[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Biofilms are thought to play an important role during colonization of the nasopharynx by Streptococcus pneumoniae, yet how they form in vivo and the determinants responsible remain unknown. Using scanning electron microscopy, we show that biofilm aggregates of increasing complexity form on murine nasal septa following intranasal inoculation. These biofilms were highly distinct from in vitro biofilms, as they were discontiguous and appeared to incorporate nonbacterial components such as intact host cells. Biofilms initially formed on the surface of ciliated epithelial cells and, as cells were sloughed off, were found on the basement membrane. The size and number of biofilm aggregates within nasal lavage fluid were digitally quantitated and revealed strain-specific capabilities that loosely correlated with the ability to form robust in vitro biofilms. We tested the ability of isogenic mutants deficient in CbpA, pneumolysin, hydrogen peroxide, LytA, LuxS, CiaR/H, and PsrP to form biofilms within the nasopharynx. This analysis revealed that CiaR/H was absolutely required for colonization, that PsrP and SpxB strongly impacted aggregate formation, and that other determinants affected aggregate morphology in a modest fashion. We determined that mice colonized with ΔpsrP mutants had greater levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-1β, and KC in nasal lavage fluid than did mice colonized with wild-type controls. This phenotype correlated with a diminished capacity of biofilm pneumococci to invade host cells in vitro despite enhanced attachment. Our results show that biofilms form during colonization and suggest that they may contribute to persistence through a hyperadhesive, noninvasive state that elicits a dampened cytokine response.
This work demonstrates the first temporal characterization of Streptococcus pneumoniae biofilm formation in vivo. Our results show that the morphology of biofilms formed by both invasive and noninvasive clinical isolates in vivo is distinct from that of formed biofilms in vitro, yet propensity to form biofilms in vivo loosely correlates with the degree of in vitro biofilm formation on a microtiter plate. We show that host components, including intact host cells, influence the formation of in vivo structures. We also found that efficient biofilm formation in vivo requires multiple bacterial determinants. While some factors are essential for in vivo biofilm formation (CiaRH, PsrP, and SpxB), other factors are less critical (CbpA, LytA, LuxS, and pneumolysin). In comparison to their planktonic counterparts, biofilm pneumococci are hyperadhesive but less invasive and elicit a weaker proinflammatory cytokine response. These findings give insight into the requirements for and potential role of biofilms during prolonged asymptomatic colonization.
[Show abstract][Hide abstract] ABSTRACT: Bacterial attachment to host surfaces is a pivotal event in the biological and infectious processes of both commensal and pathogenic bacteria, respectively. Serine-rich repeat proteins (SRRPs) are a family of adhesins in Gram-positive bacteria that mediate attachment to a variety of host and bacterial surfaces. As such, they contribute towards a wide-range of diseases including sub-acute bacterial endocarditis, community-acquired pneumonia, and meningitis. SRRPs are unique in that they are glycosylated, require a non-canonical Sec-translocase for transport, and are largely composed of a domain containing hundreds of alternating serine residues. These serine-rich repeats are thought to extend a unique non-repeat (NR) domain outward away from the bacterial surface to mediate adhesion. So far, NR domains have been determined to bind to sialic acid moieties, keratins, or other NR domains of a similar SRRP. This review summarizes how this important family of bacterial adhesins mediates bacterial attachment to host and bacterial cells, contributes to disease pathogenesis, and might be targeted for pharmacological intervention or used as novel protective vaccine antigens. This review also highlights recent structural findings on the NR domains of these proteins.
[Show abstract][Hide abstract] ABSTRACT: It is unclear whether Streptococcus pneumoniae in biofilms are virulent and contribute to development of invasive pneumococcal disease (IPD). Using electron microscopy we confirmed the development of mature pneumococcal biofilms in a continuous-flow-through line model and determined that biofilm formation occurred in discrete stages with mature biofilms composed primarily of dead pneumococci. Challenge of mice with equal colony forming units of biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Biofilm pneumococci of numerous serotypes were hyper-adhesive and bound to A549 type II pneumocytes and Detroit 562 pharyngeal epithelial cells at levels 2 to 11-fold greater than planktonic counterparts. Using genomic microarrays we examined the pneumococcal transcriptome and determined that during biofilm formation S. pneumoniae down-regulated genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide (CPS) production, and virulence. We confirmed these changes by measuring CPS by ELISA and immunoblotting for the toxin pneumolysin and the bacterial adhesins phosphorylcholine (ChoP), choline-binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP). We conclude that biofilm pneumococci were avirulent due to reduced CPS and pneumolysin production along with increased ChoP, which is known to bind C-reactive protein and is opsonizing. Likewise, biofilm pneumococci were hyper-adhesive due to selection for the transparent phase variant, reduced CPS, and enhanced production of PsrP, CbpA, and ChoP. These studies suggest that biofilms do not directly contribute to development of IPD and may instead confer a quiescent mode of growth during colonization.
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pneumoniae (the pneumococcus) is the leading cause of otitis media, community-acquired pneumonia (CAP), sepsis, and meningitis. It is now evident that S. pneumoniae forms biofilms during nasopharyngeal colonization; the former which facilitates persistence, the latter, a prerequisite for subsequent development of invasive disease. Proteomic evaluation of S. pneumoniae suggests the antigen profile available for host-recognition is altered as a consequence of biofilm growth. This has potentially meaningful implications in regards to adaptive immunity and protection from disseminated disease. We therefore examined the antigen profile of biofilm and planktonic pneumococcal cell lysates, tested their reactivity with human convalescent sera and that generated against biofilm pneumococci, and examined whether immunization with biofilm pneumococci protected mice against infectious challenge.
Biofilm pneumococci have dramatically altered protein profiles versus their planktonic counterparts. During invasive disease the humoral immune response is skewed towards the planktonic protein profile. Immunization with biofilm bacteria does not elicit a strong-cross-reactive humoral response against planktonic bacteria nor confer resistance against challenge with a virulent isolate from another serotype. We identified numerous proteins, including Pneumococcal serine-rich repeat protein (PsrP), which may serve as a protective antigens against both colonization and invasive disease.
Differential protein production by planktonic and biofilm pneumococci provides a potential explanation for why individuals remain susceptible to invasive disease despite previous colonization events. These findings also strongly suggest that differential protein production during colonization and disease be considered during the selection of antigens for any future protein vaccine.
[Show abstract][Hide abstract] ABSTRACT: Cryptococcus neoformans typically grows in a yeast-like morphology; however, under specific conditions the fungus can produce hyphae that are either dikaryotic or monokaryotic. In this study, we developed a simple method for inducing robust monokaryotic fruiting and combined the assay with Agrobacterium tumefaciens insertional mutagenesis to screen for hyphal mutants. A C. neoformans homologue of the Saccharomyces cerevisiae STE50 gene was identified and characterized. STE50 was found to be required for sexual reproduction and monokaryotic fruiting. Ste50p has conserved SAM and RA domains, as well as two SH3 domains specific to basidiomycetous Ste50 proteins. Analysis of protein-protein interaction showed that Ste50p can interact with Ste11p and Ste20p, and epistasis experiments placed STE50 between STE20 and STE11. Genetic analysis of the role of STE50 in sexual reproduction showed that it was required for all steps, from response to pheromone to production of hyphae. Analysis of the effect of individual Ste50p domains on sexual reproduction and monokaryotic fruiting revealed domain-specific effects for both processes. This study revealed that the C. neoformans STE50 gene has both conserved and novel functions during sexual reproduction and monokaryotic fruiting, and these functions are domain-dependent.
Full-text · Article · Feb 2011 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Biofilm formation has been suggested to play an important role during Streptococcus pneumoniae nasopharyngeal colonization and may facilitate progression to pneumonia. To test whether the ability of S. pneumoniae to form biofilms was important for virulence we screened the ability of 30 invasive and 22 non-invasive clinical isolates of serotype 6A and 6B to form early biofilms on polystyrene microtiter plates and infect mice following intranasal and intratracheal challenge. We first determined that no correlation existed between the ability to form early biofilms and whether isolates were collected from healthy carriers or individuals with invasive disease. A disconnect between biofilm forming ability and the capacity to colonize the nasopharynx, cause pneumonia, and enter the bloodstream was also observed in mice. Importantly, S. pneumoniae mutants deficient in the established virulence determinants pneumolysin, CbpA, and hydrogen peroxide formed biofilms normally. Incidentally, we determined that robust biofilm production was dependent on the formation and coalescing of bacterial aggregates on a thin layer of bacteria attached to the plate surface. In summary, these studies suggest that the ability to form early biofilms in vitro does not reflect virulence potential. More complex studies are required to determine if biofilm formation is important for virulence.
Full-text · Article · Mar 2010 · Microbial Pathogenesis