During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism.
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
The complement system is a crucial component of the innate immune response against invading bacterial pathogens. The human pathogen Neisseria meningitidis (Nm) is known to possess several mechanisms to evade the complement system, including binding to complement inhibitors. In this study, we describe an additional mechanism used by Nm to evade the complement system and survive in human blood. Using an isogenic NalP deletion mutant and NalP complementing strains, we show that the autotransporter protease NalP cleaves C3, the central component of the complement cascade. The cleavage occurs 4 aa upstream from the natural C3 cleavage site and produces shorter C3a-like and longer C3b-like fragments. The C3b-like fragment is degraded in the presence of the complement regulators (factors H and I), and this degradation results in lower deposition of C3b on the bacterial surface. We conclude that NalP is an important factor to increase the survival of Nm in human serum.serum resistance | immune evasion
Neisseria meningitidis is the major cause of septicemia and meningococcal meningitis. During the course of infection, the bacterium must adapt to different host environments as a crucial factor for survival and dissemination; in particular, one of the crucial factors in N. meningitidis pathogenesis is the ability to grow and survive in human blood. We recently showed that N. meningitidis alters the expression of 30% of the open reading frames (ORFs) of the genome during incubation in human whole blood and suggested the presence of fine regulation at the gene expression level in order to control this step of pathogenesis. In this work, we used a customized tiling oligonucleotide microarray to define the changes in the whole transcriptional profile of N. meningitidis in a time course experiment of ex vivo bacteremia by incubating bacteria in human whole blood and then recovering RNA at different time points. The application of a newly developed bioinformatic tool to the tiling array data set allowed the identification of new transcripts-small intergenic RNAs, cis-encoded antisense RNAs, mRNAs with extended 5= and 3= untranslated regions (UTRs), and operons-differentially expressed in human blood. Here, we report a panel of expressed small RNAs, some of which can potentially regulate genes involved in bacterial metabolism, and we show, for the first time in N. meningitidis, extensive antisense transcription activity. This analysis suggests the presence of a circuit of regulatory RNA elements used by N. meningitidis to adapt to proliferate in human blood that is worthy of further investigation.
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