Innate immunity restricts Citrobacter rodentium A/E pathogenesis initiation to an early window of opportunity

Citrobacter rodentium infection is a mouse model for the important human diarrheal infection caused by enteropathogenic E. coli (EPEC). The pathogenesis of both species is very similar and depends on their unique ability to form intimately epithelium-adherent microcolonies, also known as “attachment/effacement” (A/E) lesions. These microcolonies must be dynamic and able to self-renew by continuous re-infection of the rapidly regenerating epithelium. It is unknown whether sustained epithelial A/E lesion pathogenesis is achieved through re-infection by planktonic bacteria from the luminal compartment or local spread of sessile bacteria without a planktonic phase. Focusing on the earliest events as C. rodentium becomes established, we show here that all colonic epithelial A/E microcolonies are clonal bacterial populations, and thus depend on local clonal growth to persist. In wild-type mice, microcolonies are established exclusively within the first 18 hours of infection. These early events shape the ongoing intestinal geography and severity of infection despite the continuous presence of phenotypically virulent luminal bacteria. Mechanistically, induced resistance to A/E lesion de-novo formation is mediated by TLR-MyD88/Trif-dependent signaling and is induced specifically by virulent C. rodentium in a virulence gene-dependent manner. Our data demonstrate that the establishment phase of C. rodentium pathogenesis in vivo is restricted to a very short window of opportunity that determines both disease geography and severity.


Introduction
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) serotype O157:H7 remain important causes of human diarrhea and mortality worldwide [1,2]. Citrobacter rodentium is a natural pathogen of mice which is used to model human EPEC and EHEC infection, as it shares the pathogenic mechanism of epithelial "attachment and effacement" (A/E). These pathogens attach to and colonize the intestinal mucosal surface in the form of epithelium-adherent microcolonies, also known as A/E lesions [3], comprised of bacteria that intimately attach to the apical surface of epithelial cells and induce the local destruction ("effacement") of the epithelial brush border. This triggers reactive colonic epithelial hyperplasia, inflammation and the induction of IL-17-producing CD4 helper T (Th17) cells and IL-22-producing type 3 innate lymphoid cells (ILC3) [4,5]. C. rodentium has thus become a valued A/E infection mouse model.
Disease severity and mucosal pathology elicited by C. rodentium correlate with the extent of A/E microcolony formation. The intestinal epithelium protects its structural integrity through rapid cellular renewal driven by crypt stem cell proliferation and crypt-to-villous migration of differentiated epithelial cells, with a complete crypt turnover time of typically 3-5 days in the healthy mouse [6]. Epithelial A/E infection with C. rodentium lasts for 2-3 weeks. Thus, to compensate for the associated continuous exfoliation of infected cells, A/E microcolonies must also be dynamic and able to renew by continuous epithelial re-infection. The route of this reinfection, required for sustained epithelial A/E pathogenesis, and the dynamics of A/E lesion development remain largely unknown. Re-infection by planktonic luminal colonizers or bacteria shed from A/E microcolonies, or local spread of sessile A/E lesion-associated bacteria without planktonic phase may all contribute ( Fig 1A).
All A/E pathogens share the intestinal virulence determinant "locus of enterocyte effacement" (LEE), a genomic island that is essential for A/E lesion formation [3]. LEE encodes a type 3 secretion system that translocates a cocktail of type 3 effector proteins into enterocytes, including Tir (Translocated intimin receptor) that inserts into the enterocyte plasma membrane and functions as adhesion receptor for the LEE-encoded outer membrane protein intimin. The Tir-intimin interaction triggers actin rearrangements that cause the epithelial microvilli to vanish ("effacement") and pedestal-shaped membrane structures to emerge underneath the attached bacteria [3,7]. A/E virulence expression is regulated at the level of LEE transcription through master regulator Ler (encoded in LEE) that is in turn subject to a complex regulatory network that responds to intestinal environmental factors [3,8]. Ler-deficient bacteria are consequently avirulent [7].
Resolution of mucosal C. rodentium A/E pathology within approximately 21 days of infection depends on functional B and CD4 + T cells [9,10]. Furthermore, IL-17-producing Th17 cells, IL-22 production by type 3 innate lymphoid cells (ILC3) and Th22 CD4 + effector T cells have all been shown to contribute to C. rodentium infection control [4,5]. A complex gut microbiota acts as a biological barrier against intestinal C. rodentium colonization [11,12] and is necessary to clear C. rodentium from the intestinal lumen once mucosal A/E lesions and inflammation have resolved [13]. However, even though germ-free animals cannot clear C. rodentium from the gut lumen, they can survive and resolve C. rodentium mucosal pathology with normal kinetics (within approximately 21 days), by virulence-neutralizing adaptive humoral immune responses [13,14].
In wild-type animals, C. rodentium-induced A/E pathology progresses slowly with marked intestinal pathology not developing until after 4-5 days of infection. This delay has been assumed to result from microbiota-dependent colonization resistance decelerating intestinal colonization. Disease onset and severity are also strongly determined by innate immunity. MyD88 knockout [15,16] and MyD88-Trif double knockout mice in particular [17], which are deficient in Toll-like receptor (TLR) and IL-1 receptor (IL-1R) superfamily signaling, develop exacerbated and accelerated A/E pathology.
Previous studies have predominantly focused on the acute and resolution phases of infection. Little is known about the nature and importance of host-bacteria interactions occurring during the first hours of infection. In the present study we focused on the early dynamics and control of A/E lesion development and show that the initiation of colonic C. rodentium microcolonies is limited to a very early stage of infection. These early-established, clonally-renewing A/E lesions trigger a local innate immune response that protects the epithelium from de novo A/E lesion formation by luminal bacteria and thus critically determine disease severity and outcome.

Clonal intestinal A/E lesion development
To track and quantify C. rodentium microcolony development in vivo over the course of infection, we first inoculated SPF wild type mice with GFP (green fluorescent protein)-and mCherry (red fluorescent protein)-expressing C. rodentium wild-type bacteria. Microscopic analysis of animals that had been inoculated with 10 10 CFU of a 1:1 mixture of GFP-and mCherry-tagged bacteria at day 7 post infection revealed that only single-colored microcolonies formed in the colon (Fig 1E), showing that they develop by local clonal expansion (that is, they are derived from the local proliferation of single bacteria; Fig 1E). The fact that many of these clonal microcolonies span several neighboring epithelial cells shows that luminally colonizing bacteria do not contribute to the epithelial re-infection required for the maintenance of established epithelial A/E lesions; otherwise an amalgamation between green and red bacteria over time or a random mosaic of green and red fluorescent bacteria-infected epithelial cells would be expected, neither of which could be observed over the entire course of infection. Thus, colonic A/E lesions trace back to single bacteria and appear to depend on local cell-tocell spread and not continuous re-seeding from planktonic luminal colonizers to compensate for epithelial shedding. These data further reveal that the epithelium-adherent population of C. rodentium is derived from a very small founder population (one bacterium per microcolony) and therefore subject to a population bottleneck.

Clonal A/E lesion development is microbiota status-independent
We next addressed whether this population bottleneck is caused by the colonization barrier conferred by the gut microbiota. Murine C. rodentium infection is subject to intestinal colonization resistance conferred by a complex gut microbiota through metabolic competition for simple sugars [12,13]. In agreement with this previous work, we found that SPF animals were completely protected against colonization with an inoculum of 10 4 CFU (Fig 1B), whereas mice inoculated with a very large inoculum of 10 10 CFU became consistently, albeit still variably, colonized with mean fecal densities that gradually increased to reach a plateau by around day 5 post infection ( Fig 1B).
We hypothesized that microbiota-mediated metabolic competition not only impacts on intestinal luminal colonization density but also restricts epithelial colonization and A/E pathogenesis. Colonization resistance may be responsible for the observed population bottleneck in the A/E lesion-associated bacteria through restriction of bacterial access to the epithelium or limitation of growth and survival of attached bacteria, either through direct metabolic competition or by maturing the epithelium and inducing protective non-specific immunity. To test this hypothesis, we studied germ-free mice infected with different doses of C. rodentium. As expected, in germ-free mice fecal bacterial populations rapidly and invariably reached high densities of between 10 9 and 10 10 CFU/g, regardless of inoculum dose (Fig 1C and 1D; S1A Fig). However, the striking clonality of the A/E lesions under SPF conditions was precisely phenocopied in germ-free mice, and was hence independent of microbiota-associated luminal colonization resistance or the effects of microbiota on mucosal immunity (Fig 1F and 1H). Taken together, these experiments revealed that the population bottleneck of early C. rodentium infection is mediated independently of microbiota-mediated effects.
We next quantitated the numbers of colonic epithelial A/E microcolonies in germ-free and SPF animals over time by fluorescence microscopy (Fig 1I and 1J). Microcolony numbers correlated well with the fraction of infected epithelial area measured by computational image analysis [18] (supplementary S1C and S1D Fig). Unlike in colonization resistant SPF mice ( Fig  1E, 1G and 1I), the infection of germ-free mice with a wide range of inoculum sizes (ranging from 10 2 to 10 8 CFU) led to peak numbers of colonic A/E lesions that were similar to those observed in SPF mice infected with 10 10 CFU ( Fig 1J and S1B Fig). Thus, in absence of colonization resistance, also lower inocula led to efficient epithelial A/E infection.

Persistently reduced A/E lesion development associated with very high inocula
Remarkably however, the inoculation of germ-free mice with the extremely high dose of 10 10 CFU was associated with significantly reduced virulence, leading to significantly lowered numbers of colonic A/E lesions ( Fig 1F; compare with Fig 1H). Although the intestinal colonization kinetics in lower-dose versus high-dose infections only differed during the first <16 hours ( Fig  1D and S2B Fig), A/E lesion numbers remained significantly decreased over the entire course of infection ( Fig 1J). This was associated with significantly improved clinical disease parameters between day 8 and 21, including weight loss (Fig 2A and 2B), systemic bacteremia (manifesting in live recoverability of C. rodentium from the spleen; Fig 2C), colonic mucosal histopathology (Fig 2D and 2E), and fecal concentrations of lipocalin-2 (a fecal marker for colitis severity; Fig 2F). We considered two alternative explanations for the mechanism by which the size of the inoculum in germ-free animals could have such a persistent effect on disease severity: First, ongoing bacteria-intrinsic downregulation of virulence in luminal C. rodentium after reaching high intestinal luminal density, or second, inhibition of ongoing A/E lesion induction by an early C. rodentium-induced host immune response.
Bacteria-intrinsic virulence regulation is considered important for the in vivo fitness of this pathogen [19]. We found that colonic luminal C. rodentium ler transcription was reduced at 16 hours after inoculation in mice that had been inoculated with 10 10 CFU compared to mice inoculated with only 10 4 CFU (S2A and S2B Fig). The main difference between the infection with an inoculum of 10 10 CFU and that with lower inocula is in the early population dynamics in vivo. Upon inoculation, bacterial populations enter an early logarithmic growth phase followed by a steady-state stationary phase (see Fig 1D). In germ-free mice inoculated with 10 4 CFU, the logarithmic growth phase lasts for approximately 15.5 h ( Fig  1D). In germ-free mice inoculated with 10 10 CFU, the bacteria lack this growth phase ( Fig  1D). The associated colonic virulence gene expression differences we measured at 16 hours after inoculation could explain a delayed disease onset, but are an unlikely sole explanation for the maintained reduction in A/E lesion burden, as it is known that fully colonized germfree animals remain luminally colonized with phenotypically virulent (ler-expressing) bacteria for at least 7 days [13]. Moreover, population density-dependent virulence regulation by the croIR-encoded acyl homoserine lactone (AHL) type quorum sensing system, reported previously to regulate bacterial adhesion and virulence negatively (in an ler/LEE-independent manner) at high population density [20], did not explain our observations. Infection with 10 10 CFU of a quorum sensing-deficient croI mutant reproduced the reduced A/E lesion density phenotype (S2D Fig).
In support of a host-mediated mechanism, however, we found that grossly innate immunocompromised germ-free Myd88/Trif double-deficient mice [21], unlike wild-type animals (Figs 1F, 1H, 1J and 2), did not show a persistent disease attenuation following infection with 10 10 CFU of C. rodentium. Even though the reduction of ler expression following infection with 10 10 CFU reproduced in MyD88/Trif-deficient mice ( Fig 3A) and was associated with a transiently reduced extent of colonic A/E microcolony formation on day 2 post infection, microcolony numbers were no longer different on day 3 post infection ( Fig  3B and 3D). In an independent experiment, the weight loss of infected MyD88/Trif doubledeficient germ-free mice was followed until animals reached a defined ethical endpoint ( 80% initial body weight). Mice that were infected with 10 10 CFU continued to lose weight at a rate similar to mice infected with 10 4 CFU, but with a delay of approximately 24 hours ( Fig 3C). These data suggest that an early MyD88/Trif-dependent immune response restricts the ongoing induction of A/E lesions, rather than the maintained downregulation of bacterial virulence.
MyD88-/Trif double-knockout mice are deficient for TLR as well as IL-1R family receptor signaling. To investigate the role of TLR-and inflammasome-related pro-inflammatory signaling, we analyzed germ-free Caspase-1/11-deficient mice. Although these mice have been described as hyper-susceptible to C. rodentium infection [22], we found that they developed a persistently attenuated A/E pathogenesis in response to an infection with 10 10 CFU of C. rodentium, similar to wild-type mice (S3 Fig). Thus, mucosal innate immune activation through MyD88/Trif-TLR-signaling determines severity of ongoing A/E pathogenesis in an early phase of infection.
Taken together, our data indicated that an early host innate immune response is induced at an early stage of infection and determines ongoing disease severity by limiting the induction of A/E lesions to an early window of opportunity. The remaining course of infection would then be driven by the early induced and persisting A/E lesions. A testable prediction of this hypothesis is that C. rodentium-infected wild-type animals should become insensitive to superinfection with a congenic strain of C. rodentium after the postulated window of opportunity. Representative fluorescent microscopy images of distal colon of germ-free MyD88 -/-Trif lps/lps mice after infection with doses of 10 4 or 10 10 CFU of C. rodentium for 2 days (left) and 3 days (right). Every individual depicted was infected with a 1:1 mixture of bacteria carrying a mCherry and GFP expression plasmid, respectively. Grey, F-actin/phalloidin; green, GFP-expressing C. rodentium; red, mCherry-expressing C. rodentium. Scale bars: 200 μm and 100 μm as indicated. ns, statistically not significant; *, p < 0.05; Student's t-test. https://doi.org/10.1371/journal.ppat.1006476.g003

Induced innate immunity limits A/E lesion induction to an early window of opportunity
To test this hypothesis, we developed a strain combination infection-superinfection model, in which two differentially tagged C. rodentium strains could be administered sequentially. This allowed us to elucidate the effect of the infection with the first strain on the pathogenesis of the second and how this depended on bacterial genotype, host genotype, and time interval between primary and superinfection (see schematic in Fig 4A). The aim of this approach was (i) to demonstrate and map the duration of the proposed infection window of opportunity directly, (ii) to show definitively the inhibition of A/E lesion initiation by luminal bacteria outside the window of opportunity, and (iii) to differentiate between bacterial and host contributions to its induction.
Germ-free mice were pre-infected with a kanamycin-resistant (Kan R ) mCherry-tagged C. rodentium strain (wild-type or ler mutant), followed 18 hours later by super-infection (or in the negative-control group co-infected) with 10 4 CFU of an isogenic tetracycline-resistant (Tet R ) wild-type strain (schematized and data shown in Fig 4A). All groups were treated orally with the bacteriostatic antibiotic tetracycline starting 3 h prior to the superinfection to shift colonization from the Tet-sensitive primary strain to the Tet-resistant super-infecting strain. The luminal colonization kinetics of each strain were determined by selective bacterial plating from feces ( Fig 4A). On day 7 after superinfection the animals were sacrificed and colonic A/E lesions (all of which were derived from super-infection, and therefore mCherry-negative) were quantified after labeling of C. rodentium with an anti-C. rodentium LPS antibody (Fig 4B-4G).
Super-infection was influenced by bacterial-bacterial competition between pre-and superinfecting bacteria, since there was overlap between pre-and super-infection. The co-infection control in which pre-and super-infection inocula were administered simultaneously (Fig 4, "10 10 WT 0h") consequently presented with 2.2±3.7 -fold (mean±SD) reduced numbers of colonic A/E lesions compared to the 10 4 -CFU-super-infection-only control ( Fig 4B). This effect reduced A/E lesion density to values similar to those observed in the super-infectiononly control inoculated with 10 10 C. rodentium (Fig 4, "super-infection only 10 10 WT). However, 18-hour pre-infection with 10 10 wild-type C. rodentium significantly reduced the A/E lesion formation a further 3.3±0.3 -fold relative to the co-infection control (Fig 4B and 4D). Remarkably, 18-hour pre-infection with an isogenic avirulent ler mutant had no such additional protective effect (Fig 4B and 4E), showing that LEE-dependent (ler regulated) pathogenesis in the first 18 hours of pre-infection induced epithelial resistance to A/E lesion induction. Moreover, the effect was even more pronounced when pre-infecting with 10 4 (near-full protection, more than 25-fold reduced A/E lesion relative to co-infection control; Fig 4B and 4C) instead of 10 10 wild-type C. rodentium, indicating that the epithelial resistance induction by pre-infection correlated with infectiousness rather than bacterial load of the pre-infection inoculum.
This early mucosal resistance phenotype was associated with an early increase of mucosal protein levels of IL-1β, IL-22, KC/CXCL1 and MCP-1 (Fig 5D-5G) and elevated transcription rates of Cxcl1 and Nos2/iNos (Fig 5B and 5C) measured at 18 hours post infection. At the same time point we observed the increased recruitment of neutrophils and monocytes into blood and lamina propria, associated with an early increase of lipocalin-2 (mainly produced by neutrophils) detectable in the feces (Fig 5A and 5H-5K). Colonic histological analysis confirmed that these changes occurred prior to the onset of acute colonic histopathology (S4 Fig), although the response markers measured may overlap with those associated with the acute A/ E lesion-associated inflammation developing later. These responses were induced specifically by virulent C. rodentium, but not an avirulent ler mutant or the commensal E. coli strain HS. Thus, the virulent C. rodentium-induced resistance to epithelial A/E superinfection with luminal bacteria was associated with an early mucosal innate immune response. It was more potently induced by a low-dose (10 4 CFU) than a high-dose (10 10 CFU) infection with wildtype C. rodentium. Hence, LEE/ler-dependent C. rodentium virulence, rather than unspecific bacterial innate immune receptor agonists like LPS (expected to be increased upon administration of 10 10 bacteria and similar between C. rodentium and E. coli), appears to be the main driver of this response. Finally, the response was abrogated in MyD88-/Trif-double-deficient mice (Fig 5, open symbols). MyD88/Trif double-deficient mice consequently lacked protection against epithelial superinfection, with no protective effect of wild-type C. rodentium preinfection over the control pre-treatment with a Δler mutant (Fig 6A-6C). The specific cellular and molecular innate immune components necessary and sufficient for this protective mechanism remain to be identified.
Overall, our data show that mucosal innate immunity, specifically activated by virulent C. rodentium in an ler/LEE-driven and TLR-signaling dependent manner, very effectively limits the establishment of A/E pathogenesis of C. rodentium to a window of opportunity of <18 hours duration that determines ongoing geography and severity of infection.

Discussion
We show in this paper that the development of colonic C. rodentium A/E pathogenesis is critically dependent on very early bacteria-host interactions. The characteristic A/E lesions that determine the severity of infection originate from a limiting number of single-bacterial epithelial infection events that need to occur within the first 18 hours of infection. The ongoing infection is driven by the early induced and locally growing epithelial A/E lesions that counteract epithelial shedding by continuous localized epithelial reinfection. Whilst this localized epithelial re-infection allows A/E lesions to persist on the epithelium for more than 10 days, until an adaptive immune response clears the infection, the window of opportunity for their induction by planktonic bacteria present in the lumen is limited to the first 18 hours. This is remarkable because the colonic lumen is known to remain colonized by phenotypically virulent bacteria for at least a week [13] and these bacteria are assumed to be more infectious than invitro-grown bacteria, which is important for effective fecal-to-oral transmission [23]. Restriction of A/E lesion induction to such an early time window is due to an early host response specifically induced by virulent C. rodentium, but not by an avirulent C. rodentium mutant. Similarly, mucosal stimulation with the related Gram-negative commensal E. coli could not phenocopy the observed phenomenon. Also the physiologic mucosal conditioning with a complex microbiota does not protect against epithelial A/E pathogenesis, but only impedes intestinal colonization with this pathogen. The abolition of the pathogen-induced mucosal resistance by a deficiency in MyD88-/Trif-signaling and superinfection experiments clearly show that an innate host response, rather than bacteria-intrinsic virulence gene regulation or bacteria-bacteria interaction, is mechanistically responsible. The specific cellular and molecular innate immune components required and sufficient for this protective effect remain to be identified. Whilst highly effective against induction of A/E lesions, the MyD88-/Trif-dependent innate immunity is not sufficient to abolish the localized re-infection occurring around persisting A/ group, pooled from 3 independent experiments. (C-G) Representative confocal fluorescent microscopy images of distal colon of mice shown in panel B. Grey, F-actin/phalloidin; green, anti-O-antigen Antibody (C. rodentium total); red, mCherry-expressing C. rodentium (pre-infection strain only); Scale bars: 200 μm and 100 μm as indicated; White squares indicate origin of higher-magnification image; *, p < 0.05; **, p < 0.01; ***, P < 0.001; ns, not significant (p ! 0.05); statistical test: one-way ANOVA. https://doi.org/10.1371/journal.ppat.1006476.g004 Early restriction of Citrobacter infection Notwithstanding the fact that early host immunity is the main driver of the phenomenon described, the bacteria-intrinsic regulation of C. rodentium virulence gene expression is known to be central to the pathogenesis of this species. The altered virulence of C. rodentium following an inoculation in unphysiologically high numbers of 10 10 /dose into germ-free animals provided us with an experimental tool to demonstrate the importance of bacterial virulence during the critical early phase of infection. In this context individual A/E lesions developed normally but in persistently reduced number, with extended areas of surface epithelium remaining uninfected. The ongoing epithelial re-infection that must occur to compensate for the effect of epithelial shedding thus appears to have a remarkably short range and may depend on epithelial adherence that is already established. Exact elucidation of this specialized re-infection mechanism is beyond the scope of the present study but warrants future efforts.
Our work in gnotobiotic models demonstrates that colonic A/E lesions induced by C. rodentium in an early phase of the infection-in immunocompetent mice-limit the ongoing induction of new A/E lesions. As a consequence the survival and localized renewal of earlyinduced A/E lesions, rather than the continued exposure to virulent bacteria in the environment or intestinal lumen is the main determinant of infection severity. This was unexpected, as the virulent bacteria shed into the colonic content and consequently the feces have been shown previously to have a "hyper-infectious" phenotype [23]. However, the situation in conventional, colonization resistant, mice may be more complex: It has been shown that, in SPF monocytes were defined as CD45 + , CD11b + , CD64 -, LygG -, Ly6C high , and MHCIIpopulation (J). Neutrophils were defined as CD45 + , CD11b + , Ly6G + population (K). Quantities are expressed as percentages of leukocytes (of CD45 + population). Dotted lines represent detection limit. ns, statistically not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; groups were compared with the matching uninfected control group; statistical tests: Kruskal-Wallis (A), and 1-way ANOVA (B-K). https://doi.org/10.1371/journal.ppat.1006476.g005

Fig 6. Ongoing A/E lesion induction in mice deficient for innate signaling through MyD88 and Trif. (A) Colonic colonization dynamics in
MyD88 -/-Trif lps/lps germ-free mice consecutively infected, first with mCherry + Kan R Tet S C. rodentium wild type (WT) or an isogenic Δler mutant (10 10 CFU, red squares and triangles), and 18 hours later superinfected with mCherry -Kan S Tet R C. rodentium wild type (10 4 CFU, blue circles). Total C. rodentium counts are depicted as black symbols. Connecting lines connect means; error bars indicate standard deviation; horizontal dotted lines indicate detection limit. (B and C) Representative fluorescent microscopy images of distal colon of MyD88 -/-Trif lps/lps mice shown in panel A pre-infected with wild type (B) and Δler (C) C. rodentium, respectively. Grey, F-actin/phalloidin; green, anti-Citrobacter O-antigen antibody (pre-infection strain and superinfecting strain); red, mCherry-expressing C. rodentium (pre-infection strain only; none detectable). Scale bars: 50 μm. https://doi.org/10.1371/journal.ppat.1006476.g006 Early restriction of Citrobacter infection mice, in-vitro-grown C. rodentium initially infects and accumulates mainly at the cecal patch and only approximately two days later spreads to the colon, strongly suggesting that longitudinal cecal-to-colonic re-infection can occur [23]. This has been ascribed to an "hyper-infectiousness" phenotype of C. rodentium acquired during intestinal passage that is associated with the increased expression of LEE and related virulence determinants [24]. It has further been shown by others that virulence is required to efficiently overcome intestinal colonization resistance in SPF mice [7,13]. In contrast to the delayed colonic infection observed with in-vitrogrown C. rodentium, the inoculation of naïve SPF animals with feces from infected mice led to the more direct infection of the colon [23]. It is readily conceivable that in mice receiving an in-vitro-grown inoculum, such a "hyper-infectious" bacterial population can build up not only during the initial cecal patch infection observed in SPF mice, but also more rapidly in the noncolonization-resistant germ-free intestine. Whilst our experiments in SPF mice (also using invitro-grown bacteria) recapitulated the previously described 2-day delay in colonic infection (see Fig 1), the colonic infection kinetics in the germ-free mice was equivalent to that described for SPF mice infected via the fecal-to-oral route. Thus, in absence of microbiota-mediated colonization resistance, cecal patch colonization may not be a prerequisite for colonic infection, but we cannot rule out that cecal infection plays an important role in all models, also independent of colonization resistance.
Our observation that very high inocula of (stationary phase) C. rodentium in germ-free mice are hypovirulent is relevant for the correct interpretation of earlier work in germ-free animals. For instance, the surprising finding that germ-free mice survive and can clear C. rodentium infection with similar mucosal immune activation kinetics as SPF mice, described recently [13], was reproduced in our experiments but depended on the infectious dose. We found a very large dose of 10 10 CFU (most labs use between 10 7 and 10 9 CFU) was less virulent in germ-free than in SPF mice. Conversely, we show that an "optimal" dose of 10 4 CFU is highly pathogenic in germ-free mice, yet below the minimum infectious dose in SPF mice due to microbiota-related colonization resistance. Thus, it is not straightforward to compare C. rodentium disease kinetics between animals with different microbiota statuses.
Our findings may also be relevant for other intestinal pathogens. Clonal intraepithelial microcolony formation has recently been described for the invasive epithelial pathogenesis of Salmonella typhimurium [25,26]. Sellin et al. described an early epithelial-intrinsic protective mechanism dependent on activation of epithelial Nod-like receptor NLRC4 and Caspase-1 that leads to elimination of infected enterocytes and curbs epithelial infection within the first hours of infection [25]. C. rodentium A/E infection has been shown to be more severe in NLRC4- [27] and Caspase-1-deficient mice [22]. Although we found that Caspase-1/11-deficient animals did not phenocopy MyD88-/Trif-deficiency in our analyses, the very early mucosal innate immune response appears to be similarly important in the control of invasive intestinal pathogens.
Further studies are needed to analyze how the new information obtained with the murine C. rodentium model translates into human A/E infection. EPEC-induced acute diarrhea is primarily a neonatal and infantile infection, whereas adults are relatively protected from natural infection by intestinal colonization resistance and develop disease only after intake of very large inocula (10 8 −10 10 CFU; [28]). A recent study using a novel neonatal murine EPEC infection model revealed strikingly similar A/E microcolony formation in the neonatal small intestine, and revealed an age-dependent but microbiota-independent resistance of the adult epithelium against A/E lesion formation [29].
Our findings may have several medical implications. First, since the function of LEE is highly conserved between murine and human A/E pathogens, the observed LEE-mediated induction of epithelial resistance to super-infection may also be relevant to human infection.
We have identified the initial 18 hours of murine A/E infection as a critical time window for controlling and surviving, or potentially preventing the infection. Even incompletely or transiently neutralizing antibodies may thus be able to substantially reduce intestinal morbidity. As human A/E infection is most severe in newborns and infants, the potential benefit of maternal immunity, protective lactational antibodies and mucosal vaccination will be an important aspect of future work. Remarkably, to the authors' knowledge it is still unclear to what extent human EPEC infection generates long lasting immunity or neonate-protective maternal antibodies [30], and there is no EPEC vaccine currently available. However, we also need to consider the possibility that specific intestinal antibodies, through dampening the observed rapid innate immune activation, may alter disease progression and even worsen disease outcome.
Second, antibiotic therapy is not a recommended therapy for human A/E infection, and contraindicated in EHEC infection where antibiotic treatment is associated with an increased risk of hemolytic-uremic syndrome. Due to worldwide emergence of multi-resistant E. coli strains and the negative effect on microbiota-conferred colonization resistance, empiric antibiotic therapy is limited to severe cases. However, our findings in the murine model demonstrate that A/E microcolony formation is sensitive to antibiotic treatment, whilst epithelial re-infection by the incompletely eradicated luminal bacteria or superinfection by resistant bacteria is efficiently inhibited by mucosal innate immunity. Hence, optimized antibiotic regimens or novel selective antimicrobials, such as type 3 secretion system inhibitors [31], may have therapeutic potential in the treatment of EPEC infection.

Ethics statement
All animal experiments were performed according to protocols approved by the Bernese cantonal ethical committee for animal experiments and carried out in accordance with Swiss Federal law for animal experimentation (license numbers BE94/11 and BE91/14).

Animals
All mice had a C57BL/6 background. Germ-free animals were derived and maintained germfree in flexible film isolators in the Genaxen Foundation Clean Mouse Facility (CMF) of the University of Bern as described [32]. Myd88 -/-Ticam1/Trif lps/lps mice were kindly provided by Prof B. A. Beutler, The Scripps Research Institute (la Jolla, CA, USA) [33] and derived germfree as described [21]. Germ-free caspase-1/11-deficient mice [34] were generated by axenic embryo transfer of cryopreserved 2-cell-stage embryos (The Jackson Laboratory, Bar Harbor ME, USA). Experimental germ-free mice were aseptically transferred to autoclaved sealsafeplus individually ventilated cages (IVCs) under positive pressure (Tecniplast, Italy) in a gnotobiotic barrier unit. Cage changes were carried out under strictly aseptic conditions. In all experiments animals were provided with sterile mouse chow (Kliba 3437; autoclaved) and autoclaved water ad libitum. SPF mice were acquired from Envigo (former Harlan, the Netherlands) and housed in IVCs in the Zentrale Tierställe of the University of Bern.

In vivo infections
To generate contamination-free bacterial inocula, LB medium supplemented with appropriate antibiotics (see section bacterial culture for details) in sterile-filter-sealed flasks was aseptically inoculated from single colonies of the test bacterium and incubated, with shaking at 150 rpm at 37˚C for 16 h. Bacteria were harvested by centrifugation (7 min, 4700 × g, 4˚C) in a sterile aerosol-proof assembly, washed in autoclaved sterile PBS and concentrated to a density of 5 × 10 10 CFU/mL in sterile PBS, performed aseptically under a sterile laminar airflow. To generate lower densities cultures were diluted appropriately in PBS. The bacterial suspensions were aseptically aliquoted in autoclaved plastic tubes and sealed in a sterilized secondary containment. The sterile tubes containing the inocula and germ-free mice were aseptically imported into a sterilized laminar flow hood, and each animal inoculated with 200 μL of bacterial suspension by gavage, carried out wearing sterile surgical gowns and sterile surgical gloves. Fresh fecal pellets were collected aseptically, suspended in sterile PBS, and plated in serial dilutions on LB plates containing the appropriate antibiotics and incubated at 37˚C for 24 h. Weight of mice was measured using a hand held balance and calculated as a percentage of the body weight in the beginning of the experiment. For tetracycline treatment, mice were gavaged with 200 μL of a sterile tetracycline solution (50 mg/L) containing 2% sucrose, and the same solution was used as drinking water. The tetracycline-water was protected from light and replaced every 3 days.

Bacterial genetic engineering
All bacterial strains and plasmids used or generated in this study are specified in S1 Table. All Citrobacter rodentium strains used in this study are derivatives of the wild type strain ATCC51459 (ATCC, Manassas VA). All deletions were carried out by Lambda Red recombineering. Mutagenesis primer sequences are specified in S2 Table. (i) Strain HA526 (Δler:: tetRA) was generated by deletion of ler using recombineering plasmid pSIM5 as described [36][37][38]. A tetRA recombineering amplicon was amplified from genomic tetRA template DNA (isolated from a Tn10-containing bacterial strain) with primers CR-ler-mutF and CR-ler-mutR. (ii) The tetRA cassette was removed with the same protocol using primer CR-ler-rmvl. Tetracycline sensitive clones were selected by growth on Tetracycline counterselective plates [35,36] at 32˚C, for 2 days, yielding HA539 (Δler). (iii) Following the same protocol as (i), primers CR-croI-mutF and CR-croI-mutR were used to amplify the tetRA amplicon. The resulting amplicon was used to delete croI in C. rodentium resulting in strain HA528 (croI:: tetRA). A tetracycline-resistant strain of C. rodentium was constructed by inserting the tetRA cassette into the locus of dadX, using the primers CR-dadX-mut-F and CR-dadX-mut-R, yielding in strain HA538 (Δdadx::tetRA). Plasmid pHA500 was derived from pM965 [39], by exchanging the ampicillin resistance with a kanamycin resistance cassette. This was done using pSIM9 and lambda red recombineering. A kan amplicon was amplified using pKD4 [40] as a template with primers pM965-kan-F and pM965-kan-R. The recombineering was carried out in an E. coli K12 strain. Plasmid pHA501 was derived from pM2120 [41], by exchanging the ampicillin resistance with a kanamycin resistance. This was done using pSIM9 and lambda red recombineering. A kan amplicon was amplified using pKD4 [40] as a template with primers pM2120-kan-F and pM2120-kan-R. The recombineering was carried out in an E. coli K12 strain. All deletions were verified phenotypically and by control PCR (control primers specified in S2 Table). Plasmids (see S1 Table for a complete list) were introduced by electroporation following standard protocols.

Fluorescence microscopy
The distal 2 cm of the colon were fixed in 4% PFA in PBS for 24 h, then transferred to a 20% sucrose solution for 24-48 h. The fixed distal colon was then cut into three equally large parts and embedded in OTC media, and flash frozen with liquid nitrogen. Sections of 7 μm thickness were cut and rinsed twice in PBS and once in PBS/2%BSA. Sections were stained with a PBS/2%BSA solution containing DAPI (Sigma-Aldrich, USA, 0.01 mg/mL final concentration) and Phalloidin-Atto 647N (Sigma-Aldrich, USA, 0.04 nmol/mL final concentration) for 30min. Sections were rinsed again twice in PBS and one time in PBS/2%BSA and mounted under Vectashield (Vector laboratories, USA). Images were acquired using a Zeiss LSM710 Laser scanning microscope. In experiment with C. rodentium without a fluorescent plasmid, the bacteria were stained with an anti-E. coli O152 antiserum. Cryosections were washed twice in PBS and once in PBS/2%BSA and stained with a primary antibody (Rabbit anti E. coli O152 Antibody, Abcam, 1:400) for 1h. After washing with PBS the sections were stained with a secondary antibody (Alexa Fluor 488 AffiniPure goat anti-rabbit IgG, LucernaChem, USA, 1:400), DAPI (Sigma-Aldrich, USA, 0.01 mg/mL final concentration), and Phalloidin-Atto 647N (Sigma-Aldrich, USA, 0.04 nmol/mL final concentration) for 1h. Sections were rinsed again twice in PBS and one time in PBS/2%BSA and mounted under Vectashield (Vector laboratories, USA). Images were acquired using a Zeiss LSM710 Laser scanning microscope.

Quantitation of A/E microcolonies
Fluorescently labelled distal colon sections were used to quantify microcolonies (see section "fluorescent microscopy"). For each mouse, three different horizontal sections of the distal colon were analyzed. Profiles of microcolonies were counted manually, a microcolony being defined as a cluster of bacteria on the epithelial surface interspaced by either uninfected epithelium from the next microcolony or by a microcolony of a different color. In order to analyze the percentage of infected surface, the whole horizontal section was imaged as a tile scan using either a Zeiss LSM710 Laser scanning microscope or a Zeiss Observer standard fluorescence microscope. The image was then sliced to acquire the original individual images that composed the tile (approx. 100 images per horizontal section of colon). Sections containing parts of the epithelial surface were selected. Of this selection every fifth image was chosen for analysis, choosing the first picture to be analyzed randomly (random number between 1 and 5 was drawn). This selection yielded in approx. 20 images per mouse, originating from all three original sections. Images were then analyzed using the STEPanalyzer stereology tool [18]. As horizontal sections were chosen, and no vertical design was applied, the stereological analysis is only valid in relative numbers, not absolute.

Lipocalin-2 ELISA
Fecal lipocalin-2 was measured using the solid phase Sandwich ELISA Mouse Lipocalin-2/ NGAL DuoSet (R&D Systems, USA). The assay was performed according to the manufacturer's instructions, except for the horseradish peroxidase, HRP-SA which was used from Biolegend (USA). Fecal pellets from mice were dissolved in 0.5 mL sterile PBS and debris removed by centrifugation (microfuge 7000 rpm, 5min). The supernatant was serially diluted in triplicates (1:3 dilution steps, 6 or 12 dilutions). The standard was serially diluted in duplicates (1:3 dilution steps, 12 dilutions). The OD 405 was measured using a Thermomax microplate reader (Molecular Devices, USA) Data was analyzed using graph pad Prism program. A four parameter dose-response curve was fitted, using equal hill slopes for all samples on the same plate. Samples with low OD 405 values resulting in a curve fit with R 2 values below 0.95 were defined below detection limit. By comparison with the standard, the EC50 was used to calculate the absolute amount of Lipocalin-2 per sample.

Bacterial RT-qPCR
For validation of the method on in-vitro grown bacteria (S2C Fig), an overnight culture was prepared in LB and grown for 16 h at 37˚C, 150 rpm. The culture was then diluted 1:100 in either LB and grown at 37˚C, 150 rpm or in DMEM (Gibco, Netherlands) and grown at 37˚C, 5% CO 2 without shaking. To determine density, at the time of sampling the OD 600 was determined. Samples were removed at different time points, volumes adjusted to yield approx. 1x10 9 bacteria (OD 600 = 1). Samples were spun down (4500 g, 5 min), and resuspended in RNAprotect reagent (Qiagen, Germany). After 5 min incubation the bacterial suspension was spun again and the pellet resuspended in 200 μL lysis buffer (TE buffer containing 10 mg/mL Lysozyme, Sigma-Aldrich, Canada). The bacteria were incubated at room temperature for 10 min. RNA was isolated using the RNeasy mini kit (Qiagen, Germany), including the on column DNase digestion step. Concentration of RNA was measured using Nanodrop (Thermo-Scientific). Quality of the samples was tested with the RNA 6000 Nano kit (Agilent, USA). Colon contents were collected from mice and suspended in 5 mL sterile PBS. Large debris were sedimented for 10 sec and the supernatant transferred to a new tube. This solution containing the bacteria was spun down (4700 g, 7 min) and the pellet resuspended in 0.5 mL RNAprotect reagent prior to RNA isolation. Quantitative PCR (qPCR) was performed in duplicates or triplicates in a volume of 20 μL containing 600 ng RNA using the TaqMan RNA-to-CT 1-Step kit (Applied Biosystems, USA) and the Quant Studio 7 Flex System (ThermoFisher, USA). Following primers and probes were used: Ler-F-primer: GAG CAG GAG ATT CAA ACT GTA A; Ler-R-primer: CGT CTT CAT TAC GGT AGT ATA CC; ler-probe: CGG CGA GCA AGA GCA CCA TCA (modifications: 6-carboxyfluorescein (FAM) and Black hole quencher (BHQ-1)); rpoD-F-primer: AAG CGA AAG TCC TGC GTA TG, rpoD-R-primer: GCT TCG ATC TGA CGG ATA CG rpoD-probe: CGA TAT GAA CAC CGA CCA CAC GCT G (modifications: Yakima yellow (YYE) and BHQ-1). All probes and primers were synthesized and tested by Microsynth (Switzerland). The following program was run: 15 min 48˚C, 10 min 95˚C followed by 50 cycles of 15 s 95˚C and 1 min 60˚C. Fluorescence was measured for each cycle. C t values for both genes were averaged and the mean C t of ler was subtracted from the mean C t value of rpoD to normalize.

Gene expression analysis of distal colon tissue
Colon tissue was emptied from all fecal matter, washed in sterile PBS and immediately stored in RNAlater (Qiagen, Germany). RNA was extracted from the tissue using the RNeasy kit (Qiagen, Germany) according to the manufacturer's instructions. RNA quality was verified with the Agilent Bioanalyzer system using RNA micro chips (Agilent Technologies, USA). Using 400 ng RNA, cDNA was prepared using the RT2 Easy First Strand Kit (Qiagen) following manufacture's protocol.

Statistical tests
Statistical analysis was performed using the Prism software (GraphPad, LaJolla, CA, USA). Details for statistical tests used are given in the figure legends.
Supporting information S1  The leukocyte population in peripheral blood was defined by gating on single cells and further on CD45 + cells. Neutrophils were then defined as Ly6G + and monocytes as CD11b + and CD115 + cells. (B) The leukocyte population in caecum and colon was defined by gating on single cells and further on CD45 + cells. Neutrophils were then defined as CD11b + Ly6G + cells. The leukocyte population was further analyzed by gating on CD11b + CD64cells (immature myeloid cells), exclusion of neutrophils (Ly6G -) and separation of infiltrating monocytes (Ly6C high MHCII -) from resident monocytes. (TIF)