Although the pili of Gram-positive bacteria are putative virulence factors, little is known about their structure. Here we describe the molecular architecture of pilus-1 of Streptococcus pneumoniae, which is a major cause of morbidity and mortality worldwide. One major (RrgB) and two minor components (RrgA and RrgC) assemble into the pilus. Results from TEM and scanning transmission EM show that the native pili are approximately 6 nm wide, flexible filaments that can be over 1 lm long. They are formed by a single string of RrgB monomers and have a polarity defined by nose-like protrusions. These protrusions correlate to the shape of monomeric RrgB-His, which like RrgA-His and RrgC-His has an elongated, multi-domain structure. RrgA and RrgC are only present at the opposite ends of the pilus shaft, compatible with their putative roles as adhesin and anchor to the cell wall surface, respectively. Our structural analyses provide the first direct experimental evidence that the native S. pneumoniae pilus shaft is composed exclusively of covalently linked monomeric RrgB subunits oriented head-to-tail.

Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.MenB vaccine | selected reaction monitoring mass spectrometry | SRM-MS | fHbp antigen | antigen density

bNeisseria meningitidis is an obligate human commensal that commonly colonizes the oropharyngeal mucosa. Carriage is age dependent and very common in young adults. The relationships between carriage and invasive disease are not completely understood. In this work, we performed a longitudinal carrier study in adolescents and young adults (173 subjects). Overall, 32 subjects (18.5%) had results that were positive for meningococcal carriage in at least one visit (average monthly carriage rate, 12.1%). Only five subjects tested positive at all four visits. All meningococcal isolates were characterized by molecular and serological techniques. Multilocus sequence typing, PorA typing, and sequencing of the 4CMenB vaccine antigens were used to assess strain diversity. The majority of positive subjects were colonized by capsule null (34.4%) and capsular group B strains (28.1%), accounting for 23.5% and 29.4% of the total number of isolates, respectively. The fHbp and nhba genes were present in all isolates, while the nadA gene was present in 5% of the isolates. The genetic variability of the 4CMenB vaccine antigens in this collection was relatively high compared with that of other disease-causing strain panels. Indications about the persistence of the carriage state were limited to the time span of the study. All strains isolated from the same subject were identical or cumulated minor changes over time. The expression levels and antigenicities of the 4CMenB vaccine antigens in each strain were analyzed by the meningococcal antigen typing system (MATS), which revealed that expression can change over time in the same individual. Future analysis of antigen variability and expression in carrier strains after the introduction of the MenB vaccine will allow for a definition of its impact on nasopharyngeal/oropharyngeal carriage.

The Streptococcus pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. In vitro and in vivo assays have demonstrated that pilus-1 is involved in attachment to epithelial cells and virulence, as well as protection in mouse models of infection. Several reports suggest that pilus-1 expression is tightly regulated and involves the interplay of numerous genetic regulators, including the PI-1 positive regulator RlrA. In this report we provide evidence that pilus expression, when analyzed at the single-cell level in PI-1 positive strains, is biphasic. In fact, the strains present two phenotypically different sub-populations of bacteria, one that expresses the pilus, while the other does not. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Finally, we provide evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria. Overall, the data presented here suggest that the observed biphasic pilus expression phenotype could be an example of bistability in pneumococcus.