Environmental Acidification Drives S. pyogenes Pilus Expression and Microcolony Formation on Epithelial Cells in a FCT-Dependent Manner

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen responsible for a diverse variety of diseases, including pharyngitis, skin infections, invasive necrotizing fasciitis and autoimmune sequelae. We have recently shown that GAS cell adhesion and biofilm formation is associated with the presence of pili on the surface of these bacteria. GAS pilus proteins are encoded in the FCT (Fibronectin- Collagen-T antigen) genomic region, of which nine different variants have been identified so far. In the present study we undertook a global analysis of GAS isolates representing the majority of FCT-variants to investigate the effect of environmental growth conditions on their capacity to form multicellular communities. For FCT-types 2, 3, 5 and 6 and a subset of FCT-4 strains, we observed that acidification resulting from fermentative sugar metabolism leads to an increased ability of the bacteria to form biofilm on abiotic surfaces and microcolonies on epithelial cells. The higher biofilm forming capacity at low environmental pH was directly associated with an enhanced expression of the genes encoding the pilus components and of their transcription regulators. The data indicate that environmental pH affects the expression of most pilus types and thereby the formation of multicellular cell-adhering communities that assist the initial steps of GAS infection

Functional characterization of Streptococcus pyogenes pili

Group A Streptococcus is a Gram-positive human pathogen able to colonize both upper respiratory tract and skin. GAS is responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year (Cunningham et al., 2000). As other bacteria, GAS infections requires the capacity of the pathogen to adhere to host tissues and to form cell aggregates. The ability to persist in distinct host niches like the throat and the skin and to trigger infections is associated with the expression of different GAS virulence factors. GAS pili has been described as important virulence factors encoded by different FCT-operon regions. Based on this information, we decided to study the possible effect of environmental conditions that could regulate the pili expression. In this study we reported the influence of pH environment variations in biofilm formation for strains pertaining to a panel of different GAS FCT-types. The biofilm formation was promoted, excepted in the FCT-1 strains, by a changing in pH from physiological to acidic condition of growth in in vitro biofilm assay. By analyzing the possible association between biofilm formation and pH dependence, we have found that in FCT-2 and FCT-3 strains, the biofilm is promoted by pH reduction leading to an increase of pili expression. These data confirmed a direct link between pH dependent pilus expression and biofilm formation in GAS. As pili are a multi component structure we decided to investigate the functional role of one of its subunits, the AP-1 protein. AP-1 is highly conserved through the different FCT-types and suggests a possible essential role for the pili function. We focused our attention on the AP-1 protein encoded by the FCT-1 strains (M6). In particular this AP-1 protein contains the von Willebrand Factor A (VWFA) domain, which share an homology with the human VWFA domain that has been reported to be involved in adhesion process. We have demonstrated that the AP-1 protein binds to human epithelial cells by its VWFA domain, whereas the biofilm formation is mediated by the N-terminal region of AP-1 protein. Moreover, analyzing the importance of AP-1 in in vivo experiments we found a major capacity of tissue dissemination for the wild-type strain compared to the isogenic AP-1 deletion mutant. Pili have been also reported as potential vaccine candidates against Gram positive bacteria. For these reason we decided to investigate the relationship between cross reaction of sera raised against different GAS and GBS pilin subunits and the presence of a conserved Cna_B domain, in different pilin components. Our idea was to investigate if, using pilus conserved domains, a broad coverage vaccine against streptococcal infection could be possible

Involvement of heat shock proteins in Candida albicans biofilm formation

Hsp90 has been pursued as a target for the treatment of fungal diseases. Here we investigated the involvement of hsp90 in C. albicans biofilm. Biofilm formation was greatly reduced upon low levels of Hsp90 inhibitors 17-DMAG treatment. Further qRT-PCR experiments confirmed that hsp genes are overexpressed during the initial stage of biofilm formation. Our results suggest that hsp proteins could have a role in C.albicans biofilm development

Protective Activity of the CnaBE3 Domain Conserved among Staphylococcus aureus Sdr Proteins

Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond

Trypsin digestion of full length SdrE protein and sequence analysis of the 37 kDa resistant fragment.

<p>A) SDS-PAGE analysis of an overnight trypsin digestion of the full length SdrE protein dialyzed either in 1 mM CaCl₂ or in 1 mM EDTA. B) The C-terminal region of the full length SdrE protein is schematically depicted. The N-terminal sequence of the 37kDa resistant fragment obtained by Edman degradation (TPKYSLGDY) is shown, together with the peptides derived by trypsin digestion (black bars), and identified by analyzing the MALDI-TOF MS spectrum reported in panel C. “T” indicates trypsin autocatalytic fragment.</p

CnaBE3 domain sequence is highly conserved among phylogenetically distinct strains.

<p>A) The depicted phylogenetic tree was obtained using the Sequence Types (ST) of a panel of 59 epidemiologically relevant <i>S. aureus</i> strains. Clonal complexes 1, 5, 8 and 30 are highlighted. The eleven bacterial strains selected for conservation analysis are in bold. B) The percentages of amino acid sequence identity obtained from the comparison of Sdr proteins and the CnaBC2, D5 and E3 domain amino acid sequences of Newman strain to those of an epidemiologically relevant panel of <i>S. aureus</i> strains are reported. Dark gray color means that proteins are absent, whereas white color means present and conserved with an identity percentage ≥ 90, and light gray color means present but variable with an identity percentage ≥ 75 and ≤ 89, on at least 75% of the amino acid sequence.</p

CnaBE3 domain promotes significant bacterial load reduction in mice challenged with Newman strain and with NCTC8325 strain defective for sdrE gene.

<p>Immunized mice were intravenously infected with either <i>Staphylococcus aureus</i> Newman (N = 54, 4 independent experiments) or NCTC8325 (sdrE negative) strains (N = 48, 3 independent experiments). A) Both, mice immunized with CnaBE3 domain or SdrE protein showed a significant reduction in bacterial load in kidneys when infected with <i>S. aureus</i> Newman strain if compared to adjuvant alone immunized mice used as negative control. B) Mice immunized with CnaBE3 domain show a significant reduction in bacterial load when challenged with NCTC8325 strain as compared to adjuvant alone immunized animals. Each dot represents a single mouse, and geometric means are reported. Statistical analysis was performed using a Mann–Whitney U test (*<i>p</i>≤0.05, **<i>p</i>≤0.01, ns means not significant).</p

Antibodies against CnaBE3 and SdrE mediate opsonophagocytic killing of <i>S. aureus</i>.

<p>Sera of mice immunized either with CnaBE3 domain or with SdrE full length protein at dilution of 1:500, rabbit complement, human promyelocytic leukemia cells HL-60, and the <i>S. aureus</i> strain Newman were incubated for 1 h and plated on TSA for CFU counting. No bacterial killing was observed in the absence of serum, HL-60 cells, complement, or in presence of control serum (preimmune serum), showing the specificity of both CnaBE3 and SdrE antisera in mediating opsonophagocytic killing of the pathogen. Error bars represent standard deviation. Statistical analysis was performed by paired t test (*<i>p</i>≤0.05, ns means not significant). For calculation of the killing percentage see the material and method section.</p

CnaBE3 domain is recognized by sera of <i>S. aureus</i> infected patients.

<p>The immune reactivity of a panel of 30 human sera collected from <i>S. aureus</i> infected patients was tested against the CnaBE3 domain by ELISA assay. A panel of 46 sera collected from healthy donors was used as control. Anti-CnaBE3 IgG titers of sera collected from the patients were significantly higher than those of sera collected from healthy donors. Each dot represents a single serum, and geometric means are reported. Statistical analysis was performed with a Mann–Whitney U test. Values are expressed in lnAU (natural logarithm of Arbitrary Units), for the calculation method see the material and method section (*<i>p</i>≤0.05).</p

Schematic representation of Sdr proteins and amino acid sequence of CnaBC2 CnaBD5 and CnaBE3 domains.

<p>A) Schematic representation of Sdr proteins. A putative leader peptide (LP) sequence and an LPXTG motif are depicted in black. The A domain is reported in light gray, whereas B repeats (two, three, or five, for SdrC, SdrE, and SdrD, respectively) are shown in white, and contain putative CnaB domains shown in dark gray. Finally, at the C-terminus, the SD repeat domain is depicted in dark gray. In addition, boundaries of CnaBC2, CnaBD5 and CnaBE3 domains are reported. B) CnaBC2, CnaBD5 and CnaBE3 domain amino acid sequences are aligned. Identical residues are highlighted, and putative CnaB domains are encompassed by a black box.</p