Claudia Trappetti

Switch from planktonic to sessile life : a major event in pneumococcal pathogenesis

Two main patterns of gene expression of Streptococcus pneumoniae were observed during infection in the host by quantitative real time RT‐PCR; one was characteristic of bacteria in blood and one of bacteria in tissue, such as brain and lung. Gene expression in blood was characterized by increased expression of pneumolysin, pspA and hrcA, while pneumococci in tissue infection showed increased expression of neuraminidases, metalloproteinases, oxidative stress and competence genes. In vitro situations with similar expression patterns were detected in liquid culture and in a newly developed pneumococcal model of biofilm respectively. The biofilm model was dependent on addition of synthetic competence stimulating peptide (CSP) and no biofilm was formed by CSP receptor mutants. As one of the differentially expressed gene sets in vivo were the competence genes, we exploited competence‐specific tools to intervene on pneumococcal virulence during infection. Induction of the competence system by the quorum‐sensing peptide, CSP, not only induced biofilm formation in vitro, but also increased virulence in pneumonia in vivo. In contrast, a mutant for the ComD receptor, which did not form biofilm, also showed reduced virulence in pneumonia. These results were opposite to those found in a bacteraemic sepsis model of infection, where the competence system was downregulated. When pneumococci in the different physiological states were used directly for challenge, sessile cells grown in a biofilm were more effective in inducing meningitis and pneumonia, while planktonic cells from liquid culture were more effective in inducing sepsis. Our data enable us, using in vivo gene expression and in vivo modulation of virulence, to postulate the distinction – from the pneumococcal point of view – between two main types of disease. During bacteraemic sepsis pneumococci resemble planktonic growth, while during tissue infection, such as pneumonia or meningitis, pneumococci are in a biofilm‐like state.

Sialic acid: a preventable signal for pneumococcal biofilm formation, colonization, and invasion of the host.

The correlation between carbohydrate availability, pneumococcal biofilm formation, nasopharyngeal colonization, and invasion of the host has been investigated. Of a series of sugars, only sialic acid (i.e., N-acetylneuraminic acid) enhanced pneumococcal biofilm formation in vitro, at concentrations similar to those of free sialic acid in human saliva. In a murine model of pneumococcal carriage, intranasal inoculation of sialic acid significantly increased pneumococcal counts in the nasopharynx and instigated translocation of pneumococci to the lungs. Competition of both sialic acid-dependent phenotypes was found to be successful when evaluated using the neuraminidase inhibitors DANA (i.e., 2,3-didehydro-2-deoxy-N-acetylneuraminic acid), zanamivir, and oseltamivir. The association between levels of free sialic acid on mucosae, pneumococcal colonization, and development of invasive disease shows how a host-derived molecule can influence a colonizing microbe and also highlights a molecular mechanism that explains the epidemiologic correlation between respiratory infections due to neuraminidase-bearing viruses and bacterial pneumonia. The data provide a new paradigm for the role of a host compound in infectious diseases and point to new treatment strategies.

A random six-phase switch regulates pneumococcal virulence via global epigenetic changes

Streptococcus pneumoniae (the pneumococcus) is the world’s foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or ‘phases’) that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.

LuxS Mediates Iron-Dependent Biofilm Formation, Competence, and Fratricide in Streptococcus pneumoniae

ABSTRACT During infection, Streptococcus pneumoniae exists mainly in sessile biofilms rather than in planktonic form, except during sepsis. The capacity to form biofilms is believed to be important for nasopharyngeal colonization as well as disease pathogenesis, but relatively little is known about the regulation of this process. Here, we investigated the effect of exogenous iron [Fe(III)] as well as the role of luxS (encoding S-ribosylhomocysteine lyase) on biofilm formation by S. pneumoniae D39. Fe(III) strongly enhanced biofilm formation at concentrations of ≥50 μM, while Fe(III) chelation with deferoxamine was inhibitory. Importantly, Fe(III) also upregulated the expression of luxS in wild-type D39. A luxS-deficient mutant (D39luxS) failed to form a biofilm, even with Fe(III) supplementation, whereas a derivative overexpressing luxS (D39luxS+) exhibited enhanced biofilm formation capacity and could form a biofilm without added Fe(III). D39luxS exhibited reduced expression of the major Fe(III) transporter PiuA, and the cellular [Fe(III)] was significantly lower than that in D39; in contrast, D39luxS+ had a significantly higher cellular [Fe(III)] than the wild type. The release of extracellular DNA, which is an important component of the biofilm matrix, also was directly related to luxS expression. Similarly, genetic competence, as measured by transformation frequency as well as the expression of competence genes comD, comX, comW, cglA, and dltA and the murein hydrolase cbpD, which is associated with fratricide-dependent DNA release, all were directly related to luxS expression levels and were further upregulated by Fe(III). Moreover, mutagenesis of cbpD blocked biofilm formation. We propose that competence, fratricide, and biofilm formation are closely linked in pneumococci, and that luxS is a central regulator of these processes. We also propose that the stimulatory effects of Fe(III) on all of these parameters are due to the upregulation of luxS expression, and that LuxS provides for a positive Fe(III)-dependent amplification loop by increasing iron uptake.

Antibacterial Activity of a Competence-Stimulating Peptide in Experimental Sepsis Caused by Streptococcus pneumoniae

ABSTRACT Streptococcus pneumoniae, a major cause of human disease, produces a 17-mer autoinducer peptide pheromone (competence-stimulating peptide [CSP]) for the control of competence for genetic transformation. Due to previous work linking CSP to stress phenotypes, we set up an in vivo sepsis model to assay its effect on virulence. Our data demonstrate a significant increase in the rates of survival of mice, reductions of blood S. pneumoniae counts, and prolonged times to death for mice treated with CSP. In vitro the dose of CSP used in the animal model produced a transitory inhibition of growth. When a mutant with a mutation in the CSP sensor histidine kinase was assayed, no bacteriostatic phenotype was detected in vitro and no change in disease outcome was observed in vivo. The data demonstrate that CSP, which induces in vitro a temporary growth arrest through stimulation of its cognate histidine kinase receptor, is able to block systemic disease in mice. This therapeutic effect is novel, in that the drug-like effect is obtained by stimulation, rather than inhibition, of a bacterial drug target.

Streptococcus pneumoniae Uses Glutathione To Defend against Oxidative Stress and Metal Ion Toxicity

The thiol-containing tripeptide glutathione is an important cellular constituent of many eukaryotic and prokaryotic cells. In addition to its disulfide reductase activity, glutathione is known to protect cells from many forms of physiological stress. This report represents the first investigation into the role of glutathione in the Gram-positive pathogen Streptococcus pneumoniae. We demonstrate that pneumococci import extracellular glutathione using the ABC transporter substrate binding protein GshT. Mutation of gshT and the gene encoding glutathione reductase (gor) increases pneumococcal sensitivity to the superoxide generating compound paraquat, illustrating the importance of glutathione utilization in pneumococcal oxidative stress resistance. In addition, the gshT and gor mutant strains are hypersensitive to challenge with the divalent metal ions copper, cadmium, and zinc. The importance of glutathione utilization in pneumococcal colonization and invasion of the host is demonstrated by the attenuated phenotype of the gshT mutant strain in a mouse model of infection.

Extracellular matrix formation enhances the ability of Streptococcus pneumoniae to cause invasive disease.

During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100-fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease.

Identification of Genes That Contribute to the Pathogenesis of Invasive Pneumococcal Disease by In Vivo Transcriptomic Analysis

ABSTRACT Streptococcus pneumoniae (the pneumococcus) continues to be responsible for a high level of global morbidity and mortality resulting from pneumonia, bacteremia, meningitis, and otitis media. Here we have used a novel technique involving niche-specific, genome-wide in vivo transcriptomic analyses to identify genes upregulated in distinct niches during pathogenesis after intranasal infection of mice with serotype 4 or 6A pneumococci. The analyses yielded 28 common, significantly upregulated genes in the lungs relative to those in the nasopharynx and 25 significantly upregulated genes in the blood relative to those in the lungs in both strains, some of which were previously unrecognized. The role of five upregulated genes from either the lungs or the blood in pneumococcal pathogenesis and virulence was then evaluated by targeted mutagenesis. One of the mutants (ΔmalX) was significantly attenuated for virulence in the lungs, two (ΔaliA and ΔilvH) were significantly attenuated for virulence in the blood relative to the wild type, and two others (ΔcbiO and ΔpiuA) were completely avirulent in a mouse intranasal challenge model. We also show that the products of aliA, malX, and piuA are promising candidates for incorporation into multicomponent protein-based pneumococcal vaccines currently under development. Importantly, we suggest that this new approach is a viable complement to existing strategies for the discovery of genes critical to the distinct stages of invasive pneumococcal disease and potentially has broad application for novel protein antigen discovery in other pathogens such as S. pyogenes, Haemophilus influenzae type b, and Neisseria meningitidis.

The impact of the competence quorum sensing system on Streptococcus pneumoniae biofilms varies depending on the experimental model

BackgroundDifferent models for biofilm in Streptococcus pneumoniae have been described in literature. To permit comparison of experimental data, we characterised the impact of the pneumococcal quorum-sensing competence system on biofilm formation in three models. For this scope, we used two microtiter and one continuous culture biofilm system.ResultsIn both microtiter models the competence system influences stability and structure of biofilm in the late attachment phase and synthetic competence stimulating peptide (CSP) restored wild type phenotypes in the comC mutants unable to produce the peptide. Early attachment of single cells to well bottoms was found for both systems to be competence independent, while later phases, including microcolony formation correlated to an intact competence system. The continuous culture biofilm model was not affected by mutations in the competence locus, but deletion of capsule had a significant impact in this model.ConclusionsSince biofilm remains a largely uncharacterised multi-parameter phenotype it appears to be advisable to exploit more than one model in order to draw conclusion of possible relevance of specific genotypes on pneumococcal physiology.

Sex-Based Differences in Susceptibility to Respiratory and Systemic Pneumococcal Disease in Mice

Systemic infection with Streptococcus pneumoniae was investigated in male and female mice in models of invasive pneumonia and sepsis. Male mice were found to be more susceptible to infection, exhibiting greater weight loss, marked decrease in body temperature, and a significantly higher mortality rate compared with female mice. For pneumonia, there were significant differences in survival rates. Female mice cleared their lung infections over time, whereas male mice, compared with female mice, had significantly increased numbers of colony-forming units in early stages of infection accompanied by higher levels of neutrophil recruitment in the first 24 hours after infection. Importantly, there were significant increases in proinflammatory cytokine levels during both sepsis and pneumonia in male compared with female mice. These cytokines were indicative of T-helper 1-type responses. The data presented here describe surprising differences in survival rates, neutrophil recruitment, and proinflammatory cytokine levels, indicating a sex-based difference in susceptibility to respiratory and systemic pneumococcal disease.