IAI Accepted Manuscript Posted Online 29 February 2016 Infect. Immun. doi:10.1128/IAI.00163-16 Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Revised manuscript

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Lactobacilli reduce Helicobacter pylori attachment to host gastric epithelial cells

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by inhibiting adhesion gene expression

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Nele de Klerka, Lisa Maudsdottera, Hanna Gebreegziabhera, Sunil D. Saroj, Beatrice

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Erikssona, Olaspers Sara Erikssona, Stefan Roosb, Sara Lindénc, Hong Sjölindera, and

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Ann-Beth Jonssona#

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Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm

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University, SE-10691 Stockholm, Swedena; Department of Microbiology, Swedish

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University of Agricultural Sciences, SE-75007 Uppsala, Swedenb; Department of

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Medical Chemistry and Cell Biology, University of Gothenburg, SE-40530

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Gothenburgc

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#Corresponding author

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Ann-Beth Jonsson, Address: Department of Molecular Biosciences, The Wenner-

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Gren Institute, Stockholm University, Svante Arrhenius väg 20C, SE-10691

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Stockholm, Sweden, E-mail: [email protected], Phone: +46-8164154

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Running title: Lactobacilli inhibit H. pylori sabA expression

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ABSTRACT

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The human gastrointestinal tract, including the harsh environment of the stomach,

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harbors a large variety of bacteria of which Lactobacillus species are prominent

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members. Molecular mechanisms by which species of lactobacilli interfere with

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pathogen colonization are not fully characterized. In this study we aimed to study the

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effect of lactobacilli strains upon Helicobacter pylori initial attachment to host cells.

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Here we report a novel mechanism by which lactobacilli inhibit adherence of the

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gastric pathogen H. pylori. In a screen with Lactobacillus isolates we found that only

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a few could reduce adherence of H. pylori to gastric epithelial cells. Decreased

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attachment was not due to competition for space or due to lactobacillus-mediated

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killing of the pathogen. Instead, we show that lactobacilli act on H. pylori directly by

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an effector molecule that is released into the medium. This effector molecule acts on

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H. pylori by inhibiting expression of the adhesin-encoding gene sabA. Finally, we

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verified that inhibitory lactobacilli reduced H. pylori colonization in an in vivo model.

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In conclusion, certain Lactobacillus strains affect pathogen adherence by inhibiting

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sabA expression and thereby reducing H. pylori binding capacity.

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INTRODUCTION

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The human body is home to an extensive microbiota that outnumber our human cells

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10 to 1. This bacterial community plays a role in functions that are beneficial to the

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host, such as nutrition, immune function, development and defense against pathogens

43

(1). Even in the stomach, an organ previously thought to be sterile because of its low

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pH, the microbial load iscontains 101-103 cfu/ml gastric content bacteria, although it is

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lower than in the colon (1010-1012 cfu/ml) (2, 3). In recent years, and due to new

46

technologies that facilitate the large-scale analysis of genetic and metabolic profiles,

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the gut microbiota has been extensively studied. Healthy individuals and patients with

48

various clinical conditions differ in their microbiota composition, which strongly

49

suggests that modification of the microbiota may have an impact on health (4). A

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well-known member of the normal microbiota is the genus Lactobacillus. These lactic

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acid bacteria are considered beneficial for health and are widely studied for the

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inhibition of pathogens.

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Helicobacter pylori is a Gram negative, helical shaped, microaerophilic, human-

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specific bacterium that colonizes the stomach of more than half of the world

55

population (5). H. pylori cause chronic gastritis and when left untreated can

56

eventually lead to the development of gastroduodenal ulcers and gastric cancer in a

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subset of infected individuals (5). Although the majority of Helicobacter bacteria

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remain in the mucus layer lining the gastric epithelium (6-8), it is widely accepted that

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the bacteria in contact with epithelial cells cause disease. H. pylori produces several

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important virulence molecules that interact with epithelial cells and immune cells. The

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cag Pathogenicity Island (PAI) encodes for type 4 secretion systems that inject CagA

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into target cells upon attachment (9-11). After CagA injection, CagA undergoes

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tyrosine phosphorylation and causes actin-cytoskeletal rearrangements, proliferation

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of host cells and IL-8 release, all factors important for disease development. Another

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important virulence factor is VacA, a secreted toxin that induces vacuoles in target

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gastric cells (12). Lactobacilli have been studied in relation to H. pylori but mainly as

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a possible additive to antibiotic treatment (13). The mechanisms behind pathogen

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inhibition mediated by lactobacilli are still largely unknown.

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In this study, we investigated how lactobacilli can affect the early colonization of H.

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pylori of the gastric epithelium. Three lactobacilli strains that could reduce H. pylori

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adhesion were identified in a screen with 28 lactobacilli strains. The effector molecule

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is a component that can be released into the surroundings. The inhibitory lactobacilli

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act on H. pylori directly by reducing the expression of the SabA adhesin on a

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transcriptional level. The ability of released effector molecules from lactobacilli

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strains to reduce H. pylori attachment is intriguing. The finding opens for research to

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characterize the Lactobacillus effector molecule that reduces H. pylori attachment,

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and further investigate its mode of action. Since attachment is the first and crucial step

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to establish infection, any compound able to inhibit pathogen adherence might be a

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possible novel therapeutic agent and help battle the continued problem of

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antimicrobial resistance.

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MATERIALS AND METHODS

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Bacterial strains and cell lines.

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The gastric epithelial cell lines AGS (ATCC CRL-1739) and MKN45 (Japan Health

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Science Research Resource Bank JCRB0254) were cultured in RPMI-1640 (Life

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Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-

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Aldrich). The cells were maintained at 37°C and 5% CO2 in a humidified

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environment. The cells were seeded into tissue culture plates the day before the

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experiment to form a monolayer overnight. At the start of each experiment, the cell

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culture medium was replaced with RPMI without serum.

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The Helicobacter pylori strains J99 (ATCC 700824), J99ΔSabA (described in (14)

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and kindly provided by Thomas Borén from Umeå University), 67:21 (described in

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(15)) and SS1 (described in (16)), were grown on Colombia blood agar plates

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(Acumedia) supplemented with 8% defibrinated horse blood and 8% inactivated horse

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serum (Håtunalab) for three days at 37°C under microaerophilic conditions, i. e. in an

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incubator with 5% O2 10% CO2 and 85% N2. J99ΔSabA was grown on plates

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supplemented with chloramphenicol. The Lactobacillus strains that were used have

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been described or isolated in connection to the study of Roos et al. (17), obtained

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from culture collections, or were a gift from BioGaia AB and are listed in Table 1.

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Lactobacilli were grown on Rogosa agar plates and cultured overnight in MRS broth

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(Oxoid) at 37°C and 5% CO2 in a humidified environment.

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Urease assay.

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AGS cells in a 96-well plate were infected with H. pylori 67:21 alone or in

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combination with lactobacilli at a multiplicity of infection (MOI) of 100 for each

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bacterium. After 2 h of incubation, the unbound bacteria were washed away 3 times

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with 50 mM potassium phosphate pH 6.8. Urease assay buffer (50 mM potassium

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phosphate pH 6.8, 250 mM urea, and 20 μg/ml phenol red) was added, and the

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absorbance at 560 nm was measured every 10 min for 2 h. A dilution series with

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known amounts of bacteria was used as a standard.

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Adhesion assays by viable counts.

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H. pylori from plates were suspended to homogeneity in RPMI to an optical density

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of 0.7, i. e., 108 cfu/ml. Lactobacillus strains from overnight cultures were suspended

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in RPMI to an optical density of 1.0. Epithelial cells in 48-well plates were infected

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with H. pylori alone or together with lactobacilli at an MOI of 100 for each species.

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After 2 h of incubation, the cells were washed 3 times with PBS to remove any

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unbound bacteria. The host cells were lysed by treatment with 1% saponin in RPMI

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for 5 min. The number of adhered colony forming units was determined by serial

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dilution and spreading the lysate on agar plates. The H. pylori on blood agar plates

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was incubated for 4-7 days, and Rogosa plates with lactobacilli were incubated for 2

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days.

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Pretreatment of bacteria: Heat-killed lactobacilli were obtained by incubation at 95°C

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for 15 min. Formaldehyde-killed lactobacilli were obtained by fixing in 4%

125

formaldehyde for 15 min at room temperature. Residual formaldehyde was removed

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by 3 washing steps of resuspending the bacteria in 1 ml RPMI and centrifugation at

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10 000 × g for 1 min. Treated bacterial samples were spread on plates to confirm that

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all bacteria were dead.

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Pretreatment of host cells: Host epithelial cells were fixed by incubation with 4%

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formaldehyde in RPMI for 15 min at room temperature and subsequently washed 3

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times with RPMI to remove residual formaldehyde.

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H. pylori viability assay.

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For assessment of H. pylori viability, co-incubation of H. pylori with lactobacilli on

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the host cells was conducted as described above for 2 h. The supernatants were saved,

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and the cells were treated with 1% saponin in RPMI for 5 min and pooled with the

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supernatants. The number of viable bacteria was determined by serial dilution and

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spreading on plates as described above.

139 140

Experiments with conditioned medium (CM).

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Conditioned media (CM) from lactobacilli was prepared by incubating lactobacilli in

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RPMI at approximately 2 × 107 cfu/ml for 2 h at 37°C and 5% CO2. The suspension

143

was filtered through a 0.2 μm sterile filter to remove the bacterial cells.

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Preincubation of H. pylori in CM: H. pylori was resuspended in CM to approximately

145

2 × 107 cfu/ml and incubated for 2 h at 37°C. To remove the CM, the suspension was

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centrifuged at 4000 × g for 10 min and resuspended in RPMI. The CM-pretreated H.

147

pylori was added to AGS cells at an MOI of 100. At 2 h post-infection, the unbound

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bacteria were washed away, and the bound bacteria were plated for viable counts.

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Heat treatment of CM was done at 95°C for 15 min.

150 151

Microscopy.

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The bacteria were resuspended in PBS, and 1 μg of DyeLight NHS ester (Thermo

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Scientific) was added per 108 cfu. After incubation for 15 min at 37°C, the bacteria

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were washed with Tris-buffered saline (50 mM Tris and 150 mM NaCl) followed by a

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wash with RPMI. The AGS gastric epithelial cells, grown on poly-D-lysin coated

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coverslips, were infected with the stained bacteria to an MOI of 100. The bacteria

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were allowed to adhere to the host cells for 2 h, after which the unbound bacteria were

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washed away three times with RPMI. The cells were fixed with 4% formaldehyde for

159

10 min at room temperature and subsequently mounted in Vectashield mounting

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medium. Bright-field and fluorescence microscopy images were taken with an

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inverted Zeiss Cell Observer microscope.

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qPCR analysis.

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The H. pylori bacteria that had been incubated in conditioned media from lactobacilli

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for 2 h were resuspended in lysis buffer (30 mM Tris-HCl, 1 mM EDTA, 15 mg/ml

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lysozyme, and proteinase K) and incubated for 20 min at RT, with 10 sec vortexing

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and 2 min rest cycles. The RNA was isolated using the RNeasy kit (Qiagen)

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according to the manufacturer’s instructions. To remove the genomic DNA, the RNA

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was incubated with Turbo-DNase (Ambion) for 1 h at 37°C. The RNA was then

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purified with an RNA Cleanup & Concentrator kit (Zymo Research). The complete

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removal of genomic DNA from the RNA was confirmed by PCR with primers for the

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H. pylori housekeeping gene gyrB. SuperScript VILO Mastermix (Invitrogen) was

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used to synthesize the cDNA. Quantitave PCR was performed using a LightCycler

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480 (Roche) and SYBRGreen I Master kit (Roche). The primers used are listed in

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Table 2. The SabA fwd1 and rev1 primers were used for detection of sabA in H.

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pylori strains 67:21 and J99, the SabA P1 primer pair was used to detect sabA in the

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SS1 strain. All of the primers were designed using Primer-BLAST software, except

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for the qPCR primers for the housekeeping gene gyrB that were described in (18). The

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PCR program was a follows: initial denaturation at 95°C for 10 min followed by

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amplification for 40 cycles with denaturation at 95°C for 10 sec; annealing at 50°C

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for 20 sec and extension at 72°C for 20 sec. The melting curve analysis was as

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follows: 95°C for 5 sec, 65°C for 1 min and then increasing to 95°C with 0.08°C/sec.

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The expression was normalized against the housekeeping gene gyrB. The expression

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levels were expressed as the fold change compared to the control samples.

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Western blotting

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H. pylori that had been incubated in conditioned media from lactobacilli for 2 h were

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resuspended in sample buffer containing 5% β-mercaptoethanol, separated on

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10% SDS-PAGE gels and transferred to Immobilon-P membranes (Millipore).

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SabA was detected using a rabbit polyclonal antibody (kindly provided by

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Thomas Boren and Anna Arnqvist). For quantification of protein expression

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levels, a polyclonal AhpC antibody (19) was used as a normalization control. The

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SabA and AhpC antibodies were detected using infrared (IR)-reactive dye-

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conjugated goat anti-rabbit 800CW secondary antibodies (Li-Cor) and visualized

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using an Odyssey IR scanner (Li-Cor). ImageJ analysis software was used to

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analyze image files. Protein expression was quantified from two independent

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experiments in duplicate.

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Mouse model of infection.

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The hCD46Ge transgenic mouse line (CD46+/+) harbors the complete human CD46

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gene, expresses CD46 in a human-like pattern (20-22) and is susceptible to H. pylori

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infection (23). The mice, 5-7 weeks old, were fed ad libitum and monitored daily. To

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study the influence of inhibitory lactobacilli on the colonization of H. pylori in the

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gastric tract, the normal flora was reduced by antibiotic treatment in drinking water

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for 2 days. The antibiotic solution contained 1 g/L ampicillin, 1 g/L neomycin, 1 g/L

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metronidazole and 0.5 g/L vancomycin (Sigma Aldrich). The mice were left without

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antibiotics in water for 18 h before inoculation with 108 cfu L. gasseri 1 or L.

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salivarius 1 suspended in 100 μl PBS by gavage twice per day for two days. The mice

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(n=6) were then infected perorally with 108 cfu of the mouse adapted H. pylori strain

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SS1 in Brucella broth, alone or together with 108 cfu L. gasseri 1 or L. salivarius 1. At

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6 h post-infection, the mice were sacrificed, and the stomach tissue was collected. The

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bacterial counts were determined by plating the serial dilutions of the homogenized

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samples on selective Colombia blood agar plates containing 200 μg/ml bacitracin, 100

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μg/ml vancomycin, 10 μg/ml nalidixic acid and 3.3 μg/ml polymixin B. The

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Helicobacter pylori colonies were identified by morphology and urease activity. The

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mouse experiments described in the present study were conducted at the animal

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facility at Stockholm University. All animal care and experiments were conducted

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according to the institution's guidelines. All of the protocols were approved by the

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Swedish Ethical Committee on Animal Experiments.

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Statistical analysis.

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All of the experiments were performed at three independent occasions with triplicate

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samples, except for the qPCR, for which the results were obtained in three

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independent experiments with duplicate samples. The differences between groups

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were analyzed using ANOVA (analysis of variance) followed by the Bonferroni post

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hoc test. Statistical analysis of the ratios or relative values was performed on the log

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ratios. The data from the mouse experiment were analyzed with the Kruskal-Wallis

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test. A p-value below 0.05 was considered statistically significant. The error bars

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represent standard deviations. The statistical analysis was performed using GraphPad

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Prism 5 software.

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234 235

RESULTS

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Certain lactobacilli can inhibit adhesion of H. pylori to host gastric epithelial

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cells.

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Here, we examined whether lactobacilli can affect the early colonization of H. pylori

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to human gastric epithelial cells. In a screen using urease activity as measurement for

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adhesion, we found that 3 out of 28 Lactobacillus strains tested could reduce

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attachment of the H. pylori strain 67:21 to AGS cells (Fig. 1A). The lactobacilli used

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in this study do not express any urease activity (data not shown), but to exclude any

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false positives due to interference of the lactobacilli with H. pylori urease activity, we

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confirmed the inhibitory effect by the viable count method using five representative

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strains. In line with the results from the urease assay, L. salivarius 1 (L.sal1) and L.

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rhamnosus 1 (L.rham1) were non-inhibitory, whereas L. gasseri 1 and 2 (L.gas1 and

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L.gas2, respectively) as well as L. brevis (L.bre) inhibited H. pylori adhesion (Fig.

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1B).

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To assess whether the lactobacillus-mediated inhibition was cell line specific, we used

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the human gastric epithelial cell line MKN45. Indeed, L.gas1 and L.bre inhibited H.

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pylori 67:21 adhesion to the MKN45 cells too (Fig. 1C). To verify that these results

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were relevant to other H. pylori strains, we tested H. pylori strain J99 on the AGS

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cells. Again, L.gas1 and L.bre reduced H. pylori adhesion (Fig. 1D). In summary, 3

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out of 28 lactobacilli strains reduced H. pylori attachment to the target cells. The

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reduced adherence was not dependent on a specific cell line or H. pylori strain.

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H. pylori colonization is inhibited by lactobacilli in vivo.

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To study whether H. pylori colonization was also reduced by lactobacilli in vivo, we

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infected mice with H. pylori in the presence of the inhibitory L.gas1. We used

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transgenic mice that expressed the human protein CD46 to mimic a more human-like

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stomach, and the mouse adapted H. pylori strain SS1. The mouse model was used

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since it has been shown that H. pylori infection reduces human CD46 in gastric tissue

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(24) and that CD46 transgenic mice are susceptible to H. pylori gastric colonization

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(23).

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Before the experiments in mice, we confirmed that the inhibitory lactobacilli reduced

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the attachment of SS1 similar to 67:21 and J99 (Fig. 2A). First, the microbiota of the

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mice was reduced by antibiotic treatment for two days. Then, the mice were

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inoculated perorally with L.gas1, L. sal1 or control buffer twice a day for two days

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before infection with H. pylori. The mice treated with the inhibitory L.gas1 had less

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H. pylori bacteria in their stomach than the mice infected with H. pylori, whereas the

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H. pylori level in mice treated with the non-inhibitory L.sal1 was not reduced (Fig.

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2B). In summary, these data show that L.gas1 can also reduce the initial colonization

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of H. pylori in vivo.

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Lactobacilli do not compete for space and are not bactericidal.

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Several possible mechanisms have been described on how lactobacilli could protect

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against pathogen colonization (1). To investigate whether the inhibitory lactobacilli in

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this study could act synergistically to reduce H. pylori attachment, we used a mixture

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of two lactobacilli strains in an adhesion assay. The combination of L.gas1 and L.bre

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did not increase the inhibitory effect on H. pylori adhesion, suggesting that both

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lactobacilli strains reduce H. pylori attachment through a similar process without

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synergistic effects (Fig. 3A).

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To investigate whether steric hindrance was of importance for adhesion inhibition, we

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stained the bacteria with fluorescent dyes and used microscopy to determine the

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binding patterns of the different strains. The imaging data suggested that the

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lactobacilli and H. pylori did not adhere to the same locations on the host cells (Fig.

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3B). Although we cannot completely rule out steric hindrance, this indicates that

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competition for space is most likely not the mechanism of inhibition.

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Changes in pH can affect the binding modes of H. pylori (25-27). The lactic acid that

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lactobacilli produce and the low pH as a consequence of this production have been

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implicated in the inhibition of H. pylori (28-31). We therefore measured the pH of the

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cell culture medium after 2 h of infection with H. pylori alone or together with

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lactobacilli. The differences in pH were minimal (Fig. 3C), which indicates that the

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lactobacilli do not reduce H. pylori attachment by altering the pH of the environment.

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Lactobacilli can produce bactericidal molecules like bacteriocins and hydrogen

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peroxide (32, 33). However, the viability of H. pylori was not affected by co-

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incubation with lactobacilli (Fig. 3D), excluding killing as an anti-adhesion

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mechanism. Together, these data demonstrate that lactobacilli do not physically shield

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H. pylori from host cells and do not affect the viability of the pathogen.

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The effector molecule of lactobacilli is a released component.

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To better determine the nature of the effector component from lactobacilli, we

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compared the adhesion inhibition capacity of dead to live lactobacilli. Both heat-killed

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and formaldehyde-fixed lactobacilli reduced H. pylori attachment equally well as live

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lactobacilli (Fig. 4A). Further, heat-treatment of CM at 95°C for 15 min still inhibited

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attachment of H. pylori, indicating a heat-stable effector molecule (Fig. S1). This

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suggests that a heat- and formaldehyde-resistant component is likely responsible for

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the inhibitory effect.

311

To investigate whether the effector component is a released bacterial molecule, we

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grew lactobacilli strains in RPMI for 2 h and then filter sterilized the medium to

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remove the bacterial cells. The obtained conditioned medium (CM) was then added to

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the host cells together with H. pylori. Interestingly, CM from lactobacilli reduced

315

adhesion of H. pylori (Fig. 4D), similar to whole lactobacilli, which indicates the

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release of the inhibitory molecule into the environment. Because both formaldehyde-

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fixed lactobacilli and CM from live lactobacilli can reduce H. pylori adhesion, we

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propose that the effector molecule from inhibitory lactobacilli is a component released

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into the environment.

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Lactobacilli release an effector molecule that affects H. pylori binding capacity.

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There are two ways in which lactobacilli can inhibit H. pylori adhesion: either

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directly, by interfering with the pathogen’s binding characteristics, or indirectly by

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affecting the host cell receptors. To explore these possibilities, we fixed the AGS cells

325

with formaldehyde to stop all signaling and metabolic activity in the host cells.

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Remarkably, H. pylori bound to the fixed cells to a similar extent as untreated host

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cells, and the lactobacilli still reduced the adhesion to fixed cells (Fig. 5A). This

328

suggests that the host epithelial cells do not play a role in the adhesion-inhibition

329

process. To assess whether lactobacilli had a direct effect on H. pylori, we

330

preincubated H. pylori in CM for 2 h and then centrifuged and resuspended them in

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RPMI medium and used these H. pylori bacteria to infect the host epithelial cells. The

332

H. pylori preincubated in CM from inhibitory lactobacilli attached less to the host

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333

cells compared to H. pylori preincubated in the control medium or in CM from non-

334

inhibitory lactobacilli (Fig. 5B). These data indicate that lactobacilli have a direct

335

effect on H. pylori binding capacity and that they are not acting through the host cells.

336 337

Lactobacilli affect H. pylori attachment by inhibiting sabA expression.

338

H. pylori expresses several adhesins that mediate attachment to host surfaces. SabA,

339

BabA and AlpA/B are among the most studied adhesins of H. pylori and bind to

340

sialyl-Lewis X, Lewis B antigen and laminin, respectively (14, 34, 35). CagL uses

341

beta-integrins on the host cells as a receptor (36), whereas LabA interacts with the

342

lacdiNAc motif on gastric mucins (37). However, H. pylori also expresses other

343

putative adhesins, like OipA, HopZ and HorB for which the receptors have not yet

344

been identified (38-40). Because lactobacilli can reduce the binding capacity of H.

345

pylori, we examined the expression of adhesins by qPCR after incubation in CM from

346

live lactobacilli. We determined that several genes were differentially regulated, but

347

the most pronounced decrease was observed for the sabA gene, and this also matches

348

the inhibition pattern observed in the adhesion assays. Among the tested adhesion-

349

associated genes, some showed differences in expression but only when H. pylori was

350

incubated in CM from L.bre. Interestingly, L. bre, but not L.gas1 and L.sal1, induced

351

significant 2-fold upregulation of babA, oipA and hopZ. This indicates that certain

352

lactobacilli strains may also induce adhesion genes. However, this 2-fold gene

353

upregulation did not alter the host cell attachment level of H. pylori. Expression of

354

alpA, alpB, horB and labA was not affected (Fig. 6A). Interestingly, sabA was the

355

only gene that was downregulated by the CM from both L.gas1 and L.bre, whereas it

356

remained unaltered upon incubation with CM from the non-inhibitory L.sal1 (Fig.

357

6A). To confirm changes also in SabA protein expression, we performed a western

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358

blot using SabA antibodies. As shown in Fig. 6B, SabA protein expression as detected

359

by western blotting was also reduced after incubation with CM from lactobacilli. In

360

addition, we confirmed that inhibitory lactobacilli reduced sabA not only in 67:21 but

361

also in the strains J99 and SS1 (Fig. S2). Similar to a previous report (41), the SabA

362

mutant available in strain J99 adhered less to host epithelial cells than the wild-type

363

H. pylori (Fig. 6C), confirming the importance of SabA in adhesion. Lactobacilli were

364

unable to reduce the attachment of the SabA mutant (Fig. 6C), suggesting that

365

lactobacilli affect SabA-mediated adhesion. In conclusion, these data suggest that

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lactobacilli directly reduce H. pylori binding capacity through inhibition of the SabA

367

adhesin at a transcriptional level.

368

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369

DISCUSSION

370 371

The microbiota of the human gastrointestinal tract has an important role in protection

372

against pathogens. However, the mechanisms by which this occurs are less well

373

known. In this study, we attempted to elucidate if and how lactobacilli can inhibit the

374

colonization of the gastric pathogen H. pylori. By screening 28 different lactobacilli

375

strains, we found that only certain strains were able to reduce H. pylori adhesion to

376

gastric epithelial cells. This inhibitory action is most likely mediated by an effector

377

molecule that can be released into the environment. We also showed that the host

378

cells do not play a role in this process but rather that the lactobacilli act directly on H.

379

pylori itself. Lactobacilli reduced the expression of the adhesin encoding gene sabA,

380

thereby reducing the ability of H. pylori to bind to host cell receptors.

381

It has been reported that the inhibition of pathogen colonization by lactobacilli is

382

strain specific (42), and our results of screening different lactobacilli strains confirm

383

these reports. Two out of three L. gasseri strains had an anti-adhesive effect.

384

Interestingly, the two inhibitory strains were isolated from human gastric biopsies

385

while the third was sampled from the human vagina. This could indicate adaptation of

386

the lactobacilli strains to their environment and the pathogens they encounter.

387

However, habitat location is not a determinative factor because not all isolates from

388

gastric biopsies were able to block H. pylori adhesion.

389

We show that a released effector molecule in conditioned medium from lactobacilli

390

can reduce H. pylori attachment. The nature of the effector molecule remains to be

391

determined. It might be a surface-associated molecule that is released, or an actively

392

secreted compound not normally being part of the bacterial surface. Formaldehyde

393

fixed or heat-killed lactobacilli were still able to reduce H. pylori attachment,

18

394

indicating a fixation- and heat-resistant effector molecule.

395

We demonstrate that the inhibitory Lactobacillus isolates directly reduce H. pylori

396

adhesion capacity and that the host epithelial cells are not active in this process.

397

Additionally, competition for space on the host cells was not a contributing factor in

398

this study. This is in agreement with the fact that most bacteria in the gastrointestinal

399

tract reside in the mucus layer lining the epithelium (8, 43). The lactobacilli inhibition

400

of H. pylori adhesion would appear more effective if acting directly on H. pylori

401

rather than via the host epithelium.

402

The fact that lactobacilli reduce sabA mRNA expression and that lactobacilli cannot

403

inhibit adhesion of a sabA mutant strain of H. pylori, indicates that the lactobacilli

404

have an effect on this particular adhesin. In vivo experiments showed that inhibitory

405

lactobacilli, but not non-inhibitory, reduced gastric levels of the mouse-adapted H.

406

pylori strain SS1 at a time point of 6 h. The sabA mutant available in J99, bound less

407

in vitro to gastric AGS cells at 2 h (Fig. 6B). It is tempting to speculate that reduction

408

of sabA by certain lactobacilli might help to prevent H. pylori colonization in the

409

stomach. Interestingly, the lactobacilli CM reduced the adherence of wild-type H.

410

pylori more than the sabA mutant did, suggesting involvement of an additional factor.

411

SabA expression is regulated by different mechanisms. Gene conversion due to

412

intragenomic recombination allows variation in copy number and locus of the sabA

413

gene (44). Two simple sequence repeats mediate slipped-strand mispairing, which

414

lead to variation in expression. A dinucleotide cytosine-thymidine repeat in the 5’

415

coding region allows for phase variation, which turns the expression of SabA ‘on’ and

416

‘off’ (18). A T-tract, located at the promotor region, controls sabA transcription

417

initiation because the T-tract length influences binding of the RNA polymerase (45,

418

46). Finally, the two-component signal transduction system ArsRS mediates the

19

419

regulation of gene transcription by environmental changes. SabA has been shown to

420

be derepressed in a mutant for the histidine kinase ArsS (47), which leads to more

421

adhesion to the host cells due to higher sabA expression (18). These multiple

422

regulatory mechanisms explain why the expression of SabA is found to be so variable

423

between isolates (48, 49). Because the ArsRS system responds to environmental cues,

424

this could be a probable candidate providing the lactobacilli with a means to repress

425

sabA expression. It has been reported that an acidic pH is a key signal for the ArsRS

426

system (50). However, protein expression studies at a neutral pH with an arsS mutant

427

indicate that the ArsRS system also has a role in the regulation of the expression in

428

the absence of the low pH stimulus and that the system might be able to respond to

429

other environmental factors (51). Lactobacilli did not change the pH in our

430

experiment, but they did release a molecule into the surroundings that causes an

431

adhesion inhibitory effect. It is tempting to speculate that the ArsRS system might

432

respond to this component and thereby allow for the repression of sabA. Interestingly,

433

L. brevis induced significant 2-fold induction of babA, oipA and hopZ. This indicates

434

that certain lactobacilli strains may also induce adhesion genes. However, the 2-fold

435

induction did not alter H. pylori attachment to host cells. In the future, it would be

436

interesting to find out whether any of these are under control of ArsRS. Further, L.bre

437

slightly but significantly reduced cagL. However, there was also a trend that both

438

non-inhibitory and inhibitory lactobacilli reduced cagL, suggesting that the effect on

439

cagL is not linked to attachment levels.

440

It has been well demonstrated that the microbiota is an important component of our

441

defense against pathogens. In this study, we show that certain lactobacilli can reduce

442

initial adhesion to host epithelial cells by affecting the binding capacity of H. pylori.

443

The ability of some lactobacilli, but not all, to interfere with H. pylori virulence gene

20

444

expression is intriguing and prompts further studies to identify the lactobacillus

445

component as well its mechanism of action. In the future it would be interesting to

446

also evaluate the role of lactobacilli in long-term colonization of H. pylori using

447

available model systems. Understanding the molecular mechanisms by which the

448

microbiota inhibits pathogen colonization can reveal new knowledge of bacterial

449

pathogenesis and help in the development of novel, more effective treatment

450

strategies against bacterial infections.

21

451

FUNDING INFORMATION

452

This work was supported by the Swedish Research Council (ABJ), the Swedish

453

Cancer Society (ABJ), Torsten Söderbergs Stiftelse (ABJ), Ragnar Söderbergs

454

Stiftelse (ABJ). The funders had no role in study design, data collection and

455

interpretation, or the decision to submit the work for publication.

456 457

Disclosures

458

The authors have no conflicts of interest.

459 460

ACKNOWLEDGEMENTS

461

We thank Anna Arnqvist and Thomas Boren for providing the SabA antibody.

462

22

463

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30

638

FIGURE LEGENDS

639 640

FIG. 1. Certain lactobacilli can reduce the adhesion of H. pylori to host gastric

641

epithelial cells.

642

Attachment to gastric epithelial cells of H. pylori alone or together with lactobacilli at

643

an MOI of 100 of each strain for 2 h. A) Adhesion of H. pylori strain 67:21 to AGS

644

cells as determined by urease activity. B) Adhesion of H. pylori strain 67:21 to AGS

645

cells as determined by the viable count assay. C) Adhesion of H. pylori 67:21 to

646

MKN45 cells by viable counts. D) Adhesion of H. pylori strain J99 to AGS epithelial

647

cells by viable counts. Colony forming units (cfu)/ml were determined by serial

648

dilution and plating. Hp = H. pylori. A complete list of Lactobacillus strains is shown

649

in Table 1. The data shown is representative of three independent experiments with

650

triplicate samples. The differences between groups were analyzed using ANOVA

651

(analysis of variance) followed by the Bonferroni post hoc test. The error bars

652

represent standard deviations. An asterisk * indicates a statistically significant

653

difference (p < 0.05) compared to H. pylori alone.

654 655

FIG. 2. Lactobacilli reduce H. pylori colonization in vivo.

656

A) Adhesion of the H. pylori strain SS1 to AGS gastric epithelial cells alone or in

657

combination with lactobacilli. Colony forming units (cfu)/ml were determined by

658

serial dilution and plating. The data shown is representative of at three independent

659

experiments with triplicate samples. The differences between groups were analyzed

660

using ANOVA (analysis of variance) followed by the Bonferroni post hoc test. The

661

error bars represent standard deviations. B) Mice pretreated with L.gas1, L. sal1 or

662

PBS were perorally infected with H. pylori (108 cfu/mouse) alone or together with

31

663

L.gas1 or L.sal1 (108 cfu/mouse). H. pylori colonization of the stomach was

664

determined by viable counts at 6 h post infection. The horizontal lines represent

665

median cfu/gram tissue. The data from the mouse experiment were analyzed with the

666

Kruskal-Wallis test. An asterisk * indicates a statistically significant difference (p