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Pradeep K Singh

Publications and source records attributed to Pradeep K Singh.

13 recordsLinked to original sources
PubMed2006

Single-copy genes define a conserved order between rice and wheat for understanding differences caused by duplication, deletion, and transposition of genes.

The high-quality rice genome sequence is serving as a reference for comparative genome analysis in crop plants, especially cereals. However, early comparisons with bread wheat showed complex patterns of conserved synteny (gene content) and colinearity (gene order). Here, we show the presence of ancient duplicated segments in the progenitor of wheat, which were first identified in the rice genome. We also show that single-copy (SC) rice genes, those representing unique matches with wheat expressed sequence tag (EST) unigene contigs in the whole rice genome, show more than twice the proportion of genes mapping to syntenic wheat chromosome as compared to the multicopy (MC) or duplicated rice genes. While 58.7% of the 1,244 mapped SC rice genes were located in single syntenic wheat chromosome groups, the remaining 41.3% were distributed randomly to the other six non-syntenic wheat groups. This could only be explained by a background dispersal of genes in the genome through transposition or other unknown mechanism. The breakdown of rice-wheat synteny due to such transpositions was much greater near the wheat centromeres. Furthermore, the SC rice genes revealed a conserved primordial gene order that gives clues to the origin of rice and wheat chromosomes from a common ancestor through polyploidy, aneuploidy, centromeric fusions, and translocations. Apart from the bin-mapped wheat EST contigs, we also compared 56,298 predicted rice genes with 39,813 wheat EST contigs assembled from 409,765 EST sequences and identified 7,241 SC rice gene homologs of wheat. Based on the conserved colinearity of 1,063 mapped SC rice genes across the bins of individual wheat chromosomes, we predicted the wheat bin location of 6,178 unmapped SC rice gene homologs and validated the location of 213 of these in the telomeric bins of 21 wheat chromosomes with 35.4% initial success. This opens up the possibility of directed mapping of a large number of conserved SC rice gene homologs in wheat. Overall, only 46.4% of these SC genes code for proteins with known functional domains; the remaining 53.6% have unknown function, and hence, represent an important, but yet, under explored category of genes.

Gene Deletion↗
PubMed2006

Mucin-Pseudomonas aeruginosa interactions promote biofilm formation and antibiotic resistance.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in people suffering from cystic fibrosis (CF). In CF airways, P. aeruginosa forms surface-associated communities called biofilms. Compared with free-swimming cultures, biofilms resist clearance by the host immune system and display increased resistance to antimicrobial agents. In this study we developed a technique to coat surfaces with molecules that are abundant in CF airways in order to investigate their impact on P. aeruginosa biofilm development. We found that P. aeruginosa biofilm development proceeds differently on surfaces coated with the glycoprotein mucin compared with biofilm development on glass and surfaces coated with actin or DNA. Biofilms formed on mucin-coated surfaces developed large cellular aggregates and had increased tolerance to the antibiotic tobramycin compared with biofilms grown on glass. Analysis of selected mutant backgrounds in conjunction with time-lapse microscopy revealed that surface-associated motility was blocked on the mucin surface. Furthermore, our data suggest that a specific adhesin-mucin interaction immobilizes the bacterium on the surface. Together, these experiments suggest that mucin, which may serve as an attachment surface in CF airways, impacts P. aeruginosa biofilm development and function.

Anti-Bacterial Agents↗
PubMed2006

Bacterial neuraminidase facilitates mucosal infection by participating in biofilm production.

Many respiratory pathogens, including Hemophilus influenzae, Streptococcus pneumoniae, and Pseudomonas aeruginosa, express neuraminidases that can cleave alpha2,3-linked sialic acids from glycoconjugates. As mucosal surfaces are heavily sialylated, neuraminidases have been thought to modify epithelial cells by exposing potential bacterial receptors. However, in contrast to neuraminidase produced by the influenza virus, a role for bacterial neuraminidase in pathogenesis has not yet been clearly established. We constructed a mutant of P. aeruginosa PAO1 by deleting the PA2794 neuraminidase locus (Delta2794) and tested its virulence and immunostimulatory capabilities in a mouse model of infection. Although fully virulent when introduced i.p., the Delta2794 mutant was unable to establish respiratory infection by i.n. inoculation. The inability to colonize the respiratory tract correlated with diminished production of biofilm, as assessed by scanning electron microscopy and in vitro assays. The importance of neuraminidase in biofilm production was further demonstrated by showing that viral neuraminidase inhibitors in clinical use blocked P. aeruginosa biofilm production in vitro as well. The P. aeruginosa neuraminidase has a key role in the initial stages of pulmonary infection by targeting bacterial glycoconjugates and contributing to the formation of biofilm. Inhibiting bacterial neuraminidases could provide a novel mechanism to prevent bacterial pneumonia.

Amino Acid Sequence↗
PubMed2005

Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms.

The process of detachment, through which bacteria use active mechanisms to leave biofilms and return to the planktonic (free-living) state, is perhaps the least understood aspect of the biofilm life cycle. Like other stages of biofilm development, detachment is a dynamic, regulated process, controlled by specific genes, and induced by particular environmental cues. In previous work we discovered Pseudomonas aeruginosa variants that exhibit accelerated biofilm detachment. These hyper-detaching variants arise spontaneously from biofilms at a high frequency, and they exhibit robust detachment under different biofilm growth conditions. Here we show that these variants detach by a mechanism requiring the biosurfactant rhamnolipid and that this detachment mechanism rapidly restores antibiotic sensitivity to separating bacteria. We also show that rhamnolipids can bring about detachment in wild-type P. aeruginosa biofilms. These findings raise the possibility that this detachment mechanism may be useful as a treatment to disrupt established biofilms. Interestingly, the rhamnolipid-mediated detachment mechanism involves the formation of cavities within the centre of biofilm structures. Our data suggest a model to explain detachment that occurs via this pattern.

Bacterial Adhesion↗
PubMed2005

Pseudomonas aeruginosa acquires biofilm-like properties within airway epithelial cells.

Pseudomonas aeruginosa can notably cause both acute and chronic infection. While several virulence factors are implicated in the acute phase of infection, advances in understanding bacterial pathogenesis suggest that chronic P. aeruginosa infection is related to biofilm formation. However, the relationship between these two forms of disease is not well understood. Accumulating evidence indicates that, during acute infection, P. aeruginosa enters epithelial cells, a process viewed as either a host-mediated defense response or a pathogenic mechanism to avoid host-mediated killing. We investigated the possibility that epithelial cell entry during early P. aeruginosa-epithelial cell contact favors bacterial survival and is linked to chronic infection. Using electron microscopy and confocal microscopy to analyze primary culture airway epithelial cells infected with P. aeruginosa, we found that epithelial cells developed pod-like clusters of intracellular bacteria with regional variation in protein expression. Extracellular gentamicin added to the medium after acute infection led to the persistence of intracellular P. aeruginosa for at least 3 days. Importantly, compared to bacterial culture under planktonic conditions, the intracellular bacteria were insensitive to growth inhibition or killing by antibiotics that were capable of intraepithelial cell penetration. These findings suggest that P. aeruginosa can use airway epithelial cells as a sanctuary for persistence and develop a reversible antibiotic resistance phenotype characteristic of biofilm physiology that can contribute to development of chronic infection.

Animals↗
PubMed2005

Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology.

The opportunistic human pathogen Pseudomonas aeruginosa causes persistent airway infections in patients with cystic fibrosis (CF). To establish these chronic infections, P. aeruginosa must grow and proliferate within the highly viscous sputum in the lungs of CF patients. In this study, we used Affymetrix GeneChip microarrays to investigate the physiology of P. aeruginosa grown using CF sputum as the sole source of carbon and energy. Our results indicate that CF sputum readily supports high-density P. aeruginosa growth. Furthermore, multiple signals, which reduce swimming motility and prematurely activate the Pseudomonas quinolone signal cell-to-cell signaling cascade in P. aeruginosa, are present in CF sputum. P. aeruginosa factors critical for lysis of the common CF lung inhabitant Staphylococcus aureus were also induced in CF sputum and increased the competitiveness of P. aeruginosa during polymicrobial growth in CF sputum.

Culture Media↗
PubMed2004

Self-generated diversity produces "insurance effects" in biofilm communities.

Diversity generally protects communities from unstable environmental conditions. This principle, known as the "insurance hypothesis," has been tested in many different ecosystems. Here we show that the opportunistic pathogen Pseudomonas aeruginosa undergoes extensive genetic diversification during short-term growth in biofilm communities. The induced genetic changes are produced by a recA-dependent mechanism and affect multiple traits, including the behavior of the bacteria in biofilms. Some biofilm-derived variants exhibit an increased ability to disseminate, whereas others manifest accelerated biofilm formation. Furthermore, the presence of these functionally diverse bacteria increases the ability of biofilms to resist an environmental stress. These findings suggest that self-generated diversity in biofilms provides a form of biological insurance that can safeguard the community in the face of adverse conditions.

Biofilms↗
PubMed2003

Bacterial biofilms: an emerging link to disease pathogenesis.

The role of biofilms in the pathogenesis of some chronic human infections is now widely accepted. However, the criteria used to determine whether a given infection is caused by biofilms remain unclear. In this chapter we discuss three infections that are caused by biofilms--infectious kidney stones, bacterial endocarditis, and cystic fibrosis lung infections--and focus on the role of the biofilm in disease pathogenesis. Biofilms are also important as environmental reservoirs for pathogens, and the biofilm growth mode may provide organisms with survival advantages in natural environments and increase their virulence. The consequences of pathogens living in environmental biofilms and an analysis of some specific environmental biofilm systems are presented.

Bacterial Adhesion↗
PubMed2002

A component of innate immunity prevents bacterial biofilm development.

Antimicrobial factors form one arm of the innate immune system, which protects mucosal surfaces from bacterial infection. These factors can rapidly kill bacteria deposited on mucosal surfaces and prevent acute invasive infections. In many chronic infections, however, bacteria live in biofilms, which are distinct, matrix-encased communities specialized for surface persistence. The transition from a free-living, independent existence to a biofilm lifestyle can be devastating, because biofilms notoriously resist killing by host defence mechanisms and antibiotics. We hypothesized that the innate immune system possesses specific activity to protect against biofilm infections. Here we show that lactoferrin, a ubiquitous and abundant constituent of human external secretions, blocks biofilm development by the opportunistic pathogen Pseudomonas aeruginosa. This occurs at lactoferrin concentrations below those that kill or prevent growth. By chelating iron, lactoferrin stimulates twitching, a specialized form of surface motility, causing the bacteria to wander across the surface instead of forming cell clusters and biofilms. These findings reveal a specific anti-biofilm defence mechanism acting at a critical juncture in biofilm development, the time bacteria stop roaming as individuals and aggregate into durable communities.

Biofilms↗
PubMed2002

ExsD is a negative regulator of the Pseudomonas aeruginosa type III secretion regulon.

Expression of the Pseudomonas aeruginosa type III secretion system is induced by contact with eukaryotic cells, serum or low Ca2+ concentrations. We report that ExsD, a unique protein, is a negative regulator of the type III regulon. Localization studies indicate that ExsD is not secreted by P. aeruginosa. To determine the role of exsD, a non-polar deletion was returned to the chromosome by allelic exchange. The delta exsD mutant is competent for type III secretion and translocation of the ExoU cytotoxin to eukaryotic host cells. To examine the effect of ExsD on transcription, lacZ transcriptional reporter fusions were integrated into the chromosome. Promoters controlling transcription of genes encoding the type III secretory, regulatory and effector proteins demonstrated significant derepression in the delta exsD background. Expression of ExsD from a multicopy plasmid completely repressed transcription of the regulon. Although a mutant in pscC, encoding a structural component of the type III translocase, is repressed for expression of the regulon, a delta exsD, pscC:: omega double mutant is derepressed. Bacterial two-hybrid data indicate that ExsD binds the transcriptional activator of the regulon, ExsA. We conclude that ExsD is a negative regulator and propose that ExsD functions as an ExsA antiactivator to regulate transcription of the regulon.

Bacterial Proteins↗