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Identification of staphylococcal phage with reduced transcription in human blood through transcriptome sequencing.

Santiago-Rodriguez TM, Naidu M, Jones MB, Ly M, Pride DT - Front Microbiol (2015)

Bottom Line: We developed an ex vivo technique that involved direct inoculation of blood from subjects with invasive bloodstream infections into culture media to reduce any potential laboratory adaptation.While transposons were expressed at higher levels ex vivo, lysogenic bacteriophage had significantly higher in vitro expression.Some of the phage produced also had reduced infectivity, further supporting that phage were inhibited by blood.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Diego, CA, USA.

ABSTRACT
Many pathogenic bacteria have bacteriophage and other mobile genetic elements whose activity during human infections has not been evaluated. We investigated the gene expression patterns in human subjects with invasive Methicillin Resistant Staphylococcus aureus (MRSA) infections to determine the gene expression of bacteriophage and other mobile genetic elements. We developed an ex vivo technique that involved direct inoculation of blood from subjects with invasive bloodstream infections into culture media to reduce any potential laboratory adaptation. We compared ex vivo to in vitro profiles from 10 human subjects to determine MRSA gene expression in blood. Using RNA sequencing, we found that there were distinct and significant differences between ex vivo and in vitro MRSA gene expression profiles. Among the major differences between ex vivo and in vitro gene expression were virulence/disease/defense and mobile elements. While transposons were expressed at higher levels ex vivo, lysogenic bacteriophage had significantly higher in vitro expression. Five subjects had MRSA with bacteriophage that were inhibited by the presence of blood in the media, supporting that the lysogeny state was preferred in human blood. Some of the phage produced also had reduced infectivity, further supporting that phage were inhibited by blood. By comparing the gene expression cultured in media with and without the blood of patients, we gain insights into the specific adaptations made by MRSA and its bacteriophage to life in the human bloodstream.

No MeSH data available.


Related in: MedlinePlus

Alignments of phage 3MRA (A) and phage 23MRA (B) with various other known staphylococcal phage. The boxes represent segments of each phage that are relatively well conserved amongst the aligned phage and the lines between the boxes represent the relative position of conserved segments of each phage in the different genomes. Diagonal lines represent potential genome rearrangements. Relative locations in the genomes of each phage are demonstrated by the nucleotide numbers above each phage. The yellow lines represent the sites of the first nucleotide in the assembled 3MRA and 23MRA phage contigs. Some phage segments shown by the arrows contain conserved genes representing structural elements, DNA metabolism, lysis, lysogeny, virulence, and regulation. The height of the colors across each box represents the average conservation in that phage segment across the phage examined. attP sites, which represent the sites on phage genomes where they integrate into their host genomes, are represented by blue boxes. attP sequences were not determined for phage 3MRA and 23MRA.
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Figure 7: Alignments of phage 3MRA (A) and phage 23MRA (B) with various other known staphylococcal phage. The boxes represent segments of each phage that are relatively well conserved amongst the aligned phage and the lines between the boxes represent the relative position of conserved segments of each phage in the different genomes. Diagonal lines represent potential genome rearrangements. Relative locations in the genomes of each phage are demonstrated by the nucleotide numbers above each phage. The yellow lines represent the sites of the first nucleotide in the assembled 3MRA and 23MRA phage contigs. Some phage segments shown by the arrows contain conserved genes representing structural elements, DNA metabolism, lysis, lysogeny, virulence, and regulation. The height of the colors across each box represents the average conservation in that phage segment across the phage examined. attP sites, which represent the sites on phage genomes where they integrate into their host genomes, are represented by blue boxes. attP sequences were not determined for phage 3MRA and 23MRA.

Mentions: We sequenced the phage from subjects 3MRA and 23MRA to determine which specific phage were inhibited in culture media with blood. We isolated phage 3MRA and 23MRA starting from single plaques, then purified and sequenced the DNA directly from the virions. We produced a total of 241,857 reads for phage 3MRA, of which 234,536 (97%) assembled into a single 42,141bp contig with 1177X average coverage (Figure 6A). We also sequenced 216,080 reads for phage 23MRA, of which 197,211 (91%) assembled into a single 43,114bp contig with 961X average coverage (Figure 6B). Based on BLASTN analysis, there were no nearly identical matches for phage 3MRA, but it was similar and shared synteny with S. aureus siphoviruses phiETA and phiETA3 (Figure 7A). Phage 23MRA was virtually identical to a prophage found in the genome of S. aureus USA300 TCH1516, which also is closely related to S. aureus siphoviruses phage 77, and phiETA2 (Figure 7B). Genome alignments shows that phage 23MRA has much greater similarity to Phage 77 than to phiETA2. Each phage sequenced had homologs to structural genes (head, tail, and portal), replication machinery (polymerases), integration genes (integrases), lysis and packaging machinery (lysins and terminases), genes that control transcription (repressors/anti-repressors), and virulence genes (toxin-antitoxin, complement inhibitor, chemotaxis inhibitor, and staphylokinase). All ORFs identified in phage 3MRA and 23MRA phage had homologs to staphylococcal phage genes or genes previously identified in staphylococcal genomes. These data specifically identify prophage whose expression is inhibited by culture in media with blood, and the profound similarity between these phage and many previously identified staphylococcal prophage suggests that they may also be inhibited by human blood.


Identification of staphylococcal phage with reduced transcription in human blood through transcriptome sequencing.

Santiago-Rodriguez TM, Naidu M, Jones MB, Ly M, Pride DT - Front Microbiol (2015)

Alignments of phage 3MRA (A) and phage 23MRA (B) with various other known staphylococcal phage. The boxes represent segments of each phage that are relatively well conserved amongst the aligned phage and the lines between the boxes represent the relative position of conserved segments of each phage in the different genomes. Diagonal lines represent potential genome rearrangements. Relative locations in the genomes of each phage are demonstrated by the nucleotide numbers above each phage. The yellow lines represent the sites of the first nucleotide in the assembled 3MRA and 23MRA phage contigs. Some phage segments shown by the arrows contain conserved genes representing structural elements, DNA metabolism, lysis, lysogeny, virulence, and regulation. The height of the colors across each box represents the average conservation in that phage segment across the phage examined. attP sites, which represent the sites on phage genomes where they integrate into their host genomes, are represented by blue boxes. attP sequences were not determined for phage 3MRA and 23MRA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4447126&req=5

Figure 7: Alignments of phage 3MRA (A) and phage 23MRA (B) with various other known staphylococcal phage. The boxes represent segments of each phage that are relatively well conserved amongst the aligned phage and the lines between the boxes represent the relative position of conserved segments of each phage in the different genomes. Diagonal lines represent potential genome rearrangements. Relative locations in the genomes of each phage are demonstrated by the nucleotide numbers above each phage. The yellow lines represent the sites of the first nucleotide in the assembled 3MRA and 23MRA phage contigs. Some phage segments shown by the arrows contain conserved genes representing structural elements, DNA metabolism, lysis, lysogeny, virulence, and regulation. The height of the colors across each box represents the average conservation in that phage segment across the phage examined. attP sites, which represent the sites on phage genomes where they integrate into their host genomes, are represented by blue boxes. attP sequences were not determined for phage 3MRA and 23MRA.
Mentions: We sequenced the phage from subjects 3MRA and 23MRA to determine which specific phage were inhibited in culture media with blood. We isolated phage 3MRA and 23MRA starting from single plaques, then purified and sequenced the DNA directly from the virions. We produced a total of 241,857 reads for phage 3MRA, of which 234,536 (97%) assembled into a single 42,141bp contig with 1177X average coverage (Figure 6A). We also sequenced 216,080 reads for phage 23MRA, of which 197,211 (91%) assembled into a single 43,114bp contig with 961X average coverage (Figure 6B). Based on BLASTN analysis, there were no nearly identical matches for phage 3MRA, but it was similar and shared synteny with S. aureus siphoviruses phiETA and phiETA3 (Figure 7A). Phage 23MRA was virtually identical to a prophage found in the genome of S. aureus USA300 TCH1516, which also is closely related to S. aureus siphoviruses phage 77, and phiETA2 (Figure 7B). Genome alignments shows that phage 23MRA has much greater similarity to Phage 77 than to phiETA2. Each phage sequenced had homologs to structural genes (head, tail, and portal), replication machinery (polymerases), integration genes (integrases), lysis and packaging machinery (lysins and terminases), genes that control transcription (repressors/anti-repressors), and virulence genes (toxin-antitoxin, complement inhibitor, chemotaxis inhibitor, and staphylokinase). All ORFs identified in phage 3MRA and 23MRA phage had homologs to staphylococcal phage genes or genes previously identified in staphylococcal genomes. These data specifically identify prophage whose expression is inhibited by culture in media with blood, and the profound similarity between these phage and many previously identified staphylococcal prophage suggests that they may also be inhibited by human blood.

Bottom Line: We developed an ex vivo technique that involved direct inoculation of blood from subjects with invasive bloodstream infections into culture media to reduce any potential laboratory adaptation.While transposons were expressed at higher levels ex vivo, lysogenic bacteriophage had significantly higher in vitro expression.Some of the phage produced also had reduced infectivity, further supporting that phage were inhibited by blood.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Diego, CA, USA.

ABSTRACT
Many pathogenic bacteria have bacteriophage and other mobile genetic elements whose activity during human infections has not been evaluated. We investigated the gene expression patterns in human subjects with invasive Methicillin Resistant Staphylococcus aureus (MRSA) infections to determine the gene expression of bacteriophage and other mobile genetic elements. We developed an ex vivo technique that involved direct inoculation of blood from subjects with invasive bloodstream infections into culture media to reduce any potential laboratory adaptation. We compared ex vivo to in vitro profiles from 10 human subjects to determine MRSA gene expression in blood. Using RNA sequencing, we found that there were distinct and significant differences between ex vivo and in vitro MRSA gene expression profiles. Among the major differences between ex vivo and in vitro gene expression were virulence/disease/defense and mobile elements. While transposons were expressed at higher levels ex vivo, lysogenic bacteriophage had significantly higher in vitro expression. Five subjects had MRSA with bacteriophage that were inhibited by the presence of blood in the media, supporting that the lysogeny state was preferred in human blood. Some of the phage produced also had reduced infectivity, further supporting that phage were inhibited by blood. By comparing the gene expression cultured in media with and without the blood of patients, we gain insights into the specific adaptations made by MRSA and its bacteriophage to life in the human bloodstream.

No MeSH data available.


Related in: MedlinePlus