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PubMed1986

Studies on the transcription, translation, and structure of alpha-actinin in Dictyostelium discoideum.

A clone coding for the F-actin cross-linking protein alpha-actinin was obtained by screening a genomic library of Dictyostelium discoideum DNA in lambda gt11 with monoclonal antibodies specific for Dictyostelium alpha-actinin. The 1.2-kilobase (kb) genomic clone was confirmed as containing part of the alpha-actinin gene by comparing its nucleotide sequence with the amino acid sequence of tryptic peptides from purified alpha-actinin. The clone recognized a 3.0-kb message in a Northern blot. Hybridization to RNA isolated from different developmental stages of several D. discoideum strains indicated that the mRNA content increased during early development. A similar result was obtained when the alpha-actinin content of the cells was followed by Western blot analysis. Hybridization of the clone to DNA from different wild-type strains of D. discoideum indicated a polymorphism on the DNA level that coincided with a polymorphism on the protein level. The data suggest continuous transcription of the alpha-actinin gene throughout the development of D. discoideum, up- and down-regulation of the levels of alpha-actinin mRNA and protein with maximum levels at the onset of aggregation, and a high diversity of alpha-actinin at the DNA and protein level among different D. discoideum strains. The structural data make it conceivable that the highly conserved nature of alpha-actinin resides only at the functional sites, whereas the helical portions of the alpha-actinin molecule allow a higher level of diversity throughout evolution.

Actinin↗
PubMed1990

Studies on the sedimentation behavior of the Neurospora crassa plasma membrane H(+)-ATPase synthesized in vitro and integrated into homologous microsomal membranes.

RNA transcripts that encoded the Neurospora crassa plasma membrane H(+)-ATPase (pma+), a polytopic integral membrane protein, and the pma+344, a truncated pma+ with the amino terminal 344 amino acids, were translated in a N. crassa in vitro system. The microsomal membranes integrated products were insensitive to extraction by Na2CO3 (pH 11.5). The velocity sedimentation behavior of the in vitro synthesized pma+ were examined under various conditions. The pma+ migrated on linear sucrose gradients as aggregates which were heterogeneous in size, in the regions of 9-13 S; whereas, these values were reduced when Triton X-100 was presence in the gradients. The formation of these aggregates is interpreted to suggest a mechanism that maintains this polytopic integral membrane protein in a soluble form until it is targeted to the membranes. The sedimentation coefficient of the Triton X-100 solubilized microsomal membranes integrated pma+ corresponded roughly to a monomer of the pma+. Furthermore, a comparison of the trypsin cleavage patterns of the in vitro synthesized pma+ and of the microsomal membranes integrated pma+ suggest that they have different tertiary, or quaternary, structures. The latter did not give the characteristic trypsin cleavage patterns that have been observed for the native pma+ in the presence of its ligands MgATP and vanadate (Addison, R. and Scarborough, G.A. (1982) J. Biol. Chem. 257, 10421-10426). This was interpreted to suggest that the microsomal membranes integrated pma+ cannot interact with its substrate, suggesting that it is catalytically inactive.

Cell Membrane↗
PubMed1987

Pseudoexfoliative fibrillopathy in the conjunctiva. A relation to elastic fibers and elastosis.

Pseudoexfoliative (PSX) material in the conjunctiva was found to have a close association with elements of the elastic system in 13 eyes of patients with PSX disease studied ultrastructurally. Pseudoexfoliation aggregates were rare within vascular walls but frequent in proximity to the plasmalemma of stromal fibroblasts. No amyloid was found. A striking feature was the association of PSX fibers with the elastosis seen in many of the specimens. Typical PSX fibers were present in clumps of oxytalan and around small elastic fibers. In five cases with more advanced elastosis, the aggregates of PSX fibers were larger and their usual loose fibrogranular matrix was extensively replaced by a dense elastotic one. Morphology of PSX here varied from typical to thicker, fragmented, and sometimes nondiagnostic fibers. This intermingling of PSX and elastotic materials suggested that PSX fibrillopathy is itself a type of elastosis, possibly resulting from abnormal aggregation of components related to elastic microfibrils.

Actin Cytoskeleton↗
PubMed2000

Genomic copy number of intracellular bacterial symbionts of aphids varies in response to developmental stage and morph of their host.

Buchnera, endosymbiotic bacteria of aphids possess many genomic copies per cell. In this study, we estimated genomic copy number per Buchnera cell from host insects at various developmental stages and of two different morphs, apterae and alatae, by fluorimetry and real-time quantitative PCR. The results indicated that the genomic copy number of Buchnera increased during postembryonic development of insects to adulthood, and that it decreased during the host's ageing. In Buchnera from alatae, the genomic copy number per cell was about twice as many as in those from apterae. DAPI-staining showed that the distribution of the genomic DNA in the Buchnera cells from old insects tended to aggregate, suggesting that intracellular structure of the genomic DNA of Buchnera varies in response to the physiological conditions of their host.

Animals↗
PubMed1997

Transient aggregates in protein folding are easily mistaken for folding intermediates.

It has been questioned recently whether populated intermediates are important for the protein folding process or are artefacts trapped in nonproductive pathways. We report here that the rapidly formed intermediate of the spliceosomal protein U1A is an off-pathway artefact caused by transient aggregation of denatured protein under native conditions. Transient aggregates are easily mistaken for structured monomers and could be a general problem in time-resolved folding studies.

Artifacts↗
PubMed2002

Joint participation of mitochondria and sarcoplasmic reticulum in the formation of tubular aggregates in gastrocnemius muscle of CK-/- mice.

Tubular aggregates are specific subcellular structures that appear in skeletal muscle fibres under different pathological conditions. The origin of the tubular aggregates is generally ascribed to proliferating membranes of sarcoplasmic reticulum. There are, however, histochemical indications for the presence of mitochondrial enzymes in tubular aggregates suggesting contribution of mitochondria to the genesis of tubular aggregates. In this study we used an immunocytochemical detection technique to assess participation of mitochondria and of sarcoplasmic reticulum in derivation of tubular aggregates. The fast skeletal muscle fibres (m. gastrocnemius) of mice bearing the double invalidation for both the mitochondrial and the cytosolic isoforms of creatine kinase (CK), an enzyme involved in energetics of muscle cells, were employed as a model muscle with tubular aggregates (Steeghs et al., Cell 89, 93-103, 1997). Immunogold labelling of the bc1 complex, a specific integral protein of the inner mitochondrial membrane, provided strong signals in both the mitochondria and tubular aggregates but not in other ultrastructural components of muscle fibres. A similar strong immunogold signal was obtained when labelling for SERCA1, a specific enzyme of the sarcoplasmic reticulum membrane, in regions of typical occurrence of the sarcoplasmic reticulum and in tubular aggregates. In double labelling experiments, we found simultaneous labelling of tubular aggregates with both the bc1 and SERCA1 antibodies. It is concluded, that in CK-/- mouse both the inner mitochondrial membrane and the membrane of the sarcoplasmic reticulum participate in the formation of tubular aggregates.

Animals↗
PubMed1991

Mechanisms of amyloid deposition in Alzheimer's disease.

At the cellular level, Alzheimer's disease (AD) must be the result of neuronal dysfunction and degeneration leading to a reduction in synaptic density. Filamentous deposits of amyloid, which define the disease at the molecular level, occur within perikarya, axons, dendrites, and terminals of neurons as neurofibrillary tangles (NFT), in the extracellular neuropil as amyloid plaques (APC), and around blood vessels as amyloid congophilic angiopathy (ACA). These fibrillar amyloid protein aggregates are also found in the brain of all individuals with Down's syndrome after the age of 30 years. The amyloid deposits apparently occur in the terminal zones of neurons that develop NFT. It is suggested that amyloid deposition is of fundamental significance in AD and that a thorough understanding of amyloid formation will eventually lead to successful therapeutic intervention in AD. As elucidation of the reasons behind amyloid deposition must shed some light on the pathogenesis of AD, we review the current state of knowledge on the nature of the AD amyloid protein, its origin, and its formation. Although there is yet no agreement about the chemical nature of the amyloid protein of NFT, the major constituent of both APC and ACA has been shown to be a 4.5-kD amyloid protein originally termed "beta-protein" or "amyloid A4" which we now denote as "beta A4." Amyloid beta A4 protein is proteolytically derived from a transmembrane protein termed amyloid precursor protein (APP) which is encoded by a widely expressed gene on chromosome 21. Our present results are consistent with the possibility that amyloid formation requires membrane damage or APP molecules that are not or are incorrectly integrated into membranes. To allow the generation of the C-terminus of beta A4, one proteolytic cleavage step has to occur in the sequence that normally forms the transmembrane domain of the APP proteins. This cleavage is crucial for amyloid formation because we could show that the ability of synthetic beta A4 to form amyloid depositions is mainly based on hydrophobic parts of the sequence that have to interact with each other and build up large aggregates under physiologic conditions. Membrane association of APP is expected to interfere with this cleavage and the process of aggregation.

Aging↗
PubMed1993

devRS, an autoregulated and essential genetic locus for fruiting body development in Myxococcus xanthus.

Two Tn5 lac insertions into the Myxococcus genome at sites omega 4414 and omega 4473, which are separated by 550 nucleotides, inactivate fruiting body development. Sporulation is decreased 100- to 10,000-fold. At least two genes, devR and devS, are transcribed in this region, probably as an operon. Expression of devR begins by 6 h after starvation has initiated development. On the basis of their nucleotide sequences, devR and devS are expected to encode proteins of 302 and 214 amino acids, respectively. Dev+ function can be restored by a segment of 7.8 kb cloned from the devRS region of wild-type cells. Two experiments show that devR expression is under strong negative autoregulation. beta-Galactosidase is expressed at a higher level from a transcriptional devR::lacZ fusion when the fused operon is in a dev strain than when it is in the dev/dev+ genetic background of a partial diploid. There is more mRNA accumulation from the devRS region in the dev strain than in a rescued dev/dev+ tandem duplication strain. Sporulation rescue is correlated with some degree of negative autoregulation, even though sporulation is not inversely proportional to beta-galactosidase expression from omega 4414. A second level of regulation is suggested by complementation of dev by dev+ in duplication strains. The expression of devRS, measured by sporulation levels, differs 1,000-fold when devRS+ is moved from a distance of 20 kb to 3 Mb from the mutant devRS locus. Expression of devR is also dependent on the cell density at which development is initiated, a third level of regulation. Multiple levels of regulation suggest that devRS is a switch required to activate completion of aggregation and sporulation.

Amino Acid Sequence↗
PubMed1972

Translation of synthetic and endogenous messenger ribonucleic acid in vitro by ribosomes and polyribosomes from Clostridium pasteurianum.

Ribosomes and polyribosomes from Clostridium pasteurianum were isolated and their activities were compared with those of ribosomes from Escherichia coli in protein synthesis in vitro. C. pasteurianum ribosomes exhibited a high level of activity due to endogenous messenger ribonucleic acid (RNA). For translation of polyuridylic acid [poly(U)], C. pasteurianum ribosomes required a higher concentration of Mg(2+) and a much higher level of poly(U) than did E. coli ribosomes. Phage f2 RNA added to the system with C. pasteurianum ribosomes gave no significant stimulation of protein synthesis in a homologous system or with E. coli initiation factors. The 30S and 50S subunits prepared from C. pasteurianum ribosomes reassociated less readily than subunits from E. coli. The ability of the C. pasteurianum subunits to reassociated was found to be dependent upon the presence of a reducing agent during preparation and during analysis of the reassociation products. In heterologous combinations, E. coli 30S subunits associated readily with C. pasteurianum 50S subunits to form 70S particles, but C. pasteurianum 30S subunits and E. coli 50S subunits did not associate. In poly(U) translation, E. coli 30S subunits were active in combination with 50S subunits from either E. coli or C. pasteurianum, but C. pasteurianum 30S subunits were not active in combination with either type of 50S subunits. Polyribosomes prepared from C. pasteurianum were very active in protein synthesis, and well-defined ribosomal aggregates as large as heptamers could be seen on sucrose gradients. An attempt was made to demonstrate synthesis in vitro of ferredoxin.

Amino Acids↗
PubMed1994

Casein kinase 2 and protein substrates are released from rat liver cells nuclei by DNase or RNase digestion.

Casein kinase 2 was released from rat liver cells nuclei by digestion with DNase I plus RNase A. This treatment also released three major substrates of 50, 40-42, and 37 kDa. Casein kinase 2 and substrates were also extracted by DNase or RNase separately. However, in DNase extracts only the 37 kDa protein was phosphorylated by casein kinase 2, whereas in RNase extracts all three substrates were phosphorylated. When the DNase extracts were subsequently treated with RNase the 40-42 substrates were then phosphorylated, indicating that their interaction with RNA prevents their phosphorylation by casein kinase 2. The ratio of B: alpha subunits of casein kinase 2 present in the nuclease extracts was higher than that of the purified enzyme, which is assumed to be 1:1. A further analysis by sucrose gradient centrifugation revealed that under physiological salt conditions casein kinase 2 from nuclease extracts formed large aggregates (higher than 300 kDa) which were disrupted at 400 mM KCl. At the latter KCl concentration CK-2 activity was localized at a position corresponding to a M(r) of 230-250 kDa, which is still higher than the typical tetrameric form of the enzyme.

Animals↗
PubMed2000

Analysis of expression of prgX, a key negative regulator of the transfer of the Enterococcus faecalis pheromone-inducible plasmid pCF10.

Conjugative transfer of pCF10, a plasmid found in Enterococcus faecalis, is induced by the peptide pheromone cCF10 and the donor-recipient aggregation is mediated by PrgB. Expression of prgB in pCF10 is negatively regulated by PrgX. The prgX gene is adjacent to prgQ which provides the promoter for prgB expression. The prgX and prgQ genes are transcribed in opposite directions. A deletion spanning nucleotides+259 to +398 from the prgQ transcription initiation site abolished the prgX gene products at both RNA and protein levels. An RNA, named Qa, was found to be transcribed in the antisense direction in the prgQ region. The transcription start site and the promoter were found to be almost identical with those of the traD determinant in pAD1, another pheromone-responsive plasmid. The first inverted repeat sequence in prgQ, IRS1, was required for the full activity of the Qa promoter. Although the size of the major Qa RNA detected by Northern blot analysis was too short (ca 120 nt) to be an mRNA for PrgX protein, the transcription from the Qa promoter was shown to proceed through to prgX. Transcriptional fusion analyses showed that the transcription of prgX requires one or more trans elements. Moreover, deletion of prgX greatly reduced the level of the Qa RNA and abolished transcription of prgX. Although transcription initiation from the Qa promoter was not increased by PrgX, transcriptional readthrough to prgX was increased by the protein. Based on these data, we conclude that transcription of prgX is initiated from the Qa promoter in prgQ, and PrgX autoregulates its transcription either by mediating transcriptional readthrough or increasing mRNA stability. The PrgX protein, prgX RNA, and the Qa RNA were downregulated by cCF10 induction; however, prgX gene products were still detected for at least 40 minutes after induction.

Amino Acid Sequence↗
PubMed1988

An improved purification procedure for preparing potyviruses and cytoplasmic inclusions from the same tissue.

Twelve different potyviruses and cytoplasmic inclusion proteins were purified from a range of plant species utilizing a single purification protocol. Highly purified preparations have been obtained with yields that reflect the relative concentrations in the starting material; virus yields of up to 15 mg per 100 g of tissue were obtained. In some cases aggregation resulted in losses of significant amounts of virus to the inclusion fraction; this varied among preparations of the same virus. Preparations obtained from cesium gradients were typically unaggregated and essentially free of host materials. Purified virus was suitable for the production of antisera with high specific titers and low titers against healthy plant antigens. Both purified virus and RNA prepared from the virus retained infectivity. Purified RNA was free of detectable host plant nucleic acids, as complementary DNA preparations synthesized using virion RNA as template were highly virus-specific.

DNA↗
PubMed2026

Cholesterol dysregulation in APOE4 astrocytes promotes α-synuclein pathology in miBrains.

The pathological hallmarks of neurodegeneration are the aberrant post-translational modification and aggregation of proteins. Genetic factors, like APOE4, increase the prevalence and severity of tau, amyloid, and α-synuclein pathologies. However, the human brain is largely inaccessible during this process, limiting mechanistic understanding. Here, we developed an iPSC-based 3D model that integrates neurons, glia, myelin, and cerebrovascular cells into a human brain-like tissue ("miBrain"). Single-nucleus RNA sequencing of miBrains confirmed the presence of diverse cell populations and revealed transcriptional responses to α-synuclein pathology. Like the human brain, pathogenic α-synuclein is increased in APOE4/4 miBrains. Combinatorial experiments revealed that endolysosomal dysfunction caused by cholesterol accumulation in APOE4/4 astrocytes impairs the degradation of soluble α-synuclein leading to a pathogenic transformation that seeds α-synuclein inclusions in neurons. Collectively, this study establishes a robust model for investigating protein inclusions in human iPSC-derived brain tissue and highlights the role of astrocytes and cholesterol in APOE4-mediated pathologies.

alpha-Synuclein↗
PubMed2006

Rectal bleeding in infancy: clinical, allergological, and microbiological examination.

OBJECTIVE: Rectal bleeding is an alarming symptom and requires additional investigation. In infants it has been explained mainly by hypersensitivity. In addition to dietary antigens, intraluminal microbial agents challenge the immature gut mucosa. Although controlled in the mature gut, these antigens may induce inflammation in the developing gastrointestinal tract. The objectives of this study were to evaluate prospectively the clinical course of rectal bleeding and evaluate the impact of cow's milk allergy and aberrant gut microbiota on the condition. Because withdrawal of cow's milk antigens from the infants' diet is used as a first treatment without evidence of its efficacy, we also aimed to asses the effect of a cow's milk-elimination diet on the duration of rectal bleeding. METHODS: The study involved 40 consecutive infants (mean age: 2.7 months) with visible rectal bleeding during a 2-year period at the Tampere University Hospital Department of Pediatrics. Most of the infants (68%) were fully breastfed. At enrollment the infants were randomly allocated to receive a cow's milk-elimination diet (n = 19) or continue their previous diet (n = 21) for 1 month. Findings of colonoscopy, fecal bacterial culture, fluorescence in situ hybridization of selected gut genera, specific detection of fecal enteroviruses, rotaviruses, and adenoviruses, fecal electron microscopy for viruses, and mucosal electron microscopy for viruses were assessed. During each visit the severity of atopic eczema, if any, was assessed according to the SCORAD method. In evaluating the extent of sensitization, serum total immunoglobulin E (IgE) and specific IgE and skin-prick tests for cow's milk, egg, and wheat were studied. Cow's milk allergy was diagnosed by elimination and provocation testing. Five patients were hospitalized; all others were treated on an outpatient basis. The follow-up visits were scheduled 1 month later and at the age of 1 year. Sixty-four healthy reference infants were selected as controls according to the following criteria: age and timing of fecal sampling being identical to within 1 month. RESULTS: Altogether, 32 (80%) infants manifested bloody stools during follow-up (mean [range]: 2.1 [1-15] per day). The mean number of days with rectal bleeding on follow-up was 6. Typically, bloody stools occurred irregularly, for which reason the mean time to the last occurrence of rectal bleeding was 24 (range: 1-85) days from admission. Atopic eczema at presentation or during follow-up was diagnosed in 38% of the infants. Increased specific IgE concentrations or a positive skin-prick test were uncommon. The growth of the infants was normal on admission and during follow-up. Colonoscopy revealed typically focal mucosal erythema and aphthous ulcerations. The mucosa appeared normal in less than half of the patients. No anorectal fissures or colonic polyps were found. Light microscopy revealed that the overall architecture of the mucosa was well maintained. Acute inflammation or postinflammatory state and focal infiltration of eosinophils in the lamina propria were the most common abnormalities. A cow's milk-elimination diet did not affect the duration of rectal bleeding. Cow's milk allergy was diagnosed in 7 (18%) patients. Virus-particle aggregates were found in the microvillus layer of the colon epithelium in 8 cases. The surface epithelium of the virus-positive colon biopsy specimens regularly showed degenerative changes in the microvillus layer and epithelial cells. Electron microscopy study of the colon biopsies disclosed virus particles (30 nm in diameter) on the surface of epithelial cells. Virus particles or RNA were present in feces in only a minority of the patients. All fecal cultures were negative for Salmonella, Shigella, and Yersinia. Campylobacter jejuni was found in the feces of 1 patient, and fecal cultures were positive for Clostridium difficile in 4 patients, Staphylococcus aureus in 8 patients, and yeast in 2 patients. Fluorescence in situ hybridization revealed that at the time of admission the total numbers of bacteria and the numbers of bifidobacteria and lactobacilli in feces were lower in the patients compared with controls. The fecal concentrations of microbes characterized in this study (Bacteroides, bifidobacteria, Clostridium, lactobacilli, and enterococci) did not differ significantly between the time of admission and the second visit in the patients or controls. At the age of 1 year, 7 patients still suffered from cow's milk allergy, 5 of whom also suffered from multiple food allergies. Atopic eczema and histopathologically confirmed inflammation of the colonic mucosa at presentation were associated with persistence of cow's milk allergy at the age of 1 year. No patients exhibited gastrointestinal complaints or visible blood in stools. CONCLUSIONS: Rectal bleeding in infants is generally a benign and self-limiting disorder. Bloody stools occurred irregularly for only a few days during the following months. As in a previous report, most infants were exclusively breastfed. In the majority of the patients the cause of the condition remains unknown. An association with viruses can be seen in some patients. The microbes that commonly lead to bloody diarrhea in older children and adults, Salmonella, Shigella, and Yersinia, were absent in the present material. The low bifidobacterial numbers in fecal samples may indicate a significant aberrance that may provide a target for probiotic intervention to normalize gut microbiota. The gut microbiota overall seemed stable, because the numbers of major groups of microbiota tested did not change significantly between the time of admission and after 1 month. Cow's milk allergy among these patients is more uncommon than previously believed. Cow's milk challenge is thus essential in infants who become symptom-free during a cow's milk-free diet to reduce the number of false-positive cow's milk-allergy diagnoses.

Animals↗
PubMed2002

[Etiology, pathogenesis and management of senile inflammatory pulmonary diseases].

We studied the etiology, pathogenesis and management of therapy-resistant inflammatory pulmonary diseases. First, to understand the pathogenesis of rhinovirus (RV) infection-induced exacerbation of bronchial asthma, we infected cultured human tracheal epithelial cells with RV. The epithelial cells produced a variety of proinflammatory cytokines, intercellular adhesion molecules (ICAM-1) and low-density lipoprotein receptor, and increased the permeability across the epithelial cells. These findings suggest that these factors and the increased permeability may cause airway inflammation, resulting in the exacerbation of asthma. Glucocorticoid and bafilomycin inhibited RV infection in the epithelial cells by reducing ICAM-1 expression and RV RNA entry from the acidic endosomes to the cytoplasm. Second, we revealed the mechanisms of aspiration pneumonia induced by silent aspiration in patients with cerebral infarction. We also developed a pharmacologic treatment for preventing aspiration pneumonia with amantadine, which stimulates the dopaminergic neurons; the angiotensin-converting enzyme inhibitors, which decrease substance P catabolism; and cilostazol, which inhibits platelet aggregation and induces cerebral vasodilation. Third, we demonstrated that exhaled carbon monoxide concentrations caused by heme oxygenase-1 upregulation, may be a useful noninvasive means of monitoring airway inflammation and of controlling elderly patients with bronchial asthma. Finally, we demonstrated that microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with susceptibility to emphysema caused by cigarette smoke in Japanese patients with chronic pulmonary emphysema.

Aged↗
PubMed1994

Cloning and high level expression of gene encoding ES antigen from Trichinella spiralis muscle larvae.

The partial structure gene encoding ES antigen derived from Trichinella spiralis (TSP) muscle larvae was cloned, characterized, and expressed in E. coli. The target DNA (0.7 kb) was directly obtained from the TSP total RNA by using RNA PCR technique. Based on the analysis with the RE digestion, the fragment was cloned into the fusion expression vector pEX31C. It was shown that a kind of 37kDa fusion protein was expressed in E. coli containing the recombinant plasmid by SDS-PAGE electrophoresis. The expressed protein was over 22% of the total cell protein, and it was aggregated in the form of inclusion bodies in E. coli. The purified protein could be recognized in ELISA both by sera from swine-infected with TSP and by the monoclonal antibody against TSP. These findings suggest that the recombinant protein is a potentially valuable antigen both for immunodiagnosis and vaccine development of trichinellosis.

Animals↗
PubMed1990

YopM inhibits platelet aggregation and is necessary for virulence of Yersinia pestis in mice.

In Yersinia pestis KIM there are 11 Yops (yersinial outer membrane proteins) encoded by the low-Ca2+ response virulence plasmid pCD1. Only YopM and YopN are found in easily detectable amounts in the culture medium. In our previous work, we characterized the yopM gene. In the present study, we constructed a YopM- mutant to elucidate the role of YopM in the virulence of Y. pestis. A lacZYA sequence was inserted 126 base pairs downstream from the start codon of the yopM gene in pCD1. The YopM- mutant had the same growth properties as the parent, Y. pestis KIM5-3001. The inserted lacZ gene was regulated by the promoter of the yopM gene. Accordingly, it was expressed strongly at 37 degrees C in the absence of Ca2+ and was decreased in expression when Ca2+ was present. Northern blot (RNA blot) analysis revealed that the yopM gene was in a monocistronic operon, suggesting that the yopM insertion mutation was unlikely to have polar effects on other genes. The YopM- mutant had strongly decreased virulence in mice, with a 50% lethal dose of 3.4 x 10(5) CFU. Virulence was restored by the cloned yopM-containing 5.5-kilobase HindIII F fragment of pCD1. However, supplying a cloned 1.57-kilobase fragment containing little more than the yopM structural gene caused the yopM mutant to significantly overexpress YopM and failed to restore virulence. The infection kinetics of the YopM- mutant revealed growth in both spleens and livers from days 2 to 4 after infection, followed by a precipitous clearance of the bacteria. YopM-containing supernatant proteins of Y. pestis inhibited thrombin- or ristocetin-induced platelet aggregation, whereas there was no inhibition by supernatant proteins from the YopM- Y. pestis mutant. Accordingly, YopM may prevent platelet-mediated events and serve as an important strategy for the yersiniae in the initial stages of a plague infection.

Animals↗