Salmonella-Induced Mucosal Lectin RegIIIβ Kills Competing Gut Microbiota

Intestinal inflammation induces alterations of the gut microbiota and promotes overgrowth of the enteric pathogen Salmonella enterica by largely unknown mechanisms. Here, we identified a host factor involved in this process. Specifically, the C-type lectin RegIIIβ is strongly upregulated during mucosal infection and released into the gut lumen. In vitro, RegIIIβ kills diverse commensal gut bacteria but not Salmonella enterica subspecies I serovar Typhimurium (S. Typhimurium). Protection of the pathogen was attributable to its specific cell envelope structure. Co-infection experiments with an avirulent S. Typhimurium mutant and a RegIIIβ-sensitive commensal E. coli strain demonstrated that feeding of RegIIIβ was sufficient for suppressing commensals in the absence of all other changes inflicted by mucosal disease. These data suggest that RegIIIβ production by the host can promote S. Typhimurium infection by eliminating inhibitory gut microbiota.


Introduction
Diarrhea is an infectious disease that causes high mortality worldwide, especially among children and the elderly [1]. Salmonella spp. infection is an important cause of gastroenteritis and has been widely studied because of facile Salmonella spp. genetics and excellent animal disease models [2,3,4]. Current treatment relies mostly on water and electrolyte supplementation as Salmonellosis is often self-limiting in humans [5]. Patients which show septic Salmonellosis are treated with antibiotics, but the rise of drug resistant Salmonella strains is a current problem in efficient Salmonella spp. therapy [5,6].
The mechanism promoting inflammation-mediated overgrowth of enteric pathogens is not fully understood. Inflammation could alter intestinal nutrient availability (i.e. mucus-derived carbohy-drates) in a way that increases pathogen growth but diminishes resident microbiota ('nutrient hypothesis') [16,24]. Another reason for S. Typhimurium overgrowth in an inflamed gut is the production of tetrathionate from microbiota-derived H 2 S by the inflamed host mucosa [24]. Tetrathionate allows Salmonella enterica subspecies I serovar Typhimurium (S. Typhimurium) to perform a respiratory metabolism, which confers an advantage in the competition with the microbiota [24]. Alternatively, inflammation induces immune effector mechanisms that kill resident microbiota but not resistant pathogens ('killing hypothesis'). Indeed, the antimicrobial protein lipocalin-2, which is upregulated in inflamed mice and Rhesus macaques [25], blocks iron uptake in enteric bacteria by sequestering enterochelin. However, S. Typhimurium produces salmochelin, a glycosylated form of enterobactin, which is not bound by lipocalin-2 [26,27]. Hence, S. Typhimurium gains a selective advantage in the presence of lipocalin-2 [25]. These data suggest that there is not a single factor leading to S. Typhimurium outcompetition of the microbiota but more likely a whole range of parameters playing a role in inflammation.
Antimicrobial peptides (e.g., defensins, cathelicidins) constitute another host defence supporting the mucosal barrier. The C-type lectin RegIIIc, a member of the Reg gene family, a diverse group of secreted proteins harboring a C-type lectin carbohydrate recognition domain, has been shown to exert antimicrobial effects on commensal bacteria [28]. The closely related RegIIIb has previously been shown to be upregulated in response to inflammation and infections [29,30]. Here, we studied the properties and in vivo relevance of the C-type lectin RegIIIb in inflammation-induced microbiota-pathogen competition. This was of interest as RegIII family members are known to affect hostcommensal and host-pathogen interactions in the intestine [28,30,31]. Our results imply that RegIIIb is indeed one of the host factors explaining why S. Typhimurium can benefit from triggering gut inflammation.

Ethics statement
All animals were handled in strict accordance with good animal practice and all animal work was approved by local animal care and use committees (license 04/862 Niedersä chsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit; license 201/ 2007 Kantonales Veterinä ramt Zürich).

Bacterial strains and growth conditions
All S. Typhimurium strains used in this study were derivatives of SL1344 hisG rpsL xyl [32] (TABLE 1). The virulent and avirulent S. Typhimurium strains have been described previously [33,34] as well as S. Typhimurium DaroA [35]. Salmonella Typhimurium mutants with defined gene deletions were obtained using the Lambda phage red recombinase method [36] with primers described at http://falkow.stanford.edu/whatwedo/wanner/ (see also TABLE 2). For comparison of LPS defect mutants, Salmonella Typhimurium LT galE [37] was used.
Non-pathogenic Escherichia coli E2 (stx12 stx22 eaeA2 hlyA2 espP2 katP2 astA2 recA+ tolC+) was isolated from BALB/c mice originally obtained from Charles River and maintained at the Hannover Medical School Animal Facility for 12 weeks. Streptomycin-and rifampicin-resistant E2 derivatives were obtained by consecutive selection of spontaneous mutants on media containing rising antibiotic concentrations. Bacillus subtilis 168 was obtained from the Bacillus Genetic Stock Center . For  detailed description of gut commensal strains see TABLE 1. For mouse infections, bacterial strains were grown for 12 h at 37uC in Luria-Bertani (LB) broth (0.3 M NaCl) and subcultured for 4 h, as described [34].

Purification of recombinant Proteins
A synthetic sequence optimized for expression of murine RegIIIb (ProteinID: NP_035166.1) without signal peptide in E. coli was constructed (GENEART, Regensburg) and cloned into pET11a (Novagen, Bad Soden). Variant RegIIIb R135T was constructed using PCR mutagenesis and verified by sequencing. RegIIIb and the variant were expressed in E. coli BL21 (DE3) and purified essentially as described [38]. In brief, cells were induced with 0.1 mM IPTG for 3 h. Cells were harvested by centrifugation, washed with PBS, and frozen at 220uC. Cells were thawed on ice and resuspended in IB buffer (20 mM Tris-HCl, 10 mM EDTA, 1% Triton X-100, 0.1 mg/ml Lysozyme, pH 7.5). Cells were lysed by sonication (7620 s pulses) and RegIIIb inclusion bodies were sedimented by centrifugation. The pellet was resuspended in 25 ml IB buffer and sonification/centrifugation was repeated three times to reduce contaminations by intact cells. Purified inclusion bodies were resuspended in denaturing buffer (7 M Guanidine-HCl, 0.15 M reduced glutathione, 2 mM EDTA, 0.1 M Tris-HCl, pH 8.0). Still insoluble material was removed by centrifugation and the supernatant was slowly diluted into ice-cold refolding buffer (0.5 m arginine-HCl, 0.6 mM oxidized glutathione, 50 mM Tris-HCl, pH 8.0). After overnight incubation, insoluble material was removed by centrifugation and the supernatant was concentrated using ultrafiltration with a 10 kDa cut-off membrane (Millipore). The concentrate was dialyzed twice against binding buffer (25 mM MES-NaOH, 25 mM NaCl, pH 6.0). Insoluble material was removed by centrifugation. The final material was homogenous as judged by SDS-PAGE. We verified by omitting lysozyme in the purification protocol that it doesn't contribute to bacterial killing in the in vitro assays.
For some experiments, RegIIIb was trypsinated with 500 ng/ml Trypsin (5000 NF-U mg-1) at a concentration of 50 mg ml 21 in binding buffer at 37uC for 2 h. For fluorescence detection, RegIIIb was covalently linked to Alexa647 (Molecular Probes) following the manufacturer's instructions.
Purification of RegIIIc has been performed as described previously [38].

Animal experiments
Mice were purchased from Charles River (Sulzfeld, Germany) or bred at the RCHCI ETH Zurich, Switzerland. Eight to 12 weeks old female BALB/c or C57BL/6 mice were pre-treated with 20 mg streptomycin and 24 h later intragastrically infected with 5610 7 CFU of S. Typhimurium, as previously described [39].
Colonization was determined by plating feces on selective media (MacConkey agar with appropriate antibiotics). In contrast to many non-culturable commensals, the gut luminal cfu data of S. Typhimurium (and E. coli) obtained by plating always match the cfu data obtained in feces. This is well documented by data from plating, 16S RT PCR, in situ hybridization and immunofluorescence microscopy [9,15,39,60].
To determine the effect of RegIIIb on bacterial colonization, mice received a single intragastric dose, or daily doses of 80 mg RegIIIb or 80 mg BSA respectively, in 200 ml binding buffer.

In vitro assays
Binding and cidal activities of RegIIIb were determined using bacteria from late log liquid cultures. Bacteria were washed and resuspended in binding buffer (25 mM MES-NaOH, 25 mM NaCl, pH 6.0) at a density of 10 6 CFU ml 21 . RegIIIb was added at various concentrations and the mixture was incubated for various times at 37uC. Bacteria were then plated either on LB, Wilkins Chalgren, or MRS agar, and grown under aerobic or anaerobic conditions (7% H2, 10% CO2, 83% N2). As controls ( = 100% survival) Bacteria incubated with binding buffer only, were analyzed.
In some experiments, RegIIIb was pre-incubated for 10 min with a fivefold (weight/weigth) excess of peptidoglycan (insoluble PGN from Bacillus subtilis, Sigma) before addition to bacterial suspensions. Peptidoglycan co-precipitation was determined as described [28].
Bacteria were incubated for 40 min at 4uC with 1 mM fluorescence-labeled RegIIIb in binding buffer containing 1% bovine serum albumin to minimize unspecific binding. At this low concentration, no cidal effect of RegIIIb that could potentially affect the results has been detected. Bacteria were washed three times in binding buffer and analyzed using a Calibur flow cytometer (BD Biosciences).

Antibody generation, WB, and immunohistochemistry
A polyclonal rabbit anti-RegIIIb antibody was produced by Neosystem (Strasbourg, France) using recombinant RegIIIb. The antibody was further affinity purified with AminoLink Kit (Thermo Scientific Pierce) using recombinant RegIIIb. The antibody is specific for RegIIIb (Fig. S1).
For analysis of intestinal RegIIIb, cecal contents were resuspended in PBS. Proteins were separated by SDS-PAGE followed by Western blotting using the polyclonal anti-RegIIIb antibody and chemoluminescent detection (GE Healthcare).

Quantitative RT-PCR
RegIIIb mRNA levels were quantified using QIAGEN isolation kits, M-MLV reverse Transcriptase RNase H Minus, the QuantiTect SYBR Green PCR kit (Qiagen), and primers as described below. c t values were normalized to GAPDH [41] and represent the median of triplicate analyses compared to noninfected mice. Cycling parameters were 94uC (15 s

Statistical analysis
Statistical analysis was performed using the Student T-Test except for long-term colonization where the Mann Whitney U-test was applied. P # 0.05 was considered to be statistically significant.

S. Typhimurium infection induces intestinal RegIIIb expression
The C-type lectin RegIIIb is highly induced in rat intestinal tissues during enteric Salmonellosis [42,43]. We aimed at analyzing the role of RegIIIb in microbiota-pathogen competition in the streptomycintreated mouse model for S. Typhimurium induced gut inflammation. To initially confirm validity of previous data for this model, we performed quantitative real time PCR of RegIIIb mRNA on cecal tissue (Fig. 1A). Mice infected with an avirulent S. Typhimurium strain that fails to cause intestinal inflammation, show slightly upregulated RegIIIb mRNA levels compared to baseline levels in non-infected control mice. In contrast, mice infected with wild-type S. Typhimurium (wt) show drastically increased levels (,10-fold; Fig. 1A).
To confirm qPCR data on protein level, we analyzed intestinal contents in Western blots using an affinity-purified polyclonal antibody to RegIIIb (Fig. S2A). This antibody recognizes a major 15 kDa band and a weaker band of approx. 14 kDa, which likely correspond to RegIIIb and a proteolytically processed form (Fig. 1B). Proteolytic cleavage by trypsin has been reported for the closely related C-type lectin RegIIIc [28]. As trypsin is present in intestinal contents, RegIIIb processing is likely to occur in vivo.
Quantification using purified recombinant RegIIIb as a reference indicated that RegIIIb was present at baseline levels of less than 1 mg/g intestinal contents in uninfected mice, but increased to levels of some 350 mg/g in mice infected with virulent S. Typhimurium (Fig. S2B).
S. Typhimurium-induced RegIIIb upregulation critically depended on the innate immunity adapter protein MyD88 (Fig. 1B) as previously reported in case of RegIIIb induction by the commensal microbiota [44]. The same is true for the closely related C-type lectin RegIIIc in a L. monocytogenes infection model [30]. Immunohistochemistry of intestinal tissue sections revealed weak RegIIIb levels in S. Typhimurium infected mice, which were non-inflamed, but prominent RegIIIb expression in intestinal epithelial cells in mice with gut inflammation (Fig. 1C). These data confirmed and extended previous observations of RegIIIb upregulation in intestinal tissues in response to virulent Salmonella spp..

RegIIIb kills commensal bacteria but not S. Typhimurium
The C-type lectin RegIIIc has been reported to have bactericidal effects. To monitor the antimicrobial activity of closely related RegIIIb on different bacterial species, we produced recombinant RegIIIb. We tested its antibacterial spectrum in in vitro killing assays against S. Typhimurium wt, E. coli and B. subtilis. B. subtilis and E. coli were chosen as these strains were used in other studies assessing bacterial susceptibility to RegIIIc [28]. Since Bacillus spp. is not a normal member of the GI tract, several other Gram + bacteria typical for the GI tract were also included in the analysis (Lactobacillus spp., Enterococcus spp., Clostridium spp.). Aliquots of bacterial suspensions were incubated with recombinant RegIIIb and bacterial survival was analyzed after 30 minutes. The cfu of the negative control (no RegIIIb) was taken as 100% survival rate. Interestingly, recombinant purified RegIIIb had bactericidal activity against E. coli but not S. Typhimurium and B. subtilis. We tested an extended panel of commensal gut bacteria but found no clear killing preference for Gram + vs. Gram 2 species (Fig. 2A, B). The related intestinal C-type lectin, RegIIIc is known to selectively kill Gram + bacteria [28]. These data suggested that the two lectins may have complementary antibacterial profiles. Effective doses for bactericidal activity were below 2.5 mM (Fig. 2A, Fig. S2B), which is in the range of the RegIIIb concentrations observed in the S. Typhimurium-infected intestine (see above). Since S. Typhimurium was found to be resistant to RegIIIb, we hypothesized that S. Typhimurium-induced RegIIIb could thus affect some commensal microorganisms in the intestinal lumen upon gut inflammation, and thereby contribute to the substantial ecological changes in gut microbiota and observed pathogen overgrowth during S. Typhimurium wt infection [15,19,45].

Molecular structure correlates with bactericidal activity
To identify the mechanisms of S. Typhimurium resistance to RegIIIb we decided to determine the molecular target of RegIIIb on the bacterial cell envelope. An excess of peptidoglycan (PGN) completely blocked RegIIIb bactericidal activity (data not shown) consistent with the previously reported role of PGN as RegIIIb target [46]. Although RegIIIb did bind to bacteria containing various PGN types, including S. Typhimurium in vivo (data not shown), bactericidal activity was preferentially directed against particular PGN types. Specifically, some sensitive bacteria carried the negatively charged amino acid residue diaminopimelic acid at position 3 of the PGN pentapeptide (PGN-DAP: E. coli, Clostridium butyricum), whereas most resistant bacteria carried a neutral or positively charged amino acid at this position (B. subtilis, amidated diaminopimelic acid; Lactobacillus casei, L. murinus and Enterococcus faecalis, lysine) [47,48,49]. Interestingly, loop 2 of RegIIIb, which is homologous to the usual C-type lectin binding site but apparently not involved in binding the carbohydrate portion of peptidoglycan [46], contains a positively charged arginine (R135), while the equivalent residue is a threonine in RegIIIc which kills bacteria with PGN-Lys. In an unrelated insect peptidoglycan binding protein PGRP-LE a similar arginine -threonine substitution controls selective activity against PGN-DAP vs. PGN-LYS [50]. To test the hypothesis that arginine 135 of RegIIIb promotes selective cidal interaction with negatively charged PGN, we generated a RegIIIb R135T variant by site-directed mutagenesis. RegIIIb R135T had weaker cidal activity against E. coli but gained cidal activity against B. subtilis that was lacking in the wild-type protein (Fig. 2C) supporting the role of amino acid 135 in selective interaction with certain PGN types. The still moderate activity of RegIIIb R135T against B. subtilis suggested multiple additional interactions as observed for other PGNbinding proteins [51]. This was also evident from wild-type RegIIIb resistance of Lactobacillus reuteri despite its Lys-type peptidoglycan.

The O-antigen protects S. Typhimurium against RegIIIb killing
RegIIIb did not kill PGN-DAP containing S. Typhimurium but did kill E. coli containing the same type of PGN ( Fig. 2A). This observation was consistent with the uncompromised viability of Salmonella enterica isolated from infected gut contents [52]. As one possible resistance mechanism, Salmonella spp. outer membrane lipopolysaccharides (LPS) [53] could restrict access of RegIIIb to its target peptidoglycan in the periplasm. To test this hypothesis, we compared isogenic S. Typhimurium mutants with defined defects in LPS biosynthesis. These mutants lacked the O-antigen polymerase (rfc), the complete O-antigen (rfbP), the outer core and the Oantigen (galE) and the inner core, outer core and the O-antigen (rfaG) and certain lipid-A modifications (phoP, pagL and pagP) (Fig. 3C). Shortening of the polysaccharide component of LPS (Fig. 3A) progressively increased S. Typhimurium susceptibility to RegIIIb-mediated killing (Fig. 3B) as observed for other antimicrobial molecules [54]. Moreover, PhoP-dependent expression of pagP (involved in hepta-acetylation of lipid A with palmitate [55]) but not pagL (catalyzes the 3-O-deacylation of lipid A at position 3 [56]) was essential for S. Typhimurium resistance to RegIIIb (Fig. 3B). These data suggested that in wild-type S. Typhimurium carrying a rigid LPS layer, RegIIIb had limited access to peptidoglycan and therefore weak anti -S. Typhimurium activity. Interestingly, E. coli has similar LPS modification capabilities, but under our experimental in vitro conditions, these mechanisms were apparently insufficient to mediate resistance against RegIIIb in various E. coli strains.

RegIIIb selectively suppresses E. coli in a simple co-colonization model
The in vitro data suggested that RegIIIb killed various bacterial species but not S. Typhimurium. S. Typhimurium-induced RegIIIb could thus contribute to ecological changes in gut microbiota during S. Typhimurium infection [15,19,45]. To test this hypothesis, we established a simple experimental mouse model employing intestinal colonization of streptomycin-pretreated mice [39] with S. Typhimurium and a molecularly defined (see Material & Methods sections) non-pathogenic, streptomycin-resistant Escherichia coli strain (E. coli E2) from our mouse colony. S. Typhimurium colonization preferentially occurs in individuals with high initial E. coli abundance [9], which suggests potential relevance for S. Typhimurium/E. coli competition.
When given individually, both E. coli E2 and S. Typhimurium colonized the mouse intestine at high levels in streptomycin-treated mice (data not shown). Virulent S. Typhimurium induced intestinal inflammation as previously reported, while an avirulent S. Typhimurium invG ssrB strain as well as the E. coli E2 isolate did not induce obvious pathology (data not shown) in agreement with previous reports [15,39,57,58,59]. Interestingly, virulent S. Typhimurium wt suppressed co-administered E. coli E2 (Fig. 4A), while avirulent S. Typhimurium permitted stable high level co-colonization of both species (Fig. 4B). Our simple co-colonization model thus reproduced important aspects of S. Typhimurium-induced commensal suppression.
To test whether the host factor RegIIIb might be sufficient for E. coli E2 in vivo suppression, we administered recombinant RegIIIb to mice co-colonized by E. coli E2 and avirulent S. Typhimurium. A single dose of 80 mg RegIIIb (similar to endogenous RegIIIb levels induced by virulent S. Typhimurium wt, see above) administered at 5 h post infection rapidly suppressed E. coli but did not affect S. Typhimurium gut colonization (Fig. 4C). RegIIIb was thus sufficient for suppressing commensal E. coli E2 even in the absence of all other mechanisms that might further enhance this effect in the inflamed gut.
Typhimurium might exploit the host to suppress competing microbiota. To test this hypothesis, the simple E. coli/S. Typhimurium co-colonization model in streptomycin-pretreated mice was inappropriate. The competing microbiota is already weakened in this model. Consequently, even avirulent S. Typhimurium can efficiently colonize for several days even in the absence of intestinal inflammation.
To test if RegIIIb-induction could offer S. Typhimurium any benefit under more natural conditions, we tested conventional mice containing a normal gut microbiota (no streptomycin pretreatment). We determined the fecal density of facultative aerobic bacteria in our mouse colony by plating (Fig. 5A). This technique closely reflects the gut luminal bacterial counts based on previous data from plating, 16S RT PCR, in situ hybridization and immunofluorescence microscopy [9,15,39,60]. However, it is important to notice that luminal (or fecal) counts do not necessarily correlate with mucosal colonization levels. One group of mice was orally treated with a single dose of BSA, the other group with recombinant RegIIIb, both followed by an oral dose of S. Typhimurium (5610 7 cfu, by gavage). BSA-treated mice did not show efficient S. Typhimurium luminal colonization (Fig. 5A) as observed previously [9,39,61]. However, oral treatment with a single dose of RegIIIb suppressed endogenous Gram 2 facultative aerobic microbiota and enabled subsequent S. Typhimurium luminal colonization at moderate levels (Fig. 5A).
In another model, we followed long-term shedding of S. Typhimurium in streptomycin-pretreated mice during recolonization with competing microbiota, which terminates pathogen growth in the gut after approximately 4 days, in the absence of mucosal inflammation [15,60]. Due to its defect to trigger inflammation, the avirulent S. Typhimurium strain is overgrown by the competing commensal flora. We aimed at testing if oral supplementation of RegIIIb could interfere with reemerging microbiota-induced colonization resistance. Indeed, daily RegIIIb administration significantly prolonged S. Typhimurium fecal shedding (Fig. 5B). The combined data showed that RegIIIb can enhance S. Typhimurium gut luminal colonization in the presence of complex commensal microbiota.

Discussion
S. Typhimurium induces a complex inflammatory response in the intestinal mucosa. This inflammation has a profound impact on gut microbiota composition. Among others, microbiota that competes with S. Typhimurium is suppressed whereas S. Typhimurium colonization is promoted. The molecular mechanisms of this process are probably complex and poorly understood [10]. On one side, altered nutrient availability in the inflamed gut may lead to positive selection of the pathogen over the commensal flora [16,24]. On the other side numerous host defense factors are differentially regulated during intestinal inflammation [25,42,43,62] and could mediate selective killing of the commensal microbiota [17,20]. Their relevance for facilitating S. Typhimurium colonization has remained unclear.
Among the host factors induced by S. Typhimurium, the Ctype lectin RegIIIb is particularly strongly upregulated. RegIIIb can block mucosal infections with pathogenic Yersinia spp. although it does not inhibit Yersinia spp. directly [31]. RegIIIb is also highly related to another C-type lectin, RegIIIc that kills Gram + bacteria [28] and protects against mucosal Listeria infections [30]. For these reasons, we studied the role of RegIIIb in enteric salmonellosis.
We showed that S. Typhimurium infection induced elevated RegIIIb protein levels in the intestine. Recombinant RegIIIb bound peptidoglycan in vitro and killed a diverse set of both Gram 2 and Gram + bacteria but not S. Typhimurium. Site-directed mutagenesis of RegIIIb and comparison of various S. Typhimurium mutants suggested that peptidoglycan structure and lipopolysaccharide composition (for Gram 2 bacteria) might explain the bactericidal activity spectrum of RegIIIb. Recently published work points out that HIP/PAP (hepatointestinal pancreatic/pancreatitis associated protein), a human ortholog of RegIIIc interacts with the carbohydrate moiety of peptidoglycan which may be also the case for RegIIIb [46]. The observed antibacterial spectrum of RegIIIb was clearly different from the reported spectrum of the closely related RegIIIc suggesting that these two mucosal lectins might serve complementary functions in modulation of gut microbiota. We hypothesize that S. Typhimurium is protected against RegIIIb dependent killing by specific modifications mediating a rigid LPS layer. Possibly, differences between S. Typhimurium and E. coli in PhoP-dependent regulatory circuits [63] and/or subtle differences in LPS structure might affect the susceptibility for RegIIIb. Future work will have to address the structural mechanisms underlying differential RegIIIb killing of E. coli and S. Typhimurium.
Doses in the range of endogenous RegIIIb levels were orally administered and could efficiently suppress E. coli gut colonization and facilitate S. Typhimurium colonization. Our findings suggest that RegIIIb may contribute to inflammation-induced suppression of competing microbiota although we have no direct evidence of RegIIIb playing a role in vivo. Experiments using RegIIIb ko mice may aid clarifying this issue.
Presently, we do not know the exact effects of RegIIIb treatment on microbiota composition and function. This might be an important topic for future research. However, effects resulting from RegIIIb feeding were moderate compared to the much stronger facilitation of S. Typhimurium colonization in mice with acute intestinal inflammation. Thus, additional host factors and bacterial fitness factors apparently also contribute significantly to inflammationinduced microbiota changes and pathogen overgrowth. Possible candidates for these additional factors include the paralogue RegIIIc with a complementary bactericidal spectrum, defensins and other yet unidentified, Salmonella-encoded fitness factors. Further work will be required to elucidate the respective relevance and the unknown mechanisms, and how they might be integrated with the function of the previously identified host and bacterial factors in the microbiotapathogen interplay in the inflamed intestine.