Biomedical subjects

Ruiting Lan

Publications and source records attributed to Ruiting Lan.

16 recordsLinked to original sources

PubMed2026

Phage-encoded sRNA counteracts xenogeneic silencing in pathogenic E. coli.

Horizontal gene transfer introduces foreign DNA that can disrupt cellular processes and is therefore subject to xenogeneic silencing by nucleoid-associated proteins such as H-NS and Hha. In Enterohaemorrhagic Escherichia coli (EHEC), prophages make up a large fraction of the accessory genome and encode many virulence factors, yet their expression must overcome this silencing. We identify a prophage-encoded small RNA (sRNA), HnrS, that functions as an anti-silencing factor by targeting the H-NS paralogue Hha. HnrS is a short (66-nt) sRNA that is enriched in the locus of enterocyte effacement (LEE⁺) E. coli strains and present in up to nine copies in EHEC and Enteropathogenic Escherichia coli (EPEC) genomes. HnrS base-pairs with the hha ribosome-binding site to inhibit translation, thereby modulating Hha-H-NS repression of virulence loci including the LEE type III secretion system. Loss of HnrS alters motility, T3SS expression, and a subset of Hha-regulated genes. These findings reveal an RNA-based counter-silencing strategy encoded by prophage to relieve xenogenic silencing.

Escherichia coli Proteins↗

PubMed2025

Proteomic comparison of epidemic Australian Bordetella pertussis biofilm cells.

Bordetella pertussis causes whooping cough, a severe respiratory infectious disease. Studies have compared the currently dominant single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele, ptxP3) and previously dominant SNP cluster II (ptxP1) strains as planktonic cells. Since biofilm formation is linked with B. pertussis pathogenesis in vivo, this study compared the biofilm formation capabilities of representative strains of cluster I and cluster II. Confocal laser scanning microscopy found that the cluster I strain had a denser biofilm structure compared to the cluster II strain. Differences in protein abundance of the biofilm cells were then compared using tandem mass tagging and high-resolution multiple reaction monitoring. In total, 1,453 proteins were identified, of which 40 proteins had significant differential abundance between the two strains in biofilm conditions. Of particular interest was a large increase in the abundance of energy metabolism proteins (cytochrome proteins PetABC and BP3650) in the cluster I strain. When the abundance of these proteins was compared between six additional strains from each cluster, it was found that the protein abundance varied between all strains. These findings suggest that there are large levels of individual proteomic diversity between B. pertussis strains in biofilm conditions despite the highly conserved genome of the species. Overall, this study revealed visual differences in biofilm structure between B. pertussis strains and highlighted strain-specific variation in protein abundance that dominates potential cluster-specific changes that may be linked with the dominance of cluster I strains.IMPORTANCEBordetella pertussis causes whooping cough. The currently circulating cluster I strains have taken over previously dominant cluster II strains. It is important to understand the reasons behind this evolution to develop new strategies against the pathogen. Recent studies have shown that B. pertussis can form biofilms during infection. This study compared the biofilm formation capabilities of a cluster I and a cluster II strain and identified visual differences in the biofilms. The protein abundance between these strains grown in biofilms was compared, and proteins identified with varied abundance were measured with additional strains from each cluster. It was found that despite the highly conserved genetics of the species, there was varied protein abundance between the additional strains. This study highlights that strain-specific variation in protein abundance during biofilm conditions may dominate the cluster-specific changes that may be linked to the dominance of cluster I strains.

Bordetella pertussis↗

PubMed2006

Molecular markers with potential to replace phage typing for Salmonella enterica serovar typhimurium.

Using Amplified Fragment Length Polymorphism (AFLP) analysis of isolates from 23 phage types, we isolated 11 molecular markers that are potentially useful for molecular typing of Salmonella enterica serovar typhimurium. We tested these and 11 previously studied markers for their ability to discriminate among isolates and for correlation of their distribution with phage types. The Simpson's index of discriminatory power for the molecular markers is 0.96. One hundred and twenty one isolates from 33 phage types tested were divided into 51 types which are further grouped into 24 patterns. Eight patterns can unambiguously identify 8 phage types and a further 12 correlated with phage type distribution, showing the usefulness of these markers for molecular phage typing.

Bacteriophage Typing↗

PubMed2006

Frequent recombination and low level of clonality within Salmonella enterica subspecies I.

The genetic relationship and population structure of Salmonella enterica subspecies I strains were analysed using nucleotide sequences of four genes (mglA, proV, torC and speC). Fifteen strains from the Salmonella reference collection B (SARB), belonging to 13 serovars, were analysed. Sequence data of two housekeeping genes, mdh and mutS, of the same 15 strains reported by Brown et al. (2003) (Proc Natl Acad Sci U S A 100, 15676-15681) were also included in the analyses. Phylogenetic analysis revealed that there was a lack of congruence among the six gene trees. Split decomposition analysis resolved only five strains with a network structure, while others showed a star phylogeny. Compatibility values for the SARB strains were the lowest in comparison to those for strains representing different subspecies of S. enterica. These results showed that the genes studied have undergone frequent recombination, suggesting a low level of clonality within subspecies I of S. enterica.

Bacterial Proteins↗

PubMed2006

Sex and virulence in Escherichia coli: an evolutionary perspective.

Pathogenic Escherichia coli cause over 160 million cases of dysentery and one million deaths per year, whereas non-pathogenic E. coli constitute part of the normal intestinal flora of healthy mammals and birds. The evolutionary pathways underlying this dichotomy in bacterial lifestyle were investigated by multilocus sequence typing of a global collection of isolates. Specific pathogen types [enterohaemorrhagic E. coli, enteropathogenic E. coli, enteroinvasive E. coli, K1 and Shigella] have arisen independently and repeatedly in several lineages, whereas other lineages contain only few pathogens. Rates of evolution have accelerated in pathogenic lineages, culminating in highly virulent organisms whose genomic contents are altered frequently by increased rates of homologous recombination; thus, the evolution of virulence is linked to bacterial sex. This long-term pattern of evolution was observed in genes distributed throughout the genome, and thereby is the likely result of episodic selection for strains that can escape the host immune response.

Alleles↗

PubMed2005

Adaptation of multilocus sequencing for studying variation within a major clone: evolutionary relationships of Salmonella enterica serovar Typhimurium.

Serovar Typhimurium of Salmonella enterica is a model organism for studies of pathogenesis that exhibits phage-type variation and variation in host range and virulence, but in a recent study showed no sequence variation in four genes, indicating the clonal nature of this serovar. We determined the relationships of 46 Typhimurium isolates of nine phage types using mutational changes detected either by matching AFLP (amplified fragment length polymorphism) fragments to computer-modeled LT2 AFLP fragments or by sequencing intergenic regions. Fifty-one polymorphic sites were detected, which gave a single phylogenetic tree. Comparison with genome sequences of five other serovars, Typhi, Paratyphi A, Gallinarum, Enteritidis, and Pullorum, enabled determination of the root of the tree. Only two parallel events were observed, giving high confidence in the tree branching order. The mutation-based tree provided a high level of consistency and a clear lineage for the Typhimurium isolates studied. This enabled us to show that for seven of the nine phage types used, the isolates studied have a single origin, but that two phage types clearly have more than one independent origin. We found that sequencing intergenic regions provides a good strategy for detection of mutational polymorphisms and study of phylogenetic relationships of closely related isolates and would be applicable to many other species.

Clone Cells↗

PubMed2005

Evolutionary changes of the flhDC flagellar master operon in Shigella strains.

Shigella strains are nonmotile. The master operon of flagellar synthesis, flhDC, was analyzed for genetic damage in 46 Shigella strains representing all known serotypes. In 11 strains (B1, B3, B6, B8, B10, B18, D5, F1B, D10, F3A, and F3C) the flhDC operon was completely deleted. PCR and sequence analysis of the flhDC region of the remaining 35 strains revealed many insertions or deletions associated with insertion sequences, and the majority of the strains were found to be defective in their flhDC genes. As these genes also play a role in regulation of non-flagellar genes, the loss may have other consequences or be driven by selection pressures other than those against flagellar motility. It has been suggested that Shigella strains fall mostly into three clusters within Escherichia coli, with five outlier strains, four of which are also within E. coli (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000). The distribution of genetic changes in the flhDC region correlated very well with the three clusters and outlier strains found using housekeeping gene DNA sequences, enabling us to follow the sequence of mutational change in the flhDC locus. Two cluster 2 strains were found to have unique flhDC sequences, which are most probably due to recombination during the exchange of the adjacent O-antigen gene clusters.

Base Sequence↗

PubMed2005

Vibrio cholerae pathogenic clones.

We resolved the relationships between 2 pandemic clones of Vibrio cholerae. Using 26 housekeeping genes, we showed that the US Gulf clone, the Australian clone, and 3 El Tor strains isolated before the seventh pandemic were related to the seventh pandemic clone. The sixth pandemic clone was well separated from them.

Bacterial Proteins↗

PubMed2004

Molecular evolutionary relationships of enteroinvasive Escherichia coli and Shigella spp.

Enteroinvasive Escherichia coli (EIEC), a distinctive pathogenic form of E. coli causing dysentery, is similar in many properties to bacteria placed in the four species of Shigella. Shigella has been separated as a genus but in fact comprises several clones of E. coli. The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of Shigella strains. The EIEC strains were grouped in four clusters with one outlier strain, indicating independent derivation of EIEC several times. Three of the four clusters contain more than one O antigen type. One EIEC strain (an O112ac:H- strain) was found in Shigella cluster 3 but is not identical to the Shigella cluster 3 D2 and B15 strains with the same O antigen. Two forms of the virulence plasmid pINV have been identified in Shigella strains by using the sequences of ipgD and mxiA genes, and all but two of our EIEC strains have pINV A. The EIEC strains were grouped in two subclusters with a very low level of variation, generally not intermingled with Shigella pINV A strains. The EIEC clusters based on housekeeping genes were reflected in the plasmid gene sequences, with some exceptions. Two strains were found in the pINV B form by using the ipgD sequence, with one strain having an mxiA sequence similar to the divergent sequence of D1. Clearly, EIEC and Shigella spp. form a pathovar of E. coli.

Bacterial Proteins↗

PubMed2004

Molecular basis of the indole-negative reaction in Shigella strains: extensive damages to the tna operon by insertion sequences.

The molecular basis of the loss of tryptophan utilization (indole-negative phenotype) of Shigella strains, in effect clones of Escherichia coli, was investigated. Analysis of the tna operon of 23 Shigella strains representing each of the indole-negative serotypes revealed that insertion sequence-mediated insertion and/or deletions damaged the tna operon, leading to inability to convert tryptophan to indole. These events differ for cluster 1, cluster 3, and the outlier Shigella strains, confirming our previous observation of independent origins of these lineages from within E. coli. Parallel loss of the trait and prevalence of indole-negative strains suggest that the trait is deleterious in Shigella strains and advantages those without it.

DNA Transposable Elements↗

PubMed2003

AFLP analysis of Salmonella enterica serovar Typhimurium isolates of phage types DT 9 and DT 135: diversity within phage types and its epidemiological significance.

Amplified fragment length polymorphism (AFLP) was applied to 35 and 34 isolates, respectively, of Salmonella enterica serovar Typhimurium phage types DT 9 and DT 135, using eight primer pair combinations. Eight and 17 AFLP types were observed in DT 9 and DT 135, respectively. DT 9 is rare in the UK and common in Australia, but one AFLP form dominated with 28 isolates, comprising 22 of 25 UK isolates, four of five Australian isolates, one Jamaican and one Spanish isolate. Of the others, two UK isolates are closely related to the major form, two from elsewhere are in the major cluster and three isolates from different countries are in a separate cluster. For DT 135, two closely related AFLP types of seven and 11 isolates form the major cluster, which also includes 11 isolates, mostly in single-isolate AFLP types, while five isolates from different countries form a well-separated minor cluster. For both DTs all isolates are grouped together if only the phage type specific bands identified earlier are used, confirming their value for molecular-based 'phage typing'. Polymorphic markers identified in this study could also be used for subtyping within both phage types. The value of AFLP is in locating DNA fragments useful for typing, but implementation of a replacement typing scheme would probably involve multiplex PCR or microarray technologies.

Bacterial Typing Techniques↗

PubMed2003

Comparison of two major forms of the Shigella virulence plasmid pINV: positive selection is a major force driving the divergence.

All Shigella and enteroinvasive Escherichia coli (EIEC) strains carry a 230-kb virulence plasmid (pINV) which is essential for their invasiveness. There are two sequence forms, pINV A and pINV B, of the plasmid (R. Lan, B. Lumb, D. Ryan, and P. R. Reeves, Infect. Immun. 69:6303-6309, 2001), and the recently sequenced pINV plasmid from Shigella flexneri serotype 5 is a pINV B form. In this study we sequenced the majority of the coding region of the pINV A form from S. flexneri serotype 6 other than insertion sequence or related sequences and compared it with the pINV B form. More than half of the genes sequenced appear to be under positive selection based on their low ratio of synonymous to nonsynonymous substitutions. This high proportion of selected differences indicates that the two pINV forms have functional differences, and comparative studies of pathogenicity in different Shigella-EIEC strains could be informative. There are also genes absent in the S. flexneri serotype 6 plasmid, including the sepA gene encoding serine protease, the major secreted protein of S. flexneri serotype 2a, and the stbAB genes, which encode one of the two partition systems found in S. flexneri serotype 5. The incompatibility of the two pINV forms appears to be due to either small differences in the mvpAT postsegregational killing system or the presence of an unknown system in pINVA.

Amino Acid Sequence↗

PubMed2002

Escherichia coli in disguise: molecular origins of Shigella.

Shigella, which still stands as a genus with four species today, in reality belongs to the extremely diverse species Escherichia coli. There are several lineages of Shigella strains derived through independent acquisition of the pINV virulence plasmid. The chromosomally determined phenotypic properties of Shigella result from convergent evolution during niche adaptation, most due to loss of function, some from negative selection pressure.

Adaptation, Biological↗

PubMed2002

Pandemic spread of cholera: genetic diversity and relationships within the seventh pandemic clone of Vibrio cholerae determined by amplified fragment length polymorphism.

The seventh cholera pandemic started in 1961 and continues today. A collection of 45 seventh pandemic isolates of V. cholerae sampled over a 33-year period were analyzed by amplified fragment length polymorphism (AFLP) fingerprinting. All but four pairs and one set of three isolates were distinguished. AFLP revealed far more variation than ribotyping, which was until now the most useful method of revealing variation within the pandemic clone. Unfortunately, the ribotype variation observed is mainly due to recombination between the multiple copies of the rrn genes (R. Lan and P. R. Reeves, Microbiology 144:1213-1221, 1998), which makes changes susceptible to repeat occurrences and reversion. This AFLP study shows that particularly for the common ribotypes G and H, such events have indeed occurred. AFLP grouped most of the 45 isolates into two clusters. Cluster I consists mainly of strains from the 1960s and 1970s, while cluster II contains mainly strains from the 1980s and 1990s, revealing a temporal pattern of change in the clone. This is best seen in the relationships of the strains from Africa, which correlate with the epidemiology of epidemics on that continent. The data confirm independent introductions to Africa during the 1970s outbreak and reveal several other African introductions. In the 1991 cholera upsurge, isolates from the Southern and Eastern African epidemic focus are markedly different from those from the West African epidemic focus. An isolate from 1987 in Algeria was identical to the West epidemic isolates, suggesting that the strain was present in Africa at least 3 years before causing large outbreaks. These observations have major implications for our understanding of cholera epidemiology.

Cholera↗

PubMed2002

Fluorescent amplified fragment length polymorphism analysis of Salmonella enterica serovar typhimurium reveals phage-type- specific markers and potential for microarray typing.

Fluorescent amplified fragment length polymorphism (AFLP) was applied to 46 Salmonella enterica serovar Typhimurium isolates of Australian origin comprising nine phage types, by using the restriction enzymes MseI and EcoRI and all 16 possible MseI +1-EcoRI +1 primer pair combinations. AFLP in the present study showed a very good discrimination power with a Simpson index of diversity of 0.98, and 35 different AFLP patterns were observed in the 46 isolates. AFLP grouped most serovar Typhimurium isolates by phage type and enabled differentiation of phage types. Furthermore, 84 phage-type-specific polymorphic AFLP fragments, for which presence or absence correlated with phage type (including 25 with one exception to phage type specificity) were observed in the 46 strains studied. Eighteen phage-type-specific AFLP fragments were cloned and sequenced. Fifteen are of known genes or have a homologue in the databases. Three sequences are plasmid related, eight are phage related, and four relate to chromosomal genes. Twelve of the 18 fragments are polymorphic because the DNA is present or absent as indicated by Southern hybridization, and we see good potential to use sequences of these fragments as the basis for multiplex PCR and development of a microarray-based molecular phage-typing method for serovar Typhimurium.

Animals↗

PubMed1998

Recombination between rRNA operons created most of the ribotype variation observed in the seventh pandemic clone of Vibrio cholerae.

Individual rrn operons and their flanking regions have been analysed in a study of the molecular basis of ribotype variation in the seventh pandemic clone of Vibrio cholerae. The genome of an early isolate of the seventh pandemic clone had nine rrn operons of which two were in tandem with other rrn operons. The site for BglI, the most discriminatory enzyme used for ribotyping, was found to be present in the 16S sequence of three of the operons of the earliest isolate. This site was observed to be gained or lost in specific operons in many later isolates, presumably by recombination, and this gave most of the ribotype variation. Additional rrn recombination events were uncovered by analysis of the 16S-23S intergenic spacers associated with each operon. Spacers of 431, 509, 607 and 711 bp were found. A total of at least eight rrn recombination events were detected. Three rrn loci were primarily involved in this recombination, with four new forms generated from that in the early strains for operon B and two new forms each for operons C and G. In addition there was variation due to deletion of tandem operons. The frequency of recombination between rrn operons was very high as there were nine new ribotypes found among 47 isolates sampled over the 33 year period of study. This means that any variation could undergo precise reversion by the same recombination event within the time frame covered by the study. Recombination between rrn operons may be a factor in ribotype variation in all systems. The recombination observed is thought to be that which results in concerted evolution and the data give an indication of the rate involved.

Bacterial Typing Techniques↗