Publications by authors named "Ann-Beth Jonsson"

55 Publications

DNA Blocks the Lethal Effect of Human Beta-Defensin 2 Against .

Front Microbiol 2021 30;12:697232. Epub 2021 Jun 30.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

is a gram-negative bacterium that often asymptomatically colonizes the human nasopharyngeal tract. These bacteria cross the epithelial barrier can cause life-threatening sepsis and/or meningitis. Antimicrobial peptides are one of the first lines of defense against invading bacterial pathogens. Human beta-defensin 2 (hBD2) is an antimicrobial peptide with broad antibacterial activity, although its mechanism of action is poorly understood. Here, we investigated the effect of hBD2 on . We showed that hBD2 binds to and kills actively growing meningococcal cells. The lethal effect was evident after 2 h incubation with the peptide, which suggests a slow killing mechanism. Further, the membrane integrity was not changed during hBD2 treatment. Incubation with lethal doses of hBD2 decreased the presence of diplococci; the number and size of bacterial microcolonies/aggregates remained constant, indicating that planktonic bacteria may be more susceptible to the peptide. Meningococcal DNA bound hBD2 in mobility shift assays and inhibited the lethal effect of hBD2 in a dose-dependent manner both in suspension and biofilms, supporting the interaction between hBD2 and DNA. Taken together, the ability of meningococcal DNA to bind hBD2 opens the possibility that extracellular DNA due to bacterial lysis may be a means of to evade immune defenses.
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http://dx.doi.org/10.3389/fmicb.2021.697232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8278289PMC
June 2021

Lactate-induced dispersal of microcolonies is mediated by changes in cell density and pilus retraction and is influenced by temperature change.

Infect Immun 2021 Jun 14:IAI0029621. Epub 2021 Jun 14.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden.

is the etiologic agent of meningococcal meningitis and sepsis. Initial colonization of meningococci to the upper respiratory tract epithelium is crucial for disease development. The colonization occurs in several steps and expression of type IV pili (Tfp) is essential for both attachment and microcolony formation of encapsulated bacteria. Previously, we have shown that host-derived lactate induces synchronized dispersal of meningococcal microcolonies. In this study, we demonstrated that lactate-induced dispersal is dependent on bacterial concentration but not on the quorum sensing system autoinducer-2 or the two-component systems NarP/NarQ, PilR/PilS, NtrY/NtrX, and MisR/MisS. Further, there were no changes in expression of genes related to assembly, elongation, retraction, and modification of Tfp throughout the time course of lactate induction. By using and mutants, however, we found that lactate-induced dispersal was dependent on PilT-retraction but not on phosphoglycerol-modification of Tfp even though the PptB activity was important for preventing re-aggregation post-dispersal. Furthermore, protein synthesis was required for lactate-induced dispersal. Finally, we found that at a lower temperature, lactate-induced dispersal was delayed and unsynchronized, and bacteria reformed microcolonies. We conclude that lactate-induced microcolony dispersal is dependent on bacterial concentration, PilT-dependent Tfp retraction, and protein synthesis and influenced by environmental temperature.
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http://dx.doi.org/10.1128/IAI.00296-21DOI Listing
June 2021

Modulation of Human Beta-Defensin 2 Expression by Pathogenic Neisseria meningitidis and Commensal Lactobacilli.

Antimicrob Agents Chemother 2021 03 18;65(4). Epub 2021 Mar 18.

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

Antimicrobial peptides (AMPs) play an important role in the defense against pathogens by targeting and killing invading microbes. Some pathogenic bacteria have been shown to negatively regulate AMP expression, while several commensals may induce AMP expression. The expression of certain AMPs, such as human beta-defensin 2 (hBD2), can be induced via nuclear factor NF-κB, which, in turn, is negatively controlled by tumor necrosis factor alpha-induced protein 3 (TNFAIP3, or A20). In this work, we examined the expression of hBD1 and hBD2 during coincubation of pharyngeal epithelial cells with pathogenic and commensal lactobacilli. The strains induced hBD2 expression in human pharyngeal cells, while the pathogen did not. In coincubation experiments, meningococci were able to dampen the AMP expression induced by lactobacilli. We found that induced the NF-κB inhibitor A20. Further, RNA silencing of A20 resulted in increased hBD2 expression after meningococcal infection. Since it is known that induction of A20 reduces NF-κB activity and thus hBD2 levels, meningococcal-mediated A20 induction could be a way for the pathogen to dampen AMP expression. Finally, treatment of and lactobacilli with synthetic hBD2 reduced viability more efficiently than , explaining why maintaining low AMP levels is important for the survival of the pathogen.
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http://dx.doi.org/10.1128/AAC.02002-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097439PMC
March 2021

Role of Sortase A in Kx110A1 Adhesion to Gastric Epithelial Cells and Competitive Exclusion of .

Front Microbiol 2019 3;10:2770. Epub 2019 Dec 3.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

We have previously shown that Kx110A1, a human stomach isolate, can colonize mouse stomach and reduce the initial colonization of . Here, we investigated the role of sortase-dependent proteins (SDPs) involved in these functions by the construction of a mutant for , the gene encoding the housekeeping sortase that covalently anchors SDPs to the cell surface. The mutant showed a decrease in hydrophobicity and autoaggregation under acidic conditions, indicating the effect of SDPs on cell surface properties. Correspondingly, the mutant lost the capacity to adhere to gastric epithelial cells, thus resulting in an inability to provide a physical barrier to prevent adherence. These results indicate that sortase A is a key determinant of the cell surface properties of Kx110A1 and contributes to -mediated exclusion of . Understanding the molecular mechanisms by which lactobacilli antagonize might contribute to the development of novel therapeutic strategies that take advantage of health-promoting bacteria and reduce the burden of antibiotic resistance.
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http://dx.doi.org/10.3389/fmicb.2019.02770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902081PMC
December 2019

Suppresses the Production of Proinflammatory Cytokines in -Infected Macrophages by Inhibiting the Expression of ADAM17.

Front Immunol 2019 4;10:2326. Epub 2019 Oct 4.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

The ability of to evade the host immune system allows the bacterium to colonize the host for a lifetime. Long-term infection with causes chronic inflammation, which is the major risk factor for the development of gastric ulcers and gastric cancer. Lactobacilli are part of the human microbiota and have been studied as an adjunct treatment in eradication therapy. However, the molecular mechanisms by which lactobacilli act against infection have not been fully characterized. In this study, we investigated the anti-inflammatory effects of strains upon coincubation of host macrophages with . We found that Kx110A1 (L. gas), a strain isolated from a human stomach, but not other tested species, blocked the production of the proinflammatory cytokines TNF and IL-6 in -infected macrophages. Interestingly, L. gas also inhibited the release of these cytokines in LPS or LTA stimulated macrophages, demonstrating a general anti-inflammatory property. The inhibition of these cytokines did not occur through the polarization of macrophages from the M1 (proinflammatory) to M2 (anti-inflammatory) phenotype or through the altered viability of or host cells. Instead, we show that L. gas suppressed the release of TNF and IL-6 by reducing the expression of ADAM17 (also known as TNF-alpha-converting enzyme, TACE) on host cells. Our findings reveal a novel mechanism by which L. gas prevents the production of the proinflammatory cytokines TNF and IL-6 in host macrophages.
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http://dx.doi.org/10.3389/fimmu.2019.02326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6788455PMC
November 2020

Deletion of D-Lactate Dehydrogenase A in Promotes Biofilm Formation Through Increased Autolysis and Extracellular DNA Release.

Front Microbiol 2019 5;10:422. Epub 2019 Mar 5.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

is a Gram-negative bacterium that asymptomatically colonizes the human nasopharyngeal mucosa. Pilus-mediated initial adherence of to the epithelial mucosa is followed by the formation of three-dimensional aggregates, called microcolonies. Dispersal from microcolonies contributes to the transmission of across the epithelial mucosa. We have recently discovered that environmental concentrations of host cell-derived lactate influences microcolony dispersal. Here, we examined the ability of mutants deficient in lactate metabolism to form biofilms. A lactate dehydrogenease A () mutant had an increased level of biofilm formation. Deletion of increased the cell surface hydrophobicity and aggregation. In this study, we used FAM20, which belongs to clonal complex ST-11 that forms biofilms independently of extracellular DNA (eDNA). However, treatment with DNase I abolished the increased biofilm formation and aggregation of the -deficient mutant, suggesting a critical role for eDNA. Compared to wild-type, the -deficient mutant exhibited an increased autolytic rate, with significant increases in the eDNA concentrations in the culture supernatants and in biofilms. Within the mutant biofilm, the transcription levels of the capsule, pilus, and bacterial lysis genes were downregulated, while , which is associated with anaerobic respiration, was upregulated. These findings suggest that the absence of in promotes biofilm formation and aggregation through autolysis-mediated DNA release.
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http://dx.doi.org/10.3389/fmicb.2019.00422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411758PMC
March 2019

Quantification of Adherence to Human Epithelial Cells by Colony Counting.

Bio Protoc 2018 Feb 5;8(3):e2709. Epub 2018 Feb 5.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

To cause an infection, the human specific pathogen must first colonize the nasopharynx. Upon tight interaction with the mucosal epithelium, may cross the epithelial cellular barrier, reach the bloodstream and cause sepsis and/or meningitis. Since niche is restricted to humans the availability of relevant animal models to study host-pathogen interactions are limiting. Therefore, most findings that involve colonization derive from studies using cultured human cell lines. Human epithelial cells have been successfully used to examine and identify molecular effectors involved in initial adherence of the pathogen. Here, we describe a standard protocol to quantify the adherence of to epithelial pharyngeal FaDu cells. Colony counts of cell lysates collected after infection are used to quantify adherence to the epithelial cells.
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http://dx.doi.org/10.21769/BioProtoc.2709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8203892PMC
February 2018

Live-cell Imaging of Microcolony Dispersal Induced by Lactate or Other Molecules.

Bio Protoc 2018 Jan 20;8(2):e2695. Epub 2018 Jan 20.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

To efficiently colonize the nasopharyngeal epithelium, the human restricted pathogen follows a multistep adhesion cascade. First, the bacteria adhere to host cells and aggregate into spherical shaped structures called microcolonies. Several hours later, single bacteria start dispersing from the microcolonies and form a monolayer on top of the host cells. Once in proximity to host cells meningococci can adhere tightly to the epithelial surface or become internalized. This can eventually result in invasion of the mucosal surfaces and gain access to the bloodstream, causing a life-threatening disease. Lactate, a metabolite derived from human epithelial cells, has been previously shown to induce rapid dispersal of from microcolonies. Here, we describe a host-cell free method based on live-cell imaging to examine the effect of host derived lactate on the timing of microcolony dispersal. Although in this protocol we use lactate, it can be easily modified to test the effects of other molecules.
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http://dx.doi.org/10.21769/BioProtoc.2695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8203937PMC
January 2018

Host cell-derived lactate functions as an effector molecule in Neisseria meningitidis microcolony dispersal.

PLoS Pathog 2017 Apr 6;13(4):e1006251. Epub 2017 Apr 6.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

The development of meningococcal disease, caused by the human pathogen Neisseria meningitidis, is preceded by the colonization of the epithelial layer in the nasopharynx. After initial adhesion to host cells meningococci form aggregates, through pilus-pilus interactions, termed microcolonies from which the bacteria later detach. Dispersal from microcolonies enables access to new colonization sites and facilitates the crossing of the cell barrier; however, this process is poorly understood. In this study, we used live-cell imaging to investigate the process of N. meningitidis microcolony dispersal. We show that direct contact with host cells is not required for microcolony dispersal, instead accumulation of a host-derived effector molecule induces microcolony dispersal. By using a host-cell free approach, we demonstrated that lactate, secreted from host cells, initiate rapid dispersal of microcolonies. Interestingly, metabolic utilization of lactate by the bacteria was not required for induction of dispersal, suggesting that lactate plays a role as a signaling molecule. Furthermore, Neisseria gonorrhoeae microcolony dispersal could also be induced by lactate. These findings reveal a role of host-secreted lactate in microcolony dispersal and virulence of pathogenic Neisseria.
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http://dx.doi.org/10.1371/journal.ppat.1006251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5383330PMC
April 2017

Inhibitory role of acyl homoserine lactones in hemolytic activity and viability of Streptococcus pyogenes M6 S165.

Sci Rep 2017 03 17;7:44902. Epub 2017 Mar 17.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 10691 Stockholm, Sweden.

Streptococcus pyogenes an adapted human pathogen asymptomatically colonizes the nasopharynx, among other polymicrobial communities. However, information on the events leading to the colonization and expression of virulence markers subject to interspecies and host-bacteria interactions are limited. The interference of acyl homoserine lactones (AHLs) with the hemolytic activity and viability of S. pyogenes M6 S165 was examined. AHLs, with fatty acid side chains ≥12 carbon atoms, inhibited hemolytic activity by downregulating the expression of the sag operon involved in the production of streptolysin S. Inhibitory AHLs upregulated the expression of transcriptional regulator LuxR. Electrophoretic mobility shift assays revealed the interaction of LuxR with the region upstream of sagA. AHL-mediated bactericidal activity observed at higher concentrations (mM range) was an energy-dependent process, constrained by the requirement of glucose and iron. Ferrichrome transporter FtsABCD facilitated transport of AHLs across the streptococcal membrane. The study demonstrates a previously unreported role for AHLs in S. pyogenes virulence.
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http://dx.doi.org/10.1038/srep44902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355980PMC
March 2017

The Host Cell Transcription Factor EGR1 Is Induced by Bacteria through the EGFR-ERK1/2 Pathway.

Front Cell Infect Microbiol 2017 25;7:16. Epub 2017 Jan 25.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University Stockholm, Sweden.

The essential first step in bacterial colonization is adhesion to the host epithelial cells. The early host-responses post-bacterial adhesions are still poorly understood. Early growth response 1 (EGR1) is an early response transcriptional regulator that can be rapidly induced by various environmental stimuli. Several bacteria can induce EGR1 expression in host cells, but the involved bacterial characteristics and the underlying molecular mechanisms of this response are largely unknown. Here, we show that EGR1 can be induced in host epithelial cells by different species of bacteria independent of the adherence level, Gram-staining type and pathogenicity. However, bacterial viability and contact with host cells is necessary, indicating that an active interaction between bacteria and the host is important. Furthermore, the strongest response is observed in cells originating from the natural site of the infection, suggesting that the EGR1 induction is cell type specific. Finally, we show that EGFR-ERK1/2 and β1-integrin signaling are the main pathways used for bacteria-mediated EGR1 upregulation. In conclusion, the increase of EGR1 expression in epithelial cells is a common stress induced, cell type specific response upon host-bacteria interaction that is mediated by EGFR-ERK1/2 and β1-integrin signaling.
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http://dx.doi.org/10.3389/fcimb.2017.00016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264520PMC
September 2017

Lactobacilli Interfere with Streptococcus pyogenes Hemolytic Activity and Adherence to Host Epithelial Cells.

Front Microbiol 2016 29;7:1176. Epub 2016 Jul 29.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University Stockholm, Sweden.

Streptococcus pyogenes [Group A streptococcus (GAS)], a frequent colonizer of the respiratory tract mucosal surface, causes a variety of human diseases, ranging from pharyngitis to the life-threatening streptococcal toxic shock-like syndrome. Lactobacilli have been demonstrated to colonize the respiratory tract. In this study, we investigated the interference of lactobacilli with the virulence phenotypes of GAS. The Lactobacillus strains L. rhamnosus Kx151A1 and L. reuteri PTA-5289, but not L. salivarius LMG9477, inhibited the hemolytic activity of S. pyogenes S165. The inhibition of hemolytic activity was attributed to a decrease in the production of streptolysin S (SLS). Conditioned medium (CM) from the growth of L. rhamnosus Kx151A1 and L. reuteri PTA-5289 was sufficient to down-regulate the expression of the sag operon, encoding SLS. The Lactobacillus strains L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 inhibited the initial adherence of GAS to host epithelial cells. Intriguingly, competition with a combination of Lactobacillus species reduced GAS adherence to host cells most efficiently. The data suggest that an effector molecule released from certain Lactobacillus strains attenuates the production of SLS at the transcriptional level and that combinations of Lactobacillus strains may protect the pharyngeal mucosa more efficiently from the initial colonization of GAS. The effector molecules released from Lactobacillus strains affecting the virulence phenotypes of pathogens hold potential in the development of a new generation of therapeutics.
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http://dx.doi.org/10.3389/fmicb.2016.01176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965460PMC
August 2016

Lactobacilli Reduce Helicobacter pylori Attachment to Host Gastric Epithelial Cells by Inhibiting Adhesion Gene Expression.

Infect Immun 2016 05 22;84(5):1526-1535. Epub 2016 Apr 22.

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

The human gastrointestinal tract, including the harsh environment of the stomach, harbors a large variety of bacteria, of which Lactobacillus species are prominent members. The molecular mechanisms by which species of lactobacilli interfere with pathogen colonization are not fully characterized. In this study, we aimed to study the effect of lactobacillus strains upon the initial attachment of Helicobacter pylori to host cells. Here we report a novel mechanism by which lactobacilli inhibit adherence of the gastric pathogen H. pylori In a screen with Lactobacillus isolates, we found that only a few could reduce adherence of H. pylori to gastric epithelial cells. Decreased attachment was not due to competition for space or to lactobacillus-mediated killing of the pathogen. Instead, we show that lactobacilli act on H. pylori directly by an effector molecule that is released into the medium. This effector molecule acts on H. pylori by inhibiting expression of the adhesin-encoding gene sabA Finally, we verified that inhibitory lactobacilli reduced H. pylori colonization in an in vivo model. In conclusion, certain Lactobacillus strains affect pathogen adherence by inhibiting sabA expression and thereby reducing H. pylori binding capacity.
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http://dx.doi.org/10.1128/IAI.00163-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862695PMC
May 2016

Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence.

Infect Immun 2016 05 22;84(5):1501-1513. Epub 2016 Apr 22.

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

Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3'-5' exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis.
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http://dx.doi.org/10.1128/IAI.01463-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862713PMC
May 2016

A fusion protein derived from Moraxella catarrhalis and Neisseria meningitidis aimed for immune modulation of human B cells.

Hum Vaccin Immunother 2015 4;11(9):2223-7. Epub 2015 Jun 4.

a Clinical Microbiology; Department of Translational Medicine; Lund University ; Malmö , Sweden.

Moraxella IgD-binding protein (MID) is a well characterized trimeric autotransporter that specifically targets the IgD of B cells. We fused the membrane anchor of the meningococcal autotransporter NhhA with the IgD-binding region of MID (aa 962-1200) to create a chimeric protein designated as NID. The aim was to use this specific targeting to provide a better vaccine candidate against meningococci, in particular serogroup B by enhancing the immunogenicity of NhhA. NID was thereafter recombinantly expressed in E. coli. The NID-expressing E. coli bound to peripheral B lymphocytes that resulted in cellular activation. Furthermore, we also successfully expressed NID on outer membrane vesicles, nanoparticles that are commonly used in meningococcal vaccines. This study thus highlights the applicability of the menigococcal-Moraxella fusion protein NID to be used for specific targeting of vaccine components to the IgD B cell receptor.
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http://dx.doi.org/10.1080/21645515.2015.1034917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4635892PMC
May 2016

Characterization of motility and piliation in pathogenic Neisseria.

BMC Microbiol 2015 Apr 30;15:92. Epub 2015 Apr 30.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Svante Arrhenius väg 20C, SE-10691, Stockholm, Sweden.

Background: The type IV pili (Tfp) of pathogenic Neisseria (i.e., N. gonorrhoeae and N. meningitidis) are essential for twitching motility. Tfp retraction, which is dependent on the ATPase PilT, generates the forces that move bacteria over surfaces. Neisseria motility has mainly been studied in N. gonorrhoeae whereas the motility of N. meningitidis has not yet been characterized.

Results: In this work, we analyzed bacterial motility and monitored Tfp retraction using live-cell imaging of freely moving bacteria. We observed that N. meningitidis moved over surfaces at an approximate speed of 1.6 μm/s, whereas N. gonorrhoeae moved with a lower speed (1.0 μm/s). An alignment of the meningococcal and gonococcal pilT promoters revealed a conserved single base pair variation in the -10 promoter element that influence PilT expression. By tracking mutants with altered pilT expression or pilE sequence, we concluded that the difference in motility speed was independent of both. Live-cell imaging using total internal reflection fluorescence microscopy demonstrated that N. gonorrhoeae more often moved with fewer visible retracting filaments when compared to N. meningitidis. Correspondingly, meningococci also displayed a higher level of piliation in transmission electron microscopy. Nevertheless, motile gonococci that had the same number of filaments as N. meningitidis still moved with a lower speed.

Conclusions: These data reveal differences in both speed and piliation between the pathogenic Neisseria species during twitching motility, suggesting a difference in Tfp-dynamics.
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http://dx.doi.org/10.1186/s12866-015-0424-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449605PMC
April 2015

Nuclear trafficking, histone cleavage and induction of apoptosis by the meningococcal App and MspA autotransporters.

Cell Microbiol 2015 Jul 8;17(7):1008-20. Epub 2015 Feb 8.

School of Life Sciences, University of Nottingham, Nottingham, UK.

Neisseria meningitidis, a major cause of bacterial meningitis and septicaemia, secretes multiple virulence factors, including the adhesion and penetration protein (App) and meningococcal serine protease A (MspA). Both are conserved, immunogenic, type Va autotransporters harbouring S6-family serine endopeptidase domains. Previous work suggested that both could mediate adherence to human cells, but their precise contribution to meningococcal pathogenesis was unclear. Here, we confirm that App and MspA are in vivo virulence factors since human CD46-expressing transgenic mice infected with meningococcal mutants lacking App, MspA or both had improved survival rates compared with mice infected with wild type. Confocal imaging showed that App and MspA were internalized by human cells and trafficked to the nucleus. Cross-linking and enzyme-linked immuno assay (ELISA) confirmed that mannose receptor (MR), transferrin receptor 1 (TfR1) and histones interact with MspA and App. Dendritic cell (DC) uptake could be blocked using mannan and transferrin, the specific physiological ligands for MR and TfR1, whereas in vitro clipping assays confirmed the ability of both proteins to proteolytically cleave the core histone H3. Finally, we show that App and MspA induce a dose-dependent increase in DC death via caspase-dependent apoptosis. Our data provide novel insights into the roles of App and MspA in meningococcal infection.
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http://dx.doi.org/10.1111/cmi.12417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024080PMC
July 2015

Impact of calcium signaling during infection of Neisseria meningitidis to human brain microvascular endothelial cells.

PLoS One 2014 2;9(12):e114474. Epub 2014 Dec 2.

Department of Pediatrics, Pediatric Infectious Diseases, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.

The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114474PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4252121PMC
January 2016

Uncovering the mechanism of trapping and cell orientation during Neisseria gonorrhoeae twitching motility.

Biophys J 2014 Oct;107(7):1523-31

School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts; Department of Physics, Harvard University, Cambridge, Massachusetts. Electronic address:

Neisseria gonorrheae bacteria are the causative agent of the second most common sexually transmitted infection in the world. The bacteria move on a surface by means of twitching motility. Their movement is mediated by multiple long and flexible filaments, called type IV pili, that extend from the cell body, attach to the surface, and retract, thus generating a pulling force. Moving cells also use pili to aggregate and form microcolonies. However, the mechanism by which the pili surrounding the cell body work together to propel bacteria remains unclear. Understanding this process will help describe the motility of N. gonorrheae bacteria, and thus the dissemination of the disease which they cause. In this article we track individual twitching cells and observe that their trajectories consist of alternating moving and pausing intervals, while the cell body is preferably oriented with its wide side toward the direction of motion. Based on these data, we propose a model for the collective pili operation of N. gonorrheae bacteria that explains the experimentally observed behavior. Individual pili function independently but can lead to coordinated motion or pausing via the force balance. The geometry of the cell defines its orientation during motion. We show that by changing pili substrate interactions, the motility pattern can be altered in a predictable way. Although the model proposed is tangibly simple, it still has sufficient robustness to incorporate further advanced pili features and various cell geometries to describe other bacteria that employ pili to move on surfaces.
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http://dx.doi.org/10.1016/j.bpj.2014.07.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4190650PMC
October 2014

Beneficial antimicrobial effect of the addition of an aminoglycoside to a β-lactam antibiotic in an E. coli porcine intensive care severe sepsis model.

PLoS One 2014 28;9(2):e90441. Epub 2014 Feb 28.

Section of Infectious Diseases, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

This study aimed to determine whether the addition of an aminoglycoside to a ß-lactam antibiotic increases the antimicrobial effect during the early phase of Gram-negative severe sepsis/septic shock. A porcine model was selected that considered each animal's individual blood bactericidal capacity. Escherichia coli, susceptible to both antibiotics, was given to healthy pigs intravenously during 3 h. At 2 h, the animals were randomized to a 20-min infusion with either cefuroxime alone (n = 9), a combination of cefuroxime+tobramycin (n = 9), or saline (control, n = 9). Blood samples were collected hourly for cultures and quantitative polymerase chain reaction (PCR). Bacterial growth in the organs after 6 h was chosen as the primary endpoint. A blood sample was obtained at baseline before start of bacterial infusion for ex vivo investigation of the blood bactericidal capacity. At 1 h after the administration of the antibiotics, a second blood sample was taken for ex vivo investigation of the antibiotic-induced blood killing activity. All animals developed severe sepsis/septic shock. Blood cultures and PCR rapidly became negative after completed bacterial infusion. Antibiotic-induced blood killing activity was significantly greater in the combination group than in the cefuroxime group (p<0.001). Growth of bacteria in the spleen was reduced in the two antibiotic groups compared with the controls (p<0.01); no difference was noted between the two antibiotic groups. Bacterial growth in the liver was significantly less in the combination group than in the cefuroxime group (p<0.05). High blood bactericidal capacity at baseline was associated with decreased growth in the blood and spleen (p<0.05). The addition of tobramycin to cefuroxime results in increased antibiotic-induced blood killing activity and less bacteria in the liver than cefuroxime alone. Individual blood bactericidal capacity may have a significant effect on antimicrobial outcome.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0090441PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938751PMC
October 2014

Helicobacter pylori protein JHP0290 binds to multiple cell types and induces macrophage apoptosis via tumor necrosis factor (TNF)-dependent and independent pathways.

PLoS One 2013 1;8(11):e77872. Epub 2013 Nov 1.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

Activated macrophages at the sub-mucosal space play a major role in generating innate immune responses during H. pylori infection. Final disease outcome largely depends on how H. pylori and bacterium-derived products modulate macrophage responses. Here, we report that JHP0290, a functionally unknown protein from H. pylori, regulates macrophage functions. Recombinant purified JHP0290 (rJHP0290) had the ability to bind to several cell types including macrophages, human gastric epithelial cell lines, human monocyte-derived dendritic cells (MoDC) and human neutrophils. Exposure to rJHP0290 induced apoptosis in macrophages concurrent with release of proinflammatory cytokine tumor necrosis factor (TNF). A mutant strain of H. pylori disrupted in the jhp0290 gene was significantly impaired in its ability to induce apoptosis and TNF in macrophages confirming the role of endogenous protein in regulating macrophage responses. Intracellular signaling involving Src family of tyrosine kinases (SFKs) and ERK MAPK were required for rJHP0290-induced TNF release and apoptosis in macrophages. Furthermore, rJHP0290-induced TNF release was partly dependent on activation of nuclear transcription factor-κB (NF-κB). Neutralizing antibodies against TNF partially blocked rJHP0290-induced macrophage apoptosis indicating TNF-independent pathways were also involved. These results provide mechanistic insight into the potential role of the protein JHP0290 during H. pylori-associated disease development. By virtue of its ability to induce TNF, an acid suppressive proinflammatory cytokine and induction of macrophage apoptosis, JHP0290 possibly helps in persistent survival of the bacterium inside the stomach.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077872PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815203PMC
August 2014

Pro-inflammatory cytokines can act as intracellular modulators of commensal bacterial virulence.

Open Biol 2013 Oct 9;3(10):130048. Epub 2013 Oct 9.

School of Life Sciences, Molecular Bacteriology and Immunology Group, University of Nottingham, Nottingham NG7 2RD, UK.

Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.
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http://dx.doi.org/10.1098/rsob.130048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814720PMC
October 2013

Meningococcal resistance to antimicrobial peptides is mediated by bacterial adhesion and host cell RhoA and Cdc42 signalling.

Cell Microbiol 2013 Nov 30;15(11):1938-54. Epub 2013 Jul 30.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

Antimicrobial peptides (AMPs) constitute an essential part of the innate immune defence. Pathogenic bacteria have evolved numerous strategies to withstand AMP-mediated killing. The influence of host epithelia on bacterial AMP resistance is, however, still largely unknown. We found that adhesion to pharyngeal epithelial cells protected Neisseria meningitidis, a leading cause of meningitis and sepsis, from the human cathelicidin LL-37, the cationic model amphipathic peptide (MAP) and the peptaibol alamethicin, but not from polymyxin B. Adhesion to primary airway epithelia resulted in a similar increase in LL-37 resistance. The inhibition of selective host cell signalling mediated by RhoA and Cdc42 was found to abolish the adhesion-induced LL-37 resistance by a mechanism unrelated to the actin cytoskeleton. Moreover, N. meningitidis triggered the formation of cholesterol-rich membrane microdomains in pharyngeal epithelial cells, and host cell cholesterol proved to be essential for adhesion-induced resistance. Our data highlight the importance of Rho GTPase-dependent host cell signalling for meningococcal AMP resistance. These results indicate that N. meningitidis selectively exploits the epithelial microenvironment in order to protect itself from LL-37.
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http://dx.doi.org/10.1111/cmi.12163DOI Listing
November 2013

Identification of cell-penetrating peptides that are bactericidal to Neisseria meningitidis and prevent inflammatory responses upon infection.

Antimicrob Agents Chemother 2013 Aug 20;57(8):3704-12. Epub 2013 May 20.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

Meningococcal disease is characterized by a fast progression and a high mortality rate. Cell-penetrating peptides (CPPs), developed as vectors for cargo delivery into eukaryotic cells, share structural features with antimicrobial peptides. A screen identified two CPPs, transportan-10 (TP10) and model amphipathic peptide (MAP), with bactericidal action against Neisseria meningitidis. Both peptides were active in human whole blood at micromolar concentrations, while hemolysis remained negligible. Additionally, TP10 exhibited significant antibacterial activity in vivo. Uptake of SYTOX green into live meningococci was observed within minutes after TP10 treatment, suggesting that TP10 may act by membrane permeabilization. Apart from its bactericidal activity, TP10 suppressed inflammatory cytokine release from macrophages infected with N. meningitidis as well as from macrophages stimulated with enterobacterial and meningococcal lipopolysaccharide (LPS). Finally, incubation with TP10 reduced the binding of LPS to macrophages. This novel endotoxin-inhibiting property of TP10, together with its antimicrobial activity in vivo, indicates the possibility to design peptide-based therapies for infectious diseases.
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http://dx.doi.org/10.1128/AAC.00624-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719777PMC
August 2013

Thyroid hormone enhances nitric oxide-mediated bacterial clearance and promotes survival after meningococcal infection.

PLoS One 2012 23;7(7):e41445. Epub 2012 Jul 23.

Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden.

Euthyroid sick syndrome characterized by reduced levels of thyroid hormones (THs) is observed in patients with meningococcal shock. It has been found that the level of THs reflects disease severity and is predictive for mortality. The present study was conducted to investigate the impact of THs on host defense during meningococcal infection. We found that supplementation of thyroxine to mice infected with Neisseria meningitidis enhanced bacterial clearance, attenuated the inflammatory responses and promoted survival. In vitro studies with macrophages revealed that THs enhanced bacteria-cell interaction and intracellular killing of meningococci by stimulating inducible nitric oxide synthase (iNos)-mediated NO production. TH treatment did not activate expression of TH receptors in macrophages. Instead, the observed TH-directed actions were mediated through nongenomic pathways involving the protein kinases PI3K and ERK1/2 and initiated at the membrane receptor integrin αvβ3. Inhibition of nongenomic TH signaling prevented iNos induction, NO production and subsequent intracellular bacterial killing by macrophages. These data demonstrate a beneficial role of THs in macrophage-mediated N. meningitidis clearance. TH replacement might be a novel option to control meningococcal septicemia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041445PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402396PMC
November 2012

Loss of meningococcal PilU delays microcolony formation and attenuates virulence in vivo.

Infect Immun 2012 Jul 16;80(7):2538-47. Epub 2012 Apr 16.

Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden.

Neisseria meningitidis is a major cause of sepsis and bacterial meningitis worldwide. This bacterium expresses type IV pili (Tfp), which mediate important virulence traits such as the formation of bacterial aggregates, host cell adhesion, twitching motility, and DNA uptake. The meningococcal PilT protein is a hexameric ATPase that mediates pilus retraction. The PilU protein is produced from the pilT-pilU operon and shares a high degree of homology with PilT. The function of PilT in Tfp biology has been studied extensively, whereas the role of PilU remains poorly understood. Here we show that pilU mutants have delayed microcolony formation on host epithelial cells compared to the wild type, indicating that bacterium-bacterium interactions are affected. In normal human serum, the pilU mutant survived at a higher rate than that for wild-type bacteria. However, in a murine model of disease, mice infected with the pilT mutant demonstrated significantly reduced bacterial blood counts and survived at a higher rate than that for mice infected with the wild type. Infection of mice with the pilU mutant resulted in a trend of lower bacteremia, and still a significant increase in survival, than that of the wild type. In conclusion, these data suggest that PilU promotes timely microcolony formation and that both PilU and PilT are required for full bacterial virulence.
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http://dx.doi.org/10.1128/IAI.06354-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416451PMC
July 2012

In vivo imaging of meningococcal disease dynamics.

Methods Mol Biol 2012 ;799:153-68

Department of Genetics, Microbiology, and Toxicology (GMT), Stockholm University, Stockholm, Sweden.

Neisseria meningitidis is a human specific organism that causes severe sepsis and/or meningitis with high mortality. The disease scenario is rapid and much remains unknown about the disease process and host-pathogen interaction. In this chapter, we describe a protocol for generating a bioluminescently labeled N. meningitidis strain in order to advance our understanding of meningococcal disease progression. We also describe how in vivo bioluminescence imaging (BLI) can be used to observe novel features of the disease dynamics during meningococcal infection.
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http://dx.doi.org/10.1007/978-1-61779-346-2_10DOI Listing
February 2012

NafA negatively controls Neisseria meningitidis piliation.

PLoS One 2011 1;6(7):e21749. Epub 2011 Jul 1.

Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden.

Bacterial auto-aggregation is a critical step during adhesion of N. meningitidis to host cells. The precise mechanisms and functions of bacterial auto-aggregation still remain to be fully elucidated. In this work, we characterize the role of a meningococcal hypothetical protein, NMB0995/NMC0982, and show that this protein, here denoted NafA, acts as an anti-aggregation factor. NafA was confirmed to be surface exposed and was found to be induced at a late stage of bacterial adherence to epithelial cells. A NafA deficient mutant was hyperpiliated and formed bundles of pili. Further, the mutant displayed increased adherence to epithelial cells when compared to the wild-type strain. In the absence of host cells, the NafA deficient mutant was more aggregative than the wild-type strain. The in vivo role of NafA in sepsis was studied in a murine model of meningococcal disease. Challenge with the NafA deficient mutant resulted in lower bacteremia levels and mortality when compared to the wild-type strain. The present study reveals that meningococcal NafA is an anti-aggregation factor with strong impact on the disease outcome. These data also suggest that appropriate bacterial auto-aggregation is controlled by both aggregation and anti-aggregation factors during Neisseria infection in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021749PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128610PMC
November 2011

The complement regulator CD46 is bactericidal to Helicobacter pylori and blocks urease activity.

Gastroenterology 2011 Sep 18;141(3):918-28. Epub 2011 May 18.

Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden.

Background & Aims: CD46 is a C3b/C4b binding complement regulator and a receptor for several human pathogens. We examined the interaction between CD46 and Helicobacter pylori (a bacterium that colonizes the human gastric mucosa and causes gastritis), peptic ulcers, and cancer.

Methods: Using gastric epithelial cells, we analyzed a set of H pylori strains and mutants for their ability to interact with CD46 and/or influence CD46 expression. Bacterial interaction with full-length CD46 and small CD46 peptides was evaluated by flow cytometry, fluorescence microscopy, enzyme-linked immunosorbent assay, and bacterial survival analyses.

Results: H pylori infection caused shedding of CD46 into the extracellular environment. A soluble form of CD46 bound to H pylori and inhibited growth, in a dose- and time-dependent manner, by interacting with urease and alkyl hydroperoxide reductase, which are essential bacterial pathogenicity-associated factors. Binding of CD46 or CD46-derived synthetic peptides blocked the urease activity and ability of bacteria to survive in acidic environments. Oral administration of one CD46 peptide eradicated H pylori from infected mice.

Conclusions: CD46 is an antimicrobial agent that can eradicate H pylori. CD46 peptides might be developed to treat H pylori infection.
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http://dx.doi.org/10.1053/j.gastro.2011.05.009DOI Listing
September 2011

Lactobacilli reduce cell cytotoxicity caused by Streptococcus pyogenes by producing lactic acid that degrades the toxic component lipoteichoic acid.

Antimicrob Agents Chemother 2011 Apr 18;55(4):1622-8. Epub 2011 Jan 18.

Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden.

Lactobacilli are known to prevent colonization by many pathogens; nevertheless, the mechanisms of their protective effect are largely unknown. In this work, we investigated the role of lactobacilli during infection of epithelial cells with group A streptococci (GAS). GAS cause a variety of illnesses ranging from noninvasive disease to more severe invasive infections, such as necrotizing fasciitis and toxic shock-like syndrome. Invasion of deeper tissues is facilitated by GAS-induced apoptosis and cell death. We found that lactobacilli inhibit GAS-induced host cell cytotoxicity and shedding of the complement regulator CD46. Further, survival assays demonstrated that lactic acid secreted by lactobacilli is highly bactericidal toward GAS. In addition, lactic acid treatment of GAS, but not heat killing, prior to infection abolishes the cytotoxic effects against human cells. Since lipoteichoic acid (LTA) of GAS is heat resistant and cytotoxic, we explored the effects of lactic acid on LTA. By applying such an approach, we demonstrate that lactic acid reduces epithelial cell damage caused by GAS by degrading both secreted and cell-bound LTA. Taken together, our experiments reveal a mechanism by which lactobacilli prevent pathogen-induced host cell damage.
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http://dx.doi.org/10.1128/AAC.00770-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067128PMC
April 2011
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