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Isolation and molecular characterisation of Achromobacter phage phiAxp-3, an N4-like bacteriophage.

Ma Y, Li E, Qi Z, Li H, Wei X, Lin W, Zhao R, Jiang A, Yang H, Yin Z, Yuan J, Zhao X - Sci Rep (2016)

Bottom Line: Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles.Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3.Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.

View Article: PubMed Central - PubMed

Affiliation: College of Food Science, Henan Institute of Science and Technology, Xinxiang, 453003, China.

ABSTRACT
Achromobacter xylosoxidans, an opportunistic pathogen, is responsible for various nosocomial and community-acquired infections. We isolated phiAxp-3, an N4-like bacteriophage that infects A. xylosoxidans, from hospital waste and studied its genomic and biological properties. Transmission electron microscopy revealed that, with a 67-nm diameter icosahedral head and a 20-nm non-contractile tail, phiAxp-3 has features characteristic of Podoviridae bacteriophages (order Caudovirales). With a burst size of 9000 plaque-forming units and a latent period of 80 min, phiAxp-3 had a host range limited to only four A. xylosoxidans strains of the 35 strains that were tested. The 72,825 bp phiAxp-3 DNA genome, with 416-bp terminal redundant ends, contains 80 predicted open reading frames, none of which are related to virulence or drug resistance. Genome sequence comparisons place phiAxp-3 more closely with JWAlpha and JWDelta Achromobacter phages than with other N4 viruses. Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles. Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3. Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.

No MeSH data available.


Related in: MedlinePlus

The effects of various bacterial treatments on phiAxp-3 adsorption to host cells, as determined by residual plaque-forming unit percentages.(a) Effect of proteinase K treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. (b) Effect of periodate treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. The control (LB and “A22732 + acetate”), untreated strain (A22732), and treatment (“A22732 + ProtK” for proteinase K treatment and “A22732+IO4−” for periodate treatment) groups were tested for adsorption as indicated by the x axes. Error bars denote statistical variations. Statistical significance was determined by a Student t test for comparison between the treated and untreated groups. *P 0.05. (c) Inactivation of phage phiAxp-3 by lipopolysaccharide derived from A. xylosoxidans A22732. The percentage infectivity was determined after 1 h of incubation at 37 °C. Error bars denote statistical variations.
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f6: The effects of various bacterial treatments on phiAxp-3 adsorption to host cells, as determined by residual plaque-forming unit percentages.(a) Effect of proteinase K treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. (b) Effect of periodate treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. The control (LB and “A22732 + acetate”), untreated strain (A22732), and treatment (“A22732 + ProtK” for proteinase K treatment and “A22732+IO4−” for periodate treatment) groups were tested for adsorption as indicated by the x axes. Error bars denote statistical variations. Statistical significance was determined by a Student t test for comparison between the treated and untreated groups. *P 0.05. (c) Inactivation of phage phiAxp-3 by lipopolysaccharide derived from A. xylosoxidans A22732. The percentage infectivity was determined after 1 h of incubation at 37 °C. Error bars denote statistical variations.

Mentions: Phage infection is dependent on the presence of an attachment site on the host cell surface and any exposed component of the cell surface can potentially act as a receptor32. As a Gram-negative bacterium, the exposed surface of A. xylosoxidans consists essentially of a complex of lipopolysaccharide (LPS) and proteins32. Thus it is important to determine whether LPS and proteins are recognisable by phages during infection. To identify the host receptor for phiAxp-3, the outer membrane proteins and the carbohydrate structure of the A. xylosoxidans cell surface were destroyed by proteinase K and periodate, respectively (Fig. 6a,b). The results revealed that the absence of carbohydrate structure inhibits phage propagation, suggesting that phiAxp-3 uses the bacterial LPS layer as its specific receptor. The results were confirmed by the phage inactivation assay performed with pure LPS isolated from strain A22732. The experiments revealed a direct correlation between LPS concentration and inhibition of viral particle infectivity (Fig. 6c). LPS at 25 μg per ml was needed to inhibit the activity of 3.2 × 104 pfu phiAxp-3 by 50%, while LPS at 800 μg per ml resulted in 89% inactivation of phiAxp-3.


Isolation and molecular characterisation of Achromobacter phage phiAxp-3, an N4-like bacteriophage.

Ma Y, Li E, Qi Z, Li H, Wei X, Lin W, Zhao R, Jiang A, Yang H, Yin Z, Yuan J, Zhao X - Sci Rep (2016)

The effects of various bacterial treatments on phiAxp-3 adsorption to host cells, as determined by residual plaque-forming unit percentages.(a) Effect of proteinase K treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. (b) Effect of periodate treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. The control (LB and “A22732 + acetate”), untreated strain (A22732), and treatment (“A22732 + ProtK” for proteinase K treatment and “A22732+IO4−” for periodate treatment) groups were tested for adsorption as indicated by the x axes. Error bars denote statistical variations. Statistical significance was determined by a Student t test for comparison between the treated and untreated groups. *P 0.05. (c) Inactivation of phage phiAxp-3 by lipopolysaccharide derived from A. xylosoxidans A22732. The percentage infectivity was determined after 1 h of incubation at 37 °C. Error bars denote statistical variations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837373&req=5

f6: The effects of various bacterial treatments on phiAxp-3 adsorption to host cells, as determined by residual plaque-forming unit percentages.(a) Effect of proteinase K treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. (b) Effect of periodate treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. The control (LB and “A22732 + acetate”), untreated strain (A22732), and treatment (“A22732 + ProtK” for proteinase K treatment and “A22732+IO4−” for periodate treatment) groups were tested for adsorption as indicated by the x axes. Error bars denote statistical variations. Statistical significance was determined by a Student t test for comparison between the treated and untreated groups. *P 0.05. (c) Inactivation of phage phiAxp-3 by lipopolysaccharide derived from A. xylosoxidans A22732. The percentage infectivity was determined after 1 h of incubation at 37 °C. Error bars denote statistical variations.
Mentions: Phage infection is dependent on the presence of an attachment site on the host cell surface and any exposed component of the cell surface can potentially act as a receptor32. As a Gram-negative bacterium, the exposed surface of A. xylosoxidans consists essentially of a complex of lipopolysaccharide (LPS) and proteins32. Thus it is important to determine whether LPS and proteins are recognisable by phages during infection. To identify the host receptor for phiAxp-3, the outer membrane proteins and the carbohydrate structure of the A. xylosoxidans cell surface were destroyed by proteinase K and periodate, respectively (Fig. 6a,b). The results revealed that the absence of carbohydrate structure inhibits phage propagation, suggesting that phiAxp-3 uses the bacterial LPS layer as its specific receptor. The results were confirmed by the phage inactivation assay performed with pure LPS isolated from strain A22732. The experiments revealed a direct correlation between LPS concentration and inhibition of viral particle infectivity (Fig. 6c). LPS at 25 μg per ml was needed to inhibit the activity of 3.2 × 104 pfu phiAxp-3 by 50%, while LPS at 800 μg per ml resulted in 89% inactivation of phiAxp-3.

Bottom Line: Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles.Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3.Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.

View Article: PubMed Central - PubMed

Affiliation: College of Food Science, Henan Institute of Science and Technology, Xinxiang, 453003, China.

ABSTRACT
Achromobacter xylosoxidans, an opportunistic pathogen, is responsible for various nosocomial and community-acquired infections. We isolated phiAxp-3, an N4-like bacteriophage that infects A. xylosoxidans, from hospital waste and studied its genomic and biological properties. Transmission electron microscopy revealed that, with a 67-nm diameter icosahedral head and a 20-nm non-contractile tail, phiAxp-3 has features characteristic of Podoviridae bacteriophages (order Caudovirales). With a burst size of 9000 plaque-forming units and a latent period of 80 min, phiAxp-3 had a host range limited to only four A. xylosoxidans strains of the 35 strains that were tested. The 72,825 bp phiAxp-3 DNA genome, with 416-bp terminal redundant ends, contains 80 predicted open reading frames, none of which are related to virulence or drug resistance. Genome sequence comparisons place phiAxp-3 more closely with JWAlpha and JWDelta Achromobacter phages than with other N4 viruses. Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles. Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3. Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.

No MeSH data available.


Related in: MedlinePlus