Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein of Neisseria meningitidis and a component of the Bexsero vaccine. NHBA is characterized by the presence of a highly conserved Arg-rich region involved in binding to heparin and heparan sulphate proteoglycans present on the surface of host epithelial cells, suggesting a possible role of NHBA during N . meningitidis colonization. NHBA has been shown to be cleaved by the meningococcal protease NalP and by human lactoferrin (hLF), a host protease presents in different body fluids (saliva, breast milk and serum). Cleavage occurs upstream or downstream the Arg-rich region. Since the human nasopharynx is the only known reservoir of infection, we further investigated the susceptibility of NHBA to human proteases present in the saliva to assess whether proteolytic cleavage could happen during the initial steps of colonization. Here we show that human saliva proteolytically cleaves NHBA, and identified human kallikrein 1 (hK1), a serine protease, as responsible for this cleavage. Kallikrein-related peptidases (KLKs) have a distinct domain structure and exist as a family of 15 genes which are differentially expressed in many tissues and in the central nervous system. They are present in plasma, lymph, urine, saliva, pancreatic juices, and other body fluids where they catalyze the proteolysis of several human proteins. Here we report the characterization of NHBA cleavage by the tissue kallikrein, expressed in saliva and the identification of the cleavage site on NHBA both, as recombinant protein or as native protein, when expressed on live bacteria. Overall, these findings provide new insights on NHBA as target of host proteases, highlights thepotential role of NHBA in the Neisseria meningitidis nasopharyngeal colonization, and of kallikrein as a defensive agent against meningococcal infection.

6Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein and a component of 7 the Bexsero vaccine. NHBA is characterized by the presence of a highly conserved Arg-rich region 8 involved in binding to heparin and heparin sulphate proteoglycans present on the surface of host 9 epithelial cells, suggesting a possible role of NHBA during N. meningitidis colonization. NHBA has 10 been shown to be cleaved by the bacterial NalP protein, a meningococcal protease and by human 11 lactoferrin (hLF), a host protease present in different body fluids (saliva, breast milk and serum). 12Cleavage occurs upstream or downstream the Arg-rich region. Since the human nasopharynx is the 13 only known reservoir of infection, we further investigated the susceptibility of NHBA to human 14 proteases present in the saliva to assess whether proteolytic cleavage could happen during the initial 15 steps of colonization. Here we show that human saliva proteolytically cleaves NHBA; and 16 identified human kallikrein 1 (KLK1) as the main protease responsible for this cleavage. Kallikrein 17 is an important enzyme present in blood plasma, lymph, urine, saliva, pancreatic juices, and other 18 body fluids that catalyze the proteolysis of several human proteins. We report the in vitro 19 characterization of NHBA cleavage by kallikrein; the identification of the cleavage in the 20 recombinant NHBA protein and, on the native protein, when expressed on live bacteria. Overall, 21 this findings provide new insights on NHBA as target of host proteases, highlights a potential role 22 of NHBA in the Neisseria meningitidis nasopharyngeal colonization, and of kallikrein as a 23 defensive agent against meningococcal infection. 29 nonepidemic settings, approximately 10% of healthy individuals at any time carry Neisseria 30 meningitidis in the oropharyngeal tract. Transmission of bacteria occurs via direct contact or 31 through dispersion of respiratory droplets from a healthy carrier or an infected person to a 32 susceptible individual. Although often protected by a polysaccharide capsule, meningococci are 33 particularly sensitive to desiccation; thus, spread from one individual to another necessitates close 34 contact [4]. In fact, closed or semi-closed settings, such as residential schools and military recruit 35 camps, facilitate meningococcal transmission resulting in a dramatic increase of carriage rate, which 36 may approach 100%. 37 Occasionally, shortly after the onset of colonization, N. meningitidis penetrates the mucosal 38 membrane and enters the bloodstream, causing various forms of disease [5]. The most common 39 clinical presentations of invasive infections include meningitis and severe sepsis with an often fatal 40 outcome; other diseases, such as septic arthritis, pneumonia, purulent pericarditis, conjunctivitis, 41 otitis, sinusitis and urethritis, occur more rarely [5]. 42 Meningococcal serogroups B and C are responsible for the majority of meningitidis cases in 43 Europe, Y serogroup is relevant in the United States [8], w...