LipL32 is one of the most highly studied leptospiral proteins, because it is thought to be important during human pathogenesis. Genetic data show that the LipL32 gene is highly conserved in pathogenic species and is absent in closely related saprophytic species (11
). Proteomic studies have shown LipL32 to be the major surface-exposed outer membrane protein from laboratory-cultured strains (4
). Evidence for the direct involvement of LipL32 in mammalian pathogenesis is also strong. First, proteomics studies using leptospires isolated from an individual with the severe pulmonary form of leptospirosis that were applied to a guinea pig model of leptospirosis likewise showed that LipL32 is expressed during infection (20
). Second, immunohistochemistry studies showed that LipL32 is expressed in leptospires from kidney tissue of infected mammals (11
). Finally, anti-LipL32 antibodies are made during human infection (10
). These studies indicate selective pressure to retain this protein that dominates the cell-surface architecture of Leptospira
during laboratory culture and infection. However the function of LipL32 has proven elusive, since it has no similarity to any other protein with a known function and reports suggesting hemolytic activity (12
) have yet to be substantiated.
LipL32 was identified as an ECM-binding protein in this study, following the observation that L. interrogans
serovar Manilae cells adhered specifically to Matrigel, laminin, collagen I, and fibronectin. Previous studies showed that there was some strain-specific variation in LipL32 expression levels (11
). Therefore, it is speculated that there may be a correlation between expression and cellular interaction with ECM. Since LipL32 expression is high under laboratory culture conditions that are osmotically similar to environmental reservoirs, this may reflect the biological role of LipL32 as a protein that initiates the interaction with ECM during the transition from environment to host. However, experiments using anti-LipL32 antiserum as a potential adhesion-blocking reagent failed to block adhesion, as tested by an in vitro adhesion assay (D. E. Hoke and B. Adler, unpublished results). This is probably due to redundancy of adhesion molecules in Leptospira
(e.g., LigA, LigB, and LSA24).
rLipL32 was used to model the binding properties of native LipL32. These studies showed selective binding to Matrigel ECM and the individual components laminin and collagens I and V, but not to fibronectin. In the multiplicity of ECM ligands that it binds, LipL32 is similar to the LigA and LigB proteins, which bind collagens I and IV, laminin, and especially fibronectin and fibrinogen (3
). Work with other bacterial ECM-binding proteins that bind disparate ECM molecules showed that separate domains are responsible for these multiple interactions (26
). Future work will be aimed at determining the domains of LipL32 required for molecular interaction with different components of the ECM.
The binding curve for the interaction of rLipL32 with Matrigel ECM appears complex, with the detection of a small amount of rLipL32 bound at the 0.2 to 1 μM concentration range and 10 to 30 times more bound at the 5 to 10 μM concentration range. This is hypothesized to be due to the heterogeneous nature of this ECM preparation, in which LipL32 can bind to the major constituents of Matrigel, which are laminin and collagen IV (16
), with different affinities. While this theory requires future confirmation, including formally testing the binding activity for collagen IV, it is apparent that LipL32 displays complex binding activity consistent with its potential to bind multiple ligands within Matrigel ECM.
The first wave of bacterial genomic information was largely skewed to human pathogens. Analysis of these pathogen genomes indicated that LipL32 is restricted to the genus Leptospira
. However, recent genome projects have included environmental bacteria, allowing the identification of a highly similar gene, PTD2-05920, in the marine surface-associated bacterium P. tunicata
). Our work shows that rPTD2-05920 is immunologically and functionally similar to LipL32. Interestingly, native PTD2-05920 was not detected in broth-cultured P. tunicata
, indicating that the protein is not expressed under in vitro growth conditions. While almost nothing is known about gene or protein expression in P. tunicata
, it is logical to suggest that PTD2-05920 may be differentially expressed when required for adhesion.
was originally isolated from the marine tunicate Ciona intestinalis
, in which it is believed to protect the host surface from the colonization of a variety of fouling organisms. While little is known about this interaction, it is interesting to note that C. intestinalis
is a primitive chordate that has many of the genes necessary for ECM synthesis (14
). Therefore, it is possible that the common function of LipL32 and PTD2-05920 as ECM-binding proteins is exploited by these divergent bacteria for the common biological goal of interacting with their host. C. intestinalis
is an invertebrate that diverged most recently before the point of vertebrates, making it a model for vertebrate evolution (14
). While ECM genes have been shown to be associated with the evolution of multicellular organisms and higher organismal complexity, LipL32 may be a component of the molecular biology of bacterial evolution allowing expansion from primitive chordates to mammalian hosts.
While some bacterial MSCRAMMs have been shown to utilize similar binding domains (26
), there is nothing in the LipL32 sequence that would indicate a particular binding domain a priori. Therefore, a series of deletion constructs was made, identifying the C-terminal 72 amino acids as necessary and sufficient for binding. This region thus constitutes a novel ECM-binding sequence. A comparison of LipL32 and PTD2-05920 C termini revealed a highly conserved region of 29 amino acids with 75% identity, which is therefore a likely candidate for the minimal binding region of the two proteins.
The interaction of a bacterium with its host is the result of a response to environmental stimuli acting through multiple adhesion molecules. While the adhesive contribution of the leptospiral Lig proteins is proposed to be controlled by changes in osmolarity (3
), LipL32 function may be affected by posttranslational events, such as proteolytic cleavage. Studies have suggested that the C terminus of LipL32 is shed, while the N terminus remains attached to the cell surface (4
). Since this work implicates the C terminus of LipL32 as the region that binds ECM, cleavage may modulate the LipL32/ECM interaction. Thus, LipL32 is proposed to be a component of an adhesive program for Leptospira
and possibly other host-associated bacteria.