Small GTPases promote actin coat formation on microsporidian pathogens traversing the apical membrane of Caenorhabditis elegans intestinal cells

Many intracellular pathogens co‐opt actin in host cells, but little is known about these interactions in vivo. We study the in vivo trafficking and exit of the microsporidian Nematocida parisii, which is an intracellular pathogen that infects intestinal cells of the nematode Caenorhabditis elegans. We recently demonstrated that N. parisii uses directional exocytosis to escape out of intestinal cells into the intestinal tract. Here, we show that an intestinal‐specific isoform of C. elegans actin called ACT‐5 forms coats around membrane compartments that contain single exocytosing spores, and that these coats appear to form after fusion with the apical membrane. We performed a genetic screen for host factors required for actin coat formation and identified small GTPases important for this process. Through analysis of animals defective in these factors, we found that actin coats are not required for pathogen exit although they may boost exocytic output. Later during infection, we find that ACT‐5 also forms coats around membrane‐bound vesicles that contain multiple spores. These vesicles are likely formed by clathrin‐dependent compensatory endocytosis to retrieve membrane material that has been trafficked to the apical membrane as part of the exocytosis process. These findings provide insight into microsporidia interaction with host cells, and provide novel in vivo examples of the manner in which intracellular pathogens co‐opt host actin during their life cycle.     

Microsporidia Are Natural Intracellular Parasites of the Nematode Caenorhabditis elegans

For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.     

The purine nucleoside phosphorylase pnp-1 regulates epithelial cell resistance to infection in C. elegans

Intestinal epithelial cells are subject to attack by a diverse array of microbes, including intracellular as well as extracellular pathogens. While defense in epithelial cells can be triggered by pattern recognition receptor-mediated detection of microbe-associated molecular patterns, there is much to be learned about how they sense infection via perturbations of host physiology, which often occur during infection. A recently described host defense response in the nematode C. elegans called the Intracellular Pathogen Response (IPR) can be triggered by infection with diverse natural intracellular pathogens, as well as by perturbations to protein homeostasis. From a forward genetic screen, we identified the C. elegans ortholog of purine nucleoside phosphorylase pnp-1 as a negative regulator of IPR gene expression, as well as a negative regulator of genes induced by extracellular pathogens. Accordingly, pnp-1 mutants have resistance to both intracellular and extracellular pathogens. Metabolomics analysis indicates that C. elegans pnp-1 likely has enzymatic activity similar to its human ortholog, serving to convert purine nucleosides into free bases. Classic genetic studies have shown how mutations in human purine nucleoside phosphorylase cause immunodeficiency due to T-cell dysfunction. Here we show that C. elegans pnp-1 acts in intestinal epithelial cells to regulate defense. Altogether, these results indicate that perturbations in purine metabolism are likely monitored as a cue to promote defense against epithelial infection in the nematode C. elegans.     

Enterohaemorrhagic Escherichia coli O157:H7 Shiga‐like toxin 1 is required for full pathogenicity and activation of the p38 mitogen‐activated protein kinase pathway in Caenorhabditis elegans

Enterohaemorrhagic Escherichia coli (EHEC) causes life‐threatening infections in humans as a consequence of the production of Shiga‐like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga‐like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen‐activated protein kinase (MAPK) pathway, anevolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1‐dependent manner. Our results validate the EHEC–C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.     

Ten years of life in compost: temporal and spatial variation of North German Caenorhabditis elegans populations

The nematode Caenorhabditis elegans is a central laboratory model system in almost all biological disciplines, yet its natural life history and population biology are largely unexplored. Such information is essential for in‐depth understanding of the nematode's biology because its natural ecology provides the context, in which its traits and the underlying molecular mechanisms evolved. We characterized natural phenotypic and genetic variation among North German C. elegans isolates. We used the unique opportunity to compare samples collected 10 years apart from the same compost heap and additionally included recent samples for this and a second site, collected across a 1.5‐year period. Our analysis revealed significant population genetic differentiation between locations, across the 10‐year time period, but for only one location a trend across the shorter time frame. Significant variation was similarly found for phenotypic traits of likely importance in nature, such as choice behavior and population growth in the presence of pathogens or naturally associated bacteria. Phenotypic variation was significantly influenced by C. elegans genotype, time of isolation, and sampling site. The here studied C. elegans isolates may provide a valuable, genetically variable resource for future dissection of naturally relevant gene functions.     

A Wild C. Elegans Strain Has Enhanced Epithelial Immunity to a Natural Microsporidian Parasite

Microbial pathogens impose selective pressures on their hosts, and combatting these pathogens is fundamental to the propagation of a species. Innate immunity is an ancient system that provides the foundation for pathogen resistance, with epithelial cells in humans increasingly appreciated to play key roles in innate defense. Here, we show that the nematode C. elegans displays genetic variation in epithelial immunity against intestinal infection by its natural pathogen, Nematocida parisii. This pathogen belongs to the microsporidia phylum, which comprises a large phylum of over 1400 species of fungal-related parasites that can infect all animals, including humans, but are poorly understood. Strikingly, we find that a wild C. elegans strain from Hawaii is able to clear intracellular infection by N. parisii, with this ability restricted to young larval animals. Notably, infection of older larvae does not impair progeny production, while infection of younger larvae does. The early-life immunity of Hawaiian larvae enables them to produce more progeny later in life, providing a selective advantage in a laboratory setting—in the presence of parasite it is able to out-compete a susceptible strain in just a few generations. We show that enhanced immunity is dominant to susceptibility, and we use quantitative trait locus mapping to identify four genomic loci associated with resistance. Furthermore, we generate near-isogenic strains to directly demonstrate that two of these loci influence resistance. Thus, our findings show that early-life immunity of C. elegans against microsporidia is a complex trait that enables the host to produce more progeny later in life, likely improving its evolutionary success.     

Quantitative proteomic analysis of host epithelial cells infected by Salmonella enterica serovar Typhimurium

Systems‐level analyses have the capability to offer new insight into host–pathogen interactions on the molecular level. Using Salmonella infection of host epithelial cells as a model system, we previously analyzed intracellular bacterial proteome as a window into pathogens’ adaptations to their host environment [Infect. Immun. 2015; J. Proteome Res. 2017]. Herein we extended our efforts to quantitatively examine protein expression of host cells during infection. In total, we identified more than 5000 proteins with 194 differentially regulated proteins upon bacterial infection. Notably, we found marked induction of host integrin signaling and glycolytic pathways. Intriguingly, up‐regulation of host glucose metabolism concurred with increased utilization of glycolysis by intracellular Salmonella during infection. In addition to immunoblotting assays, we also verified the up‐regulation of PARP1 in the host nucleus by selected reaction monitoring and immunofluorescence studies. Furthermore, we provide evidence that PARP1 elevation is likely specific to Salmonella infection and independent of one of the bacterial type III secretion systems. Our work demonstrates that unbiased high‐throughput proteomics can be used as a powerful approach to provide new perspectives on host–pathogen interactions.     

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.     

TLR9 limits enteric antimicrobial responses and promotes microbiota‐based colonisation resistance during Citrobacter rodentium infection

Mammalian cells express an array of toll‐like receptors to detect and respond to microbial pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). These clinically important attaching and effacing (A/E) pathogens infect the apical surface of intestinal epithelial cells, causing inflammation as well as severe diarrheal disease. Because EPEC and EHEC are human‐specific, the related murine pathogen Citrobacter rodentium has been widely used to define how hosts defend against A/E pathogens. This study explored the role of TLR9, a receptor that recognises unmethylated CpG dinucleotides present in bacterial DNA, in promoting host defence against C. rodentium. Infected Tlr9^?/? mice suffered exaggerated intestinal damage and carried significantly higher (10–100 fold) pathogen burdens in their intestinal tissues as compared with wild type (WT) mice. C. rodentium infection also induced increased antimicrobial responses, as well as hyperactivation of NF‐κB signalling in the intestines of Tlr9^?/? mice. These changes were associated with accelerated depletion of the intestinal microbiota in Tlr9^?/? mice as compared with WT mice. Notably, antibiotic‐based depletion of the gut microbiota in WT mice prior to infection increased their susceptibility to the levels seen in Tlr9^?/? mice. Our results therefore indicate that TLR9 signalling suppresses intestinal antimicrobial responses, thereby promoting microbiota‐mediated colonisation resistance against C. rodentium infection.     

A novel co‐infection model with Toxoplasma and Chlamydia trachomatis highlights the importance of host cell manipulation for nutrient scavenging

Toxoplasma and Chlamydia trachomatis are obligate intracellular pathogens that have evolved analogous strategies to replicate within mammalian cells. Both pathogens are known to extensively remodel the cytoskeleton, and to recruit endocytic and exocytic organelles to their respective vacuoles. However, how important these activities are for infectivity by either pathogen remains elusive. Here, we have developed a novel co‐infection system to gain insights into the developmental cycles of Toxoplasma and C. trachomatis by infecting human cells with both pathogens, and examining their respective ability to replicate and scavenge nutrients. We hypothesize that the common strategies used by Toxoplasma and Chlamydia to achieve development results in direct competition of the two pathogens for the same pool of nutrients. We show that a single human cell can harbour Chlamydia and Toxoplasma. In co‐infected cells, Toxoplasma is able to divert the content of host organelles, such as cholesterol. Consequently, the infectious cycle of Toxoplasma progresses unimpeded. In contrast, Chlamydia's ability to scavenge selected nutrients is diminished, and the bacterium shifts to a stress‐induced persistent growth. Parasite killing engenders an ordered return to normal chlamydial development. We demonstrate that C. trachomatisenters a stress‐induced persistence phenotype as a direct result from being barred from its normal nutrient supplies as addition of excess nutrients, e.g. amino acids, leads to substantial recovery of Chlamydia growth and infectivity. Co‐infection of C. trachomatis with slow growing strains of Toxoplasma or a mutant impaired in nutrient acquisition does not restrict chlamydial development. Conversely, Toxoplasma growth is halted in cells infected with the highly virulent Chlamydia psittaci. This study illustrates the key role that cellular remodelling plays in the exploitation of host intracellular resources by Toxoplasma and Chlamydia. It further highlights the delicate balance between success and failure of infection by intracellular pathogens in a co‐infection system at the cellular level.     

The novel chlamydial adhesin CPn0473 mediates the lipid raft‐dependent uptake of Chlamydia pneumoniae

Chlamydiae are Gram‐negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down‐stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473‐coupled fluorescent latex beads adhere to human epithelial HEp‐2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp‐2 cells with rCPn0473 does not attenuate adhesion but promotes dose‐dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473‐dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae.     

Modulation of Caenorhabditis elegans infection sensitivity by the LIN-7 cell junction protein

In Caenorhabditis elegans, the LIN‐2/7/10 protein complex regulates the activity of signalling proteins. We found that inhibiting lin‐7 function, and also lin‐2 and lin‐10, resulted in enhanced C. elegans survival after infection by Burkholderia spp., implicating a novel role for these genes in modulating infection outcomes. Genetic experiments suggested that this infection phenotype is likely caused by modulation of the DAF‐2 insulin/IGF‐1 signalling pathway. Supporting these observations, yeast two‐hybrid assays confirmed that the LIN‐2 PDZ domain can physically bind to the DAF‐2 C‐terminus. Loss of lin‐7 activity also altered DAF‐16 nuclear localization kinetics, indicating an additional contribution by hsf‐1. Unexpectedly, silencing lin‐7 in the hypodermis, but not the intestine, was protective against infection, implicating the hypodermis as a key tissue in this phenomenon. Finally, consistent with lin‐7 acting as a general host infection factor, lin‐7 mutants also exhibited enhanced survival upon infectionby two other Gram‐negative pathogens, Pseudomonas and Salmonella spp.     

Here,there, and everywhere: How pathogenic Escherichia coli sense and respond to gastrointestinal biogeography

Gastrointestinal (GI) pathogens enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC), and related mouse pathogen Citrobacter rodentium, are referred to as attaching and effacing (AE) pathogens for the lesions they form upon colonisation of the host epithelium. EPEC, EHEC, and C. rodentium are well known to use a type III secretion system to intimately attach to intestinal cells and secrete bacterial effectors to manipulate host cell processes. Less well known is the ability of AE pathogens to overcome significant physiological and microbial barriers and target specific gut niches for initial colonisation of the host epithelium. This review considers recent work highlighting the biogeography of the GI tract as it applies to colonisation by enteric pathogens, including environmental barriers to enteric infection, signals sensed by AE pathogens for navigation of the GI tract, and the tools AE pathogens use to respond to the changing host environment.     

Extrusions are phagocytosed and promote Chlamydia survival within macrophages

The precise strategies that intracellular pathogens use to exit host cells have a direct impact on their ability to disseminate within a host, transmit to new hosts, and engage or avoid immune responses. The obligate intracellular bacterium Chlamydia trachomatis exits the host cell by two distinct exit strategies, lysis and extrusion. The defining characteristics of extrusions, and advantages gained by Chlamydia within this unique double‐membrane structure, are not well understood. Here, we define extrusions as being largely devoid of host organelles, comprised mostly of Chlamydia elementary bodies, and containing phosphatidylserine on the outer surface of the extrusion membrane. Extrusions also served as transient, intracellular‐like niches for enhanced Chlamydia survival outside the host cell. In addition to enhanced extracellular survival, we report the key discovery that chlamydial extrusions are phagocytosed by primary bone marrow‐derived macrophages, after which they provide a protective microenvironment for Chlamydia. Extrusion‐derived Chlamydia staved off macrophage‐based killing and culminated in the release of infectious elementary bodies from the macrophage. Based on these findings, we propose a model in which C. trachomatis extrusions serve as “trojan horses” for bacteria, by exploiting macrophages as vehicles for dissemination, immune evasion, and potentially transmission.     

Manipulation of the host cell death pathway by Shigella

Host cells deploy multiple defences against microbial infection. One prominent host defence mechanism, the death of infected cells, plays a pivotal role in clearing damaged cells, eliminating pathogens, removing replicative niches, exposing intracellular bacterial pathogens to extracellular immune surveillance and presenting bacteria‐derived antigens to the adaptive immune system. Although cell death can occur under either physiological or pathophysiological conditions, it acts as an innate defence mechanism against bacterial pathogens by limiting their persistent colonization. However, many bacterial pathogens, including Shigella, have evolved mechanisms that manipulate host cell death for their own benefit.     

Type I interferon promotes cell‐to‐cell spread of Listeria monocytogenes

Type I interferons (IFNs) play a critical role in antiviral immune responses, but can be deleterious to the host during some bacterial infections. Listeria monocytogenes (Lm) induces a type I IFN response by activating cytosolic antiviral surveillance pathways. This is beneficial to the bacteria as mice lacking the type I IFN receptor (IFNAR1^?/?) are resistant to systemic infection by Lm. The mechanisms by which type I IFNs promote Lm infection are unclear. Here, we show that IFNAR1 is required for dissemination of Lm within infection foci in livers of infected mice and for efficient cell‐to‐cell spread in vitro in macrophages. IFNAR1 promotes ActA polarization and actin‐based motility in the cytosol of host cells. Our studies suggest type I IFNs directly impact the intracellular life cycle of Lm and provide new insight into the mechanisms used by bacterial pathogens to exploit the type I IFN response.     

Role of the ESCRT‐III complex in controlling integrity of the Salmonella‐containing vacuole

Intracellular pathogens need to establish specialised niches for survival and proliferation in host cells. The enteropathogen Salmonella enterica accomplishes this by extensive reorganisation of the host endosomal system deploying the SPI2‐encoded type III secretion system (SPI2‐T3SS). Fusion events of endosomal compartments with the Salmonella‐containing vacuole (SCV) form elaborate membrane networks within host cells enabling intracellular nutrition. However, which host compartments exactly are involved in this process and how the integrity of Salmonella‐modified membranes is accomplished are not fully resolved. An RNA interference knockdown screen of host factors involved in cellular logistics identified the ESCRT (endosomal sorting complex required for transport) system as important for proper formation and integrity of the SCV in infected epithelial cells. We demonstrate that subunits of the ESCRT‐III complex are specifically recruited to the SCV and membrane network. To investigate the role of ESCRT‐III for the intracellular lifestyle of Salmonella, a CHMP3 knockout cell line was generated. Infected CHMP3 knockout cells formed amorphous, bulky SCV. Salmonella within these amorphous SCV were in contact with host cell cytosol, and the attenuation of an SPI2‐T3SS‐deficient mutant strain was partially abrogated. ESCRT‐dependent endolysosomal repair mechanisms have recently been described for other intracellular pathogens, and we hypothesise that minor damages of the SCV during bacterial proliferation are repaired by the action of ESCRT‐III recruitment in Salmonella‐infected host cells.