Interrelationship between polymorphisms of incA, fusogenic properties of Chlamydia trachomatis strains, and clinical manifestations in patients in The Netherlands

IncA variation among Dutch Chlamydia trachomatis isolates was investigated. Of 98 strains, two carried an incA with a premature stop codon, lacked IncA, and were nonfusogenic, while 96 contained an intact incA, expressed IncA, and were fusogenic. Among these 96 strains, nine IncA sequence types were found, of which the three most frequently encountered (88% of the strains) were randomly distributed among symptomatic and asymptomatic patients.     

Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane

The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole, termed an inclusion, throughout their developmenal cycle. When an epithelial cell is infected with multiple Chlamydia trachomatis elementary bodies, they are internalized by endocytosis into individual phagosomal vacuoles that eventually fuse to form a single inclusion. In the course of large-scale serotyping studies in which fluorescent antibody staining of infected cells was used, a minority of strains that had an alternate inclusion morphology were identified. These variants formed multiple nonfusogenic inclusions in infected cells, with the number of independent inclusions per cell varying directly with the multiplicity of infection. Overall the nonfusogenic phenotype was found in 1.5% (176 of 11,440) of independent isolates. Nonfusing variants were seen in C. trachomatis serovars B, D, D-, E, F, G, H, Ia, J, and K. The nonfusing phenotype persisted through repeated serial passage, and the phenotype was consistent in four mammalian host cell lines. Fluorescence microscopy and immunoblotting with antisera directed at proteins in the C. trachomatis inclusion membrane revealed that one such protein, IncA, was not detected in the inclusion membrane in each tested nonfusogenic strain. The distributions of other chlamydial proteins, including one additional Inc protein, were similar in wild-type and variant strains. The incA coding and upstream regions were amplified and sequenced from the prototype serovar D and two nonfusing serovar D((s)) strains. Three nucleotide changes were discovered in the D((s)) incA gene, leading to two amino acid changes within the predicted D((s)) IncA sequence. These studies demonstrate a subgroup of variant C. trachomatis isolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an altered incA sequence that modifies the characteristic hydrophobic domain of the IncA protein.     

Identification of macrolide-resistant clones of Streptococcus pyogenes in Portugal

Although the overall level of macrolide resistance (27%) has remained stable in Portugal, a rapid inversion in the dominant phenotypes has been noted, with a sharp decrease in the MLS(B) phenotype paralleled by an increase in the M phenotype. To gain further insight into these changes, 325 macrolide-resistant isolates were characterised using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The use of Cfr9I, an isoschizomer of SmaI, to digest M phenotype isolates that were refractory to SmaI digestion allowed direct comparison of MLS(B) and M isolates. The results from PFGE and MLST were highly concordant and identified eight major clones, accounting for 92% of the isolates, each of which was associated exclusively with a single macrolide resistance phenotype. Two major clones were found among MLS(B) isolates, characterised by sequence types (ST) 46 (T12/emm22) and ST52 (T28/emm28), whereas clones characterised by ST39 (T4/emm4) and ST28 (T1/emm1) dominated among M isolates. The clone defined by ST52 corresponded to a bacitracin-resistant clone circulating in Europe, and a novel variant expressing other surface antigens (T12/emm22) was detected. The presence of the four major clones has been reported previously in other European countries, suggesting Europe-wide dissemination of a few macrolide-resistant lineages.     

Molecular epidemiology of erythromycin resistance in Streptococcus pneumoniae isolates from blood and noninvasive sites

Erythromycin-resistant isolates of Streptococcus pneumoniae from blood cultures and noninvasive sites were studied over a 3-year period. The prevalence of erythromycin resistance was 11.9% (19 of 160) in blood culture isolates but 4.2% (60 of 1,435) in noninvasive-site isolates. Sixty-two of the 79 resistant isolates were available for study. The M phenotype was responsible for 76% (47 of 62) of resistance, largely due to a serotype 14 clone, characterized by multilocus sequence typing as ST9, which accounted for 79% (37 of 47) of M phenotype resistance. The ST9 clone was 4.8 times more common in blood than in noninvasive sites. All M phenotype isolates were PCR positive for mef(A), but sequencing revealed that the ST9 clone possessed the mef(A) sequence commonly associated with Streptococcus pyogenes. All M phenotype isolates with this mef(A) sequence also had sequences consistent with the presence of the Tn1207.1 genetic element inserted in the celB gene. In contrast, isolates with the mef(E) sequence normally associated with S. pneumoniae contained sequences consistent with the presence of the mega insertion element. All MLS(B) isolates carried erm(B), and two isolates carried both erm(B) and mef(E). Fourteen of the 15 MLS(B) isolates were tetracycline resistant and contained tet(M). However, six M phenotype isolates of serotypes 19 (two isolates) and 23 (four isolates) were also tetracycline resistant and contained tet(M). MICs for isolates with the mef(A) sequence were significantly higher than MICs for isolates with the mef(E) sequence (P < 0.001). Thus, the ST9 clone of S. pneumoniae is a significant cause of invasive pneumococcal disease in northeast Scotland and is the single most important contributor to M phenotype erythromycin resistance.     

Comparison of full length sequences of hepatitis B virus isolates in hepatocellular carcinoma patients and asymptomatic carriers of Korea

Relatively few genomic sequences of Korean hepatitis B virus (HBV) isolates are available. Moreover, no comparative study has been made between the full-length genomes of Korean HBV isolates and clinical status. To evaluate mutations in HBV isolates obtained from chronically infected HBV patients in terms of clinical significance, we determined the genomic sequences of HBV isolates obtained from three hepatocellular carcinoma (HCC) patients (He52, He53, and He82) and from three asymptomatic carriers (He74, He100, and He127). A comparison of sequence variations showed that the HBV isolates from the three HCC patients showed higher frequencies of mutation than the isolates from the three asymptomatic carriers. Three characteristic mutation patterns were identified in the HBV isolates from the HCC patients, which distinguished the HBV isolates from the asymptomatic carriers. First, HBV isolates from the three HCC patients both had double mutations in a core promoter (T1762/A1764) and a precore mutation (A1896). Second, although these isolates belonged to genotype C, 11 amino acids deletions in the preS1 region, specific for HBV genotype D, were detected in the isolates of two HCC patients (He52 and He82). Third, mutations (I127T/N, K130M, and V131I) at three codons in the carboxy functional region of X protein were observed in isolates from all three HCC patients. Additionally, phylogenetic analysis based on the entire HBV sequences showed that all six isolates belonged to genotype C2, as do other Korean strains.     

Characterisation of VanA and VanB elements from glycopeptide-resistant Enterococcus faecium from Greece

Ten glycopeptide-resistant Enterococcus faecium isolates from separate patients in Laikon General Hospital, Athens were studied. Eight isolates had the VanA phenotype and represented variants of three strains based on SmaI macrorestriction banding patterns. Their VanA elements were compared with the prototype element, Tn1546, by an overlapping PCR method. Three related isolates contained resistance elements indistinguishable from Tn1546 (designated Greek type I). The other five isolates all contained identical elements that differed from Tn1546 by the presence of IS1251 between vanS and vanH, by a point mutation (G --> T) at nucleotide position 8234 within vanX and by a partial loss of transposition gene orf1 (designated Greek type II). Two distinct strains of E. faecium with the VanB phenotype were obtained. HhaI digestion of an amplified fragment of the vanB gene indicated that both strains contained the vanB2 allele, and further PCR assays confirmed that the vanB2 gene cluster was located within a Tn5382-like element.     

A novel recurrent mitochondrial DNA mutation in ND3 gene is associated with isolated complex I deficiency causing Leigh syndrome and dystonia

Defects in NADH:ubiquinone oxidoreductase (complex I), the largest complex of the mitochondrial respiratory chain, account for most cases of respiratory chain deficiency in human. Complex I contains at least 45 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA). Here we report a novel 10197G>A mutation of the ND3 gene in three unrelated families with Leigh syndrome (LS) or dystonia. Variable degrees of heteroplasmy were found in all tissues tested and a high percentage of mutant mtDNA was observed in muscle. The 10197G>A mutation modifies a hydrophobic alanine residue into a hydrophilic threonine (A47T) in a highly conserved domain of ND3 subunit. Furthermore, this defect could be transferred along with the mutant mtDNAs to rho degrees lymphoblastoid cells in cybrid experiments. However, nuclear modifier genes may also play a role in the phenotypic expression and severity of the 10197G>A mutation. The association of the 10197G>A ND3 mutation with an isolated biochemical defect involving complex I and the discovery of the 10197G>A mutation with a similar phenotype in three unrelated families establish its pathogenicity and demonstrate that the amino acid position A47 is important for the function of complex I. These results show that the 10197G>A mutation in the mitochondrial ND3 gene should be considered as a common mtDNA mutation responsible for LS and dystonia.     

Analysis of the role of Mycobacterium tuberculosis kasA gene mutations in isoniazid resistance

Previous studies have suggested that Mycobacterium tuberculosis kasA G312S and G269S gene mutations may represent sequence polymorphisms of the M. tuberculosis East-African-Indian (EAI) and T families, respectively, rather than relating to isoniazid resistance. The present study examined polymorphisms of these two codons in 98 drug-susceptible M. tuberculosis isolates (68 EAI and 30 T isolates). Twenty-eight isolates belonging to a sub-lineage of the EAI family had the kasA G312S mutation, but none of the 30 T isolates had the G269S mutation. The data suggest that the kasA G312S mutation is not related to isoniazid resistance, but represents a sequence polymorphism in a sub-lineage of the EAI family.     

Sequence analysis of segment a of a field virus isolate from an outbreak of Infectious bursal disease in India

Sequence analysis of genome segment A of an Indian Infectious bursal disease virus (IBDV) field isolate (KT1/99) revealed total 95 nucleotide substitutions, resulting in 17 amino acid changes. Of these, five amino acid changes, namely F60S, T137I, I374V, V519I and E682D were unique to the KT1/99 isolate. The amino acid change P222A and the proposed hot mutation spot 680Y, reported to be present in very virulent IBDV isolates were also found in KT1/99. This isolate had nucleotide divergence of 1.1% to 4.95% from the other reported serotype 1 IBDV isolates and 19.6% from serotype 2 strain OH in polyprotein gene sequence, while divergence at amino acid level was 0.6% to 2.9% and 11.4%, respectively. Based on both nucleotide and amino acid sequence analysis, KT1/99 was grouped phylogenetically with the reported Bangladesh isolate BD3/99 in one cluster along with other reported very virulent isolates in same lineage.     

Expression of an LDL receptor allele with two different mutations (E256K and I402T).

AIMS: To investigate the disease causing event in patients with familial hypercholesterolaemia, carrying two mutations each, E256K in exon 6 and I402T in exon 9, of the gene encoding the low density lipoprotein (LDL) receptor. It was not known whether the mutations were positioned in cis or trans, or if they were each pathogenic separately or only when present together. METHODS: Polymerase chain reaction, denaturing gradient gel electrophoresis and sequencing were used to characterise the LDL receptor locus of the patients and family members. The different LDL receptor mutants, constructed in vitro by oligonucleotide directed mutagenesis, were expressed in LDL receptor deficient Chinese hamster ovary (CHO1d1A7) cells, to determine the effects of the mutations on LDL receptor function. RESULTS: The two mutations were located on the same allele of the LDL receptor gene. All mutant constructs resulted in the production of a detectable protein in CHO cells. The cells expressing only the I402T mutation, or the combination of I402T and E256K mutations, were seriously affected in mediating uptake and degradation of LDL. Contrary to initial predictions, the cells expressing only the E256K mutation showed essentially the same binding, uptake, and degradation of 125I labelled LDL as cells transfected with normal LDL receptor cDNA. These results suggest that the pathogenic mutation in the patients heterozygous for the E256K/I402T allele is the I402T mutation, and that E256K alone is a rare sequence variation, which does not affect LDL receptor protein function. E256K was not detected either in DNA from a healthy population or in DNA from other hypercholesterolaemic patients studied. CONCLUSIONS: Despite the information available on the structure-function relations between the LDL receptor and LDL receptor like proteins, predictions about the disease causing potential of a mutation are not reliable. These results suggest that the I402T mutation is pathogenic and that the substitution of E256K alone is a rare sequence variation, without a detectable phenotype modulating effect.     

Restriction fragment length polymorphism analysis of ribosomal DNA intergenic regions is useful for differentiating strains of Trichophyton mentagrophytes

Twenty isolates of Tricophyton mentagrophytes var. mentagrophytes and 47 isolates of T. mentagrophytes var. interdigitale, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sixty isolates (14 of 20 T. mentagrophytes var. mentagrophytes isolates and 46 of 47 T. mentagrophytes var. interdigitale isolates) shared an identical ITS RFLP profile and were further investigated by using a probe targeted to the rDNA nontranscribed spacer (NTS) region. Polymorphisms were observed in the NTS regions of both T. mentagrophytes var. mentagrophytes and T. mentagrophytes var. interdigitale isolates. Twenty-three individual RFLP patterns (DNA types P-1 to P-12 and A-1 to A-11) were recognized and divided into two groups depending on the presence (P) or absence (A) of a 2.5-kb band, which correlated to a large extent with the morphological variety. Eleven of 14 T. metagrophytes var. mentagrophytes isolates were A types, and all of the 46 T. mentagrophytes var. interdigitale isolates were P types. A majority of strains (23 of 60 [38.3%]) were characterized by one RFLP pattern (pattern P-1), and eight types (P-1 to P-6, P-8, and P-9) accounted for 75% (45 of 60) of all strains, including all of the T. mentagrophytes var. interdigitale isolates. The remaining 15 types were represented by one only isolate and included all of the T. mentagrophytes var. mentagrophytes isolates. We conclude that RFLP analysis of the rDNA NTS region is a valuable technique for differentiation of T. mentagrophytes strains. Furthermore, by use of this method, there appears to be a greater degree of diversity among T. mentagrophytes var. mentagrophytes isolates than among T. mentagrophytes var. interdigitale isolates.     

Polymorphism of the 5′ terminal region of Citrus tristeza virus (CTV) RNA: Incidence of three sequence types in isolates of different origin and pathogenicity

Sequences of the 5' terminal region of the genomic RNA from eight isolates of Citrus tristeza virus (CTV) were previously classified into three types (I, II and III), with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. Sequencing of an additional 58 cDNA clones from 15 virus isolates showed that all sequences could be unequivocally assigned to one of the three types previously established. The relative frequency of each sequence type was assessed in 57 CTV isolates of different geographic origin and pathogenic characteristics by RT-PCR with sets of type-specific primers using CTV dsRNA as template. None of the isolates yielded amplification of the type I or II sequences alone, but in 19 of them type III sequences were the only amplification product detected. Within isolates containing more than one sequence type, eight had type II and III sequences, 11 had type I and III sequences, and 19 had sequences of the three types. Isolates containing only type III sequences caused only mild to moderate symptoms in Mexican lime, an indicator species for most CTV isolates, whereas isolates causing stem pitting in sweet orange an/or grapefruit, generally contained sequences type II. None of the sequence types could be traced to a precise geographic area, as all types were detected in isolates from at least nine of the 12 countries from which samples were taken.     

Introduction of a strong temperature-sensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase

In 1998, an enterovirus 71 (EV71) epidemic in Taiwan resulted in 78 deaths; however, the molecular basis of EV71 pathogenicity remains poorly understood. Comparison of the deduced amino acid sequences in 3D polymerases of EV71clinical isolates showed the T251V or T251I substitution from 1986 and 1998 outbreaks. An EV71 replicon system showed that introducing an I251T mutation did not affect luciferase activities at 35 °C when compared with wild type; however, lower luciferase activities were observed when they were incubated at 39.5 °C. In addition, the I251T mutation in the EV71 infectious clone not only reduced viral replication at 39.5 °C in vitro but also decreased the virulence of the mouse adaptive strain MP4 in neonatal mice in an i.p. infection model. Therefore, these results suggested that the threonine at position 251 results in a temperature sensitivity phenotype of EV71 which may contribute to the attenuation of circulating strains.     

The phylogenetic position of Chlamydia strains isolated from monkeys and humans with Chlamydial pathology in the family Chiamydiaceae. Genotypic and phenotypic properties of this pathogen

Based on the results of the comparative analysis concerning relatedness and evolutional difference of the 16S - 23S nucleotide sequences of the middle ribosomal cluster and 23S rRNA I domain, and based on identification of phylogenetic position for Chlamydophila pneumoniae and Chlamydia trichomatis strains released from monkeys, relatedness of the above stated isolates with similar strains released from humans and with strains having nucleotide sequences presented in the GenBank electronic database has been detected for the first time ever. Position of these isolates in the Chlamydiaceae family phylogenetic tree has been identified. The evolutional position of the investigated original Chlamydia and Chlamydophila strains close to analogous strains from the GenBank electronic database has been demonstrated. Differences in the 16S - 23S nucleotide sequence of the middle ribosomal cluster and 23S rRNA I domain of plasmid and non-plasmid Chlamydia trachomatis strains released from humans and monkeys relative to different genotype groups (group B- B, Ba, D, Da, E, L1, L2, L2a; intermediate group - F, G, Ga) have been revealed for the first time ever. Abnormality in incA chromosomal gene expression resulting in Chlamydia life and development cycle disorder and decrease of Chlamydia virulence can be related to probable changes in the nucleotide sequence of the gene under consideration.     

Mutational analysis of VACM-1/cul5 exons in cancer cell lines

VACM-1, a cul-5 gene product, functions via an E3 ligase complex and when overexpressed, has an antiproliferative effect in many cell types. Overexpression of VACM-/cul5 cDNA mutated at the PKA-specific phosphorylation site at Ser730 reversed this phenotype. These effects are associated with the appearance of larger M(r) species subsequently identified as a Nedd8-modified VACM-1/cul5. Although decreased levels of VACM-1 mRNA detected in several cancers and cancer cell lines may explain the progression of cell growth, possible genetic and epigenetic changes in its sequence have not been analyzed. We hypothesized that in rapidly proliferating cells, VACM-1/cul5 may be mutated at either the PKA-specific phosphorylation site or the consensus neddylation site. We used RT-PCR and PCR, to amplify and to sequence mRNA and genomic DNA, respectively. To date we have sequenced all 19 coding exons of the VACM-1/cul5 gene in T47D breast cancer cells, U138MG glioma cells, ACHN renal cancer cells, and OVCAR-3 ovarian cancer cells. Our results indicate that in those cells VACM-1/cul5 is not mutated at the putative phosphorylation or the neddylation site. We have found one silent mutation in the genomic DNA isolated from U138MG, ACHN, and OVCAR-3 cell lines, but not from T47D cells. Our work suggests that in T47D breast cancer cells biologic activity of VACM-1/cul5 may be regulated by posttranslational modifications.     

Variations in the occurrence of the S315T mutation within the katG gene in isoniazid-resistant clinical Mycobacterium tuberculosis isolates from Kuwait

The worldwide threat of drug-resistant tuberculosis (TB) to human health has led to the development of molecular methods for rapidly determining the resistance of clinical Mycobacterium tuberculosis isolates to the two front-line antituberculous drugs, isoniazid and rifampin. The prevalence of the S315T mutation within the katG gene, which confers clinically significant resistance to isoniazid, was determined in isoniazid-resistant clinical M. tuberculosis isolates recovered from TB patients in Kuwait. A total of 67 isoniazid-resistant and 18 susceptible clinical M. tuberculosis isolates were tested. The mutation S315T was found in 46 (69%) of the 67 resistant strains, whereas none of the susceptible strains contained this mutation. The prevalence of this mutation was highest (32 of 40, 80%) in isolates recovered from patients of South Asian origin and lowest in isolates from patients of Middle Eastern origin (8 of 18, 44%). The genotyping performed on isolates carrying the S315T mutation showed that the isolates belong to several different types as they exhibited unique DNA banding patterns. The results point to a varying prevalence of the S315T mutation within the katG gene in clinical M. tuberculosis isolates recovered from patients of different ethnic groupings within the same country. The results also suggest that detection of the S315T mutation in the katG gene may be used as a rapid screening method for identifying isoniazid-resistant clinical M. tuberculosis isolates recovered from majority of patients in some ethnic groupings.