Biomedical subjects
Publications and source records attributed to M J Mahan.
In vivo expression studies reveal many bacterial genes that contribute to the fitness of the organism in the context of host ecology. This collection of virulence genes defines the unique lifestyle of a pathogen during infection, pointing to the functions that dictate host specificity, tissue tropism and disease manifestation.
recB recJ mutants of Salmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe in recD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable in recA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability. recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance, recB recJ mutants of S. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.
Lipopolysaccharide (LPS) coats the surface of gram-negative bacteria and serves to protect the cell from its environment. The O-antigen is the outermost part of LPS and is highly variable among gram-negative bacteria. Strains of Salmonella are partly distinguished by serotypic differences in their O-antigen. In Salmonella typhimurium, the O-antigen is acetylated, conferring the 05 serotype. We have previously provided evidence that this modification significantly alters the structure of the O-antigen and creates or destroys a series of conformational epitopes. Here we report the detailed mapping, cloning, and DNA sequence of the oafA gene. The locus contains one open reading frame that is predicted to encode an inner membrane protein, consistent with its role in modification of the O-antigen subunit. The OafA protein shows homology to proteins in a number of prokaryotic and one eukaryotic species, and this defines a family of membrane proteins involved in the acylation of exported carbohydrate moieties. In many of these instances, acylation defines serotype or host range and thus has a profound effect on microbe-host interaction.
We have recently described a genetic system, termed in vivo expression technology (IVET), that uses an animal as a selective medium to identify genes that pathogenic bacteria specifically express when infecting host tissues. Here, the potential utility of the IVET approach has been expanded with the development of a transcriptional-fusion vector, pIVET8, which uses antibiotics resistance as the basis for selection in host tissues. pIVET8 contains promoterless chloramphenicol acetyltransferase (cat) and lacZY genes. A pool of Salmonella typhimurium clones carrying random cat-lac transcriptional fusions, produced with pIVET8, was used to infect BALB/c mice that were subsequently treated with intraperitoneal injections of chloramphenicol. Strains that survived the selection by expressing the cat gene in the animal were then screened for those that had low-level lacZY expression on laboratory medium. These strains carry operon fusions to genes that are specifically induced in vivo (ivi genes). One of the ivi genes identified (fadB) encodes an enzyme involved in fatty acid oxidation, suggesting that this enzyme might contribute to the metabolism of bactericidal or proinflammatory host fatty acids. The pIVET8-based selection system was also used to identify S. typhimurium genes that are induced in cultured macrophages. The nature of ivi gene products will provide a more complete understanding of the metabolic, physiological, and genetic factors that contribute to the virulence of microbial pathogens.
The lipopolysaccharide (LPS) of gram-negative bacteria serves as a barrier between the cell and its environment. The LPS O antigen is the immunodominant portion of the molecule and thus has a significant effect on the interaction between a bacterial pathogen and the host organism. Antibodies directed against O antigen are vital to the immune response to infection. In this study, we have characterized the interaction between a series of monoclonal immunoglobulin A antibodies and the LPS of Salmonella typhimurium. Using one of these antibodies, we have previously shown that monoclonal immunoglobulin A is sufficient to protect against S. typhimurium infection, both in vivo and in vitro. Here, we show that recognition of LPS by the monoclonal antibodies is affected by acetylation of the O antigen on the abequose moiety, the determinant of the O5 epitope. Although recognition of LPS by several of the monoclonal antibodies is completely dependent on acetylation, the antibodies recognize clearly separable epitopes. This suggests that acetylation of O antigen affects the three-dimensional structure of the molecule and thus creates and destroys a series of conformational antigenic determinants. We have shown that a change in the acetylation state of LPS has no effect on virulence. However, acetylation has important consequences for the mucosal immune response and thus could potentially have profound implications for the ability of an immune host to respond to a subsequent infection.
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BACKGROUND/AIMS: Invasion of the intestinal epithelium is considered a critical step in Salmonella pathogenesis. Infection by Salmonella of cultured monolayers of polarized Madin-Darby canine kidney (MDCK) cells has been established as a simple in vitro system that mimics the invasion of intestinal enterocytes in vivo. This study analyzes the protective role of secretory immunoglobulin (Ig) A antibodies against epithelial invasion. METHODS: Salmonella typhimurium was applied to MDCK cell monolayers in the presence or absence of a monoclonal, polymeric IgA antibody (Sal4) directed against an antigenic determinant exposed on the surface of wild-type S. typhimurium. RESULTS: In the presence of Sal4 IgA, confluent monolayers of MDCK cells were protected against apical invasion by wild-type S. typhimurium but not against a mutant strain that lacks the Sal4 epitope. Protection was Sal4-specific, dependent on the concentration of Sal4 in the apical medium, and occurred at IgA concentrations at which agglutination of IgA-bacterial complexes was observed. When MDCK cell monolayers were formaldehyde-fixed before incubation with Salmonella to prevent bacterial invasion, adhesion of Salmonella occurred in the absence of IgA and in the presence of control IgA but not in the presence of Sal4 IgA. CONCLUSIONS: IgA alone can prevent bacterial adherence and invasion of epithelial cells in the absence of other immune or nonimmune protective mechanisms.
We have developed a genetic system, termed IVET (in vivo expression technology), designed to identify bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes should include those that encode virulence factors, products specifically required for the infection process. The system is based on complementation of an attenuating auxotrophic mutation by gene fusion, and it is designed to be of use in a wide variety of pathogenic organisms. In Salmonella typhimurium, we have successfully used the system to identify a number of genes that are induced in BALB/c mice, and that, when mutated, confer a virulence defect. The IVET system has several applications in the area of vaccine and antimicrobial drug development. The technique was designed for the identification of virulence factors and thus may lead to the discovery of new antigens useful as vaccine components. The IVET system facilitates the isolation of mutations in genes involved in virulence and, therefore, should aid in the construction of live attenuated vaccines. In addition, the identification of promoters that are optimally expressed in animal tissues provides a means of establishing in vivo regulated expression of heterologous antigens in live vaccines, an area that has been previously problematic. Finally, we expect that our methodology will be used to uncover many biosynthetic, catabolic, and regulatory genes that are required for growth of microbes in animal tissues. The elucidation of these gene products should provide new targets for antimicrobial drug development.
In order to understand the genetic regulation of bacterial genes whose products are important for pathogenesis, one needs to measure the expression of the genes during the infection process. We have devised a method to measure the transcriptional activity of such genes from bacteria recovered directly from infected host tissue. Starting with bacterial strains containing lacZ transcriptional fusions to the genes of interest, animals can be infected, with subsequent isolation of infected host tissue. Here we describe the separation of bacterial cells away from a particular host tissue and the subsequent measurement of the activity of beta-galactosidase, the product of the lacZ gene, in the bacterial cells. This assay is sensitive enough to compensate for the potentially low number of bacteria recovered from the infection site.
A genetic system was devised that positively selects for bacterial genes that are specifically induced when bacteria infect their host. With the pathogen Salmonella typhimurium, the genes identified by this selection show a marked induction in bacteria recovered from mouse spleen. Mutations in all ivi (in vivo-induced) genes that were tested conferred a defect in virulence. This genetic system was designed to be of general use in a wide variety of bacterial-host systems and has several applications in both vaccine and antimicrobial drug development.
Growth of bacteriophage P22 erf is known to require host RecA recombination function. We show that the RecA function is necessary but not sufficient to restore the plaque-forming ability of phage P22 erf; such mutant phage also requires host RecJ function. The residual efficiency of plaquing of P22 erf in a recJ background (0.03%) is completely abolished in recJ recB hosts (< 0.001%), suggesting that the RecBCD nuclease can provide an alternative function allowing phage growth. One tentative explanation is that circularization of P22 erf DNA mostly proceeds through the RecF pathway of recombination; however, less efficient circularization via the RecBCD pathway may also occur. In a recJ background, lysates obtained upon induction of an erf prophage show reduced yield (10%), suggesting that growth of P22 erf may require host RecJ in a step(s) other than circularization of phage DNA.
Five TnphoA fusions to vir-repressed genes (vrg genes) have been identified in the respiratory pathogen Bordetella pertussis. A comparison of vrg DNA sequences suggests a consensus DNA element within the coding regions of four of five vrg genes. To determine the role of this DNA sequence in vrg regulation, a nucleotide substitution mutation in the conserved region of vrg-6 was isolated. This mutant showed constitutively high levels of expression in the absence of antigenic modulators, MgSO4 and nicotinic acid, suggesting that this DNA element may be a control site for vrg repression. Moreover, Northern (RNA) analysis and transcriptional fusion analysis suggest that vrg genes are regulated at the transcriptional level. To determine whether sequences in the coding region were sufficient to respond to antigenic modulation, a vrg-6::TnphoA promoter deletion plasmid that contained a heterologous promoter driving the expression of vrg-6 coding sequences from the vrg-6 translation start site to the TnphoA fusion junction was constructed. This heterologous construct responded to modulators in a vir-dependent fashion, indicating that sequences upstream of the coding sequence are not required for antigenic modulation. Southwestern (DNA-protein) analysis and mutational studies suggest that the vrg consensus DNA sequence is specifically recognized by a 34-kDa vir-activated gene (vag) product, whose binding results in down-regulation of vrg transcript levels. We conclude, at least for the vrg::TnphoA fusion strains, that a site on the DNA that corresponds to a consensus sequence located in the vrg coding region is sufficient to confer the transcriptional regulation (repression) of vrg genes when B. pertussis strains are grown under nonmodulating conditions.
Transcriptional fusions to Salmonella typhimurium chromosomal genes were constructed by integration of a suicide fusion vector into the chromosome by homologous recombination with random cloned chromosomal fragments. We describe here a transductional method using the generalized transducing phage of S. typhimurium, P22, to clone these fusions directly from the bacterial chromosome, in a single step, without the use of restriction enzymes. In this transduction, the phage packages the chromosomal fragment containing the integrated plasmid. Once introduced into the recipient, the plasmid circularizes by homologous recombination between the duplicated region determined by the cloned fragment. Although RecA mediates the majority of these events, the plasmid can circularize in a recA recipient. However, in this case, the event occurs at a much lower frequency and only when the transduction is done at a high multiplicity of infection. In addition to integrated fusion constructs, we also show that autonomously replicating low-copy-number plasmids can be transduced. In this case, transduction is dependent on homologous recombination between the plasmid and the donor chromosome via cloned sequences, in which the transducing particle effectively traps the integrated plasmid.
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We describe recJ mutants of Salmonella typhimurium. The recJ gene maps between sufD and serA (min 62) and is transcribed counterclockwise. Unlike recJ mutants of Escherichia coli, recJ strains of S. typhimurium are sensitive to irradiation with UV light. This sensitivity is equivalent to or greater that that displayed by recBCD mutant strains. The residual ability of phage P22 abc (anti-recBCD) mutants to form plaques on recBCD+ strains is eliminated in recJ hosts. Thus host RecJ function appears to substitute for the anti-RecBCD functions of phage P22 and may serve to limit RecBCD activity.
Hybridomas producing monoclonal immunoglobulin A (IgA) antibodies against Salmonella typhimurium were generated by mucosal immunization of BALB/c mice with attenuated strains of S. typhimurium and subsequent fusion of Peyer's patch lymphoblasts with myeloma cells. To test the role of secretory IgA (sIgA) in protection against Salmonella sp., we analyzed in detail the protective capacity of a monoclonal IgA, Sal4, produced in polymeric as well as monomeric forms, that is directed against a carbohydrate epitope exposed on the surface of S. typhimurium. BALB/c mice bearing subcutaneous Sal4 hybridoma tumors and secreting monoclonal sIgA into their gastrointestinal tracts were protected against oral challenge with S. typhimurium. This protection was directly dependent on specific recognition by the monoclonal IgA, since mice secreting Sal4 IgA from hybridoma tumors were not protected against a fully virulent mutant that lacks the Sal4 epitope. Although monoclonal Sal4 IgA was present in the bloodstreams and tissues of tumor-bearing mice, it did not protect against intraperitoneal challenge and did not possess complement-fixing or bacteriocidal activity in vitro. Taken together, these results indicate that secretion of sIgA alone can prevent infection by an invasive enteric pathogen, presumably by immune exclusion at the mucosal surface.
Homologous recombination between sequences present in inverse order within the same chromosome can result in inversion formation. We have previously shown that inverse order sequences at some sites (permissive) recombine to generate the expected inversion; no inversions are found when the same inverse order sequences flank other (nonpermissive) regions of the chromosome. In hopes of defining how permissive and nonpermissive intervals are determined, we have constructed a strain that carries a large chromosomal inversion. Using this inversion mutant as the parent strain, we have determined the "permissivity" of a series of chromosomal sites for secondary inversions. For the set of intervals tested, permissivity seems to be dictated by the nature of the genetic material present within the chromosomal interval being tested rather than the flanking sequences or orientation of this material in the chromosome. Almost all permissive intervals include the origin or terminus of replication. We suggest that the rules for recovery of inversions reflect mechanistic restrictions on the occurrence of inversions rather than lethal consequences of the completed rearrangement.
The role of recBC functions has been tested for three types of chromosomal recombination events: (1) recombination between direct repeats to generate a deletion, (2) recombination between a small circular fragment and the chromosome, and (3) recombination between inversely oriented repeats to form an inversion. Deletion formation by recombination between direct repeats, which does not require a fully reciprocal exchange, is independent of recBC function. Circle integration and inversion formation are both stimulated by the recBC function; these events require full reciprocality. The results suggest that half-reciprocal exchanges can occur without recBC, but recBC functions greatly stimulate completion of a fully reciprocal exchange. We propose that chromosomal recombination is a two-step process, and recBC functions are primarily required for the second step.