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Application of a Novel "Pan-Genome"-Based Strategy for Assigning RNAseq Transcript Reads to Staphylococcus aureus Strains.

Chaves-Moreno D, Wos-Oxley ML, Jáuregui R, Medina E, Oxley AP, Pieper DH - PLoS ONE (2015)

Bottom Line: The pan-genome of S. aureus and its associated core and accessory components were compiled based on 25 genomes and comprises a total of 65,557 proteins clustering into 4,198 Orthologous Groups (OGs).The OG database generated in this study represents a useful tool to obtain a snapshot of the functional attributes of S. aureus under different in vitro and in vivo conditions.The approach proved to be advantageous to assign sequencing reads to bacterial strains when RNAseq data is derived from samples where strain information and/or the corresponding genome/s are unavailable.

View Article: PubMed Central - PubMed

Affiliation: Microbial Interactions and Processes Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany.

ABSTRACT
Understanding the behaviour of opportunistic pathogens such as Staphylococcus aureus in their natural human niche holds great medical interest. With the development of sensitive molecular methods and deep-sequencing technology, it is now possible to robustly assess the global transcriptome of bacterial species in their human habitat. However, as the genomes of the colonizing strains are often not available compiling the pan-genome for the species of interest may provide an effective method to reliably and rapidly compile the transcriptome of a bacterial species. The pan-genome of S. aureus and its associated core and accessory components were compiled based on 25 genomes and comprises a total of 65,557 proteins clustering into 4,198 Orthologous Groups (OGs). The generated gene catalogue was used to assign RNAseq-derived sequence reads to S. aureus in a variety of in vitro and in vivo samples. In all cases, the number of reads that could be assigned to S. aureus was greater using the OG database than using a reference genome. Growth of two S. aureus strains in synthetic nasal medium confirmed that both strains experienced strong iron starvation. Traits such as purine metabolism appeared to be more affected in a typical nasal colonizer than in a strain representative of the S. aureus USA300 lineage. Mapping sequencing reads from a metatranscriptome generated from the human anterior nares allowed the identification of genes highly expressed by S. aureus in vivo. The OG database generated in this study represents a useful tool to obtain a snapshot of the functional attributes of S. aureus under different in vitro and in vivo conditions. The approach proved to be advantageous to assign sequencing reads to bacterial strains when RNAseq data is derived from samples where strain information and/or the corresponding genome/s are unavailable.

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Global (pan-genome) expression map of in vitro and in vivo derived S. aureus transcriptomes.Circular ideogram depicting variations in gene expression between S. aureus strains, in vitro growth media and in vivo conditions as mapped according to the 3466 core/variable OGs and 732 unique (strain-specific) proteins defined for the S. aureus pan-genome. RNAseq generated reads (plotted as log10 expression values) were assigned to their respective OGs/proteins by rpstblastn (ordered from core–variable–unique) with each OG defined according to its major Clusters of Orthologous Groups (COG) class (outer circle). Expression values from a total of 7 conditions were mapped and represent (from outer to the inner): S. aureus USA300 in vitro exponential (EX) and stationary (ST) phase growth in Brain Heart Infusion (BHI) media; S. aureus IPL32 in vitro EX and ST phase growth in BHI; S. aureus USA300 and IPL32 in vitro EX phase growth in Synthetic Nasal Medium (SNM); and transcripts taken from an in vivo (metatranscriptomic) sample generated from the human anterior nares of an S. aureus carrier. Inner circles represent: (A) the top 25-ranked most highly expressed genes in each of the 7 conditions (based on abundance of transcripts) and plotted as a tile graph where black lines (or tiles) correspond to a highly expressed gene under a given condition (ordered according to the outer circles), with those specific to in vivo conditions marked in bold; (B) fold-change (log10) of in vitro EX growth of USA300 in SNM versus BHI media; (C) fold-change (log10) of in vitro EX growth of IPL32 in SNM versus BHI media; and (D) total S. aureus-specific read counts (log10) from the in vivo human anterior nares condition. Keys denote the color scheme used to distinguish COG classes and expression and fold-change/read count values.
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pone.0145861.g003: Global (pan-genome) expression map of in vitro and in vivo derived S. aureus transcriptomes.Circular ideogram depicting variations in gene expression between S. aureus strains, in vitro growth media and in vivo conditions as mapped according to the 3466 core/variable OGs and 732 unique (strain-specific) proteins defined for the S. aureus pan-genome. RNAseq generated reads (plotted as log10 expression values) were assigned to their respective OGs/proteins by rpstblastn (ordered from core–variable–unique) with each OG defined according to its major Clusters of Orthologous Groups (COG) class (outer circle). Expression values from a total of 7 conditions were mapped and represent (from outer to the inner): S. aureus USA300 in vitro exponential (EX) and stationary (ST) phase growth in Brain Heart Infusion (BHI) media; S. aureus IPL32 in vitro EX and ST phase growth in BHI; S. aureus USA300 and IPL32 in vitro EX phase growth in Synthetic Nasal Medium (SNM); and transcripts taken from an in vivo (metatranscriptomic) sample generated from the human anterior nares of an S. aureus carrier. Inner circles represent: (A) the top 25-ranked most highly expressed genes in each of the 7 conditions (based on abundance of transcripts) and plotted as a tile graph where black lines (or tiles) correspond to a highly expressed gene under a given condition (ordered according to the outer circles), with those specific to in vivo conditions marked in bold; (B) fold-change (log10) of in vitro EX growth of USA300 in SNM versus BHI media; (C) fold-change (log10) of in vitro EX growth of IPL32 in SNM versus BHI media; and (D) total S. aureus-specific read counts (log10) from the in vivo human anterior nares condition. Keys denote the color scheme used to distinguish COG classes and expression and fold-change/read count values.

Mentions: The differences in gene expression between S. aureus USA300 strain LAC growing in SNM versus complex medium have recently been analysed by microarray and quantitative RT-PCR [17]. Although the differences in gene expression reported in that study could be confirmed here, the RNAseq method used in this study exhibited an increased sensitivity when compared with microarray analysis [34]. The major differences between the transcriptome of cells growing exponentially in complex medium versus those growing exponentially in SNM were due to the limiting availability of iron in SNM (see Fig 3). According to the OG database, genes of the isdBACDEF gene cluster (SACOL1138, USA300HOU_1064–1068) as well as isdH (USA300HOU_1720) encoding iron regulated surface determinants were upregulated by S. aureus up to 5-fold in SNM. The sbnABCDEFGHI operon (USA300HOU_0127–0135), which encodes proteins for the biosynthesis of the staphyloferrin B siderophore [35], was upregulated by S. aureus in SNM up to 10–200 fold. An only approximately 2-fold change upregulated gene expression was determined in these genes by microarray analysis, contrasting the 40-fold upregulation determined by quantitative RT-PCR [17]. Other genes related to iron homeostasis found higly induced by S. aureus in SNM were those encoding staphyloferrin A synthesis (sfaCBAD, USA300HOU_2170–2173, upregulated 2–30 fold) [36] as well as the respective transport systems (sirABC, USA300HOU_0126–0124, upregulated 2–9 fold) and htsABC (USA300HOU_2169–2167, upregulated 2–4 fold) [37,38]. Further evidence for the need to acquire iron in SNM is provided by the high abundance of transcripts of genes encoding α-hemolysin (hly, USA300HOU_1099, upregulated 19 fold) or phenol soluble modulins β (USA300HOU_1112–1113, upregulated 74–90 fold). These are released by S. aureus to both kill the host immune cells and thereby evading the host immune defenses [39] as well as to break down red blood cells with the concomitant release of hemoglobin, which can be used by S. aureus as a source of iron [40].


Application of a Novel "Pan-Genome"-Based Strategy for Assigning RNAseq Transcript Reads to Staphylococcus aureus Strains.

Chaves-Moreno D, Wos-Oxley ML, Jáuregui R, Medina E, Oxley AP, Pieper DH - PLoS ONE (2015)

Global (pan-genome) expression map of in vitro and in vivo derived S. aureus transcriptomes.Circular ideogram depicting variations in gene expression between S. aureus strains, in vitro growth media and in vivo conditions as mapped according to the 3466 core/variable OGs and 732 unique (strain-specific) proteins defined for the S. aureus pan-genome. RNAseq generated reads (plotted as log10 expression values) were assigned to their respective OGs/proteins by rpstblastn (ordered from core–variable–unique) with each OG defined according to its major Clusters of Orthologous Groups (COG) class (outer circle). Expression values from a total of 7 conditions were mapped and represent (from outer to the inner): S. aureus USA300 in vitro exponential (EX) and stationary (ST) phase growth in Brain Heart Infusion (BHI) media; S. aureus IPL32 in vitro EX and ST phase growth in BHI; S. aureus USA300 and IPL32 in vitro EX phase growth in Synthetic Nasal Medium (SNM); and transcripts taken from an in vivo (metatranscriptomic) sample generated from the human anterior nares of an S. aureus carrier. Inner circles represent: (A) the top 25-ranked most highly expressed genes in each of the 7 conditions (based on abundance of transcripts) and plotted as a tile graph where black lines (or tiles) correspond to a highly expressed gene under a given condition (ordered according to the outer circles), with those specific to in vivo conditions marked in bold; (B) fold-change (log10) of in vitro EX growth of USA300 in SNM versus BHI media; (C) fold-change (log10) of in vitro EX growth of IPL32 in SNM versus BHI media; and (D) total S. aureus-specific read counts (log10) from the in vivo human anterior nares condition. Keys denote the color scheme used to distinguish COG classes and expression and fold-change/read count values.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696825&req=5

pone.0145861.g003: Global (pan-genome) expression map of in vitro and in vivo derived S. aureus transcriptomes.Circular ideogram depicting variations in gene expression between S. aureus strains, in vitro growth media and in vivo conditions as mapped according to the 3466 core/variable OGs and 732 unique (strain-specific) proteins defined for the S. aureus pan-genome. RNAseq generated reads (plotted as log10 expression values) were assigned to their respective OGs/proteins by rpstblastn (ordered from core–variable–unique) with each OG defined according to its major Clusters of Orthologous Groups (COG) class (outer circle). Expression values from a total of 7 conditions were mapped and represent (from outer to the inner): S. aureus USA300 in vitro exponential (EX) and stationary (ST) phase growth in Brain Heart Infusion (BHI) media; S. aureus IPL32 in vitro EX and ST phase growth in BHI; S. aureus USA300 and IPL32 in vitro EX phase growth in Synthetic Nasal Medium (SNM); and transcripts taken from an in vivo (metatranscriptomic) sample generated from the human anterior nares of an S. aureus carrier. Inner circles represent: (A) the top 25-ranked most highly expressed genes in each of the 7 conditions (based on abundance of transcripts) and plotted as a tile graph where black lines (or tiles) correspond to a highly expressed gene under a given condition (ordered according to the outer circles), with those specific to in vivo conditions marked in bold; (B) fold-change (log10) of in vitro EX growth of USA300 in SNM versus BHI media; (C) fold-change (log10) of in vitro EX growth of IPL32 in SNM versus BHI media; and (D) total S. aureus-specific read counts (log10) from the in vivo human anterior nares condition. Keys denote the color scheme used to distinguish COG classes and expression and fold-change/read count values.
Mentions: The differences in gene expression between S. aureus USA300 strain LAC growing in SNM versus complex medium have recently been analysed by microarray and quantitative RT-PCR [17]. Although the differences in gene expression reported in that study could be confirmed here, the RNAseq method used in this study exhibited an increased sensitivity when compared with microarray analysis [34]. The major differences between the transcriptome of cells growing exponentially in complex medium versus those growing exponentially in SNM were due to the limiting availability of iron in SNM (see Fig 3). According to the OG database, genes of the isdBACDEF gene cluster (SACOL1138, USA300HOU_1064–1068) as well as isdH (USA300HOU_1720) encoding iron regulated surface determinants were upregulated by S. aureus up to 5-fold in SNM. The sbnABCDEFGHI operon (USA300HOU_0127–0135), which encodes proteins for the biosynthesis of the staphyloferrin B siderophore [35], was upregulated by S. aureus in SNM up to 10–200 fold. An only approximately 2-fold change upregulated gene expression was determined in these genes by microarray analysis, contrasting the 40-fold upregulation determined by quantitative RT-PCR [17]. Other genes related to iron homeostasis found higly induced by S. aureus in SNM were those encoding staphyloferrin A synthesis (sfaCBAD, USA300HOU_2170–2173, upregulated 2–30 fold) [36] as well as the respective transport systems (sirABC, USA300HOU_0126–0124, upregulated 2–9 fold) and htsABC (USA300HOU_2169–2167, upregulated 2–4 fold) [37,38]. Further evidence for the need to acquire iron in SNM is provided by the high abundance of transcripts of genes encoding α-hemolysin (hly, USA300HOU_1099, upregulated 19 fold) or phenol soluble modulins β (USA300HOU_1112–1113, upregulated 74–90 fold). These are released by S. aureus to both kill the host immune cells and thereby evading the host immune defenses [39] as well as to break down red blood cells with the concomitant release of hemoglobin, which can be used by S. aureus as a source of iron [40].

Bottom Line: The pan-genome of S. aureus and its associated core and accessory components were compiled based on 25 genomes and comprises a total of 65,557 proteins clustering into 4,198 Orthologous Groups (OGs).The OG database generated in this study represents a useful tool to obtain a snapshot of the functional attributes of S. aureus under different in vitro and in vivo conditions.The approach proved to be advantageous to assign sequencing reads to bacterial strains when RNAseq data is derived from samples where strain information and/or the corresponding genome/s are unavailable.

View Article: PubMed Central - PubMed

Affiliation: Microbial Interactions and Processes Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany.

ABSTRACT
Understanding the behaviour of opportunistic pathogens such as Staphylococcus aureus in their natural human niche holds great medical interest. With the development of sensitive molecular methods and deep-sequencing technology, it is now possible to robustly assess the global transcriptome of bacterial species in their human habitat. However, as the genomes of the colonizing strains are often not available compiling the pan-genome for the species of interest may provide an effective method to reliably and rapidly compile the transcriptome of a bacterial species. The pan-genome of S. aureus and its associated core and accessory components were compiled based on 25 genomes and comprises a total of 65,557 proteins clustering into 4,198 Orthologous Groups (OGs). The generated gene catalogue was used to assign RNAseq-derived sequence reads to S. aureus in a variety of in vitro and in vivo samples. In all cases, the number of reads that could be assigned to S. aureus was greater using the OG database than using a reference genome. Growth of two S. aureus strains in synthetic nasal medium confirmed that both strains experienced strong iron starvation. Traits such as purine metabolism appeared to be more affected in a typical nasal colonizer than in a strain representative of the S. aureus USA300 lineage. Mapping sequencing reads from a metatranscriptome generated from the human anterior nares allowed the identification of genes highly expressed by S. aureus in vivo. The OG database generated in this study represents a useful tool to obtain a snapshot of the functional attributes of S. aureus under different in vitro and in vivo conditions. The approach proved to be advantageous to assign sequencing reads to bacterial strains when RNAseq data is derived from samples where strain information and/or the corresponding genome/s are unavailable.

Show MeSH
Related in: MedlinePlus