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PubMed2002

O(6)-alkylguanine-DNA alkyltransferase DNA repair in mycobacteria: pathogenic and non-pathogenic species differ.

SETTING: DNA repair genes assist the organism in maintaining DNA integrity in the face of environmental (mutagenic) stress. The genome sequences of M. tuberculosis and M. bovis demonstrate sequences suggestive of an O(6)-alkylguanine-DNA alkyltransferase DNA repair activity similar to that seen in almost all other bacterial and eukaryotic organisms. The near ubiquitousness of this gene implies an important function. OBJECTIVE: Our aim was to ascertain whether mycobacteria exert an alkyltransferase response to mutagen (streptozotocin) stimulation and whether alkyltransferase activity is essential for mycobacterial survival. DESIGN: Alkyltransferase activity in slow- and fast-growing mycobacterial species was determined in the presence and absence of sublethal concentrations of an alkylating agent streptozotocin. The intracellular survival and response to anti-tuberculosis drugs of an alkyltransferase knockout strain of M. bovis BCG was also determined. RESULTS: We demonstrate the presence of O(6)-alkylguanine alkyltransferase (cellular methyltransferase activity) in mycobacterial species and that there is an inducible and constitutive form in fast-growing mycobacteria (M. smegmatis), whereas only the constitutive form exists in the pathogenic or slow-growing species (M. bovis BCG) under the conditions tested. The overall activity of the constitutive form is high. We also show that intracellular growth of M. bovis BCG in macrophages is reduced when the alkyltransferase gene is absent. The presence of alkyltransferase activity appears to assist the organism in reducing the effects of isoniazid, since interruption of the gene confers sensitivity to the drug. CONCLUSIONS: We conclude that for the slow-growing mycobacteria, an inducible response is not essential as their ecological niche is stable and protected, but that the presence of the alkyltransferase activity confers a growth advantage in macrophages and offers some protection against antibiotics.

Base Sequence↗

PubMed1999

Pathogenic neisseriae: complexity of pathogen-host cell interplay.

Recent studies have provided insight into the function of important neisserial adhesins (pili and Opa) and their interaction with cellular receptors, including members of heparan sulfate proteoglycan, CD66, and integrin receptor families. These interactions not only allow colonization of the human mucosa but also stimulate cellular signaling cascades involving phosphatidylcholine-dependent phospholipase C, acidic sphingomyelinase and protein kinase C in epithelial cells, and Src-related kinases, Rac1, p21-activated kinase, and Jun N-terminal kinase in phagocytic cells. Activation of these pathways is essential for cellular entry and intracellular accommodation of the pathogens but also leads to early induction of cytokine release, thus priming the immune response. Detailed knowledge of the cellular signaling cascades that are activated by infection will aid us in applying both current and novel interfering drugs (in addition to classical antibiotic therapy) as therapy and prophylaxis for persistent or otherwise difficult-to-treat bacterial infections, including periodontal infections.

Antigens, Bacterial↗

PubMed2006

Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen.

The disease-specific (dsp) gene dspA/E of Erwinia amylovora encodes an essential pathogenicity effector of 198 kDa, which is critical to the development of the devastating plant disease fire blight. A yeast two-hybrid assay and in vitro protein pull-down assay demonstrated that DspA/E interacts physically and specifically with four similar putative leucine-rich repeat (LRR) receptor-like serine/threonine kinases (RLK) from apple, an important host of E. amylovora. The genes encoding these four DspA/E-interacting proteins of Malus xdomestica (DIPM1 to 4) are conserved in all genera of hosts of E. amylovora tested. They also are conserved in all cultivars of apple tested that range in susceptibility to fire blight from highly susceptible to highly resistant. The four DIPMs have been characterized, and they are expressed constitutively in host plants. In silico analysis indicated that the DIPMs have similar sequence structure and resemble LRR RLKs from other organisms. Evidence is presented for direct physical interaction between DspA/E and the apple proteins encoded by the four identified clones, which may act as susceptibility factors and be essential to disease development. Knowledge of DIPMs and the interaction with DspA/E thus may facilitate understanding of fire blight development and lead to new approaches to control of disease.

Amino Acid Sequence↗

PubMed1998

Cloning, expression, and role in pathogenicity of pg1 encoding the major extracellular endopolygalacturonase of the vascular wilt pathogen Fusarium oxysporum.

pg1 encoding the major in vitro extracellular endopolygalacturonase of the tomato vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici was cloned and sequenced. The deduced mature protein had a calculated molecular mass of 35.5 kDa and a pI of 6.2, and showed significant similarity with other fungal endoPGs. pg1 mRNA was induced in vitro by citrus pectin, tomato vascular tissue, 0.1% D-galacturonic acid, and polygalacturonic acid, and repressed by 1% D-galacturonic acid and 1% glucose. Reverse transcription-polymerase chain reaction revealed pg1 expression in roots and lower stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Three naturally occurring F. oxysporum f. sp. melonis isolates deficient in PG1 were transformed with the cloned gene. The PG1 enzyme secreted by the transformants had the same molecular mass, pI, and glycosylation pattern as those of the donor isolate. Polygalacturonase activity in cultures of transformants grown in vitro on citrus pectin and on melon plants, but not on glucose, increased 10- to 20-fold, compared with the PG1-deficient wild-type isolate, whereas mycelial dry weight increased two- to three-fold. Transformants exhibited the same degree of virulence toward susceptible muskmelon cultivars as the wild-type isolate and were avirulent on a resistant cultivar.

Amino Acid Sequence↗

PubMed1992

Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida.

The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 10(3) and 10(6) live cells. However, none of the strains tested were pathogenic for mice (LD50 > 10(8) cells)). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 micrograms protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 degrees C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.

Animals↗

PubMed2001

Identification of the acid phosphatase (acpA) gene homologues in pathogenic and non-pathogenic Burkholderia spp. facilitates TnphoA mutagenesis.

Burkholderia pseudomallei and Burkholderia mallei are pathogens responsible for disease in both humans and animals. Burkholderia thailandensis, while phylogenetically similar, is considered avirulent in comparison. These three species exhibit phosphatase activity when grown on media containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (XP). Tn5-OT182 mutagenesis has been utilized to isolate mutants of B. pseudomallei and B. thailandensis unable to hydrolyse XP. Sequence analysis of these mutants revealed an ORF of 1734 nucleotides demonstrating a high degree of homology to the acpA gene product of Francisella tularensis. PCR primers were designed based on the B. pseudomallei acpA gene sequence and were used to amplify an acpA homologue from B. mallei. The predicted amino acid sequence of B. pseudomallei AcpA differed from those of the predicted B. thailandensis AcpA and B. mallei AcpA by 15 and 3 amino acids, respectively. Allelic exchange was used to construct DeltaacpA mutants in each of these Burkholderia spp. These mutants were shown to be devoid of phosphatase activity and have subsequently allowed for the implementation of phoA fusion transposon mutagenesis systems. Two such systems have been successfully utilized in Burkholderia spp. for the identification of several genes encoding exported proteins.

Acid Phosphatase↗

PubMed2006

A siderophore biosynthesis gene cluster from the fish pathogen Photobacterium damselae subsp. piscicida is structurally and functionally related to the Yersinia high-pathogenicity island.

Photobacterium damselae subsp. piscicida, the causative agent of fish pasteurellosis, produces a siderophore which is distinct from that produced by P. damselae subsp. damselae. Using suppression subtractive hybridization, a subsp. piscicida-specific DNA region of 35 kb was identified in strain DI21, and 11 genes were defined: dahP, araC1, araC2, frpA, irp8, irp2, irp1, irp3, irp4, irp9 and irp5. The sequence of the predicted proteins encoded by these genes showed significant similarity with the proteins responsible for the synthesis and transport of the siderophore yersiniabactin, encoded within the Yersinia high-pathogenicity island (HPI). Southern hybridization demonstrated that this gene cluster is exclusive to some European subsp. piscicida isolates. Database searches revealed that a similar gene cluster is present in Photobacterium profundum SS9 and Vibrio cholerae RC385. An irp1 gene (encoding a putative non-ribosomal peptide synthetase) insertional mutant (CS31) was impaired for growth under iron-limiting conditions and unable to produce siderophores, and showed an approximately 100-fold decrease in degree of virulence for fish. The subsp. piscicida DI21 strain, but not CS31, promoted the growth of a Yersinia enterocolitica irp1 mutant. Furthermore, a yersiniabactin-producing Y. enterocolitica strain as well as purified yersiniabactin were able to cross-feed strains DI21 and CS31, suggesting that the subsp. piscicida siderophore might be functionally and structurally related to yersiniabactin. The differential occurrence among P. damselae strains, and the low sequence similarity to siderophore synthesis genes described in other members of the Vibrionaceae, suggest that this genetic system might have been acquired by horizontal transfer in P. damselae subsp. piscicida, and might have a common evolutionary origin with the Yersinia HPI.

Animals↗

PubMed1977

Host-Pathogen Interactions: XIII. Extracellular Invertases Secreted by Three Races of a Plant Pathogen Are Glycoproteins Which Possess Different Carbohydrate Structures.

The invertase present in the culture fluid of races 1, 2, and 3 of Phytophthora megasperma Drechs. var. sojae A. A. Hildebrand (Pms) were purified until they gave but a single band, whether stained for protein or carbohydrate, after isoelectric focusing in flat bed gels. The sugar compositions of multiple preparations of the purified invertases from each race of this fungal pathogen were determined by quantitative gas chromatography of their alditol acetates. The invertases are composed of about 25% carbohydrate. Mannose and glucosamine make up more than 97% of the carbohydrate portions of the invertases of all three Pms races analyzed, but the ratio of mannose to glucosamine is clearly not the same in each race. The glycosyl linkage compositions of the glucosamine-containing mannans of multiple preparations of the Pms invertases were determined by GC-MS analysis of the partially methylated alditol acetate derivatives. The results of these analyses demonstrate clear quantitative differences between the glycosyl components of the different Pms races. The existence of race-specific carbohydrate structures in the differentially virulent Pms races suggests that these carbohydrates may be involved in determining the specificity of hostpathogen interactions.

Journal Article↗

PubMed2003

A class-V myosin required for mating, hyphal growth, and pathogenicity in the dimorphic plant pathogen Ustilago maydis.

In the early stages of plant infection, yeast-like haploid sporidia of Ustilago maydis respond to pheromone secreted by compatible partners by forming conjugation tubes. These then fuse to generate a dikaryotic hypha that forms appressoria to penetrate the host plant. As a first step toward understanding the structural requirements for these transitions, we have identified myo5, which encodes a class-V myosin. Analysis of conditional and null mutants revealed that Myo5 plays nonessential roles in cytokinesis and morphogenesis in sporidia and is required for hyphal morphology. Consistent with a role in morphogenesis, a functional green fluorescent protein-Myo5 fusion protein localized to the bud tip and the hyphal apex as well as to the septa and the spore wall during later stages of infection. However, the loss of Myo5 did not affect the tip growth of hyphae and sporidia. By contrast, Myo5 was indispensable for conjugation tube formation. Furthermore, myo5 mutants were impaired in the perception of pheromones, which indicates a particular importance of Myo5 in the mating process. Consequently, few mutant hyphae were formed that penetrated the plant epidermis but did not continue invasive growth. These results indicate a crucial role of Myo5 in the morphogenesis, dimorphic switch, and pathogenicity of U. maydis.

Actins↗

PubMed2004

Pathogen reduction of buffy coat platelet concentrates using riboflavin and light: comparisons with pathogen-reduction technology-treated apheresis platelet products.

BACKGROUND AND OBJECTIVES: A pathogen-reduction technology (PRT) system using riboflavin and light has been developed for the treatment of platelet concentrates (PC) obtained by either buffy coat preparation (BCPC) or apheresis procedures (APPC). The aim of this study was to evaluate the effects of the treatment process on in vitro cell quality and on riboflavin conversion in PC. MATERIALS AND METHODS: BCPC were prepared with the Compomat G4 from whole blood which had been stored overnight after collection. APPC were obtained using the TRIMA apheresis procedure. Both PC products had been stored for 18-24 h prior to PRT treatment. BCPC and APPC were treated with PRT on day 2 and day 1 of shelf-life, respectively. The treated PCs were then maintained for an additional 5 days after the PRT treatment. A panel of cell quality assays and high-performance liquid chromatography (HPLC) analysis were performed. RESULTS: Cell counts and plasma lactate dehydrogenase (LDH) levels during storage indicated that PRT did not induce significant cell lysis. Acceleration of a decrease in glucose and an increase in lactate was observed for treated PCs, but no significant differences were observed between treated BCPC and APPC. The pH of treated samples remained above 7.0, although was lower than that of the control. Platelet morphology of BCPC and APPC was well preserved. P-selectin expression indicated significant platelet activation when compared with control PC (BCPC on day 6: 39% vs. 12%; APPC on day 5: 35% vs. 18%). Both P-selectin expression and microparticle formation were not significantly different between treated BCPC and APPC during storage. The JC-1 assay also displayed no loss of mitochondria integrity during the storage of treated products. Approximately 20% of riboflavin converted into photoproducts, including lumichrome. CONCLUSIONS: PRT treatment had an effect on the development of the normal platelet storage lesion at a level which seems tolerable for clinical usage.

Blood Component Removal↗

PubMed1995

[A nation-wide surveillance of pathogen in elderly patients with urinary tract infections. 1st Report: Distribution of pathogen].

A nation-wide surveillance of causative organisms was conducted on 1,699 elderly patients with UTIs aged more than 65 in 190 private clinics and 73 hospitals in August and September, 1993, in order to investigate the distribution of causative bacteria and the relationship between isolates and the background of those patients. From 1,233 of the 1699 patients, 1,636 strains were isolated. E. coli was isolated most frequently (30%). in male and older patients, less E. coli were isolated, while more attenuated Gram negative rods such as NFGNR including P. aeruginosa were found. A similar trend was observed in uncomplicated UTIs isolates in this study. More E. coli were isolated from patients with stronger subjective symptoms while more bacteria other than E. coli were isolated in those with weaker subjective symptoms. From the above findings, it was confirmed that a high recovery rate of opportunistic pathogen which is less susceptible to various antibacterials is one the refractory factors in elderly patients with UTIs. Therefore, it is necessary to select a suitable antibacterial that can be used for complicated UTIs in elderly patients even when diagnosed as uncomplicated UTIs. Regardless of the size of the clinic or hospital, when treating elderly patients, with unsatisfactory results in initial treatment and with less subjective symptoms the physician must bear this in mind.

Aged↗

PubMed1995

[A nation-wide surveillance of pathogen in elderly patients with urinary tract infections. 2nd Report: Sensitivities of pathogen for oral antibacterial agents].

A nation-wide surveillance of sensitivities of bacteria, which were isolated from elderly patients with UTIs aged more than 65 in 190 private clinics and 73 hospitals, for oral antibacterials, CPFX, OFLX, NFLX, CPDX and CCL, was conducted in August and September in 1993. Antibacterial activities of 5 agents against all 1,511 strains were intensified in CPFX, OFLX, NFLX, CPDX and CCL in that order. Major pathogens such as Enterobacteriaceae, E. faecalis, Staphylococcus spp. and P. aeruginosa were more susceptible to fluoroquinolones than cephems. The rate of resistant strains to antibacterials was higher in isolates from older patients, patients with complicated UTIs and with indwelling catheter. The representative of resistant strains was P. aeruginosa, and about 50% of the strains of P. aeruginosa were resistant to even CPFX which had the most superior antibacterial activity among the 5 agents. About 40% of the S. aureus were MRSA, which were almost resistant to the 5 agents. From the above findings, it was confirmed that a high recovery rate of strains which is less susceptible to various antibacterials is one of the refractory factors in elderly patients with UTIs, particularly in complicated UTIs and with indwelling catheter. Regardless of the size of the clinic or hospital, Fluoroquinolones are more suitable for the treatment of elderly patients with UTIs than cephems.

Aged↗

PubMed1994

Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen.

Uropathogenic Escherichia coli 536 (O6:K15:H31) carries two unstable DNA regions, which were shown to be responsible for virulence. These regions, on which the genes for hemolysin production (hly) and P-related fimbriae (prf) are located, are termed pathogenicity islands (PAI) I and II, and were mapped to positions 82 and 97, respectively, on the E. coli K-12 linkage map. Sequence analysis of the PAI region junction sites revealed sequences of the leuX and selC loci specific for leucine and selenocysteine tRNAs. The tRNA loci function as the targets for excision events. Northern (RNA) blot analysis revealed that the sites of excision are transcriptionally active in the wild-type strain and that no tRNA-specific transcripts were found in the deletion mutant. The analysis of deletion mutants revealed that the excision of these regions is specific and involves direct repeats of 16 and 18 nucleotides, respectively, on both sides of the deletions. By using DNA long-range mapping techniques, the size of PAI I, located at position 82, was calculated to be 70 kb, while PAI II, mapped at position 97, comprises 190 kb. The excision events described here reflect the dynamics of the E. coli chromosome.

Base Sequence↗

PubMed2003

A mosaic pathogenicity island made up of the locus of enterocyte effacement and a pathogenicity island of Escherichia coli O157:H7 is frequently present in attaching and effacing E. coli.

Enteropathogenic Escherichia coli (EPEC) and enterohemorragic E. coli (EHEC) possess a pathogenicity island (PAI), termed the locus of enterocyte effacement (LEE), which confers the capability to cause the characteristic attaching and effacing lesions of the brush border. Due to this common property, these organisms are also termed attaching and effacing E. coli (AEEC). Sequencing of the EHEC O157 genome recently revealed the presence of other putative PAIs in the chromosome of this organism. In this article, we report on the presence of four of those PAIs in a panel of 133 E. coli strains belonging to different pathogroups and serotypes. One of these PAIs, termed O122 in strain EDL 933 and SpLE3 in strain Sakai, was observed in most of the AEEC strains examined but not in the other groups of E. coli. It was also found to contain the virulence-associated gene efa1/lifA. In EHEC O157, PAI O122 is located 0.7 Mb away from the LEE. Conversely, we demonstrated that in many EHEC non-O157 strains and EPEC strains belonging to eight serogroups, PAI O122 and the LEE are physically linked to form a cointegrated structure. This structure can be considered a mosaic PAI that could have been acquired originally by AEEC. In some clones, such as EHEC O157, the LEE-O122 mosaic PAI might have undergone recombinational events, resulting in the insertion of the portion referred to as PAI O122 in a different location.

Chromosome Mapping↗

PubMed2005

Polyphasic characterization reveals that the human pathogen Mycobacterium peregrinum type II belongs to the bovine pathogen species Mycobacterium senegalense.

Mycobacterium peregrinum consists of two taxa: types I and II. We evaluated 43 clinical type II strains from throughout the United States. They were responsible for soft-tissue and bone infections, catheter-related infections, and possible pneumonitis. By carbohydrate utilization, they were indistinguishable from type I strains, being D-mannitol and trehalose positive. However, they had a distinct susceptibility pattern that included intermediate ciprofloxacin MICs but low clarithromycin and doxycycline MICs of < or =1 microg/ml. These features were also shared by reference isolates of Mycobacterium senegalense from African bovine cases of "farcy." By 16S rRNA gene sequencing, the type II isolates shared 100% sequence identity with M. senegalense. Partial sequencing of the type II hsp65 gene (441 bp) revealed four sequevars showing > or =98.4% identity with each other and > or =98.6% identity with the sequence of five bovine strains of M. senegalense. There was < or =97.1% identity with M. peregrinum type I isolates and other Mycobacterium fortuitum group species. Sequencing of additional gene targets including the 16S-23S rDNA internal transcribed spacer region and the rpoB gene (partial sequence) revealed a similar phylogenetic grouping. DNA-DNA hybridization showed 76 to 99% relatedness between the bovine and human strains. These studies demonstrate that type II isolates are not isolates of M. peregrinum but represent human strains of M. senegalense. This study is the first to demonstrate this species as a human pathogen. Representative human M. senegalense strains include ATCC 35755 and newly submitted strains ATCC BAA-849, ATCC BAA-850, and ATCC BAA-851.

Animals↗

PubMed2003

Molecular pathogenicity of the oral opportunistic pathogen Actinobacillus actinomycetemcomitans.

Periodontitis is mankind's most common chronic inflammatory disease. One severe form of periodontitis is localized aggressive periodontitis (LAP), a condition to which individuals of African origin demonstrate an increased susceptibility. The main causative organism of this disease is Actinobacillus actinomycetemcomitans. A member of the Pasteurellaceae, A. actinomycetemcomitans produces a number of interesting putative virulence factors including (a) an RTX leukotoxin that targets only neutrophils and monocytes and whose action is influenced by a novel type IV secretion system involved in bacterial adhesion; (b) the newly discovered toxin, cytolethal distending toxin (CDT); and (c) a secreted chaperonin 60 with potent leukocyte-activating and bone resorbing activities. This organism also produces a plethora of proteins able to inhibit eukaryotic cell cycle progression and proteins and peptides that can induce distinct forms of proinflammatory cytokine networks. A range of other proteins interacting with the host is currently being uncovered. In addition to these secreted factors, A. actinomycetemcomitans is invasive with an unusual mechanism for entering, and traveling within, eukaryotic cells. This review focuses on recent advances in our understanding of the molecular and cellular pathogenicity of this fascinating oral bacterium.

Actinobacillus Infections↗

PubMed2001

Organic acid profiles in feces of pigs with pathogenic or non-pathogenic diarrhea.

A chemical characteristic of the feces of diarrheal piglets permits differentiation among piglets receiving antibiotic treatment and those with colibacillosis or dyspepsia. A high concentration of lactic or succinic acid was observed in the diarrheic feces of piglets receiving antibiotic treatments and those with dyspepsia; however, no lactic or succinic acids were detected in piglets with colibacillosis. There was, however, little difference in the total concentration of organic acids among the three types of diarrheal illnesses. A quantitative analysis of lactic and succinic acids in diarrheic feces might provide a means for rapidly differentiating between colibacillosis and non-pathogenic diarrheas in piglets.

Animals↗

PubMedDate not supplied

Mutagenesis via insertional- or restriction enzyme-mediated-integration (REMI) as a tool to tag pathogenicity related genes in plant pathogenic fungi.

Random insertional mutagenesis is a powerful tool to investigate the molecular basis of most genetically determined processes, for example in pathogenic fungi. An improved version of this method is the insertional mutagenesis via restriction enzyme mediated integration (REMI). Transformation efficiency and mode of vector integration are species dependent and further influenced by vector conformation, restriction enzyme activity, and transformation protocol. An overview is given, covering the mutants and already identified genes obtained after REMI mutagenesis. An outlook describes the future developments in the field.

DNA Restriction Enzymes↗