Versatility of the capsular genes during biofilm formation by Streptococcus pneumoniae

Streptococcus pneumoniae forms part of the natural microbiota of the nasopharynx. For the pneumococcus to cause infection, colonization needs to occur and this process is mediated by adherence of bacteria to the respiratory epithelium. Although the capsular polysaccharide (CPS) of S. pneumoniae is known to be important for infection to occur, its role in colonization is controversial. Biofilm models are starting to emerge as a promising tool to investigate the role of CPS during nasopharyngeal carriage, which is the first step in the dissemination and initiation of a pneumococcal infection. Using a well-defined model system to analyse in vitro biofilm formation in pneumococcus, here we explore the molecular changes underlying the appearance of capsular mutants using type 3 S. pneumoniae cells. Spontaneous colony phase variants show promoter mutations, as well as duplications, deletions and point mutations in the cap3A gene, which codes for a UDP-glucose dehydrogenase (UDP-GlcDH). Increased biofilm-forming capacity could usually be correlated with a reduction both in colony size and in the relative amount of CPS present on the cell surface of each colony variant. However, a mutation in Cap3A Thr83Ile (a strictly conserved residue in bacterial UDP-GlcDHs) that resulted in very low CPS production also led to impaired biofilm formation. We propose that non-encapsulated mutants of pneumococcal type 3 strains are essentially involved in the initial stages (the attachment stage) of biofilm formation during colonization/pathogenesis.     

Biofilm formation in Streptococcus pneumoniae

Biofilm‐grown bacteria are refractory to antimicrobial agents and show an increased capacity to evade the host immune system. In recent years, studies have begun on biofilm formation by Streptococcus pneumoniae, an important human pathogen, using a variety of in vitro model systems. The bacterial cells in these biofilms are held together by an extracellular matrix composed of DNA, proteins and, possibly, polysaccharide(s). Although neither the precise nature of these proteins nor the composition of the putative polysaccharide(s) is clear, it is known that choline‐binding proteins are required for successful biofilm formation. Further, many genes appear to be involved, although the role of each appears to vary when biofilms are produced in batch or continuous culture. Prophylactic and therapeutic measures need to be developed to fight S. pneumoniae biofilm formation. However, much care needs to be taken when choosing strains for such studies because different S. pneumoniae isolates can show remarkable genomic differences. Multispecies and in vivo biofilm models must also be developed to provide a more complete understanding of biofilm formation and maintenance.     

Phenotypic characterization of Streptococcus pneumoniae biofilm development

Streptococcus pneumoniae is among the most common pathogens associated with chronic otitis media with effusion, which has been hypothesized to be a biofilm disease. S. pneumoniae has been shown to form biofilms, however, little is known about the developmental process, the architecture, and the changes that occur upon biofilm development. In the current study we made use of a continuous-culture biofilm system to characterize biofilm development of 14 different S. pneumoniae strains representing at least 10 unique serotypes. The biofilm development process was found to occur in three distinct stages, including initial attachment, cluster formation, and biofilm maturation. While all 14 pneumococcal strains displayed similar developmental stages, the mature biofilm architecture differed significantly among the serotypes tested. Overall, three biofilm architectural groups were detected based on biomass, biofilm thickness, and cluster size. The biofilm viable cell counts and total protein concentration increased steadily over the course of biofilm development, reaching approximately 8 x 10(8) cells and approximately 15 mg of protein per biofilm after 9 days of biofilm growth. Proteomic analysis confirmed the presence of distinct biofilm developmental stages by the detection of multiple phenotypes over the course of biofilm development. The biofilm development process was found to correlate not only with differential production of proteins but also with a dramatic increase in the number of detectable proteins, indicating that biofilm formation by S. pneumoniae may be a far more complex process than previously anticipated. Protein identification revealed that proteins involved in virulence, adhesion, and resistance were more abundant under biofilm growth conditions. A possible role of the identified proteins in biofilm formation is discussed.     

LuxS-based signaling affects Streptococcus mutans biofilm formation

Streptococcus mutans is implicated as a major etiological agent in human dental caries, and one of the important virulence properties of this organism is its ability to form biofilms (dental plaque) on tooth surfaces. We examined the role of autoinducer-2 (AI-2) on S. mutans biofilm formation by constructing a GS-5 luxS-null mutant. Biofilm formation by the luxS mutant in 0.5% sucrose defined medium was found to be markedly attenuated compared to the wild type. Scanning electron microscopy also revealed that biofilms of the luxS mutant formed larger clumps in sucrose medium compared to the parental strain. Therefore, the expression of glucosyltransferase genes was examined and the gtfB and gtfC genes, but not the gtfD gene, in the luxS mutant were upregulated in the mid-log growth phase. Furthermore, we developed a novel two-compartment system to monitor AI-2 production by oral streptococci and periodontopathic bacteria. The biofilm defect of the luxS mutant was complemented by strains of S. gordonii, S. sobrinus, and S. anginosus; however, it was not complemented by S. oralis, S. salivarius, or S. sanguinis. Biofilm formation by the luxS mutant was also complemented by Porphyromonas gingivalis 381 and Actinobacillus actinomycetemcomitans Y4 but not by a P. gingivalis luxS mutant. These results suggest that the regulation of the glucosyltransferase genes required for sucrose-dependent biofilm formation is regulated by AI-2. Furthermore, these results provide further confirmation of previous proposals that quorum sensing via AI-2 may play a significant role in oral biofilm formation.     

Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA

The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.     

PrgB promotes aggregation,biofilm formation,and conjugation through DNA binding and compaction

Gram‐positive bacteria deploy type IV secretion systems (T4SSs) to facilitate horizontal gene transfer. The T4SSs of Gram‐positive bacteria rely on surface adhesins as opposed to conjugative pili to facilitate mating. Enterococcus faecalis PrgB is a surface adhesin that promotes mating pair formation and robust biofilm development in an extracellular DNA (eDNA) dependent manner. Here, we report the structure of the adhesin domain of PrgB. The adhesin domain binds and compacts DNA in vitro. In vivo PrgB deleted of its adhesin domain does not support cellular aggregation, biofilm development and conjugative DNA transfer. PrgB also binds lipoteichoic acid (LTA), which competes with DNA binding. We propose that PrgB binding and compaction of eDNA facilitates cell aggregation and plays an important role in establishment of early biofilms in mono‐ or polyspecies settings. Within these biofilms, PrgB mediates formation and stabilization of direct cell‐cell contacts through alternative binding of cell‐bound LTA, which in turn promotes establishment of productive mating junctions and efficient intra‐ or inter‐species T4SS‐mediated gene transfer.     

Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes

Viridans streptococci, which include Streptococcus gordonii, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation of S. gordonii Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn916 transposon mutagenesis for defective biofilm formation. Biofilms formed optimally when bacteria were grown in a minimal medium under anaerobic conditions. Biofilm formation was affected by changes in pH, osmolarity, and carbohydrate content of the growth media. Eighteen biofilm-defective mutants of S. gordonii Challis were identified based on Southern hybridization with a Tn916-based probe and DNA sequences of the Tn916-flanking regions. Molecular analyses of these mutants showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion. These characteristics are associated with quorum sensing, osmoadaptation, and adhesion functions in oral streptococci. Only nine of the biofilm-defective mutants had defects in genes of known function, suggesting that novel aspects of bacterial physiology may play a part in biofilm formation. Further identification and characterization of biofilm-associated genes will provide insight into the molecular mechanisms of biofilm formation of oral streptococci.     

钦州市自然流产人群肺炎链球菌携带率研究

目的探讨钦州市自然流产人群肺炎链球菌携带率状况与自然流产的相关性。方法构建基于肺炎链球菌特征目标基因lyt和ply基因的实时荧光PCR检测体系,检测自然流产人群肺炎链球菌携带率,并与常规孕检的对照组进行比较。结果自然流产组人群的肺炎链球菌特征基因检出率为15.26%,对照组的肺炎链球菌特征基因检出率为15.09%,两组比较差异无统计学意义(P0.05)。结论肺炎链球菌感染与自然流产不存在相关性,不是引起流产的主要因素,但建议孕期个体应注意预防肺炎链球菌感染,避免由此带来的不良结局。     

LuxS-mediated signalling in Streptococcus anginosus and its role in biofilm formation

The autoinducer-2 signal (AI-2) produced by several Gram-positive and Gram-negative bacteria mediates interspecies communication. In this study we were able to identify an orthologue of luxS, required for the synthesis of AI-2 signals, in Streptococcus anginosus. Comparative analyses revealed conserved sequences in the predicted S. anginosus LuxS. Expression of luxS was highest during early exponential growth phase. Compared to other oral streptococci, conditioned media from growth of members of the anginosus group were the most efficient in inducing bioluminescence in Vibrio harveyi, indicative of AI-2 signalling. Disruption of luxS in S. anginosus resulted in a mutant deficient in biofilm formation, whereas no effect on planktonic growth rate was observed under various growth conditions. S. anginosus is part of the human flora found in biofilms of the oral cavity, as well as of the upper respiratory, gastrointestinal and urogenital tracts. Such habitats harbour large varieties of bacterial species, among which cell–cell communication may␣play an important role. S. anginosus has also been associated with purulent infections and cancer in the upper digestive tract. Knowledge about the molecular mechanisms involved in S.␣anginosus communication is important for understanding its commensalism and its pathogenic transition.     

Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids

A hybrid plasmid, pJS37, was made by combining pLS1, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJS140 (6.0 kb), from which pC194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4 (3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLS1. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome-binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool.     

PLA2R1 promotes DNA damage and inhibits spontaneous tumor formation during aging

Although aging is a major risk factor for most types of cancers, it is barely studied in this context. The transmembrane protein PLA2R1 (phospholipase A2 receptor) promotes cellular senescence, which can inhibit oncogene-induced tumor initiation. Functions and mechanisms of action of PLA2R1 during aging are largely unknown. In this study, we observed that old Pla2r1 knockout mice were more prone to spontaneously develop a wide spectrum of tumors compared to control littermates. Consistently, these knockout mice displayed increased Parp1, a master regulator of DNA damage repair, and decreased DNA damage, correlating with large human dataset analysis. Forced PLA2R1 expression in normal human cells decreased PARP1 expression, induced DNA damage and subsequent senescence, while the constitutive expression of PARP1 rescued cells from these PLA2R1-induced effects. Mechanistically, PARP1 expression is repressed by a ROS (reactive oxygen species)-Rb-dependent mechanism upon PLA2R1 expression. In conclusion, our results suggest that PLA2R1 suppresses aging-induced tumors by repressing PARP1, via a ROS–Rb signaling axis, and inducing DNA damage and its tumor suppressive responses.Subject terms: Cancer, Mechanisms of disease, DNA damage and repair, Ageing     

Competence-dependent endogenous DNA rearrangement and uptake of extracellular DNA give a natural variant of Streptococcus mutans without biofilm formation

The production of water-insoluble glucan (WIG) enables Streptococcus mutans to survive and persist in the oral niche. WIG is produced from sucrose by glucosyltransferase encoded tandemly by the highly homologous gtfB and gtfC genes. Conversely, a single hybrid gene from the endogenous recombination of gtfB and gtfC is easily generated using RecA, resulting in S. mutans UA159 WIG- (rate of ～1.0×10(-3)). The pneumococcus recA gene is regulated as a late competence gene. comX gene mutations did not lead to the appearance of WIG- cells. The biofilm collected from the flow cell had more WIG- cells than among the planktonic cells. Among the planktonic cells, WIG- cells appeared after 16 h and increased ～10-fold after 32 h of cultivation, suggesting an increase in planktonic WIG- cells after longer culture. The strain may be derived from the biofilm environment. In coculture with donor WIG+ and recipient WIG- cells, the recipient cells reverted to WIG+ and acquired an intact gtfBC region from the environment, indicating that the uptake of extracellular DNA resulted in the phenotypic change. Here we demonstrate that endogenous DNA rearrangement and uptake of extracellular DNA generate WIG- cells and that both are induced by the same signal transducer, the com system. Our findings may help in understanding how S. mutans can adapt to the oral environment and may explain the evolution of S. mutans.