DOI: 10.5061/dryad.1zcrjdg2p
title: A temporary cholesterol-rich diet and bacterial extracellular matrix factors favor Salmonella spp. biofilm formation in the cecum

description: Asymptomatic chronic carriers occur in approximately 5% of humans infected
 with Salmonella enterica serovar Typhi (S. Typhi) and represent a critical
 reservoir for bacterial dissemination. While chronic carriage primarily
 occurs in the gallbladder through biofilms on gallstones, additional
 anatomic sites have been suggested that could also harbor Salmonella. S.
 Typhimurium, orally-infected 129X1/SvJ mice were pre-treated with a
 cholesterol-rich diet as a gallstone model for chronic carriage. We
 observed S. Typhimurium in feces and the cecum during early and persistent
 infection. Furthermore, bacterial biofilm-like aggregates were associated
 with the cecum epithelium at 7- and 21-day post-infection (DPI) in mice on
 a lithogenic diet (Ld) and correlated with an increase in cecal
 cholesterol at 21 DPI. Salmonella’s extracellular matrix (ECM) was
 demonstrated as important in colonizing the cecum, as survival and
 aggregate formation significantly decreased when mice were infected with a
 quadruple ECM mutant strain. Gallbladder Salmonella counts were low at 36
 DPI while cecal Salmonella were high, suggesting that gallbladder
 colonization was likely not responsible for the high cecal burden. All
 cecum phenotypes were significantly diminished in mice fed a normal diet
 (Nd). Finally, we examined the capability of S. Typhi to colonize the
 cecum and showed S. Typhi in feces and in aggregates in the cecum up to 7
 DPI, with slightly higher counts in mice fed a Ld compared to Nd. Our
 findings suggest that the cecum, particularly under cholesterol-rich
 conditions, serves as an adaptive niche for Salmonella spp.
 aggregates/biofilms and is a putative site for long-term infection
description: <strong>Ethics statement. </strong>The mice used in
 this study were housed in strict adherence to the guidelines established
 by the Abigail Wexner Research Institute (AWRI) Institutional Animal Care
 and Use Committee (IACUC). All experimental procedures involving mice were
 conducted with the appropriate authorization obtained through AWRI IACUC
 protocol AR18-00080, which complied with the statutory requirements
 outlined in the <em>Guide for the Care and Use of Laboratory
 Animals.</em> <strong>Bacterial
 Strains</strong>. Previously, we described the constructions of
 <em>Salmonella</em> mutant strains deficient in biofilm
 formation capacity (35). The following bacterial strains were utilized in
 this study: <em>S.</em> Tm 14028 (<em>S.</em>
 Tm<em><sup>wt</sup></em>; JSG210),
 <em>S.</em> Tm curli mutant (<em>S.</em>
 Tm<em><sup>ΔcsgA</sup></em>; JSG3540)<em>,
 S.</em> Tm colanic acid mutant<em>
 </em>(<em>S.</em>
 Tm<em><sup>ΔwcaM</sup></em>;
 JSG3712)<em>,</em> <em>S.</em> Tm O antigen
 capsule mutant (<em>S.</em>
 Tm<em><sup>ΔyihO</sup></em>; JSG3672),
 <em>S.</em> Tm cellulose mutant (<em>S.</em>
 Tm<em><sup>ΔbcsE</sup></em>; JSG3838), the doble
 ECM mutant (<em>S.</em>
 Tm<em><sup>ΔcsAΔbcsE</sup>:
 </em>JSG3977<em>), S.</em> Tm quadruple ECM mutant
 <em>ΔwcaM, ΔcsgA, ΔyihO, ΔbcsE</em><sup>
 </sup>(<em>S.</em> Tm
 ECM<em><sup>mut</sup></em>; JSG3841),<em>
 S.</em> Tm promoter vector for curli (<em>S.</em>
 Tm<em><sup>lux:csgDEFG</sup></em>; JSG3977), and
 <em>S.</em> Typhi
 <em>rpoS</em><sup>+</sup> Ty2
 (<em>S.</em>
 Typhi<em><sup>wt</sup></em>; JSG698). Planktonic
 cultures were cultivated for 16 hours at 37°C in tryptic soy broth (TSB).
 All strains were regrown to an OD<sub>600 </sub>of 0.6 prior
 to infection. Mice were infected intragastrically with a dose of
 1x10<sup>7 </sup>for <em>S.</em> Tm or
 5x10<sup>7</sup> for <em>S.</em> Typhi.
 <strong><em>In
 vitro</em></strong><strong> bacterial growth
 curves</strong>. <em>Overnight</em> (ON) cultures of
 <em>Salmonella</em> WT and mutant strains were normalized to
 an OD<sub>600 </sub>of 1. The cultures were then diluted
 1:1000 in fresh TSB medium and incubated at 37°C. Bacterial growth was
 monitored over a 15-hour period, with OD<sub>600
 </sub>readings taken every 15 min.
 <strong>Luciferase assay</strong>: O/N cultures of
 <em>S.</em>
 Tm<em><sup>lux:csgDEFG</sup></em><em>
 </em>were normalized to an OD<sub>600 </sub>of 1. The
 cultures were subsequently diluted 1:1000 in fresh Luria-Bertani (LB)
 medium and incubated at either 26°C or 37°C in microtiter plates in the
 presence or absence of cholesterol (well coating). The strain was grown
 over a 15-hour period, with luminescence (OD<sub>490</sub>)
 readings taken every 15 min. <strong>Bacterial
 shedding and fecal collection</strong>. Fresh fecal samples were
 obtained by individually placing mice in isolation containers until
 approximately 100mg of feces were collected. The collected fecal samples
 were homogenized in 1 mL of sterile PBS and plated on selective agar XLD
 (refer to the <em>bacterial isolation</em> section), followed
 by incubation for 24h (<em>S. </em>Tm) or 48h
 (<em>S.</em> Typhi) at 37°C.
 <strong>Clinical assessment of mice</strong>: Mice
 were evaluated at 7 and 21 DPI to assess phenotypic signs of the severity
 of the infection. The evaluation included measurements of body weight,
 assessment of general health (fur, eyes, posture, clinical complications),
 and observations of motility. <strong>Mouse
 infection model and tissue CFUs.</strong> Six-week-old female
 129X1/SvJ mice (The Jackson Laboratory, ME) were utilized in this study.
 Prior to infection, the mice were fed either a normal diet or a lithogenic
 diet for 6 weeks. The LD consisted of mouse chow supplemented with 0.5%
 cholesterol and 0.5% cholic acid (Envigo, TD 140673). Two weeks after the
 diet treatment, the mice were intragastrically infected with
 <em>S.</em> Tm or S. Typhi<sup> </sup>in 200µL of
 PBS containing the bacteria. <strong>CFUs from
 feces and tissues:</strong> <em>Bacterial
 isolation</em>. Mice were euthanized at 7 and 21 DPI. The liver,
 spleen, mesenteric lymph nodes, small intestine, large intestine, and
 cecum were isolated. Approximately 150mg of tissues or feces were
 homogenized in 1 mL of sterile 1X PBS. The samples were briefly
 centrifuged for 30s, and the supernatant was filtered through a 15 mm
 strainer. The filtrate was transferred to a U-bottom 96-well plate and
 subjected to centrifugation at 1500 rpm for 8 min at 5°C. The supernatants
 were transferred to new wells (x3), followed by centrifugation at 3700 rpm
 (8 min, 5°C) to obtain a bacterial pellet. The pellet was resuspended and
 plated on selective XLD agar to determine the bacterial load of each organ
 (<strong>Fig. S3, a</strong>). The remaining tissues were
 preserved for cholesterol analysis, histology, or flow
 cytometry.
 <strong>Ba</strong><strong>cterial flow
 cytometry:</strong> Following the isolation of bacteria (as
 described above), the pellet was blocked with 5% BSA for 10 min and washed
 with 3% BSA in PBS at 3700 rpm (8 min, 5°C). Primary antibodies (5 μg
 ml<sup>-1</sup>) and secondary (2.5 μg
 ml<sup>-1</sup>) antibodies were incubated with the pellet at
 37°C for 20 min. Each antibody incubation was followed by three washes.
 The pellets were resuspended in 300µL of PBS, and the samples were
 analyzed using flow cytometry (BD LSRII), and the data were analyzed in
 FlowJo v10. The following antibodies were used: goat ⍺-<em>S.
 </em>Tm/Typhi CSA-1 (BacTrace, 5310-0322), mouse IgG2a ⍺-<em>S
 </em>Typhi LPS (Meridian Bio, C01362M), rabbit IgG ⍺-<em>S.
 </em>Tm (Thermo, PA-17244), ⍺-mouse IgG2a FITC (Invitrogen,
 11421082) and ⍺-goat AF647 (Invitrogen, A21469), and DAPI (Invitrogen,
 D1306). <strong>Cholesterol quantification:
 </strong>After the initial centrifugation of bacteria at 3700 rpm,
 50 µL of supernatant from homogenized cecum was collected, and the
 cholesterol concentration was measured using the Amplex™ Red Cholesterol
 Assay Kit following the manufacturer’s instructions.
 <strong>Histology</strong>: All tissues were fixed in
 10% neutral-buffered formalin (Fisher Scientific, MA) for 24 hours. The
 morphology core at Abigail Wexner Research Institute embedded the tissues
 in paraffin. Deparaffinization and rehydration of samples were carried out
 before staining. Permeabilization was performed by incubating the samples
 in PBS containing 1% Triton X-100 for 1h at 37°C. Histological sections of
 4μm were used for H&E staining, IHC, or IF microscopy. For IHC and
 IF, the primary antibody (Ab) incubation was performed by overnight
 incubation at 4°C in antibody dilution buffer (3% BSA and 0.35% Triton
 X-100 in PBS) with 0.5-1 μg ml<sup>-1</sup> for each Ab. The
 antibodies used included a goat IgG ⍺-<em>S. </em>Tm/Typhi
 CSA-1, mouse IgG2a ⍺-<em>S</em> Typhi LPS, rabbit IgG
 ⍺-<em>S.</em> Tm, rabbit ⍺-Villin 1 (Abclonal, A11650), rabbit
 IgG ⍺-Vi Antigen (Abcam, ab79002), Human ⍺-amyloid APP 3H3 (Creative Bio,
 TAB-0801CLV), and pAb rabbit ⍺-<em>
 </em><em>S.</em> Tm CsgA (gift from Dr. Cagla Tükel).
 For IHC, after primary Ab incubation, the VECTA Elite ABC Kit-HRP (Vector
 Lab, PK-6101 and PK6105), UltraTek Bio-HRP ⍺-goat IgG (ScyTek Labs,
 ABL/AGL015) were used, and staining was revealed using the metal enhancer
 DAB Kit (Thermo, PI34065). The slides were then counterstained with
 hematoxylin for 30 seconds, and washed in dH<sub>2</sub>O for
 1 min. Finally, coverslips were applied using ProLong mounting media
 (Thermo, P36930).
description: # A temporary cholesterol-rich diet and bacterial extracellular matrix
 factors favor *Salmonella* spp. biofilm formation in the cecum
 [https://doi.org/10.5061/dryad.1zcrjdg2p](https://doi.org/10.5061/dryad.1zcrjdg2p) ## Description of the data and file structure We investigated the adaptive survival and biofilm formation of *Salmonella* in the gastrointestinal tract of immunocompetent mice that had been pre-fed a lithogenic diet. To track bacterial presence across various tissues, we utilized CFU assays, flow cytometry, histopathology analysis, ex vivo cholesterol quantification, and immunohistochemistry/immunofluorescence. ### Files and variables #### File: Fig_1.xlsx **Description:**  This figure shows a schematic representation of the experimental strategy. 129X1/SvJ mice were pre-treated with either a lithogenic (Ld) or a normal diet (Nd) for 6 weeks and infected by the intragastric route with 1x107 CFUs of *S*. Tm*wt*. The severity level is denoted with black arrows, a score of ≤0 representing a healthy animal. Gastrointestinal tract colonization of mice was assessed by quantifying CFUs and using flow cytometry. Mesenteric lymph nodes (MLN), small intestine (SI), and large intestine (LI). Flow cytometry analyses were normalized to a PBS control. At least six mice per condition were included. The dotted line represents ± quartiles. #### File: Fig_2.xlsx  **Description:** Mice fed with an Ld were infected with *S*. Tm ECM single mutants each lacking curli (*ΔcsgA*), colanic acid (*ΔwcaM*), O antigen capsule (*ΔyihO*), cellulose (*ΔbcsE*) genes, or a quadruple mutant lacking in all the above (*ECM*mut). Bacterial burden was analyzed to quantify CFUs. CFUs were determined by culturing tissue samples on selective agar. Mesenteric lymph nodes (MLN), small intestine (SI), and large intestine (LI). Pearson correlation analysis was conducted between CFUs obtained from feces and tissues at 7 and 21 DPI. R values from Pearson’s correlation analysis are presented in the table. Bacterial burden in tissues of infected mice on a Ld at 36 DPI. A minimum of four mice per condition were utilized for these analyses. The dotted line represents ± quartiles. #### File: Fig_3.xlsx  **Description:** Anatomic and morphological changes in the cecum of infected mice were examined. H&E staining of histological sections of the cecum. Scale bar: 50μm. Zoom image: Crypt hyperplasia (#) and inflammatory cells are denoted in 1. Ulceration (#) and neutrophilic inflammation are denoted in 2, scale bar: 100μm. Histopathological and inflammatory scores were evaluated from these sections. The scores were normalized to the PBS control. Error bars represent ± SD. At least four mice per condition were included in this study. #### File: Fig_4.xlsx  **Description:** Cholesterol concentrations were determined in homogenized tissues at 21 DPI. Small intestine (SI) and large intestine (LI). Frozen samples were isolated at 21 DPI from *S.* Tm*wt*-infected mice. Staining was performed using Red Oil O (red) to identify cholesterol and DAPI (blue) as a counterstain. Cecal epithelium: cEpi. Scale bar: 100 μm. Asterisk represents the cecal lumen. Quantification of the Red Oil O signal was normalized with DAPI. RAD: Relative abundance distribution. Distribution of *S.* Tm in the cecum samples from infected mice using IHC. Scale bar: 20 μm. Visual association between *S.* Tm and cEpi was carried out using IHC. The black dashed line delineates the cEpi border, and the asterisk indicates the cEpi. Black arrows show a positive signal for S. Tm. Scale bar: 5μm. Quantification of the relative association between *S.* Tm and the cEpi. IHC of *S.* Tm curli expression in the cecum, scale bar: 50 μm. Quantification of the curli positive signal. All IHC quantifications were normalized to the PBS control and quantified using ImageJ. Error bars represent ± SD. The dotted line represents ± quartiles in c). At least four mice per condition were utilized. #### File: Fig_6.xlsx  **Description:** Mice fed with either Ld or Nd were orally administered with 5x107 CFUs of *S.* Typhi WT *rpoS+* (*S.* Typhi*wt*). The extent of colonization in mouse tissues was assessed using CFUs and flow cytometry. Mesenteric lymph nodes (MLN), small intestine (SI), and large intestine (LI). Dot plots illustrate the identification of *S.* Typhi’s population in the cecum and LI at 7 DPI. IHC images visually represent the presence of *S.* Typhi*wt* in the cecum of infected mice at 7 DPI, scale bar: 20 μm. IF to identify *S.* Typhi*wt* in the cecal lumen. Staining was performed using ⍺-Vi Antigen (ViAg), ⍺-*S.* Typhi LPS (LPS) and ⍺-CSA-1 (*S.* Typhi), scale bar: 2.5 μm. *S*. Typhi biofilms in the cecum. Biofilms were detected using the same protocol for *S*. Tm in Fig. 5. Scale bar 5μm. The flow cytometry and quantification data were normalized to the PBS control in all cases. All IHC quantifications were quantified using ImageJ. At least five mice per condition were utilized. #### File: SFig_1_.xlsx  **Description:** Bacterial growth curves of *Salmonella* cultured in TSB medium over a 15-hour period, with readings taken every 15 minutes (OD600). Quantification of the area under the curve (AUC) from the growth curves.  #### File: SFig_2.xlsx  **Description:** Gastrointestinal tract colonization was assessed by quantifying CFUs. Mice fed with either a Ld or a Nd were subsequently infected with *S.* Tm ECM single mutants each lacking curli (*ΔcsgA*), colanic acid (*ΔwcaM*), O antigen capsule (*ΔyihO*) or cellulose (*ΔbcsE*) genes or a quadruple mutant lacking in all the above (*ECM*mut). CFUs of tissue were isolated from infected mice with either a single or quadruple ECM mutant strain. Mesenteric lymph nodes (MLN), small intestine (SI), and large intestine (LI). The dotted line represents ± quartiles. A minimum of three mice per condition were utilized for these analyses. #### File: SFig_3.xlsx  **Description:** Methodology schematic. Samples were isolated from various tissues or feces to optimize the isolation of bacteria, cholesterol measurement, and flow cytometry. Quantification by serial dilution of cholesterol isolated from the cecum of infected mice at 21 DPI. Quantification of distribution of *S.* Tm in the cecum samples from infected mice using IHC. Primary Ab *S.* Tm LPS was employed. IF image showing the visualization of biofilms in the cecum from mice infected with *S.* Tmwt at 7 DPI. Asterisks represent the lumen of the cecum. The primary Abs ⍺-amyloid proteins (red, curli), ⍺-CSA-1 (yellow, *S.* Tm), ⍺-Villin 1 (green, a cecal epithelium marker), and Fluorescent Brightener 28 (blue, cellulose) were used. Scale bar: 5μm. Dot plots of colonies displaying *S.* Typhi morphology were analyzed by flow cytometry. The primary Abs ⍺-*S.* Typhi CSA-1 and ⍺-*S.* Typhi LPS were used. Microscopic examination (H&E) was analyzed to determine anatomic changes at 7 in the cecum of mice infected with *S.* Typhi*wt*. Optical magnification 40X, scale bar: 50 μm. At least four mice per condition were utilized. Error bars represent ± SD. #### File: SFig_4.xlsx  **Description:** Luciferase activity of curli (*CsgDEFG*) promoter *S.* Tm (*S.* Tm*lux:csgDEFG*) cultured in LB medium over a 15-hour period with readings taken every 15 minutes (OD490). LB+Cholesterol represents cultures grown on cholesterol-coated plates. Cultures grown under laboratory or physiological temperatures. ## Code/software Excel files can be opened with Microsoft or LibreOffice