3.1 Transcriptome sequencing analysis of Mga Spn regulatory proteins
MgaSpn is a member of the Mga/AtxA family and has high homology with the Mga protein of group A streptococcus (GAS). Mga transcribes and activates many virulence genes in Streptococcus pyogenes25,26. We previously found that MgaSpn participates in the transcriptional regulation of the cps gene cluster in the capsule and the lic1 teichoic acid synthesis-related gene cluster20. To explore whether the MgaSpn global transcriptional regulator mediates other surface virulence factors in addition to the above gene clusters, we conducted transcriptomic sequencing of the WT (D39s) and mgaSpn-deficient (D39ΔmgaSpn) strains.
The transcriptome results showed that there were 149 differential genes, with 113 upregulated genes and 36 downregulated genes above a change cutoff of 1.5-fold.(Table 3). The up-regulated genes mainly involved genes related to PTS sugar transporter, bacteriocin and de novo synthesis of uracil, the downregulated genes mainly involved ribosomal proteins and ABC transporters. What attracted us was pyrR, pyrF, carA, and carB pyrimidinede novo synthesis genes were significantly upregulated in mgaSpn-deficient strains. qRT-PCR confirmed higher transcript levels of pyrF, pyrR, carA, and carB, about 2- to 3-fold compared to WT bacteria (Fig. 1). These results were consistent with the transcriptome sequencing results, suggesting that MgaSpn maybe involved in the regulation of the uracil synthesis pathway.
Table 3
Differential gene expression detected by transcriptome sequencinga
Gene id
|
log2FoldChange
|
Gene name
|
Gene description
|
Decreased
|
|
|
|
SPD_RS00470
|
3.540844073
|
-
|
sugar ABC transporter permease && PF00528:Binding-protein-dependent transport system inner membrane component
|
SPD_RS00555
|
3.076620487
|
-
|
LysM peptidoglycan-binding domain-containing protein && PF01476:LysM domain
|
SPD_RS00580
|
2.577646712
|
-
|
transporter substrate-binding domain-containing protein
|
SPD_RS00780
|
1.758994401
|
-
|
glycosyltransferase family 2 protein
|
SPD_RS00815
|
1.794268401
|
-
|
hypothetical protein
|
SPD_RS00825
|
1.569853425
|
-
|
CPBP family intramembrane metalloprotease
|
SPD_RS00830
|
1.739055601
|
-
|
MFS transporter
|
SPD_RS01055
|
1.65768181
|
nrdG
|
anaerobic ribonucleoside-triphosphate reductase activating protein
|
SPD_RS01450
|
1.60393733
|
-
|
Cof-type HAD-IIB family hydrolase
|
SPD_RS01455
|
1.975312512
|
-
|
NCS2 family permease
|
SPD_RS01460
|
1.980699182
|
-
|
CPBP family intramembrane metalloprotease
|
SPD_RS02415
|
2.44806895
|
-
|
hypothetical protein
|
SPD_RS03785
|
2.792999234
|
-
|
DUF3270 domain-containing protein
|
SPD_RS04545
|
2.381183909
|
-
|
hypothetical protein
|
SPD_RS04555
|
1.539280353
|
-
|
ABC transporter permease
|
SPD_RS04770
|
1.699542615
|
-
|
amino acid permease
|
SPD_RS05420
|
1.609153602
|
-
|
YueI family protein
|
SPD_RS06320
|
3.167578317
|
-
|
hypothetical protein
|
SPD_RS06330
|
2.473874795
|
-
|
replication initiator protein A
|
SPD_RS07150
|
1.870233206
|
-
|
GNAT family N-acetyltransferase
|
SPD_RS07300
|
1.682021275
|
-
|
DUF5590 domain-containing protein
|
SPD_RS07520
|
3.016370291
|
-
|
OFA family MFS transporter
|
SPD_RS08460
|
7.007076022
|
-
|
helix-turn-helix domain-containing protein
|
SPD_RS08685
|
1.956176742
|
-
|
xanthine phosphoribosyltransferase
|
SPD_RS08690
|
1.766305707
|
-
|
purine permease
|
SPD_RS09375
|
1.56279407
|
-
|
HlyC/CorC family transporter
|
SPD_RS09575
|
1.66801798
|
-
|
response regulator transcription factor
|
SPD_RS09580
|
2.278029835
|
-
|
sensor histidine kinase
|
SPD_RS09585
|
1.847650149
|
-
|
ABC transporter permease
|
SPD_RS09590
|
2.663425036
|
-
|
ABC transporter ATP-binding protein
|
SPD_RS09600
|
3.955948867
|
-
|
hypothetical protein
|
SPD_RS09605
|
3.485912843
|
-
|
tRNA-Pro
|
SPD_RS09945
|
2.148818732
|
-
|
LysM peptidoglycan-binding domain-containing protein
|
SPD_RS10435
|
1.674119829
|
-
|
glycoside hydrolase family 125 protein
|
SPD_RS10725
|
1.571533956
|
-
|
thiamine-binding protein
|
SPD_RS10810
|
2.255949489
|
-
|
CHAP domain-containing protein
|
Increased
|
|
|
|
SPD_RS00145
|
-1.879533021
|
-
|
carbonic anhydrase
|
SPD_RS00185
|
-2.721181153
|
-
|
CoA-binding protein
|
SPD_RS00320
|
-1.556051641
|
-
|
beta-N-acetylhexosaminidase
|
SPD_RS00330
|
-1.917993406
|
-
|
beta-galactosidase
|
SPD_RS00495
|
-1.989274903
|
-
|
DUF4299 domain-containing protein
|
SPD_RS00565
|
-1.859739364
|
-
|
lactococcin 972 family bacteriocin
|
SPD_RS00600
|
-4.011238736
|
-
|
hypothetical protein
|
SPD_RS00605
|
-3.919982428
|
-
|
peptidase domain-containing ABC transporter
|
SPD_RS00610
|
-4.240295599
|
-
|
GyrI-like domain-containing protein
|
SPD_RS00615
|
-4.294808957
|
-
|
HlyD family efflux transporter periplasmic adaptor subunit
|
SPD_RS00620
|
-4.697260495
|
-
|
SP_0115 family bacteriocin-like peptide
|
SPD_RS00635
|
-2.722559642
|
-
|
SPH_0218 family bacteriocin-like peptide
|
SPD_RS00925
|
-1.600528958
|
-
|
6%2C7-dimethyl-8-ribityllumazine synthase
|
SPD_RS01525
|
-2.291662237
|
-
|
PTS cellobiose transporter subunit IIB
|
SPD_RS01530
|
-1.988115702
|
-
|
BglG family transcription antiterminator
|
SPD_RS01535
|
-2.749896408
|
-
|
PTS cellobiose transporter subunit IIA
|
SPD_RS01605
|
-2.34063299
|
-
|
gluconate 5-dehydrogenase
|
SPD_RS01645
|
-1.570803847
|
-
|
LacI family DNA-binding transcriptional regulator
|
SPD_RS01850
|
-1.508244909
|
rnpB
|
RNase P RNA component class B
|
SPD_RS02045
|
-2.339857524
|
-
|
enoyl-CoA hydratase
|
SPD_RS02060
|
-2.200698741
|
-
|
acyl carrier protein
|
SPD_RS02065
|
-2.26950146
|
fabK
|
enoyl-[acyl-carrier-protein] reductase FabK
|
SPD_RS02070
|
-2.833084477
|
fabD
|
ACP S-malonyltransferase
|
SPD_RS02075
|
-3.246698575
|
fabG
|
3-oxoacyl-[acyl-carrier-protein] reductase
|
SPD_RS02080
|
-3.45560506
|
fabF
|
beta-ketoacyl-ACP synthase II
|
SPD_RS02085
|
-3.11639046
|
-
|
acetyl-CoA carboxylase biotin carboxyl carrier protein
|
SPD_RS02090
|
-3.773105326
|
fabZ
|
3-hydroxyacyl-ACP dehydratase FabZ
|
SPD_RS02095
|
-3.111535703
|
accC
|
acetyl-CoA carboxylase biotin carboxylase subunit
|
SPD_RS02100
|
-2.954741497
|
-
|
acetyl-CoA carboxylase carboxyltransferase subunit beta
|
SPD_RS02105
|
-3.004358554
|
-
|
acetyl-CoA carboxylase carboxyl transferase subunit alpha
|
SPD_RS02115
|
-3.264746808
|
briC
|
biofilm-regulating peptide BriC
|
SPD_RS02125
|
-3.445879425
|
-
|
CPBP family intramembrane metalloprotease
|
SPD_RS02375
|
-1.797354206
|
-
|
CTP synthase
|
SPD_RS02470
|
-1.54647738
|
grpE
|
nucleotide exchange factor GrpE
|
SPD_RS02475
|
-1.953093782
|
dnaK
|
molecular chaperone DnaK
|
SPD_RS02480
|
-2.229957944
|
-
|
hypothetical protein
|
SPD_RS02510
|
-4.32306266
|
-
|
hypothetical protein
|
SPD_RS02530
|
-4.34995027
|
blpC
|
quorum-sensing system pheromone BlpC
|
SPD_RS02545
|
-3.257627149
|
-
|
hypothetical protein
|
SPD_RS02550
|
-3.335877819
|
-
|
CPBP family intramembrane metalloprotease
|
SPD_RS02555
|
-3.112489772
|
-
|
hypothetical protein
|
SPD_RS02565
|
-3.22119207
|
-
|
CPBP family intramembrane metalloprotease
|
SPD_RS03010
|
-1.953581792
|
-
|
S8 family serine peptidase
|
SPD_RS03025
|
-2.389396089
|
-
|
PTS galactitol transporter subunit IIC
|
SPD_RS03275
|
-1.702707644
|
pyrF
|
orotidine-5'-phosphate decarboxylase
|
SPD_RS03280
|
-1.569197499
|
-
|
orotate phosphoribosyltransferase
|
SPD_RS03475
|
-2.511480332
|
-
|
DegV family protein
|
SPD_RS03535
|
-2.413688835
|
-
|
CBS domain-containing protein
|
SPD_RS03675
|
-2.271067339
|
-
|
hypothetical protein
|
SPD_RS03695
|
-1.625360236
|
gor
|
glutathione-disulfide reductase
|
SPD_RS03840
|
-2.086302291
|
-
|
DUF1827 family protein
|
SPD_RS03940
|
-2.152351916
|
-
|
DNA topology modulation protein
|
SPD_RS04120
|
-2.042616047
|
ssrA
|
transfer-messenger RNA
|
SPD_RS04130
|
-1.710636931
|
-
|
DeoR/GlpR transcriptional regulator
|
SPD_RS04150
|
-1.831943834
|
-
|
hypothetical protein
|
SPD_RS04305
|
-2.105403339
|
-
|
PspC domain-containing protein
|
SPD_RS04310
|
-4.676754015
|
-
|
hypothetical protein
|
SPD_RS04580
|
-2.184672578
|
-
|
dihydroorotate dehydrogenase electron transfer subunit
|
SPD_RS04585
|
-2.022003954
|
-
|
dihydroorotate dehydrogenase
|
SPD_RS04590
|
-1.737808776
|
-
|
endo-beta-N-acetylglucosaminidase
|
SPD_RS04815
|
-1.786395769
|
hemH
|
ferrochelatase
|
SPD_RS04930
|
-1.505667841
|
-
|
iron-siderophore ABC transporter substrate-binding protein
|
SPD_RS05010
|
-1.918930761
|
-
|
helix-turn-helix transcriptional regulator
|
SPD_RS05165
|
-1.503059453
|
-
|
metal-sulfur cluster assembly factor
|
SPD_RS05630
|
-1.759193213
|
-
|
transcription antiterminator
|
SPD_RS05635
|
-2.003549678
|
lacD
|
tagatose-bisphosphate aldolase
|
SPD_RS05640
|
-2.458126365
|
-
|
tagatose-6-phosphate kinase
|
SPD_RS05645
|
-2.994294299
|
lacB
|
galactose-6-phosphate isomerase subunit LacB
|
SPD_RS05650
|
-2.8056828
|
lacA
|
galactose-6-phosphate isomerase subunit LacA
|
SPD_RS06035
|
-1.535668533
|
carB
|
carbamoyl-phosphate synthase large subunit
|
SPD_RS06040
|
-1.600881094
|
carA
|
glutamine-hydrolyzing carbamoyl-phosphate synthase small subunit
|
SPD_RS06050
|
-1.644316117
|
pyrR
|
bifunctional pyr operon transcriptional regulator/uracil phosphoribosyltransferase PyrR
|
SPD_RS06070
|
-1.732221049
|
-
|
ABC-F family ATP-binding cassette domain-containing protein
|
SPD_RS06090
|
-1.712128317
|
-
|
uracil transporter
|
SPD_RS06305
|
-1.590012213
|
-
|
lanthionine synthetase
|
SPD_RS06490
|
-1.939313287
|
-
|
ABC transporter ATP-binding protein
|
SPD_RS06650
|
-1.526791226
|
-
|
30S ribosomal protein S21
|
SPD_RS06750
|
-1.568313221
|
-
|
ABC transporter ATP-binding protein
|
SPD_RS06910
|
-1.806831456
|
-
|
DUF1836 domain-containing protein
|
SPD_RS06915
|
-2.426500081
|
-
|
hemolysin III family protein
|
SPD_RS07065
|
-1.548217135
|
-
|
thioredoxin
|
SPD_RS07525
|
-2.25529155
|
-
|
FAD-containing oxidoreductase
|
SPD_RS08060
|
-1.610646583
|
-
|
hypothetical protein
|
SPD_RS08110
|
-1.939427257
|
-
|
ABC transporter ATP-binding protein
|
SPD_RS08115
|
-1.655062375
|
-
|
membrane protein
|
SPD_RS08150
|
-1.793486751
|
-
|
PTS beta-glucoside transporter subunit IIBC
|
SPD_RS08330
|
-1.862798757
|
-
|
DUF4649 family protein
|
SPD_RS08335
|
-1.549051733
|
trxA
|
thioredoxin
|
SPD_RS08400
|
-2.034277727
|
-
|
type II toxin-antitoxin system HicA family toxin
|
SPD_RS08485
|
-1.805631069
|
-
|
CsbD family protein
|
SPD_RS08875
|
-2.139807301
|
treP
|
PTS system trehalose-specific EIIBC component
|
SPD_RS09185
|
-2.567762138
|
ply
|
cholesterol-dependent cytolysin pneumolysin
|
SPD_RS09190
|
-2.192158071
|
-
|
hypothetical protein
|
SPD_RS09195
|
-2.092791382
|
-
|
hypothetical protein
|
SPD_RS09200
|
-2.39262
|
-
|
DUF4231 domain-containing protein
|
SPD_RS09435
|
-2.318458004
|
-
|
acylphosphatase
|
SPD_RS09550
|
-2.260043723
|
-
|
universal stress protein
|
SPD_RS09790
|
-2.812594241
|
ulaG
|
L-ascorbate 6-phosphate lactonase
|
SPD_RS09825
|
-3.524245923
|
-
|
PTS ascorbate transporter subunit IIC
|
SPD_RS10125
|
-2.768053314
|
-
|
phosphate-binding protein
|
SPD_RS10225
|
-1.72876586
|
-
|
membrane protein
|
SPD_RS10245
|
-1.947416517
|
-
|
extracellular solute-binding protein
|
SPD_RS10405
|
-3.798523107
|
pcpA
|
choline-binding protein PcpA
|
SPD_RS10500
|
-1.757549455
|
-
|
SPFH domain-containing protein
|
SPD_RS10525
|
-2.529211255
|
-
|
PTS mannose/fructose/sorbose transporter family subunit IID
|
SPD_RS10530
|
-3.863757933
|
-
|
PTS mannose/fructose/sorbose/N-acetylgalactosamine transporter subunit IIC
|
SPD_RS10535
|
-4.532680442
|
-
|
PTS sugar transporter subunit IIB
|
SPD_RS10540
|
-4.727078471
|
-
|
PTS sugar transporter subunit IIA
|
SPD_RS10555
|
-3.778092141
|
-
|
rhamnulokinase
|
SPD_RS10630
|
-3.069439605
|
-
|
hypothetical protein
|
SPD_RS10755
|
-2.298327647
|
raiA
|
ribosome-associated translation inhibitor
|
SPD_RS10905
|
-1.809492243
|
-
|
TetR/AcrR family transcriptional regulator
|
SPD_RS10945
|
-1.897430199
|
-
|
trypsin-like peptidase domain-containing protein
|
a The reference genome comes from NCBI RefSeq assembly GCF_000014365.2. https://www.ncbi.nlm.nih.gov/nuccore/NC_008533 |
3.2. Mga Spn is involved in the regulation of the uracil synthesis pathway
To investigate if MgaSpn participates in the uracil synthesis pathway, we examined the metabolism of WT and mgaSpn-deficient bacteria cultured in C + Y medium to an OD620 of 0.5 by metabolome sequencing(Fig. 2). There were 31 different metabolites in the two strains (Table 4). Compared to WT bacteria, the contents of 15 metabolites, including limonene- 1,2-diol and L-histidine, in mgaSpn- deficient bacteria were increased. The contents of 16 metabolites, including N-acetyl-neuraminic acid, gulonic acid, UMP, pyrimidodiazepine, and D-ribose, were decreased. Of note, UMP and pyrimidodiazepine metabolites in the uracil synthesis pathway were significantly reduced by 6- to 7- fold in mgaSpn-deficient bacteria.
We previously confirmed that MgaSpn is a transcription suppressor in capsule biosynthesis20.Deletion of MgaSpn increases the capsular content in the whole bacterial lysate, and the small molecular weight proteins are concentrated in the increased capsular content20. However, the reason for the increase in the small molecular weight capsular proteins remains unclear. The uracil synthesis pathway has been shown to affect capsular polysaccharides (CPS) promoter expression and CPS production in the S. pneumoniae D39 strain7. After MgaSpn deletion, the transcriptomic results showed that the expression of uracil synthesis genes was increased, and the metabolomics results showed that the uracil synthesis pathway metabolites were decreased. These findings suggested that MgaSpn may affect capsule synthesis by regulating the uracil synthesis pathway.
Table 4
Differential metabolites detected by metabolome sequencing
|
VIP
|
log2(FC_M/D)
|
p.value
|
N-Acetyl-a-neuraminic acid
|
1.87893
|
-19.098
|
0.021071
|
Gulonic acid
|
1.860573
|
-1.7987
|
0.030383
|
UMP
|
1.836096
|
-15.964
|
0.021071
|
5-Acetamidovalerate
|
1.778424
|
-3.8623
|
0.030383
|
Dimethyl sulfone
|
1.774266
|
-1.8729
|
0.030383
|
Limonene-1,2-diol
|
1.714941
|
2.7051
|
0.030383
|
D-Ribose
|
1.713519
|
-1.0715
|
0.030383
|
Guanosine
|
1.689839
|
1.8766
|
0.030383
|
Tetracosanoic acid
|
1.68122
|
0.92923
|
0.030383
|
Lanosterin
|
1.637613
|
-1.5964
|
0.030383
|
(-)-Epigallocatechin
|
1.610466
|
-16.227
|
0.021071
|
5-Guanidino-3-methyl-2-oxopentanoate
|
1.589845
|
-2.3838
|
0.030383
|
Pyrimidodiazepine
|
1.57903
|
-2.8171
|
0.030383
|
L-Histidine
|
1.577359
|
0.96313
|
0.030383
|
Phenyl acetate
|
1.575216
|
0.47183
|
0.030383
|
Acetylcholine chloride
|
1.554901
|
2.5472
|
0.030383
|
(S)-1-Phenylethanol
|
1.547117
|
1.1176
|
0.030383
|
Imidazol-5-yl-pyruvate
|
1.544352
|
0.31394
|
0.030383
|
D-Glucuronic Acid
|
1.52796
|
1.379
|
0.030383
|
12,13-DHOME
|
1.503511
|
-0.89095
|
0.030383
|
Spermidine
|
1.493071
|
-1.1778
|
0.030383
|
L-Erythrulose
|
1.453958
|
0.79214
|
0.030383
|
Stearic acid
|
1.365478
|
-1.7833
|
0.030383
|
Adenosine diphosphate ribose
|
1.361712
|
-1.523
|
0.030383
|
L-2-Hydroxyglutaric acid
|
1.33444
|
-1.7077
|
0.030383
|
4-Oxoproline
|
1.306298
|
2.1815
|
0.030383
|
Maleimide
|
1.293903
|
-1.4198
|
0.030383
|
4-Pyridoxic acid
|
1.271316
|
2.5939
|
0.030383
|
11-Dehydrocorticosterone
|
1.196153
|
1.5698
|
0.030383
|
Dehydroepiandrosterone
|
1.189842
|
1.7868
|
0.030383
|
Methyl (indol-3-yl)acetate
|
1.185804
|
2.3809
|
0.030383
|
3.3. Mga Spn binds to the pyrR promoter region
MgaSpn, a member of the Mga/AtxA family of global transcriptional regulators, directly binds to the regulatory regions of target genes to regulate target gene expression. We found some differentially expressed genes the pyrR, pyrF, carA, and carB uracil de novo synthesis genes by transcriptomic analysis. To identify the uracil metabolism genes that MgaSpn directly regulates, we investigated the expression of the pyrR, pyrF, carA, and carB genes in WT bacteria (D39s), mgaSpn-deficient bacteria (D39∆mgaSpn),mgaSpn complement bacteria (D39∆mgaSpn::mgaSpn), and mgaSpn-overexpressing bacteria (D39::mgaSpn). As shown in Fig. 3, all genes were upregulated after mgaSpn deletion, but only pyrR was about 3-fold downregulated during mgaSpn recovery and overexpression strains, indicating that MgaSpn maybe directly involved in the regulation of pyrR.
MgaSpn is a transcriptional regulator that contains two conserved helix-turn-helix (HTH) domains, which are DNA-binding motifs, indicating that MgaSpn has the ability to bind DNA27. To explore whether MgaSpn protein directly participates in the transcriptional regulation of pyrR, EMSAs were performed using a DNA fragment probe approximately 300 bp upstream of the pyrR gene to verify the specific binding of the MgaSpn protein to the pyrR promoter. As the concentration of protein added to the reaction system gradually increased, the binding bands of the probes and proteins shifted backward, indicating that the binding of MgaSpn to the pyrR promoter was concentration-dependent (Fig. 4a). In the unlabeled probe competition lane, the unlabeled probes competed to bind to the mgaSpn- and pyrR-labeled probes. These results indicated that MgaSpn specifically binds to the pyrR promoter region.
Because MgaSpn directly binds to the pyrR promoter region, we performed DNase Ⅰ footprinting analysis to determine the specific recognition site of MgaSpn on the PpyrR probe (Fig. 4b). After adding protein (1.5 µg) to the 390 ng probe system, the following two protection areas were identified: 31 bp (5'-TAGCAATTTGTAAGATGCTACATTGAAACTT-3') and 53 bp (5'-TTGTTTAAGGAGACTTTTCTTTTTGGAGGTTTGTCATGAAAACAAAAGAAGTT-3') (Fig. 4c). To further confirm that MgaSpn specifically binds to PpyrR, we designed eight mutations in the binding site of the PpyrR promoter. The mutant pyrR probe EMSA results showed that MgaSpn lost the ability to bind the mutant probe(Figure S2). However, additional experiments are required to determine if these two binding sites are functional sites for MgaSpn transcriptional regulation. These data suggested that MgaSpn may play a key role in the co-transcription of pyrR genes.
3.4. Mga Spn negatively regulates CPS production by pyrR
We next evaluated the morphology of the capsules of each strain grown to an OD620 of 0.5 in normal C + Y medium containing 15mg L- 1 pyrimidines using transmission electron microscopy (TEM) (Fig. 5a). There was no obvious difference in capsule thickness when comparing the D39s strain to the D39∆mgaSpn and D39∆pyrR strains, but the D39∆mgaSpn∆pyrR strain had a thinner capsule than the D39s strain.The transmission electron microscopy (TEM) was used to measure the capsule thickness of 10 strain with ImageJ software, and the average capsule thicknesses of the D39s, D39∆mgaSpn, D39∆pyrR, and D39∆mgaSpn∆pyrR strains were 57.03 ± 14.73 nm, 56.51 ± 10.52 nm, 53.84 ± 17.45nm, and 30.32 ± 6.97nm, respectively (Fig. 5a).
We also detected the capsular content of the bacteria using ELISAs. When the bacteria grew to an OD620 of 0.5, the same amount of bacteria was collected by centrifugation for ELISA detection. Figure 5b shows that there was no obvious difference in the capsular content between the D39s and D39∆mgaSpn strains grown in normal C + Y medium contains 15mg L- 1 pyrimidines, and the capsular content of the D39∆pyrR-( grown in uracil-free C + Y medium) and ∆D39∆mgaSpn∆pyrR strains was 2 to 3 times lower than that of the D39s and D39∆mgaSpn strains, which was consistent with the TEM results.
In addition, capsular polysaccharides were quantified by the uronic acid method. Glucuronic acid is a specific component of S. pneumoniae capsules regulated by CpsT/F/G/L, and it exists in the form of glucuronic acid in type II capsules, with each repeating unit containing one glucuronic acid. The content of the capsule in bacteria can be measured by detecting the content of uronic acid. The capsular content of the whole bacteria (Bacteria-CPS) was detected, and the results showed that the capsular content of the D39∆mgaSpn∆pyrR strain was significantly lower than that of the WT strain and mgaSpn-deficient strains (Fig. 5c). There were fewer pods of the D39∆pyrR-deficient strain grown in uracil-free C + Y than the WT and mgaSpn-deficient strains.
We next collected the whole bacterial lysate of each strain and detected the expression of capsules by Western blot analysis (Fig. 5d). The total capsular content of the D39∆mgaSpn strain was significantly increased compared to that of the D39s strain, and the small molecular weight capsular proteins of the D39∆pyrR strain were slightly decreased compared to that of the D39s strain, which maybe due to the presence of a remedial synthesis pathway in uracil C + Y. Therefore, we detected the expression of capsules in the D39∆pyrR strain grown in uracil-free C + Y medium (D39∆pyrR-). The expression of capsules in the D39∆pyrR- and D39∆mgaSpn∆pyrR strains was significantly decreased, but the expression of capsules was recovered after supplementing the D39∆mgaSpn∆pyrR strain with pyrR. These results indicated that both mgaSpn and pyrR are involved in the regulation of capsule synthesis and mgaSpn has a positive synergistic effect on the regulation of the capsule by pyrR.
3.5. pyrR influences adhesion and pathogenicity of S. pneumoniae
To understand the effect of pyrR deficiency on the virulence of S. pneumoniae, we used the A549 lung epithelial cell line to detect the adhesion and invasion ability of the D39s, D39∆pyrR, D39∆pyrR-, D39∆mgaSpn, and D39∆mgaSpn∆pyrR::pyrR strains. As shown in Fig. 6a, the D39s and D39∆pyrR strains adhered to epithelial cells 2–3 fold greater than other strains, and the D39∆mgaSpn strain had a stronger invasion ability than the other strains. In addition, the D39∆pyrR- and D39∆mgaSpn∆pyrR strains had significantly reduced invasion and adhesion abilities compared to the other strains (Fig. 6b). Previous studies have suggested that during colonization, S. pneumoniae express low levels of CPS to enhance the exposure of cell surface proteins and promote binding to epithelial cells28. However, the invasion and adhesion of low-level capsularstrains, such as the D39∆pyrR- and D39∆mgaSpn∆pyrR strains, were reduced. These findings suggested that the simultaneous loss of mgaSpn and pyrR may also lead to changes in other adherence-related virulence factors.
Because thicker capsules help S. pneumoniae escape phagocytosis, we evaluated the anti-phagocytic ability of the strains after incubation with mouse macrophages in the absence of serum. The damaged capsules of the D39∆pyrR- and D39∆mgaSpn∆pyrR strains led to significantly reduced anti-phagocytic effects on macrophages(Fig. 6C).
3.6. pyrR is involved in systemic virulence and nasopharyngeal colonization
To explore the role of pyrR in systemic infection, the experimental mice were divided into five groups, with 6 mice in each group, and were treated with the D39s, D39∆pyrR, D39∆mgaSpn, D39∆mgaSpn∆pyrR, and D39∆mgaSpn∆pyrR::pyrR strains through nasal drops. At 48 h after bacterial infection, nasal lavage fluid, heart blood, and lung tissues were harvested and used for colony counting (Fig. 7a-c). In addition, another five groups of mice, with 12 mice in each group, were infected with the D39s, D39∆pyrR, D39∆mgaSpn,D39∆mgaSpn∆pyrR and D39∆mgaSpn∆pyrR::pyrR strains through the nasal passage, and the survival time of each group of mice was recorded for 14 days (Fig. 7d).
In vivo virulence tests showed that the bacterial load of the double deficient strain was significantly reduced in thenasopharyngeal lavage solution, lung tissues, and heart blood. In the double deficient strain, the colonization ability of the nasal cavity and lung was significantly reduced, and the survival rate was significantly increased. All abilities of pyrR were recovered after pyrR replenishment. These results indicated that MgaSpn affects the pathogenicity of S. pneumoniae through pyrR.