Quantitative Single-Cell Analysis of Host-Pathogen Interactions

During infection, microbial pathogens encounter phagocytic cells of the host innate immune system, such as macrophages and neutrophils. These encounters typically lead to uptake and killing of the bacteria by the host cell or, conversely, parasitization of the host cell by the bacteria. Microscopic analysis of these host-microbe interactions is complicated by the fact that phagocytic cells are motile and tend to stray out of the field of view. Most host-pathogen studies have therefore been undertaken at the population level. However, the success of many pathogens likely lies in their ability to generate heterogeneity within the population, facilitating adaptation to every host micro-environment and to environmental perturbations (such as application of an antibiotic). We believe that studying host-pathogen interactions at the single-cell level can reveal not only new mechanisms of pathogenesis but also why current treatments are not always successful. This thesis presents a novel microfluidic device for the long-term co-culturing of phagocytic cells and bacteria within spatially confining microchambers. Each device is patterned with thousands of microchambers, permitting high-throughput fluorescence timelapse microscopy of host-microbe interactions at single-cell resolution. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae. We observed that K. pneumoniae is readily taken up and killed by D. discoideum but some events can take ten times as long as others. The capsule, a polysaccharide layer present at the surface at the bacterial cell wall, reduces the phagocytosis efficiency by 1.5 fold, but does not influence the killing time. The opportunistic human pathogen Mycobacterium marinum was then used to study intracellular bacterial virulence. As expected, bacteria could infect host cells, replicate inside, and eventually lyse them. However, surprisingly, in the majority of the infections, the bacteria were actually released from the host, with no apparent harm to either the host or the bacteria. Less frequently, bacteria were even seen being killed. The timing of these events was highly heterogeneous, ranging from a couple of hours to several days. In summary, the tools presented in this thesis provide a new and simple approach for following individual host-microbe interactions at high spatiotemporal resolution from the onset to the end of infections. This work unveiled that the timing and outcome of host-microbe interactions are unexpectedly heterogeneous, which may have important consequences for the bacterial pathogenesis and ultimately for the development of future treatment of microbial infections.

A microfluidic cell-trapping device for single-cell tracking of host-microbe interactions

Jean-Baptiste Bureau, Matthieu Jean-Hubert Delincé, John McKinney, Thierry Soldati

The impact of cellular individuality on host-microbe interactions is increasingly appreciated but studying the temporal dynamics of single-cell behavior in this context remains technically challenging. Here we present a microfluidic platform, InfectChip, to trap motile infected cells for high-resolution time-lapse microscopy. This approach allows the direct visualization of all stages of infection, from bacterial uptake to death of the bacterium or host cell, over extended periods of time. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae or the intracellular pathogen Mycobacterium marinum. We show that the outcome of such infections is surprisingly heterogeneous, ranging from abortive infection to death of the bacterium or host cell. InfectChip thus provides a simple method to dissect the time-course of host-microbe interactions at the single-cell level, yielding new insights that could not be gleaned from conventional population-based measurements.

Royal Society of Chemistry2016

The role of immune effectors in controlling Drosophila-microbes interactions

Alice Marra

All live beings are in constant interaction with microorganisms that may be beneficial, deleterious or commensal. Insects in particular live in close contact with microorganisms. This is especially true for species, like the fruit fly Drosophila melanogaster that feed, lay eggs and develop on or close to decomposing organic matter. In contrast to vertebrates, insects did not evolve an adaptive immune system to combat pathogens selectively. They instead rely on surprisingly efficient innate defense mechanisms for the control and clearance of all microbes without any species-specific targeting. Innate immunity encompasses a wide range of mechanisms that rely on direct pathogen recognition and elimination. In addition, metabolic and behavioral responses also strongly affect the outcome of insect interactions with both pathogenic and non-pathogenic bacteria. Although the Drosophila immune system has been extensively described, little is known about the role of immune effectors in tolerating and controlling symbiotic microbes. For this reason, during my PhD studies, I investigated how Drosophila melanogaster immune effectors differentially interact with mutualistic symbionts. First, I investigated the role of some of the host antimicrobials, called antimicrobial peptides and lysozymes, in maintaining the homeostasis of the gut microbiota. I found that both antimicrobial peptides and lysozymes can actively regulate the gut microbiota composition and abundancy, especially during aging. In a second part of my thesis, I got interested in Spiroplasma, a heritable symbiotic bacterium that lives within the fly hemolymph. I characterized the role of the Drosophila iron transporter Transferrin 1 (Tsf1) during Spiroplasma-Drosophila symbiosis. I first showed that mutant flies for tsf1 have an impaired Spiroplasma load, due to iron relocation from the hemolymph to the fat body, where it becomes inaccessible for Spiroplasma. Furthermore, I demonstrated that Spiroplasma scavenges host iron only when it is bound to the protein, which points to Tsf1 and iron transport as a control mechanism for hemolymphatic symbionts. Collectively, my studies contribute to a better understanding of how the innate immune effectors interact with Drosophila microbial symbionts to both regulate and maintain stable, long-lasting, interactions.

EPFL2021

The contribution of the host stress response to the pathogenesis of Pseudomonas entomophila in the Drosophila intestine

Sveta Chakrabarti

Recently, several studies done in Drosophila have revealed that efficient and rapid recovery from bacterial infection in the gut is possible only when bacterial clearance by the immune system is coordinated with repair through renewal of the epithelium damaged by infection. In this thesis, I have analyzed how the entomopathogenic bacterium Pseudomonas entomophila affects the immune response and epithelium renewal. A microarray analysis first showed that P. entomophila is recognized by the Drosophila immune system since ingestion of this bacterium stimulated the expression of antimicrobial peptide genes by the Imd pathway. In addition, stress and damage related pathways were strongly induced by P. entomophila, which correlate with its capacity to inflict severe damage. While antibacterial genes were induced in the gut following infection with P. entomophila, the immune response was not productive due to a general inhibition of translation that affected all the newly synthesized transcripts. Furthermore, the blockage of translation also inhibited the repair program by blocking the production of JAK-STAT ligands (upd3, upd2) and growth factors, which stimulate the intestinal stem cells proliferation and differentiation. I next analyzed the pathways that link cellular damage to reduction of translation. Using a genetic approach, I showed that inhibition of translation was induced by two signaling pathways: i) the phosphorylation of elongation initiation factor 2α (eIF2α) by the stress kinase GCN2 and ii) the inhibition of the TOR pathway by the AMPK kinase. Both kinases sense metabolic deprivation, suggesting that cellular damages induced by P. entomophila induce a state of “starvation”. Inhibition of translation is usually an adaptive cellular response to adjust the metabolism to the energy status of the cells. The observation that GCN2-deficient flies survived better than wild-type flies to P. entomophila indicates that pathogenesis is linked to an over-activation of stress pathways that usually help to endure the consequence of an infection. In this in vivo model of infection, I also showed that the reduction of translation was a consequence of cellular damage to the intestine caused by host-derived reactive oxygen species (through the activity of Duox) and by the direct action of a pore-forming toxin produced by the pathogen. As a consequence of this translational arrest, flies succumbed P. entomophila infection because they were unable to repair gut damage. Finally, I showed that inhibition of translation also had a strong influence on innate immune responses observed upon P. entomophila infection. The specific activation of a systemic immune response (antimicrobial peptides produced by the fat body) observed upon P. entomophila infection could be recapitulated by feeding flies with a non-lethal pathogen and an inhibitor of translation. Hence, I showed translation inhibition could be an important feature that shapes the immune response. The p38 MAPK family is involved in stress and immunity in both mammals and Drosophila. In Drosophila, three p38-MAPK-encoding genes, p38a, p38b and p38c, have been identified but their function in the gut immune response is poorly characterized. In the second part of my thesis, I have analyzed the role of these three p38 MAPKs in Drosophila intestinal immunity, especially p38c, which is strongly enriched in the gut. I first confirmed that the three p38 are involved kinases are involved in the defense to oral infection with pathogenic (but non-lethal) bacteria such as Erwinia carotovora 15. Surprisingly, p38c mutant flies were more resistant to P. entomophila and did not show the reduction in translation observed in wild-type flies. Interestingly, the transcription of the ROS producing enzyme Duox was reduced in p38c raising the hypothesis that p38 contributes to P. entomophila pathogenicity by activating Duox. Furthermore, I have obtained preliminary data indicating that p38c and the transcription factor ATF-3 act in the same pathway contributing the host immune response and lipid metabolism. Together, my thesis reveals the complex cross-talks between stress and immune pathways during microbial infection. While, it shows that stress pathways usually contribute to endure the damage caused by infection, excessive activation of these pathways can contribute to pathogenesis.