Transcriptional regulation of capsule expression is critical for pneumococcal transition from carriage to infection, yet the underlying mechanism remains incompletely understood. Here, we describe the regulation of capsular polysaccharide, one of the most important pneumococcal virulence factor by a GntR family regulator, CpsR. Electrophoretic mobility-shift assays have shown the direct interaction between CpsR and the cps promoter (cpsp), and their interaction could be competitively interfered by glucose. DNase I footprinting assays localized the binding site to a region −146 to −114 base pairs relative to the transcriptional start site of the cps locus in S. pneumoniae D39. We found that CpsR negatively controlled the transcription of the cps locus and hence CPS production, which was confirmed by fine-tuning expression of CpsR in a ΔcpsR complemented strain. Increased expression of CpsR in complemented strain led to a decreased resistance to the whole-blood-mediated killing, suggesting a protective role for CpsR-cpsp interaction in the establishment of invasive infection. Finally, animal experiments showed that CpsR-cpsp interaction was necessary for both pneumococcal colonization and invasive infection. Taken together, our results provide a thorough insight into the regulation of capsule production mediated by CpsR and its important roles in pneumococcal pathogenesis.

Pneumococcal polysaccharide-based vaccines are effective in preventing pneumococcus infection; however, some drawbacks preclude their widespread use in developing and undeveloped countries. Here, we evaluated the protective effects of ATP-dependent caseinolytic protease (ClpP), pneumolysin mutant (⌬A146 Ply), putative lipoate-protein ligase (Lpl), or combinations thereof against pneumococcal infections in mice. Vaccinated mice were intraperitoneally and/or intranasally challenged with different pneumococcal strains. In intraperitoneal challenge models with pneumococcal strain D39 (serotype 2), the most striking protection was obtained with the combination of the three antigens. Similarly, with the intranasal challenge models, (i) additive clearance of bacteria in lungs was observed for the combination of the three antigens and (ii) a combination vaccine conferred complete protection against intranasal infections of three of the four most common pneumococcal strains (serotypes 14, 19F, and 23F) and 80% protection for pneumococcal strain 6B. Even so, immunity to this combination could confer protection against pneumococcal infection with a mixture of four serotypes. Our results showed that the combination vaccine was as effective as the currently used vaccines (PCV7 and PPV23). These results indicate that system immunization with the combination of pneumococcal antigens could provide an additive and broad protection against Streptococcus pneumoniae in pneumonia and sepsis infection models.Streptococcus pneumoniae (pneumococcus) commonly colonizes the upper respiratory tract asymptomatically and was estimated, in 2005, to kill 1.6 million people every year, most of whom were children aged Ͻ5 years in developing and undeveloped countries (36). As far as we know, 91 capsular polysaccharide serotypes have been identified in S. pneumoniae (33); among these, serotypes 23F, 19F, 14, and 6B are the four most epidemic strains worldwide (2,5,15,17,25,26,29). Moreover, and of recent concern, the widespread use of antibiotics, leading to the development of antibiotic resistance or multidrug resistance against S. pneumoniae, is increasing (9,12,26).Heptavalent protein-polysaccharide conjugate vaccine (PCV7) and 23-valent pneumococcal polysaccharide vaccine (PPV23) are the two vaccines currently being used against S. pneumoniae. Both of these vaccines are polysaccharide-based formulations and effective in preventing invasive pneumococcal infections; however, some drawbacks, such as high cost, the limited polysaccharides covered, poor immunogenicity in the very young and the very old, and serotype replacement (22,24,26,36), limit their wider use.Alternatively, in an attempt to overcome the disadvantages of polysaccharide-based vaccines, a number of studies have been focusing on the screening and evaluation of proteinbased vaccine candidates. Pneumococcal protein vaccine candidates, such as nontoxic pneumolysin derivates, pneumococcal surface proteins (PspA and PspC), pneumococcal surface adhesin (PsaA), and ATP-dependent caseino...

Pneumolysin (Ply) and its variants are protective against pneumococcal infections in animal models, and as a Toll-like receptor 4 agonist, pneumolysin has been reported to be a mucosal adjuvant. DnaJ has been approved as a useful candidate vaccine protein; we therefore designed novel fusion proteins of DnaJ with a form of Ply that has a deletion of A146 (⌬A146Ply-DnaJ [the C terminus of ⌬A146Ply connected with the N terminus of DnaJ] and DnaJ-⌬A146Ply [the C terminus of DnaJ connected with the N terminus of ⌬A146Ply]) to test whether they are protective against focal and lethal pneumococcal infections and their potential protective mechanisms. The purified proteins were used to intranasally immunize the animals without additional adjuvant. Immunization with DnaJ-⌬A146Ply or DnaJ plus ⌬A146Ply (Ply with a single deletion of A146) could significantly reduce S. pneumoniae colonization in the nasopharynx and lung relative with DnaJ alone. Additionally, we observed the best protection for DnaJ⌬A146Ply-immunized mice after challenge with lethal doses of S. pneumoniae strains, which was comparable to that achieved by PPV23. Mice immunized with DnaJ-⌬A146Ply produced significantly higher levels of anti-DnaJ IgG in serum and secretory IgA (sIgA) in saliva than those immunized with DnaJ alone. The production of IL-17A was also striking in DnaJ-⌬A146Ply-immunized mice. IL-17A knockout (KO) mice did not benefit from DnaJ-⌬A146Ply immunization in colonization experiments, and sIgA production was impaired in IL-17A KO mice. Collectively, our results indicate a mucosal adjuvant potential for ⌬A146Ply and that, without additional adjuvant, DnaJ-⌬A146Ply fusion protein exhibits extensive immune stimulation and is effective against pneumococcal challenges, properties which are partially attributed to the IL-17A-mediated immune responses.

bMucosal immunization with attenuated vaccine can protect against pneumococcal invasion infection, but the mechanism was unknown. Our study found that mucosal delivery with the live attenuated SPY1 vaccine strain can confer T cell-and B cell-dependent protection against pneumococcal colonization and invasive infection; yet it is still unclear which cell subsets contribute to the protection, and their roles in pneumococcal colonization and invasion remain elusive. Adoptive transfer of anti-SPY1 antibody conferred protection to naive MT mice, and immune T cells were indispensable to protection examined in nude mice. A critical role of interleukin 17A (IL-17A) in colonization was demonstrated in mice lacking IL-17A, and a vaccine-specific Th2 immune subset was necessary for systemic protection. Of note, we found that SPY1 could stimulate an immunoregulatory response and that SPY1-elicited regulatory T cells participated in protection against colonization and lethal infection. The data presented here aid our understanding of how live attenuated strains are able to function as effective vaccines and may contribute to a more comprehensive evaluation of live vaccines and other mucosal vaccines. Vaccination is an indispensable strategy to prevent infection caused by Streptococcus pneumoniae (S. pneumoniae), which is estimated to lead to a mortality rate of more than 50 deaths in every 1,000 births in children under 5 years of age in some countries (1). The commercially available 23-valent polysaccharide vaccine contains the most common serotypes that cause pneumococcal infection and is effective in adults but fails to protect children of less than 2 years of age, who are most vulnerable to pneumococcal infection. The recent extensive introduction of conjugated capsular polysaccharide vaccine (PCV) has drastically decreased the child morbidity and mortality caused by strains of S. pneumoniae expressing capsular serotypes included in the vaccine. However, the serotype coverage of PCV is limited, and a growing body of evidence showed that PCV could induce selective pressure and gradual replacement with nonvaccine serotypes (serotype replacement) (2, 3). The conjugated vaccine is also very expensive and is complex in design, making more difficult its application in the low-income countries that have the highest burden of S. pneumoniae infections (4).As a consequence of these shortcomings with the commercially available S. pneumoniae vaccines, other approaches have been explored, including protein antigen vaccines, killed whole-cell S. pneumoniae vaccines, or attenuated live S. pneumoniae vaccines (5, 6). The wide range of antigenic molecules present in live attenuated vaccines promises that the immune responses they induce are likely to be multiple and powerful and may also more closely mimic those obtained in natural infection than immune responses to a vaccine using a subcomponent or killed bacteria (7). Some live bacterial vaccines have been clinically used, including the Mycobacterium bovis BCG (8) and vaccines for prev...

BackgroundDue to the widespread abusage of antibiotics, antibiotic-resistance in Streptococcus pneumoniae (S. pneumoniae) has been increasing quickly in recent years, and it is obviously urgent to develop new types of antibiotics. Two-component systems (TCSs) are the major signal transduction pathways in bacteria and have emerged as potential targets for antibacterial drugs. Among the 13 pairs of TCSs proteins presenting in S. pneumoniae, VicR/K is the unique one essential for bacterium growth, and block agents to which, if can be found, may be developed as effective antibiotics against S. pneumoniae infection.ResultsUsing a structure-based virtual screening (SBVS) method, 105 compounds were computationally identified as potential inhibitors of the histidine kinase (HK) VicK protein from the compound library SPECS. Six of them were then validated in vitro to be active in inhibiting the growth of S. pneumoniae without obvious cytotoxicity to Vero cell. In mouse sepsis models, these compounds are still able to decrease the mortality of the mice infected by S. pneumoniae and one compound even has significant therapeutic effect.ConclusionTo our knowledge, these compounds are the first reported inhibitors of HK with antibacterial activity in vitro and in vivo, and are novel lead structures for developing new drugs to combat pneumococcal infection.

Objectives:To re-evaluate the epidemiology of hand, foot, and mouth disease (HFMD) in a non-vaccinated population in Zunyi, China.Methods:We used laboratory-based data from the Third Affiliated Hospital of Zunyi Medical College, Zunyi, China to assess the epidemiology of the HFMD caused by enteroviruses between January 2012 and November 2014. Real-time polymerase chain reaction was used to determine human enteroviruses from a total of 12313 probable cases enrolled in this retrospective study. All analyses were stratified by enterovirus serotype, gender, and age.Results:Virological results were available for 12313 cases of probable HFMD. A total of 5750 cases were positive for viral detection, and the positive rates of infection caused by other enteroviruses was 46.7%, EV71 9%, and CVA16 4.7%. During the study period there was a substantial increase in the occurrence of HFMD. Most of the HFMD patients (87.4%) were aged 0-59 months (median 24; range 0-59). Males showed a higher HFMD prevalence rate (62.4%) than females (37.6%).Conclusion:Enterovirus infection remains an important public health problem and other entroviruses are emerging as the major causative agent of the HFMD in Zunyi, China.

bTeichoic acid (TA), together with peptidoglycan (PG), represents a highly complex glycopolymer that ensures cell wall integrity and has several crucial physiological activities. Through an insertion-deletion mutation strategy, we show that ⌬rafX mutants are impaired in cell wall covalently attached TA (WTA)-PG biosynthesis, as evidenced by their abnormal banding patterns and reduced amounts of WTA in comparison with wild-type strains. Site-directed mutagenesis revealed an essential role for external loop 4 and some highly conserved amino acid residues in the function of RafX protein. The rafX gene was highly conserved in closely related streptococcal species, suggesting an important physiological function in the lifestyle of streptococci. Moreover, a strain D39 ⌬rafX mutant was impaired in bacterial growth, autolysis, bacterial division, and morphology. We observed that a strain R6 ⌬rafX mutant was reduced in adhesion relative to the wild-type R6 strain, which was supported by an inhibition assay and a reduced amount of CbpA protein on the ⌬rafX mutant bacterial cell surface, as shown by flow cytometric analysis. Finally, ⌬rafX mutants were significantly attenuated in virulence in a murine sepsis model. Together, these findings suggest that RafX contributes to the biosynthesis of WTA, which is essential for full pneumococcal virulence.

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