Novel Leptospira interrogans protein Lsa32 is expressed during infection and binds laminin and plasminogen

Abstract

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease of human and veterinary concern. The quest for novel antigens that could mediate host-pathogen interactions is being pursued. Owing to their location, these antigens have the potential to elicit numerous activities, including immune response and adhesion. This study focuses on a hypothetical protein of Leptospira, encoded by the gene LIC11089, and its three derived fragments: the N-terminal, intermediate and C terminus regions. The gene coding for the full-length protein and fragments was cloned and expressed in Escherichia coli BL21(SI) strain by using the expression vector pAE. The recombinant protein and fragments tagged with hexahistidine at the N terminus were purified by metal affinity chromatography. The leptospiral full-length protein, named Lsa32 (leptospiral surface adhesin, 32 kDa), adheres to laminin, with the C terminus region being responsible for this interaction. Lsa32 binds to plasminogen in a dose-dependent fashion, generating plasmin when an activator is provided. Moreover, antibodies present in leptospirosis serum samples were able to recognize Lsa32. Lsa32 is most likely a new surface protein of Leptospira, as revealed by proteinase K susceptibility. Altogether, our data suggest that this multifaceted protein is expressed during infection and may play a role in host-L. interrogans interactions.

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

“…In accordance with a potential role in host-pathogen interactions, 5 putative L. interrogans moonlighting proteins have been shown to be immunoreactive (61), suggestive of their expression during the infection process. An exoprotein (not detected in the present study) directly associated with pathogenesis has been characterized as a collagenase required for tissue invasiveness and virulence in animals (24), while another protein (Lsa32/LIC11089), detected in culture supernatants in the present study, has been characterized and demonstrates laminin and plasminogen binding capacity (62). A known Leptospira virulence factor, high-temperature protein G (HtpG) (63), was detected at a high abundance in all culture supernatants, suggesting that the extracellular presence of this protein is a factor contrib- uting to disease pathogenesis in animals through either unidentified moonlighting properties or the host inflammatory response to this protein.…”

Pathogenic <i>Leptospira</i>: Advances in understanding the molecular pathogenesis and virulence

“…Interestingly, these characteristics were not observed in saprophytes. In addition, complex proteins were shown to be expressed during infection and were thought to mediate the interaction between the susceptible host and pathogenic L. interrogans (Domingos et al, 2015). However, contrasting observations were reported where minor variations in individual sets of proteins with potential physiological and pathological roles were documented (Buyuktimkin and Saier, 2015).…”

Avaliação e caracterização de candidatos vacinais voltados para o controle da leptospirose.

“…Para isso, laminina, fibronectina celular, elastina, colágeno tipo I, colágeno tipo IV e as proteínas controles BSA e fetuína foram imobilizados em placas de microdiluição e a ligação das proteínas recombinantes foi avaliada por ELISA, após o uso dos anticorpos policlonais obtidos contra cada proteína recombinante. Várias proteínas de leptospiras com propriedades de ligação específica a laminina e a outros componentes da ECM foram identificadas pelo nosso grupo (ATZINGEN et al, 2008;ATZINGEN et al, 2009;BARBOSA et al, 2006;DOMINGOS et al, 2015;DOMINGOS et al, 2012;FERNANDES et al, 2014;FERNANDES et al, 2012;LONGHI et al, 2009;MENDES et al, 2011;OLIVEIRA et al, 2011;OLIVEIRA et al, 2010; __________________________________________________________Resultados e Discussão 113 2013; SOUZA et al, 2012;VIEIRA et al, 2010b). Outros grupos também identificaram proteínas com capacidade de adesão (CARVALHO et al, 2009;CHOY et al, 2007;HAUK et al, 2008;HOKE et al, 2008;LIN et al, 2009;STEVENSON et al, 2007).…”

“…No entanto, como essa interação não foi abolida totalmente, possivelmente epitopos não conformacionais também fazem parte dessa interação. Essa mesma faixa de redução foi observada para a proteína de L. interrogans Lsa32 quando experimentos similares foram realizados (DOMINGOS et al, 2015). Em relação à proteína Lsa77, a ligação também foi afetada após o processo de desnaturação, porém a redução da ligação foi menos expressiva (Figura 34).…”

“…Os dados representam a porcentagem de ligação das proteínas recombinantes com os componentes ± desvio padrão de uma triplicata e são representativos de dois experimentos independentes As proteínas recombinantes que não passaram pelo processo de desnaturação foi considerado como 100 % de ligação, por isso essa situação foi utilizada para comparação da ligação com as proteínas desestruturadas, pelo teste t-Student (*p<0,05). (DOMINGOS et al, 2015;FERNANDES et al, 2012;VIEIRA et al, 2010a). Dados na literatura demonstram ainda que os resíduos de lisina presentes na região C-terminal das proteínas atuam, preferencialmente, na interação ao PLG (MILES et al, 1991).…”