Streptococcal Virulence Research Articles

SPD_0410 negatively regulates capsule polysaccharide synthesis and virulence in Streptococcus pneumoniae D39.

Streptococcus pneumoniae capsular polysaccharide (CPS) is a crucial virulence factor for this pathogenic bacterium and is partially under transcriptional control. In this study, we used electrophoretic mobility shift assays and DNA enzyme footprinting to identified the hypothetical protein SPD_0410 as a negative regulator of cps locus. Our results showed that the D39Δspd0410 mutant strain exhibited significantly elevated CPS levels compared to the parental strain D39s. SPD_0410 directly binds at two specific sites on the cps promoter. The regulatory effect of SPD_0410 on CPS was weakened after the mutation of specific binding sites in the promoter. RNAseq analysis revealed that the deletion of spd0410 led to alterations in glucose metabolism. However, the altered glucose levels appeared to eliminate the regulation of CPS synthesis by SPD_0410. Deleting the spd0410 gene resulted in higher invasion and phagocytic resistance of bacteria and in vivo mouse experiments confirmed that D39Δspd0410 caused more severe systemic disease than the parental strain D39s. Our results indicated that SPD_0410 negatively regulates the synthesis of S. pneumoniae capsules and can directly alter pneumococcal virulence.

Essential role of proline synthesis and the one-carbon metabolism pathways for systemic virulence of Streptococcus pneumoniae.

Virulence screens have indicated potential roles during Streptococcus pneumoniae infection for the one-carbon metabolism pathway component Fhs and proline synthesis mediated by ProABC. To define how these metabolic pathways affect S. pneumoniae virulence, we have investigated the phenotypes, transcription, and metabolic profiles of Δfhs and ΔproABC mutants. S. pneumoniae capsular serotype 6B BHN418 Δfhs and ΔproABC mutant strains had strongly reduced virulence in mouse sepsis and pneumonia models but could colonize the nasopharynx. Both mutant strains grew normally in complete media but had markedly impaired growth in chemically defined medium, human serum, and human cerebrospinal fluid. The BHN418 ΔproABC strain also had impaired growth under conditions of osmotic and oxidative stress. The virulence role of proABC was strain specific, as the D39 ΔproABC strain could still cause septicemia and grow in serum. Compared to culture in broth, in serum, the BHN418 Δfhs and ΔproABC strains showed considerable derangement in global gene transcription that affected multiple but different metabolic pathways for each mutant strain. Metabolic data suggested that Δfhs had an impaired stringent response, and when cultured in sera, BHN418 Δfhs and ΔproABC were under increased oxidative stress and had altered lipid profiles. Loss of proABC also affected carbohydrate metabolism and the accumulation of peptidoglycan synthesis precursors in the BHN418 but not the D39 background, linking this phenotype to the conditional virulence phenotype. These data identify the S. pneumoniae metabolic functions affected by S. pneumoniae one-carbon metabolism and proline biosynthesis, and the role of these genetic loci for establishing systemic infection.IMPORTANCERapid adaptation to grow within the physiological conditions found in the host environment is an essential but poorly understood virulence requirement for systemic pathogens such as Streptococcus pneumoniae. We have now demonstrated an essential role for the one-carbon metabolism pathway and a conditional role depending on strain background for proline biosynthesis for S. pneumoniae growth in serum or cerebrospinal fluid, and therefore for systemic virulence. RNAseq and metabolomic data demonstrated that the loss of one-carbon metabolism or proline biosynthesis has profound but differing effects on S. pneumoniae metabolism in human serum, identifying the metabolic processes dependent on each pathway during systemic infection. These data provide a more detailed understanding of the adaptations required by systemic bacterial pathogens in order to cause infection and demonstrate that the requirement for some of these adaptations varies between strains from the same species and could therefore underpin strain variations in virulence potential.

The cadDX operon contributes to cadmium resistance, oxidative stress resistance, and virulence in zoonotic streptococci

Mobile genetic elements (MGEs) enable bacteria to acquire novel genes and traits. However, the functions of cargo genes within MGEs remain poorly understood. The cadmium resistance operon cadDX is present in many gram-positive bacteria. Although cadDX has been reported to be involved in metal detoxification, its regulatory mechanisms and functions in bacterial pathogenesis are poorly understood. This study revealed that cadDX contributes to cadmium resistance, oxidative stress resistance, and virulence in Streptococcus suis, an important zoonotic pathogen in pigs and humans. CadX represses cadD expression by binding to the cadDX promoter. Notably, cadX responds to H2O2 stress through an additional promoter within the cadDX operon, mitigating the harmful effect of excessive cadD expression during oxidative stress. cadDX resides within an 11 K integrative and mobilizable element that can autonomously form circular structures. Moreover, cadDX is found in diverse MGEs, accounting for its widespread distribution across various bacteria, especially among pathogenic streptococci. Transferring cadDX into another zoonotic pathogen, Streptococcus agalactiae, results in similar phenotypes, including resistance to cadmium and oxidative stresses and increased virulence of S. agalactiae in mice. The new functions and regulatory mechanisms of cadDX shed light on the importance of the cadDX system in driving evolutionary adaptations and survival strategies across diverse gram-positive bacteria.

Signal recognition particle RNA is critical for genetic competence and virulence of Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) causes multiple important infectious diseases in humans. The signal recognition particle (SRP) complex, which comprised 4.5S small cytoplasmic RNA (ScRNA), and the Ffh and FtsY proteins, mediates membrane targeting and translocation of secreted proteins in all organisms. However, the role of SRP and ScRNA has not been characterized during the induction of the competence system for genetic transformation and virulence in pneumococcus. By using a combination of genetic, biochemical, proteomic, and imaging approaches, we demonstrated that the SRP complex plays a significant role in membrane targeting of competence system-regulated effectors important for genetic transformation, virulence during bacteremia and pneumonia infections, and nutritional acquisition.

Roles of flavoprotein oxidase and the exogenous heme- and quinone-dependent respiratory chain in lactic acid bacteria

Lactic acid bacteria (LAB) are a type of bacteria that convert carbohydrates into lactate through fermentation metabolism. While LAB mainly acquire energy through this anaerobic process, they also have oxygen-consuming systems, one of which is flavoprotein oxidase and the other is exogenous heme- or heme- and quinone-dependent respiratory metabolism. Over the past two decades, research has contributed to the understanding of the roles of these oxidase machineries, confirming their suspected roles and uncovering novel functions. This review presents the roles of these oxidase machineries, which are anticipated to be critical for the future applications of LAB in industry and comprehending the virulence of pathogenic streptococci.

The Streptococcus virulence protein PepO triggers anti-tumor immune responses by reprograming tumor-associated macrophages in a mouse triple negative breast cancer model

BackgroundThe efficacy of current surgery and chemotherapy for triple negative breast cancer (TNBC) is limited due to heterogenous and immunosuppressive tumor microenvironment (TME). Tumor associated macrophages (TAMs), which are regarded as an M2 tumor-promoting phenotype, are crucial in the development of the immunosuppressive TME. Targeting TAM reprograming is a promising strategy in anti-tumor therapy since reprogramming techniques provide the opportunity to actively enhance the antitumor immunological activity of TAM in addition to eliminating their tumor-supportive roles, which is rarely applied in TNBC clinically. However, how to drive M2 macrophages reprogramming into M1 with high potency remains a challenge and the molecular mechanisms how M2 macrophages polarized into M1 are poorly understood. Here, we identified a new immunoregulatory molecular PepO that was served as an immunoregulatory molecule governed the transformation of tumor-promoting M2 to tumor-inhibitory M1 cells and represented an effective anti-tumor property.ResultsAt the present study, we identified a new immunoregulatory molecular PepO, as a harmless immunoregulatory molecule, governed the transformation of tumor-promoting M2 to tumor-inhibitory M1 cells efficiently. PepO-primed M2 macrophages decreased the expression of tumor-supportive molecules like Arg-1, Tgfb, Vegfa and IL-10, and increased the expression of iNOS, Cxcl9, Cxcl10, TNF-α and IL-6 to inhibit TNBC growth. Moreover, PepO enhanced the functions of macrophages related to cell killing, phagocytosis and nitric oxide biosynthetic process, thereby inhibiting the development of tumors in vivo and in vitro. Mechanistically, PepO reprogramed TAMs toward M1 by activating PI3K-AKT-mTOR pathway via TLR4 and suppressed the function of M2 by inhibiting JAK2-STAT3 pathway via TLR2. The PI3K inhibitor LY294002 abrogated the role of PepO in switching M2 macrophages into M1 and in inhibiting TNBC growth in vivo. And PepO failed to govern the M2 macrophages to reprogram into M1 macrophages and inhibit TNBC when TLR2 or TLR4 was deficient. Moreover, PepO enhanced the antitumor activity of doxorubicin and the combination exerted a synergistic effect on TNBC suppression.ConclusionsOur research identified a possible macrophage-based TNBC immunotherapeutic approach and suggested a novel anticancer immunoregulatory molecular called PepO.

Genomic Features and Comparative Genomic Analysis of Streptococcus sp. v1. nov., Isolated from an Endophthalmitis Patient.

Endophthalmitis is an acute inflammatory intraocular condition that can cause permanent vision loss. The treatment strategy and visual outcome partly depend on the identification of the agents of pathogens. In this study, metagenomic sequencing was conducted to investigate the microbial and antibiotic resistance genes (ARGs) composition in the vitreous (intraocular body fluid) of an endophthalmitis patient, who progressed rapidly and accompanied by severe pain. Metagenomic sequencing data revealed that the vitreous sample was predominated by Streptococcus, with a low-diversity microbiome in the vitreous. This strain harbor's the ARGs mainly against beta-lactam, macrolide-lincosamide-streptogramin, and multidrug. Additionally, metagenome-assembled genome sequence of Streptococcus sp. v1. nov. was identified. The Tetra Correlation Search (TCS) analysis uncovered that the closest relative of the Streptococcus sp. v1. nov. was Streptococcus mitis SK321. Pan/core genome analysis for Streptococcus sp. v1. nov. and TCS top 25 hits strains revealed that most unique genes of Streptococcus sp. v1. nov. were linked to ATP-binding cassette transport system, which could indicate unique virulence and pathogenic potentials of Streptococcus sp. v1. nov. In addition, a total of 7 virulence factors were identified, and the overwhelming of them were classified into "offensive virulence factors". The high pathogenicity of Streptococcus sp. v1. nov. could be a reason for the patient's rapid disease progression. Our study was first isolated an ocular pathogen with highly virulent based on metagenomic sequencing and bioinformatics analysis, which has important reference value for revealing the composition and genome characteristics of pathogens in endophthalmitis patient in the future.

Novel pneumococcal capsule type 33E results from the inactivation of glycosyltransferase WciE in vaccine type 33F

The polysaccharide (PS) capsule is essential for immune evasion and virulence of Streptococcus pneumoniae. Existing pneumococcal vaccines are designed to elicit anticapsule antibodies; however, the effectiveness of these vaccines is being challenged by the emergence of new capsule types or variants. Herein, we characterize a newly discovered capsule type, 33E, that appears to have repeatedly emerged from vaccine type 33F via an inactivation mutation in the capsule glycosyltransferase gene, wciE. Structural analysis demonstrated that 33E and 33F share an identical repeat unit backbone [→5)-β-D-Galf2Ac-(1→3)-β-D-Galp-(1→3)-α-D-Galp-(1→3)-β-D-Galf-(1→3)-β-D-Glcp-(1→], except that a galactose (α-D-Galp) branch is present in 33F but not in 33E. Though the two capsule types were indistinguishable using conventional typing methods, the monoclonal antibody Hyp33FM1 selectively bound 33F but not 33E pneumococci. Further, we confirmed that wciE encodes a glycosyltransferase that catalyzes the addition of the branching α-D-Galp and that its inactivation in 33F strains results in the expression of the 33E capsule type. Though 33F and 33E share a structural and antigenic similarity, our pilot study suggested that immunization with a 23-valent pneumococcal PS vaccine containing 33F PS did not significantly elicit cross-opsonic antibodies to 33E. New conjugate vaccines that target capsule type 33F may not necessarily protect against 33E. Therefore, studies of new conjugate vaccines require knowledge of the newly identified capsule type 33E and reliable pneumococcal typing methods capable of distinguishing it from 33F.

Type VII secretion system and its effect on group B Streptococcus virulence in isolates obtained from newborns with early onset disease and colonized pregnant women.

GBS may cause a devastating disease in newborns. In early onset disease of the newborn the bacteria are acquired from the colonized mother during delivery. We characterized type VII secretion system (T7SS), exporting small proteins of the WXG100 superfamily, in group B Streptococci (GBS) isolates from pregnant colonized women and newborns with early onset disease (EOD) to better understand T7SS contribution to virulence in these different clinical scenarios. GBS genomes [N=33, 17 EOD isolates (serotype III/ST17) and 16 colonizing isolates (12 serotype VI/ST1, one serotype VI/ST19, one serotype VI/ST6, and two serotype 3/ST19)] were analyzed for presence of T7SS genes and genes encoding WXG100 proteins. We also perform bioinformatic analysis. Galleria mellonella larvae were used to compare virulence between colonizing, EOD, and mutant EOD isolates. The EOD isolate number 118659 (III/ST17) was used for knocking out the essC gene encoding a membrane-bound ATPase, considered the driver of T7SS. Most GBS T7SS loci encoded core component genes: essC, membrane-embedded proteins (essA; essB), modulators of T7SS activity (esaA; esaB; esaC) and effectors: [esxA (SAG1039); esxB (SAG1030)].Bioinformatic analysis indicated that based on sequence type (ST) the clinicalGBS isolates encode at least three distinct subtypes of T7SS machinery. In all ST1isolates we identified two copies of esxA gene (encoding putative WXG100proteins), when only 23.5% of the ST17 isolates harbored the esxA gene. Five ST17isolates encoded two copies of the essC gene. Orphaned WXG100 molecule(SAG0230), distinct from T7SS locus, were found in all tested strains, except inST17 strains where the locus was found in only 23.5% of the isolates. In ST6 andST19 isolates most of the structure T7SS genes were missing. EOD isolates demonstrated enhanced virulence in G. mellonella modelcompared to colonizing isolates. The 118659DessC strain was attenuated in itskilling ability, and the larvae were more effective in eradicating 118659DessC. We demonstrated that T7SS plays a role during infection. Knocking out the essC gene, considered the driver of T7SS, decreased the virulence of ST17 responsible for EOD, causing them to be less virulent comparable to the virulence observed in colonizing isolates.

Identification of cis-acting elements upstream of regR gene in streptococcus pneumoniae

The identification and characterization of functional cis-acting elements is of fundamental importance for comprehending the regulatory mechanisms of gene transcription and bacterial pathogenesis. The transcription factor RegR has been demonstrated to control both competence and virulence in Streptococcus pneumoniae. Despite the clear contribution of RegR to these pathways, the mechanisms underlying its transcriptional regulation remain poorly understood. In this study, we conducted mutational analysis, gene dissection and luciferase activity assays to characterize the cis-elements situated upstream of the regR gene. Our findings revealed that a 311 bp 3′-terminal DNA sequence of the spd0300 gene represents a central region of the upstream cis-acting element of regR. Further investigations identified two structurally similar enhancer-like sequences within this region which feature prominently in the regulation of regR transcription. Furthermore, employing DNA pull-down assays allowed us to enrich the trans-acting factors with the potential to interact with these cis-acting elements. Notably, we found that the competence regulator ComE was implicated in the regulation of regR transcription, a finding which was corroborated by electrophoretic mobility shift assays (EMSA) and quantitative real-time PCR analyses (qRT-PCR). Taken together, our data thus provide fresh insight into the transcriptional regulation of regR.

Recombinant endopeptidases IdeS and IdeZ and their potential applications

IdeS and IdeZ endopeptidases are streptococcal virulence factors that specifically cleave IgG heavy chains and represent an interest from biotechnological, medical, and veterinary points of view. The genes encoding IdeS and IdeZ endopeptidases (the ideS gene was cloned from a Streptococcus pyogenes collection strain, and S. zooepidemicusideZ gene was synthesized) were cloned and expressed in Escherichia coli heterologous expression system, and 6His-tag affinity domain was introduced into the amino acid sequence of each endopeptidase. IdeS and IdeZ enzymes were isolated and purified by metal affinity chromatography. The resulting IdeS and IdeZ are apparently homogeneous on polyacrylamide gel electrophoresis and are active against human and animal IgG. The specificity of human IgG cleavage by endopeptidases IdeS and IdeZ was confirmed by polyacrylamide gel electrophoresis. Application of recombinant IdeZ was demonstrated for immunological analysis of equine “strangles” (diagnostics and specific antibodies’ titer determination in the blood). Thus, IdeZ can be used in veterinary medicine and sanitary microbiology for the diagnosis of infections caused by S. equi and S. zooepidemicus in addition to their applications in medicine and biotechnology.

Membrane vesicle delivery of a streptococcal M protein disrupts the blood-brain barrier by inducing autophagic endothelial cell death.

M family proteins are critical virulence determinants of Streptococci. Streptococcus equi subsp. zooepidemicus (SEZ) are Group C streptococci that cause meningitis in animals and humans. SzM, the M protein of SEZ, has been linked to SEZ brain invasion. Here, we demonstrate that SzM is important in SEZ disruption of the blood-brain barrier (BBB). SEZ release SzM-bound membrane vesicles (MVs), and endocytosis of these vesicles by human brain endothelial microvascular cells (hBMECs) results in SzM-dependent cytotoxicity. Furthermore, administration of SzM-bound MVs disrupted the murine BBB. A CRISPR screen revealed that SzM cytotoxicity in hBMECs depends on PTEN-related activation of autophagic cell death. Pharmacologic inhibition of PTEN activity prevented SEZ disruption of the murine BBB and delayed mortality. Our data show that MV delivery of SzM to host cells plays a key role in SEZ pathogenicity and suggests that MV delivery of streptococcal M family proteins is likely a common streptococcal virulence mechanism.

Recombinant Endopeptidases IdeS and IdeZ and Their Potential Application.

Endopeptidases IdeS and IdeZ (streptococcal virulence factors that specifically cleave IgG heavy chains) are of particular interest because of their potential use in biotechnology, medicine, and veterinary. Genes encoding these enzymes were cloned and expressed in Escherichia coli heterologous expression system (ideS was cloned from a Streptococcus pyogenes collection strain; ideZ from Streptococcus zooepidemicus was synthesized). The 6His-tag was introduced into the amino acid sequence of each endopeptidase, and IdeS and IdeZ were purified by metal affinity chromatography to an apparent homogeneity (according to polyacrylamide gel electrophoresis). Purified enzymes were active against human and animal IgGs; their specificity toward human IgGs was confirmed by polyacrylamide gel electrophoresis. Recombinant IdeZ was used for immunological analysis of equine strangles infection (diagnostics and determination of the titer of specific antibodies in blood). Hence, IdeZ can be used in veterinary and sanitary microbiology to diagnose infections caused by Streptococcus equi and S. zooepidemicus in addition to its application in medicine and biotechnology.

TCS01 Two-Component System Influenced the Virulence of Streptococcus pneumoniae by Regulating PcpA.

Streptococcus pneumoniae relies on two-component systems (TCSs) to regulate the processes of pathogenicity, osmotic pressure, chemotaxis, and energy metabolism. The TCS01 system of S. pneumoniae is composed of HK01 (histidine kinase) and RR01 (response regulator). Previous studies have reported that an rr01 mutant reduced the pneumococcal virulence in rat pneumonia, bacteremia, a nasopharyngeal model, and infective endocarditis. However, the mechanism of TCS01 (HK/RR01) regulating pneumococcal virulence remains unclear. Here, pneumococcal mutant strains Δrr01, Δhk01, and Δrr01&hk01 were constructed, and bacterial adhesion and invasion to A549 cells were compared. RNA sequencing was performed in D39 wild-type and Δrr01 strains, and transcript profile changes were analyzed. Differentially expressed virulence genes in the Δrr01 strain were screened out and identified by quantitative real-time PCR (qRT-PCR). Our results showed that pneumococcal mutant strains exhibited attenuated adhesion and invasion to A549 cells and differential transcript profiles. Results of qRT-PCR identification showed that the differential virulence genes screened out were downregulated. Among those changed virulence genes in the Δrr01 strain, the downregulated expression level of choline binding protein pcpA was the most obvious. Complementation of rr01 and overexpression of pcpA in the Δrr01 strain partially restored both pneumococcal adhesion and invasion, and rr01 complementation made the expression of pcpA upregulated. These findings revealed that rr01 influenced pneumococcal virulence by regulating pcpA.

Group B streptococcus virulence factors associated with different clinical syndromes: Asymptomatic carriage in pregnant women and early-onset disease in the newborn.

Group B streptococcus (GBS) harbors many virulence factors but there is limited data regarding their importance in colonization in pregnancy and early-onset disease (EOD) in the newborn. We hypothesized that colonization and EOD are associated with different distribution and expression of virulence factors. We studied 36 GBS EOD and 234 GBS isolates collected during routine screening. Virulence genes (pilus-like structures-PI-1, PI-2a, PI-2b; rib and hvgA) presence and expression were identified by PCR and qRT-PCR. Whole genome sequencing (WGS) and comparative genomic analyses were used to compare coding sequences (CDSs) of colonizing and EOD isolates. Serotype III (ST17) was significantly associated with EOD and serotype VI (ST1) with colonization. hvgA and rib genes were more prevalent among EOD isolates (58.3 and 77.8%, respectively; p < 0.01). The pilus loci PI-2b and PI-2a were more prevalent among EOD isolates (61.1%, p < 0.01), while the pilus loci PI-2a and PI-1 among colonizing isolates (89.7 and 93.1% vs. 55.6 and 69.4%, p < 0.01). qRT PCR analysis revealed that hvgA was barely expressed in colonizing isolates, even though the gene was detected. Expression of the rib gene and PI-2b was two-fold higher in EOD isolates compared to colonizing isolates. Transcription of PI-2a was three-fold higher in colonizing isolates compared to EOD isolates. ST17 isolates (associated with EOD) had a smaller genome size compared ST1 and the genome was more conserved relative to the reference strain and ST17 isolates. In a multivariate logistic regression analysis virulence factors independently associated with EOD were serotype 3, and PI-1 and PI-2a was protective. There was a significant difference in the distribution of hvg A, rib, and PI genes among EOD (serotype III/ST17) and colonizing (serotype VI/ST1) isolates suggesting an association between invasive disease and these virulence factors. Further study is needed to understand the contribution of these genes to GBS virulence.

Glucose levels affect MgaSpn regulation on the virulence and adaptability of Streptococcus pneumoniae.

Streptococcus pneumoniae can regulate virulence gene expression by sensing environmental changes, which is key to its pathogenicity. The global transcription regulator MgaSpn of Streptococcus pneumoniae regulates virulence genes expression by directly binding to the promoter regions, but its role in response to different environments remains unclear. In this study, we found that glucose levels could affect phosphocholine content, which was mediated by MgaSpn. MgaSpn can also alter its anti-phagocytosis ability, depending on the availability of glucose. In addition, transcriptome analysis of wild-type D39s in low and high glucose concentrations revealed that MgaSpn was also involved in the regulation of carbon metabolism inhibition (carbon catabolite repression; CCR) and translation processes, which made S. pneumoniae highly competitive in fluctuating environments. In conclusion, MgaSpn is closely related to the virulence and environmental adaptability of Streptococcus pneumoniae.

Correlation Analysis between the Antimicrobial Resistance and Virulence of Pathogenic Streptococcus Isolates from Cows

Correlation Analysis between the Antimicrobial Resistance and Virulence of Pathogenic Streptococcus Isolates from Cows

Implications of oral streptococcal bacteriophages in autism spectrum disorder

Growing evidence suggests altered oral and gut microbiota in autism spectrum disorder (ASD), but little is known about the alterations and roles of phages, especially within the oral microbiota in ASD subjects. We enrolled ASD (n = 26) and neurotypical subjects (n = 26) with their oral hygiene controlled, and the metagenomes of both oral and fecal samples (n = 104) are shotgun-sequenced and compared. We observe extensive and diverse oral phageome comparable to that of the gut, and clear signals of mouth-to-gut phage strain transfer within individuals. However, the overall phageomes of the two sites are widely different and show even less similarity in the oral communities between ASD and control subjects. The ASD oral phageome exhibits significantly reduced abundance and alpha diversity, but the Streptococcal phages there are atypically enriched, often dominating the community. The over-representation of Streptococcal phages is accompanied by enriched oral Streptococcal virulence factors and Streptococcus bacteria, all exhibiting a positive correlation with the severity of ASD clinical manifestations. These changes are not observed in the parallel sampling of the gut flora, suggesting a previously unknown oral-specific association between the excessive Streptococcal phage enrichment and ASD pathogenesis. The findings provide new evidence for the independent microbiome-mouth-brain connection, deepen our understanding of how the growth dynamics of bacteriophages and oral microbiota contribute to ASD, and point to novel effective therapeutics.

Quorum Sensing Regulation of a Major Facilitator Superfamily Transporter Affects Multiple Streptococcal Virulence Factors.

Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes, including all streptococci. In Streptococcus pyogenes, or group A strep (GAS), one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes, including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit, and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells. IMPORTANCE The production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple virulence factors and accounts for QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport.

Investigation on the virulence of non-encapsulated Streptococcus pneumoniae using liquid agar pneumonia model

IntroductionSince the introduction of pneumococcal conjugate vaccine, there have been warnings of an increase in infections caused by non-vaccine type of Streptococcus pneumoniae strains. Among them, nonencapsulated Streptococcus pneumoniae (NESp) has been reported to cause invasive infections, especially in children and the elderly. Due to low virulence, however, basic experimental reports on invasive infections are limited. MethodsWe applied a liquid-agar method to establish a mouse model of invasive NESp infection. Mice were intratracheally administered a bacterial suspension including agar. With this technique, we investigated the pathogenicity of NESp and the effect of Pneumococcal surface protein K (PspK), a specific surface protein antigen of NESp.NESp wild-type strain (MNZ11) and NESp pspK-deleted mutant strain (MNZ1131) were used in this study. The survival rate, number of bacteria, cytokine/chemokine levels in the bronchoalveolar lavage fluid, and histology of the lung tissue were evaluated. ResultsMice that were intratracheally administered MNZ11 developed lethal pneumonia with bacteremia within 48 h. Conversely, MNZ1131 showed predominantly low lethality without significant pro-inflammatory cytokine production. NESp was found to cause severe pneumonia and bacteremia upon reaching the lower respiratory tract, and PspK was a critical factor of NESp for developing invasive infections. ConclusionsThe current study demonstrated the ability of NESp to develop invasive diseases, especially in connection with PspK by use of a mouse pneumonia model.