Abstract
Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enabled the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms enabled E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof-of-concept for the extension to other phage-bacterial pairs of interest.
Current strategies for manipulating the microbiome either lack species- or strain-level precision1, 2 or require the introduction of an exogenous bacterium into the host3, 4. Pioneering studies of pathogenic bacteria that colonize the skin5–7 and gut8 support the potential for the use of engineered bacteriophage carrying an exogenous CRISPR-Cas system that could be directed to any target of interest; however, these methods have yet to be applied to the human or mouse microbiome. Given the tremendous diversity within both the bacterial9 and viral10 components of the human gut microbiota, we sought to establish a tripartite model system that builds upon tools for the genetic manipulation of a bacteriophage and its bacterial target coupled to an experimentally tractable mammalian host.
We focused on M13, a single-stranded DNA (ssDNA) filamentous inovirus11, 12 able to replicate and release virions without causing cell lysis13. M13 infects E. coli and related Enterobacteriaceae carrying the F sex factor necessary to form the conjugative F pilus14, 15. M13 phagemid vectors combine the advantages of plasmid DNA manipulation with the ability to easily package recombinant DNA into virions16. M13 has been used to target E. coli17, 18 and Helicobacter pylori19, including engineered M13 carrying CRISPR-Cas9 in an insect model of bacterial infection5. However, the use of M13 to deliver genetic constructs (including CRISPR-Cas systems) to cells within the mammalian gastrointestinal tract had not been previously demonstrated. We also leveraged the streptomycin (Sm)-treated mouse model for E. coli colonization within the mouse gut microbiota20, providing a robust and accessible model that could be used by any group with access to a mouse colony.
Results
Bacteriophage M13 enables the delivery of DNA to the gut microbiome
We utilized phagemid pBluescript II21 carrying the bla (β-lactamase) gene and a β-lactam antibiotic in the drinking water to select for successfully infected E. coli. pBluescript II conferred in vitro resistance to ampicillin and the semi-synthetic analogue carbenicillin at concentrations exceeding 1 mg/ml (Supplementary Fig. 1). We used streptomycin-resistant (SmR) E. coli to colonize the GI tract of Sm-treated mice. As expected, streptomycin altered gut microbial community structure while decreasing diversity and colonization level (Supplementary Figs. 2a-d). SmR E. coli colonized at a high proportion (median 18% of the gut microbiota; range 1.4-43%) four days after gavage (Supplementary Fig. 2e). We introduced a SmR E. coli population that was a mixture of 99.9% AmpS (no plasmid) and 0.1% AmpR cells (pBluescript II), split the mice into two groups with access to water containing only streptomycin or both streptomycin and ampicillin, and tracked both total E. coli and AmpR E. coli in mouse feces. At 6 h post-E. coli introduction, the percentage of AmpR E. coli in the feces of all mice was at or close to 0.1%, consistent with the gavaged mixture transiting through the GI tract. Within 1-2 days, mice on water containing ampicillin exhibited an increase in the percent of AmpR E. coli by 3 orders of magnitude, reaching complete or near complete colonization (Fig. 1a). In contrast, the AmpR subpopulation was lost in mice on water without ampicillin. These results demonstrate that β-lactam antibiotics can be used to select for resistant E. coli.
Antibiotics were capable of eradicating a sensitive population of E. coli that had established stable colonization in the mouse gut. We colonized Sm-treated mice with SmR E. coli MG1655 or W1655 F+ and tracked colonization levels during treatment with the β-lactam antibiotic carbenicillin. Carbenicillin decreased the median E. coli colonization level from 9.6 × 109 to 2.0 × 103 CFU/gram feces in the first day, and levels decreased to below our limit of detection (∼102 CFU/g) in all mice over the course of treatment (Fig. 1b). When selection was lifted on Day 7, recolonization was observed for 5/6 mice. When carbenicillin was reintroduced on Day 13, colonization again dropped below our limit of detection. The low background of E. coli in the gut during carbenicillin treatment, as well as the lack of spontaneous resistant cells able to expand, supports the utility of this model for assessing the phage-mediated delivery of a resistance gene.
Next, we tested our ability to deliver an antibiotic resistance gene to E. coli within the gut. We colonized Sm-treated mice with either SmR E. coli W1655 F+ (M13S) or W1655 F− (M13R as a control), and dosed each animal with either live or heat-inactivated M13 carrying pBluescript II (Fig. 1c). After dosing the mice with 1×1014 M13(pBluescript II), we immediately transferred them to water containing carbenicillin and tracked both total E. coli and CarbR E. coli in the feces. E. coli colonization fell rapidly and stayed near or below the limit of detection in control mice that were either colonized with F− and given live phage or colonized with F+ but given heat-inactivated phage. In contrast, mice colonized with F+ and dosed with live phage had a transient drop in colonization on the first day, during which the rise of CarbR cells occurred, and colonization was re-established within one day by an E. coli population resistant to carbenicillin (Fig. 1c). These results suggest that orally dosed M13 phage were able to infect E. coli in the gut and deliver a plasmid conferring resistance to carbenicillin.
We replicated M13-mediated pBluescript II delivery to E. coli in the gut in an independent animal experiment. Sm-treated mice were colonized with SmR E. coli W1655 F+ and orally dosed with ten-fold serial dilutions of M13(pBluescript II). Colonization by CarbR E. coli was consistent at high doses but variable at lower doses representing a significantly higher probability of successful colonization with increasing phage dose (Fig. 1d; P=0.009, odds ratio=2.5, logistic regression). Plasmid DNA of the expected size was detected in fecal CarbR E. coli isolates from all 11 mice that were successfully colonized (Supplementary Fig. 3a). Genome sequencing confirmed the presence of pBluescript II in these 11 isolates, which was undetectable in the parent strain (Supplementary Fig. 3b). These results indicate that plasmid DNA was transferred from M13 phage into recipient E. coli colonizing the GI tract.
Finally, we repeated this experiment in the absence of carbenicillin selection. We colonized mice with SmR E. coli, gavaged each mouse with M13(pBluescript II), and tracked both infected (CarbR) and total (SmR) E. coli in feces. The fraction of phage-infected CarbR E. coli was low, reaching a maximum of 0.1% of the total population (Supplementary Fig. 4), potentially indicative of poor phage survival during GI transit. We gavaged mice with M13(pBluescript II) and assayed for viable phage in the feces. The median output of viable M13(pBluescript II) was reduced to 1×106 relative to an input of 6×1013 (Supplementary Figs. 5a,b). M13(pBluescript II) is acid tolerant in vitro (Supplementary Fig. 5c), suggesting that additional factors may be responsible for the low in vivo viability and emphasizing the benefits of pairing gene delivery with antibiotic selection.
M13 carrying CRISPR-Cas9 can target E. coli in vitro
We generated two fluorescently marked isogenic derivatives of SmR W1655 F+ using the mcherry (red fluorescence) or the sfgfp (green fluorescence) marker gene. Next, we constructed M13-compatible non-targeting (NT) and GFP-targeting (GFPT) CRISPR-Cas9 vectors by cloning the spacers sequences, bla gene, and f1 origin of replication into the previously described low-copy vector pCas922, generating pCas9-NT-f1A/B and pCas9-GFPT-f1A/B (Supplementary Fig. 6). The bla and f1 ori were cloned as a fragment from pBluescript II in both possible orientations (A or B) to make possible M13 ssDNA packaging of either strand of vector DNA. We packaged these phagemids into M13 using a helper strain and called the resulting phage NT-M13 or GFPT-M13. The two phage were used to infect the GFP+ or mCherry+ strains and cells were diluted and spotted on solid media containing carbenicillin to select for the transferred phagemid. GFP+ E. coli infected with GFPT-M13 exhibited impaired colony growth relative to the NT-M13 control (Fig. 2a and Supplementary Fig. 7). Total CFUs were not markedly affected, indicating that cells can recover from M13-delivered CRISPR-Cas9 targeting.
Analysis of the surviving cells provided mechanistic insights. Colonies arising from infection with NT-M13 or GFPT-M13 were streak purified, allowing us to pick a mixture of bright and dim colonies. Of 16 GFPT clones analyzed, 11 were non-fluorescent (Fig. 2b). PCR amplification of sfgfp confirmed the intact gene in 4 NT controls and all 5 GFPT clones that retained fluorescence (Fig. 2c). Sanger sequencing revealed that 1 GFPT clone had a point mutation in the sfgfp target (Supplementary Fig. 8) while the 4 others had lost the spacer in the CRISPR-Cas9 phagemid that leads to targeting (Supplementary Fig. 9). All 11 non-fluorescent GFPT clones retained the spacer (Supplementary Fig. 9) and had chromosomal deletions at the target locus: 10 were PCR negative (Fig. 2c) and 1 had a small deletion within sfgfp (Supplementary Fig. 8). Finally, we used whole genome sequencing to define the size of each deletion, which ranged from 45 bp to 82.6 kb (Fig. 2d), consistent with prior work demonstrating that E. coli can repair Cas9-induced double-stranded breaks through homologous recombination23.
These results led us to hypothesize that targeted cells would be less able to recover during competitive growth. We co-cultured GFP+ and mCherry+ E. coli, adding either NT-M13 or GFPT-M13 followed by carbenicillin to select for phage infection. GFPT-M13 decreased the frequency of GFP+ colonies by 4 h, relative to the NT-M13 control (Fig. 3a). At later timepoints (16-24 h), healthy GFP+ colonies increased in abundance, consistent with low levels of carbenicillin after 4 h in cultures expressing the β-lactamase resistance gene (Fig. 3b). We confirmed the loss of selection for the phagemid by re-analyzing our colonies on selective media (Supplementary Fig. 10). Next, we used flow cytometry to better quantify the two strains in an independent experiment. Compared to the NT-M13 control, GFPT-M13 co-cultures exhibited fewer GFP+ events (Fig. 3c) and a bimodal distribution of fluorescence (Fig. 3d). Counts of GFP+ cells were higher by flow cytometry than on solid media for the same co-cultures (Fig. 3c inset), consistent with an impaired growth of these cells. GFP+ events further decreased at 24 h in the GFPT-M13 group (Supplementary Fig. 11). Taken together, these results suggest that competitive growth can increase the efficiency of targeting a strain for depletion due to the resulting growth impairments in the targeted strain.
Sequence-specific depletion of E. coli within the mouse gut microbiota
We co-colonized Sm-treated mice with both SmR F+ sfgfp and SmR F+ mcherry strains, orally dosed them with either 1011 NT-M13 or GFPT-M13, and added carbenicillin in the water to select for phage infection. After one week of treatment, carbenicillin was removed from the water and mice were followed for an additional week to determine whether phage-induced changes would persist in the absence of maintaining selection (Fig. 4a). Flow cytometry on mouse stool samples revealed that the GFP+ strain outcompeted the mCherry+ strain in the NT-M13 group (Figs. 4b,c and Supplementary Figs. 12,13). In contrast, GFP+ events in the GFPT-M13 group exhibited a sharp decrease on Day 2, followed by a recovery on Days 7 and 14 to levels below the NT-M13 group (Figs. 4b,c). Culturing from mouse stool confirmed the decreased GFP+ events on Day 2 (Supplementary Fig. 14). In 4 mice that received GFPT-M13, the mCherry+ strain fixed in the population (GFP+ events were below background), an outcome that was not observed for any mouse in the NT-M13 group (Fig. 4d). Despite lifting the carbenicillin selection for 1 week, endpoint GFP+ events remained significantly lower in the GFPT-M13 group relative to NT-M13 controls (Fig. 4e; P=0.0002, Mann-Whitney test). These data support the utility of M13-delivered CRISPR-Cas9 for sequence-specific depletion of an otherwise isogenic bacterial strain in the mouse gut.
M13-delivered CRISPR-Cas9 induces chromosomal deletions in the gut microbiome
We constructed a double-marked SmR F+ sfgfp mcherry strain to quantify the efficiency of gene deletion. We introduced this strain into Sm-treated mice, orally dosed each mouse with either 1011 NT-M13 or GFPT-M13, and added carbenicillin in the water. After one week, we removed carbenicillin and followed the mice for a final week (Fig. 5a). GFP-mCherry+ events were detectable in GFPT-M13 but not NT-M13 mice, indicative of successful CRISPR-Cas9 delivery and gene deletion (Fig. 5b and Supplementary Fig. 15). By the final timepoint, GFP-mCherry+ events were detected in 3/8 mice (Fig. 5c and Supplementary Fig. 16a). The relative abundance of GFP-mCherry+ cells varied from 12-96% (Fig. 5c). Culturing on solid media confirmed the presence of viable red fluorescent colonies in proportions consistent with flow cytometry results (Fig. 5d and Supplementary Fig. 16b).
To more definitively assess the presence or absence of the targeted genomic locus and the CRISPR-Cas system, we isolated GFP-mCherry+ and GFP+ mCherry+ E. coli from Day 2 mouse stool. All of the GFP+ mCherry+ isolates from the NT-M13 group and the GFP-mCherry+ isolates from the GFPT-M13 group had an intact spacer sequence (Supplementary Fig. 17a). In contrast, 4/5 GFP+ mCherry+ isolates from the GFPT-M13 group had lost the spacer (Supplementary Fig. 17a). Of note, the remaining isolate lost cas9, and parts of the CRISPR array and tracrRNA (Supplementary Figs. 17b,c). Whole genome sequencing was used to confirm putative chromosomal deletions and to quantify their size. Two representative colonies were analyzed from each of the 3 mice with detectable GFP-mCherry+ cells, revealing a wide range in deletion sizes that were not observed in a control GFP+ mCherry+ isolate from each animal (Fig. 5e). These results indicate that while it is possible for CRISPR-Cas9-induced genomic deletion events to occur in vivo, resultant deletion strains may or may not outcompete the parent strain due to the potential to evade targeting through loss of some or all of the exogenous CRISPR-Cas system.
Discussion
Our results emphasize that foundational, reductionist, and highly controlled studies will be necessary to assess the feasibility, utility, and limitations of phage-based gene delivery as a tool for microbiome editing. While our results provide a proof-of-principle for strain-specific targeting within the GI tract, the full eradication of the targeted strain was difficult to achieve due to the ability of bacterial cells to survive Cas9-induced double-stranded breaks by homologous recombination24. We propose that CRISPR-Cas9 may be better suited to induce targeted genomic deletions, leveraging the conserved DNA repair pathways present in bacteria. An advantage of this approach is that the deletion of a single genomic locus is unlikely to have as large an impact on the rest of the gut microbiota than if the strain were to be removed entirely. Remarkably, we detected a wide range of deletion sizes (379–68,321 bp), highlighting the ability of bacteria to survive large deletions and opening up the potential for the in vivo removal of entire biosynthetic gene clusters or pathogenicity islands. In turn, our data suggests that it may also be feasible to deliver more complex genetic circuits to E. coli, with the goal of boosting metabolic pathways beneficial to its mammalian host. Furthermore, the size of resulting chromosomal deletions could be controlled by providing a DNA repair template alongside CRISPR-Cas922.
There are several limitations of our current approach that could be refined through iterative “design-build-test” cycles. Perhaps most importantly, we identified multiple mechanisms through which cells can escape CRISPR-Cas9 targeting during in vitro growth and/or within the GI tract, including the loss of the spacer in the CRISPR array, target site mutations, and even one case in which the entire CRISPR-Cas9 system was deleted. Spacer loss could be reduced through the use of single guide RNAs (sgRNAs)25 or through the mutation of the repeat sequences that flank each spacer26. Target site mutation could be countered by targeting multiple sites simultaneously and by prioritizing conserved regions of the target genes essential for activity.
Another important caveat is that antibiotics were used to select for successful gene delivery, which is not ideal due to potential disruptions to the gut microbiota1 and/or selection for antibiotic resistance. Removing this selection resulted in a low penetrance of cells carrying the delivered cargo, similar to that observed for delivery to bacteria in a soil community in the absence of selection27. Low rates of gene delivery are likely driven by the observed loss of viable M13 bacteriophage during transit through the GI tract. We confirmed that M13 is acid tolerant; however, it is sensitive to artificial gastric juice28, suggesting that gastric proteases may be responsible for low phage survival. The aggregative behaviour of filamentous phage in response to microbial and/or host polymers29 may also be important to consider. Encapsulating phage for oral transit may be able to circumvent these limitations or alternatively, it may be possible to use non-antibiotic selection strategies such as dietary supplementation of an exclusive substrate3 that can only be used by phage-infected strains.
In conclusion, this work provides a valuable step towards establishing a modular toolkit for microbiome editing. The extension of these approaches to enable the genetic manipulation of a more diverse panel of bacteria found within the human microbiota will require a renewed effort to isolate and characterize bacteriophage that target strains of interest30. Robust in vitro methods to study and genetically modify novel bacteriophage are also needed, given that most of their host bacteria remain genetically intractable31. Finally, our work emphasizes the value of model systems for understanding the rules of engagement, complementing ongoing efforts to advance candidate phage-based therapies into the clinic.
Methods
Strains, plasmids, phage, and oligonucleotides
Bacterial strains, plasmids, and phage used in this study, including descriptions and sources, are provided in Supplementary Table 1. Oligonucleotides used in this study are provided in Supplementary Table 2.
Minimum inhibitory concentration (MIC) assay
Cells were prepared by standardizing an overnight culture to an OD600 of 0.1 using saline (0.85% NaCl), and further diluted ten-fold in saline then ten-fold in LB. The drug was prepared by dissolving the antibiotic in vehicle (sterile distilled water) and filter-sterilizing, then serially diluting two-fold in vehicle to prepare 100× stock solutions, and finally diluting ten-fold in LB for 10× stock. To wells of a 96-well plate, 60 µl of LB, 15 µl of drug, and 75 µl of cells were added and mixed well. Final drug concentrations ranged between 0.002 µg/ml to 1000 µg/ml for ampicillin and 0.24 µg/ml to 2000 µg/ml for carbenicillin. The plate was incubated overnight at 37°C without shaking and OD600 was measured the following morning after agitation.
16S rRNA gene sequencing
Mouse fecal pellets were stored at −80°C. DNA was extracted from single pellets using a ZymoBIOMICS 96 MagBead DNA Kit and 16S rRNA gene sequencing was performed using a dual indexing strategy32. Briefly, a 22-cycle primary PCR was performed using KAPA HiFi Hot Start DNA polymerase (KAPA KK2502) and V4 515F/806R Nextera primers. The reaction was diluted in UltraPure DNase/RNase-free water (Life Tech 0977-023) and used as template for a 10-cycle secondary (indexing) PCR using sample-specific dual indexing primers. The reactions were normalized using a SequelPrep Normalization plate (Life Tech A10510-01) and the DNA was eluted and pooled. To purify and concentrate the DNA, 5 volumes of PB Buffer (Qiagen 28004) were added, mixed, and purified using a QIAquick PCR Purification Kit (Qiagen 28106). The DNA was gel extracted using a MinElute Gel Extraction Kit (Qiagen 28604), quantified by qPCR using a KAPA Library Quantification Kit for Illumina Platforms (KAPA KK4824), and paired-end sequenced on the Illumina MiSeq platform. Data were processed using a 16S rRNA gene analysis pipeline (https://github.com/jbisanz/AmpliconSeq) based on QIIME233 incorporating DADA234, and analyzed using R packages qiime2R (v0.99.23; https://github.com/jbisanz/qiime2R), phyloseq (v1.33.0)35, and phylosmith (v1.0.4)36. See Data Availability for more information.
Construction of streptomycin-resistant E. coli strains
Strains resistant to the antibiotic streptomycin were generated by either selection for spontaneous resistance or by lambda Red recombineering37, 38. Spontaneous resistant mutants were selected by plating overnight cultures on LB supplemented with 500 µg/ml streptomycin. Lambda Red recombineering was later used to introduce a specific allele for genetic consistency between strains as different mutations in the rpsL gene can confer resistance to streptomycin39. Briefly, cells were transformed with the CarbR temperature-sensitive plasmid pSIJ838, and electrocompetent cells were prepared from cells grown in LB carbenicillin at 30°C to early exponential phase and lambda Red recombinase genes were induced by addition of L-arabinose to 7.5 mM. Cells were electroporated with an rpsL-SmR PCR product amplified from a spontaneous streptomycin-resistant mutant of MG1655 using primers PS-rpsL1 and PS-rpsL2, and recombinants were selected on LB supplemented with 500 µg/ml streptomycin. The pSIJ8 plasmid was cured by culturing in liquid at 37°C in the absence of carbenicillin, plating for single colonies, and confirming CarbS. The rpsL gene of SmR strains was confirmed by Sanger sequencing.
Construction of fluorescently marked E. coli strains
P1 lysates were generated of AV01::pAV01 and AV01::pAV02 carrying clonetegrated sfgfp and mcherry, respectively40. Briefly, 150 µl of overnight culture in LB supplemented with 12.5 µg/ml kanamycin was mixed with 1 µl to 25 µl P1 phage (initially propagated from ATCC on MG1655). The mixture was incubated for 10 min at 30°C to aid adsorption, added to 4 ml LB 0.7% agar, and overlaid on pre-warmed LB agar supplemented with 25 µg/ml kanamycin 10 mM MgSO4. Plates were incubated overnight at 30°C, and phage were harvested by adding 5 ml SM buffer, incubating at room temperature for 10 min, and breaking and scraping off the top agar into a conical tube. Phage suspensions were centrifuged to pellet agar; the supernatant was passed through a 100 µm cell strainer, then through a 0.45 µm syringe filter, and lysates were stored at 4°C. For transduction, 1-2 ml of recipient overnight culture was pelleted and resuspended in 1/3 volume LB 10 mM MgSO4 5 mM CaCl2. 100 µl of cells was mixed with 1 µl to 10 µl P1 lysate and incubated at 30°C for 60 minutes. To minimize secondary infections, 200 µl 1 M sodium citrate was added, followed by 1 ml of LB. The mixture was incubated at 30°C for 2 h, then plated on LB 10 mM sodium citrate 25 µg/ml kanamycin to select for transductants. For excision of the vector backbone including the kanamycin resistance gene and heat-inducible integrase, cells were electroporated with pE-FLP41; transformants were selected on carbenicillin and confirmed for KmS. pE-FLP was cured by culturing in liquid at 37°C in the absence of carbenicillin, plating for single colonies, and confirming CarbS. Strains were subsequently grown routinely at 37°C. For imaging fluorescent strains on agar, plates were typically incubated at 37°C overnight, transferred to room temperature to allow fluorescence intensity to increase, and then imaged.
Mouse experiments with E. coli colonization, antibiotic water, and phage treatment
Animal procedures were approved by the University of California, San Francisco (UCSF) Institutional Animal Care and Use Committee (IACUC), and animal experiments performed were in compliance with ethical regulations. Specific pathogen free female BALB/c mice from the vendor Taconic were used for all mouse experiments. Streptomycin water was prepared by dissolving USP grade streptomycin sulfate (VWR 0382) in autoclaved tap water to a final concentration of 5 mg/ml and passing through 0.45 µm filtration units. Mice were provided streptomycin water for 1 day, followed by oral gavage of 0.2 ml containing approximately 109 CFU of streptomycin-resistant E. coli. Mice were kept on streptomycin water thereafter to maintain colonization. For selection with β-lactam antibiotics, USP grade ampicillin sodium salt (Teknova A9510) or USP grade carbenicillin disodium salt (Teknova C2110) was also dissolved in the water to a final concentration of 1 mg/ml; carbenicillin was preferred for its increased stability over ampicillin42. Drinking water containing streptomycin was prepared fresh weekly; with the addition of a β-lactam antibiotic, it was prepared fresh every 3-4 days. For phage treatment, filtered phage solutions stored at −80°C were thawed and used directly for oral gavage. Unfiltered phage solutions were precipitated by diluting approximately 5-fold in PBS, adding 0.2 volumes phage precipitation solution (20% PEG-8000, 2.5 M NaCl), incubating for 15 min on ice, pelleting at 15,000–21,000g for 15 min at 4°C, resuspending in PBS, centrifuging to pellet insoluble matter, and filtering through 0.45 µm. Heat-inactivated phage were prepared by incubating 1 ml aliquots at 95°C in a water bath for 30 min. Streptomycin-treated mice colonized with SmR E. coli were orally gavaged with 0.2 ml of phage and placed on drinking water containing both streptomycin and carbenicillin.
Enumeration and culture of E. coli from mouse feces
Fecal pellets were collected from individual mice and CFU counts were performed on the same day to determine CFU per gram feces. Briefly, fecal samples (typically 10-40 mg) were weighed on an analytical balance and 250 µl to 500 µl PBS or saline was added. Samples were incubated for 5 min at room temperature and suspended by manual mixing and vortexing. Large particulate matter was pelleted by centrifuging at 100g, ten-fold serial dilutions were made in PBS, and 5 µl of each dilution was spotted on Difco MacConkey agar (BD 212123) supplemented with the appropriate antibiotics, i.e., streptomycin (100 µg/ml) or carbenicillin (50 µg/ml). For qualitative assessment of the fluorescent strains in feces, samples were spotted onto LB supplemented with the appropriate antibiotics. For isolating E. coli from fecal samples for genomic or plasmid DNA analysis, the fecal suspension was streaked on agar, and single colonies were further streak-purified.
Construction of CRISPR-Cas9 phagemid vectors
Cultures were grown in LB or TB media supplemented with the appropriate antibiotics. Plasmid DNA was prepared by QIAprep Spin Miniprep Kit (Qiagen 27106), eluted in TE buffer, and incubated at 60°C for 10 min. Samples were quantified using a NanoDrop One spectrophotometer. The vector pCas922 was digested with BsaI (NEB R0535) and gel extracted with a QIAquick Gel Extraction Kit (Qiagen 28706). Spacers were generated by annealing and phosphorylating the two oligos (PSP116 and PSP117 for GFPT; PSP120 and PSP121 for NT40) at 10 µM each in T4 ligation buffer (NEB B0202S) with T4 polynucleotide kinase (NEB M0201S) by incubating at 37°C for 2 h, 95°C for 5 min, and ramping down to 20°C at 5°C/min. The annealed product was diluted 1 in 200 in sterile distilled water and used for directional cloning by ligating (Thermo Scientific FEREL0011) to 60 ng of BsaI-digested, gel extracted pCas9 overnight at room temperature. Ligations were used to transform NEB 5-alpha competent cells (NEB C2987H) and the cloned spacer was verified by Sanger sequencing using primer PSP108. The trailing repeat was later confirmed to lack the starting 5’G, which did not interfere with GFP-targeting function. The 1.8-kb fragment carrying the f1 origin of replication and β-lactamase gene (f1-bla) was amplified from pBluescript II with SalI adapters using primers KL215 and KL216 and KOD Hot Start DNA polymerase (Millipore 71842-3). The PCR product was purified using a QIAquick PCR Purification Kit (Qiagen 28104), digested with SalI (Thermo Fisher FD0644), gel extracted, and used to ligate to SalI-digested, FastAP-dephosphorylated (Thermo Fisher FEREF0651) vector. Ligations were used to transform DH5α and clones were screened by restriction digest for both possible insert orientations (A or B) using XbaI (Thermo Scientific FD0684) and one of each orientation was saved for both the GFPT and NT phagemids.
Preparation of M13 carrying pBluescript II
This protocol was adapted from those to generate phage display libraries43. XL1-Blue MRF’ was transformed with pBluescript II (Agilent 212208). An overnight culture of this strain was prepared in 5 ml LB supplemented with tetracycline (5 µg/ml) and carbenicillin (50 µg/ml) and subcultured the following day 1-in-100 into 5 ml 2YT supplemented with the same antibiotics. At an OD600 of 0.8, cells were infected with helper phage M13KO7 (NEB N0315S) or VCSM13 (Agilent 200251) at a multiplicity of infection of approximately 10-to-1 for 1 h at 37°C The infected cells were used to seed 2YT supplemented with carbenicillin (100 µg/ml) and kanamycin (25 µg/ml) at 1-in-100, and the culture was grown overnight to produce phage. Cells were pelleted at 10,000g for 15 min, and the supernatant containing phage was transferred. Phage were precipitated by adding 0.2 volumes phage precipitation solution, inverting to mix well, and incubating for 30 min on ice. Phage were pelleted at 15,000g for 15 min at 4°C and the supernatant was discarded. The phage pellet was resuspended in PBS at 1-4% of the culture volume. The resuspension was centrifuged to pellet insoluble material and transferred to a new tube. Glycerol was added to a final concentration of 10-15%. Phage preparations were aliquoted into cryovials and stored at −80°C.
Preparation of M13 carrying CRISPR-Cas9 phagemids
DH5α(HP4_M13)44 was transformed with the GFPT phagemid (pCas9-GFPT-f1A or pCas9-GFPT-f1B) or the NT phagemid (pCas9-GFPT-f1A or pCas9-GFPT-f1B) and plated on LB media containing carbenicillin and kanamycin. Transformants were inoculated into 5 ml 2YT supplemented with 100 µg/ml carbenicillin and 25 µg/ml kanamycin, incubated overnight, used 1-in-100 to seed 250 ml of the same media, and incubated overnight. Cells were pelleted at 10,000g for 15 min, and the supernatant containing phage was transferred. Phage were precipitated by adding 0.2 volumes phage precipitation solution, inverting to mix well, and incubating for 30 min on ice. Phage were pelleted at 20,000g for 20 min at 4°C with slow deceleration. The supernatant was completely removed, phage were resuspended in PBS at 1% of the culture volume, and glycerol was added to a final concentration of 10-15%. The phage solution was centrifuged at 21,000g to pellet insoluble matter, filtered through 0.45 µm, and stored at −80°C.
Titration of M13 phage carrying phagemid DNA
Phage titer was determined using indicator strain XL1-Blue MRF’ or SmR W1655 F+. An overnight culture of the indicator strain in LB supplemented with the appropriate antibiotics was subcultured 1-in-100 or 1-in-200 into fresh media and grown to an OD600 of 0.8. To estimate titer, serial ten-fold dilutions of the phage preparation were made in PBS, and 10 µl of each dilution was used to infect 90 µl of cells. After incubating at 37°C for 30 min with shaking, 10 µl of the infection mix was spotted onto LB supplemented with carbenicillin. For more accurate titration, 100 µl of phage dilutions were mixed with 900 µl cells in culture tubes, incubated at 37°C for 30 min with shaking, and 100 µl was plated on LB carbenicillin.
Enumeration of viable M13 from mouse feces. Mice were orally gavaged with 6×10 13 M13(pBluescript II) or as negative controls, heat-inactivated phage or PBS. Approximately 100 mg of feces were collected at 0, 3, 6, 9, and 24 h post-gavage, and samples at each timepoint were processed i mmediately. 5 00 μl P BS w as added, samples were i ncubated for 5 min at room temperature, then suspended by manual mixing and vortexing. Samples were centrifuged at 21,000g for 1 min, the supernatant was transferred to a new tube, and phage titer was determined against i ndicator strain XL1-Blue MRF’ by diluting samples i n PBS, i ncubating with cells, and plating on LB supplemented with carbenicillin. For all dilutions and the undiluted suspension, 10 μl was used to i nfect 90 μl cells; additionally, for the undiluted suspension, 100 μl was used to i nfect 900 μl cells to maximize the l imit of detection.
Assay for acid survival. Phage M13(pBluescript II) stored i n PBS was diluted 1-in-100 i n saline. Solutions varying i n pH (1.2, 2, 3, 4, 5, 6, and 7) were prepared by mixing different ratios of 0.2 M sodium phosphate dibasic and 0.1 M citric acid and adjusting with concentrated HCl. 200 μl of each pH solution was transferred to the wells of a microtiter plate, and 10 μl of phage was added containing 1×10 9 M13(pBluescript II). Phage were i ncubated i n the solution, and 10 μl was sampled at 5, 15, and 60 min. Samples were diluted 1-in-100 i n PBS to make acidic samples neutral and phage titer was determined against i ndicator strain XL1-Blue MRF’ by plating on LB supplemented with carbenicillin. Solution-only controls were assayed simultaneously and cells were plated on LB to confirm viability of the i ndicator strain i n the presence of samples originating from an acidic pH.
Targeting experiments in vitro with M13 CRISPR-Cas9
Overnight cultures of fluorescently marked SmR W1655 F+ sfgfp and mcherry were prepared in LB supplemented with streptomycin, subcultured 1 in 200 into fresh media, and grown to an OD600 of 0.8. 900 µl cells (approximately 1×109) was transferred to a culture tube, 100 µl phage (approximately 1×1010 for f1A vectors and approximately 5×1010 for f1B vectors) was added, and the tube was incubated at 37°C for 30 min. The infection culture was transferred to a microfuge tube, cells were pelleted at 21,000g for 1 min, and the supernatant was removed. Cells were washed twice by adding 1 ml PBS, vortexing, pelleting cells, and removing supernatant. Cells were resuspended in 1 ml PBS, and ten-fold serially diluted in PBS. 10 µl of each dilution was spotted onto LB supplemented with carbenicillin and 100 µl was plated on larger plates for isolating single colonies for analysis. Colonies were picked and streak-purified four times to ensure phenotypic homogeneity and clonality.
Co-culture experiments with sfgfp and mcherry-marked strains infected with M13 CRISPR-Cas9
Overnight cultures of fluorescently marked SmR W1655 F+ sfgfp and mcherry were prepared in LB supplemented with streptomycin. For each culture, three serial ten-fold dilutions were made in PBS, followed by a fourth ten-fold dilution into LB. Equal volumes of each were combined and 5 ml aliquots were transferred to culture tubes. Using a CFU assay, the input was determined to be 6×106 CFU of each strain or 1×107 CFU total. 10 µl (5×109) M13 carrying CRISPR-Cas9 was added, the co-culture was incubated at 37°C for 30 min, and carbenicillin was added to a final concentration of 100 µg/ml. The co-culture was sampled for the t = 0 timepoint and then incubated for 24 h with further sampling every 4 h. At each timepoint, 200 µl was taken; 100 µl was used to assay carbenicillin in the media (see section: Carbenicillin bioassay) and the remaining 100 µl was used for plating as follows. To the 100 µl sample of culture, 900 µl was added and cells were washed by vortexing. Cells were pelleted by centrifuging at 21,000g for 1 min, and 900 µl of the supernatant was removed. To remove residual phage and antibiotic, the wash was repeated once more by adding 900 µl PBS, vortexing, pelleting cells, and removing 900 µl. Cells were resuspended in the remaining 100 µl. Serial ten-fold dilutions were made in PBS and 10 µl of each dilution was spotted onto LB or LB carbenicillin.
Carbenicillin bioassay
Cultures were sampled over time, cells were pelleted at 21,000g for 1 min, and the supernatant was transferred to a new tube and frozen at −20°C until all timepoints were collected. The supernatants were thawed and assayed using a Kirby-Bauer disk diffusion test. An overnight culture of the indicator organism (Bacillus subtilis 168) was diluted in saline to an OD600 of 0.1. A cotton swab was dipped into this dilution and spread across LB agar, antibiotic sensitivity disks (Fisher Scientific S70150A) were overlaid using tweezers, and 20 µl of the supernatant was applied to the disk. At the same time, carbenicillin standards were prepared from 1 µg/ml to 100 µg/ml and also applied to discs. Plates were incubated overnight at 37°C and imaged the following morning.
Flow cytometry
For turbid in vitro cultures, samples were diluted 1-in-10,000 in PBS. For mouse fecal pellets, samples were used fresh or thawed from −80°C, and suspended in 500 µl PBS by manual mixing and vortexing. Fecal suspensions were incubated aerobically at 4°C overnight to improve fluorescence signal (Supplementary Fig. 18). Samples were vortexed to mix, large particulate matter was pelleted by centrifuging at 100g for 30 seconds, and the sample was diluted 1-in-100 in PBS. Samples were run on a BD LSRFortessa flow cytometer using a 530/30 nm filter for GFP fluorescence and 610/20 nm for mCherry fluorescence, with the following voltages: 750 V for FSC, 400 V for SSC, 700 V for mCherry, and 700-800 V (in vivo) or 650 V (in vitro) for GFP. Flow cytometry data were analyzed in R using packages flowCore (v1.52.1)45, Phenoflow (v1.1.2)46, and ggcyto (v1.14.0)47. Typically, between 10,000 and 100,000 events were collected per sample, and data were rarefied after gating on FSC and SSC. Background events were accounted for on a per-mouse basis. For co-colonization with the sfgfp-marked and mcherry-marked strains, GFP+ and mCherry+ events from Day −3 (pre-E. coli) were used to subtract background at subsequent timepoints. For colonization with the double-marked strain, GFP+ mCherry+ events from Day −5 (pre-E. coli) were used to subtract background of double fluorescence at subsequent timepoints, and GFP-mCherry+ events from Day 0 (pre-phage) were used to subtract background of red fluorescence at subsequent timepoints. For exclusion of timepoints due to lack of colonization, the background threshold was calculated as the maximum background observed for that population across all timepoints multiplied by a factor of three. See Code Availability for more information.
Quick extraction and PCR analysis of genomic DNA from in vitro or in vivo isolates
Genomic DNA was extracted crudely to use as template for PCR. Briefly, 1.5 ml to 3 ml of culture was transferred to a microfuge tube, cells were pelleted by centrifuging, and the supernatant was discarded. The pellet was frozen, allowed to thaw on ice, resuspended in 100 µl TE, and incubated at 100°C for 15 min in an Eppendorf ThermoMixer. Samples were cooled on ice, cell debris was pelleted by centrifuging at 21,000g for 1 min, the supernatant was transferred to a new tube, and diluted 1-in-100 in TE to use as template DNA. PCR was performed using KOD Hot Start DNA polymerase (Millipore 71842-3) using primers KL207/KL200 for the sfgfp gene and primers BAC338F/BAC805R for the 16S rRNA gene48.
Extraction of DNA for hybrid assembly
E. coli strains KL68 (W1655 F+ or ATCC 23590), KL114 (W1655 F+ rpsL-SmR sfgfp), and KL204 (W1655 F+ rpsL-SmR sfgfp mcherry) were cultured in 50 ml LB supplemented with streptomycin. Cells were collected by centrifuging at 6,000g for 10 min at room temperature, washed in 10 ml 10 mM Tris 25 mM EDTA (pH 8.0), and resuspended in 4 ml of the same buffer. 12.5 mg lysozyme (Sigma-Aldrich L6876), 100 µl 5 M NaCl, and 50 µl 10 mg/ml RNase A (Thermo-Fisher EN0531) were added and the mixture was incubated at 37°C for 15 min. To lyse cells, 350 µl 5 M NaCl, 20 µl 20 mg/ml Proteinase K (Ambion AM2546), and 500 µl 10% SDS were added, and the mixture was incubated at 60°C for 1 h with gentle inversions. 2.75 ml of 7.5 M ammonium acetate was added, and the mixture was incubated on ice 20 min to precipitate proteins. Debris was removed by centrifuging 20,000g for 10 min and the supernatant was transferred to a new tube. To extract, an equal volume of chloroform was added and mixed; phases were separated by centrifuging at 2,000g for 10 min, and the aqueous phase was transferred to a new tube. To precipitate the DNA, 1 volume of isopropanol was added, and the tube was inverted until a white precipitate formed. The DNA was pelleted by centrifuging at 2,000g for 10 min and the supernatant was removed. The pellet was washed with 500 µl ice-cold 70% ethanol, allowed to dry, 1 ml TE was added, and the pellet allowed to dissolve overnight at 4°C. To further remove RNA, 250 µl of the genomic prep was transferred to a new tube, 12.5 µl 10 mg/ml RNase A was added, and the mixture was incubated at 37°C for 2 h with mixing every 30 min. To precipitate the DNA, 0.1 volume of 3 M sodium acetate was added followed by 3 volumes of 100% ethanol, and the mixture was inverted until a white precipitate formed. DNA was pelleted by centrifuging at 2,000g for 10 min, the supernatant was removed, the pellet washed with 100 µl 70% ethanol, allowed to dry, and resuspended in 100 µl TE. Samples were quantified by Qubit dsDNA BR Assay and DNA integrity was confirmed by 0.4% agarose gel electrophoresis using GeneRuler High Range DNA Ladder (Thermo-Fisher FERSM1353). DNA was used for both Oxford Nanopore sequencing and Illumina sequencing.
Illumina whole genome sequencing. DNA concentration was quantified using PicoGreen (ThermoFisher). Genomic DNA was normalized to 0.18 ng/ μl f or l ibrary preparation. Nextera XT libraries were constructed i n 384-well plates using a custom, miniaturized version of the standard Nextera XT protocol. Small volume l iquid handlers such as the Mosquito HTS (TTP LabTech) and Mantis (Formulatrix) were used to aliquot precise reagent volumes of <1.2 μl to generate a total of 4 μl per l ibrary. Libraries were normalized and 1.2 μl of each normalized library was pooled and sequenced on the Illumina NextSeq or MiSeq platform using 2×146 bp configurations. 12 bp unique dual i ndices were used to avoid i ndex hopping, a phenomenon known to occur on ExAmp based Illumina technologies. See Data Availability for more information.
Oxford Nanopore sequencing and hybrid Nanopore/Illumina assembly
PCR-free long read libraries were prepared using the Ligation Sequencing Kit (SQK-LSK109), multiplexed using the Native Barcoding Kit (EXP-NBD114), and sequenced on the MinION platform using flow cell version MIN106 (Oxford Nanopore Technologies). Basecalling of MinION raw signals was done using Guppy (v2.2.2, Oxford Nanopore Technologies). Reads were demultiplexed with qcat (v1.1.0, Oxford Nanopore Technologies). Quality control was achieved using porechop (v0.2.3 seqan2.1.1) (https://github.com/rrwick/Porechop) using the discard middle option. Reads were filtered using NanoFilt (v2.6.0)49 with the following parameters: minimum average read quality score of 10 (-q 10) and minimum read length of 100 (-l 100). Illumina reads were quality filtered using fastp (v0.20.1)50 with the following parameters: cut front, cut tail, cut window size 4, cut mean quality 20, length required 60. Filtered MinION and Illumina reads were then provided to Unicycler (v0.4.8)51 for hybrid assembly; default parameters were used unless otherwise noted. See Data Availability for more information.
Analysis of isolates after in vitro or in vivo M13-mediated delivery of phagemid
Isolates were cultured on LB or Difco MacConkey agar plates supplemented with carbenicillin or both carbenicillin and streptomycin. Isolates from in vitro GFP-targeting experiments were streak purified 4 times on agar to ensure clonality. Isolates from in vivo experiments were obtained by suspending a fecal pellet in 500 µl PBS, streaking the suspension onto agar, followed by streak purification of single colonies. For DNA extraction, single colonies were inoculated into LB or TB supplemented with the appropriate antibiotics. For analysis of the phagemid, plasmid DNA was extracted using a QIAprep Spin Miniprep Kit (Qiagen 27106), eluted in TE buffer, and incubated at 60°C for 10 min. DNA was quantified using a NanoDrop One spectrophotometer and 200-600 ng was digested with FastDigest restriction enzymes (KpnI, Thermo Scientific FD0524; XbaI, Thermo Scientific FD0684) for 10 min at 37°C followed by gel electrophoresis. Spacer sequences on phagemids were confirmed by Sanger sequencing using primer PSP108. For genome sequencing, genomic DNA was either extracted using a DNeasy Blood & Tissue Kit (Qiagen 69506) or an in-house protocol. Briefly, isolates were cultured in 3 ml TB supplemented with streptomycin and carbenicillin. Cells were pelleted and resuspended in 460 ul of freshly prepared buffer [per sample: 400 ul 10 mM Tris (pH 8.0) 25 mM ETDA, 50 µl 5 M NaCl, and 10 µl 10 mg/ml RNase A (Thermo-Fisher EN0531)]. 50 µl 10% SDS was added, mixed well, and samples were incubated at 60°C for 1 h with periodic inversions. 260 µl of 7.5 M ammonium acetate was added, and the mixture was incubated on ice 20 min to precipitate proteins. Precipitate was removed by centrifuging 21,000g for 5 min and the supernatant was transferred to a new tube. To extract, an equal volume of chloroform was added and mixed; phases were separated by centrifuging at 21,000g for 2.5 min. The aqueous phase was transferred to a new tube, centrifuged at 21,000g for 2.5 min, and 500 ul was transferred to a new tube. To precipitate the DNA, 500 µl isopropanol was added, and the tube was inverted until a white precipitate formed. Using a pipette tip, the clump was transferred to a new tube, washed with 100 µl cold 70% ethanol, and allowed to dry. 50 µl TE was added and the pellet was allowed to dissolve at 4°C overnight. DNA integrity was confirmed by gel electrophoresis and used for Illumina whole genome sequencing (see section: Illumina whole genome sequencing). Sequence reads were quality filtered using fastp (v0.20.1)50 and reads were aligned using bowtie2 (v2.3.5.1)52 to reference genomes and phagemid sequences; complete reference genomes were generated using hybrid assembly (see section: Oxford Nanopore sequencing and hybrid Nanopore/Illumina assembly). For CarbR isolates obtained after delivery of the pBluescript II phagemid, reads were simultaneously aligned to the genome of strain KL68 (W1655 F+) and the pBluescript II sequence (NCBI accession X52329.1). For CarbR isolates obtained after delivery of CRISPR-Cas9 phagemids, reads were simultaneously aligned to the genome of the strain used for in vitro (KL114; sfgfp) or in vivo (KL204; sfgfp mcherry) experiments and the sequence of the delivered phagemid. For isolates from GFP-targeting experiments, deletions were visualized by using samtools (v1.9)53 to filter multi-mapping and low-quality read alignments with MAPQ<2 (view -q 2), and depth was calculated using a sliding window of 20; breseq (v0.35.4)54 was used to assess deletion size. See Data Availability for more information.
Data availability
All raw sequence data have been deposited at the NCBI Sequence Read Archive under BioProject PRJNA642411. For 16S rRNA gene sequencing, Illumina sequence data have been deposited with accession numbers SRR12118792 to SRR12118959. For sequencing of isolates from mouse fecal samples after delivery of pBluescript II, Illumina sequence data deposited with accession numbers SRR14278062 to SRR14278073. For Nanopore/Illumina hybrid assembly of reference strain KL68 (W1655 F+ or ATCC 23590) as well as SmR fluorescent derivatives KL114 (sfgfp) and KL204 (sfgfp mcherry), Illumina sequence data have been deposited with accession numbers SRR14296642 to SRR14296644 and Oxford Nanopore MinION data with accession numbers SRR14297452 to SRR14297454. For sequencing of isolates after targeting with phage-delivered CRISPR-Cas9, Illumina sequence data deposited with accession numbers SRR14289086 to SRR14289109. Supporting data have also have been made publicly available on Github: https://github.com/turnbaughlab/2021_Lam_M13_CRISPRCas9
Code availability
Bash scripts and R Markdown documents for flow cytometry, 16S rRNA gene sequencing, and genomic deletion analyses have been made publicly available on GitHub: https://github.com/turnbaughlab/2021_Lam_M13_CRISPRCas9
Author contributions
KNL and PJT conceived the ideas. KNL supervised laboratory work and analyzed the data. KNL, PS, PSP, and PJT designed the experiments. KNL and PSP constructed plasmids. KNL constructed E. coli strains with assistance from PS. KNL performed phage and animal experiments with assistance from PSP. KL prepared samples and PS performed antibiotic assays. KNL, PSP and MN prepared samples, FBY performed 16S rRNA gene sequencing, and KNL and JEB analyzed the data. MA performed flow cytometry and KNL analyzed the data. KNL analyzed plasmid DNA from mouse fecal isolates with assistance from MN. KNL prepared samples, FBY and AMW performed Nanopore and Illumina whole genome sequencing, RRN performed the hybrid assembly of reference genomes, and KNL analyzed the genomic deletion data. PJT provided reagents and materials. KNL made the figures. KNL and PJT wrote the manuscript; PS, PSP, MA, JEB, and FBY assisted with editing.
Competing Interests
KNL, PS, and PJT are listed inventors on a U.S. provisional patent application related to this work (33167/55262P1). PJT is on the scientific advisory boards for Kaleido, Pendulum, Seres, and SNIPRbiome. All other authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to PJT.
SUPPLEMENTARY INFORMATION
Supplementary Figures
Supplementary Tables
Supplementary Table 1. Strains, plasmids, and phage.
Supplementary Table 2. Oligonucleotides.
Acknowledgments
We thank the UCSF Gnotobiotics Core staff (Jessie Turnbaugh, Kimberly Ly, and Jolie Ma) for animal care support and assistance with learning animal procedures. We are grateful to Daryll Gempis, Bernarda Lopez, and Ernesto Valencia of the UCSF G.W. Hooper Foundation for laboratory and administrative support. We thank Katja Engel (University of Waterloo) for help in translating the original publication on the isolation of M13 written in German. We are grateful to Antoine Vigouroux (Institut Pasteur) for generously sharing MG1655 derivatives carrying sfgfp and mcherry marker genes. We thank the Chan-Zuckerberg BioHub for sequencing through the Microbiome Initiative. We are grateful to Joseph Bondy-Denomy and Oren Rosenberg (UCSF) for constructive criticism of the manuscript. The graphic of a laboratory mouse was adapted from a Wikimedia Commons graphic distributed under CC-BY-SA-4.0 by Gwilz. KNL and PS were both supported by a postdoctoral fellowship from the Canadian Institutes of Health Research (CIHR), JEB by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada (NSERC), MA by an F32 fellowship from the National Institutes of Health (F32AI147456-01), and PSP by a scholarship from the UCSF Discovery Fellows Program. This work was supported by the National Institutes of Health (PJT, R01HL122593, R01AT011117; RRN, K08AR073930). PJT held an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund, was a Chan Zuckerberg Biohub investigator, and was a Nadia’s Gift Foundation Innovator supported, in part, by the Damon Runyon Cancer Research Foundation (DRR-42-16) and the Searle Scholars Program (SSP-2016-1352).