The lpf Gene Cluster for Long Polar Fimbriae Is Not Involved in Adherence of Enteropathogenic Escherichia coli or Virulence of Citrobacter rodentium

ABSTRACT Using the enteropathogenic Escherichia coli (EPEC) genome sequence, we found that EPEC E2348/69 has an lpfABCDE gene cluster homologous (about 60% identical at the protein level) to the Salmonella long polar fimbria (LPF) operon. To determine whether this operon is essential for adherence, the lpfABCDE2 3 genes were deleted from EPEC strain E2348/69 by allelic exchange. Analysis of the resulting EPECΔlpfABCDE23 strain showed no change in adherence to HeLa cells or to human intestinal biopsy cells in the in vitro organ culture (IVOC) system compared to the wild type. Sera from volunteers experimentally infected with E2348/69 showed no antibody response to the major subunit protein, LpfA. These results suggested that the lpfE23 gene cluster is not necessary for EPEC adherence and attaching/effacing (A/E) lesion formation on human biopsy samples and is not expressed during human infection. We also identified an lpf gene cluster in Citrobacter rodentium strain ICC168 (lpfcr ). A ΔlpfAcr mutant of ICC168 retained wild-type adherence and A/E lesion-forming activity on HeLa cells. C3H/HeJ mice were infected with a wild-type C. rodentium strain and its lpfAcr isogenic mutant. Both strains were recovered at high levels in stools, and there were no significant differences between the groups both in terms of the number of CFU/organ (colon and cecum) and in terms of the amount of hyperplasia, as measured by weight. Similar results were observed in a second mouse strain, C57BL/6. These data suggest that in addition to playing no apparent role in EPEC pathogenesis, lpfcr is not required for C. rodentium virulence in either the C3H/HeJ or C57BL/6 mouse model.

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Multiple Elements Controlling Adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa Cells

Infection and Immunity ◽

10.1128/iai.71.9.4985-4995.2003 ◽

2003 ◽

Vol 71 (9) ◽

pp. 4985-4995 ◽

Cited By ~ 108

Author(s):

Alfredo G. Torres ◽

James B. Kaper

Keyword(s):

Escherichia Coli ◽

Hela Cells ◽

Culture Conditions ◽

Enterohemorrhagic Escherichia Coli ◽

Differentially Expressed ◽

Wild Type ◽

Transposon Insertion ◽

Reduced Adherence ◽

Insertion Mutants

ABSTRACT Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated tcdA gene. An isogenic ompA mutant showed reduced adherence compared to the parent strain. Disruption of the ompA gene in the tdcA mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and tdcA mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.

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Characterization of a Haemophilus ducreyi Mutant Deficient in Expression of Cytolethal Distending Toxin

Infection and Immunity ◽

10.1128/iai.67.8.3900-3908.1999 ◽

1999 ◽

Vol 67 (8) ◽

pp. 3900-3908 ◽

Cited By ~ 49

Author(s):

Marla K. Stevens ◽

Jo L. Latimer ◽

Sheryl R. Lumbley ◽

Christine K. Ward ◽

Leslie D. Cope ◽

...

Keyword(s):

Gene Cluster ◽

Hela Cells ◽

Culture Supernatant ◽

Haemophilus Ducreyi ◽

Cytolethal Distending Toxin ◽

Supernatant Fluid ◽

Hacat Keratinocytes ◽

Wild Type ◽

Insertional Inactivation

ABSTRACT Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that kills HeLa, HEp-2, and other human epithelial cells in vitro. H. ducreyi CDT activity is encoded by a three-gene cluster (cdtABC), and antibody to the cdtC gene product can neutralize CDT activity in vitro (L. D. Cope, S. R. Lumbley, J. L. Latimer, J. Klesney-Tait, M. K. Stevens, L. S. Johnson, M. Purven, R. S. Munson, Jr., T. Lagergard, J. D. Radolf, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 94:4056–4061, 1997). Culture supernatant fluid from a recombinant Escherichia colistrain containing the H. ducreyi cdtABC gene cluster readily killed both HeLa cells and HaCaT keratinocytes and had a modest inhibitory effect on the growth of human foreskin fibroblasts. Insertional inactivation of the cdtC gene in this recombinant E. coli strain eliminated the ability of this strain to kill HeLa cells and HaCaT keratinocytes. This mutatedH. ducreyi cdtABC gene cluster was used to construct an isogenic H. ducreyi cdtC mutant. Monoclonal antibodies against the H. ducreyi CdtA, CdtB, and CdtC proteins were used to characterize protein expression by this cdtCmutant. Culture supernatant fluid from this H. ducreyi cdtCmutant did not detectably affect any of the human cells used in this study. The presence of the wild-type H. ducreyi cdtC gene in trans in this H. ducreyi mutant restored its ability to express a CDT that killed both HeLa cells and HaCaT keratinocytes. The isogenic H. ducreyi cdtC mutant was shown to be as virulent as its wild-type parent strain in the temperature-dependent rabbit model for experimental chancroid. Lack of expression of the H. ducreyi CdtC protein also did not affect the ability of this H. ducreyi mutant to survive in the skin of rabbits.

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Interleukin-1 (IL-1) Signaling in Intestinal Stromal Cells Controls KC/CXCL1 Secretion, Which Correlates with Recruitment of IL-22-Secreting Neutrophils at Early Stages of Citrobacter rodentium Infection

Infection and Immunity ◽

10.1128/iai.00670-15 ◽

2015 ◽

Vol 83 (8) ◽

pp. 3257-3267 ◽

Cited By ~ 24

Author(s):

Yong-Soo Lee ◽

Hyungjun Yang ◽

Jin-Young Yang ◽

Yeji Kim ◽

Su-Hyun Lee ◽

...

Keyword(s):

Public Health ◽

Escherichia Coli ◽

Bacterial Infection ◽

Stromal Cells ◽

Interleukin 1 ◽

Citrobacter Rodentium ◽

Wild Type ◽

Content Type ◽

Health Concerns

Attaching and effacing pathogens, including enterohemorrhagicEscherichia coliin humans andCitrobacter rodentiumin mice, raise serious public health concerns. Here we demonstrate that interleukin-1 receptor (IL-1R) signaling is indispensable for protection againstC. rodentiuminfection in mice. Four days after infection withC. rodentium, there were significantly fewer neutrophils (CD11b+Ly6C+Ly6G+) in the colons of IL-1R−/−mice than in wild-type mice. Levels of mRNA and protein of KC/CXCL1 were also significantly reduced in colon homogenates of infected IL-1R−/−mice relative to wild-type mice. Of note, infiltrated CD11b+Ly6C+Ly6G+neutrophils were the main source of IL-22 secretion afterC. rodentiuminfection. Interestingly, intestinal stromal cells isolated from IL-1R−/−mice secreted lower levels of KC/CXCL1 than stromal cells from wild-type mice duringC. rodentiuminfection. Similar effects were found when mouse intestinal stromal cells and human nasal polyp stromal cells were treated with IL-1R antagonists (i.e., anakinra)in vitro. These results suggest that IL-1 signaling plays a pivotal role in activating mucosal stromal cells to secrete KC/CXCL1, which is essential for infiltration of IL-22-secreting neutrophils upon bacterial infection.

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Differential Suppression of priA2::kan Phenotypes in Escherichia coli K-12 by Mutations in priA, lexA, and dnaC

Genetics ◽

10.1093/genetics/143.1.5 ◽

1996 ◽

Vol 143 (1) ◽

pp. 5-13 ◽

Cited By ~ 21

Author(s):

Steven J Sandler ◽

Hardeep S Samra ◽

Alvin J Clark

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Mutant Allele ◽

Wild Type ◽

Suppressor Mutations ◽

Dnab Helicase ◽

K 12 ◽

Extragenic Suppressors ◽

Assembly Activity

Abstract First identified as an essential component of the ϕX174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec−, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.

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Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3468-3474.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3468-3474 ◽

Cited By ~ 19

Author(s):

Gyeong Tae Eom ◽

Jae Kwang Song ◽

Jung Hoon Ahn ◽

Yeon Soo Seo ◽

Joon Shick Rhee

Keyword(s):

Escherichia Coli ◽

Directed Evolution ◽

Abc Transporter ◽

Microtiter Plate ◽

Single Amino Acid ◽

Wild Type ◽

E Coli ◽

Single Amino Acid Change ◽

Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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Interactions of Enteropathogenic Escherichia coli with Pediatric and Adult Intestinal Biopsy Specimens during Early Adherence

Infection and Immunity ◽

10.1128/iai.00686-09 ◽

2009 ◽

Vol 77 (10) ◽

pp. 4463-4468 ◽

Cited By ~ 6

Author(s):

Romney M. Humphries ◽

Christopher C. M. Waterhouse ◽

George Mulvey ◽

Paul Beck ◽

Glen D. Armstrong

Keyword(s):

Escherichia Coli ◽

Age Groups ◽

Watery Diarrhea ◽

Intestinal Biopsy ◽

Epec Strain ◽

Type Iv ◽

Biopsy Specimens ◽

Cultured Epithelial Cells ◽

Glycan Receptors

ABSTRACT Enteropathogenic Escherichia coli (EPEC) strains cause watery diarrhea almost exclusively in young children. The basis for this age discrimination has never been determined, but it may be related to host cell receptors. During infection, EPEC strains express type IV bundle-forming pili composed of repeating subunits of the protein called bundlin. The very first interaction between EPEC and in vitro-cultured epithelial cells is mediated by the binding of α-bundlin to a carbohydrate receptor that contains, at a minimum, the N-acetyllactosamine (LacNAc) glycan sequence. However, bundlins expressed from the β-bundlin allele do not bind LacNAc glycan sequences. Herein, we investigated whether EPEC strains use α-bundlin to mediate early adherence to human intestinal biopsy specimens cultured in vitro by assessing the ability of isogenic EPEC mutants expressing either the α1- or β1-bundlin allele or a bundlin-deficient EPEC strain to bind to these specimens. Furthermore, we directly compared the abilities of a wild-type EPEC strain to bind to the epithelial surfaces of both human adult and pediatric biopsy specimens. Our results demonstrate that β-bundlin does not act as an adhesin during early EPEC adherence to adult duodenal biopsy specimens. The results also indicate that EPEC binds equally well to adult and pediatric biopsy specimens in an early adherence assay. This result is supported by the finding that the early adherence of EPEC to both adult and pediatric biopsy specimens was inhibited by LacNAc neoglycoconjugates, suggesting that organisms expressing α-bundlin-type bundle-forming pili initially bind to related glycan receptors in both age groups.

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Identification and Regulation of a Novel Citrobacter rodentium Gut Colonization Fimbria (Gcf)

Journal of Bacteriology ◽

10.1128/jb.02486-14 ◽

2015 ◽

Vol 197 (8) ◽

pp. 1478-1491 ◽

Cited By ~ 2

Author(s):

Gustavo G. Caballero-Flores ◽

Matthew A. Croxen ◽

Verónica I. Martínez-Santos ◽

B. Brett Finlay ◽

José L. Puente

Keyword(s):

Escherichia Coli ◽

Intestinal Epithelium ◽

Pathogenic Bacteria ◽

Critical Role ◽

Growth Conditions ◽

Citrobacter Rodentium ◽

Content Type ◽

Gut Colonization ◽

Successful Infection

ABSTRACTThe Gram-negative enteric bacteriumCitrobacter rodentiumis a natural mouse pathogen that has been extensively used as a surrogate model for studying the human pathogens enteropathogenic and enterohemorrhagicEscherichia coli. All three pathogens produce similar attaching and effacing (A/E) lesions in the intestinal epithelium. During infection, these bacteria employ surface structures called fimbriae to adhere and colonize the host intestinal epithelium. ForC. rodentium, the roles of only a small number of its genome-carried fimbrial operons have been evaluated. Here, we report the identification of a novelC. rodentiumcolonization factor, calledgutcolonizationfimbria (Gcf), which is encoded by a chaperone-usher fimbrial operon. AgcfAmutant shows a severe colonization defect within the first 10 days of infection. Thegcfpromoter is not active inC. rodentiumunder severalin vitrogrowth conditions; however, it is readily expressed in aC. rodentiumΔhns1mutant lacking the closest ortholog of theEscherichia colihistone-like nucleoid structuring protein (H-NS) but not in mutants with deletion of the other four genes encoding H-NS homologs. H-NS binds to the regulatory region ofgcf, further supporting its direct role as a repressor of thegcfpromoter that starts transcription 158 bp upstream of the start codon of its first open reading frame. Thegcfoperon possesses interesting novel traits that open future opportunities to expand our knowledge of the structure, regulation, and function during infection of these important bacterial structures.IMPORTANCEFimbriae are surface bacterial structures implicated in a variety of biological processes. Some have been shown to play a critical role during host colonization and thus in disease. Pathogenic bacteria possess the genetic information for an assortment of fimbriae, but their function and regulation and the interplay between them have not been studied in detail. This work provides new insights into the function and regulation of a novel fimbria called Gcf that is important for early establishment of a successful infection byC. rodentiumin mice, despite being poorly expressed underin vitrogrowth conditions. This discovery offers an opportunity to better understand the individual role and the regulatory mechanisms controlling the expression of specific fimbrial operons that are critical during infection.

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SlyA and H-NS Regulate Transcription of the Escherichia coli K5 Capsule Gene Cluster, and Expression of slyA in Escherichia coli Is Temperature-dependent, Positively Autoregulated, and Independent of H-NS

Journal of Biological Chemistry ◽

10.1074/jbc.m703465200 ◽

2007 ◽

Vol 282 (46) ◽

pp. 33326-33335 ◽

Cited By ~ 38

Author(s):

David Corbett ◽

Hayley J. Bennett ◽

Hamdia Askar ◽

Jeffrey Green ◽

Ian S. Roberts

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Regulation Of Transcription ◽

Temperature Dependent ◽

Mobility Shift ◽

E Coli ◽

Dnase I Footprinting ◽

Nucleoprotein Complex ◽

Electrophoretic Mobility Shift Assays

In this paper, we present the first evidence of a role for the transcriptional regulator SlyA in the regulation of transcription of the Escherichia coli K5 capsule gene cluster and demonstrate, using a combination of reporter gene fusions, DNase I footprinting, and electrophoretic mobility shift assays, the dependence of transcription on the functional interplay between H-NS and SlyA. Both SlyA and H-NS bind to multiple overlapping sites within the promoter in vitro, but their binding is not mutually exclusive, resulting in a remodeled nucleoprotein complex. In addition, we show that expression of the E. coli slyA gene is temperature-regulated, positively autoregulated, and independent of H-NS.

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SodA Contributes to the Virulence of Avian PathogenicEscherichia coliO2 Strain E058 in Experimentally Infected Chickens

Journal of Bacteriology ◽

10.1128/jb.00625-18 ◽

2019 ◽

Vol 201 (6) ◽

Cited By ~ 1

Author(s):

Qingqing Gao ◽

Le Xia ◽

Xiaobo Wang ◽

Zhengqin Ye ◽

Jinbiao Liu ◽

...

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Hydrogen Peroxide ◽

Virulence Factor ◽

Infection Process ◽

Strain Identification ◽

Bacterial Disease ◽

Wild Type ◽

Content Type

ABSTRACTStrains of avian pathogenicEscherichia coli(APEC), the common pathogen of avian colibacillosis, encounter reactive oxygen species (ROS) during the infection process. Superoxide dismutases (SODs), acting as antioxidant factors, can protect against ROS-mediated host defenses. Our previous reports showed that thesodAgene (encoding a Mn-cofactor-containing SOD [MnSOD]) is highly expressed during the septicemic infection process of APEC.sodAhas been proven to be a virulence factor of certain pathogens, but its role in the pathogenicity of APEC has not been fully identified. In this study, we deleted thesodAgene from the virulent APEC O2 strain E058 and examined thein vitroandin vivophenotypes of the mutant. ThesodAmutant was more sensitive to hydrogen peroxide in terms of both its growth and viability than was the wild type. The ability to form a biofilm was weakened in thesodAmutant. ThesodAmutant was significantly more easily phagocytosed by chicken macrophages than was the wild-type strain. Chicken infection assays revealed significantly attenuated virulence of thesodAmutant compared with the wild type at 24 h postinfection. The virulence phenotype was restored by complementation of thesodAgene. Quantitative real-time reverse transcription-PCR revealed that the inactivation ofsodAreduced the expression of oxidative stress response geneskatE,perR, andosmCbut did not affect the expression ofsodBandsodC. Taken together, our studies indicate that SodA is important for oxidative resistance and virulence of APEC E058.IMPORTANCEAvian colibacillosis, caused by strains of avian pathogenicEscherichia coli, is a major bacterial disease of severe economic significance to the poultry industry worldwide. The virulence mechanisms of APEC are not completely understood. This study investigated the influence of an antioxidant protein, SodA, on the phenotype and pathogenicity of APEC O2 strain E058. This is the first report demonstrating that SodA plays an important role in protecting a specific APEC strain against hydrogen peroxide-induced oxidative stress and contributes to the virulence of this pathotype strain. Identification of this virulence factor will enhance our knowledge of APEC pathogenic mechanisms, which is crucial for designing successful strategies against associated infections and transmission.

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Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ

Journal of Bacteriology ◽

10.1128/jb.182.14.3965-3971.2000 ◽

2000 ◽

Vol 182 (14) ◽

pp. 3965-3971 ◽

Cited By ~ 98

Author(s):

Zonglin Hu ◽

Joe Lutkenhaus

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Functional Domains ◽

Wild Type ◽

Terminal Domain ◽

Ring Assembly ◽

Z Ring ◽

Ftsz Assembly

ABSTRACT In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by themin system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts minfunction, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

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