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.

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.

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.

Elucidation of independently modulated genes in Streptococcus pyogenes reveals carbon sources that control its expression of hemolytic toxins.

Streptococcus pyogenes can cause a wide variety of acute infections throughout the body of its human host. An underlying transcriptional regulatory network (TRN) is responsible for altering the physiological state of the bacterium to adapt to each unique host environment. Consequently, an in-depth understanding of the comprehensive dynamics of the S. pyogenes TRN could inform new therapeutic strategies. Here, we compiled 116 existing high-quality RNA sequencing data sets of invasive S. pyogenes serotype M1 and estimated the TRN structure in a top-down fashion by performing independent component analysis (ICA). The algorithm computed 42 independently modulated sets of genes (iModulons). Four iModulons contained the nga-ifs-slo virulence-related operon, which allowed us to identify carbon sources that control its expression. In particular, dextrin utilization upregulated the nga-ifs-slo operon by activation of two-component regulatory system CovRS-related iModulons, altering bacterial hemolytic activity compared to glucose or maltose utilization. Finally, we show that the iModulon-based TRN structure can be used to simplify the interpretation of noisy bacterial transcriptome data at the infection site. IMPORTANCE S. pyogenes is a pre-eminent human bacterial pathogen that causes a wide variety of acute infections throughout the body of its host. Understanding the comprehensive dynamics of its TRN could inform new therapeutic strategies. Since at least 43 S. pyogenes transcriptional regulators are known, it is often difficult to interpret transcriptomic data from regulon annotations. This study shows the novel ICA-based framework to elucidate the underlying regulatory structure of S. pyogenes allows us to interpret the transcriptome profile using data-driven regulons (iModulons). Additionally, the observations of the iModulon architecture lead us to identify the multiple regulatory inputs governing the expression of a virulence-related operon. The iModulons identified in this study serve as a powerful guidepost to further our understanding of S. pyogenes TRN structure and dynamics.

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.

Construction of Human Three-Dimensional Lung Model Using Layer-by-Layer Method.

The respiratory tract is one of the frontline barriers for biological defense. Lung epithelial intercellular adhesions provide protection from bacterial and viral infections and prevent invasion into deep tissues by pathogens. Dysfunction of lung epithelial intercellular adhesion caused by pathogens is associated with development of several diseases, such as acute respiratory distress syndrome, pneumonia, and asthma. To elucidate the pathological mechanism of respiratory infections, two-dimensional cell cultures and animal models are commonly used, although are not useful for evaluating host specificity or human biological response. With the rapid progression and worldwide spread of severe acute respiratory syndrome coronavirus-2, there is increasing interest in the development of a three-dimensional (3D) in vitro lung model for analyzing interactions between pathogens and hosts. However, some models possess unclear epithelial polarity or insufficient barrier functions and need the use of complex technologies, have high cost, and long cultivation terms. We previously reported about the fabrication of 3D cellular multilayers using a layer-by-layer (LbL) cell coating technique with extracellular matrix protein, fibronectin (FN), and gelatin (G). In the present study, such a LbL cell coating technique was utilized to construct a human 3D lung model in which a monolayer of the human lower airway epithelial adenocarcinoma cell line Calu-3 cells was placed on 3D-cellular multilayers composed of FN-G-coated human primary pulmonary fibroblast cells. The 3D lung model thus constructed demonstrated an epithelial-fibroblast layer that maintained uniform thickness until 7 days of incubation. Moreover, expressions of E-cadherin, ZO-1, and mucin in the epithelial layer were observed by immunohistochemical staining. Epithelial barrier integrity was evaluated using transepithelial electrical resistance values. The results indicate that the present constructed human 3D lung model is similar to human lung tissues and also features epithelial polarity and a barrier function, thus is considered useful for evaluating infection and pathological mechanisms related to pneumonia and several pathogens. Impact statement A novel in vitro model of lung tissue was established. Using a layer-by-layer cell coating technique, a three-dimensional cultured lung model was constructed. The present novel model was shown to have epithelial polarity and chemical barrier functions. This model may be useful for investigating interaction pathogens and human biology.

Oral mitis group streptococci reduce infectivity of influenza A virus via acidification and H2O2 production.

Members of the mitis group streptococci are the most abundant inhabitants of the oral cavity and dental plaque. Influenza A virus (IAV), the causative agent of influenza, infects the upper respiratory tract, and co-infection with Streptococcus pneumoniae is a major cause of morbidity during influenza epidemics. S. pneumoniae is a member of mitis group streptococci and shares many features with oral mitis group streptococci. In this study, we investigated the effect of viable Streptococcus oralis, a representative member of oral mitis group, on the infectivity of H1N1 IAV. The infectivity of IAV was measured by a plaque assay using Madin-Darby canine kidney cells. When IAV was incubated in growing culture of S. oralis, the IAV titer decreased in a time- and dose-dependent manner and became less than 100-fold, whereas heat-inactivated S. oralis had no effect. Other oral streptococci such as Streptococcus mutans and Streptococcus salivarius also reduced the viral infectivity to a lesser extent compared to S. oralis and Streptococcus gordonii, another member of the oral mitis group. S. oralis produces hydrogen peroxide (H2O2) at a concentration of 1-2 mM, and its mutant deficient in H2O2 production showed a weaker effect on the inactivation of IAV, suggesting that H2O2 contributes to viral inactivation. The contribution of H2O2 was confirmed by an inhibition assay using catalase, an H2O2-decomposing enzyme. These oral streptococci produce short chain fatty acids (SCFA) such as acetic acid as a by-product of sugar metabolism, and we also found that the inactivation of IAV was dependent on the mildly acidic pH (around pH 5.0) of these streptococcal cultures. Although inactivation of IAV in buffers of pH 5.0 was limited, incubation in the same buffer containing 2 mM H2O2 resulted in marked inactivation of IAV, which was similar to the effect of growing S. oralis culture. Taken together, these results reveal that viable S. oralis can inactivate IAV via the production of SCFAs and H2O2. This finding also suggests that the combination of mildly acidic pH and H2O2 at low concentrations could be an effective method to inactivate IAV.

Biological Evaluation of the Effect of Root Canal Sealers Using a Rat Model.

Gutta-percha points and root canal sealers have been used for decades in endodontics for root canal obturation. With techniques such as single cone methods, the amount of sealer is larger, making their properties more critical. However, relatively few reports have comprehensively evaluated their biological effects. To this end, we evaluated three types of sealers, zinc oxide-fatty acid-, bio-glass- and methacrylate resin-containing sealers were considered. Their biological effects were evaluated using a rat subcutaneous implantation model. Each sealer was loaded inside a Teflon tube and implanted subcutaneously in the backs of rats. Inflammatory cells were observed around all samples 7 days after implantation and reduced after 28 days. Our results revealed that all samples were in contact with the subcutaneous tissue surrounding the sealer. Additionally, Ca and P accumulation was observed in only the bio-glass-containing sealer. Furthermore, each of the three sealers exhibited unique immune and inflammatory modulatory effects. In particular, bio-glass and methacrylate resin sealers were found to induce variable gene expression in adjacent subcutaneous tissues related to angiogenesis, wound healing, muscle tissue, and surrounding subcutaneous tissue. These results may help to understand the biological impacts of root canal sealers on surrounding biological tissues, guiding future research and comparisons with new generations of materials.

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.

Pneumococcal BgaA Promotes Host Organ Bleeding and Coagulation in a Mouse Sepsis Model.

Streptococcus pneumoniae is a major cause of invasive diseases such as pneumonia, meningitis, and sepsis, with high associated mortality. Our previous molecular evolutionary analysis revealed that the S. pneumoniae gene bgaA, encoding the enzyme ß-galactosidase (BgaA), had a high proportion of codons under negative selection among the examined pneumococcal genes and that deletion of bgaA significantly reduced host mortality in a mouse intravenous infection assay. BgaA is a multifunctional protein that plays a role in cleaving terminal galactose in N-linked glycans, resistance to human neutrophil-mediated opsonophagocytic killing, and bacterial adherence to human epithelial cells. In this study, we performed in vitro and in vivo assays to evaluate the precise role of bgaA as a virulence factor in sepsis. Our in vitro assays showed that the deletion of bgaA significantly reduced the bacterial association with human lung epithelial and vascular endothelial cells. The deletion of bgaA also reduced pneumococcal survival in human blood by promoting neutrophil-mediated killing, but did not affect pneumococcal survival in mouse blood. In a mouse sepsis model, mice infected with an S. pneumoniae bgaA-deleted mutant strain exhibited upregulated host innate immunity pathways, suppressed tissue damage, and blood coagulation compared with mice infected with the wild-type strain. These results suggest that BgaA functions as a multifunctional virulence factor whereby it induces host tissue damage and blood coagulation. Taken together, our results suggest that BgaA could be an attractive target for drug design and vaccine development to control pneumococcal infection.

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.

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.

The group A Streptococcus interleukin-8 protease SpyCEP promotes bacterial intracellular survival by evasion of autophagy.

Autophagy serves an innate immune function in defending the host against invading bacteria, including group A Streptococcus (GAS). Autophagy is regulated by numerous host proteins, including the endogenous negative regulator calpain, a cytosolic protease. Globally disseminated serotype M1T1 GAS strains associated with high invasive disease potential express numerous virulence factors and resist autophagic clearance. Upon in vitro infection of human epithelial cell lines with representative wild-type GAS M1T1 strain 5448 (M1.5448), we observed increased calpain activation linked to a specific GAS virulence factor, the IL-8 protease SpyCEP. Calpain activation inhibited autophagy and decreased capture of cytosolic GAS in autophagosomes. In contrast, the serotype M6 GAS strain JRS4 (M6.JRS4), which is highly susceptible to host autophagy-mediated killing, expresses low levels of SpyCEP and does not activate calpain. Overexpression of SpyCEP in M6.JRS4 stimulated calpain activation, inhibited autophagy and significantly decreased bacterial capture in autophagosomes. These paired loss- and gain-of-function studies reveal a novel role for the bacterial protease SpyCEP in enabling GAS M1 evasion of autophagy and host innate immune clearance.

Unique virulence role of post-translocational chaperone PrsA in shaping Streptococcus pyogenes secretome.

Streptococcus pyogenes (group A Streptococcus, GAS) is a strict human pathogen causing a broad spectrum of diseases and a variety of autoimmune sequelae. The pathogenesis of GAS infection mostly relies on the production of an extensive network of cell wall-associated and secreted virulence proteins, such as adhesins, toxins, and exoenzymes. PrsA, the only extracellular parvulin-type peptidyl-prolyl isomerase expressed ubiquitously in Gram-positive bacteria, has been suggested to assist the folding and maturation of newly exported proteins to acquire their native conformation and activity. Two PrsA proteins, PrsA1 and PrsA2, have been identified in GAS, but the respective contribution of each PrsA in GAS pathogenesis remains largely unknown. By combining comparative proteomic and phenotypic analysis approaches, we demonstrate that both PrsA isoforms are required to maintain GAS proteome homeostasis and virulence-associated traits in a unique and overlapping manner. The inactivation of both PrsA in GAS caused remarkable impairment in biofilm formation, host adherence, infection-induced cytotoxicity, and in vivo virulence in a murine soft tissue infection model. The concordance of proteomic and phenotypic data clearly features the essential role of PrsA in GAS full virulence.

GP96 Drives Exacerbation of Secondary Bacterial Pneumonia following Influenza A Virus Infection.

Influenza A virus (IAV) infection predisposes the host to secondary bacterial pneumonia, known as a major cause of morbidity and mortality during influenza virus epidemics. Analysis of interactions between IAV-infected human epithelial cells and Streptococcus pneumoniae revealed that infected cells ectopically exhibited the endoplasmic reticulum chaperone glycoprotein 96 (GP96) on the surface. Importantly, efficient pneumococcal adherence to epithelial cells was imparted by interactions with extracellular GP96 and integrin αV, with the surface expression mediated by GP96 chaperone activity. Furthermore, abrogation of adherence was gained by chemical inhibition or genetic knockout of GP96 as well as addition of RGD peptide, an inhibitor of integrin-ligand interactions. Direct binding of extracellular GP96 and pneumococci was shown to be mediated by pneumococcal oligopeptide permease components. Additionally, IAV infection induced activation of calpains and Snail1, which are responsible for degradation and transcriptional repression of junctional proteins in the host, respectively, indicating increased bacterial translocation across the epithelial barrier. Notably, treatment of IAV-infected mice with the GP96 inhibitor enhanced pneumococcal clearance from lung tissues and ameliorated lung pathology. Taken together, the present findings indicate a viral-bacterial synergy in relation to disease progression and suggest a paradigm for developing novel therapeutic strategies tailored to inhibit pneumococcal colonization in an IAV-infected respiratory tract. IMPORTANCE Secondary bacterial pneumonia following an influenza A virus (IAV) infection is a major cause of morbidity and mortality. Although it is generally accepted that preceding IAV infection leads to increased susceptibility to secondary bacterial infection, details regarding the pathogenic mechanism during the early stage of superinfection remain elusive. Here, we focused on the interaction of IAV-infected cells and Streptococcus pneumoniae, which revealed that human epithelial cells infected with IAV exhibit a cell surface display of GP96, an endoplasmic reticulum chaperon. Notably, extracellular GP96 was shown to impart efficient adherence for secondary infection by S. pneumoniae, and GP96 inhibition ameliorated lung pathology of superinfected mice, indicating it to be a useful target for development of therapeutic strategies for patients with superinfection.

Respiratory FimA-Specific Secretory IgA Antibodies Upregulated by DC-Targeting Nasal Double DNA Adjuvant Are Essential for Elimination of Porphyromonas gingivalis.

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4+ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients.

Streptococcus pyogenes upregulates arginine catabolism to exert its pathogenesis on the skin surface.

The arginine deiminase (ADI) pathway has been found in many kinds of bacteria and functions to supplement energy production and provide protection against acid stress. The Streptococcus pyogenes ADI pathway is upregulated upon exposure to various environmental stresses, including glucose starvation. However, there are several unclear points about the advantages to the organism for upregulating arginine catabolism. We show that the ADI pathway contributes to bacterial viability and pathogenesis under low-glucose conditions. S. pyogenes changes global gene expression, including upregulation of virulence genes, by catabolizing arginine. In a murine model of epicutaneous infection, S. pyogenes uses the ADI pathway to augment its pathogenicity by increasing the expression of virulence genes, including those encoding the exotoxins. We also find that arginine from stratum-corneum-derived filaggrin is a key substrate for the ADI pathway. In summary, arginine is a nutrient source that promotes the pathogenicity of S. pyogenes on the skin.

Epidemiological analysis of pneumococcal strains isolated at Yangon Children's Hospital in Myanmar via whole-genome sequencing-based methods.

Streptococcus pneumoniae causes over one million deaths from lower respiratory infections per annum worldwide. Although mortality is very high in Southeast Asian countries, molecular epidemiological information remains unavailable for some countries. In this study, we report, for the first time, the whole-genome sequences and genetic profiles of pneumococcal strains isolated in Myanmar. We isolated 60 streptococcal strains from 300 children with acute respiratory infection at Yangon Children's Hospital in Myanmar. We obtained whole-genome sequences and identified the species, serotypes, sequence types, antimicrobial resistance (AMR) profiles, virulence factor profiles and pangenome structure using sequencing-based analysis. Average nucleotide identity analysis indicated that 58 strains were S. pneumoniae and the other 2 strains were Streptococcus mitis. The major serotype was 19F (11 strains), followed by 6E (6B genetic variant; 7 strains) and 15 other serotypes; 5 untypable strains were also detected. Multilocus sequence typing analysis revealed 39 different sequence types, including 11 novel ones. In addition, genetic profiling indicated that AMR genes and mutations spread among pneumococcal strains in Myanmar. A minimum inhibitory concentration assay indicated that several pneumococcal strains had acquired azithromycin and tetracycline resistance, whereas no strains were found to be resistant against levofloxacin and high-dose penicillin G. Phylogenetic and pangenome analysis showed various pneumococcal lineages and that the pneumococcal strains contain a rich and mobile gene pool, providing them with the ability to adapt to selective pressures. This molecular epidemiological information can help in tracking global infection and supporting AMR control in addition to public health interventions in Myanmar.

Role of BgaA as a Pneumococcal Virulence Factor Elucidated by Molecular Evolutionary Analysis.

Streptococcus pneumoniae is a major cause of pneumonia, sepsis, and meningitis. Previously, we identified a novel virulence factor by investigating evolutionary selective pressure exerted on pneumococcal choline-binding cell surface proteins. Herein, we focus on another pneumococcal cell surface protein. Cell wall-anchoring proteins containing the LPXTG motif are conserved in Gram-positive bacteria. Our evolutionary analysis showed that among the examined genes, nanA and bgaA had high proportions of codon that were under significant negative selection. Both nanA and bgaA encode a multi-functional glycosidase that aids nutrient acquisition in a glucose-poor environment, pneumococcal adherence to host cells, and evasion from host immunity. However, several studies have shown that the role of BgaA is limited in a mouse pneumonia model, and it remains unclear if BgaA affects pneumococcal pathogenesis in a mouse sepsis model. To evaluate the distribution and pathogenicity of bgaA, we performed phylogenetic analysis and intravenous infection assay. In both Bayesian and maximum likelihood phylogenetic trees, the genetic distances between pneumococcal bgaA was small, and the cluster of pneumococcal bgaA did not contain other bacterial orthologs except for a Streptococcus gwangjuense gene. Evolutionary analysis and BgaA structure indicated BgaA active site was not allowed to change. The mouse infection assay showed that the deletion of bgaA significantly reduced host mortality. These results indicated that both nanA and bgaA encode evolutionally conserved pneumococcal virulence factors and that molecular evolutionary analysis could be a useful alternative strategy for identification of virulence factors.

Detection of Fibronectin-Binding Proteins of Streptococcus pyogenes Using Ligand Blot Analysis.

Streptococcus pyogenes utilizes extracellular cellular matrix (ECM) proteins to adhere to human tissues and internalize into host cells. Fibronectin (Fn) is one of the most abundant ECM proteins and targeted by a wide variety of secreted Fn-binding proteins (Fbps) of S. pyogenes. However, prior to detailed kinetic analysis of that binding process, evaluations of the ability of S. pyogenes strains to bind to Fn as well as interactions of target molecules with Fn are required. In this chapter, we present routine procedures for ligand blot analysis with labeled human Fn, using bacterial cell wall extracts prepared by either enzymatic digestion of cells or extraction with a denaturing agent.