Biomedical subjects

Andrew Camilli

Publications and source records attributed to Andrew Camilli.

At least 19 recordsLinked to original sources

PubMed2006

Bacterial small-molecule signaling pathways.

Bacteria use diverse small molecules for extra- and intracellular signaling. They scan small-molecule mixtures to access information about both their extracellular environment and their intracellular physiological status, and based on this information, they continuously interpret their circumstances and react rapidly to changes. Bacteria must integrate extra- and intracellular signaling information to mount appropriate responses to changes in their environment. We review recent research into two fundamental bacterial small-molecule signaling pathways: extracellular quorum-sensing signaling and intracellular cyclic dinucleotide signaling. We suggest how these two pathways may converge to control complex processes including multicellularity, biofilm formation, and virulence. We also outline new questions that have arisen from recent studies in these fields.

4-Butyrolactone↗

PubMed2006

Cholera stool bacteria repress chemotaxis to increase infectivity.

Factors that enhance the transmission of pathogens are poorly understood. We show that Vibrio cholerae shed in human 'rice-water' stools have a 10-fold lower oral infectious dose in an animal model than in vitro grown V. cholerae, which may aid in transmission during outbreaks. Furthermore, we identify a bacterial factor contributing to this enhanced infectivity: The achievement of a transient motile but chemotaxis-defective state upon shedding from humans. Rice-water stool V. cholerae have reduced levels of CheW-1, which is essential for chemotaxis, and were consequently shown to have a chemotaxis defect when tested in capillary assays. Through mutational analyses, such a state is known to enhance the infectivity of V. cholerae. This is the first report of a pathogen altering its chemotactic state in response to human infection in order to enhance its transmission.

Bacterial Proteins↗

PubMed2006

Differences in gene expression between the classical and El Tor biotypes of Vibrio cholerae O1.

Differences in whole-genome expression patterns between the classical and El Tor biotypes of Vibrio cholerae O1 were determined under conditions that induce virulence gene expression in the classical biotype. A total of 524 genes (13.5% of the genome) were found to be differentially expressed in the two biotypes. The expression of genes encoding proteins required for biofilm formation, chemotaxis, and transport of amino acids, peptides, and iron was higher in the El Tor biotype. These gene expression differences may contribute to the enhanced survival capacity of the El Tor biotype in environmental reservoirs. The expression of genes encoding virulence factors was higher in the classical than in the El Tor biotype. In addition, the vieSAB genes, which were originally identified as regulators of ctxA transcription, were expressed at a fivefold higher level in the classical biotype. We determined the VieA regulon in both biotypes by transcriptome comparison of wild-type and vieA deletion mutant strains. VieA predominantly regulates gene expression in the classical biotype; 401 genes (10.3% of the genome), including those encoding proteins required for virulence, exopolysaccharide biosynthesis, and flagellum production as well as those regulated by sigmaE, are differentially expressed in the classical vieA deletion mutant. In contrast, only five genes were regulated by VieA in the El Tor biotype. A large fraction (20.8%) of the genes that are differentially expressed in the classical versus the El Tor biotype are controlled by VieA in the classical biotype. Thus, VieA is a major regulator of genes in the classical biotype under virulence gene-inducing conditions.

Amino Acids↗

PubMed2006

Contribution of hemagglutinin/protease and motility to the pathogenesis of El Tor biotype cholera.

Vibrio cholerae is a highly motile organism that secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HapA). HapA has been shown to have mucinase activity and contribute to the reactogenicity of live vaccine candidates, but its role in cholera pathogenesis is not yet clear. The contribution of motility to pathogenesis is not fully understood, since conflicting results have been obtained with different strains, mutants, and animal models. The objective of this work was to determine the contribution of HapA and motility to the pathogenesis of El Tor biotype cholera. To this end we constructed isogenic motility (motY) and mucinase (hapA) single and double mutants of an El Tor biotype V. cholerae strain. Mutants were characterized for the expression of major virulence factors in vitro and in vivo. The motility mutant showed a remarkable increase in cholera toxin (CT), toxin coregulated pilus major subunit (TcpA), and HapA production in vitro. Increased TcpA and CT production could be explained by increased transcription of tcpA, ctxA, and toxT. No effect was detected on the transcription of hapA in the motility mutant. The sodium ionophore monensin diminished production of HapA in the parent but not in the motility mutant. Phenamil, a specific inhibitor of the flagellar motor, diminished CT production in the wild-type and motY strains. The hapA mutant showed increased binding to mucin. In contrast, the motY mutation diminished adherence to biotic and abiotic surfaces including mucin. Lack of HapA did not affect colonization in the suckling mouse model. The motility and mucinase defects did not prevent induction of ctxA and tcpA in the mouse intestine as measured by recombinase-based in vivo expression technology. Analysis of mutants in the rabbit ileal loop model showed that both V. cholerae motility and HapA were necessary for full expression of enterotoxicity.

Animals↗

PubMed2006

RrgA and RrgB are components of a multisubunit pilus encoded by the Streptococcus pneumoniae rlrA pathogenicity islet.

The rlrA pathogenicity islet in Streptococcus pneumoniae TIGR4 encodes three surface proteins, RrgA, RrgB, and RrgC, and three sortase enzymes. Using transmission electron microscopy, cell fractionation, cell wall sorting signal domain swapping, and Western blotting, we show that RrgA and RrgB are incorporated into a multisubunit pilus in S. pneumoniae.

Aminoacyltransferases↗

PubMed2006

Transcriptome and phenotypic responses of Vibrio cholerae to increased cyclic di-GMP level.

Vibrio cholerae, the causative agent of cholera, is a facultative human pathogen with intestinal and aquatic life cycles. The capacity of V. cholerae to recognize and respond to fluctuating parameters in its environment is critical to its survival. In many microorganisms, the second messenger, 3',5'-cyclic diguanylic acid (c-di-GMP), is believed to be important for integrating environmental stimuli that affect cell physiology. Sequence analysis of the V. cholerae genome has revealed an abundance of genes encoding proteins with either GGDEF domains, EAL domains, or both, which are predicted to modulate cellular c-di-GMP concentrations. To elucidate the cellular processes controlled by c-di-GMP, whole-genome transcriptome responses of the El Tor and classical V. cholerae biotypes to increased c-di-GMP concentrations were determined. The results suggest that V. cholerae responds to an elevated level of c-di-GMP by increasing the transcription of the vps, eps, and msh genes and decreasing that of flagellar genes. The functions of other c-di-GMP-regulated genes in V. cholerae are yet to be identified.

Biofilms↗

PubMed2005

The EAL domain protein VieA is a cyclic diguanylate phosphodiesterase.

The newly recognized bacterial second messenger 3',5'-cyclic diguanylic acid (cyclic diguanylate (c-di-GMP)) has been shown to regulate a wide variety of bacterial behaviors and traits. Biosynthesis and degradation of c-di-GMP have been attributed to the GGDEF and EAL protein domains, respectively, based primarily on genetic evidence. Whereas the GGDEF domain was demonstrated to possess diguanylate cyclase activity in vitro, the EAL domain has not been tested directly for c-di-GMP phosphodiesterase activity. This study describes the analysis of c-di-GMP hydrolysis by an EAL domain protein in a purified system. The Vibrio cholerae EAL domain protein VieA has been shown to inversely regulate biofilm-specific genes (vps) and virulence genes (ctxA), presumably by decreasing the cellular pool of c-di-GMP. VieA was maximally active at neutral pH, physiological ionic strength, and ambient temperatures and demonstrated c-di-GMP hydrolytic activity with a Km of 0.06 microM. VieA was unable to hydrolyze cGMP. The putative metal coordination site of the EAL domain, Glu170, was demonstrated to be necessary for VieA activity. Furthermore, the divalent cations Mg2+ and Mn2+ were necessary for VieA activity; conversely, Ca2+ and Zn2+ were potent inhibitors of the VieA phosphodiesterase. Calcium inhibition of the VieA EAL domain provides a potential mechanism for regulation of c-di-GMP degradation.

Bacterial Proteins↗

PubMed2005

Going against the grain: chemotaxis and infection in Vibrio cholerae.

Chemotaxis is the process by which motile cells move in a biased manner both towards favourable and away from unfavourable environments. The requirement of this process for infection has been examined in several bacterial pathogens, including Vibrio cholerae. The single polar flagellum of Vibrio species is powered by a sodium-motive force across the inner membrane, and can rotate to produce speeds of up to 60 cell-body lengths (approximately 60microm) per second. Investigating the role of the chemotactic control of rapid flagellar motility during V. cholerae infection has revealed some unexpected and intriguing results.

Chemotaxis↗

PubMed2005

Cyclic diguanylate regulates Vibrio cholerae virulence gene expression.

The cyclic dinucleotide second messenger cyclic diguanylate (c-diGMP) has been implicated in regulation of cell surface properties in several bacterial species, including Vibrio cholerae. Expression of genes required for V. cholerae biofilm formation is activated by an increased intracellular c-diGMP concentration. The response regulator VieA, which contains a domain responsible for degradation of c-diGMP, is required to maintain a low concentration of c-diGMP and repress biofilm formation. The VieSAB three-component signal transduction system was, however, originally identified as a regulator of ctxAB, the genes encoding cholera toxin (CT). Here we show that the c-diGMP phosphodiesterase activity of VieA is required to enhance CT production. This regulation occurred at the transcriptional level, and ectopically altering the c-diGMP concentration by expression of diguanylate cyclase or phosphodiesterase enzymes also affected ctxAB transcription. The c-diGMP phosphodiesterase activity of VieA was also required for maximal transcription toxT but did not influence the activity of ToxR or expression of TcpP. Finally, a single amino acid substitution in VieA that increases the intracellular c-diGMP concentration led to attenuation in the infant mouse model of cholera. Since virulence genes including toxT and ctxA are repressed by a high concentration of c-diGMP, while biofilm genes are activated, we suggest that c-diGMP signaling is important for the transition of V. cholerae from the environment to the host.

Bacterial Proteins↗

PubMed2005

Catabolite control protein A (CcpA) contributes to virulence and regulation of sugar metabolism in Streptococcus pneumoniae.

We characterized the role of catabolite control protein A (ccpA) in the physiology and virulence of Streptococcus pneumoniae. S. pneumoniae has a large percentage of its genome devoted to sugar uptake and metabolism, and therefore, regulation of these processes is likely to be crucial for fitness in the nasopharynx and may play a role during invasive disease. In many bacteria, carbon catabolite repression (CCR) is central to such regulation, influencing hierarchical sugar utilization and growth rates. CcpA is the major transcriptional regulator in CCR in several gram-positive bacteria. We show that CcpA functions in CCR of lactose-inducible beta-galactosidase activity in S. pneumoniae. CCR of maltose-inducible alpha-glucosidase, raffinose-inducible alpha-galactosidase, and cellobiose-inducible beta-glucosidase is unaffected in the ccpA strain, suggesting that other regulators, possibly redundant with CcpA, control these systems. The ccpA strain is severely attenuated for nasopharyngeal colonization and lung infection in the mouse, establishing its role in fitness on these mucosal surfaces. Comparison of the cell wall fraction of the ccpA and wild-type strains shows that CcpA regulates many proteins in this compartment that are involved in central and intermediary metabolism, a subset of which are required for survival and multiplication in vivo. Both in vitro and in vivo defects were complemented by providing ccpA in trans. Our results demonstrate that CcpA, though not a global regulator of CCR in S. pneumoniae, is required for colonization of the nasopharynx and survival and multiplication in the lung.

Animals↗

PubMed2004

Both chemotaxis and net motility greatly influence the infectivity of Vibrio cholerae.

The role of chemotaxis in the virulence of gastrointestinal pathogens is ill defined. Counterintuitively, nonchemotactic mutants of the polarly flagellated pathogen Vibrio cholerae greatly out-compete the wild-type strain during infection of the small intestine. We show that the out-competition phenotype is dependent on the direction of flagellar rotation and independent of Toxin Co-regulated Pilus function. Specifically, the out-competition associated with the loss of chemotaxis required the presence of counterclockwise-biased flagellar rotation and smooth straight runs by the bacteria. In contrast, a nonchemotactic strain with clockwise-biased flagellar rotation was confined to small-scale net movement and was attenuated for infection. The significance of the out-competition phenotype was examined and was shown to correlate with a true increase in infectivity. Counterclockwise-biased mutants are aberrantly distributed throughout the infant mouse small intestine and we find that the expression of virulence factors occurs normally in all segments. Thus, alteration of the chemotactic properties of V. cholerae allows it to exploit additional niches in the host intestine.

Bacterial Proteins↗

PubMed2004

Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation.

While studying virulence gene regulation in Vibrio cholerae during infection of the host small intestine, we identified VieA as a two-component response regulator that contributes to activating expression of cholera toxin. Here we report that VieA represses transcription of Vibrio exopolysaccharide synthesis (vps) genes involved in biofilm formation by a mechanism independent of its phosphorelay and DNA-binding activities. VieA controls the intracellular concentration of the cyclic nucleotide second messenger cyclic diguanylate (c-di-GMP) using an EAL domain that functions as a c-di-GMP phosphodiesterase. Two-dimensional thin layer chromatography of nucleotide extracts confirmed that VieA reduces the concentration of c-di-GMP, opposing the action of c-di-GMP synthetase proteins. Expression of unrelated V. cholerae c-di-GMP synthetase or phosphodiesterae proteins also modulated c-di-GMP concentration and vps gene expression. We propose that c-di-GMP synthetase and phosphodiesterase domain-containing proteins contribute to regulating biofilm formation by controlling c-di-GMP concentration.

Biofilms↗

PubMed2004

Spatiotemporal analysis of acid adaptation-mediated Vibrio cholerae hyperinfectivity.

Acid adaptation has previously been shown to increase the infectivity of Vibrio cholerae in the infant mouse model. To better understand this phenomenon, we monitored the spatial distribution and temporal changes in the ratios of acid-adapted cells to unadapted V. cholerae cells in the small intestine, as well as the timing of virulence factor expression. We found that the competitive advantage afforded by acid adaptation does not become manifest until greater than 3 h postinfection; thus, acid adaptation does not increase V. cholerae passage through the gastric acid barrier. Additionally, acid-adapted and unadapted V. cholerae cells colonize the same sections of the small intestine and show similar kinetics of transcriptional induction of the virulence genes tcpA and ctxA. These studies suggest that the increased infectivity of acid-adapted V. cholerae is due to a more rapid onset of multiplication and/or to an increased multiplication rate within the infant mouse intestine.

Adaptation, Physiological↗

PubMed2004

The ompU Paralogue vca1008 is required for virulence of Vibrio cholerae.

We made single and combined mutations in ompU, ompT, and the two putative porin genes vca1008 and vc0972. The fitness of the strains was tested in vitro and in the infant mouse model of intestinal infection. We also studied the transcriptional induction of vca1008 in vitro and during mouse infection. We show that vca1008 is induced during infection and is necessary and sufficient (in the absence of ompU, ompT, and vc0972) for infection.

Adhesins, Bacterial↗

PubMed2003

From nose to lung: the regulation behind Streptococcus pneumoniae virulence factors.

Streptococcus pneumoniae probably possesses a redundant set of factors required for colonization of the nasopharynx and invasive disease, because of its strict relationship with its human host and relatively small genome size (approximately 2.1 Mb). Nevertheless, transcriptional regulation of genes encoding factors required for in vivo growth is predicted to be important on two fronts: in the transition from carriage to invasive disease and within different microniches of the nasopharynx. The importance of both serotype-specific and host tissue-specific virulence factors during infection and disease has been highlighted by the recent identification of novel virulence factors in this organism coupled with the release of complete genome sequences from two strains. These studies add to the foundation of knowledge of classical S. pneumoniae virulence factors such as polysaccharide capsule and pneumolysin, which have well-documented roles in pathogenesis.

Bacterial Proteins↗

PubMed2003

Transcriptional regulation in the Streptococcus pneumoniae rlrA pathogenicity islet by RlrA.

The proper temporal expression of virulence genes during infection is crucial to the infectious life cycle of microbial pathogens, particularly in pathogens that encounter a multitude of environments in eukaryotic hosts. Streptococcus pneumoniae normally colonizes the nasopharynges of healthy adults but can cause a range of diseases at a variety of host sites. Transcriptional regulators that are essential for full virulence of S. pneumoniae in different animal models have been identified. One such regulator, rlrA, is required for colonization of the nasopharynx and lung infection but is dispensable for systemic infection. Previous work has shown that rlrA lies in a 12-kb pathogenicity islet, divergently opposed to three putative sortase-anchored surface proteins and three sortase enzymes. In addition to rlrA, one of the putative surface proteins and one of the sortases have also been shown to be essential for lung infection. In this work, we demonstrate that RlrA is a positive regulator of all seven genes in the rlrA pathogenicity islet, with transcriptional activation occurring at four different promoters in the islet with AT-rich sequences. These promoters direct the expression of rlrA itself, the three sortases, rrgA, and rrgBC. These data are consistent with the model whereby the rlrA pathogenicity islet acts in an autonomous manner to alter the bacterial surface components that interact with the pulmonary and nasopharyngeal environments.

Bacterial Proteins↗

PubMed2003

MgrA, an orthologue of Mga, Acts as a transcriptional repressor of the genes within the rlrA pathogenicity islet in Streptococcus pneumoniae.

Streptococcus pneumoniae normally resides in the human nasopharynx in a nondisease state. In response to unknown triggers this organism can descend to the lower respiratory tract and/or invade the bloodstream. Regulation and activation of virulence genes play essential roles in this process of disease development. Characterization of S. pneumoniae regulatory networks has been a recent area of interest, but despite inroads little is known about regulation of virulence genes in this pathogen. A putative transcriptional regulator in S. pneumoniae, mgrA, which exhibits homology to the virulence gene activator mga of group A streptococcus, was previously identified as a regulator that is required for development of pneumonia in a murine model. In this study we confirmed that mgrA plays a role in both nasopharyngeal carriage and pneumonia. Transcriptional profiling by microarray technology was used to show that mgrA acts as a repressor of the previously characterized rlrA pathogenicity islet. This is manifested phenotypically by a decrease in adherence to epithelial cells in tissue culture since the rlrA pathogenicity islet contains genes mediating adherence.

Animals↗

PubMed2002

Host-induced epidemic spread of the cholera bacterium.

The factors that enhance the transmission of pathogens during epidemic spread are ill defined. Water-borne spread of the diarrhoeal disease cholera occurs rapidly in nature, whereas infection of human volunteers with bacteria grown in vitro is difficult in the absence of stomach acid buffering. It is unclear, however, whether stomach acidity is a principal factor contributing to epidemic spread. Here we report that characterization of Vibrio cholerae from human stools supports a model whereby human colonization creates a hyperinfectious bacterial state that is maintained after dissemination and that may contribute to epidemic spread of cholera. Transcriptional profiling of V. cholerae from stool samples revealed a unique physiological and behavioural state characterized by high expression levels of genes required for nutrient acquisition and motility, and low expression levels of genes required for bacterial chemotaxis.

Animals↗