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PubMed2005

Specificity and polymorphism of the PlcR-PapR quorum-sensing system in the Bacillus cereus group.

The expression of extracellular virulence factors in various species of the Bacillus cereus group is controlled by the plcR and papR genes, which encode a transcriptional regulator and a cell-cell signaling peptide, respectively. A processed form of PapR, presumably a pentapeptide, specifically interacts with PlcR to facilitate its binding to its DNA targets. This activating mechanism is strain specific, with this specificity being determined by the first residue of the pentapeptide. We carried out in vivo complementation assays and compared the PlcR-PapR sequences of 29 strains from the B. cereus group. Our findings suggested that the fifth amino acid of the pentapeptide is also involved in the specificity of activation. We identified four classes of PlcR-PapR pairs, defining four distinct pherotypes in the B. cereus group. We used these findings to look at the evolution of the PlcR-PapR quorum-sensing system with regard to the phylogeny of the species forming the B. cereus group.

Amino Acid Sequence↗
PubMed2004

Evolutionary dynamics of insertion sequences in Helicobacter pylori.

Prokaryotic insertion sequence (IS) elements behave like parasites in terms of their ability to invade and proliferate in microbial gene pools and like symbionts when they coevolve with their bacterial hosts. Here we investigated the evolutionary history of IS605 and IS607 of Helicobacter pylori, a genetically diverse gastric pathogen. These elements contain unrelated transposase genes (orfA) and also a homolog of the Salmonella virulence gene gipA (orfB). A total of 488 East Asian, Indian, Peruvian, and Spanish isolates were screened, and 18 and 14% of them harbored IS605 and IS607, respectively. IS605 nucleotide sequence analysis (n = 42) revealed geographic subdivisions similar to those of H. pylori; the geographic subdivision was blurred, however, due in part to homologous recombination, as indicated by split decomposition and homoplasy tests (homoplasy ratio, 0.56). In contrast, the IS607 populations (n = 44) showed strong geographic subdivisions with less homologous recombination (homoplasy ratio, 0.2). Diversifying selection (ratio of nonsynonymous change to synonymous change, >>1) was evident in approximately 15% of the IS605 orfA codons analyzed but not in the IS607 orfA codons. Diversifying selection was also evident in approximately 2% of the IS605 orfB and approximately 10% of the IS607 orfB codons analyzed. We suggest that the evolution of these elements reflects selection for optimal transposition activity in the case of IS605 orfA and for interactions between the OrfB proteins and other cellular constituents that potentially contribute to bacterial fitness. Taken together, similarities in IS elements and H. pylori population genetic structures and evidence of adaptive evolution in IS elements suggest that there is coevolution between these elements and their bacterial hosts.

DNA Transposable Elements↗
PubMed2001

Complete genomic sequence of Pasteurella multocida, Pm70.

We present here the complete genome sequence of a common avian clone of Pasteurella multocida, Pm70. The genome of Pm70 is a single circular chromosome 2,257,487 base pairs in length and contains 2,014 predicted coding regions, 6 ribosomal RNA operons, and 57 tRNAs. Genome-scale evolutionary analyses based on pairwise comparisons of 1,197 orthologous sequences between P. multocida, Haemophilus influenzae, and Escherichia coli suggest that P. multocida and H. influenzae diverged approximately 270 million years ago and the gamma subdivision of the proteobacteria radiated about 680 million years ago. Two previously undescribed open reading frames, accounting for approximately 1% of the genome, encode large proteins with homology to the virulence-associated filamentous hemagglutinin of Bordetella pertussis. Consistent with the critical role of iron in the survival of many microbial pathogens, in silico and whole-genome microarray analyses identified more than 50 Pm70 genes with a potential role in iron acquisition and metabolism. Overall, the complete genomic sequence and preliminary functional analyses provide a foundation for future research into the mechanisms of pathogenesis and host specificity of this important multispecies pathogen.

Base Sequence↗
PubMed2002

Analysis of the genetic variability of genes encoding the RNA III-activating components Agr and TRAP in a population of Staphylococcus aureus strains isolated from cows with mastitis.

The expression of Staphylococcus aureus virulence proteins is under the control of RNA III, a central pleiotropic regulator transcribed from the agr locus. RNA III is activated by at least two two-component systems, one encoded by the agr locus (AgrC-AgrA) and another encoded outside of this locus (TRAP-RAP). In this work, we developed new typing methods based on genes encoding these two systems, which we used to characterize a nonclonal population of S. aureus bovine mastitis isolates. Twelve agr restriction types were identified in this population, but the majority of strains (56.3%) were grouped in the R III-A1 type. No strain isolated from humans, whose agr sequence is available from GenBank, was found to belong to this major type. Restriction maps constructed for all of those agr variants allowed the linking of all types in an evolution scheme and their grouping in one of the four agr interference groups. This analysis indicates that groups 2, 3, and 4 probably evolved from the more frequently encountered type, which belongs to group 1. agr group 1 was also found to be the most prevalent (69.0% of the strains) and the most polymorphic interference group. By developing an agr group-specific multiplex PCR, we confirmed the above classification of strains in the agr interference groups. Four allelic variants of trap were also identified, indicating that this two-component system is also polymorphic. The majority of strains was grouped in the trap 1 type (71.8%). Whereas no relationships between agr group and trap types were found, strains of similar agr restriction type were also of similar trap type (with the exception of strains belonging to the agr R IV-A5 and R VI-A8 types). Our analysis indicates that S. aureus isolated from cows has predominantly a clonal structure and that the highly prevalent agr R III-A1, trap 1 type (56.3% of the strains) probably possesses a genetic background which endows it with superior ability to infect the bovine mammary gland.

Animals↗
PubMed2004

Surveillance of methicillin-resistant Staphylococcus aureus in a pediatric hospital in Mexico City during a 7-year period (1997 to 2003): clonal evolution and impact of infection control.

Between 1997 and 2000 a single multidrug-susceptible methicillin-resistant Staphylococcus aureus clone, M (sequence type 30 [ST30]-staphylococcal cassette chromosome mec [SCCmec] type IV), was present in a pediatric hospital in Mexico City, Mexico. In 2001 the international multidrug-resistant New York-Japan clone (ST5-SCCmec type II) was introduced into the hospital, completely replacing clone M by 2002.

Child↗
PubMed1987

Immunological localization of the GTP-binding protein Go in different tissues of vertebrates and invertebrates.

Bovine brain contains two GTP-binding proteins, Gi and Go, which are substrates for ADP ribosylation by pertussis toxin. The Gi protein mediates hormone and GTP inhibition of adenylate cyclase, but the function and the precise tissue distribution of Go are unknown. To immunologically probe the localization of Go, we have purified the Go alpha and G beta, gamma subunits of Go and have raised antibodies against them. The polyclonal anti-Go alpha antibodies obtained were very selective for Go alpha compared to Gi alpha or Gs alpha. The positive Go alpha and G beta, gamma immunoreactivities were investigated in different tissues of vertebrates and invertebrates on immunoblots after gel electrophoresis of the crude membranes. The anti-G beta, gamma antibodies recognized a 35-36-kDa protein in brain of vertebrates such as mammals (rat), avians (pigeon), amphibians (frog), fish (trout), and reptiles (turtle) but not in the invertebrates such as molluscs (snail) and insects (locust). With the anti-Go alpha antibodies a high level of immunoreactivity was detected at molecular weights of 39,000-40,000 in the brain of invertebrates as well as in the central nervous system of vertebrates. Moreover, ADP ribosylation with pertussis toxin occurred in the nervous system of invertebrates. These results suggest that the GTP-binding proteins of invertebrates either are devoid of G beta, gamma subunit or, more probably, possess immunologically different G beta, gamma subunits when compared to those of vertebrates. In the vertebrates, Go alpha immunoreactivity was also present in the peripheral nervous system in areas such as the superior cervical ganglia and sciatic nerve. When examined with the anti-Go alpha antibodies, the neuro-and adenohypophysis exhibited a similar immunoreactivity which was about 6 times lower than in brain. Our antibodies also recognized a 40-kDa protein in human adipocytes but at a concentration 17 times lower than that recognized in brain. Taken together, these data show that the Go alpha subunit is well conserved through evolution and, furthermore, confirm that Go alpha is not strictly limited to the nervous system. This suggests that the protein Go ensures a function required for neuronal activity but also present in some other non-nervous tissues.

Adenosine Diphosphate Ribose↗
PubMed2002

Genomics of Helicobacter pylori.

During this review period, we have definitely entered into the genomic era. The Helicobacter pylori studies reported here illustrate the use of most of the technologies currently available to globally interrogate the genome of a pathogen. Global analysis of the gene content of H. pylori strains gives insight into the extent of its genetic diversity and its in vivo evolution. Our understanding of the particularities of H. pylori as a gastric pathogen colonizing a unique niche has been improved by studies aimed at: (i) the identification of H. pylori-specific genes; (ii) the establishment of correlations between the presence of one or a group of genes (or proteins) with clinical outcome; and (iii) the analysis of global regulatory circuits or responses to the extracellular signals. The response of host cells to H. pylori infection will be developed in the chapter 'H. pylori and gastric malignancies' by Sepulveda and Coehlo. Despite our knowledge of the H. pylori genome, the function of about one third of its total proteins is still unknown. Functional genomics are straightforward approaches for the identification of new gene functions or metabolic pathways as well as for the understanding of cellular processes and the detection of new virulence factors. In silico studies combined with experimental work will undoubtedly continue to develop. To date, the expansion of proteomics with refinements in mass spectrometry technology has illustrated that through immunoproteomics and comparative studies, relevant novel antigens can be identified. Genomics not only provides invaluable information on H. pylori but also opens new perspectives for diagnostic or therapeutic applications.

Genome, Bacterial↗
PubMed2003

[Genome-wide non-sequencing strategies for bacterial genome comparison: the necessity and an analysis of the variable bacterial world].

A tremendous success in bacterial genome sequencing has been achieved during the recent years; it resulted in making available, for analysis, multiple sequences of different bacterial genomes, including such pathogens as causative agents of syphilis, typhus, and tuberculosis as well as such organisms like archaebacterias living under extreme conditions. A comparative analysis of bacterial genomes leads to conclusions, which have a general biological value, and, in particular, to the conclusions about mechanisms and evolution rate as well as about the variability of genomes and interrelation between organisms and their habitat. On the other hand, the analysis reveals specific features of separate bacterial species responsible for their pathogenicity and ability to avoid the destruction of the host immune system as well as for adaptation to exist within a certain ecological niche. However, the variability of bacterial genomes is so high that methods, which enable to evaluate the variability without full genome sequencing, are needed to depict adequately the evolution and ecological characteristics of the prokaryotic world and to develop new effective therapeutics and diagnostic tools. The survey covers two approaches to such comparative analysis, i.e. DNA arrays and subtractive hybridization. The advantages and disadvantages of each approach are discussed and the necessity in a new approach combining the positive features of the two mentioned approaches is substantiated.

DNA, Bacterial↗
PubMed1999

Inhibition of Shigella flexneri-induced transepithelial migration of polymorphonuclear leucocytes by cadaverine.

Dysentery caused by Shigella species is characterized by infiltration of polymorphonuclear leucocytes (PMNs) into the colonic mucosa. Shigella spp. evolved into pathogens by the acquisition of virulence genes and by the deletion of 'antivirulence' genes detrimental to its pathogenic lifestyle. An example is cadA (encoding lysine decarboxylase), which is uniformly absent in Shigella spp., whereas it is present in nearly all isolates of the closely related non-pathogen Escherichia coli. Here, using monolayers of T84 cells to model the human intestinal epithelium, we determined that the introduction of cadA into S. flexneri and the expression of lysine decarboxylase attenuated the bacteria's ability to induce PMN influx across model intestinal epithelium. Such inhibition was caused by cadaverine generated from the decarboxylation of lysine. Cadaverine treatment of model intestinal epithelia specifically inhibited S. flexneri induction of PMN transepithelial migration, while having no effect on the ability of Salmonella or enteropathogenic E. coli (EPEC) to induce PMN migration. These observations not only provide insight into mechanisms of S. flexneri pathogen evolution and pathogenesis, but also suggest a potential for the use of cadaverine in the treatment of dysentery.

Actins↗
PubMed2004

Highly divergent RfaH orthologs from pathogenic proteobacteria can substitute for Escherichia coli RfaH both in vivo and in vitro.

The transcriptional enhancer protein RfaH positively regulates production of virulence factors in Escherichia coli and Salmonella enterica serovar Typhimurium via a cis element, ops. Genes coding for RfaH orthologs were identified in conceptually translated genomes of bacterial pathogens, including Vibrio and Yersinia spp. We cloned the rfaH genes from Vibrio cholerae, Yersinia enterocolitica, S. enterica serovar Typhimurium, and Klebsiella pneumoniae into E. coli expression vectors. Purified RfaH orthologs, including the most divergent one from V. cholerae, were readily recruited to the E. coli transcription elongation complex. Postrecruitment stimulation of transcript elongation appeared to vary with the degree of similarity to E. coli RfaH. V. cholerae RfaH was particularly defective in reducing downstream pausing and termination; this defect was substantially alleviated by an increase in its concentration. When overexpressed episomally, all of the rfaH genes complemented the disruption of the chromosomal copy of the E. coli gene. Thus, despite the apparently accelerated divergent evolution of the RfaH proteins, the mechanism of their action is conserved well enough to make them transcriptionally active in the E. coli system.

Amino Acid Sequence↗
PubMed1998

Short-sequence DNA repeats in prokaryotic genomes.

Short-sequence DNA repeat (SSR) loci can be identified in all eukaryotic and many prokaryotic genomes. These loci harbor short or long stretches of repeated nucleotide sequence motifs. DNA sequence motifs in a single locus can be identical and/or heterogeneous. SSRs are encountered in many different branches of the prokaryote kingdom. They are found in genes encoding products as diverse as microbial surface components recognizing adhesive matrix molecules and specific bacterial virulence factors such as lipopolysaccharide-modifying enzymes or adhesins. SSRs enable genetic and consequently phenotypic flexibility. SSRs function at various levels of gene expression regulation. Variations in the number of repeat units per locus or changes in the nature of the individual repeat sequences may result from recombination processes or polymerase inadequacy such as slipped-strand mispairing (SSM), either alone or in combination with DNA repair deficiencies. These rather complex phenomena can occur with relative ease, with SSM approaching a frequency of 10(-4) per bacterial cell division and allowing high-frequency genetic switching. Bacteria use this random strategy to adapt their genetic repertoire in response to selective environmental pressure. SSR-mediated variation has important implications for bacterial pathogenesis and evolutionary fitness. Molecular analysis of changes in SSRs allows epidemiological studies on the spread of pathogenic bacteria. The occurrence, evolution and function of SSRs, and the molecular methods used to analyze them are discussed in the context of responsiveness to environmental factors, bacterial pathogenicity, epidemiology, and the availability of full-genome sequences for increasing numbers of microorganisms, especially those that are medically relevant.

Adhesins, Bacterial↗
PubMed2004

Recombination in the genome of Chlamydia trachomatis involving the polymorphic membrane protein C gene relative to ompA and evidence for horizontal gene transfer.

Genome sequencing of Chlamydia trachomatis serovar D has identified polymorphic membrane proteins (Pmp) that are a newly recognized protein family unique to the Chlamydiaceae family. Cumulative data suggest that these diverse proteins are expressed on the cell surface and might be immunologically important. We performed phylogenetic analyses and statistical modeling with 18 reference serovars and 1 genovariant of C. trachomatis to examine the evolutionary characteristics and comparative genetics of PmpC and pmpC, the gene that encodes this protein. We also examined 12 recently isolated ocular and urogenital clinical samples, since reference serovars are laboratory adapted and may not represent strains that are presently responsible for human disease. Phylogenetic reconstructions revealed a clear distinction for disease groups, corresponding to levels of tissue specificity and virulence of the organism. Further, the most prevalent serovars, E, F, and Da, formed a distinct clade. According to the results of comparative genetic analyses, these three genital serovars contained two putative insertion sequence (IS)-like elements with 10- and 15-bp direct repeats, respectively, while all other genital serovars contained one IS-like element. Ocular trachoma serovars also contained both insertions. Previously, no IS-like elements have been identified for Chlamydiaceae. Surprisingly, 7 (58%) of 12 clinical isolates revealed pmpC sequences that were identical to the sequences of other serovars, providing clear evidence for a high rate of whole-gene recombination. Recombination and the differential presence of IS-like elements among distinct disease and prevalence groups may contribute to genome plasticity, which may lead to adaptive changes in tissue tropism and pathogenesis over the course of the organism's evolution.

Bacterial Outer Membrane Proteins↗
PubMed2001

Salmonella host cell invasion emerged by acquisition of a mosaic of separate genetic elements, including Salmonella pathogenicity island 1 (SPI1), SPI5, and sopE2.

Salmonella spp. possess a conserved type III secretion system encoded within the pathogenicity island 1 (SPI1; centisome 63), which mediates translocation of effector proteins into the host cell cytosol to trigger responses such as bacterial internalization. Several translocated effector proteins are encoded in other regions of the Salmonella chromosome. It remains unclear how this complex chromosomal arrangement of genes for the type III apparatus and the effector proteins emerged and how the different effector proteins cooperate to mediate virulence. By Southern blotting, PCR, and phylogenetic analyses of highly diverse Salmonella spp., we show here that effector protein genes located in the core of SPI1 are present in all Salmonella lineages. Surprisingly, the same holds true for several effector protein genes located in distant regions of the Salmonella chromosome, namely, sopB (SPI5, centisome 20), sopD (centisome 64), and sopE2 (centisomes 40 to 42). Our data demonstrate that sopB, sopD, and sopE2, along with SPI1, were already present in the last common ancestor of all contemporary Salmonella spp. Analysis of Salmonella mutants revealed that host cell invasion is mediated by SopB, SopE2, and, in the case of Salmonella enterica serovar Typhimurium SL1344, by SopE: a sopB sopE sopE2-deficient triple mutant was incapable of inducing membrane ruffling and was >100-fold attenuated in host cell invasion. We conclude that host cell invasion emerged early during evolution by acquisition of a mosaic of genetic elements (SPI1 itself, SPI5 [sopB], and sopE2) and that the last common ancestor of all contemporary Salmonella spp. was probably already invasive.

Animals↗
PubMed2004

The evolutionary history of quorum-sensing systems in bacteria.

Communication among bacterial cells through quorum-sensing (QS) systems is used to regulate ecologically and medically important traits, including virulence to hosts. QS is widespread in bacteria; it has been demonstrated experimentally in diverse phylogenetic groups, and homologs to the implicated genes have been discovered in a large proportion of sequenced bacterial genomes. The widespread distribution of the underlying gene families (LuxI/R and LuxS) raises the questions of how often QS genes have been transferred among bacterial lineages and the extent to which genes in the same QS system exchange partners or coevolve. Phylogenetic analyses of the relevant gene families show that the genes annotated as LuxI/R inducer and receptor elements comprise two families with virtually no homology between them and with one family restricted to the gamma-Proteobacteria and the other more widely distributed. Within bacterial phyla, trees for the LuxS and the two LuxI/R families show broad agreement with the ribosomal RNA tree, suggesting that these systems have been continually present during the evolution of groups such as the Proteobacteria and the Firmicutes. However, lateral transfer can be inferred for some genes (e.g., from Firmicutes to some distantly related lineages for LuxS). In general, the inducer/receptor elements in the LuxI/R systems have evolved together with little exchange of partners, although loss or replacement of partners has occurred in several lineages of gamma-Proteobacteria, the group for which sampling is most intensive in current databases. For instance, in Pseudomonas aeruginosa, a transferred QS system has been incorporated into the pathway of a native one. Gene phylogenies for the main LuxI/R family in Pseudomonas species imply a complex history of lateral transfer, ancestral duplication, and gene loss within the genus.

Bacteria↗
PubMed1994

Evaluation of the efficacy of human antimeningococcal immunoglobulin G in infant rats experimentally infected with Neisseria meningitidis group B.

Infants rats, a well known model for the experimental reproduction of bacterial meningitis, were used by us to test the protective potential of antibodies developed in humans who had been vaccinated with the Cuban antimeningitis vaccine (VA-MENGOCBC). Newborn rats were inoculated by the intraperitoneal and intranasal routes with suspensions of Neisseria meningitidis group B bacteria. Bacteremia kinetics were evaluated from blood and brain-spinal fluid cultures. Samples of the central nervous system were taken and smears of backbone fluids prepared for histopathologic evaluations. Characterization of bacteremia evolution, as well as the mean lethal dose of germs and histopathologic features, were determined. After standardization of the model, therapeutic schemes were applied using passive immunization pre- and post-infection with N. meningitidis. A significant level of protection was obtained in relation to control animals that received the same challenge doses.

Animals↗
PubMed2005

Human natural killer cells: a comprehensive review.

The senior author of this comprehensive review observed and reported in 1969 that his lymphocytes killed allogeneic tumor cells in vitro. Some of his research associates and technicians and other healthy individuals also yielded such killer lymphocytes. The team considered pre-immunization to cancer occurring in individuals after in-family or professional exposure to patients with cancer (in an era when the concept of viral etiology of cancer was receiving major support); or that lymphocytes can acquire through blastic transformation immune reactivity to allogeneic cells anew in vitro. The phenomenon was eventually referred to as 'lymphocytes practicing Burnet's immunosurveillance.' Project site visitors of the USA NCI first viewed these observations as a matter of 'in vitro artifacts' being in opposition to strong tumor- specific cytotoxicity of tumor-bearing patients' lymphocytes recognized in the vast majority of other assays. After NCI funds were released for intramural studies on the phenomenon of non-specific cytotoxicity by lymphocytes, recipients (other than the senior author) of these NCI funds later characterized (1973-1975) the 'indiscriminately' cytotoxic lymphocyte populations as those of 'natural killer (NK) cells.' In this article, the original observations made in 1969-1971 are reviewed based on genuine material preserved by the senior author and are explained in view of recent discoveries that were not available at the time of the original observations. NK cells display a fascinating history arising first in urochordates during the cambrian explosion. At that level, NK cells protected their hosts from incompatible cell colony fusions and against intracellular, especially viral, pathogens. Since then, viruses evolved evasive maneuvers to escape NK cell attack on the infected cells. NK cells persisted after the evolution of adaptive immunity in cartilaginous fish fitting seamlessly into the new system. In mammals, NK cells assumed the role of chief arbitrators between the fetal trophoblast and maternal immune reactions to the semi-allograft fetus. Tumors induce in NK cells the same (inactivating; mediating Th2-type immunity) reactions the fetal trophoblast engenders in utero, but NK cells may overcome the host's tolerance to its tumor and kill tumor cells, especially when converted into lymphokine-activated killer (LAK) cells by molecular mediators of Th1-type immunity. The authors prepare and utilize LAK cells and IL-2 for adoptive immunotherapy of patients with metastatic melanoma and kidney carcinoma. A patient with malignant ascites due to ovarian carcinoma entered remission on LAK cell therapy. Just as dendritic cells, the major antigen presentors, may undergo malignant transformation, NK cells are also subject to transformation into FasL-producer virulent lymphoma-leukemia cells. The senior author reported in 1970 a patient with 'lymphosarcoma cell leukemia' whose circulating lymphoma cells killed indiscriminately human sarcoma and carcinoma cells. The exemplary case history of another patient with NK cell lymphoma-leukemia treated by the authors is presented.

Adult↗
PubMed2001

Evolutionary dynamics of plant R-genes.

Plant R-genes involved in gene-for-gene interactions with pathogens are expected to undergo coevolutionary arms races in which plant specificity and pathogen virulence continually adapt in response to each other. Lending support to this idea, the solvent-exposed amino acid residues of leucine-rich repeats, a region of R-genes involved in recognizing pathogens, often evolve at unusually fast rates. But within-species polymorphism is also common in R-genes, implying that the adaptive substitution process is not simply one of successive selective sweeps. Here we document these features in available data and discuss them in light of the evolutionary dynamics they likely reflect.

Alleles↗
PubMed1987

Size variation in group A streptococcal M protein is generated by homologous recombination between intragenic repeats.

M protein, a major surface protein and virulence factor for the group A streptococcus, exhibits extraordinary size variation in strains of the same serotype (Fischetti et al. 1985). RNA sequence analysis of spontaneous M protein size variants shows that deletion mutations arise in a single strain by homologous recombination events between intragenic tandem repeats. Similar deletion and duplication events also occur in serial streptococcal isolates from a single patient and among related strains in a recent outbreak. We discuss how homologous recombination events can lead to the generation of antigenic variation.

Antigens, Bacterial↗