A human lectin array for characterizing host-pathogen interactions

Sugars on the surface of bacteria, viruses, fungi and parasites that inhabit and infect human hosts form an important means of identifying these micro-organisms. Lectins, which are sugar-binding proteins found on human cells and in the blood, can distinguish between the different sugar structures on human cells and those on many types of micro-organisms. This form of recognition can be useful as a means of differentiating self from non-self, which can in turn serve as a basis for innate immunity. For example, soluble lectins in the blood bind sugars on bacteria and activate a special pathway of complement fixation to directly attack and kill them. Cell-surface lectins on macrophages bind sugars on bacteria and viruses, causing them to be internalized and destroyed. Lectins can also initiate protective inflammatory responses in which immune cells are recruited to sites of infection.

To fulfil these functions, lectins in the human system are hard-wired to recognize and attack potential pathogens based on their surface sugars. However, some bacteria, such as commensals that constitute the microbiota, are not attacked. Some microbes have also developed the ability to hijack host lectins as a way to enter and attack host cells. By a combination of biochemistry, structural biology, genetics and genomics, multiple families of human lectins have been defined and many are well characterized. However, knowing the receptors and the types of sugars that they interact with is not sufficient to allow us to predict which receptors will bind to what micro-organisms and what the consequences will be. The key problem is that sugars on micro-organisms are very diverse and many are not fully characterized.

The work that we propose is designed to address this problem by developing a lectin array as a tool for screening the human repertoire of lectins against micro-organisms. The array will consist of the immobilized carbohydrate-binding domains from the major families of human lectins, so that the panel of lectins can be probed with fluorescently-labelled viruses, bacteria and fungi. In a single experiment, it will then be possible to see which receptors are able to recognize and bind to each individual microbe. We have recently created the first mammalian lectin array, containing cow lectins, to demonstrate the feasibility of this approach. The proposed studies will provide our first overall view of how the human host interacts with sugars on both pathogenic and non-pathogenic micro-organisms so that it can respond differently to different challenges.

The lectin array to be developed is a discovery tool that will help to identify receptors that may be responsible for early responses to micro-organisms. Once the array is set up, one potential application will be to screen novel and emergent pathogens quickly to see which receptors they can bind to, in order to provide information about how they may enter cells and whether the innate immune response may effectively control an infection. Comparison of the human array with our prototype animal lectin array will also provide some indication of common types of interactions that might help micro-organisms move between species.

Genetic variations in the human population result in changes in the amino acid sequences of some of the sugar-binding receptors, which can affect their sugar-binding properties and thus change the way that individuals respond to particular microbes. In a further development of the human lectin array, we will examine the effects of sequence variations, identified in large-scale genomic studies, on interactions of the panel of human lectins. We will also follow up results of array screening by examining which sugars on the surfaces of the target micro-organisms interact with specific lectins, filling important gaps in our knowledge of how these sugar-binding receptors distinguish between self and non-self.

Mammalian sugar-binding receptors, known as animal lectins, bind both endogenous mammalian glycans and pathogen glycans. Our understanding of these receptors has been dramatically enhanced by glycan array analysis combined with structural analysis to determine what sugars they bind and how they bind. Up to now, the focus has largely been on binding of endogenous mammalian glycans on cells and circulating glycoproteins. However, many of the receptors form part of the way that hosts interact with diverse glycans found on micro-organisms. The goal of these studies is to provide a novel tool that will provide insight into how binding of non-self glycans by receptors can distinguish micro-organisms from self in order to initiate appropriate innate immune responses. We will populate a first-generation human lectin array with biotin-tagged receptor fragments representing the complete set of sugar-binding receptors from the three major structural groups. We will initiate screening of the array with fluorescently-labelled bacteria, viruses and fungi to identify which receptor-mediated pathogen recognition events may underlie sugar-based innate immune responses to these organisms. The molecular basis for novel and unexpected interactions of receptors will be investigated quantitatively to identify sugar determinants used by receptors to distinguish self from non-self. Pathogen binding to the human array will be compared with our prototype bovine array to assess the implications for zoonotic infections. In the final portion of the project, we will generate a second-generation array to probe the effects of variations in the human population on the interactions with pathogens. The array will be supplemented with additional structural groups of lectins as well as variant forms of the glycan-binding receptors that reflect polymorphisms in the human population. An alternative micro-array format will also be investigated.