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This article discusses mechanisms of host resistance to fungal infection and those conditions, both natural and iatrogenic, that predispose to fungal infection. Specific topics discussed include barrier defenses, bacterial antagonism, nonimmune host factors, cellular and humoral immunity, and underlying and iatrogenic medical conditions.
Inversion of the G-segment of Escherichia coli phage Mu was studied in vitro. The reaction requires the Gin recombination protein, which was purified to near homogeneity from overproducing cells. Upon purification the protein lost activity, which was restored by addition of an extract from uninfected E. coli cells. The stimulatory host factor is a small heat-stable protein and was purified from E. coli cells. Full recombination required both proteins, but Gin alone promoted some recombination by itself, particularly at high concentrations. Relaxation of negative supercoils and recombination of a substrate with two recombination sites in an inverted orientation both have the same specificity for Gin and the host factor. The Gin-associated topoisomerase activity appears tightly coupled to its recombination activity.
As reported previously, Integration Host Factor (IHF) stimulates cII expression but the stimulatory effect is prevented by the NusA protein (Peacock and Weissbach, 1985, Biochem. Biophys. Res. Commun. 127, 1026-1031). The interaction between IHF and the NusA protein has been investigated further in studies on the in vitro expression of the genes for the beta (rpoB) and sigma (rpoD) subunits of RNA polymerase, both known to be stimulated by NusA. The NusA stimulation of rpoD expression can be prevented by IHF, but IHF has no effect by itself on rpoD expression. IHF does not influence rpoB expression either in the presence or absence of NusA.
The integration host factor (IHF) is a DNA-binding and -bending protein with roles in local DNA structural organization and transcriptional regulation in Gram-negative bacteria. This heterodimeric protein is composed of the two highly homologous subunits IHFalpha and IHFbeta. DNA microarray analysis was used to define the regulon of genes subject to IHF control in Salmonella enterica serovar Typhimurium (S. Typhimurium). The transcription profile of the wild type was compared with those of mutants deficient in IHFalpha, IHFbeta, or both IHFalpha and IHFbeta. Our data reveal a new connection between IHF and the expression of genes required by the bacterium to undergo the physiological changes associated with the transition from exponential growth to stationary phase. When a mutant lacking IHF entered stationary phase, it displayed downregulated expression of classic stationary-phase genes in the absence of any concomitant change in expression of the RpoS sigma factor. Purified IHF was found to bind to the regulatory regions of stationary-phase genes indicating an auxiliary and direct role for IHF in RpoS-dependent gene activation. Loss of IHF also had a profound influence on expression of the major virulence genes and epithelial cell invasion, indicating a role in co-ordinating regulation of the pathogenic traits with adaptation to stationary phase. Although the three mutants showed considerable overlaps in the genes affected by the ihf lesions, the observed patterns were not identical, showing that S. Typhimurium has not one but three overlapping IHF regulons.
Integration host factor (IHF) of E. coli positively regulates both early and repressor transcription of bacteriophage Mu. In this paper we show that although binding of IHF to the same binding site is responsible for both types of transcription regulation, the mechanisms by which these regulations occur are different: Activation of transcription from the early promoter (Pe) requires a helix-dependent orientation of IHF- and RNA polymerase binding sites on the DNA helix with a limited distance between both sites. Activation of repressor transcription shows no helix dependency between promoter and IHF binding site and the distance between both sites can be enlarged at least by 100 base pairs without affecting the positive control. A possible mechanism for both types of transcription stimulation will be discussed.
Previous work has shown that integration host factor (IHF) mutants have increased expression and altered osmoregulation of OmpF, a major Escherichia coli outer membrane protein. By in vitro analysis the possibility was investigated that IHF interacts directly with the ompF promoter region. Gel retardation assays and DNase I protection experiments showed that IHF binds to two sites in the ompF promoter region centered at positions -180 and -60 relative to the start of transcription. Gel electrophoresis studies with circularly permuted ompF promoter fragments indicated that IHF binding strongly increased a small intrinsic bend in the ompF promoter region. The addition of IHF to a purified in vitro transcription system strongly and specifically inhibited ompF transcription. This inhibition was reversed by increasing the concentration of OmpR, a positive activator required for ompF expression, suggesting that IHF may inhibit ompF transcription by altering how OmpR interacts with the ompF promoter.
The interaction of E. coli integration host factor (IHF) with the cohesive end sites (cos's) of phages lambda and 21 has been studied by the DNAase I footprinting technique. Six potential sites in cos lambda differ from the consensus IHF binding sequence by 1 to 3 base pairs. Of the six, one site, I1, binds IHF strongly. The I1 segment protected by IHF contains two sequences that closely match the IHF consensus binding sequence. Another site, I2, binds IHF moderately well, and three sites: 10', 13 and 14 bind IHF very weakly. The 10 site does not bind IHF under the conditions used here. In phage 21 the DNA segment extending to the right from the cohesive ends, which contains three potential IHF binding sites, was examined. Two sites bind IHF well; I1, the 21 analogue of one of the lambda I1 sites, and I0, a site not analogous to a lambda site. The third 21 site, I2, binds IHF moderately well, as does the analogous I2 site in lambda. The significance of the results for lambda DNA packaging is discussed.
We have investigated the role of integration host factor (IHF) in the replication of plasmid R6K by studying the maintainance of the plasmid in a strain of Escherichia coli that lacks both subunits of IHF and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of IHF; however, loss of IHF reduced the copy number of those replicons initiating solely from ori gamma by 5-fold. Concomitant loss of direct repeats within the origin that bind the R6K replication initiator protein, Pi, resulted in a further reduction in copy number. Using gel mobility shift analysis, we showed that IHF bound specifically only to one site within the A/T rich region of the minimal origin adjacent to the Pi binding sites. The origin region possessed no intrinsic DNA curvature although IHF induced a strong bend upon binding. Combination footprinting with different orders of addition of Pi and IHF suggested that there was no cooperativity between the two proteins with regard to DNA binding. Hydroxyl-radical footprinting revealed hypersensitive asymmetric periodic cleavage sites within the origin region in the presence of IHF that extended over 200 base pairs and a localized perturbation of cleavage chemistry. The presence of periodic cleavages was dependent upon the presence of the wild type R6K origin sequence and was not observed when the IHF binding site was positioned adjacent to a heterologous sequence. We observed that the conformational changes induced by IHF upon binding to the R6K origin were negatively correlated with the observed decrease in copy number, and therefore, origin conformation altered by protein-DNA interaction may play an important role in the regulation of replication initiation.
Graft-versus-host disease (GVHD) induced by organ transplantation is dependent on both the quality and quantity of lymphocytes included in the graft. Changes in the host immune function after surgery are also important factors in the development of this disease. Based on our experimental data, GVHD after organ transplantation is reviewed To test the reactivity of graft lymphocytes in in vivo, small bowel, liver pancreas/spleen, and heart/lung transplantations were performed using a rat in parent-to-F 1 hybrid combination. All of the lymphoid organ transplants induced lethal GVHD, while liver graft did not accelerated the disease after the transplantation procedure. Lymphocytes in liver grafts had a unique population and showed a low response against alloantigens in vivo. Surgical treatments also increased the susceptibility to GVHD, especially the ischemic-reperfusion procedure; Endogenous endotoxemia triggered by reperfusion promoted the development of lethal GVHD.
The minimal replication region of the mycobacterial plasmid pAL5000 encompasses the replication origin (ori) and two tandemly organized replication genes, repA and repB, the functions of which are not clearly known. It was observed that when the repA and repB genes were expressed in Escherichia coli, a strong ori binding activity was generated in the host cells. Inactivation of repB led to a complete loss of activity, whereas inactivation of repA had a partial effect, indicating that while repB plays an important role in the process, its activity is stimulated through coexpression of repA. However, this stimulatory effect could be demonstrated only when expression of repA and that of repB were coupled. At a relatively high concentration (1,000 nM), the purified RepB protein was found to form an ori complex with low specificity, which was sensitive to high salt concentrations and challenge by a nonspecific competitor. In contrast, the complex formed by an extract of repA-repB-expressing cells was highly specific and was resistant to both types of challenges. At a 10-fold-lower concentration, RepB did not exhibit ori binding activity, but it could nevertheless form a salt-resistant ori complex in vitro, provided that host factors were present. Antibody supershift experiments indicated that RepB is a key component of the specific complex formed by extracts prepared from E. coli cells expressing the repA and repB genes and also from mycobacterial cells harboring pAL5000-derived vectors. The results indicate that in vivo RepB interacts with host factors and forms an ori complex, but this activity is maximal only when there is coupled expression of repA.
In order to evaluate bacterial factors which might predispose to P. aeruginosa colonization or bacteremia in the granulocytopenic patient, 132 isolates recovered from 44 oncology patients were evaluated for antigenic serotype, iron correctable sensitivity to pooled human serum, antibiotic susceptibility, production of lecithinase, elastase, protease, gelatinase, pyocyanin and pyoverdin. Similarly, potential host factors, primarily total iron binding capacity, were evaluated in a subpopulation of acute leukemia patients composed of 13 control patients without P. aeruginosa cultured during their hospital course, 11 colonization only patients and 15 P. aeruginosa bacteremia patients. No significant differences were observed between strains recovered from bacteremia vs. colonization patients for extracellular enzyme activity, pigment production, serum sensitivity and antigenic serotype. Significant differences were observed between bacteremia and colonizing strains for antibiotic susceptibility to ticarcillin, 40% vs. 76% (P less than 0.002); piperacillin, 44% vs. 86% (P less than 0.006); and cefsulodin, 60% vs. 90% (P less than 0.02). Of the host factors evaluated in the acute leukemia patients, significant differences were observed between the TIBC nadir of control patients and both colonization patients (P less than 0.0002) and bacteremia patients (P less than 0.0004). P. aeruginosa bacteremia was associated with the temporal occurrence of TIBC nadir and the detection of the organism. These data suggest a possible role for beta-lactam antibiotic resistance and host iron binding capacity as determinants and possible predictors of P. aeruginosa sepsis in the granulocytopenic patient.
Integration host factor (IHF) of Escherichia coli is a DNA-binding protein involved in gene expression and other cellular functions in E. coli and some of its bacteriophages and plasmids. We report here that IHF is a direct negative effector of the ompC operon of E. coli. IHF binds to ompC DNA and protects a region of 35 base pairs located upstream from the ompC promoters. The addition of IHF to a purified in vitro transcription system inhibited transcription from two of the three ompC promoters. In vivo experiments suggest that the in vitro results are physiologically relevant. IHF mutants show increased expression of OmpC. In addition, the OmpC- phenotype of certain strains is completely suppressed by a mutation in IHF.
Site-specific recombination by phages lambda and P22 is carried out by multiprotein-DNA complexes. Integration host factor (IHF) facilitates lambda site-specific recombination by inducing DNA bends necessary to form an active recombinogenic complex. Mutants lacking IHF are over 1,000-fold less proficient in supporting lambda site-specific recombination than wild-type cells. Although the attP region of P22 contains strong IHF binding sites, in vivo measurements of integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid integration assay showed that integrative recombination occurs equally well in wild-type and ihfA mutant cells. P22 integrative recombination is also efficient in Escherichia coli in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another factor(s) can substitute for IHF in the formation of complexes.
Binding specificity of integration host factor (IHF) to oligo DNAs has been studied by circular dichroism (CD) spectroscopy and filter binding experiment. CD difference spectra of IHF-DNA complexes demonstrated that a conformational change in DNA was induced by binding of IHF when DNA had a consensus sequence for the binding sites of IHF, but that such conformational change was not observed for consensus DNA 20 mer as well as nonconsensus DNA 45 mer. Dissociation constants for IHF-DNA complexes determined by filter binding assay showed that IHF has indeed stronger affinity to DNA with the consensus binding site than to nonconsensus DNA, but the difference in its affinity between consensus and nonconsensus DNAs was rather small, 3.4-fold. It was, therefore, concluded that the flanking regions of the consensus sequence are important for the specific binding of IHF and that its binding specificity is well characterized by the induced conformational change in DNA rather than by dissociation constants for IHF-DNA complexes.
Antibiotic-associated diarrhoea (AAD) represents a clinical entity leading to prolonged hospital stays and diagnostic and therapeutic procedures, and results in additional costs. The aim of the present study was to assess the prevalence and characteristics of different bacteria in stools of patients with AAD. The reliability of diagnostic procedures under routine conditions was evaluated. Host factors were also analysed. From June 2002 to April 2003 89 cases of diarrhoea were reported at a hospital unit for internal medicine. Clostridium difficile and Clostridium perfringens toxin enzyme-immunoassays (EIAs), and culture for C. difficile, C. perfringens and Staphylococcus aureus were performed on stool samples from all patients. Toxin production was determined in isolated S. aureus strains. In vitro susceptibility of S. aureus for oxacillin and of C. difficile for vancomycin, metronidazole, linezolid, fusidic acid and tetracycline was tested. Host factors, such as age, comorbidities, antibiotic exposure and contact with other patients, were evaluated. Twenty-six stools were positive for C. difficile toxins by an EIA technique, while C. difficile was cultured from 39. C. difficile was isolated from 21 stools that were EIA negative. Additionally, from 28 stools S. aureus and/or C. perfringens could be isolated. Nine samples contained only S. aureus and/or C. perfringens. Thirty-one stools were negative in all tests. All C. difficile isolates were susceptible to vancomycin and metronidazole. Age >60 years, and diseases of the vascular system, the heart, the kidneys and the lungs were identified as risk factors for acquiring C. difficile in this setting (P values < 0.05). Stool culture for C. difficile was shown to be more sensitive than toxin EIA in this study. Risk factors for the acquisition of C. difficile in outbreak situations seem to differ from risk factors in the normal hospital setting. The role of toxin-producing S. aureus in cases of AAD needs further investigation.
Histone-like proteins, such as HU and the integration host factor (IHF), are small, dimeric, DNA-bending proteins which play a role in maintaining constrained DNA structures and hence in regulating gene expression. Two different strategies were used to isolate the genes coding for Pseudomonas aeruginosa (Pa) HU and IHF, two proteins that we have previously isolated from a mucoid strain. By use of a PCR-based technique with oligodeoxyribonucleotides (oligos) designed from the N-terminal amino acid (aa) sequences of HU and the beta-subunit of IHF, and Southern blot analyses, hupB and himD, encoding HU and IHF beta, respectively, have been cloned. The himA gene of Pa, encoding the alpha-subunit of IHF, was isolated using himA of Escherichia coli (Ec) as a probe in Southern blot analyses. The deduced hupB product (90 aa, 9 kDa) is 79% identical to HU beta and 61% to HU alpha of Ec. The predicted products of himA (100 aa, 11.5 kDa) and of himD (94 aa, 10.6 kDa) share 77 and 70% identity with IHF alpha and IHF beta of Ec, respectively. The promoter region of himD contains an IHF consensus sequence, as is the case for Ec himD.
Efficient transcription and replication of the influenza virus genome are dependent upon host-derived factors. Using an in vitro RNA synthesis system, we have purified and identified Hsp90 as one of the host factors that stimulate viral RNA polymerase activity. Hsp90 interacted with the PB2 subunit of the viral RNA polymerase through the amino-terminal chaperone domain and the middle region containing a highly acidic domain. The acidic middle region was also responsible for its stimulatory activity. We found that a portion of Hsp90 is re-localized to the cell nucleus after viral infection. A PB2 fragment containing a Hsp90 binding domain inhibited viral gene expression in a dominant-negative manner. These results suggest that Hsp90 is a host factor for the influenza virus RNA polymerase.
The specific DNA-binding protein integration host factor (IHF) of Escherichia coli stimulates the site-specific recombination reaction between the attP site of bacteriophage HP1 and the attB site of its host, Haemophilus influenzae, in vitro and also appears to regulate the expression of HP1 integrase. IHF interacts specifically with DNA segments containing the att sites and the integrase regulatory region, as judged by IHF-dependent retardation of relevant DNA fragments during gel electrophoresis. The locations of the protein-binding sites were identified by DNase I protection experiments. Three sites in the HP1 attP region bound IHF, two binding sites were present in the vicinity of the attB region, and one region containing three partially overlapping sites was present in the HP1 integrase regulatory segment. The binding sites defined in these experiments all contained sequences which matched the consensus IHF binding sequences first identified in the lambda attP region. An activity which stimulated the HP1 site-specific integration reaction was found in extracts of H. influenzae, suggesting that an IHF-like protein is present in this organism.