Specific Involvement of Pilus Type 2a in Biofilm Formation in Group B Streptococcus

Streptococcus agalactiae is the primary colonizer of the anogenital mucosa of up to 30% of healthy women and can infect newborns during delivery and cause severe sepsis and meningitis. Persistent colonization usually involves the formation of biofilm and increasing evidences indicate that in pathogenic streptococci biofilm formation is mediated by pili. Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands. Here we have investigated the capacity of these strains to form biofilms. We have found that most of the biofilm-formers carry pilus 2a, and using insertion and deletion mutants we have confirmed that pilus type 2a, but not pilus types 1 and 2b, confers biofilm-forming phenotype. We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype. Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells

Protein Array Profiling of Tic Patient Sera Reveals a Broad Range and Enhanced Immune Response against Group A Streptococcus Antigens

The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders

A Novel Computational Method Identifies Intra- and Inter-Species Recombination Events in <em>Staphylococcus aureus</em> and <em>Streptococcus pneumoniae</em>

<div><p>Advances in high-throughput DNA sequencing technologies have determined an explosion in the number of sequenced bacterial genomes. Comparative sequence analysis frequently reveals evidences of homologous recombination occurring with different mechanisms and rates in different species, but the large-scale use of computational methods to identify recombination events is hampered by their high computational costs. Here, we propose a new method to identify recombination events in large datasets of whole genome sequences. Using a filtering procedure of the gene conservation profiles of a test genome against a panel of strains, this algorithm identifies sets of contiguous genes acquired by homologous recombination. The locations of the recombination breakpoints are determined using a statistical test that is able to account for the differences in the natural rate of evolution between different genes. The algorithm was tested on a dataset of 75 genomes of <em>Staphylococcus aureus</em> and 50 genomes comprising different streptococcal species, and was able to detect intra-species recombination events in <em>S. aureus</em> and in <em>Streptococcus pneumoniae</em>. Furthermore, we found evidences of an inter-species exchange of genetic material between <em>S. pneumoniae</em> and <em>Streptococcus mitis</em>, a closely related commensal species that colonizes the same ecological niche. The method has been implemented in an R package, <em>Reco</em>, which is freely available from supplementary material, and provides a rapid screening tool to investigate recombination on a genome-wide scale from sequence data.</p> </div

Recombination in <i>S. aureus</i> CC5.

<p>a) Comparison of <i>S. aureus</i> JH1 (ST5, CC5) strain with all the other <i>S. aureus</i> genomes. b) Filtered data. Red boxes represent the four regions not conserved in most of CC5 genomes, corresponding to phage encoding islands acquired by a JH1 ancestor after the diversification from the other CC5 strains.</p

Recombination in <i>S. aureus</i> ST239.

<p>a) Heatmap representation of the percentage of conservation of the genes of <i>S. aureus</i> TW20 strain (ST239) against all the other strains. Each row represents the percentage of identity of one gene in TW20 in all other strains. Each column represents one reference genome. The origin of replication of TW20 is at the bottom of the figure, and rows are ordered according to the gene order in TW20. Columns are ordered according to a whole-genome phylogenetic tree (upper panel). The color code and a histogram of the percentage of identity of the TW20 genes in the reference genomes are shown in the inset. The blue box identifies a phage shared by TW20 and MRGR3 and not present in other strains. b) Filtered data. Yellow and black dots indicate conserved and non-conserved genes, respectively. White dots indicate missing genes. Red boxes highlight two regions that TW20 acquired from one CC30. Due to the circular nature of the chromosome of <i>S. aureus</i>, these two regions have probably been acquired in a single recombination event.</p

Schema of the artificial recombinant genome.

<p>a) The recombinant genome has been designed identical to a major parent (recipient) and containing segments acquired from donor_1 (red) and donor_2 (blue) in a variable number. b) Recombination events detected for different sizes of the sliding window (top to bottom, <i>l</i> = 10, <i>l</i> = 30, <i>l</i> = 80).</p

Recombination in the <i>S. pneumoniae</i>-<i>S. mitis</i> complex.

<p>a) Comparison of <i>S. pneumoniae</i> Taiwan19F-14 (ST236, CC271) strain against all the other streptococci. b) Filtered data. Red box represents a region of Taiwan19F-14 shared with <i>S. mitis</i> SK564 with a percentage of conservation higher than many strains of <i>S. pneumoniae</i>. This region includes a transposon coding for erythromycin resistance and represents an event of horizontal transfer between two closely related species both colonizing the nasopharynx of human hosts.</p

Recombination events in <i>S. pneumoniae</i> strain INV104B identified using RDP.

<p>a) RDP detected four regions acquired from strain P1031. Strain P1031 belongs to CC217 and it could be the putative donor strain for INV104B. b) <i>Reco</i> grouped the recombination events of INV104B in a single event (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002668#pcbi-1002668-g005" target="_blank">Fig. 5b</a> region (1)).</p

Borders of the recombinant region in ST239.

<p>a) P-values of the Fisher test performed on the filtered matrix obtained from the comparison of TW20 (ST239) against all the other <i>S. aureus</i> strains. b) P-values of the Fisher test of TW20 against 552053 (CC30). Minima in the p-values (green dots) identified the breakpoint positions of the recombination event shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002668#pcbi-1002668-g002" target="_blank">Fig. 2b</a>.</p