Analysis of FctB3 crystal structure and insight into its structural stabilization and pilin linkage mechanisms.

Streptococcus pyogenes harboring an FCT type 3 genomic region display pili composed of three types of pilins. In this study, the structure of the base pilin FctB from a serotype M3 strain (FctB3) was determined at 2.8 Å resolution. In accordance with the previously reported structure of FctB from a serotype T9 strain (FctB9), FctB3 was found to consist of an immunoglobulin-like domain and proline-rich tail region. Data obtained from structure comparison revealed main differences in the omega (Ω) loop structure and the proline-rich tail direction. In the Ω loop structure, a differential hydrogen bond network was observed, while the lysine residue responsible for linkage to growing pili was located at the same position in both structures, which indicated that switching of the hydrogen bond network in the Ω loop without changing the lysine position is advantageous for linkage to the backbone pilin FctA. The difference in direction of the proline-rich tail is potentially caused by a single residue located at the root of the proline-rich tail. Also, the FctB3 structure was found to be stabilized by intramolecular large hydrophobic interactions instead of an isopeptide bond. Comparisons of the FctB3 and FctA structures indicated that the FctA structure is more favorable for linkage to FctA. In addition, the heterodimer formation of FctB with Cpa or FctA was shown to be mediated by the putative chaperone SipA. Together, these findings provide an alternative FctB structure as well as insight into the interactions between pilin proteins.

Involvement of ribonuclease Y in pilus production by M49 Streptococcus pyogenes strain via modulation of messenger RNA level of transcriptional regulator.

Streptococcus pyogenes displays a wide variety of pili, which is largely dependent on serotype. A distinct subset of S. pyogenes strains that possess the Nra transcriptional regulator demonstrates thermoregulated pilus production. Findings obtained in the present study of an Nra-positive serotype M49 strain revealed involvement of conserved virulence factor A (CvfA), also referred to as ribonuclease Y (RNase Y), in virulence factor expression and pilus production, while a cvfA deletion strain showed reduced pilus production and adherence to human keratinocytes as compared with wild-type and revertant strains. Furthermore, transcript levels of pilus subunits and srtC2 genes were decreased by cvfA deletion, which was remarkable at 25°C. Likewise, both messenger RNA (mRNA) and protein levels of Nra were remarkably decreased by cvfA deletion. Whether the expression of other pilus-related regulators, including fasX and CovR, was subject to thermoregulation was also examined. While the mRNA level of fasX, which inhibits cpa and fctA translation, was decreased by cvfA deletion at both 37°C and 25°C, CovR mRNA and protein levels, as well as its phosphorylation level were not significantly changed, suggesting that neither fasX nor CovR is necessarily involved in thermosensitive pilus production. Phenotypic analysis of the mutant strains revealed that culture temperature and cvfA deletion had varied effects on streptolysin S and SpeB activities. Furthermore, bactericidal assay data showed that cvfA deletion decreased the rate of survival in human blood. Together, the present findings indicate that CvfA is involved in regulation of pilus production and virulence-related phenotypes of the serotype M49 strain of S. pyogenes.

Contribution of Streptococcus pyogenes M87 protein to innate immune resistance and virulence.

Streptococcus pyogenes is a pre-eminent human pathogen, and classified by the hypervariable sequence of the emm gene encoding the cell surface M protein. Among a diversity of M/emm types, the prevalence of the M/emm87 strain has been steadily increasing in invasive S. pyogenes infections. Although M protein is the major virulence factor for globally disseminated M/emm1 strain, it is unclear if or how the corresponding M protein of M/emm87 strain (M87 protein) functions as a virulence factor. Here, we use targeted mutagenesis to show that the M87 protein contributes to bacterial resistance to neutrophil and whole blood killing and promotes the release of mature IL-1ß from macrophages. While deletion of emm87 did not influence epithelial cell adherence and nasal colonization, it significantly reduced S. pyogenes-induced mortality and bacterial loads in a murine systemic infection model. Our data suggest that emm87 is involved in pathogenesis by modulating the interaction between S. pyogenes and innate immune cells.

Oral mitis group streptococci: A silent majority in our oral cavity.

Members of the oral mitis group streptococci including Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii are the most abundant inhabitants of human oral cavity and dental plaque, and have been implicated in infectious complications such as bacteremia and infective endocarditis. Oral mitis group streptococci are genetically close to Streptococcus pneumoniae; however, they do not produce cytolysin (pneumolysin), which is a key virulence factor of S. pneumoniae. Similar to S. pneumoniae, oral mitis group streptococci possess several cell surface proteins that bind to the cell surface components of host mammalian cells. S. sanguinis expresses long filamentous pili that bind to the matrix proteins of host cells. The cell wall-anchored nuclease of S. sanguinis contributes to the evasion of the neutrophil extracellular trap by digesting its web-like extracellular DNA. Oral mitis group streptococci produce glucosyltransferases, which synthesize glucan (glucose polymer) from sucrose of dietary origin. Neuraminidase (NA) is a virulent factor in oral mitis group streptococci. Influenza type A virus (IAV) relies on viral NA activity to release progeny viruses from infected cells and spread the infection, and NA-producing oral streptococci elevate the risk of IAV infection. Moreover, oral mitis group streptococci produce hydrogen peroxide (H2 O2 ) as a by-product of sugar metabolism. Although the concentrations of streptococcal H2 O2 are low (1-2 mM), they play important roles in bacterial competition in the oral cavity and evasion of phagocytosis by host macrophages and neutrophils. In this review, we intended to describe the diverse pathogenicity of oral mitis group streptococci.

Secondary streptococcal infection following influenza.

Secondary bacterial infection following influenza type A virus (IAV) infection is a major cause of morbidity and mortality during influenza epidemics. Streptococcus pneumoniae has been identified as a predominant pathogen in secondary pneumonia cases that develop following influenza. Although IAV has been shown to enhance susceptibility to the secondary bacterial infection, the underlying mechanism of the viral-bacterial synergy leading to disease progression is complex and remains elusive. In this review, cooperative interactions of viruses and streptococci during co- or secondary infection with IAV are described. IAV infects the upper respiratory tract, therefore, streptococci that inhabit or infect the respiratory tract are of special interest. As many excellent reviews on the co-infection of IAV and S. pneumoniae have already been published, this review is intended to describe the unique interactions between other streptococci and IAV. Both streptococcal and IAV infections modulate the host epithelial barrier of the respiratory tract in various ways. IAV infection directly disrupts epithelial barriers, though at the same time the virus modifies the properties of infected cells to enhance streptococcal adherence and invasion. Mitis group streptococci produce neuraminidases, which promote IAV infection in a unique manner. The studies reviewed here have revealed intriguing mechanisms underlying secondary streptococcal infection following influenza.

Thermosensitive pilus production by FCT type 3 Streptococcus pyogenes controlled by Nra regulator translational efficiency.

Streptococcus pyogenes produces a diverse variety of pili in a serotype-dependent manner and thermosensitive expression of pilus biogenesis genes was previously observed in a serotype M49 strain. However, the precise mechanism and biological significance remain unclear. Herein, the pilus expression analysis revealed the thermosensitive pilus production only in strains possessing the transcriptional regulator Nra. Experimental data obtained for nra deletion and conditional nra-expressing strains in the background of an M49 strain and the Lactococcus heterologous expression system, indicated that Nra is a positive regulator of pilus genes and also highlighted the importance of the level of intracellular Nra for the thermoregulation of pilus expression. While the nra mRNA level was not significantly influenced by a temperature shift, the Nra protein level was concomitantly increased when the culture temperature was decreased. Intriguingly, a putative stem-loop structure within the coding region of nra mRNA was a factor related to the post-transcriptional efficiency of nra mRNA translation. Either deletion of the stem-loop structure or introduction of silent chromosomal mutations designed to melt the structure attenuated Nra levels, resulting in decreased pilus production. Consequently, the temperature-dependent translational efficacy of nra mRNA influenced pilus thermoregulation, thereby potentially contributing to the fitness of nra-positive S. pyogenes in human tissues.

Streptococcus pyogenes Transcriptome Changes in the Inflammatory Environment of Necrotizing Fasciitis.

Streptococcus pyogenes is a major cause of necrotizing fasciitis, a life-threatening subcutaneous soft-tissue infection. At the host infection site, the local environment and interactions between the host and bacteria have effects on bacterial gene expression profiles, while the gene expression pattern of S. pyogenes related to this disease remains unknown. In this study, we used a mouse model of necrotizing fasciitis and performed RNA-sequencing (RNA-seq) analysis of S. pyogenes M1T1 strain 5448 by isolating total RNA from infected hind limbs obtained at 24, 48, and 96 h postinfection. RNA-seq analysis results identified 483 bacterial genes whose expression was consistently altered in the infected hindlimbs compared to their expression under in vitro conditions. Genes showing consistent enrichment during infection included 306 encoding molecules involved in virulence, carbohydrate utilization, amino acid metabolism, trace-metal transport, and the vacuolar ATPase transport system. Surprisingly, drastic upregulation of 3 genes, encoding streptolysin S precursor (sagA), cysteine protease (speB), and secreted DNase (spd), was noted in the present mouse model (log2 fold change, >6.0, >9.4, and >7.1, respectively). Conversely, the number of consistently downregulated genes was 177, including those associated with the oxidative stress response and cell division. These results suggest that in necrotizing fasciitis, S. pyogenes shows an altered metabolism, decreased cell proliferation, and upregulation of expression of major toxins. Our findings are considered to provide critical information for developing novel treatment strategies and vaccines for necrotizing fasciitis.IMPORTANCE Necrotizing fasciitis, a life-threatening subcutaneous soft-tissue infection, is principally caused by S. pyogenes The inflammatory environment at the site of infection causes global gene expression changes for survival of the bacterium and pathogenesis. However, no known study regarding transcriptomic profiling of S. pyogenes in cases of necrotizing fasciitis has been presented. We identified 483 bacterial genes whose expression was consistently altered during infection. Our results showed that S. pyogenes infection induces drastic upregulation of the expression of virulence-associated genes and shifts metabolic pathway usage. In particular, high-level expression of toxins, such as cytolysins, proteases, and nucleases, was observed at infection sites. In addition, genes identified as consistently enriched included those related to metabolism of arginine and histidine as well as carbohydrate uptake and utilization. Conversely, genes associated with the oxidative stress response and cell division were consistently downregulated during infection. The present findings provide useful information for establishing novel treatment strategies.

Antibacterial activity of hinokitiol against both antibiotic-resistant and -susceptible pathogenic bacteria that predominate in the oral cavity and upper airways.

Hinokitiol, a component of the essential oil isolated from Cupressaceae, possesses antibacterial and antifungal activities and has been used in oral care products. In this study, the antibacterial activities of hinokitiol toward various oral, nasal and nasopharyngeal pathogenic bacteria, including Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, methicillin-resistant and -susceptible Staphylococcus aureus, antibiotic-resistant and -susceptible Streptococcus pneumoniae, and Streptococcus pyogenes were examined. Growth of all these bacterial strains was significantly inhibited by hinokitiol, minimal inhibitory concentrations of hinokitiol against S. mutans, S. sobrinus, P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, methicillin-resistant S. aureus, methicillin-susceptible S. aureus, antibiotic-resistant S. pneumoniae isolates, antibiotic-susceptible S. pneumoniae, and S. pyogenes being 0.3, 1.0, 1.0, 30, 0.5, 50, 50, 30, 0.3-1.0, 0.5, and 0.3 µg/mL, respectively. Additionally, with the exception of P. gingivalis, hinokitiol exerted bactericidal effects against all bacterial strains 1 hr after exposure. Hinokitiol did not display any significant cytotoxicity toward the human gingival epithelial cell line Ca9-22, pharyngeal epithelial cell line Detroit 562, human umbilical vein endothelial cells, or human gingival fibroblasts, with the exception of treatment with 500 µg/mL hinokitiol, which decreased numbers of viable Ca9-22 cells and gingival fibroblasts by 13% and 12%, respectively. These results suggest that hinokitiol exhibits antibacterial activity against a broad spectrum of pathogenic bacteria and has low cytotoxicity towards human epithelial cells.

Streptococcus pyogenes Endopeptidase O Contributes to Evasion from Complement-mediated Bacteriolysis via Binding to Human Complement Factor C1q.

Streptococcus pyogenes secretes various virulence factors for evasion from complement-mediated bacteriolysis. However, full understanding of the molecules possessed by this organism that interact with complement C1q, an initiator of the classical complement pathway, remains elusive. In this study, we identified an endopeptidase of S. pyogenes, PepO, as an interacting molecule, and investigated its effects on complement immunity and pathogenesis. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis findings revealed that S. pyogenes recombinant PepO bound to human C1q in a concentration-dependent manner under physiological conditions. Sites of inflammation are known to have decreased pH levels, thus the effects of PepO on bacterial evasion from complement immunity was analyzed in a low pH condition. Notably, under low pH conditions, PepO exhibited a higher affinity for C1q as compared with IgG, and PepO inhibited the binding of IgG to C1q. In addition, pepO deletion rendered S. pyogenes more susceptible to the bacteriocidal activity of human serum. Also, observations of the morphological features of the pepO mutant strain (ΔpepO) showed damaged irregular surfaces as compared with the wild-type strain (WT). WT-infected tissues exhibited greater severity and lower complement activity as compared with those infected by ΔpepO in a mouse skin infection model. Furthermore, WT infection resulted in a larger accumulation of C1q than that with ΔpepO. Our results suggest that interaction of S. pyogenes PepO with C1q interferes with the complement pathway, which enables S. pyogenes to evade complement-mediated bacteriolysis under acidic conditions, such as seen in inflammatory sites.

Pneumococcal DNA-binding proteins released through autolysis induce the production of proinflammatory cytokines via toll-like receptor 4.

Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis. We demonstrated that recombinant (r) DnaK, rEF-Tu, and rGAPDH induced significantly higher levels of interleukin-6 and tumor necrosis factor production in peritoneal macrophages and THP-1-derived macrophage-like cells via toll-like receptor 4. Furthermore, the DNA-binding activity of these proteins was confirmed by surface plasmon resonance assay. We demonstrated that pneumococcal DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in macrophages, and might cause host tissue damage and affect the development of pneumococcal diseases.

Streptococcus pyogenes CAMP factor promotes calcium ion uptake in RAW264.7 cells.

Streptococcus pyogenes is a bacterium that causes systemic diseases such as pharyngitis and toxic shock syndrome. S. pyogenes produces molecules that inhibit the function of the human immune system, thus allowing growth and spread of the pathogen in tissues. It is known that S. pyogenes CAMP factor induces vacuolation in macrophages; however, the mechanism remains unclear. In the current study, the mechanism by which CAMP factor induces vacuolation in macrophages was investigated. CAMP factor was found to induce calcium ion uptake in murine macrophage RAW264.7 cells. In addition, EDTA inhibited calcium ion uptake and vacuolation in the cells. The L-type voltage-dependent calcium ion channel blockers nifedipine and verapamil reduced vacuolation. Furthermore, the phosphoinositide 3-kinase inhibitors LY294002 and wortmannin also inhibited the vacuolation induced by CAMP factor. Fluorescent microscopy revealed that clathrin localized to the vacuoles. These results suggest that the vacuolation is related to calcium ion uptake by RAW264.7 cells via L-type voltage-dependent calcium ion channels. Therefore, it was concluded that the vacuoles induced by S. pyogenes CAMP factor in macrophages are clathrin-dependent endosomes induced by activation of the phosphoinositide 3-kinase signaling pathway through calcium ion uptake.

Streptococcus oralis Induces Lysosomal Impairment of Macrophages via Bacterial Hydrogen Peroxide.

Streptococcus oralis, an oral commensal, belongs to the mitis group of streptococci and occasionally causes opportunistic infections, such as bacterial endocarditis and bacteremia. Recently, we found that the hydrogen peroxide (H2O2) produced by S. oralis is sufficient to kill human monocytes and epithelial cells, implying that streptococcal H2O2 is a cytotoxin. In the present study, we investigated whether streptococcal H2O2 impacts lysosomes, organelles of the intracellular digestive system, in relation to cell death. S. oralis infection induced the death of RAW 264 macrophages in an H2O2-dependent manner, which was exemplified by the fact that exogenous H2O2 also induced cell death. Infection with either a mutant lacking spxB, which encodes pyruvate oxidase responsible for H2O2 production, or Streptococcus mutans, which does not produce H2O2, showed less cytotoxicity. Visualization of lysosomes with LysoTracker revealed lysosome deacidification after infection with S. oralis or exposure to H2O2, which was corroborated by acridine orange staining. Similarly, fluorescent labeling of lysosome-associated membrane protein-1 gradually disappeared during infection with S. oralis or exposure to H2O2 The deacidification and the following induction of cell death were inhibited by chelating iron in lysosomes. Moreover, fluorescent staining of cathepsin B indicated lysosomal destruction. However, treatment of infected cells with a specific inhibitor of cathepsin B had negligible effects on cell death; instead, it suppressed the detachment of dead cells from the culture plates. These results suggest that streptococcal H2O2 induces cell death with lysosomal destruction and then the released lysosomal cathepsins contribute to the detachment of the dead cells.

Lysine and Threonine Biosynthesis from Aspartate Contributes to Staphylococcus aureus Growth in Calf Serum.

Staphylococcus aureus is a human pathogen, and S. aureus bacteremia can cause serious problems in humans. To identify the genes required for bacterial growth in calf serum (CS), a library of S. aureus mutants with randomly inserted transposons were analyzed for growth in CS, and the aspartate semialdehyde dehydrogenase (asd)-inactivated mutant exhibited significantly reduced growth in CS compared with the wild type (WT). The mutant also exhibited significantly reduced growth in medium, mimicking the concentrations of amino acids and glucose in CS. Asd is an essential enzyme for the biosynthesis of lysine, methionine, and threonine from aspartate. We constructed inactivated mutants of the genes for lysine (lysA), methionine (metE), and threonine (thrC) biosynthesis and found that the inactivated mutants of lysA and thrC exhibited significantly lower growth in CS than the WT, but the growth of the metE mutant was similar to that of the WT. The reduced growth of the asd mutant was recovered by addition of 100 µg/ml lysine and threonine in CS. These results suggest that S. aureus requires lysine and threonine biosynthesis to grow in CS. On the other hand, the asd-, lysA-, metE-, and thrC-inactivated mutants exhibited significantly reduced growth in mouse serum compared with the WT. In mouse bacteremia experiments, the asd-, lysA-, metE-, and thrC-inactivated mutants exhibited attenuated virulence compared with WT infection. In conclusion, our results suggest that the biosynthesis of de novo aspartate family amino acids, especially lysine and threonine, is important for staphylococcal bloodstream infection. IMPORTANCE: Studying the growth of bacteria in blood is important for understanding its pathogenicity in the host. Staphylococcus aureus sometimes causes bacteremia or sepsis. However, the factors responsible for S. aureus growth in the blood are not well understood. In this study, using a library of 2,914 transposon-insertional mutants in the S. aureus MW2 strain, we identified the factors responsible for bacterial growth in CS. We found that inactivation of the lysine and threonine biosynthesis genes led to deficient growth in CS. However, the inactivation of these genes did not affect S. aureus growth in general medium. Because the concentration of amino acids in CS is low compared to that in general bacterial medium, our results suggest that lysine and threonine biosynthesis is important for the growth of S. aureus in CS. Our findings provide new insights for S. aureus adaptation in the host and for understanding the pathogenesis of bacteremia.

Molecular and genomic characterization of pathogenic traits of group A Streptococcus pyogenes.

Group A streptococcus (GAS) or Streptococcus pyogenes causes various diseases ranging from self-limiting sore throat to deadly invasive diseases. The genome size of GAS is 1.85-1.9 Mb, and genomic rearrangement has been demonstrated. GAS possesses various surface-associated substances such as hyaluronic capsule, M proteins, and fibronectin/laminin/immunoglobulin-binding proteins. These are related to the virulence and play multifaceted and mutually reflected roles in the pathogenesis of GAS infections. Invasion of GAS into epithelial cells and deeper tissues provokes immune and non-immune defense or inflammatory responses including the recruitment of neutrophils, macrophages, and dendritic cells in hosts. GAS frequently evades host defense mechanisms by using its virulence factors. Extracellular products of GAS may perturb cellular and subcellular functions and degrade tissues enzymatically, which leads to the aggravation of local and/or systemic disorders in the host. In this review, we summarize some important cellular and extracellular substances that may affect pathogenic processes during GAS infections, and the host responses to these.

Group A streptococcal cysteine protease cleaves epithelial junctions and contributes to bacterial translocation.

BACKGROUND: Group A Streptococcus (GAS) translocates across the host epithelial barrier. RESULTS: Streptococcal pyrogenic exotoxin B (SpeB) directly cleaves junctional proteins. CONCLUSION: The proteolytic efficacy of SpeB allows GAS to translocate across the epithelial barrier. SIGNIFICANCE: SpeB-mediated dysfunction of the epithelial barrier may have important implications for not only bacterial invasion but also dissemination of other virulence factors throughout intercellular spaces. Group A Streptococcus (GAS) is an important human pathogen that possesses an ability to translocate across the epithelial barrier. In this study, culture supernatants of tested GAS strains showed proteolytic activity against human occludin and E-cadherin. Utilizing various types of protease inhibitors and amino acid sequence analysis, we identified SpeB (streptococcal pyrogenic exotoxin B) as the proteolytic factor that cleaves E-cadherin in the region neighboring the calcium-binding sites within the extracellular domain. The cleaving activities of culture supernatants from several GAS isolates were correlated with the amount of active SpeB, whereas culture supernatants from an speB mutant showed no such activities. Of note, the wild type strain efficiently translocated across the epithelial monolayer along with cleavage of occludin and E-cadherin, whereas deletion of the speB gene compromised those activities. Moreover, destabilization of the junctional proteins was apparently relieved in cells infected with the speB mutant, as compared with those infected with the wild type. Taken together, our findings indicate that the proteolytic efficacy of SpeB in junctional degradation allows GAS to invade deeper into tissues.

Cysteine proteinase from Streptococcus pyogenes enables evasion of innate immunity via degradation of complement factors.

Streptococcus pyogenes is an important human pathogen that causes invasive diseases such as necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome. We investigated the function of a major cysteine protease from S. pyogenes that affects the amount of C1-esterase inhibitor (C1-INH) and other complement factors and aimed to elucidate the mechanism involved in occurrence of streptococcal toxic shock syndrome from the aspect of the complement system. First, we revealed that culture supernatant of a given S. pyogenes strain and recombinant SpeB degraded the C1-INH. Then, we determined the N-terminal sequence of the C1-INH fragment degraded by recombinant SpeB. Interestingly, the region containing one of the identified cleavage sites is not present in patients with C1-INH deficiency. Scanning electron microscopy of the speB mutant incubated in human serum showed the abnormal superficial architecture and irregular oval structure. Furthermore, unlike the wild-type strain, that mutant strain showed lower survival capacity than normal as compared with heat-inactivated serum, whereas it had a significantly higher survival rate in serum without the C1-INH than in normal serum. Also, SpeB degraded multiple complement factors and the membrane attack complex. Flow cytometric analyses revealed deposition of C9, one of the components of membrane the attack complex, in greater amounts on the surface of the speB mutant, whereas lower amounts of C9 were bound to the wild-type strain surface. These results suggest that SpeB can interrupt the human complement system via degrading the C1-INH, thus enabling S. pyogenes to evade eradication in a hostile environment.

Pleiotropic virulence factor - Streptococcus pyogenes fibronectin-binding proteins.

Streptococcus pyogenes causes a broad spectrum of infectious diseases, including pharyngitis, skin infections and invasive necrotizing fasciitis. The initial phase of infection involves colonization, followed by intimate contact with the host cells, thus promoting bacterial uptake by them. S. pyogenes recognizes fibronectin (Fn) through its own Fn-binding proteins to obtain access to epithelial and endothelial cells in host tissue. Fn-binding proteins bind to Fn to form a bridge to α5 ß1 -integrins, which leads to rearrangement of cytoskeletal actin in host cells and uptake of invading S. pyogenes. Recently, several structural analyses of the invasion mechanism showed molecular interactions by which Fn converts from a compact plasma protein to a fibrillar component of the extracellular matrix. After colonization, S. pyogenes must evade the host innate immune system to spread into blood vessels and deeper organs. Some Fn-binding proteins contribute to evasion of host innate immunity, such as the complement system and phagocytosis. In addition, Fn-binding proteins have received focus as non-M protein vaccine candidates, because of their localization and conservation among different M serotypes.Here, we review the roles of Fn-binding proteins in the pathogenesis and speculate regarding possible vaccine antigen candidates.

Respiratory tract barrier dysfunction in viral-bacterial co-infection cases.

A preceding viral infection of the respiratory tract predisposes the host to secondary bacterial pneumonia, known as a major cause of morbidity and mortality. However, the underlying mechanism of the viral-bacterial synergy that leads to disease progression has remained elusive, thus hampering the production of effective prophylactic and therapeutic intervention options. In addition to viral-induced airway epithelial damage, which allows dissemination of bacteria to the lower respiratory tract and increases their invasiveness, dysfunction of immune defense following a viral infection has been implicated as a factor for enhanced susceptibility to secondary bacterial infections. Given the proximity of the oral cavity to the respiratory tract, where viruses enter and replicate, it is also well-established that oral health status can significantly influence the initiation, progression, and pathology of respiratory viral infections. This review was conducted to focus on the dysfunction of the respiratory barrier, which plays a crucial role in providing physical and secretory barriers as well as immune defense in the context of viral-bacterial synergy. Greater understanding of barrier response to viral-bacterial co-infections, will ultimately lead to development of effective, broad-spectrum therapeutic approaches for prevention of enhanced susceptibility to these pathogens.

Hybrid sequence-based analysis reveals the distribution of bacterial species and genes in the oral microbiome at a high resolution.

Bacteria in the oral microbiome are poorly identified owing to the lack of established culture methods for them. Thus, this study aimed to use culture-free analysis techniques, including bacterial single-cell genome sequencing, to identify bacterial species and investigate gene distribution in saliva. Saliva samples from the same individual were classified as inactivated or viable and then analyzed using 16S rRNA sequencing, metagenomic shotgun sequencing, and bacterial single-cell sequencing. The results of 16S rRNA sequencing revealed similar microbiota structures in both samples, with Streptococcus being the predominant genus. Metagenomic shotgun sequencing showed that approximately 80 % of the DNA in the samples was of non-bacterial origin, whereas single-cell sequencing showed an average contamination rate of 10.4 % per genome. Single-cell sequencing also yielded genome sequences for 43 out of 48 wells for the inactivated samples and 45 out of 48 wells for the viable samples. With respect to resistance genes, four out of 88 isolates carried cfxA, which encodes a ß-lactamase, and four isolates carried erythromycin resistance genes. Tetracycline resistance genes were found in nine bacteria. Metagenomic shotgun sequencing provided complete sequences of cfxA, ermF, and ermX, whereas other resistance genes, such as tetQ and tetM, were detected as fragments. In addition, virulence factors from Streptococcus pneumoniae were the most common, with 13 genes detected. Our average nucleotide identity analysis also suggested five single-cell-isolated bacteria as potential novel species. These data would contribute to expanding the oral microbiome data resource.

Two-component regulatory system TCS08 of a serotype 4 strain in pneumococcal pneumonia pathogenesis.

OBJECTIVES: Streptococcus pneumoniae, a human respiratory pathogen, causes diseases with severe morbidity and mortality rates worldwide. The two-component regulatory system (TCS) is an important signaling pathway that enables regulation of gene expression in response to environmental cues, thereby allowing an organism to adapt to a variety of host niches. Here we examined the contribution of pneumococcal TCS08 to bacterial colonization, the development of pneumonia, and pulmonary dysfunction. METHODS: We employed an hk08 knockout mutant (Δhk08) with a background of the TIGR4 wild-type (WT) strain to verify whether TCS08 is associated with bacterial colonization and the development of pneumonia in a murine infection model. To clarify the association of hk08 inactivation-induced phenotypic changes with their virulence, we examined pneumococcal capsule production, colony morphology, and surface-displayed protein profiles. RESULTS: Pneumococcal TCS08 was involved in bacterial colonization in the respiratory tract. Interruption of the signaling pathway of TCS08 by hk08 inactivation impaired mouse survival and increased the bacterial burden within the respiratory tract. Furthermore, a histopathological examination revealed massive inflammatory cell infiltration, edema formation, and diffuse alveolar damage in the lung tissues of mice infected with Δhk08 versus the WT or complemented strain. Interestingly, virulence-associated phenotype changes, including capsule production, increased chain length, and surface-displayed protein profile, were observed in the Δhk08 strain. CONCLUSIONS: The present findings indicate that TCS08 contributes to pneumococcal colonization and pulmonary dysfunction by assisting adaptation to the respiratory tract milieu, leading to the development of pneumonia.