Amyloids have been identified as functional components of the extracellular matrix of bacterial biofilms. Streptococcus mutans is an established aetiologic agent of dental caries and a biofilm dweller. In addition to the previously identified amyloidogenic adhesin P1 (also known as AgI/II, PAc), we show that the naturally occurring antigen A derivative of S. mutans wall-associated protein A (WapA) and the secreted protein SMU_63c can also form amyloid fibrils. P1, WapA and SMU_63c were found to significantly influence biofilm development and architecture, and all three proteins were shown by immunogold electron microscopy to reside within the fibrillar extracellular matrix of the biofilms. We also showed that SMU_63c functions as a negative regulator of biofilm cell density and genetic competence. In addition, the naturally occurring C-terminal cleavage product of P1, C123 (also known as AgII), was shown to represent the amyloidogenic moiety of this protein. Thus, P1 and WapA both represent sortase substrates that are processed to amyloidogenic truncation derivatives. Our current results suggest a novel mechanism by which certain cell surface adhesins are processed and contribute to the amyloidogenic capability of S. mutans. We further demonstrate that the polyphenolic small molecules tannic acid and epigallocatechin-3-gallate, and the benzoquinone derivative AA-861, which all inhibit amyloid fibrillization of C123 and antigen A in vitro, also inhibit S. mutans biofilm formation via P1-and WapA-dependent mechanisms, indicating that these proteins serve as therapeutic targets of anti-amyloid compounds.

We introduced pairs of cysteine residues into the passenger sequence of pertactin, an AT virulence protein from Bordetella pertussis, and show that OM secretion of the passenger domain stalls due to the formation of a disulphide bond. We further show that the C-terminus of the pertactin passenger domain b-helix crosses the OM first, followed by the N-terminal portions of the virulence protein. In vivo proteolytic digestion shows that the C-terminus is exposed to the extracellular milieu during stalling, and forms stable structure. These AT secretion and folding features can potentially facilitate efficient secretion.

Summary Autotransporter (AT) proteins are the largest class of extracellular virulence proteins secreted from Gram-negative bacteria. The mechanism by which AT proteins cross the bacterial outer membrane (OM), in the absence of ATP or another external energy source, is unknown. Here we demonstrate a linear correlation between localized regions of stability (ΔGfolding) in the mature virulence protein (the AT “passenger”) and OM secretion efficiency. Destabilizing the C-terminal β-helical domain of a passenger reduced secretion efficiency. In contrast, destabilizing the globular N-terminal domain of a passenger produced a linearly correlated increase in secretion efficiency. Thus, C-terminal passenger stability facilitates OM secretion, whereas N-terminal stability hinders it. The contributions of regional passenger stability to OM secretion demonstrate a crucial role for the passenger itself in directing its secretion, suggesting a novel type of ATP-independent, folding-driven transporter.

In the many areas where human malaria and helminthiases are co-endemic, schoolchildren often harbour the heaviest infections and suffer much of the associated morbidity, especially when co-infected. In one such area, the Buea district, in south-western Cameroon, two cross-sectional surveys, together covering 263 apparently healthy schoolchildren aged 4-12 years, were recently conducted. The prevalences of fever, malarial parasitaemia and intestinal helminth infections, the seroprevalences of anti-Plasmodium falciparum IgG and IgE and anti-glycosylphosphatidylinositol (anti-GPI) IgG, plasma concentrations of total IgE, and the incidence of anaemia were all investigated. The mean (S.D.) age of the study children was 7.56 (1.82) years. Overall, 156 (59.3%) of the children were found parasitaemic, with a geometric mean parasitaemia of 565 parasites/microl. Parasitaemia and fever were significantly associated (P=0.042). The children who lived at low altitude, attending schools that lay 400-650 m above sea level, had significantly higher parasitaemias than their high-altitude counterparts (P<0.01). At low altitude, the children attending government schools had significantly higher parasitaemias than their mission-school counterparts (P=0.010). Of the 31 children (11.9%) found anaemic, 22 (70.4%) had mild anaemia and none had severe anaemia. A significant negative correlation (r=-0.224; P=0.005) was observed between haemoglobin concentration and level of parasitaemia. Infection with Plasmodium appeared to reduce erythrocyte counts (P=0.045), a condition that was exacerbated by co-infection with helminths (P=0.035). Plasma concentrations of total IgE were higher in the children found to be excreting helminth eggs than in those who appeared helminth-free, while levels of anti-P. falciparum IgE were higher in the children with low-grade parasitaemias than in those with more intense parasitaemias. Levels of anti-GPI IgG increased with age and were relatively high in the children who lived at low altitude and in those who were aparasitaemic. The survey results confirm that asymptomatic malarial parasitaemia frequently co-exists with helminth infections in schoolchildren and indicate links with fever, altitude and school type. Immunoglobulin E may play a role in immune protection against helminthiasis whereas anti-GPI antibodies may be important in the development of antimalarial immunity in such children. In Cameroon, as in other areas with endemic malaria, control programmes to reduce the prevalences of infections with intestinal helminths and malarial parasites in schoolchildren, which may effectively reduce the incidence of anaemia, are clearly needed.

Background: P1 is an adhesin on the surface of Streptococcus mutans. Results: Adhesive P1 on the surface of S. mutans exhibits a macromolecular ultrastructure. Conclusion:The architecture of P1 on the surface of S. mutans plays a critical role in adherence. Significance: Recognizing the macromolecular assembly of P1 on the surface of S. mutans is critical to understanding the adhesive function of the molecule.