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Jason Hinds

Publications and source records attributed to Jason Hinds.

31 records · Page 2Linked to original sources
PubMed2004

The influence of reduced oxygen availability on pathogenicity and gene expression in Mycobacterium tuberculosis.

We investigated how Mycobacterium tuberculosis responded to a reduced oxygen tension in terms of its pathogenicity and gene expression by growing cells under either aerobic or low-oxygen conditions in chemostat culture. The chemostat enabled us to control and vary the oxygen tension independently of other environmental parameters, so that true cause-and-effect relationships of reduced oxygen availability could be established. Cells grown under low oxygen were more pathogenic for guinea pigs than those grown aerobically. The effect of reduced oxygen on global gene expression was determined using DNA microarray. Spearman rank correlation confirmed that microarray expression profiles were highly reproducible between repeat cultures. Using microarray analysis we have identified genes that respond to a low-oxygen environment without the influence of other parameters such as nutrient depletion. Some of these genes appear to be involved in the biosynthesis of cell wall precursors and their induction may have contributed to increased infectivity in the guinea pig. This study has shown that a combination of chemostat culture and microarray presents a biologically robust and statistically reliable experimental approach for studying the effect of relevant and specific environmental stimuli on mycobacterial virulence and gene expression.

Anaerobiosis↗
PubMed2004

Genome structure in the vole bacillus, Mycobacterium microti, a member of the Mycobacterium tuberculosis complex with a low virulence for humans.

Mycobacterium microti, a member of the Mycobacterium tuberculosis complex, is phylogenetically closely related to M. tuberculosis, differing in a few biochemical properties. However, these species have different levels of virulence in different hosts; most notably M. microti shows lower virulence for humans than M. tuberculosis. This report presents genomic comparisons using DNA microarray analysis for an extensive study of the diversity of M. microti strains. Compared to M. tuberculosis H37Rv, 13 deletions were identified in 12 strains of M. microti, including the regions RD1 to RD10, which are also missing in Mycobacterium bovis BCG. In addition, four new deleted regions, named MiD1, RD1beta, MiD2 and MiD3, were identified. DNA sequencing was used to define the extent of most of the deletions in one strain. Although RD1 of M. bovis BCG and M. microti is thought to be crucial for attenuation, in this study, three of the four M. microti strains that were isolated from immunocompetent patients had the RD1 deletion. In fact, only the RD3 deletion was present in all of the strains examined, although deletions RD7, RD8 and MiD1 were found in almost all the M. microti strains. These deletions might therefore have some relation to the different host range of M. microti. It was also noticeable that of the 12 strains studied, only three were identical; these strains were all isolated from immunocompetent humans, suggesting that they could have arisen from a single source. Thus, this study shows that it is difficult to ascribe virulence to any particular pattern of deletion in M. microti.

Animals↗
PubMed2004

DNA microarrays for virus detection in cases of central nervous system infection.

A low-density, high-resolution diagnostic DNA microarray comprising 38 gene targets for 13 viral causes of meningitis and encephalitis was constructed. The array has been used for the detection of multiplex PCR-amplified viruses in cerebrospinal fluid (CSF) and non-CSF specimens. A total of 41 clinical specimens were positive for echoviruses (23 samples), herpes simplex virus type 2 (4 samples), varicella-zoster virus (4 samples), human herpesvirus 7 (1 sample), human herpesvirus 6A (1 sample) and 6B (2 samples), Epstein-Barr virus (three samples), polyomavirus JC (1 sample), and cytomegalovirus (2 samples). Probes for herpes simplex virus type 1, polyomavirus BK, and mumps and measles viruses were also included on the array. Three samples were false negative by the microarray assay due to discordant results between the multiplex PCR for all 13 viruses simultaneously and the virus-specific PCR alone. Fifteen CSF specimens were true negative. The clinical sensitivity, specificity, and negative and positive predictive values of the assay were 93, 100, 100, and 83%, respectively, when the results were compared to those of the single-virus PCR, which was used as the "gold standard." The microarray-based virus detection assay is qualitative and provides a single-format diagnostic tool for the detection of panviral CNS infections.

Central Nervous System Viral Diseases↗
PubMed2004

Use of genome level-informed PCR as a new investigational approach for analysis of outbreak-associated Mycobacterium tuberculosis isolates.

Mycobacterium tuberculosis strain CH, the index isolate linked to a major tuberculosis outbreak associated with high levels of transmissibility and virulence, was characterized by microarray analysis by use of a PCR product array representative of the genome of M. tuberculosis strain H37Rv. Seven potential genomic deletions were identified in CH, five of which were confirmed by PCR analysis across the predicted deletion points. The panel of five PCRs required to individually interrogate these loci was collectively referred to as the genome level-informed PCR (GLIP) assay. GLIP analysis was performed with CH, 12 other epidemiologically linked isolates, and 43 recent, non-outbreak-associated isolates derived from patients within the local area. All 13 outbreak-linked isolates showed a profile corresponding to the presence of all five deletions. These 13 isolates were also found to share common variable-number tandem repeat and mycobacterial interspersed repetitive unit profiles. None of the 43 non-outbreak-associated isolates exhibited the five-deletion profile. Although three individual deletions were present in upwards of 44% of the non-outbreak-associated isolates, no single-deletion isolates were detected. Interestingly, none of these deletions had been previously recognized, and sequence analysis of the immediate flanking regions in CH failed to identify a likely mechanism of deletion for four of the five loci. The GLIP assay also proved valuable in ongoing surveillance of the outbreak, rapidly identifying a further two outbreak-associated cases months after the initial cluster and, importantly, dismissing a further 12 epidemiologically suspect cases, which allowed the optimum deployment of public health resources.

Base Sequence↗
PubMed2003

The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA.

In many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction. We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis. Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA. Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild-type M. tuberculosis and a recA deletion strain of M. tuberculosis using microarrays. This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain. Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction. Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination. Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M. tuberculosis.

DNA Damage↗
PubMed2003

Analysis of whole-genome microarray replicates using mixed models.

MOTIVATION: Microarray experiments are inherently noisy. Replication is the key to estimating realistic fold-changes despite such noise. In the analysis of the various sources of noise the dependency structure of the replication needs to be taken into account. RESULTS: We analyzed replicate data sets from a Mycobacterium tuberculosis trcS mutant in order to identify differentially expressed genes and suggest new methods for filtering and normalizing raw array data and for imputing missing values. Mixed ANOVA models are applied to quantify the various sources of error. Such analysis also allows us to determine the optimal number of samples and arrays. Significance values for differential expression are obtained by a hierarchical bootstrapping scheme on scaled residuals. Four highly upregulated genes, including bfrB, were analyzed further. We observed an artefact, where transcriptional readthrough from these genes led to apparent upregulation of adjacent genes. AVAILABILITY: All methods and data discussed are available in the package YASMAhttp://www.cryst.bbk.ac.uk/wernisch/yasma.html for the statistical data analysis system R (http://www.R-project.org).

Algorithms↗
PubMed2003

Application of DNA microarrays to study the evolutionary genomics of Yersinia pestis and Yersinia pseudotuberculosis.

Yersinia pestis, the causative agent of plague, diverged from Yersinia pseudotuberculosis, an enteric pathogen, an estimated 1500-20,000 years ago. Genetic characterization of these closely related organisms represents a useful model to study the rapid emergence of bacterial pathogens that threaten mankind. To this end, we undertook genome-wide DNA microarray analysis of 22 strains of Y. pestis and 10 strains of Y. pseudotuberculosis of diverse origin. Eleven Y. pestis DNA loci were deemed absent or highly divergent in all strains of Y. pseudotuberculosis. Four were regions of phage origin, whereas the other seven included genes encoding a vitamin B12 receptor and the insect toxin sepC. Sixteen differences were identified between Y. pestis strains, with biovar Antiqua and Mediaevalis strains showing most divergence from the arrayed CO92 Orientalis strain. Fifty-eight Y. pestis regions were specific to a limited number of Y. pseudotuberculosis strains, including the high pathogenicity island, three putative autotransporters, and several possible insecticidal toxins and hemolysins. The O-antigen gene cluster and one of two possible flagellar operons had high levels of divergence between Y. pseudotuberculosis strains. This study reports chromosomal differences between species, biovars, serotypes, and strains of Y. pestis and Y. pseudotuberculosis that may relate to the evolution of these species in their respective niches.

Adaptation, Physiological↗
PubMed2003

Genomic analysis of Mycobacterium tuberculosis complex strains used for production of purified protein derivative.

The genomes of the tuberculin production strains Mycobacterium bovis AN5 and Mycobacterium tuberculosis DT were compared to genome-sequenced tubercle bacilli by using DNA microarrays. Neither the AN5 nor DT strain suffered extensive gene deletions during in vitro passage. This suggests that bovine tuberculin made from M. bovis AN5 is suitable to detect infection with presently prevalent M. bovis strains.

Gene Deletion↗
PubMed2002

Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays.

Regulation of the expression of heat-shock proteins plays an important role in the pathogenesis of Mycobacterium tuberculosis. The heat-shock response of bacteria involves genome-wide changes in gene expression. A combination of targeted mutagenesis and whole-genome expression profiling was used to characterize transcription factors responsible for control of genes encoding the major heat-shock proteins of M. tuberculosis. Two heat-shock regulons were identified. HspR acts as a transcriptional repressor for the members of the Hsp70 (DnaK) regulon, and HrcA similarly regulates the Hsp60 (GroE) response. These two specific repressor circuits overlap with broader transcriptional changes mediated by alternative sigma factors during exposure to high temperatures. Several previously undescribed heat-shock genes were identified as members of the HspR and HrcA regulons. A novel HspR-controlled operon encodes a member of the low-molecular-mass alpha-crystallin family. This protein is one of the most prominent features of the M. tuberculosis heat-shock response and is related to a major antigen induced in response to anaerobic stress.

Bacterial Proteins↗
PubMed1999

Enhanced gene replacement in mycobacteria.

Allelic replacement will be a vital tool for understanding gene function in mycobacteria. Disruption of the chromosomal hisD gene of Mycobacterium smegmatis by standard gene replacement methods was surprisingly difficult, with most products being caused by illegitimate recombination (IR) events. A recombination assay was therefore developed and used to optimize conditions for homologous recombination (HR) in M. smegmatis. Treatment of competent cells with UV, hydrogen peroxide or mitomycin C did not improve the frequency of HR; however, treatment of the DNA with alkali or UV enhanced recombination frequency, while boiling did not. Applying these observations to allele replacement, UV and alkali treatment of transforming DNA increased HR events with pyrF and hisD, while the level of IR was unchanged. The introduction of ss phagemid DNA improved the level of HR and abolished IR. In Mycobacterium intracellulare the use of alkali-denatured DNA increased the numbers of recombinants obtained with an inactivated 19Ag gene, while in Mycobacterium tuberculosis, inactivation of a putative haemolysin gene, tlyA, was achieved using both UV-irradiated DNA and ss phagemid DNA. Significantly, IR, which has been reported to be a problem in this species, was not observed. Thus, four genes in three species were successfully knocked-out using non-replicating DNA pretreated with alkali, UV or in an ss form. The use of these methods to enhance HR will greatly facilitate experiments to inactivate other genes in these important species.

Alcohol Oxidoreductases↗