Bacteriophages infect an estimated 10
to 10
bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though ...phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub "superspreaders," releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications.
Bacteriophages (phages), viruses that infect bacteria, are the planet's most numerous biological entities and kill vast numbers of bacteria in natural environments. Many of these bacteria carry plasmids, extrachromosomal DNA elements that frequently encode antibiotic resistance. However, it is largely unknown whether plasmids are destroyed during phage infection or released intact upon phage lysis, whereupon their encoded resistance could be acquired and manifested by other bacteria (transformation). Because phages are being developed to combat antibiotic-resistant bacteria and because transformation is a principal form of horizontal gene transfer, this question has important implications for biomedicine and microbial evolution alike. Here we report the isolation and characterization of two novel Escherichia coli phages, dubbed "superspreaders," that promote extensive plasmid transformation and efficiently disperse antibiotic resistance genes. Our work suggests that phage superspreaders are not suitable for use in medicine but may help drive bacterial evolution in natural environments.
Adoptively transferred tumor-infiltrating T lymphocytes (TILs) that mediate complete regression of metastatic melanoma have been shown to recognize mutated epitopes expressed by autologous tumors. ...Here, in an attempt to develop a strategy for facilitating the isolation, expansion, and study of mutated antigen-specific T cells, we performed whole-exome sequencing on matched tumor and normal DNA isolated from 8 patients with metastatic melanoma. Candidate mutated epitopes were identified using a peptide-MHC-binding algorithm, and these epitopes were synthesized and used to generate panels of MHC tetramers that were evaluated for binding to tumor digests and cultured TILs used for the treatment of patients. This strategy resulted in the identification of 9 mutated epitopes from 5 of the 8 patients tested. Cells reactive with 8 of the 9 epitopes could be isolated from autologous peripheral blood, where they were detected at frequencies that were estimated to range between 0.4% and 0.002%. To the best of our knowledge, this represents the first demonstration of the successful isolation of mutation-reactive T cells from patients' peripheral blood prior to immune therapy, potentially providing the basis for designing personalized immunotherapies to treat patients with advanced cancer.
Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier ...integrity and increases susceptibility to infections. Herein, we hypothesize that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from 33 controls and 143 cancer cases, we conduct 16S ribosomal RNA (rRNA) bacterial gene sequencing, with RNA-sequencing data from lung cancer cases in The Cancer Genome Atlas serving as the validation cohort.
Overall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma specifically, a separate group of taxa are identified, in which Acidovorax is enriched in smokers. Acidovorax temporans is identified within tumor sections by fluorescent in situ hybridization and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibit higher abundance among the subset of squamous cell carcinoma cases with TP53 mutations, an association not seen in adenocarcinomas.
The results of this comprehensive study show both microbiome-gene and microbiome-exposure interactions in squamous cell carcinoma lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can impair epithelial function, have a unique bacterial consortium that is higher in relative abundance in smoking-associated tumors of this type. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.
Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three‐dimensional (3D) bioreactor system where ...cancer cells are grown in Matrigel in modified six‐well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3–0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional two‐dimensional monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.
Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three‐dimensional (3D) bioreactor system where cancer cells are grown in Matrigel and oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.
Inside Cover Image, Volume 234, Number 11, November 2019 Wulftange, William J.; Rose, Michelle A.; Garmendia‐Cedillos, Marcial ...
Journal of cellular physiology,
November 2019, 2019-11-00, 20191101, Letnik:
234, Številka:
11
Journal Article
Recenzirano
Inside Cover: The cover image is based on the Original Research Article Spatial control of oxygen delivery to 3D cultures alters cancer cell growth and gene expression by William J. Wulftange et al., ...DOI: 10.1002/jcp.28665.
Following publication of the original paper 1, the authors submitted a new Additional file 5 to replace the one containing formatting issues. The updated Additional file 5 is published in this ...correction.
We sequenced a naturally competent bacterial isolate, WY10, cultured from a Wyoming soil sample. Sequence analysis revealed that WY10 is a novel strain of
To our knowledge, WY10 is the first
strain ...to be characterized as naturally competent for DNA uptake by transformation.
Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three-dimensional bioreactor system where cancer ...cells are grown in Matrigel in modified 6-well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3% to 0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional 2D monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.
BK polyomavirus (BKV) frequently causes nephropathy (BKVN) in kidney transplant recipients (KTRs). BKV has also been implicated in the etiology of bladder and kidney cancers. We characterized BKV ...variants from two KTRs who developed BKVN followed by renal carcinoma. Both patients showed a swarm of BKV sequence variants encoding non-silent mutations in surface loops of the viral major capsid protein. The temporal appearance and disappearance of these mutations highlights the intra-patient evolution of BKV. Some of the observed mutations conferred resistance to antibody-mediated neutralization. The mutations also modified the spectrum of receptor glycans engaged by BKV during host cell entry. Intriguingly, all observed mutations were consistent with DNA damage caused by antiviral APOBEC3 cytosine deaminases. Moreover, APOBEC3 expression was evident upon immunohistochemical analysis of renal biopsies from KTRs. These results provide a snapshot of in-host BKV evolution and suggest that APOBEC3 may drive BKV mutagenesis in vivo.
Display omitted
•BKV variants sequenced from kidney transplant patients during development of nephropathy•BKV major capsid protein acquires surface loop mutations over time•Mutations confer resistance to neutralizing antibodies and modify glycan receptor usage•Mutational signatures match APOBEC3 activity, and renal biopsies are positive for APOBEC3
BK polyomavirus causes nephropathy in kidney transplant patients. Peretti et al. show that, during the development of nephropathy, BKV acquires mutations that confer a selective advantage to the virus. The sequence changes are suggestive of a role for cellular APOBEC3 DNA cytosine deaminases in driving BKV mutagenesis in vivo.
Several immunodeficiencies are associated with high susceptibility to persistent and progressive human papillomavirus (HPV) infection leading to a wide range of cutaneous and mucosal lesions. ...However, the HPV types most commonly associated with such clinical manifestations in these patients have not been systematically defined. Here, we used virion enrichment, rolling circle amplification, and deep sequencing to identify circular DNA viruses present in skin swabs and/or wart biopsy samples from 48 patients with rare genetic immunodeficiencies, including patients with warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, or epidermodysplasia verruciformis (EV). Their profiles were compared with the profiles of swabs from 14 healthy adults and warts from 6 immunologically normal children. Individual patients were typically infected with multiple HPV types; up to 26 different types were isolated from a single patient (multiple anatomical sites, one time point). Among these, we identified the complete genomes of 83 previously unknown HPV types and 35 incomplete genomes representing possible additional new types. HPV types in the genus
were common in WHIM patients, whereas EV patients mainly shed HPVs from the genus
Preliminary evidence based on three WHIM patients treated with plerixafor, a leukocyte mobilizing agent, suggest that longer-term therapy may correlate with decreased HPV diversity and increased predominance of HPV types associated with childhood skin warts.
Although some members of the viral family
cause benign skin warts (papillomas), many human papillomavirus (HPV) infections are not associated with visible symptoms. For example, most healthy adults chronically shed
(
) virions from apparently healthy skin surfaces. To further explore the diversity of papillomaviruses, we performed viromic surveys on immunodeficient individuals suffering from florid skin warts. Our results nearly double the number of known
HPV types and suggest that WHIM syndrome patients are uniquely susceptible to
HPV-associated skin warts. Preliminary results suggest that treatment with the drug plerixafor may promote resolution of the unusual
HPV skin warts observed in WHIM patients.
PDF
