Biomedical subjects

A Camilli

Publications and source records attributed to A Camilli.

At least 19 recordsLinked to original sources

PubMed2005

Second-generation recombination-based in vivo expression technology for large-scale screening for Vibrio cholerae genes induced during infection of the mouse small intestine.

We have constructed an improved recombination-based in vivo expression technology (RIVET) and used it as a screening method to identify Vibrio cholerae genes that are transcriptionally induced during infection of infant mice. The improvements include the introduction of modified substrate cassettes for resolvase that can be positively and negatively selected for, allowing selection of resolved strains from intestinal homogenates, and three different tnpR alleles that cover a range of translation initiation efficiencies, allowing identification of infection-induced genes that have low-to-moderate basal levels of transcription during growth in vitro. A transcriptional fusion library of 8,734 isolates of a V. cholerae El Tor strain that remain unresolved when the vibrios are grown in vitro was passed through infant mice, and 40 infection-induced genes were identified. Nine of these genes were inactivated by in-frame deletions, and their roles in growth in vitro and fitness during infection were measured by competition assays. Four mutant strains were attenuated >10-fold in vivo compared with the parental strain, demonstrating that infection-induced genes are enriched in genes essential for virulence.

Animals↗

PubMed2001

Selection for in vivo regulators of bacterial virulence.

We devised a noninvasive genetic selection strategy to identify positive regulators of bacterial virulence genes during actual infection of an intact animal host. This strategy combines random mutagenesis with a switch-like reporter of transcription that confers antibiotic resistance in the off state and sensitivity in the on state. Application of this technology to the human intestinal pathogen Vibrio cholerae identified several regulators of cholera toxin and a central virulence gene regulator that are operative during infection. These regulators function in chemotaxis, signaling pathways, transport across the cell envelope, biosynthesis, and adherence. We show that phenotypes that appear genetically independent in cell culture become interrelated in the host milieu.

Animals↗

PubMed2001

Isolation and characterization of a temperature-sensitive generalized transducing bacteriophage for Vibrio cholerae.

CP-T1 is the only described generalized transducing bacteriophage for the intestinal pathogen Vibrio cholerae, yet many of its basic biological parameters remain unknown. Due to low frequencies of transduction and pseudolysogen formation, CP-T1 has not been widely used as a genetic tool. To overcome these limitations, we have isolated a conditional mutant of CP-T1 that exhibits temperature-sensitive plaque formation. Several biological properties of CP-T1ts were determined, including its restrictive temperature, adsorbance profile to host cells, burst time, and burst size. Based on these properties, an optimized transduction protocol was designed which resulted in several fold higher transduction frequencies for a variety of genetic markers from a number of chromosomal loci. Generalized transduction was also demonstrated between classical and E1 Tor biotype strains of V. cholerae.

Bacteriophages↗

PubMed2001

The ToxR-mediated organic acid tolerance response of Vibrio cholerae requires OmpU.

It was previously demonstrated that the intestinal pathogen Vibrio cholerae could undergo an adaptive stress response known as the acid tolerance response (ATR). The ATR is subdivided into two branches, inorganic ATR and organic ATR. The transcriptional regulator ToxR, while not involved in inorganic ATR, is required for organic ATR in a ToxT-independent manner. Herein, we investigate the effect of organic acid stress on global protein synthesis in V. cholerae and show by two-dimensional gel electrophoresis that the stress response alters the expression of more than 100 polypeptide species. The expression of more than 20 polypeptide species is altered in a toxR strain compared to the wild type. Despite this, ectopic expression of the porin OmpU from an inducible promoter is shown to be sufficient to bypass the toxR organic ATR defect. Characterization of the effect of organic acid stress on ompU and ompT transcription reveals that while ompU transcription remains virtually unaffected, ompT transcription is repressed in a ToxR-independent manner. These transcript levels are similarly reflected in the extent of accumulation of OmpU and OmpT. Possible roles for OmpU in organic acid resistance are discussed.

Adhesins, Bacterial↗

PubMed2000

Detection and analysis of gene expression during infection by in vivo expression technology.

Many limitations associated with the use of in vitro models for study of bacterial pathogenesis can be overcome by the use of technologies that detect pathogen gene expression during the course of infection within an intact animal. In vivo expression technology (IVET) accomplishes this with versatility: it has been developed with a variety of reporter systems which allow for either in vivo selection or ex vivo screening. Selectable gene fusion systems generally allow for the complementation of a bacterial metabolic defect that is lethal in vivo, or for antibiotic resistance during the course of in vivo antibiotic challenge. In contrast, the screenable gene fusion system uses a site-specific DNA recombinase that, when expressed in vivo, excises a selectable gene cassette from the bacterial chromosome. Loss of this cassette can then be either screened or selected for ex vivo. The recombinase-based IVET can be used to detect genes that are transcriptionally induced during infection, including those expressed transiently or at low levels and, in addition, can be used to monitor the spatial and temporal expression of specific genes during the course of infection.

Adaptation, Physiological↗

PubMed2000

IVET and RIVET: use of gene fusions to identify bacterial virulence factors specifically induced in host tissues.

IVET was designed to identify those bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes encode virulence factors, products specifically required for the infection process. The paradigm IVET system is based on complementation of an attenuating auxotrophic mutation by gene fusion and is designed to be of use in a wide variety of pathogenic organisms. In S. typhimurium, we have used this system successfully to identify a number of genes that are induced in a BALB/c mouse and that, when mutated, confer a virulence defect. The RIVET system is based on recombinase gene fusions, which, on induction during infection, mediate a site-specific recombination, the product of which can be screened for after recovery of bacteria from host tissues. In V. cholerae, we have used this system successfully to identify genes that are induced transcriptionally during infection of the gastrointestinal tract of infant mice. RIVET is also uniquely designed for postidentification analysis of in vivo-induced genes: (1) it has been used to analyze the temporal and spatial patterns of virulence gene induction during infection and (2) it has been used to dissect the regulatory requirements of in vivo induction with respect to both bacterial regulatory factors and host-inducing environments. The IVET system has several applications in the area of vaccine and antimicrobial drug development. This 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 expressed optimally in animal tissues provides a means of establishing in vivo-regulated expression of heterologous antigens in live vaccines, an area that has been problematic previously. Finally, we expect that our methodology will 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.

Animals↗

PubMed2000

Novel approaches to monitor bacterial gene expression in infected tissue and host.

Elucidating the complex and dynamic host-microbe interactions during infection requires, among other things, detailed knowledge of microbial gene expression in vivo. Recently, advances in fluorescence and bioluminescence detection techniques, as well as recombinase-based in vivo expression technology, have rendered monitoring virulence gene expression in vivo a feasible task. These techniques have been adapted by several laboratories to study the spatial and temporal patterns of virulence gene expression by organisms such as Salmonella typhimurium, Listeria monocytogenes, Yersinia entercolitica and Vibrio cholerae during infection of tissue culture or animal models of infection.

Animals↗

PubMed2000

Vibrio cholerae requires rpoS for efficient intestinal colonization.

Vibrio cholerae is a facultative intestinal pathogen that lives in aquatic environments, often in association with planktonic species. In the suckling mouse, oral inoculation with V. cholerae leads to intestinal colonization and symptoms of diarrheal disease. Results reported here indicate a role for the alternative sigma factor, RpoS, in intestinal colonization in this model of cholera. We constructed within rpoS multiple independent mutations which consistently resulted in a fivefold decrease in colonization ability as assessed by competition assays. These mutations had no detectable effect on the in vitro growth of V. cholerae in a rich medium. The occurrence of spontaneous suppressor mutations potentially required for viability of rpoS strains was ruled out by determination of the frequency of insertional inactivation of rpoS in comparison to two other nonessential loci. Finally, both the in vitro and in vivo mutant phenotypes of rpoS strains were fully complemented by providing rpoS in trans or by allelic reversion, indicating that the observed decrease in colonization fitness was indeed due to the loss of functional RpoS.

Animals↗

PubMed2000

Random transposon mutagenesis of Campylobacter jejuni.

Genetic studies of Campylobacter jejuni have been limited due to the lack of a transposon mutagenesis method. Here, we describe a novel technique for random transposon mutagenesis using a mariner-based transposon into C. jejuni strain 480. Insertions were random, as demonstrated by Southern blot analysis and insertional junction sequencing. We have demonstrated, for the first time, random in vivo transposon mutagenesis of C. jejuni.

Campylobacter jejuni↗

PubMed2000

Regulation of vibrio cholerae genes required for acid tolerance by a member of the "ToxR-like" family of transcriptional regulators.

The ability of the intestinal pathogen Vibrio cholerae to undergo an adaptive stress response, known as the acid tolerance response (ATR), was previously shown to enhance virulence. An essential component of the ATR is CadA-mediated lysine decarboxylation. CadA is encoded by the acid- and infection-induced gene cadA. Herein, cadA is shown to be the second gene in an operon with cadB, encoding a lysine/cadaverine antiporter. cadC, which is 5' of cadB, encodes an acid-responsive, positive transcriptional regulator of cadBA. Unlike in Escherichia coli, V. cholerae cadB and cadA are also transcribed monocistronically. Of note, bicistronic cadBA is transcribed at low constitutive levels in an acid- and CadC-independent manner. CadC represents a new member of the "ToxR-like" family of transcriptional regulators in V. cholerae and, in addition, exhibits extensive amino acid and functional similarity to E. coli CadC. The amino-terminal, putative DNA binding domains of ToxR and CadC are highly conserved, as are the putative promoter elements recognized by these transcription factors.

Acids↗

PubMed2000

Postchemotherapy late recurrence of non metastatic gestational trophoblastic disease following a partiale mole. A case report.

The authors describe a case of a 35-year-old woman who showed elevation of betahCG 13 months after the complete regression of betahCG values following chemotherapy for an incomplete mole. This case outlines the necessity for careful monitoring of betahCG levels in low risk gestational trophoblastic diseases for a period of time longer than one year after achieving the first clinical remission.

Abortion, Spontaneous↗

PubMed1999

Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection.

The temporal expression patterns of the critical Vibrio cholerae virulence genes, tcpA and ctxA, were determined during infection using a recombinase reporter. TcpA was induced biphasically in two temporally and spatially separable events in the small intestine, whereas ctxA was induced monophasically only after, and remarkably, dependent upon, tcpA expression; however, this dependence was not observed during in vitro growth. The requirements of the virulence regulators, ToxR, TcpP, and ToxT, for expression of tcpA and ctxA were determined and were found to differ significantly during infection versus during growth in vitro. These results illustrate the importance of examining virulence gene expression in the context of bona fide host-pathogen interactions.

Animals↗

PubMed1999

Transformation of a type 4 encapsulated strain of Streptococcus pneumoniae.

Streptococcus pneumoniae strain JNR.7/87 is a highly virulent, type 4 encapsulated Gram-positive bacterium whose transformability has not been tested previously, and whose genome is currently being sequenced. The strain was transformed at very low efficiency by addition of exogenous competence-stimulating peptide: However, the efficiency was too low and irreproducible to be useful in many genetic studies. Therefore, the effects on transformation efficiency of changing different components of competence-stimulating peptide-induced transformation have been examined. Screening of growth media was followed by optimization of pre-induction culture acidification, glycine concentration, and induction time. An optimized protocol was developed whereby S. pneumoniae strain JNR.7/87 was transformed reproducibly with a streptomycin resistance (SmR) marker at an efficiency of approximately 10(5) colony forming units per 10(8) cells.

Bacterial Proteins↗

PubMed1999

The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance.

Vibrio cholerae is a facultative pathogen of humans that must survive exposure to inorganic and organic acids in the stomach and small intestine. To learn more about the mechanisms by which this pathogen colonizes the intestinal tract, we used a recombinase gene fusion reporter to identify transcripts induced during infection in an adult rabbit model of cholera. One of the genes identified was cadA, which encodes an inducible lysine decarboxylase. CadA was also induced during infections of the suckling and adult mouse intestines, and in vitro under conditions of low pH and high lysine concentration. We show that V. cholerae is capable of mounting an acid tolerance response (ATR) to both inorganic and organic acid challenges. Mutational analyses revealed a significant role for cadA, but not for speF, which encodes an ornithine decarboxylase, in both inorganic and organic ATR. Potential roles for toxR, toxT and rpoS in ATR were examined, and it was found that toxR plays a ToxT-independent role in mediating organic ATR, whereas rpoS played no detectable role in either ATR. Transcriptional analysis showed that the toxR defect in ATR is not caused by decreased cadA transcription. Despite induction of cadA in these animal models, competition assays revealed that neither cadA nor speF alone or together were required for colonization of suckling or adult mice. However, acid-adapted wild-type V. cholerae exhibited a major competitive advantage over unadapted cells during colonization of suckling mice.

Animals↗

PubMed1999

Vibrio cholerae intestinal population dynamics in the suckling mouse model of infection.

The suckling mouse has been used as a model to identify Vibrio cholerae intestinal colonization factors for over two decades, yet little is known about the location of recoverable organisms along the gastrointestinal (GI) tract following intragastric inoculation. In the present study, we determined the population dynamics of wild-type and avirulent mutant derivatives of both classical and El Tor biotype strains throughout the entire suckling mouse GI tract at various times after intragastric inoculation. Wild-type strains preferentially colonized the middle small bowel with a sharp demarcation between more proximal segments which had manyfold-fewer recoverable cells. Surprisingly, large and stable populations of viable cells were also recovered from the cecum and large bowel. Strains lacking toxin-coregulated pili (TCP(-)) were cleared from the small bowel; however, an El Tor TCP(-) strain colonized the cecum and large bowel almost as well as the wild-type strain. Strains lacking lipopolysaccharide O antigen (OA(-)) were efficiently cleared from the small bowel at early times but then showed net growth for the remainder of the infections. Moreover, large populations of the OA(-) strains were maintained in the large bowel. These results show that for the El Tor biotype neither TCP nor OA is required for colonization of the suckling mouse large bowel. Finally, similar percent recoveries of wild-type, TCP(-), and OA(-) strains from the small bowel at an early time after infection suggest that TCP and OA are not required for strains of either biotype to resist bactericidal mechanisms in the suckling mouse GI tract.

Animals↗

PubMed1998

Systematic identification of essential genes by in vitro mariner mutagenesis.

Although the complete DNA sequences of several microbial genomes are now available, nearly 40% of the putative genes lack identifiable functions. Comprehensive screens and selections for identifying functional classes of genes are needed to convert sequence data into meaningful biological information. One particularly significant group of bacterial genes consists of those that are essential for growth or viability. Here, we describe a simple system for performing transposon mutagenesis on naturally transformable organisms along with a technique to rapidly identify essential or conditionally essential DNA segments. We show the general utility of this approach by applying it to two human pathogens, Haemophilus influenzae and Streptococcus pneumoniae, in which we detected known essential genes and assigned essentiality to several ORFs of unknown function.

Base Sequence↗

PubMed1998

Neither the Bvg- phase nor the vrg6 locus of Bordetella pertussis is required for respiratory infection in mice.

In Bordetella species, the BvgAS sensory transduction system mediates an alteration between the Bvg+ phase, characterized by expression of adhesins and toxins, and the Bvg- phase, characterized by the expression of motility and coregulated phenotypes in Bordetella bronchiseptica and by the expression of vrg loci in Bordetella pertussis. Since there is no known environmental or animal reservoir for B. pertussis, the causative agent of whooping cough, it has been assumed that this phenotypic alteration must occur within the human host during infection. Consistent with this hypothesis was the observation that a B. pertussis mutant, SK6, containing a TnphoA insertion mutation in a Bvg-repressed gene (vrg6) was defective for tracheal and lung colonization in a mouse model of respiratory infection (D. T. Beattie, R. Shahin, and J. Mekalanos, Infect. Immun. 60:571-577, 1992). This result was inconsistent, however, with the observation that a Bvg+ phase-locked B. bronchiseptica mutant was indistinguishable from the wild type in its ability to establish a persistent respiratory infection in rabbits and rats (P. A. Cotter and J. F. Miller, Infect. Immun. 62:3381-3390, 1994; B. J. Akerley, P. A. Cotter, and J. F. Miller, Cell 80:611-620, 1995). To directly address the role of Bvg-mediated signal transduction in B. pertussis pathogenesis, we constructed Bvg+ and Bvg- phase-locked mutants and compared them with the wild type for their ability to colonize the respiratory tracts of mice. Our results show that the Bvg+ phase of B. pertussis is necessary and sufficient for respiratory infection. By constructing a strain with a deletion in the bvgR regulatory locus, we also show that ectopic expression of Bvg- phase phenotypes decreases the efficiency of colonization, underscoring the importance of Bvg-mediated repression of gene expression in vivo. Finally, we show that the virulence defect present in strain SK6 cannot be attributed to the vrg6 mutation. These data contradict an in vivo role for the Bvg- phase of B. pertussis.

Animals↗

PubMed1998

Nucleotide sequence and spatiotemporal expression of the Vibrio cholerae vieSAB genes during infection.

The iviVII gene of Vibrio cholerae was previously identified by a screen for genes induced during intestinal infection. In the present study, nucleotide sequence analysis revealed that iviVII is a 1,659-bp open reading frame, herein designated vieB, that is predicted to be last in a tricistronic operon (vieSAB). The deduced amino acid sequence of VieS exhibited similarity to the sensor kinase component, and those of VieA and VieB were similar to the response regulator components, respectively, of the two-component signal transduction family. Analysis of transcriptional fusions to a site-specific DNA recombinase reporter, tnpR, revealed that vieS and vieA are transcribed during in vitro growth in a vieAB-independent and vieA-dependent manner, respectively. In contrast, transcription of vieB occurred exclusively during infection and was not dependent upon VieB. We conclude that the vieSAB genes are differentially regulated, at least during laboratory growth. Use of a V. cholerae strain harboring a vieB::tnpR transcriptional fusion allowed the kinetics and location of vieB expression within the intestine to be determined. We found that vieB transcription is induced shortly after infection of the proximal and mid-small intestine.

Amino Acid Sequence↗