Hfq- and sRNA-mediated regulation in Neisseria meningitidis

Neisseria meningitidis, also known as the meningococcus, is a human-specific pathogen that commonly colonises the nasopharynx without causing disease. For reasons unknown, N. meningitidis can traverse the nasopharyngeal epithelium and enter the bloodstream, causing invasive meningococcal disease manifesting in septicaemia and/or meningitis. Meningococci are divided into serogroups based on the serological response to their different polysaccharide capsule. They can also be subdivided into clonal complexes based on multi locus sequence typing of seven housekeeping genes. An interesting feature of N. meningitidis is the compact genome with frequent numbers of repeat elements present throughout the genome. DNA uptake sequences, repeat sequences, neisserial intergenic mosaic elements, Correia repeat enclosed elements, tandem repeats and insertion sequences are all examples of such repeat elements. The presence of these repeat sequence elements is attributed to the great genome flexibility that N. meningitidis possess. Repeat sequence and genome flexibility allows meningococci to switch gene expression in an ON/OFF manner as well as acquiring new genetic elements beneficial to the bacterium.

N. meningitidis utilises several virulence factors in its arsenal to overcome the host immune system. Polysaccharide capsule shields the bacterium from a variety of antimicrobial agents and complement system. Capsule can also prevent opsonophagocytosis as well as cover protein epitopes targeted by antibodies. Type IV pilus is another virulence factor that facilitate twitching motility, enables uptake of exogenous DNA, and adhesion to both bacteria and host cells. N. meningitidis is a human-specific bacteria in part due to the bacterium having specialised in acquiring iron from human iron sequestering proteins such as transferrin, lactoferrin, haemoglobin and haemoglobin-haptoglobin. These iron sequestering proteins are all part of the host nutritional immunity, directed to deprive bacteria from essential nutrients. After iron is extracted from human proteins, N. meningitidis utilises an iron transport system to shuttle iron from the outer membrane to the cytoplasm. The protein FbpA is known to perform the iron transport through the periplasm.

Recent findings have elucidated the increasing importance of sRNAs as possible mediators for N. meningitidis sudden switch from a passive coloniser to lethal pathogen. The RNA chaperone Hfq is often required for the function of a class of sRNA, called trans-encoded sRNAs, and acts as a matchmaker between sRNA and its target mRNA. There are also Hfq-independent sRNAs, called cis-encoded sRNA, that usually enact their functions on the same transcript in which they are transcribed.

The work presented in this thesis aims to further the knowledge of Hfq-dependent and independent sRNAs within N. meningitidis as well as explore Hfq/RNA regulation impact on invasive meningococcal disease. The structure of Hfq as well as two point mutated variants of Hfq have been determined through X ray crystallography. Several RNAs were used to investigate RNA binding patterns in wild-type Hfq in comparison with point mutated variants of Hfq predicted to affect Hfq binding capabilities to RNA species. In this research, a previously Hfq-dependent uncharacterised sRNA, termed NirF, was shown to be a negative regulator of fbpA. NirF is expressed during iron replete conditions and is thought to act in coordination with the iron-responsive transcriptional regulator Fur to prevent possible hazardous intake of excess iron.

The studies presented herein continues by investigation of a phenotype in a Hfq knock-out strain of N. meningitidis. The Hfq knock-out strain revealed a visual increase in surface blebbing which at a closer inspection was deemed to be membrane vesicles These vesicles were smaller in size and accounted for a hundred times increased yield when compared to membrane vesicles harvested from the wild-type counterpart. Proteomic analysis revealed similar protein content but with distinct differences. Of note, the membrane vesicles from the Hfq-knock out strain contained fewer numbers of different proteins which resulted in an enrichment of immunogenic proteins such as Opc, PorA, PorB and App. While studying the phenotypic effects of Hfq knock-out in N. meningitidis, an impact on the regulation of iga, the gene encoding the IgA1P protein was discovered. IgA1P is classically known to cleave IgA1 antibodies. Further investigation revealed that a specific type of IgA1P can also cleave IgG3 with the possibility to enhance N. meningitidis survival in the human host. In fact, this additional cleaving type including IgG3 was significantly associated with invasive disease isolates of N. meningitidis in comparison with carrier isolates. The polysaccharide capsule is a vital virulence factor for the development of invasive meningococcal disease. Further research was dedicated to investigating sRNA dependent regulation of capsule production. A previously known RNA thermosensor in the capsule biosynthesis operon was studied for potential variations which could have an impact on capsule regulation. Five novel RNA thermosensor variants were discovered which all resulted in a hypercapsulation phenotype. These new RNA thermosensors, together with previously discovered RNA thermosensors increasing capsule production, were all sufficient to protect N. meningitidis from high exposure to human serum. The abnormal capsule regulation by disrupted RNA thermosensors were significantly associated with invasive meningococcal disease isolates. Furthermore, RNA thermosensor disruption was identified to be the singular feature which significantly differentiated two closely related meningococcal isolates whereas the isolate having the disrupted RNA thermosensor resulted in invasive meningococcal disease.

The works included in this thesis show several new features of Hfq-dependent and independent sRNA regulations and the impact on N. meningitidis adaptation to environmental factors and virulence gene expression. Further research is needed to fully understand the triggering switch between meningococcal colonisation and pathogenesis, but the work included herein has contributed to one step closer to this goal.

List of scientific papers

I. Jens Karlsson, Francesco Righetti, Yuri Yoshimasu, Martin Moche, Edmund Loh. Structure of RNA chaperone Hfq in Neisseria meningitidis and characterisation of an sRNA repressing FbpA expression. [Manuscript]

II. Jens Karlsson, John Boss, Ryoma Nakao, Edmund Loh. Characterisation of membrane vesicles from a Hfq mutant strain of Neisseria meningitidis. [Manuscript]

III. Christian Spoerry. Jens Karlsson, Marie-Stephanie Aschtgen, Edmund Loh. Neisseria meningitidis IgA1-specific serine protease exhibits novel cleavage activity against IgG3. Virulence. 2021; 12(1): e389-403.
https://doi.org/10.1080/21505594.2021.1871822

IV. Jens Karlsson, Hannes Eichner, Cecilia Andersson, Susanne Jacobsson, Edmund Loh. Novel hypercapsulation RNA thermosensor variants in Neisseria meningitidis and their association with invasive meningococcal disease: a genetic and phenotypic investigation and molecular epidemiological study. The Lancet Microbe. 2020; 1(8): e319-327.
https://doi.org/10.1016/S2666-5247(20)30146-4