Structural and functional relationships among the RTX toxin determinants of Gram-negative bacteria

Abstract The RTX (repeats in toxin) cytolytic toxins r represent a family of important virulence factors that have disseminated widely among Gram-negative bacteria. They are characterised by a series of glycine-rich repeat units at the C-terminal end of each protein. They also have other features in common. Secretion from the cell occurs without a periplasmic intermediate by a novel mechanism which involves recognition of a signal sequence at the C-terminus of the toxin by membrane-associated proteins that export the toxin directly to the outside of the cell. The structural gene for each protein encodes an inactive toxin which is modified post-translationally to an active cytotoxic form by another gene product before secretion. The genes for toxin synthesis, activation and secretion are for the most part grouped together on the chromosome and form an operon. The toxins all create pores in the cell membrane of target cells leading to eventual cell lysis and they appear to require Ca²⁺ for cytotoxic activity. Although the toxins have a similar mode of action, they vary in target cell specificity. Some are cytotoxic for a wide variety of eukaryotic cell types while others exhibit precise target cell specificity and are only active against leukocytes from certain host species. The characteristic glycine-rich repeat units have been identified in other exoproteins besides those with cytotoxic activity and it is likely that the novel secretory mechanism has been harnessed by a variety of pathogens to release important virulence-associated factors from the cell or to locate them on the cell surface.     

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes.

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.     

Structural and functional characterization of an essential RTX subdomain of Bordetella pertussis adenylate cyclase toxin

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells by a unique mechanism that consists in a calcium-dependent, direct translocation of the CyaA catalytic domain across the plasma membrane of the target cells. CyaA possesses a series of a glycine- and aspartate-rich nonapeptide repeats (residues 1006-1613) of the prototype GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX (repeat in toxin) family of bacterial cytolysins. These repeats are arranged in a tandem fashion and may fold into a characteristic parallel beta-helix or beta-roll motif that constitutes a novel type of calcium binding structure, as revealed by the three-dimensional structure of the Pseudomonas aeruginosa alkaline protease. Here we have characterized the structure-function relationships of various fragments from the CyaA RTX subdomain. Our results indicate that the RTX functional unit includes both the tandem repeated nonapeptide motifs and the adjacent polypeptide segments, which are essential for the folding and calcium responsiveness of the RTX module. Upon calcium binding to the RTX repeats, a conformational rearrangement of the adjacent non-RTX sequences may act as a critical molecular switch to trigger the CyaA entry into target cells.     

Plasmids and genetic determinants of antibiotic resistance of gram-negative bacteria

Distribution of plasmids and genetic determinants of antibiotic resistance was studied in 129 strains of Pseudomonas, Klebsiella, Serratia and Enterobacter isolated from oncological patients. It was shown that 56 isolates contained the plasmids, 9 conjugative plasmids being plasmids with broad bacterial host spectrum. A significant part of the strains contained genes controlling production of APH (3"), type II APH (3'), type I and II DHPS and type type II DHFR. Genetic determinants of tetracycline resistance of classes D and E were detected for the first time in the strains of Klebsiella, Serratia and Pseudomonas aeruginosa.     

Structural and functional relationships between aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases can be divided in two groups of equal size on the basis of differences in the structure of their active sites. The core of class I synthetases is the classical nucleotide-binding domain with its characteristic Rossmann fold. In contrast, the active site of class II synthetases is built around an antiparallel beta-sheet, to which the substrates bind. This classification, which is based on structural data (amino acid sequences and tertiary structures), can be rationalized in functional terms.     

Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria.

Comparative 16S rRNA sequencing was used to evaluate phylogenetic relationships among selected strains of ammonia- and nitrite-oxidizing bacteria. All characterized strains were shown to be affiliated with the proteobacteria. The study extended recent 16S rRNA-based studies of phylogenetic diversity among nitrifiers by the comparison of eight strains of the genus Nitrobacter and representatives of the genera Nitrospira and Nitrospina. The later genera were shown to be affiliated with the delta subdivision of the proteobacteria but did not share a specific relationship to each other or to other members of the delta subdivision. All characterized Nitrobacter strains constituted a closely related assemblage within the alpha subdivision of the proteobacteria. As previously observed, all ammonia-oxidizing genera except Nitrosococcus oceanus constitute a monophyletic assemblage within the beta subdivision of the proteobacteria. Errors in the 16S rRNA sequences for two strains previously deposited in the databases by other investigators (Nitrosolobus multiformis C-71 and Nitrospira briensis C-128) were corrected. Consideration of physiology and phylogenetic distribution suggested that nitrite-oxidizing bacteria of the alpha and gamma subdivisions are derived from immediate photosynthetic ancestry. Each nitrifier retains the general structural features of the specific ancestor's photosynthetic membrane complex. Thus, the nitrifiers, as a group, apparently are not derived from an ancestral nitrifying phenotype.     

Structural and functional relationships between h- and l-caldesmons.

Two different Mr forms of caldesmon as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr values in the range of 120,000-150,000, h-caldesmon and 70,000-80,000, l-caldesmon) have been already identified. h-Caldesmon is predominantly expressed in smooth muscle cells, whereas l-caldesmon widely distributes in non-muscle cells. Most recently, the molecular cloning of h-caldesmon has been reported (Hayashi, K., Kanda, K., Kimizuka, F., Kato, I., and Sobue, K. (1989) Biochem. Biophys. Res. Commun. 164, 503-511; Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G., and Lin, W-G. (1989) J. Biol. Chem. 264, 13873-13879). The calculated Mr of this protein from its primary structure is 88,743. Here, the nucleotide and deduced amino acid sequences of l-caldesmon have been determined by cloning and sequencing the cDNA from chick brain and compared with those of h-caldesmon. The l-caldesmon cDNA encodes a sequence of 517 amino acids with the calculated Mr of 58,844. Two isoforms of caldesmon conserve the completely identical sequences in the NH2- and COOH-terminal domains except for the insertion of Ala-508 in l-caldesmon. Interestingly, the central repeating sequence of h-caldesmon (residues 201-447) is deleted in the l-caldesmon molecule. The short NH2-terminals of two caldesmons individually show the unique sequences. The results of Northern and Southern blot analyses suggest that two mRNAs (4.8 and 4.1 kilo-bases) coding for caldesmon isoforms may be generated from a single gene by alternative splicing. Using a series of truncated caldesmons expressed in Escherichia coli, the common calmodulin-, tropomyosin-, and actin-binding sites and the minimum regulatory domains, which are involved in the Ca2(+)-dependent regulation of actin-myosin interaction, have been identified within the limited consensus sequences (residues 381-433 for l-caldesmon and residues 636-688 for h-caldesmon).     

Structural and functional relationships of FAN1

FANCD2/FANCI-associated nuclease (FAN1) is a 5′ flap structure-specific endonuclease and 5′ to 3′ exonuclease. This nuclease can resolve interstrand cross-links (ICLs) independently of the Fanconi anemia (FA) pathway and controls the progression of stalled replication forks in an FA-dependent manner, thereby maintaining chromosomal stability. Several FAN1 mutations are observed in various cancers and degenerative diseases. Recently, several crystal structures of the FAN1-DNA complexes have been reported, and to date, these represent the only structures for a DNA bound ICL-repair nuclease. Puzzlingly, human FAN1 forms two different quaternary structures with different DNA binding modes, and based on these structures, two ICL-repair mechanisms have been proposed. In one mechanism, monomeric FAN1 recognizes the 5′ flap terminal phosphate via a basic pocket and successively cleaves at every third nucleotide of the DNA substrates. In the other mechanism, dimeric FAN1 scans, latches, and unwinds the postnick duplex of the substrate DNA to direct the scissile phosphodiester group to the active site. In this review, we discuss the structures, function, and proposed mechanisms of FAN1 nuclease, and provide the insights into its role in ICL repair and in processing of stalled replication forks.     

Evolutionary relationships among photosynthetic bacteria

To understand the evolution of photosynthetic bacteria it is necessary to understand how the main groups within Bacteria have evolved from a common ancestor, a critical issue that has not been resolved in the past. Recent analysis of shared conserved inserts or deletions (indels) in protein sequences has provided a powerful means to resolve this long-standing problem in microbiology. Based on a set of 25 indels in highly conserved and widely distributed proteins, all main groups within bacteria can now be defined in clear molecular terms and their relative branching orders logically deduced. For the 82 presently completed bacterial genomes, the presence or absence of these signatures in various proteins was found to be almost exactly as predicted by the indel model, with only 11 exceptions observed in 1842 observations. The branching order of different bacterial groups as deduced using this approach is as follows: low G+C Gram-positive (Heliobacterium chlorum) ↔ high G+C Gram-positive ↔ Clostridium–Fusobacterium–Thermotoga ↔ Deinococcus–Thermus ↔ green nonsulfur bacteria (Chloroflexus aurantiacus) ↔ Cyanobacteria ↔ Spirochetes ↔ Chlamydia–Cytophaga–Flavobacteria–green sulfur bacteria (Chlorobium tepidum) ↔ Aquifex ↔ Proteobacteria (δ and ∈) ↔ Proteobacteria (α) ↔ Proteobacteria (β) and ↔ Proteobacteria (γ). The Heliobacterium species, which contain an Fe–S type of reaction center (RC 1) and represent the sole photosynthetic phylum from the Gram-positive or monoderm bacteria (i.e., bounded by only a single membrane), is indicated to be the most ancestral of the photosynthetic lineages. Among the Gram-negative or diderm bacteria (containing both inner and outer cell membranes) the green nonsulfur bacteria, which contain a pheophytin-quinone type of reaction center (RC 2), are indicated to have evolved first. The later emerging photosynthetic groups which contain either one or both of these reaction centers could have acquired such genes from the earlier branching lineages by either direct descent or by means of lateral gene transfer. This revised version was published online in August 2006 with corrections to the Cover Date.     

Vibrio vulnificus RTX toxin kills host cells only after contact of the bacteria with host cells

Vibrio vulnificus causes acute cell death and a fatal septicaemia. In this study, we show that contact with host cells is a prerequisite to the acute cytotoxicity. We screened transposon mutants defective in the contact-dependent cytotoxicity . Two mutants had insertions within two open reading frames in a putative RTX toxin operon, the rtxA1 or rtxD encoding an RTX toxin (4701 amino acids) or an ABC type transporter (467 amino acids). An rtxA1 mutation resulted in a cytotoxicity defect, which was fully restored by in trans complementation. The expression of RtxA1 toxin increased after host cell contact in a time-dependent manner. The RtxA1 toxin induced cytoskeletal rearrangements and plasma membrane blebs, which culminated in a necrotic cell death. RtxA1 colocalized with actin and caused actin aggregation coinciding with a significant decrease in the F/G actin ratio. The RtxA1 toxin caused haemolysis through pore formation (radius 1.63 nm). The rtxA1 deletion mutant was defective in invading the blood stream from ligated ileal loops of CD1 mice. The rtxA1 null mutation resulted in over 100-fold increase in both intragastric and intraperitoneal LD₅₀s against mice. Overall, these results show that the RtxA1 toxin is a multifunctional cytotoxin and plays an essential role in the pathogenesis of V. vulnificus infections.     

Structural determinants of Clostridium difficile toxin A glucosyltransferase activity

The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB, homologous glucosyltransferases capable of inactivating small GTPases within the host cell. We present crystal structures of the TcdA glucosyltransferase domain in the presence and absence of the co-substrate UDP-glucose. Although the enzymatic core is similar to that of TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is comparable with TcdB in its modification of Rho family substrates and that, unlike TcdB, TcdA is also capable of modifying Rap family GTPases both in vitro and in cells. The glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but can be restored with autoproteolytic activation and glucosyltransferase domain release. These studies highlight the importance of cellular activation in determining the array of substrates available to the toxins once delivered into the cell.     

Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases.

The Bacillus subtilis phage luminal diameter 29 DNA polymerase, involved in protein-primed viral DNA replication, was inhibited by phosphonoacetic acid (PAA), a known inhibitor of alpha-like DNA polymerases, by decreasing the rate of elongation. Three highly conserved regions of amino acid homology, found in several viral alpha-like DNA polymerases and in the luminal diameter 29 DNA polymerase, one of them proposed to be the PAA binding site, were also found in the T4 DNA polymerase. This prokaryotic enzyme was highly sensitive to the drugs aphidicolin and the nucleotide analogues butylanilino dATP (BuAdATP) and butylphenyl dGTP (BuPdGTP), known to be specific inhibitors of eukaryotic alpha-like DNA polymerases. Two potential DNA polymerases from the linear plasmid pGKL1 from yeast and the S1 mitochondrial DNA from maize have been identified, based on the fact that they contain the three conserved regions of amino acid homology. Comparison of DNA polymerases from prokaryotic and eukaryotic origin showed extensive amino acid homology in addition to highly conserved domains. These findings reflect evolutionary relationships between hypothetically unrelated DNA polymerases.     

Analysis of functional regions of YPM, a superantigen derived from gram-negative bacteria.

The bacterial superantigens, staphylococcal enterotoxins and streptococcal pyrogenic exotoxins, are grouped in a family by the conservation of amino acid sequence and polypeptide folding patterns. In the case of Yersinia pseudotuberculosis-derived mitogen (YPM), however, there is no noticeable homology with this family, although many of the in vitro functional features conform to the criteria for a superantigen. To study the mode of action of YPM at the molecular level, we first generated a number of YPM point mutants with reduced T-cell proliferative activity using random mutagenesis and localized the amino acid positions involved in either major histocompatibility complex class II or T-cell receptor Vbeta-interaction. Plotting the elucidated positions on the hydrophilicity profile suggested that they reside mostly on the outer portion of the molecule. We also report that the two cysteines positioned almost at opposing ends of the YPM molecule are connected by an S-S bond the destruction of which causes fatal damage. Finally, we obtained evidence that YPM partially competes with staphylococcal enterotoxin E for human leukocyte antigen-DR binding. This raises the question of whether these different types of superantigens have acquired the same function by genetic convergence or originated from a common ancestral gene.     

Molecular and evolutionary relationships among enteric bacteria

Classification of bacterial species into genera has traditionally relied upon variation in phenotypic characteristics. However, these phenotypes often have a multifactorial genetic basis, making unambiguous taxonomic placement of new species difficult. By designing evolutionarily conserved oligonucleotide primers, it is possible to amplify homologous regions of genes in diverse taxa using the polymerase chain reaction and determine their nucleotide sequences. We have constructed a phylogeny of some enteric bacteria, including five species classified as members of the genus Escherichia, based on nucleotide sequence variation at the loci encoding glyceraldehyde-3-phosphate dehydrogenase and outer membrane protein 3A, and compared this genealogy with the relationships inferred by biotyping. The DNA sequences of these genes defined congruent and robust phylogenetic trees indicating that they are an accurate reflection of the evolutionary history of the bacterial species. The five species of Escherichia were found to be distantly related and, contrary to their placement in the same genus, do not form a monophyletic group. These data provide a framework which allows the relationships of additional species of enteric bacteria to be inferred. These procedures have general applicability for analysis of the classification, evolution, and epidemiology of bacterial taxa.     

Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.     

Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.