scholarly journals Lipidation of an FlrC-Dependent Protein Is Required for Enhanced Intestinal Colonization by Vibrio cholerae

2007 ◽  
Vol 190 (1) ◽  
pp. 231-239 ◽  
Author(s):  
David C. Morris ◽  
Fen Peng ◽  
Jeffrey R. Barker ◽  
Karl E. Klose
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Wild Type Strain ◽  
Intestinal Colonization ◽  
Class Iii ◽  
Wild Type ◽  
Colonization Factor ◽  
Dependent Protein ◽  
Fusion Analysis ◽  
Flagellar Genes

ABSTRACT Vibrio cholerae, the causative agent of cholera, has a sheathed, polar flagellum, and motility has been linked to virulence. An operon with two genes, flgO and flgP (VC2207 and VC2206), is positively regulated by FlrC, the activator of class III flagellar genes. Deletion of flgP results in a nonmotile phenotype, demonstrating the requirement of this gene for V. cholerae motility. V. cholerae ΔflgP cells synthesize fragile and defective flagella but transcribe flagellar genes similar to the wild-type strain. PhoA fusion analysis indicated that the putative lipoprotein FlgP is localized external to the cytoplasm, and fractionation demonstrated that it was localized to the outer membrane. Mutagenesis of the site of lipidation of FlgP (C18G) prevented [3H]palmitate incorporation and outer membrane localization. Interestingly, FlgP with the mutation C18G [FlgP(C18G)] could complement the ΔflgP mutant for motility, and the cells synthesized wild-type flagella. The ΔflgP mutant strain was defective for intestinal colonization (∼20-fold), but FlgP(C18G) was unable to complement this defect, demonstrating that lipidation of FlgP is essential for its role in intestinal colonization but not flagellar synthesis. FlgP thus represents a novel V. cholerae intestinal colonization factor that is regulated by the flagellar transcription hierarchy.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ryan W. Bogard ◽  
Bryan W. Davies ◽  
John J. Mekalanos
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Current Knowledge ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Pathogenic Potential ◽  
Content Type ◽  
Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

scholarly journals Role of the Helicobacter hepaticus Flagellar Sigma Factor FliA in Gene Regulation and Murine Colonization

2008 ◽  
Vol 190 (19) ◽  
pp. 6398-6408 ◽  
Author(s):  
Torsten Sterzenbach ◽  
Lucie Bartonickova ◽  
Wiebke Behrens ◽  
Birgit Brenneke ◽  
Jessika Schulze ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Sigma Factor ◽  
Intestinal Inflammation ◽  
Gall Stone ◽  
Helicobacter Hepaticus ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Chronic Intestinal Inflammation ◽  
Flagellar Genes

ABSTRACT The enterohepatic Helicobacter species Helicobacter hepaticus colonizes the murine intestinal and hepatobiliary tract and is associated with chronic intestinal inflammation, gall stone formation, hepatitis, and hepatocellular carcinoma. Thus far, the role of H. hepaticus motility and flagella in intestinal colonization is unknown. In other, closely related bacteria, late flagellar genes are mainly regulated by the sigma factor FliA (σ28). We investigated the function of the H. hepaticus FliA in gene regulation, flagellar biosynthesis, motility, and murine colonization. Competitive microarray analysis of the wild type versus an isogenic fliA mutant revealed that 11 genes were significantly more highly expressed in wild-type bacteria and 2 genes were significantly more highly expressed in the fliA mutant. Most of these were flagellar genes, but four novel FliA-regulated genes of unknown function were identified. H. hepaticus possesses two identical copies of the gene encoding the FliA-dependent major flagellin subunit FlaA (open reading frames HH1364 and HH1653). We characterized the phenotypes of mutants in which fliA or one or both copies of the flaA gene were knocked out. flaA_1 flaA_2 double mutants and fliA mutants did not synthesize detectable amounts of FlaA and possessed severely truncated flagella. Also, both mutants were nonmotile and unable to colonize mice. Mutants with either flaA gene knocked out produced flagella morphologically similar to those of wild-type bacteria and expressed FlaA and FlaB. flaA_1 mutants which had flagella but displayed reduced motility did not colonize mice, indicating that motility is required for intestinal colonization by H. hepaticus and that the presence of flagella alone is not sufficient.

scholarly journals Role of β-Lactamases and Porins in Resistance to Ertapenem and Other β-Lactams in Klebsiella pneumoniae

2004 ◽  
Vol 48 (8) ◽  
pp. 3203-3206 ◽  
Author(s):  
George A. Jacoby ◽  
Debra M. Mills ◽  
Nancy Chow
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Type Strain ◽  
Wild Type Strain ◽  
Outer Membrane Proteins ◽  
Wild Type ◽  
Resistant Mutants ◽  
High Level ◽  
Level Resistance

ABSTRACT High-level resistance to ertapenem was produced by β-lactamases of groups 1, 2f, and 3 in a strain of Klebsiella pneumoniae deficient in Omp35 and Omp36. From a wild-type strain producing ACT-1 β-lactamase, ertapenem-resistant mutants for which the ertapenem MICs were up to 128 μg/ml and expression of outer membrane proteins was diminished could be selected.

scholarly journals The Vibrio cholerae ToxR-Regulated Porin OmpU Confers Resistance to Antimicrobial Peptides

2004 ◽  
Vol 72 (6) ◽  
pp. 3577-3583 ◽  
Author(s):  
Jyoti Mathur ◽  
Matthew K. Waldor
Keyword(s):  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Surface Protein ◽  
Polymyxin B ◽  
Common Mechanism ◽  
Antimicrobial Protein ◽  
Wild Type ◽  
Human Gastrointestinal Tract ◽  
Serovar Typhimurium

ABSTRACT BPI (bactericidal/permeability-increasing) is a potent antimicrobial protein that was recently reported to be expressed as a surface protein on human gastrointestinal tract epithelial cells. In this study, we investigated the resistance of Vibrio cholerae, a small-bowel pathogen that causes cholera, to a BPI-derived peptide, P2. Unlike in Escherichia coli and Salmonella enterica serovar Typhimurium, resistance to P2 in V. cholerae was not dependent on the BipA GTPase. Instead, we found that ToxR, the master regulator of V. cholerae pathogenicity, controlled resistance to P2 by regulating the production of the outer membrane protein OmpU. Both toxR and ompU mutants were at least 100-fold more sensitive to P2 than were wild-type cells. OmpU also conferred resistance to polymyxin B sulfate, suggesting that this porin may impart resistance to cationic antibacterial proteins via a common mechanism. Studies of stationary-phase cells revealed that the ToxR-repressed porin OmpT may also contribute to P2 resistance. Finally, although the mechanism of porin-mediated resistance to antimicrobial peptides remains elusive, our data suggest that the BPI peptide sensitivity of OmpU-deficient V. cholerae is not attributable to a generally defective outer membrane.

scholarly journals Mutation in the peb1A Locus ofCampylobacter jejuni Reduces Interactions with Epithelial Cells and Intestinal Colonization of Mice

1998 ◽  
Vol 66 (3) ◽  
pp. 938-943 ◽  
Author(s):  
Zhiheng Pei ◽  
Christophe Burucoa ◽  
Bernadette Grignon ◽  
Shahida Baqar ◽  
Xiao-Zhe Huang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Type Strain ◽  
Wild Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kanamycin Resistance ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Cell Binding ◽  
Binding Factor

ABSTRACT Campylobacter jejuni is one of the leading causes of bacterial diarrhea throughout the world. We previously found that PEB1 is a homolog of cluster 3 binding proteins of bacterial ABC transporters and that a C. jejuni adhesin, cell-binding factor 1 (CBF1), if not identical to, contains PEB1. A single protein migrating at approximately 27 to 28 kDa was recognized by anti-CBF1 and anti-PEB1. To determine the role that the operon encoding PEB1 plays inC. jejuni adherence, peb1A, the gene encoding PEB1, was disrupted in strain 81-176 by insertion of a kanamycin resistance gene through homologous recombination. Inactivation of this operon completely abolished expression of CBF1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. In comparison to the wild-type strain, the mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture. Mouse challenge studies showed that the rate and duration of intestinal colonization by the mutant were significantly lower and shorter than with the wild-type strain. In summary, PEB1 is identical to a previously identified cell-binding factor, CBF1, in C. jejuni, and the peb1A locus plays an important role in epithelial cell interactions and in intestinal colonization in a mouse model.

scholarly journals Characterization of Two Outer Membrane Proteins, FlgO and FlgP, That Influence Vibrio cholerae Motility

2009 ◽  
Vol 191 (18) ◽  
pp. 5669-5679 ◽  
Author(s):  
Raquel M. Martinez ◽  
Madushini N. Dharmasena ◽  
Thomas J. Kirn ◽  
Ronald K. Taylor
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Outer Membrane Proteins ◽  
Sigma 54 ◽  
Colonization Factor ◽  
Motile Cells ◽  
Outer Membrane Lipoprotein ◽  
Important Virulence Factor

ABSTRACT Vibrio cholerae is highly motile by the action of a single polar flagellum. The loss of motility reduces the infectivity of V. cholerae, demonstrating that motility is an important virulence factor. FlrC is the sigma-54-dependent positive regulator of flagellar genes. Recently, the genes VC2206 (flgP) and VC2207 (flgO) were identified as being regulated by FlrC via a microarray analysis of an flrC mutant (D. C. Morris, F. Peng, J. R. Barker, and K. E. Klose, J. Bacteriol. 190:231-239, 2008). FlgP is reported to be an outer membrane lipoprotein required for motility that functions as a colonization factor. The study reported here focuses on the characterization of flgO, the first gene in the flgOP operon. We show that FlgO and FlgP are important for motility, as strains with mutations in the flgOP genes have reduced motility phenotypes. The flgO and flgP mutant populations display fewer motile cells as well as reduced numbers of flagellated cells. The flagella produced by the flgO and flgP mutant strains are shorter in length than the wild-type flagella, which can be restored by inhibiting rotation of the flagellum. FlgO is an outer membrane protein that localizes throughout the membrane and not at the flagellar pole. Although FlgO and FlgP do not specifically localize to the flagellum, they are required for flagellar stability. Due to the nature of these motility defects, we established that the flagellum is not sufficient for adherence; rather, motility is the essential factor required for attachment and thus colonization by V. cholerae O1 of the classical biotype. This study reveals a novel mechanism for which the outer membrane proteins FlgO and FlgP function in motility to mediate flagellar stability and influence attachment and colonization.

scholarly journals Novel Toluene Elimination System in a Toluene-Tolerant Microorganism

2000 ◽  
Vol 182 (22) ◽  
pp. 6451-6455 ◽  
Author(s):  
Hideki Kobayashi ◽  
Katsuyuki Uematsu ◽  
Hisako Hirayama ◽  
Koki Horikoshi
Keyword(s):  
Membrane Proteins ◽  
Cell Membrane ◽  
Outer Membrane ◽  
Parent Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Wild Type ◽  
Sensitive Mutant

ABSTRACT In studies of Pseudomonas putida IH-2000, a toluene-tolerant microorganism, membrane vesicles (MVs) were found to be released from the outer membrane when toluene was added to the culture. These MVs were found to be composed of phospholipids, lipopolysaccharides (LPS), and very low amounts of outer membrane proteins. The MVs also contained a higher concentration of toluene molecules (0.172 ± 0.012 mol/mol of lipid) than that found in the cell membrane. In contrast to the wild-type strain, the toluene-sensitive mutant strain 32, which differs from the parent strain in LPS and outer membrane proteins, did not release MVs from the outer membrane. The toluene molecules adhering to the outer membrane are eliminated by the shedding of MVs, and this system appears to serve as an important part of the toluene tolerance system of IH-2000.

scholarly journals OpnS, an Outer Membrane Porin of Xenorhabdus nematophila, Confers a Competitive Advantage for Growth in the Insect Host

2009 ◽  
Vol 191 (17) ◽  
pp. 5471-5479 ◽  
Author(s):  
Ransome van der Hoeven ◽  
Steven Forst
Keyword(s):  
Competitive Advantage ◽  
Outer Membrane ◽  
Type Strain ◽  
Wild Type Strain ◽  
Entomopathogenic Nematode ◽  
Growth Defect ◽  
Insect Host ◽  
Xenorhabdus Nematophila ◽  
Wild Type ◽  
Defined Culture

ABSTRACT The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded β-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the ΔopnS strain. Coinjection of the wild-type and ΔopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or ΔopnS strain were colonized by the wild-type strain. In addition, the ΔopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The ΔopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment.

scholarly journals Leptospira interrogans lpxDHomologue Is Required for Thermal Acclimatization and Virulence

2015 ◽  
Vol 83 (11) ◽  
pp. 4314-4321 ◽  
Author(s):  
Azad Eshghi ◽  
Jeremy Henderson ◽  
M. Stephen Trent ◽  
Mathieu Picardeau
Keyword(s):  
Outer Membrane ◽  
Type Strain ◽  
Lipid A ◽  
Wild Type Strain ◽  
Infection Model ◽  
Leptospira Interrogans ◽  
Wild Type ◽  
Content Type ◽  
Physiological Temperature ◽  
Animal Infection

ABSTRACTLeptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. PathogenicLeptospirabacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered byLeptospiralikely requires various adaptive mechanisms. Little is known aboutLeptospiraouter membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts.Leptospirabacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamineN-acyltransferase genes (la0512 and la4326 [lpxD1andlpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in thelpxD1mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, anin transcomplementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated aslpxD1inLeptospira interrogansplays an important role in temperature adaptation and virulence in the animal infection model.

Sign in / Sign up

Export Citation Format

Share Document