Type VI secretion requires a dynamic contractile phage tail-like structure.

Type VI secretion systems are bacterial virulence-associated nanomachines composed of proteins that are evolutionarily related to components of bacteriophage tails. Here we show that protein secretion by the type VI secretion system of Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally homologous to contractile phage tail sheath. Time-lapse fluorescence light microscopy reveals that sheaths of the type VI secretion system cycle between assembly, quick contraction, disassembly and re-assembly. Whole-cell electron cryotomography further shows that the sheaths appear as long tubular structures in either extended or contracted conformations that are connected to the inner membrane by a distinct basal structure. These data support a model in which the contraction of the type VI secretion system sheath provides the energy needed to translocate proteins out of effector cells and into adjacent target cells.

Lipoprotein e(P4) is essential for hemin uptake by Haemophilus influenzae.

Heme uptake is a common means of iron and porphyrin acquisition by many pathogenic bacteria. The genus Haemophilus includes several important pathogenic bacterial species that characteristically require hemin-, protoporphyrin-, or heme-substituted proteins as essential growth factors under aerobic conditions. However, the mechanism of heme transport is not understood for Haemophilus. We have cloned a DNA fragment from H. influenzae that allows an Escherichia coli hemA mutant to employ exogenous hemin or protoporphyrin IX as sole sources of porphyrin. DNA sequencing of the cloned DNA fragment suggested that a previously characterized gene (hel) encoding an antigenic, outer membrane lipoprotein e(P4) was responsible for the complementation activity. Construction of hel insertion mutations in strain H. influenzae Rd demonstrated that hel is essential for growth under aerobic conditions but not under anaerobic conditions. The aerobic growth defect of hel mutants could be reversed by providing exogenous hemin in the presence of outer membrane. The analysis of hybrids between e(P4) and beta-lactamase demonstrated that a domain of e(P4) near its NH2' terminus was required for its function in hemin use. Within this domain is a short amino acid sequence that displays similarity to H. influenzae hemin binding protein HbpA, hemin-binding motifs present in eukaryotic transcription activator heme-activated protein, and the heme containing proteins hemoglobin (alpha-chain) and cytochrome C3, suggesting that this region may be involved in hemin binding and/or transport.

Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans.

Isogenic mutant strains of V. cholerae O1 lacking elements of a genetic regulon controlled by toxR and implicated in virulence were tested in volunteers. A deletion mutation in ctxA, the gene encoding the A subunit of cholera toxin, markedly attenuated disease symptoms without affecting intestinal colonization. Deletion of toxR, the gene encoding the cholera toxin-positive regulatory protein resulted in a diminution in colonizing capacity. A deletion mutation in tcpA, encoding the major subunit of the toxin coregulated pilus (regulated by toxR), abolished the colonizing capacity of this strain. These results show for the first time the role of a specific pilus structure in colonization of the human intestine by V. cholerae O1 and exemplify the significance of a genetic regulon in pathogenesis.

Lysogenic conversion by a filamentous phage encoding cholera toxin.

Vibrio cholerae, the causative agent of cholera, requires two coordinately regulated factors for full virulence: cholera toxin (CT), a potent enterotoxin, and toxin-coregulated pili (TCP), surface organelles required for intestinal colonization. The structural genes for CT are shown here to be encoded by a filamentous bacteriophage (designated CTXphi), which is related to coliphage M13. The CTXphi genome chromosomally integrated or replicated as a plasmid. CTXphi used TCP as its receptor and infected V. cholerae cells within the gastrointestinal tracts of mice more efficiently than under laboratory conditions. Thus, the emergence of toxigenic V. cholerae involves horizontal gene transfer that may depend on in vivo gene expression.

Staphylococcal enterotoxin A is encoded by phage.

The gene for staphylococcal enterotoxin A (entA), in two wild-type strains, is carried by related temperate bacteriophages. Hybridization analysis of DNA from entA-converting phage PS42-D and its bacterial host suggests that this phage integrates into the bacterial chromosome by circularization and reciprocal crossover (the Campbell model) and that the entA gene is located near the phage attachment site. DNA from three of eight staphylococcal strains that did not produce enterotoxin A and seven wild-type enterotoxin A-producing (EntA+) strains had extensive homology to the entA-converting phage PS42-D DNA, although there was a high degree of restriction-fragment length polymorphisms. At least one EntA+ strain did not produce detectable viable phage after induction. These data indicate that a polymorphic family of Staphylococcus aureus phages (some of which may be defective) can carry the entA gene.

Coordinate regulation and sensory transduction in the control of bacterial virulence.

Genes and operons that encode bacterial virulence factors are often subject to coordinate regulation. These regulatory systems are capable of responding to various environmental signals that may be encountered during the infectious cycle. For some pathogens, proteins that mediate sensory transduction and virulence control are similar to components of other bacterial information processing systems. Understanding the molecular mechanisms governing global regulation of pathogenicity is essential for understanding bacterial infectious diseases.

Selection of bacterial virulence genes that are specifically induced in host tissues.

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.

Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae.

Cholera caused by toxigenic Vibrio cholerae is a major public health problem confronting developing countries, where outbreaks occur in a regular seasonal pattern and are particularly associated with poverty and poor sanitation. The disease is characterized by a devastating watery diarrhea which leads to rapid dehydration, and death occurs in 50 to 70% of untreated patients. Cholera is a waterborne disease, and the importance of water ecology is suggested by the close association of V. cholerae with surface water and the population interacting with the water. Cholera toxin (CT), which is responsible for the profuse diarrhea, is encoded by a lysogenic bacteriophage designated CTXPhi. Although the mechanism by which CT causes diarrhea is known, it is not clear why V. cholerae should infect and elaborate the lethal toxin in the host. Molecular epidemiological surveillance has revealed clonal diversity among toxigenic V. cholerae strains and a continual emergence of new epidemic clones. In view of lysogenic conversion by CTXPhi as a possible mechanism of origination of new toxigenic clones of V. cholerae, it appears that the continual emergence of new toxigenic strains and their selective enrichment during cholera outbreaks constitute an essential component of the natural ecosystem for the evolution of epidemic V. cholerae strains and genetic elements that mediate the transfer of virulence genes. The ecosystem comprising V. cholerae, CTXPhi, the aquatic environment, and the mammalian host offers an understanding of the complex relationship between pathogenesis and the natural selection of a pathogen.

TnAraOut, a transposon-based approach to identify and characterize essential bacterial genes.

Identification of genes that encode essential products provides a promising approach to validation of new antibacterial drug targets. We have developed a mariner-based transposon, TnAraOut, that allows efficient identification and characterization of essential genes by transcriptionally fusing them to an outward-facing, arabinose-inducible promoter, PBAD, located at one end of the transposon. In the absence of arabinose, such TnAraOut fusion strains display pronounced growth defects. Of a total of 16 arabinose-dependent TnAraOut mutants characterized in Vibrio cholerae, four were found to carry insertions upstream of known essential genes (gyrB, proRS, ileRS, and aspRS) whereas the other strains carried insertions upstream of known and hypothetical genes not previously shown to encode essential gene products. One of the essential genes identified by this analysis appears to be unique to V. cholerae and thus may represent an example of a species-specific drug target.

Transposon-based approaches to identify essential bacterial genes.

Transposons are a powerful tool for identifying genes essential for bacterial viability. The availability of many bacterial genome sequences and the large number of genes of unknown function therein have inspired the generation of a variety of different approaches. These methods are described and their advantages and disadvantages are discussed.

Detection of NAD:arginine ADPribosyltransferases in animal tissues using 125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor.

125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. and Romig, W.R. (1979) J. Biol. Chem. 254, 5849-5854) was utilized to identify NAD:arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and 125I-labeled guanyltyramine as ADPribose acceptor, formation of ADPribosyl 125I-guanyltyramine was linear with time and enzyme concentration. The product was indistinguishable on both thin-layer and high-performance liquid chromatography from that formed by choleragen. Using 125I-guanyltyramine, ADPribosyltransferase activity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractionated, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase product by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free 125I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl 125I-guanyltyramine and other metabolites of 125I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl 125I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, 125I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.

Live bacterial vaccines: environmental aspects.

Recombinant DNA technology has greatly accelerated the development of live attenuated bacterial vaccines for cholera, typhoid, and shigellosis. Significant attenuation has been achieved by deleting genes for various virulence determinants, biosynthetic genes, and regulatory genes. As these vaccine candidates move from closed-ward clinical studies to outpatient and field trials, a variety of concerns needs to be addressed about the safety of these vaccines, not only for the vaccinee, but also for the community and the environment. In the case of Vibrio cholerae, specific deletions (delta attRS1 and delta recA) have been introduced into some live vaccine candidates, rendering them incapable of performing homologous and site-specific recombination events that could lead to reacquisition of active cholera toxin genes. Mutations in recA might also limit the persistence of the live vaccine candidate in the environment.

Quartz crystal microbalance detection of Vibrio cholerae O139 serotype.

A piezoelectric (PZ) quartz crystal microbalance (QCM) biosensor for the rapid detection of Vibrio cholerae serotype O139 has been developed. The antibody to this serotype was immobilized on the gold transducer surface of a 10 MHz AT cut PZ crystal. Solutions containing known antigen concentrations were then incubated for 1 h on the antibody-bound transducer. The biosensor was able to detect 10(5) cells per ml of O139 versus a background of O1 (Ogawa) serotype.

In vivo expression technology for selection of bacterial genes specifically induced in host tissues.

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.

Measurement of transcriptional activity in pathogenic bacteria recovered directly from infected host tissue.

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.

Helicobacter pylori mutagenesis by mariner in vitro transposition.

We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation. Transformants were selected by plating on kanamycin. Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ-marked transposons. Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H. pylori in mixed competition experiments.

Type 6 secretion dynamics within and between bacterial cells.

The bacterial type 6 secretion system (T6SS) functions as a virulence factor capable of attacking both eukaryotic and prokaryotic target cells by a process that involves protein transport through a contractile bacteriophage tail-like structure. The T6SS apparatus is composed, in part, of an exterior sheath wrapped around an interior tube. Here, we report that in living cells the cytoplasmic adenosine triphosphatase called ClpV specifically recognizes the contracted T6SS sheath structure, causing its disassembly within seconds. ClpV imaging allowed spatial and temporal documentation of cell-cell interactions (termed T6SS dueling) that likely mark the location of repeated T6SS-mediated protein translocation events between bacterial cells.