AcrAB-TolC is a constitutively expressed, tripartite efflux transporter complex that functions as the primary resistance mechanism to lipophilic drugs, dyes, detergents, and bile acids in Escherichia coli. TolC is an outer membrane channel, and AcrA is an elongated lipoprotein that is hypothesized to span the periplasm and coordinate efflux of such substrates by AcrB and TolC. AcrD is an efflux transporter of E. coli that provides resistance to aminoglycosides as well as to a limited range of amphiphilic agents, such as bile acids, novobiocin, and fusidic acid. AcrB and AcrD belong to the resistance nodulation division superfamily and share a similar topology, which includes a pair of large periplasmic loops containing more than 300 amino acid residues each. We used this knowledge to test several plasmid-encoded chimeric constructs of acrD and acrB for substrate specificity in a marR1 ⌬acrB ⌬acrD host. AcrD chimeras were constructed in which the large, periplasmic loops between transmembrane domains 1 and 2 and 7 and 8 were replaced with the corresponding loops of AcrB. Such constructs provided resistance to AcrB substrates at levels similar to native AcrB. Conversely, AcrB chimeras containing both loops of AcrD conferred resistance only to the typical substrates of AcrD. These results cannot be explained by simply assuming that AcrD, not hitherto known to interact with AcrA, acquired this ability by the introduction of the loop regions of AcrB, because (i) both AcrD and AcrA were found, in this study, to be required for the efflux of amphiphilic substrates, and (ii) chemical cross-linking in intact cells efficiently produced complexes between AcrD and AcrA. Since AcrD can already interact with AcrA, the alterations in substrate range accompanying the exchange of loop regions can only mean that substrate recognition (and presumably binding) is determined largely by the two periplasmic loops.
Piliated Neisseria gonorrhoeae are known to be transformed less readily if transforming DNA competes with DNA containing the 10-bp sequence GCCGTCTGAA. It has been postulated that the 10-bp sequence is a recognition sequence which is required for efficient DNA uptake. We show that the presence of various forms of this 10-bp sequence results in increased uptake of double-stranded DNA into a DNase-resistant state and allows genetic transformation by an otherwise nontransformable plasmid.Transformation of Neisseria gonorrhoeae (the gonococcus) has been studied extensively (1-7, 16, 17), but the mechanisms involved in this process remain unclear. The observation that gonococci are transformed efficiently by their own but not foreign DNA cannot be totally accounted for by restriction barriers (2, 3, 7). High levels of competency are strongly associated with piliation, but there is no evidence that pili are directly involved in uptake (1, 12). Competent gonococci are known to preferentially take up certain plasmid fragments into a DNase-resistant state (3), suggesting a structural basis for uptake specificity. Recently, Goodman and Scocca showed that DNA fragments as small as 93 bp efficiently compete against chromosomal DNA for transformation if they contain the 10-bp sequence GCCGTC TGAA (7). Goodman and Scocca postulate that the mechanism of the inhibition of transformation was at the stage of uptake but provide no direct evidence for increased DNA uptake. Genetic transformation is a complex, multistep phenomenon, and the observed competition could have occurred during other steps of transformation. In this study, we examine the effect of several forms of this uptake sequence on gonococcal DNA uptake and demonstrate that it does allow efficient entry of transforming DNA.Strain F62 piliated gonococci was used throughout this study and was grown on clear typing medium (GC medium base; Difco) as previously described (3). Gonococci of the piliated phenotype were clonally picked for uptake experiments, using the criteria of Kellogg et al. (9). Uptake experiments were performed essentially as described by . Briefly, gonococci were suspended to 5 x 107 CFU/ml in GC broth containing 10 mM MgCl2 at 37°C. Radiolabeled, PstI-linearized plasmid DNA was added (50 to 200 ng), and incubation continued for the indicated time periods. DNase I (Sigma) was added to a final concentration of 160 ,ug/ml to degrade extracellular DNA, and the gonococci were immediately either placed on ice or kept at 37°C as described. Gonococci were then pelleted and washed three times, and the total DNA was extracted (3). Agarose gel electrophoresis was performed in the presence of ethidium bromide, and the gels were photographed to vistially compare relative amounts of extracted DNA. The gels were dried and autoradiographed for 8 to 48 h at -70°C, using an * Corresponding author. intensifying screen. In experiments involving preincubation by unlabeled DNAs, 4 ,ug of the appropriate unlabeled competitor DNA was added for 15 min prior to 100 ng of labele...
Haemophilus ducreyi is resistant to killing by normal serum antibody and complement. We discovered an H. ducreyi outer membrane protein required for expression of serum resistance and termed it DsrA (for "ducreyi serum resistance A"). The dsrA locus was cloned, sequenced, and mutagenized. An isogenic mutant (FX517) of parent strain 35000 was constructed and characterized, and it was found to no longer express dsrA. FX517 was at least 10-fold more serum susceptible than 35000. DsrA was expressed by all strains of H. ducreyi tested, except three naturally occurring, avirulent, serum-sensitive strains. FX517 and the three naturally occurring dsrA-nonexpressing strains were complemented in trans with a plasmid expressing dsrA. All four strains were converted to a serum-resistant phenotype, including two that contained truncated lipooligosaccharide (LOS). Therefore, serum resistance in H. ducreyi does not require expression of full-length LOS but does require expression of dsrA. The dsrA locus from eight additional H. ducreyi strains was sequenced, and the deduced amino acid sequences were more than 85% identical. The major difference between the DsrA proteins was due to the presence of one, two, or three copies of the heptameric amino acid repeat NTHNINK. These repeats account for the variability in apparent molecular mass of the monomeric form of DsrA (28 to 35 kDa) observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since DsrA is present in virulent strains, is highly conserved, and is required for serum resistance, we speculate that it may be a virulence factor and a potential vaccine candidate.Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease transmitted by sexual contact (reviewed in references 2 and 50). Chancroid has gained importance recently because it has been implicated as an independent risk factor for the heterosexual transmission of human immunodeficiency virus
Neisseria gonorrhoeae isolated from patients with disseminated infection are often of the porin (Por1A) serotype and resist killing by nonimmune normal human serum. The molecular basis of this resistance (termed stable serum resistance) in these strains has not been fully defined but is not related to sialylation of lipooligosaccharide. Here we demonstrate that Por1A bearing gonococcal strains bind more factor H, a critical downregulator of the alternative complement pathway, than their Por1B counterparts. This results in a sevenfold reduction in C3b, which is >75% converted to iC3b. Factor H binding to isogenic gonococcal strains that differed only in their porin serotype, confirmed that Por1A was the acceptor molecule for factor H. We identified a surface exposed region on the Por1A molecule that served as the binding site for factor H. We used gonococcal strains with hybrid Por1A/B molecules that differed in their surface exposed domains to localize the factor H binding site to loop 5 of Por1A. This was confirmed by inhibition of factor H binding using synthetic peptides corresponding to the putative exposed regions of the porin loops. The addition of Por1A loop 5 peptide in a serum bactericidal assay, which inhibited binding of factor H to the bacterial surface, permitted 50% killing of an otherwise completely serum resistant gonococcal strain. Collectively, these data provide a molecular basis to explain serum resistance of Por1A strains of N. gonorrhoeae.
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