Dissecting host-microbe interactions with modern functional genomics

Abstract

Interrogation of host-microbe interactions has long been a source of both basic discoveries and benefits to human health. Here, we review the role that functional genomics approaches have played in such efforts, with an emphasis on recent examples that have harnessed technological advances to provide mechanistic insight at increased scale and resolution. Finally, we discuss how concurrent innovations in model systems and genetic tools have afforded opportunities to interrogate additional types of host-microbe relationships, such as those in the mammalian gut. Bringing these innovations together promises many exciting discoveries ahead.

Figure 1:. Functional genomics approaches for identifying host factors that impact pathogen infection.

A) Overview of canonical CRISPR screens. Populations of cells are transduced individually (arrayed) or in a pool with lentivirus containing sgRNAs. The cells are then exposed to a pathogen. For arrayed screens, host cell viability is measured for each population to evaluate the impact of the genetic perturbation on pathogen susceptibility. For pooled screens, sgRNA abundances are quantified before and after infection to evaluate the impact of all genetic perturbations on pathogen susceptibility. B) Using pathogen-encoded genetic perturbations to identify host factors that modify mid- to late- stage infection. Unlike classical screens in which the host cell encodes both the (d)Cas9 and the sgRNA, encoding (d)Cas9 in the pathogen necessitates successful pathogen entry prior to genetic perturbation. This screening strategy may enrich for host factors important for late-stage infection, such as a host factor that blocks viral egress. C) Using high-content readouts to identify host factors that affect infection progression. Unlike classical screens in which host cell survival of infection is the primary readout, high-content readouts provide multiple metrics to evaluate screen outcomes and identify hits that affect specific aspects of infection. This screen strategy can reveal subtle phenotypes that are not captured in screens with survival or cell death as a readout. Possible high-content readouts include microscopy (shown in the figure) as well as measurements of transcriptomes, protein levels, epigenomes, and others.

Figure 2:. Functional genomics approaches for identifying microbial factors that impact host infection.

A) Overview of screening strategies available for studying microbial factors important for host infection. Depending on the pathogen of interest, genetic perturbation may be possible via saturating transposon mutagenesis or via CRISPR technologies. These tools generate libraries of mutant pathogens which can then be applied to various infection models. Pathogens that successfully infect the model of interest are sequenced to identify their genetic perturbation. Comparing the representation of mutants in the original library to the library that succeeds in infection can reveal relevant genes. B) Using CRISPRi-based hypomorph screening to identify vulnerable genes. A sgRNA library is designed with several guides targeting each gene with variable knockdown strength. The predicted expression level of the target gene is compared to the effect on pathogen fitness to identify genes for which even mild knockdown leads to a significant reduction in fitness. These “vulnerable” genes are likely high priority targets for antimicrobials. C) Using inducible genetic perturbations to overcome in vivo colonization bottlenecks. During conventional Tn-seq screens, a mutant library is generated prior to infection and a colonization bottleneck may select mutants at random rather than as a function of fitness. Using an inducible-transposon system allows for generation of the mutant library once the infection is established, allowing for accurate quantification of fitness of individual mutants.

Figure 3:. Functional genomics approaches for identifying factors in host-symbiont interactions.

A) In systems in which a single bacterial species dominates a particular host niche, such as the squid light organ, a library of bacterial mutants can be introduced and evaluated for successful colonization. B) In systems in which diverse microbial species colonize a host, such as the mammalian gut, gnotobiotic models comprising minimal microbial communities provide a setting for functional genomics screens. Here, mutant libraries of one or more of the microbial members can be introduced along with unperturbed species to identify fitness determinants in individual species and to evaluate whether different species (or strains) have different fitness determinants. C) In systems that are stable over time, such as the commensal microbiome, genetic screens can identify colonization determinants that vary over time. Here, one or more genetically perturbed microbes are introduced, and survival of mutants is measured over time. This can lead to important insights into the dynamic adaptation of microbes in a complex environment, such as the mouse gut.

Figure 4:. Opportunities afforded by recent innovations.

Advances in technologies that may expand the use of functional genomics to dissect host-microbe interactions. In situ editing of cells in a host organ is now possible via delivery of host-targeting sgRNA libraries. Additionally, examples of in situ editing of gut microbes via phage or conjugation systems may be expanded to delivering genetic perturbations like mobile-CRISPR to complex model microbial communities. In parallel to in situ editing, high-content readouts like scRNA-seq and MERFISH may capture comprehensive phenotypes.