The role of the NadR regulator during infection and its implication for the coverage of a new Meningococcus B vaccine

Background: Neisseria meningitides represents a major cause of meningitis and sepsis. The meningococcal regulator NadR was previously shown to repress the expression of the Neisserial Adhesin A (NadA) and play a major role in its phase-variation. NadA is a surface exposed protein involved in epithelial cell adhesion and colonization and a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B infection. The NadR mediated repression of NadA is attenuated by 4-HPA, a natural molecule released in human saliva. Results: In this thesis we investigated the global role of NadR during meningogoccal infection, identifying through microarray analysis the NadR regulon. Two distinct types of NadR targets were identified, differing in their promoter architectures and 4HPA responsive activities: type I are induced, while type II are co-repressed in response to the same 4HPA signal. We then investigate the mechanism of regulation of NadR by 4-HPA, generating NadR mutants and identifying classes or residues involved in either NadR DNA binding or 4HPA responsive activities. Finally, we studied the impact of NadR mediated repression of NadA on the vaccine coverage of 4CMenB. A selected MenB strains is not killed by sera from immunized infants when the strain is grown in vitro, however, in an in vivo passive protection model, the same sera protected infant rats from bacteremia. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h post-infection. Conclusions: Our results suggest that NadR coordinates a broad transcriptional response to signals present in the human host, enabling the meningococcus to adapt to the relevant host niche. During infectious disease the effect of the same signal on NadR changes between different targets. In particular NadA expression is induced in vivo, leading to efficient killing of meningococcus by anti-NadA antibodies elicited by the 4CMenB vaccine.

The silent survival of Staphylococcus aureus phagocyted by HL-60 derived neutrophils

Staphylococcus aureus is a Gram positive pathogen that causes various human infections and represents one of the most common causes of bacteremia. S. aureus is able to invade a variety of non-professional phagocytes and that can survive engulfment by neutrophils, producing both secreted and surface components that compromise innate immune responses. In the contest of our study we evaluated the functional activity of vaccine specific antibodies by opsonophagocytosis killing assay (OPKA). Interestingly a low level of killing of the staphylococcal cells has been observed. In the meanwhile intracellular survival studies showed that S. aureus persisted inside phagocytes for several hours until a burst of growth after 5 hours in the supernatant. These data suggest that the strong ability of S. aureus to survive in the phagocytes could be the cause of the low killing measured by OPKA. Moreover parallel studies on HL-60 cells infected with S. aureus done by using transmission electron microscopy (TEM) interestingly showed that staphylococcal cells have an intracellular localization (endosomal vacuoles) and that they are able not only to maintain the integrity of their membrane but also to replicate inside vacuolar compartments. Finally in order to generate 3D volume of whole bacteria when present inside neutrophilic vacuoles, we collected a series of tomographic two-dimensional (2D) images by using a transmission electron microscope, generating 5 different tomograms. The three-dimensional reconstruction reveals the presence of intact bacteria within neutrophil vacuoles. The S. aureus membrane appears completely undamaged and integral in contrast with the physiological process of phagosytosis through vacuoles progression. S. aureus bacteria show a homogenous distribution of the density in all the three dimensions (X, Y, Z). All these evidences definitely explain the ability of the pathogen to survive inside the endosomal vacuoles and should be the cause of the low killing level.

Regulatory networks of Neisseria meningitidis and their implications for pathogenesis

Neisseria meningitidis, the leading cause of bacterial meningitis, can adapt to different host niches during human infection. Both transcriptional and post-transcriptional regulatory networks have been identified as playing a crucial role for bacterial stress responses and virulence. We investigated the N. meningitidis transcriptional landscape both by microarray and by RNA sequencing (RNAseq). Microarray analysis of N. meningitidis grown in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. In particular, we identified a glucose-responsive hexR-like transcriptional regulator in N. meningitidis. Deletion analysis showed that the hexR gene is accountable for a subset of the glucose-responsive regulation, and in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of meningococcus, by targeting a DNA region overlapping putative regulatory sequences. Our results indicate that HexR coordinates the central metabolism of meningococcus in response to the availability of glucose, and N. meningitidis strains lacking the hexR gene are also deficient in establishing successful bacteremia in a mouse model of infection. In parallel, RNAseq analysis of N. meningitidis cultured under standard or iron-limiting in vitro growth conditions allowed us to identify novel small non-coding RNAs (sRNAs) potentially involved in N. meningitidis regulatory networks. Manual curation of the RNAseq data generated a list of 51 sRNAs, 8 of which were validated by Northern blotting. Deletion of selected sRNAs caused attenuation of N. meningitidis infection in a murine model, leading to the identification of the first sRNAs influencing meningococcal bacteraemia. Furthermore, we describe the identification and initial characterization of a novel sRNA unique to meningococcus, closely associated to genes relevant for the intracellular survival of pathogenic Neisseriae. Taken together, our findings could help unravel the regulation of N. meningitidis adaptation to the host environment and its implications for pathogenesis.