Transcriptomic Analysis of Streptococcus pyogenes Colonizing the Vaginal Mucosa Identifies hupY, an MtsR-Regulated Adhesin Involved in Heme Utilization

Abstract

Streptococcus pyogenes (group A streptococcus [GAS]) is a serious human pathogen with the ability to colonize mucosal surfaces such as the nasopharynx and vaginal tract, often leading to infections such as pharyngitis and vulvovaginitis. We present genome-wide transcriptome sequencing (RNASeq) data showing the transcriptomic changes GAS undergoes during vaginal colonization. These data reveal that the regulon controlled by MtsR, a master metal regulator, is activated during vaginal colonization. This regulon includes two genes highly expressed during vaginal colonization, hupYZ Here we show that HupY binds heme in vitro, affects intracellular concentrations of iron, and is essential for proper growth of GAS using hemoglobin or serum as the sole iron source. HupY is also important for murine vaginal colonization of both GAS and the related vaginal colonizer and pathogen Streptococcus agalactiae (group B streptococcus [GBS]). These data provide essential information on the link between metal regulation and mucosal colonization in both GAS and GBS.IMPORTANCE Colonization of the host requires the ability to adapt to an environment that is often low in essential nutrients such as iron. Here we present data showing that the transcriptome of the important human pathogen Streptococcus pyogenes shows extensive remodeling during in vivo growth, resulting in, among many other differentially expressed genes and pathways, a significant increase in genes involved in acquiring iron from host heme. Data show that HupY, previously characterized as an adhesin in both S. pyogenes and the related pathogen Streptococcus agalactiae, binds heme and affects intracellular iron concentrations. HupY, a protein with no known heme binding domains, represents a novel heme binding protein playing an important role in bacterial iron homeostasis as well as vaginal colonization.

Keywords: Streptococcus; adherence; colonization; heme; iron uptake.

FIG 1

Differentially expressed genes during GAS growth in vivo. The volcano plot shows transcriptomic changes in GAS genes during growth in liquid versus colonizing the murine vaginal tract with –log₁₀ false-discovery rate (FDR) adjusted P value on the y axis and log₂ fold change during murine vaginal growth on the x axis. Genes in blue were significantly differentially expressed with an FDR adjusted P value of <0.05 and a fold change of >2. Genes known to be regulated by the metal regulator MtsR are shown in red, and hupY (spy49_0661)/hupZ (spy49_0662) are specifically marked.

FIG 2

Differential regulation of MtsR-regulated operons in GAS and GBS and MtsR-regulated promoter alignment. (A) Select MtsR-regulated operons of GAS and GBS are shown. Red genes are hupYZ homologs and blue genes are nrdF.2I.2E.2 homologs. Fold change during in vivo growth is shown in white. Values from GBS A909 were previously reported (16). (B) Promoter alignment of select MtsR-regulated genes in both GAS and GBS.

FIG 3

Differential absorption spectroscopy of heme-HupY complex. An increase of heme bound to HupY (17 μM) as increasing concentrations of heme were added to the protein is shown by the sharp peak at 414 nm. The inset displays the changes in absorbance at 414 nm plotted against heme concentration. The data are representative of at least two independent spectroscopic analyses, 0 μM line at axis.

FIG 4

HupY affects intracellular iron concentrations. Shown is growth (20 h) of NZ131 wild-type (blue), ΔhupY mutant (red), and complemented (ΔhupY/pLC007 [gray]) strains in THYB or in THYB containing streptonigrin. The data are from two independent experiments done in technical triplicates, with SD shown. The asterisk indicates significance (P < 0.05, Student’s t test, equal variance).

FIG 5

ΔhupY mutants are impaired in growth with serum or hemoglobin as the sole iron source. Shown is growth (20 h) of NZ131 wild-type (blue), ΔhupY mutant (red), and complemented (ΔhupY/pLC007 [gray]) strains in THYB, THYB with dipyridyl (DP), or THYB-DP supplemented with human serum (5 to 20% final volume) (A) or human hemoglobin (B). The data are from two independent experiments done in technical triplicates, with SD shown. The asterisk indicates significance (P < 0.05, Student’s t test, equal variance).

FIG 6

ΔhupY mutants in both GAS and GBS are impaired in the ability to colonize the murine vaginal tract. WT and isogenic ΔhupY mutants in GAS (A) or GBS (B) were used to colonize the murine vaginal tract. In both cases, the GAS hupY gene was used to complement the mutant strains. Both NZ131 ΔhupY and A909 ΔhupY mutants were attenuated for vaginal colonization compared to WT GAS and GBS, and both mutants were able to be at least partially complemented by the addition of GAS hupY on a plasmid. Statistical significance was assessed using a one-way analysis of variance (ANOVA) and nonparametric Kruskal-Wallis test; the asterisk indicates significance (P < 0.05). n.s., not significant.

FIG 7

Model of the role of HupY in GAS colonization and heme utilization on mucosal surfaces. In the presence of high levels of iron, iron-bound MtsR represses a large number of genes, including the sia operon, which includes the genes for the siaABC heme importer as well as shr, and shp heme binding proteins. In addition, MtsR represses the manganese and iron mtsABC transporter, hupZ, and hupY. MtsR also downregulates the mtsABC gene cluster in a manganese-dependent manner (; not shown in the model). Under iron-depleted conditions such as on mucosal surfaces, MtsR repression is relieved, leading to upregulation of these operons. Shr captures heme (from host hemoproteins or the environment) and delivers it to Shp and subsequently to SiaABC for import into the cell. Once inside the cell, iron can be liberated from heme by the HupZ enzyme. HupY, coregulated with HupZ, is a surface protein that has the ability to bind heme. We propose that HupY binds heme to allow transport to HupZ, either through the Shp/SiaABC heme import pathway or through another mechanism. Open arrows indicate possible heme transfer pathways. HupY also plays a role in mucosal colonization, possibly as an adhesin important for binding to host cells.