Streptococcus pyogenes interaction with the lymphatic hyaluronan receptor LYVE-1 as a mechanism for lymphatic dissemination and disease progression

Streptococcus pyogenes, often known as 'group A strep' normally causes infections such as sore throat, tonsillitis and impetigo, commonly among children. However, it can also spread from person to person rapidly, to cause life-threatening infections such as necrotising fasciitis, maternal sepsis, and toxic shock, most commonly in those who were otherwise previously fit and healthy. The most virulent, or dangerous, types of group A strep often demonstrate a thick capsule, or outer coating, when grown from the blood stream. Heavily encapsulated strains of group A strep have also been associated with outbreaks of the immune disorder, rheumatic fever, the leading cause of valvular heart disease worldwide. Coupled with the relevance to lethal invasive strep infections, there is a major imperative to understand the interaction of group A strep with the immune system focussing in particular on the role of the capsule.

The group A strep capsule is made from a substance known as hyaluronic acid or hyaluronan, a large sugar thought to facilitate wound healing, immune cell migration and cancer spread. Much of our own body is filled with hyaluronan, and group A strep may use this capsule to cloak itself from our immune system. Lymphatics are channels distributed throughout the body, through which fluid known as lymph flows to lymph glands, taking with it, the specialised white blood cells required for immune responses and also, hyaluronic acid - which is itself broken down in lymph nodes. The cells lining lymphatic channels actually have a receptor or 'hook' for hyaluronan to latch onto, presumably helping the hyaluronic acid to enter the channel although little is known about how the hyaluronan moves along the channels.

We believe that group A strep exploits the very same hyaluronan 'hook' on the cells lining the lymphatic channel in order to enter lymphatics and reach lymph glands. The lymph glands are "depots" where microbes arising from infections elsewhere in the body are conveyed, so that they can be exhibited to the immune system to generate a protective immune response. We suspect that the group A strep hyaluronan capsule not only helps the microorganism to get to the lymph nodes but also protects it from being recognised and killed by the immune system. In this project we will find out whether group A strep does use this system to reach the lymph glands, and whether that has adverse consequences for people with infection, both those with tonsillitis and those with more dangerous infections such as necrotising fasciitis. In the work proposed, we will test strains that are currently causing disease in the UK for their ability to be trafficked in lymphatics, as well as strains that have been altered in the laboratory to express greater or lesser amounts of capsule. If we were able to show a deleterious effect of group A strep interacting with lymphatics, it might then be possible to block or prevent the interaction between the lymphatic cells and the streptococcus from occurring, and to develop treatments to prevent invasive infections from progressing, or to prevent triggering of inflammation.

The polysaccharide capsule of the virulent human pathogen Streptococcus pyogenes is comprised of hyaluronan (HA), a large mucopolysaccharide in extracellular matrix and lymph. Building on our previous work demonstrating dissemination of S. pyogenes from a focus of infection to local lymph node, we have now shown that HA-encapsulated S. pyogenes can bind to human lymphatic endothelial cells via the lymphatic endothelial cell (LEC) hyaluronan receptor LYVE-1, providing a potential conduit for extracellular transport of S. pyogenes to lymph nodes.

We have three broad questions
(1) What is the impact of S. pyogenes binding to LYVE-1 on LEC in vitro?
(2) What is the impact of the binding in vivo?
(3) Is there evidence of binding in clinical tissues?

In this proposal we will determine the impact of S. pyogenes capsular binding to primary human dermal (HD)LEC via LYVE-1, to determine if the interaction promotes internalisation by LEC or invasion/transmigration, as assessed by quantitative microbiology, confocal and 2 photon microscopy. The response of LEC to S. pyogenes binding will also be investigated by confocal microscopy and whole genome transcriptomics followed by targeted analysis of enriched gene products. The specificity of interaction will be underpinned by use of isogenic S. pyogenes HA mutants from leading clinical lineages and by use of LYVE-1 blocking strategies. We will go on to determine the functional significance of S. pyogenes-LYVE-1 binding in upper respiratory tract and soft tissue infections in refined experimental models exploiting availability of LYVE-1 blocking antibodies and homozygous LYVE1-/- knockout mice. Finally we will investigate the clinical relevance of our observations by appropriate immunostaining of tissues from patients that are infected with S. pyogenes and by determining the capacity of S. pyogenes to invade lymphatics of tonsil and soft tissue samples ex vivo.

The earliest impact of this project will, in the first 2-3 years, be felt by the research communities outlined above, including those working in microbiology, cancer cell biology, immunology, and matrix biology. The investigators' teams, in particular the research staff employed by the award, will benefit considerably from the collaboration across two institutions and each will acquire training and knowledge from the academic partner, for example in the application of the 3Rs to modelling infection and the use of confocal and intravital microscopy in cell biology, notwithstanding the differing research environments. We anticipate that the careers of those supported by the grant will be developed to the extent that they are able to apply for independent research fellowships, building a cadre of researchers with complementary skills in cell biology and infection. The data generated from the study will also contribute to subsequent PhD studentship projects and clinical fellowships within the term of the award.

The earliest impact outside the academic field may be in the field of public health. Prediction of disease phenotypes associated with extreme capsular phenotypes may be possible as a result of the study. Epidemiological changes in invasive disease strain lineage are already the subject of ongoing national surveillance. If excess or absent capsule is shown to be a key determinant of any specific harmful disease phenotype, then this would provide a rationale for initiating surveillance of streptococcal pharyngitis, or for measurement of capsule by the reference laboratory, since epidemiological changes in pharyngitis isolates precede surges in invasive disease cases. This could allow prediction and alert systems to be developed, as well as, if indicated, a targeted campaign of treatment aimed at patients with symptomatic infections.

Identification of the functional relevance of S. pyogenes binding to lymphatics may provide a rationale for therapeutic modulation of binding, for clinical benefit. Although speculative, together with appropriately-used antibacterial agents, this could impact on future sufferers of streptococcal infections or the complications thereof, such as rheumatic fever. S. pyogenes represents a paradigm for bacteria with a hyaluronan capsule and the research could be extrapolated to several veterinary bacterial infections, in particular equine strangles which is characterised by painful lymphadenopathy and suppuration. Clinically-relevant interventions identified may therefore impact on the veterinary community and equine populations, as well as the sporting and leisure sectors where Strangles is a major concern.

The work may also impact upon the development of vaccines for bacteria that produce hyaluronan. There is no clinically licensed vaccine for S. pyogenes and the basis for naturally-acquired immunity is not understood. S. pyogenes-LYVE1 binding may circumvent normal immune responses and may in some circumstances lead to an abnormal immune response. Vaccine development and efficacy of any vaccine developed could well be influenced by knowledge of the dissemination routes taken by non-encapsulated encapsulated S. pyogenes.

Finally, but perhaps most importantly, the project has broad relevance to LYVE1 biology, as the impact of the LYVE1-HA interaction is poorly understood. HA is used widely in the cosmetics and tissue regeneration fields. Recognition of any harmful or beneficial effects of HA will be of importance in both the industrial and clinical settings. As LYVE1 is believed important to leucocyte trafficking in lymphatics, improved understanding of the outcome of the HA-LYVE1 interaction and development of any strategy to impede or influence the interaction of HA with lymphatics may impact on strategies to control inflammation and autoimmunity, as well as to optimize delivery of protective vaccines against disease.