Contribution of biofilm regulatory protein A of Streptococcus mutans, to systemic virulence

Streptococcus mutans is occasionally isolated from the blood of patients with bacteremia and infective endocarditis (IE), and the possibility that it could be pathogenic for those diseases has been discussed. The initial important step for the involvement of bacterial pathogens in the virulence of IE is thought to be survival in blood for an extended period. Recently, the brpA gene encoding biofilm regulatory protein A (BrpA) of S. mutans was cloned and sequenced, after which it was shown that inactivation of brpA in an isogenic mutant strain resulted in longer chain formation than in the parental strain. In the present study, a BrpA-defective isogenic mutant strain (MT8148BRD) was constructed from strain MT8148. In an analysis of its susceptibility to phagocytosis by human polymorphonuclear leukocytes (PMNs), the phagocytosis rate of MT8148BRD was shown to be significantly lower than that of MT8148 (P < 0.01). Next, strains with various chain lengths were produced by culturing MT8148 in media with various initial pH levels, which revealed that there was a statistically negative correlation between phagocytosis susceptibility and chain length (P < 0.01). Further, MT8148BRD was found to possess higher platelet aggregation properties than MT8148 (P < 0.05). In addition, injection of MT8148BRD into the jugular vein of specific pathogen-free Sprague-Dawley rats resulted in a longer duration of bacteremia, which prolonged systemic inflammation for a longer period than in those infected with MT8148. These results indicate that S. mutans BrpA is associated with virulence in blood, due to its correlation to phagocytosis susceptibility and platelet aggregation properties.     

Staphylococcus aureus Host Cell Invasion and Virulence in Sepsis Is Facilitated by the Multiple Repeats within FnBPA

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to α₅β₁ integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis.     

The Streptococcus sanguinis competence regulon is not required for infective endocarditis virulence in a rabbit model

Streptococcus sanguinis is an important component of dental plaque and a leading cause of infective endocarditis. Genetic competence in S. sanguinis requires a quorum sensing system encoded by the early comCDE genes, as well as late genes controlled by the alternative sigma factor, ComX. Previous studies of Streptococcus pneumoniae and Streptococcus mutans have identified functions for the >100-gene com regulon in addition to DNA uptake, including virulence. We investigated this possibility in S. sanguinis. Strains deleted for the comCDE or comX master regulatory genes were created. Using a rabbit endocarditis model in conjunction with a variety of virulence assays, we determined that both mutants possessed infectivity equivalent to that of a virulent control strain, and that measures of disease were similar in rabbits infected with each strain. These results suggest that the com regulon is not required for S. sanguinis infective endocarditis virulence in this model. We propose that the different roles of the S. sanguinis, S. pneumoniae, and S. mutans com regulons in virulence can be understood in relation to the pathogenic mechanisms employed by each species.     

Contribution of cell surface protein antigen PAc of Streptococcus mutans to bacteremia

Streptococcus mutans, a major cariogenic bacterium, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis. Mutant strains of S. mutans MT8148, defective in the major surface proteins glucosyltransferase (GTF) B-, C-, and D-, and protein antigen c (PAc), were constructed by insertional inactivation of each respective gene with an antibiotic resistant cassette. Susceptibility to phagocytosis was determined by analyses of interactions of the bacteria with human polymorphonuclear leukocytes, and the PAc-defective mutant strain (PD) showed the lowest rate of phagocytosis. Further, when PD and MT8148 were separately injected into the jugular veins of Sprague-Dawley rats, PD was recovered in significantly larger numbers and for a longer duration, and caused more severe systemic inflammation than MT8148, indicating that S. mutans PAc is associated with its systemic virulence in blood. Next, 100 S. mutans clinical isolates from 100 Japanese children and adolescents were analyzed by Western blotting using antisera raised against recombinant PAc, generated based on the pac sequence of MT8148. Four of the 100 strains showed no positive band and each exhibited a significantly lower phagocytosis rate than that of 25 randomly selected clinical strains (P < 0.01). In addition, three of the 100 strains possessed a lower molecular weight PAc and a significantly lower rate of phagocytosis than the 25 reference strains (P < 0.05). These results suggest that S. mutans PAc may be associated with phagocytosis susceptibility to human polymorphonuclear leukocytes, with approximately 7% of S. mutans clinical isolates possible high-risk strains for the development of bacteremia.     

Ecto-5'-nucleotidase: a candidate virulence factor in Streptococcus sanguinis experimental endocarditis

Streptococcus sanguinis is the most common cause of infective endocarditis (IE). Since the molecular basis of virulence of this oral commensal bacterium remains unclear, we searched the genome of S. sanguinis for previously unidentified virulence factors. We identified a cell surface ecto-5'-nucleotidase (Nt5e), as a candidate virulence factor. By colorimetric phosphate assay, we showed that S. sanguinis Nt5e can hydrolyze extracellular adenosine triphosphate to generate adenosine. Moreover, a nt5e deletion mutant showed significantly shorter lag time (P<0.05) to onset of platelet aggregation than the wild-type strain, without affecting platelet-bacterial adhesion in vitro (P?=?0.98). In the absence of nt5e, S. sanguinis caused IE (4 d) in a rabbit model with significantly decreased mass of vegetations (P<0.01) and recovered bacterial loads (log(10)CFU, P?=?0.01), suggesting that Nt5e contributes to the virulence of S. sanguinis in vivo. As a virulence factor, Nt5e may function by (i) hydrolyzing ATP, a pro-inflammatory molecule, and generating adenosine, an immunosuppressive molecule to inhibit phagocytic monocytes/macrophages associated with valvular vegetations. (ii) Nt5e-mediated inhibition of platelet aggregation could also delay presentation of platelet microbicidal proteins to infecting bacteria on heart valves. Both plausible Nt5e-dependent mechanisms would promote survival of infecting S. sanguinis. In conclusion, we now show for the first time that streptococcal Nt5e modulates S. sanguinis-induced platelet aggregation and may contribute to the virulence of streptococci in experimental IE.     

Enterococcal Infective Endocarditis following Periodontal Disease in Dogs

In humans, one of the major factors associated with infective endocarditis (IE) is the concurrent presence of periodontal disease (PD). However, in veterinary medicine, the relevance of PD in the evolution of dogs’ endocarditis remains poorly understood. In order to try to establish a correlation between mouth-associated Enterococcus spp. and infective endocarditis in dogs, the present study evaluated the presence and diversity of enterococci in the gum and heart of dogs with PD. Samples were collected during necropsy of 32 dogs with PD and visually diagnosed with IE, which died of natural causes or euthanasia. Enterococci were isolated, identified and further characterized by Pulsed-Field Gel Electrophoresis (PFGE); susceptibility to antimicrobial agents and pathogenicity potential was also evaluated. In seven sampled animals, PFGE-patterns, resistance and virulence profiles were found to be identical between mouth and heart enterococci obtained from the same dog, allowing the establishment of an association between enterococcal periodontal disease and endocarditis in dogs. These findings represent a crucial step towards understanding the pathogenesis of PD-driven IE, and constitute a major progress in veterinary medicine.     

The two-component system ScnRK of Streptococcus mutans affects hydrogen peroxide resistance and murine macrophage killing

To survive macrophage killing is critical in the pathogenesis of viridians streptococci-induced infective endocarditis (IE). Streptococcus mutans, an opportunistic IE pathogen, generally does not survive well phagocytic killing in murine macrophage RAW 264.7 cells. A putative two-component system (TCS), ScnR/ScnK from S. mutans, was investigated to elucidate the mechanisms underlying bacteria-cellular interaction in this study. Both the wild-type and mutant strains were phagocytosed by RAW 264.7 cells at a comparable rate and an increased intracellular susceptibility during a 5 h incubation period was observed with the scnRK-null mutants. The amount of reactive oxygen species (ROS) in activated macrophages was reduced significantly after ingesting wild-type, but not scnRK-null mutant strains, suggesting that increased macrophage killing of these mutants is due to the impaired ability of S. mutans to counteract ROS. Additionally, both scnR- or scnRK-null mutants were more susceptible to hydrogen peroxide. Interestingly, scnRK expression was unaffected by hydrogen peroxide. These experimental results indicate that scnRK is important in counteracting oxidative stress in S. mutans, and decreased susceptibility to phagocytic killing is at least partly attributable to inhibition of intracellular ROS formation.     

Two-Component System Response Regulators Involved in Virulence of Streptococcus pneumoniae TIGR4 in Infective Endocarditis

Streptococci resident in the oral cavity have been linked to infective endocarditis (IE). While other viridans streptococci are commonly studied in relation to IE, less research has been focused on Streptococcus pneumoniae. We established for the first time an animal model of S. pneumoniae IE, and examined the virulence of the TIGR4 strain in this model. We hypothesized that two-component systems (TCS) may mediate S. pneumoniae TIGR4 strain virulence in IE and examined TCS response regulator (RR) mutants of TIGR4 in vivo with the IE model. Thirteen of the 14 RR protein genes were mutagenized, excluding only the essential gene SP_1227. The requirement of the 13 RRs for S. pneumoniae competitiveness in the IE model was assessed in vivo through use of quantitative real-time PCR (qPCR) and competitive index assays. Using real-time PCR, several RR mutants were detected at significantly lower levels in infected heart valves compared with a control strain suggesting the respective RRs are candidate virulence factors for IE. The virulence reduction of the ΔciaR mutant was further confirmed by competitive index assay. Our data suggest that CiaR is a virulence factor of S. pneumoniae strain TIGR4 for IE.     

Pleiotropic virulence factor – Streptococcus pyogenes fibronectin‐binding proteins

Streptococcus pyogenes causes a broad spectrum of infectious diseases, including pharyngitis, skin infections and invasive necrotizing fasciitis. The initial phase of infection involves colonization, followed by intimate contact with the host cells, thus promoting bacterial uptake by them. S. pyogenes recognizes fibronectin (Fn) through its own Fn‐binding proteins to obtain access to epithelial and endothelial cells in host tissue. Fn‐binding proteins bind to Fn to form a bridge to α₅β₁‐integrins, which leads to rearrangement of cytoskeletal actin in host cells and uptake of invading S. pyogenes. Recently, several structural analyses of the invasion mechanism showed molecular interactions by which Fn converts from a compact plasma protein to a fibrillar component of the extracellular matrix. After colonization, S. pyogenes must evade the host innate immune system to spread into blood vessels and deeper organs. Some Fn‐binding proteins contribute to evasion of host innate immunity, such as the complement system and phagocytosis. In addition, Fn‐binding proteins have received focus as non‐M protein vaccine candidates, because of their localization and conservation among different M serotypes.Here, we review the roles of Fn‐binding proteins in the pathogenesis and speculate regarding possible vaccine antigen candidates.     

Rise of CC398 Lineage of Staphylococcus aureus among Infective Endocarditis Isolates Revealed by Two Consecutive Population-Based Studies in France

Staphylococcus aureus isolates from two prospective studies on infective endocarditis (IE) conducted in 1999 and 2008 and isolated from non-IE bacteremia collected in 2006 were spa-typed and their virulence factors were analyzed with a microarray. Both populations were genetically diverse, with no virulence factors or genotypes significantly more associated with the IE isolates compared with the non-IE isolates. The population structure of the IE isolates did not change much between 1999 and 2008, with the exception of the appearance of CC398 methicillin-susceptible Staphylococcus aureus (MSSA) isolates responsible for 5.6% of all cases in 2008. In 1999, this lineage was responsible for no cases. The increasing prevalence of S. aureus in IE is apparently not the result of a major change in staphylococcal population structure over time, with the exception of the emerging CC398 MSSA lineage.     

Inhibition of fibronectin binding of some bacterial cells by subtle pH increase within the physiological range

The fibronectin (Fn)-binding ability of microorganisms is considered to be involved in their pathogenicities. Granulicatella adiacens, a member of the oral flora and a causative agent of culture-negative infective endocarditis, showed nearly maximum binding to immobilized Fn at pH 7.2 but greatly reduced binding at a slightly higher pH 7.4 and almost no binding at pH 7.6 in the presence of physiological concentration of NaCl (0.15 M). A similar pH-sensitive Fn-binding property was noted with Escherichia coli and Abiotrophia defectiva, but not with Streptococcus pyogenes nor Staphylococcus aureus. In contrast, bindings to laminin and fibrinogen observed for some of these strains were unaffected by the same pH changes. This fastidious pH-dependency of Fn-binding abilities of some bacteria warns that the pH condition must be seriously considered in the in vitro assay of bacterial adherence to fibronectin.     

Genome-based approaches to develop epitope-driven subunit vaccines against pathogens of infective endocarditis

Infective endocarditis (IE) has emerged as a public health problem due to changes in the etiologic spectrum and due to involvement of resistant bacterial strains with increased virulence. Developing potent vaccine is an important strategy to tackle IE. Complete genome sequences of eight selected pathogens of IE paved the way to design common T-cell driven subunit vaccines. Comparative genomics and subtractive genomic analysis were applied to identify adinosine tri phosphate (ATP)-binding cassette (ABC) transporter ATP-binding protein from Streptococcus mitis (reference organism) as common vaccine target. Reverse vaccinology technique was implemented using computational tools such as ProPred, SYFPEITHI, and Immune epitope database. Twenty-one T-cell epitopes were predicted from ABC transporter ATP-binding protein. Multiple sequence alignment of ABC transporter ATP-binding protein from eight selected IE pathogens was performed to identify six conserved T-cell epitopes. The six selected T-cell epitopes were further evaluated at structure level for HLA-DRB binding through homology modeling and molecular docking analysis using Maestro v9.2. The proposed six T-cell epitopes showed better binding affinity with the selected HLA-DRB alleles. Subsequently, the docking complexes of T-cell epitope and HLA-DRBs were ranked based on XP Gscore. The T-cell epitope (208-LNYITPDVV-216)–HLA-DRB1^?0101 (1T5?W) complex having the best XP Gscore (?13.25?kcal/mol) was assessed for conformational stability and interaction stability through molecular dynamic simulation for 10?ns using Desmond v3.2. The simulation results revealed that the HLA-DRB–epitope complex was stable throughout the simulation time. Thus, the epitope would be ideal candidate for T-cell driven subunit vaccine design against infective endocarditis.     

Role of Streptococcus sanguinis sortase A in bacterial colonization

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, has low cariogenicity, though colonization on tooth surfaces by this bacterium initiates aggregation by other oral bacteria and maturation of dental plaque. Additionally, S. sanguinis is frequently isolated from infective endocarditis patients. We investigated the functions of sortase A (SrtA), which cleaves LPXTG-containing proteins and anchors them to the bacterial cell wall, as a possible virulence factor of S. sanguinis. We identified the srtA gene of S. sanguinis by searching a homologous gene of Streptococcus mutans in genome databases. Next, we constructed an srtA-deficient mutant strain of S. sanguinis by insertional inactivation and compared it to the wild type strain. In the case of the mutant strain, some surface proteins could not anchor to the cell wall and were partially released into the culture supernatant. Furthermore, adherence to saliva-coated hydroxyapatite beads and polystyrene plates, as well as adherence to and invasion of human epithelial cells were reduced significantly in the srtA-deficient strain when compared to the wild type. In addition, antiopsonization levels and bacterial survival of the srtA-deficient mutant were decreased in human whole blood. This is the first known study to report that SrtA contributes to antiopsonization in streptococci. Our results suggest that SrtA anchors surface adhesins as well as some proteins that function as antiopsonic molecules as a means of evading the human immune system. Furthermore, they demonstrate that SrtA of S. sanguinis plays important roles in bacterial colonization.     

The uptake of apoptotic cells drives Coxiella burnetii replication and macrophage polarization: a model for Q fever endocarditis

Patients with valvulopathy have the highest risk to develop infective endocarditis (IE), although the relationship between valvulopathy and IE is not clearly understood. Q fever endocarditis, an IE due to Coxiella burnetii, is accompanied by immune impairment. Patients with valvulopathy exhibited increased levels of circulating apoptotic leukocytes, as determined by the measurement of active caspases and nucleosome determination. The binding of apoptotic cells to monocytes and macrophages, the hosts of C. burnetii, may be responsible for the immune impairment observed in Q fever endocarditis. Apoptotic lymphocytes (AL) increased C. burnetii replication in monocytes and monocyte-derived macrophages in a cell-contact dependent manner, as determined by quantitative PCR and immunofluorescence. AL binding induced a M2 program in monocytes and macrophages stimulated with C. burnetii as determined by a cDNA chip containing 440 arrayed sequences and functional tests, but this program was in part different in monocytes and macrophages. While monocytes that had bound AL released high levels of IL-10 and IL-6, low levels of TNF and increased CD14 expression, macrophages that had bound AL released high levels of TGF-beta1 and expressed mannose receptor. The neutralization of IL-10 and TGF-beta1 prevented the replication of C. burnetii due to the binding of AL, suggesting that they were critically involved in bacterial replication. In contrast, the binding of necrotic cells to monocytes and macrophages led to C. burnetii killing and typical M1 polarization. Finally, interferon-gamma corrected the immune deactivation induced by apoptotic cells: it prevented the replication of C. burnetii and re-directed monocytes and macrophages toward a M1 program, which was deleterious for C. burnetii. We suggest that leukocyte apoptosis associated with valvulopathy may be critical for the pathogenesis of Q fever endocarditis by deactivating immune cells and creating a favorable environment for bacterial persistence.     

Effects of oxygen on biofilm formation and the AtlA autolysin of Streptococcus mutans

The Streptococcus mutans atlA gene encodes an autolysin required for biofilm maturation and biogenesis of a normal cell surface. We found that the capacity to form biofilms by S. mutans, one of the principal causative agents of dental caries, was dramatically impaired by growth of the organism in an aerated environment and that cells exposed to oxygen displayed marked changes in surface protein profiles. Inactivation of the atlA gene alleviated repression of biofilm formation in the presence of oxygen. Also, the formation of long chains, a characteristic of AtlA-deficient strains, was less evident in cells grown with aeration. The SMu0629 gene is immediately upstream of atlA and encodes a product that contains a C-X-X-C motif, a characteristic of thiol-disulfide oxidoreductases. Inactivation of SMu0629 significantly reduced the levels of AtlA protein and led to resistance to autolysis. The SMu0629 mutant also displayed an enhanced capacity to form biofilms in the presence of oxygen compared to that of the parental strain. The expression of SMu0629 was shown to be under the control of the VicRK two-component system, which influences oxidative stress tolerance in S. mutans. Disruption of vicK also led to inhibition of processing of AtlA, and the mutant was hyperresistant to autolysis. When grown under aerobic conditions, the vicK mutant also showed significantly increased biofilm formation compared to strain UA159. This study illustrates the central role of AtlA and VicK in orchestrating growth on surfaces and envelope biogenesis in response to redox conditions.     

Interaction of culture-derived macrophages with the fibroblast-binding domain of fibronectin is a necessary but inefficient signal for fibronectin enhancement of CR1-mediated phagocytosis

Human plasma fibronectin (Fn) enhances ingestion of opsonized particles through its interaction with phagocytic cells. To better characterize the site or sites on Fn responsible for this effect, we subjected Fn to limited proteolytic cleavage by chymotrypsin and used affinity and gel filtration chromatography to isolate a 110,000 dalton cell-binding fragment, a 60,000 dalton fragment that bound both heparin and gelatin, and 50,000 and 45,000 dalton fragments that bound to gelatin but not heparin. The cell-binding fragment mediated adhesion and spreading of fibroblasts on glass slides, whereas the heparin-gelatin and gelatin-binding fragments failed to cause fibroblast spreading. At high concentrations, the cell-binding fragment doubled phagocytosis of C4b-coated sheep erythrocytes by human monocyte-derived macrophages, whereas equal concentrations of the other fragments had minimal enhancing effect on phagocytosis. Interestingly, the effect of the cell-binding fragment on CR1-mediated phagocytosis was always less than the effect of intact Fn, even when a 40-fold higher molar concentration of the cell-binding fragment was used. Fab of a monoclonal anti-Fn, HFn 7.1, which recognizes the 110,000 dalton cell-binding fragment of Fn and inhibits fibroblast binding, blocked enhancement of CR1-mediated phagocytosis by intact Fn. Fab of Fn 8, a monoclonal anti-Fn that binds the heparin-gelatin-binding fragment, failed to inhibit the Fn effect. These data suggest that interaction of the macrophage with the cell-binding domain of Fn is critical for the Fn effect on CR1-mediated phagocytosis. In addition, there may be other domains of the Fn molecule that have a role in augmenting the Fn-phagocyte interaction.     

Relapse of polymicrobial endocarditis in an intravenous drug user

A 26-year-old male intravenous drug user (IDU) presented twice within 6 months with relapsed polymicrobial infective endocarditis (IE) due to Eikenella corrodens and Streptococcus constellatus after completing two courses of appropriate antimicrobial therapy. This report points to relapsing endocarditis as a clinical entity that warrants attention in IDUs when E. corrodens or S. constellatus are causative agents of IE.     

Complete genome sequence of the serotype k Streptococcus mutans strain LJ23

Streptococcus mutans is the major pathogen of dental caries and occasionally causes infective endocarditis. Here we report the complete genome sequence of serotype k S. mutans strain LJ23, which was recently isolated from the oral cavity of a Japanese patient.