Development of a Tetracycline-Inducible Gene Expression System for the Study of Helicobacter pylori Pathogenesis

ABSTRACTDeletion mutants and animal models have been instrumental in the study ofHelicobacter pyloripathogenesis. Conditional mutants, however, would enable the study of the temporal gene requirement duringH. pyloricolonization and chronic infection. To achieve this goal, we adapted theEscherichia coliTn10-derived tetracycline-inducible expression system for use inH. pylori. TheureApromoter was modified by inserting one or twotetoperators to generate tetracycline-responsive promoters, nameduPtetO, and these promoters were then fused to the reportergfpmut2 and inserted into different loci. The expression of the tetracycline repressor (tetR) was placed under the control of one of three promoters and inserted into the chromosome. Conditional expression of green fluorescent protein (GFP) in strains harboringtetRanduPtetO-GFPwas characterized by measuring GFP activity and by immunoblotting. The twotet-responsiveuPtetOpromoters differ in strength, and induction of these promoters was inducer concentration and time dependent, with maximum expression achieved after induction for 8 to 16 h. Furthermore, the chromosomal location of theuPtetO-GFPconstruct and the nature of the promoter driving expression oftetRinfluenced the strength of theuPtetOpromoters upon induction. Integration ofuPtetO-GFPandtetRconstructs at different genomic loci was stablein vivoand did not affect colonization. Finally, we demonstrate tetracycline-dependent induction of GFP expressionin vivoduring chronic infection. These results open new experimental avenues for dissectingH. pyloripathogenesis using animal models and for testing the roles of specific genes in colonization of, adaptation to, and persistence in the host.

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Measurement of Internal pH inHelicobacter pyloriby Using Green Fluorescent Protein Fluorimetry

Journal of Bacteriology ◽

10.1128/jb.00178-18 ◽

2018 ◽

Vol 200 (14) ◽

Cited By ~ 1

Author(s):

Yi Wen ◽

David R. Scott ◽

Olga Vagin ◽

Elmira Tokhtaeva ◽

Elizabeth A. Marcus ◽

...

Keyword(s):

Helicobacter Pylori ◽

Green Fluorescent Protein ◽

Fusion Proteins ◽

Acidic Medium ◽

Fluorescent Protein ◽

Medium Ph ◽

Content Type ◽

H Pylori ◽

Green Fluorescent ◽

Acid Acclimation

ABSTRACTHelicobacter pyloriis an organism known to colonize the normal human stomach. Previous studies have shown that the bacterium does this by elevating its periplasmic pH via the hydrolysis of urea. However, the value of the periplasmic pH was calculated indirectly from the proton motive force equation. To measure the periplasmic pH directly inH. pylori, we fused enhanced green fluorescent protein (EGFP) to the predicted twin-arginine signal peptides of HydA and KapA fromH. pyloriand TorA fromEscherichia coli. The fusion proteins were expressed in theH. pylorigenome under the control of thecagApromoter. Confocal microscopic and cell fractionation/immunoblotting analyses detected TorA-EGFP in the periplasm and KapA-EGFP in both the periplasm and cytoplasm, while the mature form of HydA-EGFP was seen at low levels in the periplasm, with major cytoplasmic retention of the precursor form. WithH. pyloriexpressing TorA-EGFP, we established a system to directly measure periplasmic pH based on the pH-sensitive fluorimetry of EGFP. These measurements demonstrated that the addition of 5 mM urea has little effect on the periplasmic pH at a medium pH higher than pH 6.5 but rapidly increases the periplasmic pH to pH 6.1 at an acidic medium pH (pH 5.0), corresponding to the opening of the proton-gated channel, UreI, and confirming the basis of gastric colonization. Measurements of the periplasmic pH in an HP0244 (FlgS)-deficient mutant ofH. pyloriexpressing TorA-EGFP revealed a significant loss of the urea-dependent increase in the periplasmic pH at an acidic medium pH, providing additional evidence that FlgS is responsible for recruitment of urease to the inner membrane in association with UreI.IMPORTANCEHelicobacter pylorihas been identified as the major cause of chronic superficial gastritis and peptic ulcer disease. In addition, persistent infection withH. pylori, which, if untreated, lasts for the lifetime of an infected individual, predisposes one to gastric malignancies, such as adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. A unique feature of the neutralophilic bacteriumH. pyloriis its ability to survive in the extremely acidic environment of the stomach through its acid acclimation mechanism. The presented results on measurements of periplasmic pH inH. pyloribased on fluorimetry of fully active green fluorescent protein fusion proteins exported with the twin-arginine translocase system provide a reliable and rapid tool for the investigation of acid acclimation inH. pylori.

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Systems Modeling of the Role of Interleukin-21 in the Maintenance of Effector CD4+ T Cell Responses during Chronic Helicobacter pylori Infection

mBio ◽

10.1128/mbio.01243-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 36

Author(s):

Adria Carbo ◽

Danyvid Olivares-Villagómez ◽

Raquel Hontecillas ◽

Josep Bassaganya-Riera ◽

Rupesh Chaturvedi ◽

...

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

T Cell ◽

Wild Type ◽

Content Type ◽

Interleukin 21 ◽

H Pylori ◽

Deficient Mice

ABSTRACTThe development of gastritis duringHelicobacter pyloriinfection is dependent on an activated adaptive immune response orchestrated by T helper (Th) cells. However, the relative contributions of the Th1 and Th17 subsets to gastritis and control of infection are still under investigation. To investigate the role of interleukin-21 (IL-21) in the gastric mucosa duringH. pyloriinfection, we combined mathematical modeling of CD4+T cell differentiation within vivomechanistic studies. We infected IL-21-deficient and wild-type mice withH. pyloristrain SS1 and assessed colonization, gastric inflammation, cellular infiltration, and cytokine profiles. ChronicallyH. pylori-infected IL-21-deficient mice had higherH. pyloricolonization, significantly less gastritis, and reduced expression of proinflammatory cytokines and chemokines compared to these parameters in infected wild-type littermates. Thesein vivodata were used to calibrate anH. pyloriinfection-dependent, CD4+T cell-specific computational model, which then described the mechanism by which IL-21 activates the production of interferon gamma (IFN-γ) and IL-17 during chronicH. pyloriinfection. The model predicted activated expression of T-bet and RORγt and the phosphorylation of STAT3 and STAT1 and suggested a potential role of IL-21 in the modulation of IL-10. Driven by our modeling-derived predictions, we found reduced levels of CD4+splenocyte-specifictbx21androrcexpression, reduced phosphorylation of STAT1 and STAT3, and an increase in CD4+T cell-specific IL-10 expression inH. pylori-infected IL-21-deficient mice. Our results indicate that IL-21 regulates Th1 and Th17 effector responses during chronicH. pyloriinfection in a STAT1- and STAT3-dependent manner, therefore playing a major role controllingH. pyloriinfection and gastritis.IMPORTANCEHelicobacter pyloriis the dominant member of the gastric microbiota in more than 50% of the world’s population.H. pyloricolonization has been implicated in gastritis and gastric cancer, as infection withH. pyloriis the single most common risk factor for gastric cancer. Current data suggest that, in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization and chronic infection. This study uses a combined computational and experimental approach to investigate how IL-21, a proinflammatory T cell-derived cytokine, maintains the chronic proinflammatory T cell immune response driving chronic gastritis duringH. pyloriinfection. This research will also provide insight into a myriad of other infectious and immune disorders in which IL-21 is increasingly recognized to play a central role. The use of IL-21-related therapies may provide treatment options for individuals chronically colonized withH. pylorias an alternative to aggressive antibiotics.

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Dynamics of Lewis b Binding and Sequence Variation of the babA Adhesin Gene during Chronic Helicobacter pylori Infection in Humans

mBio ◽

10.1128/mbio.02281-14 ◽

2014 ◽

Vol 5 (6) ◽

Cited By ~ 26

Author(s):

Sandra Nell ◽

Lynn Kennemann ◽

Sandra Schwarz ◽

Christine Josenhans ◽

Sebastian Suerbaum

Keyword(s):

Helicobacter Pylori ◽

Amino Acid ◽

Outer Membrane ◽

Chronic Infection ◽

Human Infection ◽

Complete Loss ◽

Blood Group Antigen ◽

Content Type ◽

Strain Pair ◽

H Pylori

ABSTRACTHelicobacter pyloriundergoes rapid microevolution during chronic infection, but very little is known about how this affects host interaction factors. The best-studied adhesin ofH. pyloriis BabA, which mediates binding to the blood group antigen Lewis b [Le(b)]. To study the dynamics of Le(b) adherence during human infection, we analyzed pairedH. pyloriisolates obtained sequentially from chronically infected individuals. A complete loss or significant reduction of Le(b) binding was observed in strains from 5 out of 23 individuals, indicating that the Le(b) binding phenotype is quite stable during chronic human infection. Sequence comparisons ofbabAidentified differences due to mutation and/or recombination in 12 out of 16 strain pairs analyzed. Most amino acid changes were found in the putative N-terminal extracellular adhesion domain. One strain pair that had changed from a Le(b) binding to a nonbinding phenotype was used to study the role of distinct sequence changes in Le(b) binding. By transformations of the nonbinding strain with ababAgene amplified from the binding strain,H. pyloristrains with mosaicbabAgenes were generated. Recombinants were enriched for a gain of Le(b) binding by biopanning or for BabA expression on the bacterial surface by pulldown assay. With this approach, we identified several amino acid residues affecting the strength of Le(b) binding. Additionally, the data showed that the C terminus of BabA, which is predicted to encode an outer membrane β-barrel domain, plays an essential role in the biogenesis of this protein.IMPORTANCEHelicobacter pyloricauses a chronic infection of the human stomach that can lead to ulcers and cancer. The bacterium can bind to gastric epithelial cells with specialized outer membrane proteins. The best-studied protein is the BabA adhesin which binds to the Lewis b blood group antigen. SinceH. pyloriis a bacterium with very high genetic variability, we asked whetherbabAevolves during chronic infection and how mutations or recombination inbabAaffect binding. We found that BabA-mediated adherence was stable in most individuals but observed a complete loss of binding or reduced binding in 22% of individuals. One strain pair in which binding was lost was used to generatebabAsequences that were mosaics of a functional allele and a nonfunctional allele, and the mosaic sequences were used to identify amino acids critically involved in binding of BabA to Lewis b.

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Cholesterol Enhances Helicobacter pylori Resistance to Antibiotics and LL-37

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00016-11 ◽

2011 ◽

Vol 55 (6) ◽

pp. 2897-2904 ◽

Cited By ~ 63

Author(s):

David J. McGee ◽

Alika E. George ◽

Elizabeth A. Trainor ◽

Katherine E. Horton ◽

Ellen Hildebrandt ◽

...

Keyword(s):

Helicobacter Pylori ◽

Lipid A ◽

Polymyxin B ◽

Defined Medium ◽

Vital Role ◽

Content Type ◽

The Two Cultures ◽

H Pylori ◽

The Impact

ABSTRACTThe human gastric pathogenHelicobacter pyloristeals host cholesterol, modifies it by glycosylation, and incorporates the glycosylated cholesterol onto its surface via a cholesterol glucosyltransferase, encoded bycgt. The impact of cholesterol onH. pyloriantimicrobial resistance is unknown.H. pyloristrain 26695 was cultured in Ham's F12 chemically defined medium in the presence or absence of cholesterol. The two cultures were subjected to overnight incubations with serial 2-fold dilutions of 12 antibiotics, six antifungals, and seven antimicrobial peptides (including LL-37 cathelicidin and human alpha and beta defensins). Of 25 agents tested, cholesterol-grownH. pyloricells were substantially more resistant (over 100-fold) to nine agents than wereH. pyloricells grown without cholesterol. These nine agents included eight antibiotics and LL-37.H. pyloriwas susceptible to the antifungal drug pimaricin regardless of cholesterol presence in the culture medium. Acgtmutant retained cholesterol-dependent resistance to most antimicrobials but displayed increased susceptibility to colistin, suggesting an involvement of lipid A. Mutation oflpxE, encoding lipid A1-phosphatase, led to loss of cholesterol-dependent resistance to polymyxin B and colistin but not other antimicrobials tested. Thecgtmutant was severely attenuated in gerbils, indicating that glycosylation is essentialin vivo. These findings suggest that cholesterol plays a vital role in virulence and contributes to the intrinsic antibiotic resistance ofH. pylori.

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In Vitro and In Vivo Antibacterial Activities of Patchouli Alcohol, a Naturally Occurring Tricyclic Sesquiterpene, against Helicobacter pylori Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00122-17 ◽

2017 ◽

Vol 61 (6) ◽

Cited By ~ 16

Author(s):

Y. F. Xu ◽

D. W. Lian ◽

Y. Q. Chen ◽

Y. F. Cai ◽

Y. F. Zheng ◽

...

Keyword(s):

Helicobacter Pylori ◽

Bacterial Resistance ◽

Clinical Isolates ◽

Potential Candidate ◽

Necrosis Factor Alpha ◽

Content Type ◽

Patchouli Alcohol ◽

H Pylori

ABSTRACT This study further evaluated the in vitro and in vivo anti-Helicobacter pylori activities and potential underlying mechanism of patchouli alcohol (PA), a tricyclic sesquiterpene. In the in vitro assay, the capacities of PA to inhibit and kill H. pylori were tested on three standard strains at different pH values and on 12 clinical isolates. The effects of PA on H. pylori adhesion (and its alpA, alpB, and babA genes), motility (and its flaA and flaB genes), ultrastructure, and flagellation were investigated. Moreover, the H. pylori resistance to and postantibiotic effect (PAE) of PA were determined. Furthermore, the in vivo effects of PA on H. pylori eradication and gastritis were examined. Results showed that MICs of PA against three standard strains (pH 5.3 to 9) and 12 clinical isolates were 25 to 75 and 12.5 to 50 μg/ml, respectively. The killing kinetics of PA were time and concentration dependent, and its minimal bactericidal concentrations (MBCs) were 25 to 75 μg/ml. In addition, H. pylori adhesion, motility, ultrastructure, and flagellation were significantly suppressed. PA also remarkably inhibited the expression of adhesion genes (alpA and alpB) and motility genes (flaA and flaB). Furthermore, PA treatment caused a longer PAE and less bacterial resistance than clarithromycin and metronidazole. The in vivo study showed that PA can effectively eradicate H. pylori, inhibit gastritis, and suppress the expression of inflammatory mediators (COX-2, interleukin 1β, tumor necrosis factor alpha, and inducible nitric oxide synthase [iNOS]). In conclusion, PA can efficiently kill H. pylori, interfere with its infection process, and attenuate gastritis with less bacterial resistance, making it a potential candidate for new drug development.

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Natural Competence Promotes Helicobacter pylori Chronic Infection

Infection and Immunity ◽

10.1128/iai.01042-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 209-215 ◽

Cited By ~ 31

Author(s):

Marion S. Dorer ◽

Ilana E. Cohen ◽

Tate H. Sessler ◽

Jutta Fero ◽

Nina R. Salama

Keyword(s):

Helicobacter Pylori ◽

Chronic Infection ◽

Acute Infection ◽

Host Cells ◽

High Rate ◽

Natural Competence ◽

Content Type ◽

Type Iv ◽

Competence Factor ◽

H Pylori

Animal models are important tools for studies of human disease, but developing these models is a particular challenge with regard to organisms with restricted host ranges, such as the human stomach pathogenHelicobacter pylori. In most cases,H. pyloriinfects the stomach for many decades before symptoms appear, distinguishing it from many bacterial pathogens that cause acute infection. To model chronic infection in the mouse, a human clinical isolate was selected for its ability to survive for 2 months in the mouse stomach, and the resulting strain, MSD132, colonized the mouse stomach for at least 28 weeks. During selection, thecagYcomponent of the Cag type IV secretion system was mutated, disrupting a key interaction with host cells. Increases in both bacterial persistence and bacterial burden occurred prior to this mutation, and a mixed population ofcagY+andcagYmutant cells was isolated from a single mouse, suggesting that mutations accumulate during selection and that factors in addition to the Cag apparatus are important for murine adaptation. Diversity in both alleles and genes is common inH. pyloristrains, and natural competence mediates a high rate of interstrain genetic exchange. Mutations of the Com apparatus, a membrane DNA transporter, and DprA, a cytosolic competence factor, resulted in reduced persistence, although initial colonization was normal. Thus, exchange of DNA between genetically heterogeneousH. pyloristrains may improve chronic colonization. The strains and methods described here will be important tools for defining both the spectrum of mutations that promote murine adaptation and the genetic program of chronic infection.

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Helicobacter pylori Stores Nickel To Aid Its Host Colonization

Infection and Immunity ◽

10.1128/iai.00858-12 ◽

2012 ◽

Vol 81 (2) ◽

pp. 580-584 ◽

Cited By ~ 21

Author(s):

Stéphane L. Benoit ◽

Erica F. Miller ◽

Robert J. Maier

Keyword(s):

Helicobacter Pylori ◽

Gastric Mucosa ◽

Physiological Role ◽

Wild Type ◽

Rank Tests ◽

Content Type ◽

Host Mouse ◽

H Pylori ◽

Very High

ABSTRACTThe transition metal nickel (Ni) is critical for the pathogenicity ofHelicobacter pylori. Indeed the element is a required component of two enzymes, hydrogenase and urease, that have been shown to be important forin vivocolonization of the host gastric mucosa. Urease accounts for up to 10% of the total cellularH. pyloriprotein content, and therefore the bacterial Ni demand is very high.H. pyloripossess two small and abundant histidine-rich, Ni-binding proteins, Hpn and Hpn-like, whose physiological role in the host have not been investigated. In this study, special husbandry conditions were used to control Ni levels in the host (mouse), including the use of Ni-free versus Ni-supplemented food. The efficacy of each diet was confirmed by measuring the Ni concentrations in sera of mice fed with either diet. Colonization levels (based on rank tests) of theΔhpn Δhpn-like double mutants isolated from the mice provided Ni-deficient chow were statistically lower than those for mice given Ni in their diet. In contrast,H. pyloriwild-type colonization levels were similar in both host groups (e.g., regardless of Ni levels). Our results indicate that the gastric pathogenH. pylorican utilize stored Ni via defined histidine-rich proteins to aid colonization of the host.

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Anti-Helicobacter pylori Potential of Artemisinin and Its Derivatives

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00407-12 ◽

2012 ◽

Vol 56 (9) ◽

pp. 4594-4607 ◽

Cited By ~ 24

Author(s):

Suchandra Goswami ◽

Rajendra S. Bhakuni ◽

Annalakshmi Chinniah ◽

Anirban Pal ◽

Sudip K. Kar ◽

...

Keyword(s):

Helicobacter Pylori ◽

Artemisia Annua ◽

Infection Model ◽

Strong Activity ◽

Content Type ◽

Content Development ◽

Candidate Drug ◽

Resistant Strains ◽

H Pylori

ABSTRACTThe antimalarial drug artemisinin fromArtemisia annuademonstrated remarkably strong activity againstHelicobacter pylori, the pathogen responsible for peptic ulcer diseases. In an effort to develop a novel antimicrobial chemotherapeutic agent containing such a sesquiterpene lactone endoperoxide, a series of analogues (2 natural and 15 semisynthetic molecules), including eight newly synthesized compounds, were investigated against clinical and standard strains ofH. pylori. The antimicrobial spectrum against 10H. pyloristrains and a few other bacterial and fungal strains indicated specificity against the ulcer causing organism. Of five promising molecules, a newly synthesized ether derivative β-artecyclopropylmether was found to be the most potent compound, which exhibited MIC range, MIC90, and minimum bactericidal concentration range values of 0.25 to 1.0 μg/ml, 1.0 μg/ml, and 1 to 16 μg/ml, respectively, against both resistant and sensitive strains ofH. pylori. The molecule demonstrated strong bactericidal kinetics with extensive morphological degeneration, retained functional efficacy at stomach acidic pH unlike clarithromycin, did not elicit drug resistance unlike metronidazole, and imparted sensitivity to resistant strains. It is not cytotoxic and exhibitsin vivopotentiality to reduce theH. pyloriburden in a chronic infection model. Thus, β-artecyclopropylmether could be a lead candidate for anti-H. pyloritherapeutics. Since the recurrence of gastroduodenal ulcers is believed to be mainly due to antibiotic resistance of the commensal organismH. pylori, development of a candidate drug from this finding is warranted.

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Hydrogen Peroxide-Mediated Oxygen Enrichment Eradicates Helicobacter pylori In Vitro and In Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02192-19 ◽

2020 ◽

Vol 64 (5) ◽

Author(s):

Jia Di ◽

Jun Zhang ◽

Lei Cao ◽

Ting-ting Huang ◽

Jun-xia Zhang ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Helicobacter Pylori ◽

Bacterial Cell ◽

Cell Membranes ◽

Enriched Environment ◽

Mongolian Gerbils ◽

Content Type ◽

H Pylori

ABSTRACT Helicobacter pylori is an important risk factor for gastric ulcers. However, antibacterial therapies increase the resistance rate and decrease the eradication rate of H. pylori. Inspired by the microaerophilic characteristics of H. pylori, we aimed at effectively establishing an oxygen-enriched environment to eradicate and prevent the recurrence of H. pylori. The effect and the mechanism of an oxygen-enriched environment in eradicating H. pylori and preventing the recurrence were explored in vitro and in vivo. During oral administration and after drug withdrawal, H. pylori counts were evaluated by Giemsa staining in animal cohorts. An oxygen-enriched environment in which H. pylori could not survive was successfully established by adding hydrogen peroxide into several solutions and rabbit gastric juice. Hydrogen peroxide effectively killed H. pylori in Columbia blood agar and special peptone broth. Minimum inhibition concentrations and minimum bactericidal concentrations of hydrogen peroxide were both relatively stable after promotion of resistance for 30 generations, indicating that hydrogen peroxide did not easily promote resistance in H. pylori. In models of Mongolian gerbils and Kunming mice, hydrogen peroxide has been shown to significantly eradicate and effectively prevent the recurrence of H. pylori without toxicity and damage to the gastric mucosa. The mechanism of hydrogen peroxide causing H. pylori death was related to the disruption of bacterial cell membranes. The oxygen-enriched environment achieved by hydrogen peroxide eradicates and prevents the recurrence of H. pylori by damaging bacterial cell membranes. Hydrogen peroxide thus provides an attractive candidate for anti-H. pylori treatment.

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Mutant Brucella abortus Membrane Fusogenic Protein Induces Protection against Challenge Infection in Mice

Infection and Immunity ◽

10.1128/iai.02790-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1458-1464 ◽

Cited By ~ 6

Author(s):

Job Alves de Souza Filho ◽

Vicente de Paulo Martins ◽

Priscila Carneiro Campos ◽

Juliana Alves-Silva ◽

Nathalia V. Santos ◽

...

Keyword(s):

Fluorescent Protein ◽

Economic Losses ◽

Macrophage Infection ◽

Content Type ◽

Live Vaccines ◽

Interferon Regulatory Factor 1 ◽

Potential Vaccine ◽

Green Fluorescent

Brucellaspecies can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence ofBrucellaspp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of theBrucellamembrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed thatB. abortusΔmfp::kanand Δomp19::kandeletion mutant strains have reduced persistencein vivoin C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kanor Δomp19::kanstrain expressing green fluorescent protein (GFP) approximately 80% or 65% ofBrucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments.B. abortusΔomp19::kanwas attenuatedin vivobut had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kanstrain had a lower virulence in these same mouse models. Furthermore, Δmfp::kanand Δomp19::kanstrains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kanstrain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kanstrain induced protection similar to that of RB51. Thus, these results demonstrate thatBrucellaMfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kanmutant may serve as a potential vaccine candidate in future studies.

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