In vivo expression of Neisseria meningitidis proteins homologous to the Haemophilus influenzae Hap and Hia autotransporters.

The genome sequences of Neisseria meningitidis serogroup B strain MC58 and serogroup A strain Z2491 were systematically searched for open reading frames (ORFs) encoding autotransporters. Eight ORFs were identified, six of which were present in both genomes, whereas two were specific for MC58. Among the identified ORFs was the gene encoding the known autotransporter IgA1 protease. The deduced amino acid sequences of the other identified ORFs were homologous to known autotransporters and found to contain an N-terminal signal sequence and a C-terminal domain that could constitute a beta-barrel in the outer membrane. The ORFs NMB1985 and NMB0992, encoding homologs of the Hap (for Haemophilus adhesion and penetration protein) and Hia (for Haemophilus influenzae adherence protein) autotransporters of H. influenzae, were cloned from serogroup B strain H44/76 and expressed in Escherichia coli. Western blots revealed that all sera of patients (n=14) and healthy carriers (n=3) tested contained antibodies against at least one of the recombinant proteins. These results indicate that both genes are widely distributed among N. meningitidis isolates and expressed during colonization and infection.

Cloning of genes of nontypeable Haemophilus influenzae involved in penetration between human lung epithelial cells.

Haemophilus influenzae penetrates between epithelial cells via an unknown mechanism. A chromosomal library of nonencapsulated H. influenzae strain A960053 DNA was constructed in Escherichia coli DH5alpha to identify bacterial genes contributing to this paracytosis. Two E. coli clones that contained open reading frames (ORFs) homologous to HI0636 to HI0641 of H. influenzae strain Rd and that showed an increased penetration in epithelial cell layers of the human bronchial epithelial cell line NCI-H292 were identified. ORFs HI0636 and HI0638, encoding two small proteins of unknown functions, were further investigated. The clone containing ORFs HI0636 and HI0637 as well as the clone containing ORF HI0638 showed a significant increase in penetration. Disruption of HI0638 by kanamycin box insertion in H. influenzae strain A960053 resulted in loss of penetration into the epithelial cell layers. Disruption of HI0636 had no effect on penetration in this model system. Since a role for HI0637 in the paracytosis of H. influenzae is very unlikely because it encodes TrpS, we conclude that the protein encoded by ORF HI0638 may function as a paracytin, while that encoded by HI0636 may have an auxiliary function.

Characterization of adherence of nontypeable Haemophilus influenzae to human epithelial cells.

The adherence of 58 nontypeable Haemophilus influenzae isolates obtained from patients with otitis media or chronic obstructive pulmonary disease (COPD) and obtained from the throats of healthy individuals to Chang and NCI-H292 epithelial cells was compared. Otitis media isolates, but not COPD isolates, adhered significantly more to both cell lines than did throat isolates. Since high-molecular-weight (HMW) proteins are major adhesins of nontypeable H. influenzae, the isolates were screened for HMW protein expression by Western blotting with two polyclonal sera and PCR with hmw-specific primers. Twenty-three of the 32 adhering isolates (72%) and only 1 of the 26 nonadherent strains were HMW protein or hmw gene positive. Among the 32 isolates adhering to either cell line, 5 different adherence patterns were distinguished based on the inhibiting effect of dextran sulfate. Using H. influenzae strain 12 expressing two well-defined HMW proteins (HMW1 and HMW2) and its isogenic mutants as a reference, we observed HMW1-like adherence to both cell lines for 16 of the 32 adherent isolates. Four others showed HMW2-like adherence to NCI-H292. Of the three other patterns of adherence, one probably also involved HMW protein. Screening of the isolates with six HMW-specific monoclonal antibodies in a whole-cell enzyme-linked immunosorbent assay showed that the HMW proteins of COPD isolates and carrier isolates were more distinct from the HMW proteins from H. influenzae strain 12 than those from otitis media isolates. Characterization of the HMW protein of a COPD isolate by adherence and DNA sequence analysis showed that despite large sequence diversity in the hmwA gene, probably resulting in the antigenic differences, the HMW protein mediated the HMW2-like adherence of this strain.

Haemophilus influenzae localized in epithelial cell layers is shielded from antibiotics and antibody-mediated bactericidal activity.

Nonencapsulated Haemophilus influenzae frequently persists in the lungs of chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) patients for prolonged periods of time. The bacteria are not eradicated by antibiotic treatment of the patients or by specific antibodies that are found in the sputum and sera of these patients. We investigated whether H. influenzae, when localized in lung epithelial cell layers, is shielded from antibiotics and from antibody-mediated bactericidal activity of specific antibodies. An in vitro model system consisting of lung epithelial NCI-H292 cells on permeable supports was developed to allow long term association of H. influenzae with the cells. Microscopic examination showed increasing numbers of H. influenzae bacteria between the epithelial cells up to 24 h of incubation. Coinciding with the microscopic observations the maximum number of cell-associated bacteria surviving gentamicin treatment of the cell layers was obtained after 24 h of incubation. All H. influenzae strains, and one Haemophilus parainfluenzae strain tested penetrated into the cell layer as determined by gentamicin killing. Cell-associated bacteria were shielded from the bactericidal activity of several antibiotics and from antibody-mediated bactericidal activity. After prolonged incubation in the cell system in the presence of a specific bactericidal antibody against major outer membrane protein (MOMP) P2, antigenic variation occurred due to a point mutation in the MOMP P2 gene, similar to point mutations observed in vivo. We conclude that penetration of H. influenzae between lung epithelial cells results in shielding the bacteria from killing by antibody dependent defense mechanisms and by antibiotics. Therefore, penetration of H. influenzae between epithelial cells may contribute to the persistence of this microorganism in COPD and CF patients.

Variation in the composition and pore function of major outer membrane pore protein P2 of Haemophilus influenzae from cystic fibrosis patients.

We investigated the relationship between susceptibility to beta-lactam antibiotics and variation in the major outer membrane protein P2 (OmpP2; also called porin) of persistent nonencapsulated Haemophilus influenzae isolated from cystic fibrosis patients. Nine OmpP2 variants were selected from two distinct H. influenzae strains from two patients extensively treated with beta-lactam antibiotics. The variants differed in their susceptibilities to at least two beta-lactam antibiotics. By detergent extraction and column chromatography, OmpP2 was purified from two variants that were derived from strain 70 and that differed notably in their susceptibilities to beta-lactam antibiotics. The proteins were reconstituted into black lipid membranes for measurement of porin function. OmpP2 from the more resistant isolate (isolate 70b) had a smaller channel conductance than OmpP2 of the more susceptible isolate (isolate 70f). DNA sequencing of ompP2 of these isolates revealed single nonsynonymous base differences; there were changes in the amino acid sequence corresponding to surface-exposed loops 4, 5, 6, and 8. Changes in loops 4, 5, and 6 were previously shown to result in antigenic differences. Beside these mutations, variants of strain 70 showed additional mutations in loop 1 and nonexposed loop 3. Taken together, our results suggest that in variants of strain 70, nonsynonymous point mutations accumulated both in the sequences of ompP2 coding for antigen-variable loops and in other loops, notably, loops 1 and 3. The latter changes are suggested to affect the permeability of the porin channel.

The integration host factor-DNA complex upstream of the early promoter of bacteriophage Mu is functionally symmetric.

Inversion of the ihf site in the promoter region of the early promoter of bacteriophage Mu did not influence the integration host factor (IHF)-mediated functions. IHF bound to this inverted site could counteract H-NS-mediated repression, directly activate transcription, and support lytic growth of bacteriophage Mu. This implies that the IHF heterodimer and its asymmetrical binding site form a functionally symmetrical complex.

Function of the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase in basal expression and integration host factor-mediated activation of the early promoter of bacteriophage Mu.

Integration host factor (IHF) can activate transcription from the early promoter (Pe) of bacteriophage Mu both directly and indirectly. Indirect activation occurs through alleviation of H-NS-mediated repression of the Pe promoter (P. Van Ulsen, M. Hillebrand, L. Zulianello, P. Van de Putte, and N. Goosen, Mol. Microbiol. 21:567-578, 1996). The direct activation involves the C-terminal domain of the alpha subunit (alphaCTD) of RNA polymerase. We investigated which residues in the alphaCTD are important for IHF-mediated activation of the Pe promoter. Initial in vivo screening, using a set of substitution mutants derived from an alanine scan (T. Gaal, W. Ross, E. E. Blatter, T. Tang, X. Jia, V. V. Krishnan, N. Assa-Munt, R. Ebright, and R. L. Gourse, Genes Dev. 10:16-26, 1996; H. Tang, K. Severinov, A. Goldfarb, D. Fenyo, B. Chait, and R. H. Ebright, Genes Dev. 8:3058-3067, 1994), indicated that the residues, which are required for transcription activation by the UP element of the rrnB P1 promoter (T. Gaal, W. Ross, E. E. Blatter, T. Tang, X. Jia, V. V. Krishnan, N. Assa-Munt, R. Ebright, and R. L. Gourse, Genes Dev. 10:16-26, 1996), are also important for Pe expression in the presence of IHF. Two of the RNA polymerase mutants, alphaR265A and alphaG296A, that affected Pe expression most in vivo were subsequently tested in in vitro transcription experiments. Mutant RNA polymerase with alphaR265A showed no IHF-mediated activation and a severely reduced basal level of transcription from the Pe promoter. Mutant RNA polymerase with alphaG296A resulted in a slightly reduced transcription from the Pe promoter in the absence of IHF but could still be activated by IHF. These results indicate that interaction of the alphaCTD with DNA is involved not only in the IHF-mediated activation of Pe transcription but also in maintaining the basal level of transcription from this promoter. Mutational analysis of the upstream region of the Pe promoter identified a sequence, positioned from -39 to -51 with respect to the transcription start site, that is important for basal Pe expression, presumably through binding of the alphaCTD. The role of the alphaCTD in IHF-mediated stimulation of transcription from the Pe promoter is discussed.

Integration host factor alleviates the H-NS-mediated repression of the early promoter of bacteriophage Mu.

Integration host factor (IHF), which is a histone-like protein, has been shown to positively regulate transcription in two different ways. It can either help the formation of a complex between a transcription factor and RNA polymerase or it can itself activate RNA polymerase without the involvement of other transcription factors. In this study, we present a third mechanism for IHF-stimulated gene expression, by counteracting the repression by another histone-like protein, H-NS. The early (Pe) promoter of bacteriophage Mu is specifically inhibited by H-NS, both in vivo and in vitro. For this inhibition, H-NS binds to a large DNA region overlapping the Pe promoter. Binding of IHF to a binding site just upstream of Pe alleviates the H-NS-mediated repression of transcription. This same ihf site is also involved in the direct activation of Pe by IHF. In contrast to the direct activation by IHF, however, the alleviating effect of IHF appears not to be dependent on the relevant position of the ihf site on the DNA helix, and it also does not require the presence of the C-terminal domain of the alpha subunit of RNA polymerase. Footprint analysis shows that binding of IHF to the ihf site destabilizes the interaction of H-NS with the DNA, not only in the IHF-binding region but also in the DNA regions flanking the ihf site. These results suggest that IHF disrupts a higher-order nucleoprotein complex that is formed by H-NS and the DNA.

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.

The HimA and HimD subunits of integration host factor can specifically bind to DNA as homodimers.

Integration host factor (IHF) is a heterodimeric protein from Escherichia coli which specifically binds to an asymmetric consensus sequence. We have isolated the individual subunits of IHF, HimA and HimD, and show that an active IHF protein can be reconstituted from these subunits. The HimA and HimD polypeptides alone are capable of specifically recognizing the same ihf sequence. The mobilities of the protein-DNA complexes in a gel-retardation assay suggest that the proteins bind as homodimers. The stability of the HimD-DNA complex is approximately 100-fold lower than that of the IHF-DNA complex. The HimA-DNA complex is even less stable and is only observed when a large excess of HimA is used. This instability is possibly due to the inability of HimA to form stable homodimers. By domain swapping between HimA and HimD, we have constructed an IHF fusion protein which has the putative DNA-binding domains of only HimA. This fusion protein forms stable dimers and makes specific protein-DNA complexes with a high efficiency. A comparable fusion protein with only the DNA-binding domains of HimD forms less stable complexes, suggesting that sequence-specific contacts between IHF and the ihf consensus are mainly provided by the HimA subunit.

Escherichia coli SecB, SecA, and SecY proteins are required for expression and membrane insertion of the bacteriocin release protein, a small lipoprotein.

The SecB, SecA, and SecY dependency of a small outer membrane lipoprotein in Escherichia coli, the bacteriocin release protein (BRP), was studied. The detrimental effect of BRP expression on the culture turbidity (quasi-lysis) was strongly reduced in the sec mutants. Immunoblotting and radioactive labeling experiments showed that the expression, membrane insertion, and processing of the BRP precursor are dependent on SecB, SecA, and SecY. Labeling experiments with hybrid BRP gene constructs revealed that the mature part of the BRP precursor and not its stable signal sequence is important for its SecB dependency.