Transcriptome Analysis of Neisseria meningitidis in Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in Blood Survival

Abstract

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.

Neisseria meningitidis NalP cleaves human complement C3, facilitating degradation of C3b and survival in human serum

“…The infrequency of the nalP deletion suggests that it is disadvantageous for the bacteria and that, in the nalP-negative strain, the function could be compensated by other factors or other mechanisms. This hypothesis recalls the redundancy in fH binding by Nm, where strains lacking fHbp can use NspA to bind fH (13,14) to avoid complement activation on bacterial surface.…”

“…In a previous study of Nm transcriptome analysis in human blood, we showed that the nalP gene was up-regulated during incubation in human blood and that survival of a nalP deletion mutant (named MCΔnalP) in human blood was impaired (14). To further characterize the NalP phenotype, we…”

“…The experiments of survival in human blood and human serum were performed as previously described (14). Bacteria were grown until midexponential phase and then added to human whole blood or human serum at 40% in D-PBS.…”

“…Nm has evolved several redundant mechanisms to evade the host innate responses at sites of colonization and during systemic growth, including expression of fHbp (10,11) and NspA (12,13), that facilitate binding of fH, lipooligosaccharide (LOS) sialic acid, which inhibits complement deposition (12), and Opc protein, which binds to vitronectin (2). In a recent functional genomic study, we identified factors involved in Nm survival in human blood (14). One of these factors is NalP, which is an autotransporter with subtilisin-like serine protease activity that is involved in autoproteolytic processing, resulting in secretion of the NalP passenger domain into the bacterial supernatant (15,16).…”

FKBPs in bacterial infections

“…Surface-exposed Mip-like proteins were also found in the important human pathogens Neisseria gonorrhoeae and N. meningitidis where they mediate persistence in macrophages or improve bacterial survival in the blood, respectively (Echenique-Rivera et al, 2011;Leuzzi et al, 2005). In the caries-associated periodontal pathogen Aggregatibacter actinomycetemcomitans a Mip-like protein was found to be up-regulated in primary isolates.…”