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.

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.

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.

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.

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.

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.

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.

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.

Involvement of T6 pili in biofilm formation by serotype M6 Streptococcus pyogenes.

The group A streptococcus (GAS) Streptococcus pyogenes is known to cause self-limiting purulent infections in humans. The role of GAS pili in host cell adhesion and biofilm formation is likely fundamental in early colonization. Pilus genes are found in the FCT (fibronectin-binding protein, collagen-binding protein, and trypsin-resistant antigen) genomic region, which has been classified into nine subtypes based on the diversity of gene content and nucleotide sequence. Several epidemiological studies have indicated that FCT type 1 strains, including serotype M6, produce large amounts of monospecies biofilm in vitro. We examined the direct involvement of pili in biofilm formation by serotype M6 clinical isolates. In the majority of tested strains, deletion of the tee6 gene encoding pilus shaft protein T6 compromised the ability to form biofilm on an abiotic surface. Deletion of the fctX and srtB genes, which encode pilus ancillary protein and class C pilus-associated sortase, respectively, also decreased biofilm formation by a representative strain. Unexpectedly, these mutant strains showed increased bacterial aggregation compared with that of the wild-type strain. When the entire FCT type 1 pilus region was ectopically expressed in serotype M1 strain SF370, biofilm formation was promoted and autoaggregation was inhibited. These findings indicate that assembled FCT type 1 pili contribute to biofilm formation and also function as attenuators of bacterial aggregation. Taken together, our results show the potential role of FCT type 1 pili in the pathogenesis of GAS infections.

Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier.

Group A Streptococcus pyogenes (GAS) is a human pathogen that causes local suppurative infections and severe invasive diseases. Systemic dissemination of GAS is initiated by bacterial penetration of the epithelial barrier of the pharynx or damaged skin. To gain insight into the mechanism by which GAS penetrates the epithelial barrier, we sought to identify both bacterial and host factors involved in the process. Screening of a transposon mutant library of a clinical GAS isolate recovered from an invasive episode allowed identification of streptolysin S (SLS) as a novel factor that facilitates the translocation of GAS. Of note, the wild type strain efficiently translocated across the epithelial monolayer, accompanied by a decrease in transepithelial electrical resistance and cleavage of transmembrane junctional proteins, including occludin and E-cadherin. Loss of integrity of intercellular junctions was inhibited after infection with a deletion mutant of the sagA gene encoding SLS, as compared with those infected with the wild type strain. Interestingly, following GAS infection, calpain was recruited to the plasma membrane along with E-cadherin. Moreover, bacterial translocation and destabilization of the junctions were partially inhibited by a pharmacological calpain inhibitor or genetic interference with calpain. Our data indicate a potential function of SLS that facilitates GAS invasion into deeper tissues via degradation of epithelial intercellular junctions in concert with the host cysteine protease calpain.

Assembly mechanism of FCT region type 1 pili in serotype M6 Streptococcus pyogenes.

The human pathogen Streptococcus pyogenes produces diverse pili depending on the serotype. We investigated the assembly mechanism of FCT type 1 pili in a serotype M6 strain. The pili were found to be assembled from two precursor proteins, the backbone protein T6 and ancillary protein FctX, and anchored to the cell wall in a manner that requires both a housekeeping sortase enzyme (SrtA) and pilus-associated sortase enzyme (SrtB). SrtB is primarily required for efficient formation of the T6 and FctX complex and subsequent polymerization of T6, whereas proper anchoring of the pili to the cell wall is mainly mediated by SrtA. Because motifs essential for polymerization of pilus backbone proteins in other Gram-positive bacteria are not present in T6, we sought to identify the functional residues involved in this process. Our results showed that T6 encompasses the novel VAKS pilin motif conserved in streptococcal T6 homologues and that the lysine residue (Lys-175) within the motif and cell wall sorting signal of T6 are prerequisites for isopeptide linkage of T6 molecules. Because Lys-175 and the cell wall sorting signal of FctX are indispensable for substantial incorporation of FctX into the T6 pilus shaft, FctX is suggested to be located at the pilus tip, which was also implied by immunogold electron microscopy findings. Thus, the elaborate assembly of FCT type 1 pili is potentially organized by sortase-mediated cross-linking between sorting signals and the amino group of Lys-175 positioned in the VAKS motif of T6, thereby displaying T6 and FctX in a temporospatial manner.

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.

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.

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.

Pili of oral Streptococcus sanguinis bind to salivary amylase and promote the biofilm formation.

Streptococcus sanguinis is a member of oral streptococci and one of the most abundant species found in oral biofilm called dental plaque. Colonization of the oral streptococci on the tooth surface depends on the adhesion of bacteria to salivary components adsorbed to the tooth surface. Recently, we identified unique cell surface long filamentous structures named pili in this species. Herein, we investigated the role of S. sanguinis pili in biofilm formation. We found that pili-deficient mutant, in which the genes encoding the three pilus proteins PilA, PilB and PilC have been deleted, showed an impaired bacterial accumulation on saliva-coated surfaces. Confocal microscopic observations suggested that the mutant was incapable of producing typical three-dimensional layer of biofilm. Ligand blot analysis showed that the ancillary pilus proteins PilB and PilC bound to human whole saliva. Additional analysis demonstrated that PilC bound to multiple salivary components, and one of which was found to be salivary α-amylase. These results indicate that pilus proteins are members of saliva-binding proteins of oral S. sanguinis, and suggest the involvement of pili in its colonization on saliva-coated tooth surfaces and in the human oral cavity.

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.