs T lymphocyte dynamics in vivo

The Gram-negative enteroinvasive bacterium Shigella flexneri is responsible for the endemic form of bacillary dysentery, an acute rectocolitis in humans. S. flexneri uses a type III secretion system to inject effector proteins into host cells, thus diverting cellular functions to its own benefit. Protective immunity to reinfection requires several rounds of infection to be elicited and is short-lasting, suggesting that S. flexneri interferes with the priming of specific immunity. Considering the key role played by T-lymphocyte trafficking in priming of adaptive immunity, we investigated the impact of S. flexneri on T-cell dynamics in vivo. By using two-photon microscopy to visualize bacterium–T-cell cross-talks in the lymph nodes, where the adaptive immunity is initiated, we provide evidence that S. flexneri, via its type III secretion system, impairs the migration pattern of CD4⁺ T cells independently of cognate recognition of bacterial antigens. We show that bacterial invasion of CD4⁺ T lymphocytes occurs in vivo, and results in cell migration arrest. In the absence of invasion, CD4⁺ T-cell migration parameters are also dramatically altered. Signals resulting from S. flexneri interactions with subcapsular sinus macrophages and dendritic cells, and recruitment of polymorphonuclear cells are likely to contribute to this phenomenon. These findings indicate that S. flexneri targets T lymphocytes in vivo and highlight the role of type III effector secretion in modulating host adaptive immune responses.     

A tethered ligand assay to probe SARS-CoV-2:ACE2 interactions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are initiated by attachment of the receptor-binding domain (RBD) on the viral Spike protein to angiotensin-converting enzyme-2 (ACE2) on human host cells. This critical first step occurs in dynamic environments, where external forces act on the binding partners and avidity effects play an important role, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using magnetic tweezers and atomic force spectroscopy as highly complementary single-molecule force spectroscopy techniques, we investigate the RBD:ACE2 interaction over the whole physiologically relevant force range. We combine the experimental results with steered molecular dynamics simulations and observe and assign fully consistent unbinding and unfolding events across the three techniques, enabling us to establish ACE2 unfolding as a molecular fingerprint. Measuring at forces of 2 to 5 pN, we quantify the force dependence and kinetics of the RBD:ACE2 bond in equilibrium. We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical stability, larger binding free energy, and a lower dissociation rate compared to SARS-CoV-1, which helps to rationalize the different infection patterns of the two viruses. By studying how free ACE2 outcompetes tethered ACE2, we show that our assay is sensitive to prevention of bond formation by external binders. We expect our results to provide a way to investigate the roles of viral mutations and blocking agents for targeted pharmaceutical intervention.

A subset of coronaviruses (CoV) causes severe acute respiratory syndrome (SARS) in humans. We have seen three major recent outbreaks, including the first SARS pandemic from 2002 to 2004 (SARS-CoV-1), Middle East respiratory syndrome that emerged in 2012, and the ongoing COVID-19 pandemic (SARS-CoV-2). SARS-CoV-2 particles carry ∼100 copies of the trimeric viral glycoprotein Spike (S) on their surface (1), giving the appearance of an eponymous corona around the virus. Like SARS-CoV-1, SARS-CoV-2 attaches to human host cells by S binding to angiotensin-converting enzyme-2 (ACE2) (2–6) (Fig. 1A). Specifically, each of the three S1 subunits in an S trimer carries a receptor-binding domain (RBD) at its tip, which is presented in an up or down conformation and can bind ACE2 in the up conformation (Fig. 1B) (7). Binding of the virus to host cells occurs in dynamic environments (8, 9) where external forces act on the virus particle. In particular, in the respiratory tract, coughing, sneezing, and mucus clearance exert mechanical forces (10, 11) that the virus must withstand for productive infection. The magnitude and dynamics of these forces are not known precisely and are likely variable. A rough estimate from fluid dynamics suggests an upper limit of forces in the range of ∼2 pN to 2 nN (estimates are provided in SI Appendix).Open in a separate windowFig. 1.Single-molecule assays to probe the SARS-CoV-2 RBD:ACE2 interface under force. Motivation and overview of our tethered ligand assay for equilibrium and dynamic SMFS measurements in MT and AFM. (A) Schematic of a SARS-CoV-2 virus particle (green) presenting spike protein trimers (gray) that can bind to human ACE2 (red) on the cell surface via their RBDs (blue). The bond between RBD and ACE2 is formed in a dynamic environment, where it must withstand external mechanical forces (indicated by the red arrow), for example, caused by coughing or sneezing in the respiratory tract, in order to allow efficient infection of the human host cell (orange). (B) Crystal structure of the RBD:ACE2 complex (PDB ID: 6m0j). The N and C termini of the RBD (blue) and ACE2 (red) are indicated with yellow dots. (C) Schematic (not to scale) of the tethered ligand assay in MT. The tethered ligand construct consists of the ACE2 ectodomain (red square) and RBD (blue triangle) joined by a flexible polypeptide linker (black line) of 85 aa (31 nm contour length) or 115 aa (42 nm contour length). The tethered ligand construct is attached with one end covalently to the surface via an ELP linker (33) and with the other end to a superparamagnetic bead via a biotin–streptavidin bond. Permanent magnets above the flow cell enable the application of precisely calibrated stretching forces. (D) Tethered ligand construct in the absence of force, where the RBD remains bound to ACE2. (E) Stylized measurement of the tethered ligand construct in the MT. (Bottom) To probe RBD:ACE2 bond dynamics, time traces of the tether extension are recorded at different levels of applied force (indicated at the bottom). At low forces, reversible transitions between the bound configuration, with the RBD:ACE2 interface engaged, and a dissociated configuration, where the interface is broken and the peptide linker connecting the domains is stretched, are observed as jumps between two extension levels (red and blue dashed lines). At higher forces, further upward steps in the extension trace correspond to unfolding events of protein (sub)domains. (Top) From the MT time traces, both the fraction of time spent in the dissociated state and the dwell times in the bound and dissociated state can be determined as a function of applied force. (F) Schematic (not to scale) of the tethered ligand construct in the AFM. Here, covalent attachment to the surface uses a heterobifunctional PEG spacer, and the coupling to the AFM cantilever is accomplished via an Fgɣ tag on the protein that binds with very high force stability to the ClfA protein handle on the cantilever. (G) Stylized AFM measurement. (Bottom) The cantilever is retracted with constant velocity, and the force response to the applied extension is shown as a force-extension curve. With increasing extension, the RBD:ACE2 interface ruptures, protein subdomains unfold, and, finally, the ClfA:Fgɣ bond ruptures, giving rise to distinct peaks in the force-extension curve. Comparing two constructs with different linker lengths (31 nm black solid line and 42 nm gray/lilac alternative first peak) joining RBD and ACE2 allows assignment of the RBD:ACE2 interface rupture and unfolding of parts of the RBD to the first increment. Histograms of rupture forces (Top) are compiled from multiple measurements. The blue star refers to the RBD:ACE2 interface rupture.The SARS-CoV-2 S protein and its interaction with ACE2 have been the target of intense research activity, as they are critical in the first steps of SARS-CoV-2 infection, and S constitutes a major drug and the key vaccine target in the current fight against COVID-19. Further, differences in binding between ACE2 and the SARS-CoV-1 and SARS-CoV-2 RBDs have been linked to the different observed patterns in lower and upper respiratory tract infections by the two viruses (5). Despite their importance, many questions about RBD:ACE2 interactions, particularly about their stability under external forces, are unresolved. Consequently, there is an urgent need for assays that can probe the affinity and kinetics of the interaction under a wide range of external forces. In nature, receptor–ligand pairs are often held in spatial proximity by neighboring interactions, creating high effective concentrations. Engagement of multiple interactions has been suggested to be important in other viral infections, including influenza, rabies, and HIV (12–16). Since conventional affinity measurements do not take into account these effects, there is a need for novel in vitro assays mimicking these effects when measuring bond characteristics.Here, we present a tethered ligand assay to determine RBD interactions with ACE2 at the single-molecule level subject to defined levels of applied force. Our assay utilizes fusion protein constructs of SARS-CoV-1 or SARS-CoV-2 RBD and the human ACE2 ectodomain joined by flexible peptide linkers. To probe the linkage under a large range of mechanical forces and loading rates, we used two highly complementary single-molecule force spectroscopy (SMFS) approaches: an atomic force microscope (AFM) and magnetic tweezers (MT) (Fig. 1 C–G). We complemented the experiments with steered molecular dynamics (SMD) simulations to provide microscopic insights that are inaccessible experimentally.AFM force spectroscopy can probe molecular interactions and protein stability dynamically (15–29), typically measuring at constant loading rate, and can investigate even the most stable high-force host–pathogen interactions (at forces F > 2,000 pN) (18). In AFM experiments, the molecular construct of interest is stretched between a surface and the tip of an AFM cantilever. The cantilever is retracted at a constant velocity, and the force is monitored from the cantilever deflection. Molecular rupture or protein (sub)domain unfolding events give rise to a sawtooth-like pattern in the force vs. extension traces (Fig. 1G). In contrast, MT typically operate at constant force and can resolve very low forces (19, 20), down to F < 0.01 pN. In MT, molecules are tethered between a flow cell surface and magnetic beads. External magnets apply defined and constant stretching forces, and the tether extension is monitored by video microscopy. In MT, unbinding or unfolding events give rise to steps in the extension vs. time trace (Fig. 1E).Tethered ligand assays have provided insights into a range of critical molecular interactions under mechanical load (21–29). Under constant force, they allow observation of repeated interactions of the same binding partners, which are held in spatial proximity under mechanical control. Therefore, they can provide information on affinity, avidity, on and off rates, and mechanical stability (21, 23). Conversely, AFM force spectroscopy can perform dynamic measurements in a highly automated fashion and can reveal characteristic protein unfolding patterns, which can serve as molecular fingerprints (30) to select only properly folded and assembled molecular constructs for further analysis.Probing our tethered ligand construct by AFM force spectroscopy, we reveal the dynamic force stability of the assembly. In combination with SMD simulations, we assign the increments revealed by force spectroscopy and establish the ACE2 unfolding pattern as a molecular fingerprint to select properly assembled tethers. Using MT, we measure the on and off rates at different levels of mechanical load and extrapolate to the thermodynamic stability at zero load. We compare the stability of the SARS-CoV-1 and SARS-CoV-2 RBD:ACE2 interactions in all three assays and consistently find a lower force stability for SARS-CoV-1 across the different techniques.     

Phylogenetic analysis of beak and feather disease virus across a host ring-species complex

Pathogens have been hypothesized to play a major role in host diversity and speciation. Susceptibility of hybrid hosts to pathogens is thought to be a common phenomenon that could promote host population divergence and subsequently speciation. However, few studies have tested for pathogen infection across animal hybrid zones while testing for codivergence of the pathogens in the hybridizing host complex. Over 8 y, we studied natural infection by a rapidly evolving single-strand DNA virus, beak and feather diseases virus (BFDV), which infects parrots, exploiting a host-ring species complex (Platycercus elegans) in Australia. We found that host subspecies and their hybrids varied strikingly in both BFDV prevalence and load: both hybrid and phenotypically intermediate subspecies had lower prevalence and load compared with parental subspecies, while controlling for host age, sex, longitude and latitude, as well as temporal effects. We sequenced viral isolates throughout the range, which revealed patterns of genomic variation analogous to Mayr’s ring-species hypothesis, to our knowledge for the first time in any host–pathogen system. Viral phylogeny, geographic location, intraspecific host density, and parrot community diversity and composition did not explain the differences in BFDV prevalence or load between subpopulations. Overall, our analyses suggest that functional host responses to infection, or force of infection, differ between subspecies and hybrids. Our findings highlight the role of host hybridization and clines in altering host–pathogen interactions, dynamics that can have important implications for models of speciation with gene flow, and offer insights into how pathogens may adapt to diverging host populations.A long-standing puzzle in evolutionary ecology concerns the processes that promote speciation, and particularly the factors that favor or constrain genetic divergence in the absence of physical barriers to gene flow (1, 2). Coevolution between pathogens and their hosts is considered a fundamental interaction that influences microevolutionary changes in both the host and pathogen, and could potentially mediate gene flow between populations and consequently speciation (3, 4). Parasites have the potential to influence incipient speciation of their hosts by differentially influencing the fitness of diverging or intermediate host lineages, and thus the genetic exchange between host populations (4–6). Conversely, differing selection pressures exerted by host populations may lead to specialization and subsequent speciation of their parasites, especially if transmission between host populations is limited (4). Excellent opportunities to study such phenomena are provided by clinal and hybridizing populations, which offer natural laboratories in which to investigate population divergence and the early stages of speciation (2, 7). Parasitism may either promote or penalize hybridization, depending on a range of host, parasite, or environmental factors (8). Currently, our understanding of how host–parasite coevolution proceeds in diverging or hybridizing populations and its role in speciation is limited, in large part because of the small number of studies that examine variation in both hosts and parasites over sufficient spatial or temporal scales, or in hybridizing communities (8, 9).To date, studies of host–parasite interactions in hybridizing species have been overwhelmingly focused on plants (8–10). Moulia and Joly (9) identified only eight animal hybridization models where parasitism has been studied under natural conditions. Overall, both plant and animal studies suggest that higher parasite loads in hybrids compared with parental forms is the norm (8, 9), suggesting that hybrids are typically more susceptible to parasites compared with their parental species, and may therefore restrict gene flow between parental populations. For example, a hybrid population between two subspecies of house mice (Mus domesticus) were found to have higher helminth loads (6, 9, 11), suggesting that parasites could be selecting against hybridization. A similar but more complex pattern was found in hybridogenetic water frogs (Rana lessonae and Rana esculenta). Joly et al. (12) reported a higher load of lung flukes in hybrids, but this pattern varied depending on the particular parasite being tested, as this study also demonstrated that parental frogs had higher loads of lung roundworms. A separate study supporting this claim on the same system reported no differences in prevalence in loads of a nematode or two trematode species between hybrid and parental frogs (13). Baird et al. (14) recently found, in the same house mouse system mentioned above, that hybrids between two subspecies have lower helminth loads, the opposite pattern to what was previously found. This finding questions whether parasitic selection against hybrids in this system is consistent enough to prevent gene flow between the parental subspecies. Furthermore, doubt has been raised over whether helminth parasites have a fitness cost on hybrid mice (15). These studies are indicative of a dynamic interaction between hosts and parasites. Most studies have attempted to explain differences in infection levels across diverging host populations in terms of host genes or environmental variation (8). However, in general exogenous selection from environmental variation and differences in host architecture arising from hybridization have not provided satisfactory explanations for the infection scenarios observed (8). Recent explanations for discrepancies between studies have invoked the possibility of Red Queen dynamics leading to dynamic infection scenarios in hybridizing communities over space or time (8, 13, 15), although the empirical data required to fully test this has been inadequate both in spatial and temporal terms (8). Notably, few field or laboratory studies of hybrid parasitism have examined genetic variation in the parasite (8, 15; but see ref. 16). Experimental infections using different house mouse strains have demonstrated that host genotype affects host–protozoa interactions, but these experiments only used a single parasite strain. This is potentially a significant shortcoming, because parasites can evolve faster than their hosts (17) and host populations may be subject to specific parasite variants early in the process of divergence, potentially leading to variation in virulence when transmitted to a different population. Thus, parasite divergence may play a crucial role in host divergence and incipient speciation of their hosts.We studied geographic variation in the prevalence, infection load, and genetic variation of a virus (beak and feather disease virus, BFDV) infecting a parrot species complex (crimson rosella, Platycercus elegans). The P. elegans complex is a long-postulated example of a “circular overlap” or “ring species,” of which only about 25 have been proposed worldwide (2, 18–20), because it features clinally diverging populations with ongoing gene flow (21, 22) in an approximate horse shoe-shaped distribution, which culminate in a zone of overlap between the most divergent taxa (terminal forms). Such species complexes offer powerful and unique insights into coevolution of traits, population divergence, and speciation (e.g., refs. 20, 23, and 24), but surprisingly, the opportunity presented by such systems has not yet been used to understand host–parasite interactions (20). BFDV occurs in many wild and captive parrot populations worldwide, with the potential to cause high mortality (25, 26). Accordingly, it is considered a significant conservation threat and has been implicated in parrot declines in Australia and globally (27–30). BFDV possesses a single-stranded DNA genome of ∼2,000 nucleotides (31). Like most small single-stranded DNA and RNA viruses, BFDV shows high levels of genetic variation and recombination (27, 32, 33), and evolves rapidly in novel conditions (34), with multiple variant infections present in individual animals (29). This parrot–virus system is thus an excellent candidate to study how pathogens interact with diverging and hybridizing hosts.We investigated the prevalence and infection load of BFDV over 8 y across a 1,200-km-wide study area, which included the three main host subspecies (Platycercus elegans elegans, Platycercus elegans flaveoulus, Platycercus elegans adelaidae), and a zone of hybridization (Western Slopes or WS hybrid) where the most phenotypically distinct host subspecies overlap (Fig. 1). In this way we could determine the role of host factors (subspecies, sex, age) and ecology (host population density, host community diversity, and composition, temporal, and spatial location) on both viral prevalence and viral load. We also sequenced the virus throughout the host range to determine how it differs in response to host divergence and hybridization, and how it may differ phylogenetically from BFDV virus in other host species. We used these data to test whether BFDV phylogeography supports the hypothesis that P. elegans is a ring species.Open in a separate windowFig. 1.Map of Platycercus elegans geographic distribution in south eastern Australia. Colors indicate the approximate range of each subspecies based on observational data from The New Atlas of Australian Birds (53); P. e. melanoptera was not used in this study.     

Structure of a PE–PPE–EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed “type VII.” How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE–PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.Tuberculosis is a major public health challenge, and new interventions are needed to control emerging, highly drug-resistant strains (1). Sequencing of the Mycobacterium tuberculosis (Mtb) genome revealed the presence of two mysterious, highly expanded protein families in pathogenic mycobacteria (2), named PE and PPE, due to the presence of N-terminal domains with conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) sequence motifs. The Mtb genome encodes ∼100 PE and ∼70 PPE genes, accounting for ∼10% of the genome’s coding capacity (2), whereas nonpathogenic mycobacteria harbor relatively few PE and PPE genes. Both protein families are highly polymorphic, localize to the cell surface or are secreted (3, 4), and are expressed during infection (5), leading to hypotheses that they are involved in virulence, antigenic diversity, or immune evasion (6). Although many PE-PPE proteins are recognized by the immune system during infection (7), it remains unclear whether they are involved in antigenic variation (8). Outside the N-terminal core PE or PPE domain, the C-terminal segments vary widely (2, 9), sometimes encoding putative enzymatic domains or large peptide repeat arrays (several greater than 1,000 aa in length). PE and PPE genes often form operons, suggesting PE and PPE proteins interact with each other, and the crystal structure of a PE–PPE protein complex showed directly that they form a heterodimer (10). However, the PE and PPE domains have no detectable sequence or structural homology to other protein families, and their function remains unknown.Intriguingly, secretion of PE and PPE proteins has been linked to a set of related, specialized protein export pathways of mycobacteria called the 6-kDa early secreted antigenic target (ESAT-6) secretion system (ESX) (11, 12). “Type VII” secretion systems distantly related to mycobacterial ESX systems have been identified in numerous other Gram-positive bacteria (13–17). The protein components of type VII secretion systems differ considerably between bacterial species but generally include the following: (i) one or more small helical proteins of the WXG100 protein family (e.g., ESAT-6, YukE), (ii) an FtsK/SpoIII-type ATPase that is thought to drive protein secretion (e.g., EccC, YukB), and (iii) a multipass transmembrane protein that may form the pore of the translocon (e.g., EccD, YueB). Along with additional species/lineage-specific factors, such as the mycobacterial PE and PPE proteins, these components have been proposed to assemble in the plasma membrane, where they translocate specific protein substrates to the cell surface (or to the periplasmic space of mycobacteria) (figure 4B of ref. 18). The Mtb genome encodes five distinct but evolutionarily related type VII/ESX systems (ESX-1 to ESX-5) at different loci around the genome, and the primary attenuating mutation in the Mycobacterium bovis bacillus Calmette–Guérin vaccine strain is a deletion of a large segment of the ESX-1 locus (19). Each ESX system is thought to secrete a distinct complement of proteins, including the cognate ESAT-6 and 10-kDa culture filtrate protein (CFP-10) homologs encoded in each specific locus (19, 20). All ESX gene clusters, with the exception of ESX-4, also encode at least one PE-PPE gene pair, and all of the PE-PPE proteins tested so far are secreted in an ESX-dependent manner (11, 21), suggesting that PE-PPE secretion may be an important function of the ESX, although many PE-PPE proteins are encoded outside of ESX loci. Recently, PPE proteins were reported to interact with ESX secretion-associated protein G (EspG) (22), another ESX-encoded protein of unknown structure and function. However, the molecular basis for this interaction and its functional importance in PPE secretion remain unclear.Here, we report the crystal structures of three EspGs from Mtb and Mycobacterium smegmatis (Msmeg), and a ternary complex between an Mtb PE-PPE pair and its cognate EspG. These structures define the key elements of the PPE–EspG interaction and, coupled with bioinformatics and biochemical interaction studies, suggest that EspGs function as adaptors to deliver PE–PPE complexes to their cognate ESX system for translocation across the plasma membrane. Moreover, our work shows that the vast majority of PE-PPE proteins in Mtb interact with EspG from the ESX-5 secretion system, which has been hypothesized to play a major role in PE-PPE protein secretion (9, 20, 23).     

Host genotype structures the microbiome of a globally dispersed marine phytoplankton

Phytoplankton support complex bacterial microbiomes that rely on phytoplankton-derived extracellular compounds and perform functions necessary for algal growth. Recent work has revealed sophisticated interactions and exchanges of molecules between specific phytoplankton–bacteria pairs, but the role of host genotype in regulating those interactions is unknown. Here, we show how phytoplankton microbiomes are shaped by intraspecific genetic variation in the host using global environmental isolates of the model phytoplankton host Thalassiosira rotula and a laboratory common garden experiment. A set of 81 environmental T. rotula genotypes from three ocean basins and eight genetically distinct populations did not reveal a core microbiome. While no single bacterial phylotype was shared across all genotypes, we found strong genotypic influence of T. rotula, with microbiomes associating more strongly with host genetic population than with environmental factors. The microbiome association with host genetic population persisted across different ocean basins, suggesting that microbiomes may be associated with host populations for decades. To isolate the impact of host genotype on microbiomes, a common garden experiment using eight genotypes from three distinct host populations again found that host genotype influenced microbial community composition, suggesting that a process we describe as genotypic filtering, analogous to environmental filtering, shapes phytoplankton microbiomes. In both the environmental and laboratory studies, microbiome variation between genotypes suggests that other factors influenced microbiome composition but did not swamp the dominant signal of host genetic background. The long-term association of microbiomes with specific host genotypes reveals a possible mechanism explaining the evolution and maintenance of complex phytoplankton–bacteria chemical exchanges.

Interactions between marine phytoplankton and bacteria can exert a profound influence on ecosystem function and biogeochemical cycling, impacting rates of primary production, phytoplankton aggregation, organic carbon export, and nutrient cycling (1–3). As an aquatic analog of the plant rhizosphere, the most intimate relationships between phytoplankton and bacteria exist in the phycosphere, the region immediately surrounding a phytoplankton cell, where molecules can be exchanged despite the effects of turbulence and diffusion (4). Relationships between phytoplankton and bacteria in the phycosphere range from cooperative to competitive (5). For example, during exponential growth, phytoplankton actively secrete amino acids that are taken up by bacteria, despite potentially significant energy costs (6). In turn, some bacteria synthesize essential vitamins and growth hormones that stimulate phytoplankton productivity (7, 8). During phytoplankton senescence, formerly “friendly” bacteria can become pathogenic, producing algicides that lyse phytoplankton cells and release organic carbon to the environment (9). These complex ecological interactions have been investigated in the laboratory for specific phytoplankton–bacteria pairs (3). However, the persistence and phylogenetic breadth of these relationships for both host and microbiome remain open questions (10–12).In terrestrial habitats, clear linkages exist between the genetic background (i.e., host genotype and population genetic structure) of foundational plant species and the organisms that rely on them. For example, genetic variation within tree species can influence the structure of associated epiphytic, mycorrhizal, and invertebrate communities (13–15) through mutualism, parasitism, commensalism, facilitation, and competition (reviewed in ref. 16). These associations extend to plant-associated bacteria, whose abundance, composition, and diversity reflect intraspecific trait variation among host genotypes (17). Because microbes regulate processes such as decomposition, nutrient dynamics, and energy flow, the influence of intraspecific genetic variation in plants on their associated bacteria extends the effects of community genetics to ecosystem processes (18). In contrast to terrestrial habitats, seawater allows both bacteria and their phytoplankton hosts to drift with nearly unlimited dispersal across the global ocean. It is unknown whether planktonic communities mirror those in terrestrial habitats, where host genetics can shape bacterial community composition and influence ecosystem processes (16), or whether dispersal in the dilute marine environment overwhelms the formation of close and specific relationships between phytoplankton and their associated microbiota. Although phytoplankton drift freely across the global ocean with nearly unlimited dispersal and divide primarily asexually, they still possess clear genetic structure. Phytoplankton species are organized into genetically distinct populations that possess phenotypic trait variation, are associated with specific environmental conditions, and undergo large increases in abundance, known as blooms (19–23). Furthermore, genetically distinct phytoplankton populations persist on time scales of decades to centuries (23, 24), providing ample opportunity for populations to develop specific relationships with other microbes. Few studies have evaluated the microbiomes of multiple strains within a phytoplankton species. Some studies found that individual phytoplankton species may possess a core microbiome, with a consistent set of bacterial phylotypes and metabolic potentials (25, 26), while others found that phytoplankton strains supported distinct microbiomes (10) and differed in their growth responses to the same bacterial strain (7, 27, 28). These intriguing findings have not been rigorously examined in light of the genetic background of the host phytoplankton species. Given that plant genotype often (29–31) but not always (32) drives host microbiomes and given the differences between terrestrial and planktonic habitats, understanding to what extent the genetic background of phytoplankton species influences microbiome composition is critical to understanding the nature of their interactions and parsing the roles of both partners in global biogeochemical cycles.Here, we assessed host–microbe interactions using the model marine phytoplankton Thalassiosira rotula, a cosmopolitan species characterized by high genotypic and phenotypic diversity, which is subdivided into genetically distinct populations (24, 33). We examined whether the T. rotula microbiome was influenced by host genetic background, either at the genotype or population level. We combined a study of the microbiomes of 81 environmental genotypes (representing eight genetically distinct populations) sampled from around the world (Fig. 1 and Open in a separate windowFig. 1.Global sampling locations of the phytoplankton host T. rotula populations [symbol colors denote populations identified in Whittaker and Rynearson (24), and black indicates populations identified in this study] and their associated microbiomes (SI Appendix, Table S1). Three sites (Wa, Nb, and Fr) were resampled (symbols with two colors), and whole seawater was collected twice from Narragansett Bay (Nb) for microbiome comparisons with the in situ whole seawater bacterial community (asterisks). The base map is a composite of log annual average chlorophyll a concentrations (milligrams/meter⁻³, 2010) (https://oceandata.sci.gsfc.nasa.gov/directaccess/MODIS-Aqua/Mapped/Annual/9km/chlor_a/).Table 1.Global sampling site information for T. rotula and associated bacteria

Genome Editing Strategies to Protect Livestock from Viral Infections

The livestock industry is constantly threatened by viral disease outbreaks, including infections with zoonotic potential. While preventive vaccination is frequently applied, disease control and eradication also depend on strict biosecurity measures. Clustered regularly interspaced palindromic repeats (CRISPR) and associated proteins (Cas) have been repurposed as genome editors to induce targeted double-strand breaks at almost any location in the genome. Thus, CRISPR/Cas genome editors can also be utilized to generate disease-resistant or resilient livestock, develop vaccines, and further understand virus–host interactions. Genes of interest in animals and viruses can be targeted to understand their functions during infection. Furthermore, transgenic animals expressing CRISPR/Cas can be generated to target the viral genome upon infection. Genetically modified livestock can thereby reduce disease outbreaks and decrease zoonotic threats.     

Manipulation of Host Microtubule Networks by Viral Microtubule-Associated Proteins

Diverse DNA and RNA viruses utilize cytoskeletal networks to efficiently enter, replicate, and exit the host cell, while evading host immune responses. It is well established that the microtubule (MT) network is commonly hijacked by viruses to traffic to sites of replication after entry and to promote egress from the cell. However, mounting evidence suggests that the MT network is also a key regulator of host immune responses to infection. At the same time, viruses have acquired mechanisms to manipulate and/or usurp MT networks to evade these immune responses. Central to most interactions of viruses with the MT network are virally encoded microtubule-associated proteins (MAPs) that bind to MTs directly or indirectly. These MAPs associate with MTs and other viral or cellular MAPs to regulate various aspects of the MT network, including MT dynamics, MT-dependent transport via motor proteins such as kinesins and dyneins, and MT-dependent regulation of innate immune responses. In this review, we examine how viral MAP interactions with the MT network facilitate viral replication and immune evasion.     

SIK2 orchestrates actin-dependent host response upon Salmonella infection

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Salmonella enterica is a gram-negative, facultative intracellular human pathogen, annually causing more than 100 million food- and waterborne infections worldwide. Salmonella Typhimurium causes severe gastroenteritis, which could turn into a systemic infection in children, immune-compromised, or elderly people (1, 2). Concurrently, multidrug resistant bacteria are globally emerging and threatening our health systems, calling for a better understanding of the underlying virulence mechanism and host response.Pathogenic bacteria have evolved the inherent ability to infect and to establish their niche within host cells. For colonizing nonphagocytic cells such as epithelial cells, Salmonella uses a trigger mechanism–based entry mode. Bacterial virulence factors are then injected via a Type III-secretion system (T3SS) into the host cell to induce cytoskeletal rearrangements leading to membrane ruffling and macropinocytosis-driven internalization into a sealed phagosome (3, 4). This specialized compartment is referred to as the Salmonella-containing vacuole (SCV) and serves as the intracellular replicative niche by hiding the bacteria from the humoral and cell-autonomous immune response (5). Salmonella invasion requires a cooperative action of several bacterial effector proteins hijacking multiple host targets. One of the main targets forcing Salmonella´s uptake is the actin cytoskeleton by subverting the host Rho GTPases system. Bacterial effector proteins such as SopE/SopE2 mimic host nucleotide exchange factors (GEFs) to stimulate Rac1 and CDC42 activity (6, 7). Once GTP-activated, Rho GTPases stimulate downstream pathways to drive actin filament (F-actin) assembly and rearrangement.The actin cytoskeleton network is regulated by actin-binding proteins (ABPs), which orchestrate assembly and disassembly of actin in higher networks (8). Monomeric, globular actin (G-actin) is nucleated into new actin filaments, or the existing F-actin is elongated, stabilized, or disassembled by ABPs. The major actin nucleation factor is the multimeric Arp2/3 complex, which generates branched actin filament networks. Formins generate long unbranched actin filaments and represent another actin nucleation family. Together with actin nucleation-promoting factors, small Rho GTPases control ABPs in a spatiotemporal manner. Actin polymerization and membrane ruffling are necessary for Salmonella invasion. Following Salmonella internalization, the SCV undergoes SPI-1–dependent biogenesis and is transported to a juxtanuclear position at 1 to 2 h postinfection (pi). At later time-points (4 to 6 h pi), SPI-2–dependent effector proteins are expressed to further mature the SCV, allowing bacterial proliferation. Pioneering work described that, at later stages of the infection (≥6 h pi), an actin meshwork around the SCV stabilizes and protects the vacuolar niche (9–13).Here, we report SIK2 as a Salmonella resistance factor and a regulator of the actin cytoskeleton. SIK2 belongs to the AMPK kinase family and was named after its homolog SIK1, found to be expressed upon high-salt diet-induced stress in rats (14, 15). SIK2 has been implicated into multiple biological roles including melanogenesis, cancer progression, and gluconeogenesis (16–18). SIK2 depletion results in a loss of SCV integrity and bacterial escape into the host cytosol, causing intracellular Salmonella hyperproliferation. Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. Hence, SIK2 may represent a cellular safeguard, which controls the actin cytoskeleton and SCV integrity, thereby serving as a prime regulator of Salmonella proliferation subsequent to cellular internalization.