Glycan-based interactions of Streptococcus pneumoniae and the host

Streptococcus pneumoniae is commonly found as an asymptomatic colonizer of the nasopharynx of children, but it can also translocate to normally sterile body sites and cause severe diseases, like pneumonia, septicemia or meningitis. Pneumococci spread via aerosols. Upon entry into the upper respiratory tract of the host, glycoconjugates with terminal sialic acids (Sias) are among the first structures pneumococci encounter. Hence, they play an important role in pneumococcal pathogenesis. Moreover, glycans are also implicated in the recognition of microbial pathogens by the innate immune system, as many ligands of Tolllike receptors are glycoconjugates. Both aspects of glycan-based pneumococcal-host interactions were studied in this thesis. In most mammals, the Sia N-acetylneuraminic acid (Neu5Ac) is converted into Nglycolylneuraminic acid (Neu5Gc) by the cytidine-monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). However, humans lack Neu5Gc due to a deletion in CMAH, instead they overproduce Neu5Ac. We reported a faster disease progression in Cmah-/- versus wildtype (wt) mice after pneumococcal challenge and an upregulation of pneumococcal sialidase NanA and the main sialic acid transporter SatABC in response to Neu5Ac as compared with Neu5Gc, which was mediated by the response regulator CiaR. Moreover, we detected higher pneumococcal adhesion rates to cells presenting Neu5Ac than Neu5Gc. In vitro, higher bacterial adherence downregulated IL-8 secretion, and in vivo, pneumococcal pyruvate oxidase (SpxB) and pneumolysin contributed to a reduced immune response in Cmah-/- compared with wt mice after intranasal challenge. Influenza infections lead to changes in the pulmonary environment and sensitize for a pneumococcal infection. We observed higher protein concentrations, increased numbers of dead cells as well as upregulated hydrogen peroxide concentrations in bronchoalveolar lavages of influenza- versus mock-infected mice. The increased virus-mediated stress in the lower respiratory tract mediated an upregulation of the pneumococcal serine protease HtrA during influenza/pneumococcal coinfection. A mutant of HtrA was severely attenuated in a murine coinfection model, suggesting an important role of HtrA in pneumococcal outgrowth following primary influenza infection. Dendritic cells link the innate with the adaptive immune system. We found an RNA-mediated recognition of pneumococci by TLR3 in dendritic cells, which induced the secretion of the cytokine IL-12. Moreover, in influenza/pneumococcal coinfections, the virus upregulated TLR3 expression, which led to an enhanced production of IL-12 by dendritic cells. In summary, we show that glycan-mediated interactions of S. pneumoniae and the host play a major role in pneumococcal host tropism and strongly affect pneumococcal virulence, as well as innate immune responses

Proton Motive Force Disruptors Block Bacterial Competence and Horizontal Gene Transfer

Streptococcus pneumoniae is a commensal of the human nasopharynx that can also cause severe antibiotic-resistant infections. Antibiotics drive the spread of resistance by inducing S. pneumoniae competence, in which bacteria express the transformation machinery that facilitates uptake of exogenous DNA and horizontal gene transfer (HGT). We performed a high-throughput screen and identified potent inhibitors of S. pneumoniae competence, called COM-blockers. COM-blockers limit competence by inhibiting the proton motive force (PMF), thereby disrupting export of a quorum-sensing peptide that regulates the transformation machinery. Known chemical PMF disruptors and alterations in pH homeostasis similarly inhibit competence. COM-blockers limit transformation of clinical multi-drug-resistant strains and HGT in infected mice. At their active concentrations, COM-blockers do not affect growth, compromise antibiotic activity, or elicit detectable resistance. COM-blockers provide an experimental tool to inhibit competence and other PMF-involved processes and could help reduce the spread of virulence factors and antibiotic resistance in bacteria. VIDEO ABSTRACT.</p

Pilus Adhesin RrgA Interacts with Complement Receptor 3, Thereby Affecting Macrophage Function and Systemic Pneumococcal Disease

10.1128/mBio.00535-12Pneumococcal pili have been shown to influence pneumococcal colonization, disease development, and the inflammatory response in mice. The role of the pilus-associated RrgA adhesin in pneumococcal interactions with murine and human macrophages was investigated. Expression of pili with RrgA enhanced the uptake of pneumococci by murine and human macrophages that was abolished by antibodies to complement receptor 3 (CR3) and not seen in CR3-deficient macrophages. Recombinant RrgA, but not pilus subunit RrgC, promoted CR3-mediated phagocytosis of coated beads by murine and human macrophages. Flow cytometry showed that purified CR3 binds pneumococcal cells expressing RrgA, and purified RrgA was shown to interact with CR3 and its I domain. In vivo, RrgA facilitated spread of pneumococci from the upper airways and peritoneal cavity to the bloodstream. Earlier onset of septicemia and more rapidly progressing disease was observed in wild-type mice compared to CR3-deficient mice challenged intranasally or intraperitoneally with pneumococci. Motility assays and time-lapse video microscopy showed that pneumococcal stimulation of macrophage motility required RrgA and CR3. These findings, together with the observed RrgA-dependent increase of intracellular survivors up to 10 h following macrophage infection, suggest that RrgA-CR3-mediated phagocytosis promotes systemic pneumococcal spread from local sites.IMPORTANCE Streptococcus pneumoniae is a major contributor to morbidity and mortality in infectious diseases globally. Symptomatology is mainly due to pneumococcal interactions with host cells leading to an inflammatory response. However, we still need more knowledge on how pneumococci talk to immune cells and the importance of this interaction. Recently, a novel structure was identified on the pneumococcal surface, an adhesive pilus found in about 30% of clinical pneumococcal isolates. The pilus has been suggested to be important for successful spread of antibiotic-resistant pneumococcal clones globally. Here we sought to identify mechanisms for how the pneumococcal pilin subunit RrgA contributes to disease development by interacting with host immune cells. Our data suggest a new way for how pneumococci may cross talk with phagocytic cells and affect disease progression. An increased understanding of these processes may lead to better strategies for how to treat these common infections.Peer reviewe

The basic keratin 10-binding domain of the virulence-associated pneumococcal serine-rich protein PsrP adopts a novel MSCRAMM fold.

International audienceStreptococcus pneumoniae is a major human pathogen, and a leading cause of disease and death worldwide. Pneumococcal invasive disease is triggered by initial asymptomatic colonization of the human upper respiratory tract. The pneumococcal serine-rich repeat protein (PsrP) is a lung-specific virulence factor whose functional binding region (BR) binds to keratin-10 (KRT10) and promotes pneumococcal biofilm formation through self-oligomerization. We present the crystal structure of the KRT10-binding domain of PsrP (BR187-385) determined to 2.0 Å resolution. BR187-385 adopts a novel variant of the DEv-IgG fold, typical for microbial surface components recognizing adhesive matrix molecules adhesins, despite very low sequence identity. An extended β-sheet on one side of the compressed, two-sided barrel presents a basic groove that possibly binds to the acidic helical rod domain of KRT10. Our study also demonstrates the importance of the other side of the barrel, formed by extensive well-ordered loops and stabilized by short β-strands, for interaction with KRT10

Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method

Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections