Pneumolysin contributes to dysfunction of nasal epithelial barrier for promotion of pneumococcal dissemination into brain tissue.

Streptococcus pneumoniae is one of the major pathogens responsible for bacterial meningitis and neurological sequelae. The present study was conducted to identify a non-hematogenous route used by S. pneumoniae to gain access to brain tissue without causing bacteremia or pneumonia, as well as bacterial and host factors involved in this process. To investigate the molecular mechanisms and dissemination pathways of pneumococcal infection in brain tissue, mice were intranasally inoculated with S. pneumoniae strain EF3030, a clinical isolate from a patient with otitis media. Pneumococci were isolated from the frontal olfactory bulb, caudal cerebrum, and cerebellum, with neither bacteremia nor pneumonia observed in the present model. Immunostaining imaging revealed the presence of S. pneumoniae organisms in olfactory nerve fibers. Knockout of the ply gene encoding pneumolysin (PLY) markedly compromised the ability of the bacterial organisms to disseminate into brain tissue, whereas the dissemination efficiency of the complemented strain was restored to nearly the same level as the wild type. Notably, distinct upregulation of Gli1 and Snail1, which are involved in the transcriptional repression of junctional proteins, along with downregulation of E-cadherin, was detected in nasal lavage samples from mice infected with the wild-type or complemented strain, but not in those from mice infected with the ply mutant. Taken together, the present findings indicate that PLY induces Gli1-Snail1-dependent dysfunction of the nasal epithelial barrier, thus allowing pneumococcal dissemination to brain tissue that occurs in a non-hematogenous manner.IMPORTANCEBacterial meningitis, considered to be caused by bacteremia, can lead to blood-brain barrier disruption and bacterial dissemination into the central nervous system. Despite the availability of intravenously administered antibiotics with cerebrospinal fluid transferability, bacterial meningitis remains associated with high rates of morbidity and mortality. Here, we utilized Streptococcus pneumoniae strain EF3030, clinically isolated from otitis media, for the construction of a murine infection model to investigate the molecular mechanisms by which nasally colonized pneumococci disseminate into brain tissue. The obtained findings indicate that pneumolysin (PLY) induces Gli1-Snail1-dependent dysfunction of the nasal epithelial barrier, which facilitates pneumococcal dissemination to brain tissue in a non-hematogenous manner. Our results support the existence of an alternative route by which S. pneumoniae can reach the central nervous system and indicate the need for the development of novel therapeutic strategies, which would be an important contribution to the clinical management of bacterial meningitis.

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.

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.

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.

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.

Intravascular large B-cell lymphoma causing hypopituitarism and respiratory failure due to infiltration into pulmonary capillaries.

We report a rare case of intravascular large B-cell lymphoma (IVLBCL) with hypopituitarism and respiratory failure. A man in his 80s presented with hypotension and respiratory failure but was unsuccessfully treated for septic shock. Biological investigations were performed, and he was diagnosed with hypopituitarism due to insufficient secretion of anterior pituitary hormone. Although his condition temporarily improved following hormone replacement therapy, he eventually died of progressive respiratory failure. The lymphoma was only discovered during the autopsy, where it was observed to have spread to the pituitary gland and lung capillaries. Therefore, we concluded that the lymphoma had caused respiratory failure and hypopituitarism. The patient was thus diagnosed with IVLBCL posthumously. In conclusion, IVLBCL can cause hypopituitarism and respiratory failure due to pituitary and pulmonary capillary invasion by lymphoma 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.

Complete sequences of epidermin and nukacin encoding plasmids from oral-derived Staphylococcus epidermidis and their antibacterial activity.

Staphylococcus epidermidis is a commensal bacterium in humans. To persist in the bacterial flora of the host, some bacteria produce antibacterial factors such as the antimicrobial peptides known as bacteriocins. In this study, we tried to isolate bacteriocin-producing S. epidermidis strains. Among 150 S. epidermidis isolates from the oral cavities of 287 volunteers, we detected two bacteriocin-producing strains, KSE56 and KSE650. Complete genome sequences of the two strains confirmed that they carried the epidermin-harboring plasmid pEpi56 and the nukacin IVK45-like-harboring plasmid pNuk650. The amino acid sequence of epidermin from KSE56 was identical to the previously reported sequence, but the epidermin synthesis-related genes were partially different. The prepeptide amino acid sequences of nukacin KSE650 and nukacin IVK45 showed one mismatch, but both mature peptides were entirely similar. pNuk650 was larger and had an additional seven ORFs compared to pIVK45. We then investigated the antibacterial activity of the two strains against several skin and oral bacteria and found their different activity patterns. In conclusion, we report the complete sequences of 2 plasmids coding for bacteriocins from S. epidermidis, which were partially different from those previously reported. Furthermore, this is the first report to show the complete sequence of an epidermin-carrying plasmid, pEpi56.

Comprehensive characterization of sortase A-dependent surface proteins in Streptococcus mutans.

Streptococcus mutans, a cariogenic pathogen, adheres to the tooth surface and forms a biofilm. Bacterial cell surface proteins are associated with adherence to substrates. Sortase A (SrtA) mediates the localization of proteins with an LPXTG motif-containing proteins to the cell surface by covalent binding to peptidoglycan. In S. mutans UA159, six SrtA-dependent proteins, SpaP, WapA, WapE, DexA, FruA, and GbpC, were identified. Although some of these proteins were characterized, a comprehensive analysis of the six proteins has not been reported. In this study, we constructed mutants deficient in each of these proteins and the SrtA-deficient mutant. The SrtA-deficient mutant showed drastically decreased binding to salivary components, biofilm formation, bacterial coaggregation activity, hydrophobicity, and cellular matrix binding (collagen type I, fibronectin, and laminin). The SpaP-deficient mutant showed significantly reduced binding to salivary components and partially increased coaggregation with Porphyromonas gingivalis, and decreased hydrophobicity, and collagen binding. The WapA-deficient mutant showed slightly decreased coaggregation with Fusobacterium nucleatum. Although the SrtA-deficient mutant showed drastically altered phenotypes, all SrtA-dependent protein-deficient mutants, except the SpaP-deficient mutant, did not show considerable alterations in binding to salivary components. These results indicate that the six proteins may coordinately contribute to these activities. In addition, using genomic data of 125 S. mutans strains, the amino acid sequences of each surface protein were compared and many variations were found among strains, which may affect the phenotype of cell surface proteins in S. mutans.

Small regulatory RNAs of oral streptococci and periodontal bacteria.

Small regulatory RNAs (sRNAs) belong to a family of non-coding RNAs, and many of which regulate expression of genes via interaction with mRNA. The recent popularity of high-throughput next generation sequencers have presented abundant sRNA-related data, including sRNAs of several different oral bacterial species. Some sRNA candidates have been validated in terms of their expression and interaction with target mRNAs. Since the oral cavity is an environment constantly exposed to various stimuli, such as fluctuations in temperature and pH, and osmotic pressure, as well as changes in nutrient availability, oral bacteria require rapid control of gene expression for adaptation to such diverse conditions, while regulation via interactions of sRNAs with mRNA provides advantages for rapid adaptation. This review summarizes methods effective for identification and validation of sRNAs, as well as sRNAs identified to be associated with oral bacterial species, including cariogenic and periodontal pathogens, together with their confirmed and putative target genes.

Comprehensive analysis of bacteriocins in Streptococcus mutans.

Streptococcus mutans produces bacteriocins that show antibacterial activity against several bacteria. However, comprehensive analysis of these bacteriocins has not been well done. In this study, we isolated 125 S. mutans strains from volunteers and determined their whole genome sequence. Based on the genome analysis, the distribution of each bacteriocin gene (mutacins I-IV, K8 and Smb) was investigated. We found 17, 5, and 2 strains showing 100% matches with mutacin I, mutacin II and mutacin III, respectively. Five mutacin III-positive strains had 2 mismatches compared to mature mutacin III. In 67 mutacin IV-positive strains, 38 strains showed 100% match with mutacin IV, while 29 strains showed some variations. In 23 mutacin K8- and 32 mutacin Smb-positive strains, all except one mutacin K8-positive strain showed 100% match with the mature peptides. Among 125 strains, 84 (65.1%), 26 (20.2%), and 5 (3.9%) strains were positive for one, two and three bacteriocin genes, respectively. Then, the antibacterial activity against oral streptococci and other oral bacterial species was investigated by using bacteriocin gene single-positive strains. Each bacteriocin gene-positive strain showed a different pattern of antibacterial activity. These results speculate that individual S. mutans strains may affect the bacterial composition of dental plaques.

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.

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.

Genetics, Structure, and Function of Group A Streptococcal Pili.

Streptococcus pyogenes (Group A Streptococcus; GAS) is an exclusively human pathogen. This bacterial species is responsible for a large variety of infections, ranging from purulent but mostly self-limiting oropharynx/skin diseases to streptococcal sequelae, including glomerulonephritis and rheumatic fever, as well as life-threatening streptococcal toxic-shock syndrome. GAS displays a wide array of surface proteins, with antigenicity of the M protein and pili utilized for M- and T-serotyping, respectively. Since the discovery of GAS pili in 2005, their genetic features, including regulation of expression, and structural features, including assembly mechanisms and protein conformation, as well as their functional role in GAS pathogenesis have been intensively examined. Moreover, their potential as vaccine antigens has been studied in detail. Pilus biogenesis-related genes are located in a discrete section of the GAS genome encoding fibronectin and collagen binding proteins and trypsin-resistant antigens (FCT region). Based on the heterogeneity of genetic composition and DNA sequences, this region is currently classified into nine distinguishable forms. Pili and fibronectin-binding proteins encoded in the FCT region are known to be correlated with infection sites, such as the skin and throat, possibly contributing to tissue tropism. As also found for pili of other Gram-positive bacterial pathogens, GAS pilin proteins polymerize via isopeptide bonds, while intramolecular isopeptide bonds present in the pilin provide increased resistance to degradation by proteases. As supported by findings showing that the main subunit is primarily responsible for T-serotyping antigenicity, pilus functions and gene expression modes are divergent. GAS pili serve as adhesins for tonsillar tissues and keratinocyte cell lines. Of note, a minor subunit is considered to have a harpoon function by which covalent thioester bonds with host ligands are formed. Additionally, GAS pili participate in biofilm formation and evasion of the immune system in a serotype/strain-specific manner. These multiple functions highlight crucial roles of pili during the onset of GAS infection. This review summarizes the current state of the art regarding GAS pili, including a new mode of host-GAS interaction mediated by pili, along with insights into pilus expression in terms of tissue tropism.

Expression of virulence factors under different environmental conditions in Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a facultative anaerobic Gram-negative bacterium associated with periodontal diseases, especially aggressive periodontitis. The virulence factors of this pathogen, including adhesins, exotoxins, and endotoxin, have been extensively studied. However, little is known about their gene expression mode in the host. Herein, we investigated whether culture conditions reflecting in vivo environments, including serum and saliva, alter expression levels of virulence genes in the strain HK1651, a JP2 clone. Under aerobic conditions, addition of calf serum (CS) into a general medium induced high expression of two outer membrane proteins (omp100 and omp64). The high expression of omp100 and omp64 was also induced by an iron-limited medium. RNA-seq analysis showed that the gene expressions of several factors involved in iron acquisition were increased in the CS-containing medium. When HK1651 was grown on agar plates, genes encoding many virulence factors, including the Omps, cytolethal distending toxin, and leukotoxin, were differentially expressed. Then, we investigated their expression in five other A. actinomycetemcomitans strains grown in general and CS-containing media. The expression pattern of virulence factors varied among strains. Compared with the other five strains, HK1561 showed high expression of omp29 regardless of the CS addition, while the gene expression of leukotoxin in HK1651 was higher only in the medium without CS. HK1651 showed reduced biofilm in both CS- and saliva-containing media. Coaggregation with Fusobacterium nucleatum was remarkably enhanced using HK1651 grown in the CS-containing medium. Our results indicate that the expression of virulence factors is altered by adaptation to different conditions during infection.

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.

Association of Epstein-Barr virus with regression after withdrawal of immunosuppressive drugs and subsequent progression of iatrogenic immunodeficiency-associated lymphoproliferative disorders in patients with autoimmune diseases.

Patients with autoimmune diseases (AIDs) may develop lymphoproliferative disorders (LPDs) during treatment with immunosuppressive agents (IS) such as methotrexate (MTX), biological agents, or tacrolimus. Some LPDs in patients with AIDs (AID-LPDs) regress after withdrawal of IS, and a high incidence of Epstein-Barr virus (EBV) positivity in such patients has been reported. To identify characteristics and factors predictive of the response to treatment and disease progression, we retrospectively analyzed clinical and histopathological data for 81 patients with AID-LPDs. Almost all of them (96%) had been treated with MTX. Diffuse large B cell lymphoma was the most common LPD type (61%) and seven patients (9%) had classical Hodgkin lymphoma (CHL). EBV was detected by in situ hybridization with an EBV-encoded small RNA (EBER) probe in 43% of the examined cases. In 59 patients, IS was discontinued as the initial treatment, resulting in regression of LPDs in 69% of them, and multivariate analysis showed that EBER positivity was an independent factor predictive of such regression (p = 0.022). Two-year progression-free survival (PFS) and overall survival for the patients overall were 63% and 83%, respectively. Poor PFS was associated with advanced stage (p = 0.024), worse performance status (PS, p = 0.031), CHL histology (p = 0.013), and reactivation of EBV-related antibodies (p = 0.029). In conclusion, EBV positivity demonstrated using an EBER probe is useful for prediction of successful regression after withdrawal of IS in patients with AID-LPDs. Patients with advanced stage disease, worse PS, CHL histology, or reactivation of EBV-related antibodies should be closely monitored after initial treatment.

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.