2.2. Phenotypic Characterization of SH10001st-2
Growth curves of SH10001st-2 revealed that this mutant grew slower than parent SH1000 at three temperatures (25 °C, 37 °C, and 42 °C) (
Figure 1). However, respiration measurements revealed that SH1000 produced a similar amount of CO
2 (178 ± 24.8 uL CO
2/h/OD unit) and consumed a similar amount of O
2 (159.2 ± 16.4 uL O
2/h/OD unit) as SH10001st-2 (166.2 ± 19.74 uL CO
2/h/OD unit; 154.6 ± 24.47 uL O
2/h/OD unit;
n = 6,
p > 0.05).
SH1000 demonstrated no hemolytic activity on blood agar, whereas SH10001st-2 produced zones of hemolysis 7.8 ± 0.4 mm in diameter (
n = 3). Suspensions of SH10001st-2 and plasma required 195 ± 0 min for plasma clots to appear versus only 75 ± 0 min with SH1000 (
n = 3,
p < 0.05). The fitness costs represented by the reduced growth rates and coagulase activity observed in SH10001st-2 have previously been reported to result from fusidic acid resistance-mediating amino acid substitutions in FusA [
15]. Compensatory
fusA intragenic mutations that correct these fitness costs have also been reported [
15,
16]. Based on the reduced growth rate and coagulase activity observed in SH10001st-2, it is not likely that the four SNPs in the DUF1381 superfamily gene represent fitness-compensating mutations.
2.3. Overview of the SH10001st-2 Transcriptome
A summary of transcriptomic results by 16 gene product functional categories is presented in
Table 3. Of the genes upregulated in SH10001st-2, 45.1% were represented by three functional categories: cell envelope, regulatory functions, and transport and binding proteins. Among genes downregulated in SH10001st-2, 36.3% of genes were from four categories: cell envelope, transport and binding proteins, biosynthesis of cofactors, prosthetic groups and carriers, and DNA metabolism. On both the upregulated and downregulated lists, the cell envelope and transport and binding proteins categories included the greatest number of characterized gene products, together accounting for 36.2% of upregulated and 27.5% of downregulated gene sets (
Table 3). Genes encoding hypothetical uncharacterized proteins collectively represented 37.7% of upregulated and 36.9% of downregulated genes in SH10001st-2.
Protein synthesis genes accounted for 3.1% of upregulated and 3.1% of downregulated genes, and the associated transcription and protein fate categories accounted for 5.2% of both the upregulated and downregulated genes (
Table 3). Interestingly, the protein synthesis gene
prfC was upregulated in SH10001st-2 by a factor of 2.26 (
Table S1). This gene encodes peptide chain release factor 3, which shares significant sequence homology with EF-G, and is involved with the fidelity of protein synthesis [
17,
18].
2.4. Highly Upregulated and Downregulated Genes in SH10001st-2
Table 4 lists all genes upregulated by ≥10-fold in SH10001st-2 compared to parent SH1000. Virulence factors and virulence regulatory elements dominated the 26 upregulated genes list (
Table 4), including all 8 genes in the cell envelope functional category: seven encoding capsular polysaccharide synthesis enzymes plus the delta-hemolysin encoded by RNAIII of the
agr global quorum sensing/regulatory system that is directly involved with virulence gene expression by
S. aureus [
19,
20]. The upregulated virulence genes also included 2 lipase genes and two genes (SACOL0212 and SACOL214) encoding gene products involved with fatty acid oxidation. In addition, 4 regulatory virulence genes encoded by RNAII of the
agrBCDA operon and 2 regulatory genes encoding the
kdpDE two-component regulatory system were also on this list. More than 150 genes have been identified as regulated by the
agr system [
20]. Finally, three of the four genes in the transport and binding proteins category encoded components of the KdpFABC system, and one of the four hypothetical protein category gene products was a putative phenol-soluble modulin secreted virulence factor (
Table 4). KdpDE in
S. aureus acts as a transcriptional regulator of virulence factors by means of a mechanism that possibly involves sensing external potassium levels via KdpFABC; the Kdp system itself is upregulated by the
agr system [
21,
22].
Table 5 lists the 11 genes downregulated by ≥10-fold in SH10001st-2 versus parent SH1000. These consisted of 3 genes encoding cell envelope-associated virulence factors (
isaB, SACOL0089 and
sspB) as well as 3 genes associated with nitrogen metabolism (
narK,
nirR, and
narG). Virulence regulators were notably absent from the list of highly downregulated genes.
2.5. SH10001st-2 Expression of Virulence Factors
Virulence-associated genes differentially expressed in SH10001st-2 compared to parent strain SH1000 are found in
Table S3. Upregulated genes for secreted proteins, 10 of which encode degradative enzymes (e.g. proteases, hemolysins, or lipases), outnumbered the downregulated genes by more than 3:1 (13 upregulated versus 4 downregulated). All 11 genes encoding surface adhesion proteins were downregulated in SH10001st-2 (
Table S3). A majority of the surface proteins not involved in adhesion have immune evasion functions. More than twice as many genes encoding non-adhesion surface proteins were upregulated as downregulated (ratio 19:8) in SH10001st-2, almost entirely represented by the upregulation of all 16 genes of the
cap operon (
capA-capP). The
cap operon, as well as many secreted enzymes, are upregulated by the
agr system [
20] and KdpDE also regulates capsular polysaccharide production [
23]. Expression of genes encoding several global transcriptional regulators of virulence from the SarA family [
24] were differentially regulated (e.g.,
sarR,
sarS, and
rot) (
Tables S2 and S3), notably the repressor of toxins (
rot) which was downregulated.
rot plays a role in the regulation of the
kdp system by
agr [
21]. It has previously been reported that the
agr operon was upregulated by FA challenge and that the
agr operon [
13] and the virulence regulatory gene
sarA are required for the full expression of intrinsic low-level FA resistance [
25]. The increased hemolysis and reduced coagulase activity exhibited by SH10001st-2 can be explained by the upregulation of three hemolysin-encoding genes (
hla,
hlb, and
hld) and the downregulation of the gene encoding staphylocoagulase (
coa) in SH10001st-2 (
Table S3).
Iron acquisition is a key virulence strategy employed to counter iron sequestration, a common host defense against pathogens [
26]. The most highly downregulated gene in SH10001st-2 encodes an iron compound transporter, SmpB (
Table 5), and there were 9 iron/heme transport-associated genes that were upregulated (e.g.,
isdC,
isdG,
srtB, and heme permeases) (
Table S1 and
Table 5). A gene encoding oleate hydratase, which is a virulence factor that promotes immune evasion by modifying host fatty acids provided by secreted lipases [
27], was also downregulated (
Table 5). We note that these gene expression patterns were inconsistent with a strictly upregulated virulence response interpretation.
2.6. Metabolomics of SH1000 vs SH10001st-2
Fifty-three characterized metabolites were significantly altered in SH10001st-2 when compared to parent strain SH1000 (
p < 0.05).
Table 6 lists the 42 metabolites present at greater concentrations in SH10001st-2, 20 of which demonstrated ≥2-fold increases.
Table 7 lists the 12 metabolites present at lower concentrations in SH10001st-2, 4 of which demonstrated ≥2-fold decreases. The 17 amino acids and 13 sugars in
Table 6 together accounted for more than 60% of the metabolites that increased in concentration in SH10001st-2. Five metabolites had ≥10-fold increased concentrations in SH10001st-2: glucosamine and four amino acids, including
N-acetyl-serine with a 70-fold increase and serine with a striking 221-fold increase. Nine metabolites were not detected in SH1000 but accumulated in SH10001st-2, including three amino acids (asparagine, homocysteine, and threonine) and five sugars that included glucose-6-P, fructose, ribitol, and ribose (
Table 6). Only two metabolites were detected in SH1000 but not in SH10001st-2; arginine and linoleic acid (
Table 7).
A number of alterations in the expression of genes encoding transport and binding proteins likely contributed, or partially contributed, to the increase in amino acids and sugars in SH10001st-2. While 11 genes encoding amino acid permeases and uptake systems (
aapA,
brnQ3,
gltT, SACOL1367, SACOL1392, SAR1419, SAS2274, and SAV1380) and oligopeptide transporters (SAV0727, SAV1380, and SAV1381) were upregulated in SH10001st-2, 8 other genes in this category (SACOL1476, SACOL2453, SAR2503, SAV0722, SAS0283, SAV2412,
opuCA, and
proP) were downregulated (
Tables S1 and S2). At the same time, there were more amino acid biosynthesis genes downregulated (
argF,
asd,
cysE,
glyA,
hisF, SAS0418, and SAS2563) than upregulated (SACOL2044 and SAV1737) in SH10001st-2 (
Tables S1 and S2). With regard to carbohydrate transporters, 3 (SACOL2146, SACOL2552, and
treP) were downregulated and another 2 (SAV0192 and
ptaA) were upregulated (
Tables S1 and S2).
The downregulation of SH10001st-2 genes directly involved in pyruvate metabolism (e.g.,
ddh, encoding D-lactate dehydrogenase, and
adh1, encoding alcohol dehydrogenase) (
Table S2 and
Table 5) may explain the elevated levels of pyruvic acid in SH10001st-2 (
Table 6). The amino sugar glucosamine is a key building block for capsular polysaccharide and cell wall biosynthesis that is associated with central intermediary carbohydrate metabolism [
28]. The 11-fold concentration increase in glucosamine in SH10001st-2 may be required to support the anabolic demands associated with the upregulated expression of genes encoding capsular biosynthetic enzymes.
The serine family of amino acids are prominent among those with increased concentrations in SH10001st-2. Downregulation of
cysE (
Table S2), which participates in the conversion of serine to cysteine, would contribute to the accumulation of free serine, as would upregulation of
aapA (
Table S1), which encodes a D-serine/D-alanine/glycine transporter. However, downregulation of
glyA (
Table S2), which catalyzes the production of serine from glycine, would contribute to the observed accumulation of glycine but at the expense of the serine pool. Furthermore, pyruvate can be synthesized directly from serine [
29]. In Gram- negative organisms,
N-acetyl-serine, which is produced from the cysteine precursor
O-acetyl-serine whose own synthesis is catalyzed by CysE, serves a regulatory function with respect to sulfur assimilation and metabolism [
30]. Unfortunately, the relationship between amino acid metabolism and metabolite pools is not well understood in staphylococci, although the regulation of amino acid and carbohydrate metabolism involves the
agr system’s response regulator AgrA [
31].