15 research outputs found
Role of mutation in pseudomonas aeruginosa biofilm development.
No full textThe survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations
GFP fluorescence-inducing mutations (FIMS) occur during <i>P. aeruginosa</i> pMDGFP biofilm development.
No full text<p>FIMS were observed exclusively within microcolony structures, but not within unstructured ‘carpet’ regions of the biofilm as in (A), which shows bright field (BF) and fluorescence (FL) images of the same field of view. FIMS were observed as individual GFP-expressing cells (A), or as clusters of GFP cells within microcolonies possibly due to clonal expansion following GPF reversion (B, C). Microcolonies comprised wholly of GFP-expressing cells were occasionally observed (D).</p
Microevolutionary model for microbial biofilm growth.
No full text<p>(A) A bacterial cell attached to a substratum acquires a primary mutation (•) that provides a growth advantage on the surface; (B) mutation selection and clonal expansion occurs; some cells acquire beneficial secondary mutations (×), for example due to exogenous DNA damaging stresses localized to microcolonies; (C) subsequent waves of clonal expansion contribute to microcolony growth and proliferation.</p
<i>P. aeruginosa</i> Δ<i>mutS</i> and Δ<i>mutL</i> strains exhibit enhanced microcolony initiation and development (A), and also show increased total biofilm volume (B) and maximum microcolony thickness (C).
No full text<p>Biofilm development of <i>P. aeruginosa</i> PAO1 WT, Δ<i>mutS</i>, Δ<i>mutL</i>, Δ<i>mutS</i>+pUCPMS and Δ<i>mutL</i>+pUCmutL grown in continuous culture flow cells over a 10 day period and examined using confocal scanning laser microscopy. Asterisks indicate a significant difference compared with WT or related complemented strain (P<0.05).</p
Influence of mutagen dose levels on mutation rate in <i>P. aeruginosa</i> assessed by pMDGFP FIM fluorescent colony assays.
No full text<p>Data for each of three replicate experiments are shown.</p
Representative confocal scanning laser microscopy images showing biofilms of <i>P. aeruginosa</i> wild-type, Δ<i>mutS</i>, Δ<i>mutL</i> and complemented strains after 10 days of biofilm development.
No full text<p>The central images show a top-down view of the biofilm; side panels are vertical sections. Scale bar represents 150 µm.</p
Sensitive testing of plasma HIV-1 RNA and Sanger sequencing of cellular HIV-1 DNA for the detection of drug resistance prior to starting first-line antiretroviral therapy with etravirine or efavirenz
No full textOBJECTIVES:
This study investigated strategies that may increase the yield of drug resistance testing prior to starting antiretroviral therapy (ART), and whether transmitted and polymorphic resistance-associated mutations (RAMs) correlated with virological outcomes.
METHODS:
We carried out retrospective testing of baseline samples from patients entering the SENSE trial of first-line ART in Europe, Russia and Israel. Prior to randomization to etravirine or efavirenz plus two nucleos(t)ide reverse transcriptase inhibitors (NRTIs), plasma samples underwent routine Sanger sequencing of HIV-1 RT and protease ((plasma)SS) in order to exclude patients with transmitted RAMs. Retrospectively, Sanger sequencing was repeated with HIV-1 DNA from baseline peripheral blood mononuclear cells (PBMCSS); baseline plasma samples were retested by allele-specific PCR targeting seven RT RAMs (AS-PCR) and ultra-deep RT sequencing (UDS).
RESULTS:
By (plasma)SS, 16/193 (8.3%) patients showed ≥ 1 transmitted RAM affecting the NRTIs (10/193, 5.2%), non-nucleoside reverse transcriptase inhibitors (4/193, 2.1%) or protease inhibitors (2/193, 1.0%). No additional RAMs were detected by AS-PCR (n = 152) and UDS (n = 24); PBMCSS (n = 91) yielded two additional samples with one RAM each. Over 48 weeks, 4/79 (5.1%) patients on etravirine and 7/78 (9.0%) on efavirenz experienced virological failure; none had baseline RAMs. Conversely, 11/79 (13.9%) patients randomized to etravirine had one polymorphic RAM from the etravirine score in baseline plasma (V90I, V106I or E138A), without any impact on virological outcomes.
CONCLUSIONS:
The detection of resistance increased marginally with PBMC testing but did not increase with sensitive plasma testing. A careful consideration is required of the cost-effectiveness of different strategies for baseline HIV drug resistance testing
VPS35 Mutations in Parkinson Disease
No full textThe identification of genetic causes for Mendelian disorders has been based on the collection of multi-incident families, linkage analysis, and sequencing of genes in candidate intervals. This study describes the application of next-generation sequencing technologies to a Swiss kindred presenting with autosomal-dominant, late-onset Parkinson disease (PD). The family has tremor-predominant dopa-responsive parkinsonism with a mean onset of 50.6 ± 7.3 years. Exome analysis suggests that an aspartic-acid-to-asparagine mutation within vacuolar protein sorting 35 (VPS35 c.1858G>A; p.Asp620Asn) is the genetic determinant of disease. VPS35 is a central component of the retromer cargo-recognition complex, is critical for endosome-trans-golgi trafficking and membrane-protein recycling, and is evolutionarily highly conserved. VPS35 c.1858G>A was found in all affected members of the Swiss kindred and in three more families and one patient with sporadic PD, but it was not observed in 3,309 controls. Further sequencing of familial affected probands revealed only one other missense variant, VPS35 c.946C>T; (p.Pro316Ser), in a pedigree with one unaffected and two affected carriers, and thus the pathogenicity of this mutation remains uncertain. Retromer-mediated sorting and transport is best characterized for acid hydrolase receptors. However, the complex has many types of cargo and is involved in a diverse array of biologic pathways from developmental Wnt signaling to lysosome biogenesis. Our study implicates disruption of VPS35 and retromer-mediated trans-membrane protein sorting, rescue, and recycling in the neurodegenerative process leading to PD
Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution
No full textImmune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens
