Dynamic Changes in the Streptococcus pneumoniae Transcriptome during Transition from Biofilm Formation to Invasive Disease upon Influenza A Virus Infection

Abstract

f Streptococcus pneumoniae is a leading cause of infectious disease globally. Nasopharyngeal colonization occurs in biofilms and precedes infection. Prior studies have indicated that biofilm-derived pneumococci are avirulent. However, influenza A virus (IAV) infection releases virulent pneumococci from biofilms in vitro and in vivo. Triggers of dispersal include IAV-induced changes in the nasopharynx, such as increased temperature (fever) and extracellular ATP (tissue damage). We used whole-transcriptome shotgun sequencing (RNA-seq) to compare the S. pneumoniae transcriptome in biofilms, bacteria dispersed from biofilms after exposure to IAV, febrile-range temperature, or ATP, and planktonic cells grown at 37°C. Compared with biofilm bacteria, actively dispersed S. pneumoniae, which were more virulent in invasive disease, upregulated genes involved in carbohydrate metabolism. Enzymatic assays for ATP and lactate production confirmed that dispersed pneumococci exhibited increased metabolism compared to those in biofilms. Dispersed pneumococci also upregulated genes associated with production of bacteriocins and downregulated colonization-associated genes related to competence, fratricide, and the transparent colony phenotype. IAV had the largest impact on the pneumococcal transcriptome. Similar transcriptional differences were also observed when actively dispersed bacteria were compared with avirulent planktonic bacteria. Our data demonstrate complex changes in the pneumococcal transcriptome in response to IAV-induced changes in the environment. Our data suggest that disease is caused by pneumococci that are primed to move to tissue sites with altered nutrient availability and to protect themselves from the nasopharyngeal microflora and host immune response. These data help explain pneumococcal virulence after IAV infection and have important implications for studies of S. pneumoniae pathogenesis.

Directed vaccination against pneumococcal disease

“…Through the combination of an in vitro biofilm model ( Fig. 2A) and transcriptional analysis of the bacterial populations comprising this model (23,36), antigens were identified that were significantly up-regulated in biofilm-released pneumococci demonstrating increased virulence. In addition to providing target antigens, the biofilm model served another key purpose in this study: Namely, S. pneumoniae is a human pathogen that, with the exception of a few strains, cannot cause invasive disease in mice.…”

“…Namely, antigen targets up-regulated in biofilm-released bacteria were prepared as recombinant proteins and tested for protection relative to the well-established S. pneumoniae surface protein antigen (PspA), which is one of the best-studied protein protective vaccine candidates and also is up-regulated during virulence transition ( Fig. 2E) (36)(37)(38). Under these conditions, all antigens except DexB showed protection comparable to PspA, with two antigens, GlpO (an α-glycerophosphate oxidase) and PncO (a bacteriocin ABC transporter transmembrane protein), demonstrating promising individual protection surpassing that of PspA.…”

Viral–bacterial interactions in the respiratory tract

“…Influenza neuraminidase cleaves sialic acid from sialylated airway mucins enabling pneumococcal strains that are able to metabolize these sugars to increase their rates of division (Siegel et al, 2014). Moreover, elevated temperature and extracellular ATP occurring during viral infection can trigger the release of pneumococci from biofilms and induce changes in the bacterial transcriptome, associated with improved bacterial stress responses, altered metabolism and increased virulence (Marks et al, 2013;Pettigrew et al, 2014). Viral infections differentially impact the formation and maintenance of P. aeruginosa biofilms: while the release of extracellular iron and transferrin stimulates biofilm formation after RSV infection, the release of hydrogen peroxide during HRV infection triggers the release of P. aeruginosa and facilitates transmigration of bacteria through the epithelial layer and might thus contribute to the dissemination of infection (Chattoraj et al, 2011b;Hendricks et al, 2016).…”

Viral/bacterial respiratory tract co-infections in children

“…pneumoniae and S. pneumoniae after direct binding to RSV, both of which were associated with increased pneumococcal virulence in mice (128,134). …”

“…Temperature increases modelling fever have been found to trigger the expression of immune evasion factors in the opportunistic pathogen Neisseria meningitidis (132). Fever and other host cell damage signals induced by IAV infection have been shown to stimulate S. pneumoniae biofilm dispersal, leading to bacteria with an increased ability to disseminate from the nasopharynx of mice, resulting in infection of sites such as the lung and the blood (133,134). Corroborating these studies, Leung et al (135) found the microbiome of IAV positive patients contained significantly higher expression levels of genes involved in cell motility and signal transduction, such as chemotaxis and flagellum assembly, compared to those of IAV negative pneumonia patients.…”