Abstract

   We performed in vivo RNA-sequencing analysis of Staphylococcus aureus
   in infected mouse liver using the 2-step cell-crush method. We compared
   the transcriptome of S. aureus at 6, 24, and 48 h post-infection
   (h.p.i) in mice and in culture medium. Genes related to anaerobic
   respiration were highly upregulated at 24 and 48 h.p.i. The gene
   expression patterns of virulence factors differed depending on the type
   of toxin. For example, hemolysins, but not leukotoxins and serine
   proteases, were highly upregulated at 6 h.p.i. Gene expression of metal
   transporters, such as iron transporters, gradually increased at 24 and
   48 h.p.i. We also analyzed the transcriptome of mouse liver infected
   with S. aureus. Hypoxia response genes were upregulated at 24 and
   48 h.p.i., and immune response genes were upregulated from 6 h.p.i.
   These findings suggest that gene expression of S. aureus in the host
   changes in response to changes in the host environment, such as the
   oxygenation status or immune system attacks during infection.

   Subject terms: Pathogens, Infection
     __________________________________________________________________

   An in vivo transcriptomic analysis of Staphylococcus aureus infection
   in mice provides further insight into how this pathogen responds to
   changes in the host environment over time.

Introduction

   The rapid emergence of multi-antimicrobial resistant strains has
   created an urgent need for the development of novel therapeutic agents.
   The number of antibacterial agents with novel mechanisms of action
   recently approved by the FDA is limited^[36]1, however, suggesting the
   need for new strategies to discover target molecules that can be
   developed as novel antimicrobials^[37]2. Antimicrobials are typically
   screened on the basis of their antimicrobial activity in vitro. As
   recently pointed out^[38]3, [39]4, however, pathogen behavior exhibited
   during in vitro culture is much different from that in vivo in the
   host; thus, it is important to identify antimicrobial targets expected
   to be more efficient in the host.

   Staphylococcus aureus has successfully adapted to the environmental
   conditions of the human body. It can survive and respond to various
   conditions in the human body, and causes a wide range of diseases, such
   as acne, pneumonia, and bacteremia^[40]5, despite its relatively small
   genome size^[41]6. In addition, S. aureus easily acquires multidrug
   resistance^[42]7. The estimated number of deaths caused annually by
   infection with methicillin-resistant S. aureus is 10,000 in the United
   States and 4000 in Japan^[43]8. S. aureus secretes various kinds of
   toxins, such as hemolysins, leukotoxins, and proteases^[44]9, and grows
   under both aerobic and anaerobic conditions^[45]10. Furthermore, S.
   aureus has at least 5 iron acquisition systems and many metal
   transporters that are essential for its colonization and pathogenesis
   under host conditions^[46]11,[47]12. Many genes have been identified as
   pathogenic factors of S. aureus, and analysis of their transcriptomes
   under host infection conditions is crucial. By performing RNA
   sequencing (RNA-Seq) analysis, we revealed the in vivo transcriptome of
   S. aureus Newman strain at various time-points in the liver of mice
   systemically infected with S. aureus, in contrast to previous studies
   in which analysis was performed at only one specific time-point after
   systemic infection, or at acute and chronic time-points in local
   infection. For example, the transcriptional profile of S. aureus in the
   kidneys was examined at 48 h post-infection (h.p.i.) following
   intravenous inoculation in mice with decreased susceptibility^[48]13;
   acutely in a local prosthetic joint infection^[49]14; and at acute (7
   days) and chronic (28 days) time-points in murine osteomyelitis^[50]15.
   In the present study, we selected a highly virulent strain that killed
   the animals within 72 h.p.i., and evaluated the transcriptome at
   6 h.p.i., when mice exhibited no symptoms, and at 24 and 48 h.p.i.,
   when mice exhibited a phenotype of reduced locomotion and some
   individuals begin to die. We recently established an improved in vivo
   RNA-Seq analysis applicable to a smaller S. aureus population size in
   infected organs by taking advantage of the fact that Gram-positive
   bacteria can be separated from host cells by mechanical disruption due
   to the presence of a strong cell wall. This technique has been
   successfully applied to in vivo RNA-Seq for Streptococcus
   pyogenes^[51]16, and in the present study, we applied the method to S.
   aureus in a systemic mouse infection model.

Results and discussion

In vivo RNA-Seq analysis for S. aureus infection in mouse liver

   We previously reported that the 2-step cell-crush method was applicable
   to in vivo RNA-Seq analysis of S. pyogenes, which have a rigid cell
   wall like S. aureus, in necrotizing fasciitis^[52]16. In this method,
   the first step is to use large beads to crush and lyse the host tissue
   in lysis buffer, followed by the use of small beads to crush enriched
   S. aureus cells in mouse organs to prepare enriched bacterial RNA for
   RNA-Seq analysis (Fig. [53]1). In the present study, we performed in
   vivo RNA-Seq analysis of S. aureus grown in organs of mice systemically
   infected with S. aureus. Injection of S. aureus Newman strain into the
   mouse tail vein killed half of the mice within 48 h.p.i., and all the
   mice within 72 h.p.i. (Fig. [54]2a). Under this condition, the number
   of bacterial cells per organ increased exponentially in the kidney and
   heart within 24 h.p.i., and reached 10^7 colony-forming units (CFU)/mg
   in the liver at 6 h.p.i., with more than 10^6 CFU/mg in most
   individuals remaining at 48 h.p.i. (Fig. [55]2b). The number of
   bacteria in the blood, on the other hand, was not high at any time
   during the experiment. Thus, we performed in vivo RNA-Seq analysis of
   S. aureus in the liver at 6, 24, and 48 h.p.i., and obtained
   approximately 160 to 700 thousand uniquely mapped reads on the S.
   aureus genome (Supplementary Table [56]1). The number of genes with no
   mapped reads was 97 (3% of all genes) for RNA extracted from in vitro
   culture and 374 (12% of all genes) in S. aureus Newman strain isolated
   from liver 6 h.p.i. Therefore, most of the genes in the S. aureus
   genome were successfully analyzed by this method (Supplementary
   Data [57]1). We found 472, 545, and 616 genes with significant
   upregulation (>2-fold) and 637, 558, and 655 genes with significant
   downregulation (>2-fold) at 6, 24, and 48 h.p.i., respectively.

Fig. 1. The 2-step cell-crush method for in vivo RNA-Seq analysis of S.
aureus in mouse liver.

   [58]Fig. 1
   [59]Open in a new tab

   C57BL/6J mice were infected with S. aureus Newman strain injected
   through the tail vein, and organs were harvested at 6, 24, and
   48 h.p.i. Tissue and bacterial cells were separately crushed and lysed
   in lysis buffer using beads of 2 different sizes.

Fig. 2. Conditions of systemic S. aureus infection model in this analysis.

   [60]Fig. 2
   [61]Open in a new tab

   a Survival of mice infected with S. aureus Newman strain. Bacterial
   cells (5.6 × 10^7 CFU) were injected into the tail vein (n = 5). Data
   represent the mean ± SEM. b Number of S. aureus cells in each organ
   after infection with (5.3 × 10^7 CFU, n = 2 to 11). c Histopathological
   analysis of liver from mice infected with S. aureus Newman strain
   (6.8 × 10^7 CFU). Photographs are representative of the results for 2
   mice. The bars in the pictures were indicated as 10 µm. d–f Analysis of
   hematological parameters of infected mice. AST (d), ALT (e) and CK (f)
   parameters in the plasma collected from mice (n = 5 for 0 and 6 h.p.i.,
   n = 7 for 24 h.p.i. and n = 9 for 48 h.p.i.) infected with S. aureus
   Newman strain (6.8 × 10^7 CFU) were measured. Data represent the
   mean ± SEM and statistical analysis was performed using a
   Kruskal-Wallis test with Dunn’s multiple comparisons test.

Pathway analysis of the genes altered after infection with S. aureus

   To elucidate the trend of S. aureus gene expression in the host
   environment, we performed a KEGG pathway enrichment analysis to compare
   the in vivo gene expression with that in culture medium in vitro
   (Table [62]1). The expression of genes involved in carbon metabolism
   (glycolysis) and TCA cycle pathways was significantly upregulated at
   6 h.p.i., but not at 48 h.p.i (Table [63]1). Expression of genes
   involved in beta-oxidation (responsible for the production of
   acetyl-CoA from fatty acids), and the PTS system (required for
   incorporation of phosphorylated saccharide) was significantly
   upregulated starting at 24 h.p.i. Expression of genes required for iron
   acquisition, such as the biosynthesis of various secondary
   metabolites—staphyloferrin A and B—was not upregulated at 6 h.p.i., but
   was upregulated after 24 h.p.i. In addition, expression of ABC
   transporters (required for the acquisition of nickel and manganese) was
   upregulated after 24 h.p.i. On the other hand, expression of genes
   involved in terpenoid backbone biosynthesis (required for cell wall
   synthesis, pigment, menaquinone, and peptidoglycan biosynthesis) was
   downregulated from 24 h.p.i. As for the host side, we performed RNA-Seq
   analysis using RNA of liver organs mixed in a sample that mapped
   uniquely on the mouse genome (Supplementary Data [64]2) and selected
   genes with a significant 5-fold difference (false discovery rate [FDR]
   p-value < 0.05) in liver infected with S. aureus compared with liver
   injected with phosphate-buffered saline (PBS) to perform the GO term
   enrichment analysis (Reactome, Supplementary Data [65]3). The results
   suggested that genes involved in the induction of innate immunity and
   inflammation, and those related to metal sequestration were
   significantly upregulated (Supplementary Table [66]2). On the other
   hand, reactomes related to ATP synthesis, such as respiratory electron
   transport, ATP synthesis by chemiosmotic coupling, and heat production
   by uncoupling proteins, were downregulated at all time-points
   (Supplementary Table [67]2). In addition, hif1a and hif3a, which are
   involved in the hypoxia response, were upregulated after 24 h.p.i.,
   suggesting that the host environment was low in oxygen at the late
   stage of infection. To further confirm the host condition, we performed
   a histopathological analysis and biochemical tests to evaluate liver
   function. We observed a number of abscesses at 24 and 48 h.p.i., but
   none at 6 h.p.i. In addition, the number of bacteria per abscess
   increased in a time-dependent manner (Fig. [68]2c). Furthermore,
   hematological parameters, such as aspartate aminotransferase activity
   (AST), alanine aminotransferase activity (ALT), and creatine kinase
   activity (CK) in the plasma, which are indicators of liver and heart
   damage, were significantly increased at 24 and 48 h.p.i.
   (Fig. [69]2d–f), suggesting that the liver and heart were damaged
   within 24 h after S. aureus infection. These results well correlated
   with the trend of the S. aureus gene expression changes in the host and
   suggested that carbon metabolism, metal metabolism, and cell wall
   synthesis of S. aureus were highly influenced by the host condition.

Table 1.

   KEGG pathway enrichment analysis for upregulated and downregulated
   genes of S. aureus-infected liver compared with culture medium.
   Hours Pathway ID Description GeneRatio BgRatio FDR p-value
   Upregulated
   6 h sae00020 Citrate cycle (TCA cycle) 15/157 22/963 0.0000
   sae01120 Microbial metabolism in diverse environments 43/157 132/963
   0.0000
   sae01200 Carbon metabolism 29/157 80/963 0.0001
   24 h sae00997 Biosynthesis of various secondary metabolites—part 3
   9/208 10/963 0.0004
   sae01120 Microbial metabolism in diverse environments 48/208 132/963
   0.0006
   sae00290 Valine, leucine, and isoleucine biosynthesis 9/208 11/963
   0.0006
   sae00020 Citrate cycle (TCA cycle) 13/208 22/963 0.0018
   sae01210 2-Oxocarboxylic acid metabolism 11/208 18/963 0.0033
   sae00650 Butanoate metabolism 10/208 16/963 0.0041
   sae05150 Staphylococcus aureus infection 17/208 38/963 0.0077
   sae02060 Phosphotransferase system (PTS) 12/208 23/963 0.0077
   sae00071 Fatty acid degradation 7/208 10/963 0.0085
   sae01100 Metabolic pathways 125/208 497/963 0.0204
   sae02010 ABC transporters 28/208 85/963 0.0396
   48 h sae00220 Arginine biosynthesis 15/253 18/963 0.0000
   sae02010 ABC transporters 39/253 85/963 0.0008
   sae00997 Biosynthesis of various secondary metabolites—part 3 9/253
   10/963 0.0008
   sae00340 Histidine metabolism 12/253 16/963 0.0008
   sae00290 Valine, leucine, and isoleucine biosynthesis 9/253 11/963
   0.0020
   sae01230 Biosynthesis of amino acids 42/253 100/963 0.0021
   sae01210 2-Oxocarboxylic acid metabolism 12/253 18/963 0.0027
   sae02060 Phosphotransferase system (PTS) 14/253 23/963 0.0030
   sae02024 Quorum sensing 28/253 61/963 0.0032
   sae05150 Staphylococcus aureus infection 19/253 38/963 0.0068
   sae00052 Galactose metabolism 12/253 20/963 0.0069
   sae00071 Fatty acid degradation 7/253 10/963 0.0216
   sae01100 Metabolic pathways 148/253 497/963 0.0284
   sae02020 Two-component system 29/253 75/963 0.0398
   Downregulated
   6 h sae03010 Ribosome 47/297 73/963 0.0000
   sae00230 Purine metabolism 25/297 43/963 0.0041
   24 h sae03010 Ribosome 46/311 73/963 0.0000
   sae00240 Pyrimidine metabolism 18/311 28/963 0.0125
   sae00900 Terpenoid backbone biosynthesis 9/311 11/963 0.0195
   48 h sae03010 Ribosome 56/460 73/963 0.0000
   sae00900 Terpenoid backbone biosynthesis 11/460 11/963 0.0065
   sae00550 Peptidoglycan biosynthesis 20/460 24/963 0.0065
   sae00240 Pyrimidine metabolism 22/460 28/963 0.0115
   [70]Open in a new tab

Energy metabolism

   We further analyzed the gene expression changes in each S. aureus
   pathway in mouse liver after infection. Expression of genes involved in
   the glycolysis pathway was suppressed in mouse liver compared with the
   culture medium conditions (Supplementary Fig. [71]1) throughout the
   infection period. Expression of genes involved in the TCA cycle was
   relatively upregulated until 24 h.p.i. and downregulated at 48 h.p.i.
   On the other hand, fermentation-related genes such as pflB, ldh, and
   adhE were highly upregulated at 24 and 48 h.p.i. In addition, the genes
   involved in nitrate respiration, such as narK, which is required for
   nitrate uptake; the narGHJI operon encoding respiratory nitrate
   reductase; and the nirBD operon encoding assimilatory nitrite
   reductase, which produces nitrate (NO[3]^−), an electron acceptor
   instead of oxygen in anaerobic conditions in the electron transport
   chain (Fig. [72]3a)^[73]17, were upregulated at 24 h.p.i. and highly
   upregulated at 48 h.p.i. (Fig. [74]3b). These results suggest that
   nitrate respiration was upregulated at the late stage of infection,
   during which the bacterial number was increasing in liver abscesses,
   and damage was occurring in the liver and heart (Fig. [75]2c–f). The
   increased nitrate respiration may be a response of S. aureus in the
   liver to an oxygen insufficiency associated with abscess formation and
   liver tissue damage. We disrupted the narK gene to evaluate the
   requirement of nitrate respiration in the virulence of S. aureus and
   found the disruptant strain reduced virulence, although, the
   complemented strain did not recover the virulence of the disruptant
   strain (Supplementary Fig. [76]2a). Further studies are needed to
   clarify the requirement of genes involved in nitrate respiration for S.
   aureus virulence. On the other hand, the mouse-killing ability of the
   pflB gene-disrupted mutant was not significantly reduced (Supplementary
   Fig. [77]2b). PflB is involved in synthesizing acetyl-CoA from pyruvate
   in anaerobic conditions, although other enzymes also cover this
   enzymatic reaction. Thus, an alternative pathway might compensate for
   its function in the pflB gene disruption mutant.

Fig. 3. Gene expression changes regarding nitrate respiration of S. aureus
infected in liver.

   [78]Fig. 3
   [79]Open in a new tab

   a Metabolic pathway of nitrate in S. aureus. b Expression changes in
   genes involved in the nitrogen respiration pathway. The values in the
   box show fold-change compared with S. aureus cultured in the TSB medium
   and the boxes. The asterisk *. **, and *** indicate FDR p-values
   < 0.05, <0.01, and <0.001, respectively.

Lipid metabolism

   KEGG pathway analysis suggested the upregulation of genes regulating
   fatty acid degradation. The genes fadABDE are involved in lipid beta
   oxidation and required for acetyl-CoA production utilized in the TCA
   cycle. These genes were upregulated in the host liver from the initial
   stage of infection compared with culture medium conditions
   (Table [80]2). In contrast, expression of the fabIH genes, required for
   type II fatty acid synthetase, a target for antimicrobial agent
   development^[81]18, was significantly downregulated in the later stage
   of infection (Table [82]2). These findings suggest that S. aureus
   infected in mouse liver obtained a part of its energy from fatty acid
   degradation.

Table 2.

   Gene expression change of S. aureus lipid metabolism in the host liver
   compared with culture medium.
   Gene KEGG pathway 6 h.p.i. 24 h.p.i. 48 h.p.i.
   Fold change FDR p-value Fold change FDR p-value Fold change FDR p-value
   fadA Fatty acid degradation^a 12.0 <0.001 11.5 <0.001 6.7 <0.001
   fadB 26.3 <0.001 16.8 <0.001 7.3 <0.001
   fadD 84.7 <0.001 61.9 <0.001 9.7 <0.001
   fadE 23.4 <0.001 30.0 <0.001 7.7 <0.001
   fab Fatty acid elongation^b −1.8 0.050 −2.3 0.006 −5.3 <0.001
   fabD −1.9 0.030 −2.3 0.005 −3.7 <0.001
   fabG −1.8 0.024 −2.8 <0.001 −3.2 <0.001
   fabH −5.3 <0.001 −4.7 <0.001 −4.3 <0.001
   fabI −1.1 0.959 −1.5 0.214 −3.2 <0.001
   fabZ −2.7 <0.001 −4.1 <0.001 −4.5 <0.001
   [83]Open in a new tab

   ^a[84]https://www.kegg.jp/kegg-bin/show_pathway?sae00071.

   ^b[85]https://www.kegg.jp/kegg-bin/show_pathway?sae00061.

Metal acquisition system

   Iron acquisition is essential for pathogen growth in the host^[86]19.
   S. aureus has at least 5 iron acquisition systems^[87]20,[88]21, and
   the genes involved in these systems are known to be upregulated in the
   host because the pathogen-infected host hides iron by increasing metal
   sequestration proteins, as shown in Supplementary Table [89]2. Although
   the expression of iron acquisition system genes other than sbnCD was
   not upregulated at 6 h.p.i., it was highly increased at 24 and
   48 h.p.i. (Fig. [90]4a), a pattern that did not correspond to that of
   the host’s metal sequestration proteins, which were upregulated from
   6 h.p.i. It might be that S. aureus obtained iron from hemocytes lysed
   by hemolysins, which were highly upregulated at the initial infection
   stage in the host, as described below.

Fig. 4. Gene expression change in S. aureus metal acquisition system in the
host liver compared with culture medium.

   [91]Fig. 4
   [92]Open in a new tab

   a, b Time course of expression changes in the iron acquisition systems
   (a) and the divalent cation acquisition systems (b) in S.
   aureus-infected liver compared with culture medium. The values in the
   box show fold-change compared with S. aureus cultured in the TSB medium
   and boxes filled with red indicate >40-fold change. The asterisk *. **,
   and *** indicate FDR p-values < 0.05, <0.01, and <0.001, respectively.
   c, e Genome structure around mntA gene (c) and cntK and cntE (e). d, f,
   g Mouse-killing ability of a disruption mutant of the mntA (d), cntE
   (f), and cntK gene (g), respectively. Bacterial suspensions were
   intravenously injected. The Data were presented combined independent
   two experiments (n = 12 for d, g, n = 11 for f). Injection doses were
   3.8 × 10^7 for wild-type of d, g, 3.5 × 10^7 CFU for wild-type of f,
   each 3.8 × 10^7 CFU for ∆mntA, 4.0 × 10^7 CFU for ∆cntE and 3.8 × 10^7
   CFU for ∆cntK, respectively. Statistical analyses were performed by
   Log-rank (Mantel-Cox) test (c: p = < 0.0001, chi square = 24.72,
   df = 1, d: p = < 0.0001, chi square = 24.38, df = 1, e: p = < 0.0001,
   chi square = 22.78, df = 1).

   S. aureus has 2 manganese transporters: the mntABC genes encoding the
   ABC transporter, and the mntH gene encoding a proton-ion coupled
   transporter. Disruption mutants of both genes are reported to have
   reduced virulence against mice^[93]22. In this analysis, we found that
   mntABC genes and not the mntH gene were highly upregulated in the host
   compared with the culture medium condition (Fig. [94]4b). In addition,
   we revealed that disruption of the mntABC gene operon (Fig. [95]4c) in
   S. aureus with an intact mntH gene reduced virulence against mice
   (Fig. [96]4d), and the virulence was partially recovered by
   complementation of this operon (Supplementary Fig. [97]3a). These
   results indicated that the MntABC transporter significantly contributes
   to S. aureus virulence. In addition, a staphylopine-mediated transport
   system related to the acquisition of broad metal ions such as iron,
   zinc, copper, nickel, and cobalt was recently reported^[98]12. The
   expression level of the cntABCDF operon (Fig. [99]4e) encoding the ABC
   transporter increased from 24 to 48 h.p.i., and the expression level of
   the cntKLM gene, which is involved in staphylopine synthesis, increased
   several-fold as the infection progressed. Disruption of the cntK gene,
   which is involved in staphylopine synthesis, and the cntE gene, which
   is involved in the secretion of staphylopine from bacterial cells,
   significantly reduces virulence (Fig. [100]4f, g). Furthermore, the
   virulence of cntE and cntK disruptant mutants were partially and almost
   completely recovered in complemented strains, suggesting that this
   metal transporter is essential for the virulence of S. aureus
   (Supplementary Fig. [101]3b, c). Further studies are needed to clarify
   the functions of these genes in the full virulence of S. aureus and to
   identify the responsible genes since these genes are organized into
   operons (Fig. [102]4c, e).

   A gradual increase in the expression of copper and cobalt metal
   transporters, copA and cobI, respectively, was observed after infection
   (Fig. [103]4b). We observed no difference in the expression of the mgtE
   gene and modABC, a transporter of magnesium and molybdenum, in the host
   compared with that in the culture medium condition (Fig. [104]4b).

Virulence factors and their regulators

   S. aureus possesses a wide variety of toxins, and the expression of
   these toxins, such as hemolysin, increases after infection^[105]13. In
   this study, we revealed the time course of these gene expression
   changes (Fig. [106]5). Expression of genes encoding hemolysins such as
   hla and hlgABC was highly upregulated from the initial stage of
   infection. We assume that expression of these genes would contribute to
   iron acquisition by S. aureus in the host at the early stage of
   infection. The expression level of leukotoxin genes such as lukDE was
   increased from 6 h.p.i. Expression of superantigen genes, which are
   involved in evading the immune system^[107]23, increased starting at
   6 h.p.i., although significant expression increments were observed from
   24 h.p.i. It is uncertain why the expression of genes corresponding to
   the host’s immune response was delayed even though the innate immune
   system of the mice was already activated at 6 h.p.i.

Fig. 5. Time course of expression changes in virulence factors in S.
aureus-infected liver compared with culture medium.

   [108]Fig. 5
   [109]Open in a new tab

   Time course of expression changes for toxins (a), proteases (b), and
   superantigens (c). Values in the boxes show fold-change compared with
   S. aureus cultured in the TSB medium and the boxes filled with red
   indicate >40-fold change. The asterisk *. **, and *** indicate FDR
   p-values < 0.05, <0.01, and <0.001, respectively. lukG and lukH genes
   in (a) are annotated as lukS and lukF in Refseq ([110]NZ_CP023390.1),
   although we annotated these as lukGH genes as these genes are not
   related to the PVL phage and are similar to the lukGH gene.

   We further evaluated the expression of 2-component regulatory systems
   identified in S. aureus required for environmental responses and toxin
   regulators. The expression of agr and sae genes, which are necessary
   for toxin expression^[111]24, did not increase in the host, but rather
   tended to decrease at 48 h.p.i. (Table [112]3). These findings are
   consistent with findings in other models^[113]3,[114]25. Several
   transcription factors are considered to influence agr gene regulation,
   which regulates toxin production^[115]24,[116]26,[117]27. The
   expression levels of sarH1 and sarX, which negatively regulate agr gene
   expression, were decreased, while expression of rsr, a repressor of agr
   expression, was increased. On the other hand, the expression levels of
   sarA, sarZ, ccpA, and mgrA, which positively regulate agr expression,
   tended to decrease. Furthermore, expression of sarH1 and sarT, which
   are negative regulators of agr and sarA genes, decreased at 6 h.p.i,
   but increased after 24 h.p.i. These findings suggest that the
   expression of transcription factors involved in the regulation of
   virulence gene expression in the host condition was not consistent with
   the interpretation of the regulation of toxin production and agr gene
   expression based on in vitro analysis. Furthermore, expression of the
   tcs7RS gene, a 2-component regulatory system with unknown function, and
   kdpDE genes, a 2-component regulatory system for potassium homeostasis,
   was also upregulated throughout the infection process. In addition, we
   recently reported a novel virulence regulator, the yjbH gene, which
   regulates the expression of an iron transporter, and several virulence
   factors, such as spa and leukotoxin, and oxidative stress response
   genes^[118]28 were upregulated until 24 h.p.i., suggesting that yjbH
   contributes to the expression of virulence-related genes in the early
   stage of infection in mice. These results suggest that transcription of
   the agr gene was not positively regulated and other genes such as yjbH
   gene regulate the expression of virulence-related genes.

Table 3.

   Gene expression change in the S. aureus 2-component system and
   virulence-related transcription factors in the host liver compared with
   culture medium.
   Gene Function 6 h.p.i. 24 h.p.i. 48 h.p.i.
   Fold FDR p-value Fold FDR p-value Fold FDR p-value
   Two-component factors^[119]37 agrC Quorum sensing control of adhesion
   and virulence factors 1 1 −1.2 0.765 −2.3 0.013
   agrA 1.2 0.696 1.2 0.672 −1.9 0.026
   saeS Virulence factors regulation (toxins, enzymes) 1.1 0.743 1.1 0.848
   −4.7 <0.001
   saeR 1 0.996 −1.1 0.918 −4.2 <0.001
   vraR Cell wall -affecting antibiotic resistance, cell wall biosynthesis
   2.3 <0.001 1.7 0.024 −2 0.009
   vraS 2.1 0.001 1.3 0.279 −2.6 0.005
   graX AMP resistance, growth at low pH 1.9 0.007 1.1 0.951 1 0.996
   graR 2.7 <0.001 −1 0.998 −1.3 0.545
   graS 1.9 0.007 1.3 0.464 −1.2 0.694
   braS Antimicrobial peptide resistance 1.2 0.588 1.3 0.365 −1.2 0.581
   braR −1.4 0.468 −1.4 0.54 1.1 0.885
   arlS Pathogenesis mechanisms: autolysis, adhesion, biofilm 1.4 0.199
   −1.2 0.576 −2 0.016
   arlR −1.5 0.18 −2.1 0.022 −1.9 0.023
   WalR cell wall maintenance, cell viability −1.2 0.612 −1.2 0.583 −1.3
   0.613
   WalK 1 1 −1.2 0.738 −1.4 0.166
   hptS intercellular survival, uptake of hexose phosphate −2.2 0.115 1.1
   0.922 1.6 0.21
   hptR −3.3 0.012 −2.8 0.029 1.9 0.04
   tcs7R Uncharacterized function 4.9 <0.001 3 0.001 1.9 0.085
   tcs7S 3.2 <0.001 2.7 0.005 1.4 0.371
   srrB Anaerobic respiration, metabolism, growth at low temperature −1.8
   0.027 −1.5 0.141 1.3 0.443
   srrA −1.4 0.303 −1.4 0.316 2.1 0.03
   phoR Phosphate uptake and homeostasis 5.3 <0.001 3.4 <0.001 1.2 0.486
   phoP 1.2 0.555 −1.1 0.871 1.6 0.252
   airR Oxidative stress response −1.3 0.634 1.6 0.272 −1.8 0.089
   airS 1.1 0.802 1 0.998 −1.1 3831
   kdpD Potassium homeostasis regulation 1.4 0.545 3.5 <0.0001 3.7 <0.001
   kdpE 2.1 0.123 3.1 0.005 4.4 <0.001
   hssR Heme metabolism regulation −1.7 0.317 −5.3 0.018 1.7 0.228
   hssS 1.8 0.058 −1.1 0.888 −1.1 0.826
   nreC Response to low oxygen, nitrate reduction 1.3 0.337 1.2 0.576 1.2
   0.56
   nreB 1.1 0.872 1.2 0.635 1.6 0.177
   Transcription factor^[120]27 sarA Positive to agr expression, induction
   of exoproteins and repression of spa −1.6 0.051 1.7 0.118 −5.4 <0.001
   sarH1 Repressor of agr, sarA −13.6 <0.001 −8.7 <0.001 −4.3 <0.001
   sarU Positive to agr expression, −5.2 0.925 18.6 0.001 4.3 <0.001
   sarR Negative to agr, sarA 1.1 0.792 1.1 0.801 −5.2 <0.001
   sarT Negative to agr, hla, sarU expression −9.4 0.868 6.9 0.039 5.3
   <0.001
   sarX Negative to agr −8.6 0.001 −1.2 0.778 −1.7 0.255
   sarZ Positive to agr^[121]38 −1.7 0.041 −1.5 0.136 −2.6 0.009
   CcpA Positive to agr 1.8 0.015 −1.1 0.797 −2.3 0.011
   codY Negative to agr 1.8 0.001 1.4 0.257 −1.1 0.783
   mgrA Cytoplasmic regulator; induction of efflux pumps and capsule
   expression; repress of surface proteins, Positive to agr, sarZ, sarX
   −1.9 0.017 1.1 0.966 −4.6 <0.001
   rsr Repressor of agr^[122]39 4.9 <0.001 2.3 0.003 −1.9 0.092
   sigB Stress response −2.4 <0.001 −2.6 <0.001 −2.2 <0.001
   yjbH Virulence factor regulator^[123]28 5.6 <0.001 2.4 <0.001 −1.4
   0.139
   [124]Open in a new tab

Conclusions

   In the present study, S. aureus gene expression changes in the mouse
   liver after systemic infection were analyzed over time by in vivo
   RNA-Seq. The results suggested that S. aureus responds to changes in
   oxygenation and environmental influences associated with abscess
   formation of S. aureus in the liver, thereby causing liver and heart
   damage. Further, only a few metal transporters were upregulated at
   6 h.p.i., but many metal transport systems increased remarkedly after
   that time. Our results also revealed the contribution of the manganese
   transporter mntABC alone and staphylopine to the pathogenicity of the
   broad metal transport system. As for the expression of pathogenic
   toxins, the timing of the upregulation differed depending on the toxin.
   Blood hemolytic toxin significantly increased from the early stage of
   infection, while the expression levels of leukocyte toxin,
   superantigens, and serine proteases increased in the late stage of
   infection. By regulating gene expression, S. aureus exhibits a
   sophisticated response to changes in environmental conditions in the
   host during infection.

Methods

Ethics statement

   The mouse experiments were performed at the University of Tokyo and
   Teikyo University following the regulations for animal care and use and
   approved by the Animal Use Committee at the Graduate School of
   Pharmaceutical Science at the University of Tokyo (P27-4) and Teikyo
   University (21-027).

S. aureus infection and organ collection for RNA isolation

   S. aureus Newman strain was grown for 20 h on TSB medium at 37 °C. The
   full growth was diluted 100-fold with 5 ml TSB and regrown for 24 h,
   and then the cells were centrifuged and suspended in PBS pH 7.2. The
   cells (5.6 × 10^7 CFU) were injected into C57BL/6J mice via the tail
   vein. At 6, 24, or 48 h.p.i., mice were killed to isolate the liver.
   The organs were immediately placed in liquid nitrogen and maintained at
   −80 °C until RNA extraction. A part of the mouse organs were
   homogenized to calculate viable cell numbers in each organ. Each
   experiment was conducted with 3 animals.

Enrichment of S. aureus Newman RNA from infected mouse organs and removal of
Ribosomal RNA

   Mouse livers (n = 3 for each time-point, ~50 mg each) were homogenized
   and lysed in 1.5 mL of buffer RLT using an RNeasy Mini Kit (QIAGEN,
   Hilden, Germany) with one ф 5-mm zirconia bead by shaking in a 2.0-mL
   screw cap microtube using a Bead Crusher (μT-12, Taitec, Saitama,
   Japan, tube holders were chilled at −20 °C until use) at 2500 rpm for
   1 min. The samples were centrifuged at 20,000 × g at 4 ˚C for 1 min,
   and precipitants were washed with an equal volume of PBS by
   centrifugation at 20,000 × g at 4 ˚C for 1 min. Bacterial cell
   precipitates were suspended in 200 µl of TE buffer containing 0.2 mg/mL
   lysostaphin and incubated at room temperature for 30 min. After adding
   700 µl of RLT buffer and 200 µl volume of ф 0.5-mm zirconia beads, the
   samples were shaken at 2500 rpm for 5 min, and debris was removed by
   centrifugation at 5000 × g at room temperature for 10 min. The RNA was
   purified according to the manufacturer’s instructions. Ribosomal RNA in
   the above samples was removed using a Ribo-Zero rRNA Removal Kit
   (Human/Mouse/Rat) (Illumina, San Diego, CA) according to the
   manufacturer’s protocol. For extraction of RNA from cultures in TSB
   medium, S. aureus Newman strain was grown for 20 h on TSB medium at
   37 ˚C. The full growth was diluted 100-fold with 5 ml TSB and regrown
   until A[600] = 1.0 (late log phase). The cells were then centrifuged
   and suspended in PBS pH 7.2, and library preparation was performed as
   described below. Four independent cultures were used for the analysis.

Library preparation and RNA-sequencing

   RNA-Seq analysis for differential expression analysis was performed
   with the HiSeq platform (Illumina) or Ion Proton system (Thermo Fisher
   Scientific) according to the manufacturer’s instructions. Briefly, for
   Hiseq, the RNA-Seq libraries were prepared using the TruSeq RNA sample
   preparation kit (version 2; Illumina), except the poly(A) selection
   procedure was omitted. The double-stranded PCR products were purified
   and size-fractionated using a bead-mediated method with AMPure XP
   (Beckman Coulter, California, CA). The RNA-Seq libraries were
   quantified by a bioanalyzer (Agilent, California, CA). Thirty-six
   base-pair single-end sequencing was conducted on a HiSeq 2000 or 2500
   platform, using a TruSeq SR Cluster Kit v3-cBot-HS and a TruSeq SBS kit
   (version 3-HS). For the Ion Proton system, library preparation for
   RNA-Seq was performed using an Ion Total RNA-Seq Kit v2 following the
   manufacturer’s instructions. Briefly, the ribosome depleted RNA was
   then fragmented by RNase III, reverse transcribed, and amplified. The
   size distribution and yield of the amplified library was confirmed in
   the bioanalyzer, and the libraries were enriched in an Ion PI Chip v2
   using the Ion Chef (Thermo Fisher Scientific). Subsequent sequencing
   was performed in the Ion Proton System.

Differential gene expression analysis

   All data were analyzed using CLC Genomics Workbench software, version
   12 (CLC Bio, Aarhus, Denmark). Reads were aligned to the Newman genome
   (Accession No. [125]NC_009641) and the mouse genome
   (Mus_musculus.GRCm38) allowing a minimum length fraction of 0.95 and
   minimum similarity fraction of 0.95. Differential gene expression
   analysis was performed using edgeR analysis^[126]29 for a normalized
   dataset by scaling using the TMM method^[127]30. Genes with an FDR
   p < 0.05 using the Benjamini and Hochberg’s algorithm^[128]31 were
   classified as having significantly different expression.

Histopathology and hematological parameter analysis

   After C57BL/6J mice were infected with S. aureus Newman strain, livers
   were harvested at 6, 24, and 48 h.p.i. Histopathological analysis was
   performed by Advantec Co., Ltd (Osaka, Japan). Briefly,
   histopathological specimens were prepared by paraffin-embedding and
   cutting into 4-µm-thick slices. Hematoxylin and eosin staining and Gram
   staining (Hacker variant) were performed to evaluate the histopathology
   and the presence of bacterial nests in specimens. At the same time, we
   collected blood from the heart with a heparin-treated syringe and
   prepared a plasma fraction by centrifugation at 5000 × g for 10 min at
   4 ˚C. The samples were frozen at −80 ˚C until measurement.
   Hematological parameters such as ALT, AST, and CL were analyzed using a
   7180 clinical analyzer (Hitachi High-Tech Corporation, Tokyo, Japan) by
   Ina Research Inc. (Nagano, Japan).

Construction of S. aureus mutants

   Single cross-over recombination; gene disruptions were performed as
   previously described^[129]32. In summary, the internal regions within
   the open reading frames of the gene were amplified by PCR (Prime Star
   Max DNA polymerase, Takara, Tokyo, Japan) using the primers listed in
   Supplementary Table [130]3, and the PCR product was cloned into
   integration vector pCK20^[131]32. The plasmid was then transformed to
   S. aureus RN4220^[132]26 by electroporation using Bio-Rad Genepulser
   Xcell (0.2 mm cubet, 2.3 kV, 100 ohm, 25 µFD). Double cross-over
   recombination; gene disruptions were performed as previously
   described^[133]33. Briefly, the genome DNA regions upstream and
   downstream of the target region were amplified by PCR using the listed
   primers, and then overlap extension-PCR was performed using these 2 DNA
   fragments together with the aph gene amplified from the pSF151 vector
   (primers: KmF; 5′-AGCGAACCATTTGAGGTGAT-3′ and KmR; 5′-GGGACCCCT
   ATCTAGCGAAC-3′). The PCR product was cloned into the pKOR3a
   vector^[134]33 and introduced into the RN4220 strain by
   electroporation. Integration of the mutant cassette in the genome was
   confirmed by PCR and further transformed into S. aureus Newman^[135]34
   by phage transduction using phage 80α^[136]35. We confirmed that the
   established deletion mutants showed a similar growth curve as the
   wild-type (Supplementary Fig. [137]4). To establish the complementary
   strains, we amplified the mntABC operon, narK gene, cntE gene, and
   cntLMK operon from the genome of the Newman strain using the primers
   listed in Supplementary Table [138]3. For narK, mntABC, and cntLMK
   genes, native promoters were used and introduced into the pHY300-erm
   vector, in which the tetracycline-resistant gene of pHY300plk was
   substituted with the erythromycin-resistant gene after digestion by the
   restriction enzymes Hind III and BamH I. For the cntE gene, we
   introduced this gene following the fbaA gene promoter in
   pHY300-erm-fbaA, a vector that harbors the EcoRI- and BamHI-digested
   DNA region from the pND50-fbaA vector^[139]36, as this gene is located
   at the end of the operon. The constructed plasmids were transduced to
   the RN4220 strain and the plasmids were transferred to each
   gene-disrupted mutant using bacteriophage 80α^[140]36.

Mouse survival assay

   S. aureus Newman wild-type and mutant strains were grown overnight on
   TSB medium supplemented with antibiotics on a rotary shaker maintained
   at 37 °C for 20 h to obtain full growth. The full growth was diluted
   100-fold with TSB and cultured for 24 h on the same shaker, and then
   the cells were centrifuged and resuspended in PBS pH 7.2 to an optical
   density of 0.7 at 600 nm. From this, 200 μl of the cells was injected
   intravenously into C57BL/6J mice, and mouse survival was determined.

Statistics and reproducibility

   Unless stated otherwise, statistical analysis was performed using
   GraphPad Prism version 9.0 (GraphPad Software). We used Log-rank
   (Mantel-Cox) test for mice survival assay and a Kruskal-Wallis test
   with Dunn’s multiple comparisons test for the comparison of the
   hematological parameters. We performed more than 2 times of infection
   experiments as described in figure legends, except for the survival
   assay of complement strains in Supplementary Figs. [141]2a and [142]3.

Reporting summary

   Further information on research design is available in the [143]Nature
   Research Reporting Summary linked to this article.

Supplementary information

   [144]Peer Review File^ (1MB, pdf)
   [145]Supplementary information^ (1.4MB, pdf)
   [146]42003_2022_3674_MOESM3_ESM.pdf^ (485.6KB, pdf)

   Description of Additional Supplementary Files
   [147]Supplementary Data 1^ (438.8KB, xlsx)
   [148]Supplementary Data 2^ (2.5MB, xlsx)
   [149]Supplementary Data 3^ (21.4KB, xlsx)
   [150]Supplementary Data 4^ (17.5KB, xlsx)
   [151]Reporting summary^ (1.2MB, pdf)

Acknowledgements