Synergism and antagonism of bacterial-viral co-infection in the upper respiratory tract

ABSTRACTStreptococcus pneumoniae (the pneumococcus) is a leading cause of pneumonia in children under five years old. Co-infection by pneumococci and respiratory viruses enhances disease severity. Little is known about pneumococcal co-infections with Respiratory Syncytial Virus (RSV). Here, we developed a novel infant mouse model of co-infection using Pneumonia Virus of Mice (PVM), a murine analogue of RSV, to examine the dynamics of co-infection in the upper respiratory tract, an anatomical niche that is essential for host-to-host transmission and progression to disease. Coinfection increased damage to the nasal tissue and increased production of the chemokine CCL3. Pneumococcal nasopharyngeal density and shedding in nasal secretions were increased by co-infection. In contrast, co-infection reduced PVM loads in the nasopharynx, an effect that was independent of pneumococcal strain and the order of infection. We showed this ‘antagonistic’ effect was abrogated using a pneumococcal mutant deficient in capsule production and incapable of nasopharyngeal carriage. The pneumococcal-mediated reduction in PVM loads was caused by accelerated viral clearance from the nasopharynx. Although these synergistic and antagonistic effects occurred with both wild-type pneumococcal strains used in this study, the magnitude of the effects was strain dependent. Lastly, we showed that pneumococci can also antagonize influenza virus. Taken together, our study has uncovered multiple novel facets of bacterial-viral co-infection. Our findings have important public health implications, including for bacterial and viral vaccination strategies in young children.

Invasive pneumococcal strain distributions and isolate clusters associated with persons experiencing homelessness during 2018

Objectives We aimed to characterize invasive pneumococcal disease (IPD) isolates collected from multistate surveillance in the USA during 2018 and examine within-serotype propensities of isolates to form related clusters. Methods We predicted strain features using whole genome sequence obtained from 2885 IPD isolates obtained through the Center for Disease Control and Prevention’s Active Bacterial Core surveillance (ABCs) that has a surveillance population of approximately 34.5 million individuals distributed among 10 states. Phylogenetic analysis was provided for serotypes accounting for >27 isolates. Results Thirteen-valent conjugate vaccine (PCV13) serotypes together with 6C accounted for 23/105 (21.9%) of isolates from children aged <5 years and 820/2780 (29.5%) isolates from those aged >5 years. The most common serotypes from adult IPD isolates were serotypes 3 (413/2780, 14.9%), 22F (291/2780, 10.5%) and 9N (191/2780, 6.9%). Among children IPD isolates, serotypes 15BC (18/105, 17.1%), 3 (11/105, 10.5%) and 33F (10/105, 9.5%) were most common. Serotypes 4, 12F, 20, and 7F had the highest proportions of isolates that formed related clusters together with highest proportions of isolates from persons experiencing homelessness (PEH). Among 84 isolates from long-term care facilities, two instances of highly related isolate pairs from co-residents were identified. Conclusions Non-PCV13 serotypes accounted for more than 70% of IPD in ABCs, however PCV13 serotype 3 is the most common IPD serotype overall. Serotypes most common among PEH were more often associated with temporally related clusters identified both among PEH and among persons not reportedly experiencing homelessness.

Adenosine Triphosphate Neutralizes Pneumolysin-Induced Neutrophil Activation

Background In tissue infections, adenosine triphosphate (ATP) is released into extracellular space and contributes to purinergic chemotaxis. Neutrophils are important players in bacterial clearance and are recruited to the site of tissue infections. Pneumococcal infections can lead to uncontrolled hyperinflammation of the tissue along with substantial tissue damage through excessive neutrophil activation and uncontrolled granule release. We aimed to investigate the role of ATP in neutrophil response to pneumococcal infections. Methods Primary human neutrophils were exposed to the pneumococcal strain TIGR4 and its pneumolysin-deficient mutant or directly to different concentrations of recombinant pneumolysin. Neutrophil activation was assessed by measurement of secreted azurophilic granule protein resistin and profiling of the secretome, using mass spectrometry. Results Pneumococci are potent inducers of neutrophil degranulation. Pneumolysin was identified as a major trigger of neutrophil activation. This process is partially lysis independent and inhibited by ATP. Pneumolysin and ATP interact with each other in the extracellular space leading to reduced neutrophil activation. Proteome analyses of the neutrophil secretome confirmed that ATP inhibits pneumolysin-dependent neutrophil activation. Conclusions Our findings suggest that despite its cytolytic activity, pneumolysin serves as a potent neutrophil activating factor. Extracellular ATP mitigates pneumolysin-induced neutrophil activation.

Pneumococci Can Become Virulent by Acquiring a New Capsule From Oral Streptococci

Pneumococcal conjugate vaccines have been successful, but their use has increased infections by nonvaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). Although this has not been observed in nature, if pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire. In the current study, we showed that oral Streptococcus strain SK95 and pneumococcal strain D39 both produce structurally identical capsular PS, and their genetic backgrounds influence the amount of capsule production and shielding from nonspecific killing. SK95 is avirulent in a well-established in vivo mouse model. When acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice. Thus, cps from oral Streptococcus strains can make acapsular pneumococcus virulent, and interspecies cps transfer should be considered a potential mechanism of serotype replacement. Our findings, along with publications from the US Centers for Disease Control and Prevention, highlight potential limitations of the 2013 World Health Organization criterion for studying pneumococcal serotypes carried without isolating bacteria. We show that an oral streptococcal strain, SK95, and a pneumococcal strain, D39, both produce chemically identical capsular PS. We also show that transferring SK95 cps into noncapsulated, avirulent pneumococcus gave it the capacity for virulence in a mouse model.

Characterization of Streptococcus pneumoniae phage-like element SpnCI reveals an enhanced virulent phenotype in the acute invertebrate infection model Galleria mellonella

Phage-like elements are found in a multitude of streptococcal species, including pneumococcal strain Hungary19A-6 (SpnCI). The aim of our research was to investigate the role of phage-like element SpnCI in enhanced virulence and phenotypic modulation within Streptococcus pneumoniae. SpnCI was found to significantly enhance virulence within the invertebrate infection model Galleria mellonella. Infections with SpnCI led to a lower mean health score (1.6) and survival percentage (20%) compared to SpnCI null TIGR4 infections (3.85 mean health score and 50% survival). SpnCI remained integrated throughout growth, conferring greater sensitivity to UV irradiation. Change in transcriptional patterns occurred, including downregulation of operons involved with cell surface modelling in the SpnCI containing strain of TIGR4. Kanamycin-tagged SpnCI strain in Hungary19A-6 was inducible and isolated from lysate along with both annotated prophages. No phages were identified by PCR nor electron microscopy (EM) following induction of TIGR4 SpnCIΔstrA suggesting helper-phage dependence for dissemination. EM of lysate showed typical siphoviridae morphology with an average capsid size of 60 nm. Two of sixty capsids were found to be smaller, suggesting SpnCI disseminates using a similar mechanism described for Staphylococcus aureus phage-like element SaPI. SpnCI from lysate infected capsule null strain T4R but was incapable of infecting the encapsulated TIGR4 strain suggesting that capsule impedes phage infection. Our work demonstrates that SpnCI can modulate virulence, UV susceptibility, alter transcriptional patterns, and furthermore, can disseminate via infection within pneumococcus. Further research is necessary to elucidate how SpnCI modulates virulence and what genes are responsible for the enhanced virulence phenotype.ImportanceAlthough vaccines have limited the scope of pneumococcal infections, Streptococcus pneumoniae still remains an important human pathogen. Understanding novel elements, such as SpnCI, that enhance virulence can lead to the development of more targeted therapeutic and diagnostic tools within the clinical realm.

Effectiveness of the 13-valent pneumococcal conjugate vaccine against adult pneumonia in Italy: a case–control study in a 2-year prospective cohort

ObjectivesCurrent strategies to prevent adult pneumococcal disease have been recently reviewed in Italy. We did a postlicensure study to estimate the direct vaccine effectiveness (VE) of the 13-valent pneumococcal conjugate vaccine (PCV13) against adult pneumococcal community-acquired pneumonia (pCAP).Study designBetween 2013 and 2015, a 2-year prospective cohort study of adults with CAP was conducted in the Apulia region of Italy where the average vaccine uptake of PCV13 was 32% among adults ≥65 years. The test-negative design was used to estimate VE against all episodes of confirmed pCAP and vaccine-type (VT)-CAP. VE in a subgroup of patients managed in the community was also estimated using a matched case–control design. VE was calculated as one minus the OR times 100%.ResultsThe overall VE of PCV13 was 33.2% (95% CI −106.6% to 82%) against pCAP irrespective of serotype and 38.1% (95% CI −131.9% to 89%) against VT-CAP in the cohort of adults ≥65 years. The VE was 42.3% (95% CI −244.1% to 94.7%) against VT-CAP in the age group at higher vaccine uptake. For the subgroup of cases managed in the community, the overall VE against disease due to any pneumococcal strain was 88.1% (95% CI 4.2% to 98.5%) and 91.7% (95% CI 13.1% to 99.2%) when we controlled for underlying conditions.ConclusionsAlthough our results are non-significant, PCV13 promises to be effective against all confirmed pCAP already with modest levels of uptake in the population of adults ≥65 years of age. Larger studies are needed to confirm the direct vaccine benefits.

Expression of Toll-Like Receptor 2 by Dendritic Cells Is Essential for the DnaJ-ΔA146Ply-Mediated Th1 Immune Response against Streptococcus pneumoniae

ABSTRACT The fusion protein DnaJ-ΔA146Ply could induce cross-protective immunity against pneumococcal infection via mucosal and subcutaneous immunization in mice in the absence of additional adjuvants. DnaJ and Ply are both Toll-like receptor 4 (TLR4) but not TLR2 ligands. However, we found that TLR2 −/− mice immunized subcutaneously with DnaJ-ΔA146Ply showed significantly lower survival rates and higher bacterial loads in nasal washes than did wild-type (WT) mice after being challenged with pneumococcal strain D39 or 19F. The gamma interferon (IFN-γ) level in splenocytes decreased in TLR2 −/− mice, indicating that Th1 immunity elicited by DnaJ-ΔA146Ply was impaired in these mice. We explored the mechanism of protective immunity conferred by DnaJ-ΔA146Ply and the role of TLR2 in this process. DnaJ-ΔA146Ply effectively promoted dendritic cell (DC) maturation via TLR4 but not the TLR2 signaling pathway. In a DnaJ-ΔA146Ply-treated DC and naive CD4 + T cell coculture system, the deficiency of TLR2 in DCs resulted in a significant decline of IFN-γ production and Th1 subset differentiation. The same effect was observed in adoptive-transfer experiments. In addition, TLR2 −/− DCs showed remarkably lower levels of the Th1-polarizing cytokine IL-12p70 than did WT DCs, suggesting that TLR2 was indispensable for DnaJ-ΔA146Ply-induced IL-12 production and Th1 proliferation. Thus, our findings illustrate that dendritic cell expression of TLR2 is essential for optimal Th1 immune response against pneumococci in mice immunized subcutaneously with DnaJ-ΔA146Ply.

Genetic, Biochemical, and Serological Characterization of a New Pneumococcal Serotype, 6H, and Generation of a Pneumococcal Strain Producing Three Different Capsular Repeat Units

ABSTRACTStreptococcus pneumoniaeclinical isolates were recently described that produced capsular polysaccharide with properties of both serotypes 6A and 6B. Their hybrid serological property correlated with mutations affecting the glycosyltransferase WciP, which links rhamnose to ribitol by an α(1-3) linkage for serotypes 6A and 6C and an α(1-4) linkage for serotypes 6B and 6D. The isolates had mutations in the triad residues of WciP that have been correlated with enzyme specificity. The canonical triad residues of WciP are Ala192-Ser195-Arg254 for serotypes 6A and 6C and Ser192-Asn195-Gly254 for serotypes 6B and 6D. To prove that the mutations in the triad residues are responsible for the hybrid serotype, we introduced the previously described Ala192-Cys195-Arg254 triad into a 6A strain and found that the change made WciP bispecific, resulting in 6A and 6B repeat unit expression, although 6B repeat unit production was favored over production of 6A repeat units. Likewise, this triad permitted a 6C strain to express 6C and 6D repeat units. With reported bispecificity in WciN, which adds either glucose or galactose as the second sugar in the serogroup 6 repeat unit, the possibility exists for a strain to simultaneously produce all four serogroup 6 repeat units; however, when genes encoding both bispecific enzymes were introduced into a 6A strain, only 6A, 6B, and 6D repeat units were detected serologically. Nonetheless, this may be the first example of a bacterial polysaccharide with three different repeat units. This strategy of expressing multiple repeat units in a single polymer is a novel approach to broadening vaccine coverage by eliminating the need for multiple polysaccharide sources to cover multiple serogroup members.

Immunization with a Combination of Three Pneumococcal Proteins Confers Additive and Broad Protection against Streptococcus pneumoniae Infections in Mice

ABSTRACT 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 (ΔA146 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.