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PubMed1995

In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell.

Adenosine Triphosphatases↗

PubMed2004

Molecular biology of gastric cancer: Helicobacter infection and gastric adenocarcinoma: bacterial and host factors responsible for altered growth signaling.

Gastric cancer remains the second most common cause of cancer-related mortality worldwide. The single most common cause of gastric cancer is chronic infection with the gram-negative microaerophilic spiral bacterium: Helicobacter pylori. Recent advances in this field have identified host factors which predispose to gastric cancer formation via modulation of the host immune response. In addition, recent work has explored bacterial virulence factors which may directly cause tissue damage, and lead to gastric carcinogenesis, as well as factors responsible for enhanced immune response. Environmental factors, long associated with a predilection for gastric cancer, are recognized as modifiers of key growth signalling pathways within the gastric mucosa and as such lead to growth alterations. This review focuses on exploring new advances in our understanding of bacterial factors, host genetic polymorphisms and the interaction between the bacterium and host at the level of the immune response and the regulation of proliferative and apoptotic signal transduction cascades. Modulation of the pivotal balance between cell growth and cell death leads to the formation of gastric adenocarcinoma.

Adenocarcinoma↗

PubMed2025

Identification of glycogen synthase kinase 3alpha/beta as a host factor required for HBV transcription using high-throughput screening.

BACKGROUND AND AIMS: HBV leads to severe liver diseases, such as cirrhosis and HCC. Identification of host factors that regulate HBV replication can provide new therapeutic targets. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV entry receptor has enabled the establishment of hepatic cell lines for analyzing HBV infection and propagation. Using this new system, studies aimed at identifying host factors that regulate HBV propagation have increased. APPROACH AND RESULTS: We established an HBV-based-reporter gene expression system that mimics HBV replication from transcription to virus egress. Using this approach, we screened 1827 Food and Drug Administration-approved compounds and identified glycogen synthase kinase 3 (GSK3)alpha/beta inhibitors, including AZD1080, CHIR-98014, CHIR-98021, BIO, and AZD2858, as anti-HBV compounds. These compounds suppressed HBeAg and HBsAg production in HBV-infected human primary hepatocytes. Proteome analysis revealed that GSK3alpha/beta phosphorylated forkhead box K1/2 (FOXK1/2)s. A double-knockout of FOXK1/2 in HBV-infected HepG2-NTCP cells reduced HBeAg and HBsAg production. The rescue of FOXK2 expression, but not FOXK1 expression, in FOXK1/2-double-knockout cells restored HBeAg and HBsAg production. Importantly, phosphorylation of FOXK2 at Ser 424 is required for GSK3alpha/beta-mediated HBeAg and HBsAg production. We observed the binding of FOXK2 to HBV DNA in HepG2-NTCP cells. CONCLUSIONS: Our recombinant HBV-based screening system enables the discovery of new targets. Using our approach, we identified GSK3 inhibitors as potential anti-HBV agents.

Humans↗

PubMed1984

Reduced expression of the isoleucine and valine enzymes in integration host factor mutants of Escherichia coli.

The level of the isoleucine and valine (Ilv) enzymes specified by the ilvB and ilvGEDA operons is reduced in integration host factor mutants (himA and himD) of Escherichia coli K-12. Growth inhibition of these strains in minimal medium can be explained by the decreased amounts of one of the Ilv enzymes, acetohydroxy acid synthase I (AHASI). No growth inhibition, or reduction in AHASI activity, was found in a himA derivative of a mutant strain containing high constitutive levels of AHASI. A strong correlation was observed in himA strains between the reduced amount of the Ilv enzymes and of Ilv-specific messenger RNA. These data suggest that integration host factor may be a positive effector for transcription of the ilvB and ilvGEDA operons.

Acetolactate Synthase↗

PubMed2026

Genome-wide CRISPR screen identifies RNF24 as a critical host factor for foot-and-mouth disease virus entry.

BACKGROUND: Foot-and-mouth disease virus (FMDV) causes substantial economic losses in global livestock production; however, the key host factors supporting its early infection process remain poorly characterized. METHODS: In this study, we performed an unbiased genome-wide CRISPR/Cas9 knockout screening using porcine cells to screen and identify host factors involved in FMDV infection. RESULTS: We identified that the E3 ubiquitin ligase RNF24 supports efficient FMDV entry. RNF24 depletion inhibits viral entry and replication, whereas its overexpression enhances viral infectivity. Mechanistically, RNF24 preferentially promotes K27-linked non-degradative polyubiquitination of leupaxin (LPXN) at lysine 162, driving LPXN's trafficking to the plasma membrane. At the membrane, LPXN assembles a ternary integrin-LPXN-VP1 complex that strengthens virus-receptor interactions and promotes viral adsorption and entry. Disruption of this ubiquitination event via the LPXN K162R mutation impairs complex formation and compromises viral entry. CONCLUSION: Together, our study reveals a ubiquitin-dependent RNF24-LPXN regulatory axis that supports FMDV entry, highlights the role of non-degradative ubiquitination in viral pathogenesis, and proposes this interface as a potential target for antiviral intervention.

CRISPR screening↗

PubMed1996

Host factors associated with protection against rotavirus disease: the skies are clearing.

Over the past several years, a number of studies have clarified aspects of rotavirus immunology and vaccinology previously considered controversial. In this review, studies that address the following questions will be summarized: Which host factors are responsible for recovery from acute rotavirus infection? Are the host factors responsible for recovery from acute infection the same as those necessary for prevention of infection? What is the relative importance of the nature of the inoculum (e.g., homologous or heterologous host virus and live or inactivated virus), route of inoculation, or virus serotype in vaccine development? What is the immunologic basis by which infection with 1 viral serotype protects against challenge with another serotype (heterotypic protection)?

Animals↗

PubMed1988

Integration host factor binds specifically to sites in the ilvGMEDA operon in Escherichia coli.

Integration host factor (IHF) of Escherichia coli is a histone-like protein that is involved both in site-specific recombination and in regulating the expression of a number of phage and bacterial genes. We have shown previously that transcription of the ilvGMEDA operon in E. coli is greatly reduced in IHF mutants. We report here that IHF specifically protects two sites within the ilvGMEDA promoter-regulatory region against DNase I digestion. These sites are located upstream from the promoter and in the leader region just prior to the sequence that specifies the attenuator. The footprinting experiments and gel retardation assays show that these sites have strong affinity for IHF. These data and results with ilvGMEDA-lac promoter fusions suggest a direct role for IHF in expression of the ilvGMEDA operon.

Bacterial Proteins↗

PubMed1994

Growth phase variation of integration host factor level in Escherichia coli.

We have measured the intracellular abundance of integration host factor (IHF), a site-specific, heterodimeric DNA-binding protein, in exponential- and stationary-phase cultures of Escherichia coli K-12. Western immunoblot analysis showed that cultures that had been growing exponentially for several generations contained 0.5 to 1.0 ng of IHF subunits per microgram of total protein and that this increased to 5 to 6 ng/microgram in late-stationary-phase cultures. IHF is about one-third to one-half as abundant in exponentially growing cells as HU, a structurally related protein that binds DNA with little or no site specificity. Wild-type IHF is metabolically stable, but deletion mutations that eliminated one subunit reduced the abundance of the other when cells enter stationary phase. We attribute this reduction to the loss of stabilizing interactions between subunits. A mutation that inactivates IHF function but not subunit interaction increased IHF abundance, consistent with results of previous work showing that IHF synthesis is negatively autoregulated. We estimate that steady-state exponential-phase cultures contain about 8,500 to 17,000 IHF dimers per cell, a surprisingly large number for a site-specific DNA-binding protein with a limited number of specific sites. Nevertheless, small reductions in IHF abundance had significant effects on several IHF-dependent functions, suggesting that the wild-type exponential phase level is not in large excess of the minimum required for occupancy of physiologically important IHF-binding sites.

Amino Acid Sequence↗

PubMed1995

Participation of the flank regions of the integration host factor protein in the specificity and stability of DNA binding.

The heterodimeric integration host factor (IHF) protein is a site-specific DNA-binding protein from Escherichia coli that strongly bends the DNA. It has been proposed (Yang, C., and Nash, H.A. (1989) Cell 57, 869-880; Granston, A. E., and Nash, H. A. (1993) J. Mol. Biol 234, 45-59; Lee, E. C., Hales, L. M., Gumport, R. I., and Gardner, J. F. (1992) EMBO J. 11, 305-313) that the wrapping of the DNA around the protein is stabilized through interactions between the flanks of the protein and the DNA. In order to elucidate which domains of the IHF protein are involved in these interactions, we have constructed mutant proteins in which the C-terminal part of one of the subunits has been deleted. We observed that the C-terminal alpha 3 helix of HimD is involved in the stability of DNA binding, but not in the specificity. In contrast the corresponding alpha 3 helix of HimA is essential for the sequence specificity, since an IHF mutant lacking this domain only binds to the DNA in a non-specific way. The possible role of the two C-terminal alpha-helical structures in complex formation will be discussed. We also examined the properties of an IHF mutant that has an amino acid substitution between beta sheets beta 1 and beta 2 of the HimD subunit (R46H). The occupancy of the ihf site by the mutant and wild type proteins differ in the 3' part of the ihf site and as a result the bend introduced in the DNA by the mutant protein is less pronounced. We propose that the arginine 46 in the HimD subunit is in vicinity of the TTR region of the consensus and that through contacts within the minor groove the DNA bend introduced by IHF is stabilized.

Amino Acid Sequence↗

PubMed1994

Physiopathology of primary periodontitis associated with plaque. Microbial and host factors. A review. Part 2.

To a considerable extent, host factors condition the initiation and progression of periodontitis. The integrity of the periodontal structures and the immune elements in the gingival fluid may act as a defensive barrier against microorganisms. However, immune over-response may lead to tissue damage and bone resorption from anaphylactic reactions, immunocomplex reactions, type IV hypersensitivity, complement hyperactivation via the alternative pathway, or direct action of leukocytes and macrophages. Moreover, insufficient host response allows bacteria to escape control, allowing more rapid progression of the disease.

Bacteria↗

PubMed1989

Physical and biological consequences of interactions between integration host factor (IHF) and coliphage lambda late p'R promoter and its mutants.

The integration host factor (IHF) binds to a site (ihf) that overlaps the -35 region of the phage lambda late rightward promoter (p'R). This interaction represses p'R-promoted transcription, both in vivo and in vitro. In vivo repression was observed when a plasmid carrying both p'R and the galK reporter gene was transfected into IHF+ or IHF- hosts. In vitro repression of transcription by IHF was observed only with linear, but not with supercoiled wild-type p'R templates. When binding to ihf, IHF imposes a strong bend on the DNA and protects this site from cleavage by neocarzinostatin, pancreatic DNase I, and hydroxyl radicals, as assessed by footprinting experiments. Both the functional and nonfunctional p'R mutants, in which the upstream part of the -35 region was replaced by an EcoRI linker, show modified behavior toward IHF. Some are more sensitive to IHF-mediated repression, even in the supercoiled form, while others have lost their affinity for IHF. We conclude that IHF binding depends not only on the consensus ihf sequence, but also on a suitable combination of the sequences of both ihf and neighboring regions, together with the DNA conformation, which includes both natural and imposed bends in DNA and the degree of supercoiling. Based on most of the present data, it is difficult to predict the relationship between the ihf sequence and IHF interaction, since two very different sequences (less than 50% homology) show strong IHF binding, whereas very similar sequences (80-87% homology) show a very different behavior. However, the hydroxylradical footprinting data show that three A + T-rich sequences are protected by IHF: the central sequence, which overlaps the -35 region of p'R, and two flanking sequences removed by one helix turn. All three sequences are located on the same face of the helix, and the amino acid side chains of IHF seem to occupy the narrow minor groove. A novel consensus sequence is proposed.

Bacterial Proteins↗

PubMed1995

Pyrimidine regulation of the Escherichia coli and Salmonella typhimurium carAB operons: CarP and integration host factor (IHF) modulate the methylation status of a GATC site present in the control region.

By measuring the protection against Dam methylase modification of a GATC sequence located 106 bp upstream of the startpoint of promoter P1 in the control region of the carAB operon (encoding carbamoylphosphate synthetase) we have obtained evidence for a direct correlation between the degree of in vivo occupancy of a specific regulatory target site and the repressibility of the P1 promoter by pyrimidine residues. A high uridine nucleotide pool as well as binding of the carP (alias xerB/pepA) gene product and of the integration host factor (IHF) to the carAB control region are prerequisites to observe this in vivo protection. Purified CarP binds in vitro to the carAB control region and protects against DNase I two approximately 25 bp long stretches, one of which is located just downstream of the GATC sequence. Mutations in this site strongly impair the pyrimidine regulation of the P1 promoter and the interference with Dam methylase modification. These processes are also strongly impaired in the absence of integration host factor and in mutants affected in the IHF site located some 200 bp upstream of this Dam methylase modification site. IHF therefore exerts at least part of its antagonistic effects on P1, i.e. increased expression in minimal medium but increased repression in the presence of pyrimidine residues, indirectly by influencing the formation or the stability of a particular protein-DNA complex. Furthermore, we demonstrate that the distance separating the IHF and Dam methylase target sites is crucial for the in vivo protection and for pyrimidine-mediated regulation of the promoter expression. Mutations altering this distance result in severe reductions of the degree of in vivo protection and, concomitantly, of the repressibility by pyrimidine residues of promoter P1 activity in a way indicative of the formation of a complex nucleoprotein structure. Since neither IHF nor CarP require pyrimidine residues to bind to the carAB control region, at least not in vitro, it is tempting to suggest that IHF and CarP-induced bending and looping provide changes in DNA topology that are required for assembling a specific pyrimidine-dependent nucleoprotein complex that modulates P1 activity.

Adenine↗

PubMed1994

P1 plasmid partition: binding of P1 ParB protein and Escherichia coli integration host factor to altered parS sites.

The Escherichia coli integration host factor (IHF) participates in P1 plasmid partition by assisting the interaction of P1 ParB protein with its specific site, parS. Together they form an extremely high-affinity protein-DNA complex, in which parS DNA is wrapped around a core of ParB and IHF protein in a precise three-dimensional conformation. We have investigated the interaction of ParB and IHF with mutant DNA sites, to examine protein specificity and cooperativity. The results indicate that ParB specifically recognizes two separate types of sequence repeats in its minimal binding site in one half of the parS site. The affinity of ParB or IHF for parS is much greater in the presence of the other protein. Mutations that decrease ParB or IHF binding to parS have relatively minor defects in vivo, because each protein still binds well to parS in the presence of the other protein. We observed that ParB acts better when provided in cis than in trans to parS in vivo. Our experiments suggest that in vivo, the local concentration of ParB protein near the plasmid is high, so that ParB can act reasonably well to promote partition in cells without IHF. However, this activity is lower than in wild-type cells, indicating that IHF is essential for long-term plasmid stability.

Bacterial Proteins↗

PubMed1980

Host factors in the formation of abscesses.

The role of host factors in the formation of subcutaneous abscesses due to Bacteroides fragilis or Staphylococcus aureus was investigated in athymic nude mice, mice deficient in the fifth component of complement (C5D), and mice pretreated with cobra venom factor (CVF). CVF-treated mice, inoculated with B. fragilis, had abscesses 62% larger than those of control mice at day 4 (P < 0.01), but no significant difference was observed when S. aureus was used. Athymic nude mice had significantly smaller abscesses than control animals at both days 4 and 7 after inoculation with either organism. There were no differences in abscess size or in the accumulation of neutrophils at day 4 when C5D mice were compared with control animals for either organism. The results suggested that the activity of the alternative complement pathway may influence abscess size and that chemotactic factors other than the fifth component of complement can supervene in C5D mice to produce encapsulated abscesses. The absence of thymic factors did not prevent containment of abscesses.

Abscess↗

PubMed1979

Host factors affecting perinatal carcinogenesis by resorptive alkylnitrosoureas in rats.

Host factors are operative in transplacental carcinogenesis. There are species, strain, sex, and individual differences in susceptibility to carcinogens. Rat fetuses are over 50 times more susceptible than adults to the oncogenic effects of ENU, yet are less susceptible to tumor induction with DMN. Immune mechanisms do not seem to affect the latency period, incidence, location, and type of neurogenic neoplasms, although tumor-specific antigens are demonstrable in neoplastic neuroectodermal cells. Although placental barriers, DNA repair capacities, age at exposure, hormonal interaction, and immune responsiveness may be singled out as possible causes for species, strain, sex, and individual variations in susceptibility to the neuro-oncogenic potential of ENU, no definitive proof of the mechanism of action of any of these factors has yet been presented.

Animals↗

PubMed1996

Effects of integration host factor and DNA supercoiling on transcription from the ilvPG promoter of Escherichia coli.

Integration host factor (IHF) activates transcription from the ilvPG promoter by severely distorting the DNA helix in an upstream region of a supercoiled DNA template in a way that alters the structure of the DNA in the downstream promoter region and facilitates open complex formation. In this report, the in vivo and in vitro influence of DNA supercoiling on transcription from this promoter is examined. In the absence of IHF, promoter activity increases with increased DNA supercoiling. In the presence of IHF, the same increases in superhelical DNA densities result in larger increases in promoter activity until a maximal activation of 5-fold is obtained. However, the relative transcriptional activities of the promoter in the presence and absence of IHF at any given DNA superhelical density remains the same. Thus, IHF and increased DNA supercoiling activate transcription by different mechanisms. Also, IHF binds with equal affinities to its target site on linear and supercoiled DNA templates. Therefore, IHF binding does not activate transcription simply by increasing the local negative supercoiling of the DNA helix in the downstream promoter region or by differential binding to relaxed and supercoiled DNA templates.

Allosteric Regulation↗

PubMed1994

Determining the DNA sequence elements required for binding integration host factor to two different target sites.

Binding sites for the Escherichia coli protein integration host factor (IHF) include a set of conserved bases that can be summarized by the consensus sequence WATCAANNNNTTR (W is dA or dT, R is dA or dG, and N is any nucleotide). However, additional 5'-proximal bases, whose common feature is a high dA+dT content, are also thought to be required for binding at some sites. We examine the relative contribution of these two sequence elements to IHF binding to the H' and H1 sites in attP of bacteriophage lambda by using the bacteriophage P22-based challenge-phage system. IHF was unable to act as a repressor in the challenge-phage assay at H' sites containing the core consensus element but lacking the dA+dT-rich element. This indicates that both elements are required for IHF to bind to the H' site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site, which lacks an upstream dA+dT-rich region. Fifty mutants that decreased or eliminated IHF binding to the H1 site were isolated. Sequence analysis showed changes in the bases in the core consensus element only, further indicating that this determinant is sufficient for IHF binding to the H1 site. We found that placement of a dA+dT-rich element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT-rich element enhances IHF binding.

Bacterial Proteins↗

PubMed1992

The isolation and characterization of mutants of the integration host factor (IHF) of Escherichia coli with altered, expanded DNA-binding specificities.

The integration host factor (IHF) of Escherichia coli is a small, basic protein that is required for lambda site-specific recombination and a variety of cellular processes. It is composed of two subunits, alpha and beta, that are encoded by the himA and hip (himD) genes, respectively. IHF is a sequence-specific DNA-binding protein and bends the DNA when it binds. We have used the bacteriophage P22-based challenge phage selection to isolate suppressor mutants with altered, expanded DNA binding specificities. The suppressors were isolated by selecting mutants that recognize variants of the phage lambda H'IHF recognition site. Two of the mutants recognize both the wild-type and a single variant site and contain amino acid substitutions at positions 64 (Pro to Leu) or 65 (Lys to Ser) of the alpha subunit. These substitutions are in a region of the protein that is predicted to contain a flexible arm that interacts with DNA. Three other mutants, which recognize the wild-type and a different variant site, contain amino acid substitutions at position 44 (Glu to Lys, Val or Gly) of the beta subunit. These substitutions are in the middle of a predicted beta-strand of the subunit. We discuss the possible mechanisms of suppression by the mutants in terms of a model of the IHF-DNA complex proposed by Yang and Nash [Cell, 57, 869-880 (1989)].

Amino Acid Sequence↗