It has long been appreciated that Leptospira
species adapt to and survive in vastly different environments, but little is known about the molecular nature of these adaptations. The leptospiral outer membrane lipoprotein LipL36 has provided one example of environmentally regulated protein expression. Immunohistochemical staining demonstrated expression of LipL36 during in vitro growth at 30°C but not in infected tissue or at culture temperatures of 37°C, indicating an adaptive response by the organism to infection which included the diminution of expression of LipL36 (7
We have recently reported that a human SPFL isolate causes the severe pulmonary form of leptospirosis in guinea pigs and chronic asymptomatic carriage in rats (13
). Since large numbers of Leptospira
organisms can be found in the livers of infected guinea pigs, a procedure was developed for extracting intact motile Leptospira
organisms from infected host tissue. The completed genomes of L. interrogans
serovar Lai and serovar Copenhageni are predicted to have 3,728 and 4,727 protein-coding genes, respectively (17
). In order to reduce the complexity of proteins for sample analysis, both IVCL and HTL samples were extracted with 2% TX-114 which also enriches for hydrophobic protein antigens, as previously described (9
). This has the added advantage of identifying putative vaccinogen and diagnostic antigens associated with the outer membrane of Leptospira
during infection. Two-dimensional immunoblotting of TX-114-extracted IVCL indicated that CRS reacts with several antigens of the outer membrane of IVCL, which is confirmed by their reactivity with monospecific antiserum for LipL32, LipL21, LipL41, and Loa22. Mass spectrometry confirmed the identity of two additional CRS-reactive antigens as the putative lipoprotein NT03LI2251. Several other antigens have yet to be identified. By definition, all of these antigens are expressed during the chronic infection process of rats and in sufficient amounts to generate an antibody response. Similarly, CRS detected several antigens in HTL samples derived from the liver tissue of acutely infected guinea pigs. These antigens include several also expressed in IVCL including the aforementioned LipL32, LigA, LipL21, LipL41, and Loa22.
While absolute quantification of the amounts of each antigen present in each sample is not provided, these findings do provide an accurate view of the expression of antigens relative to each other in HTL and IVCL preparations. For example, Loa 22 is expressed in large amounts in HTL and is the only antigen whose expression appears to be significantly up-regulated during disease relative to detection of other antigens in the same sample. In contrast to Loa22, the relative amounts of LigA, LipL41, and LipL21 are reduced in HTL compared to IVCL. There are also a number of unidentified antigens detected with CRS or OMV serum, and their expression appears reduced in HTL relative to their representation in IVCL. Significant amounts of LipL32 are detected both during in vitro growth and disease relative to the diminution of several other known outer membrane antigens.
Expression of LipL32, LipL21, and Loa22 was detected with antiserum specific for OMVs of IVCL and serum from chronically infected rats. Some antigens were detected only with anti-OMV, including the putative lipoprotein NT03LI0010 and the conserved hypothetical protein NT03LIA0039, indicating that they are expressed in IVCL but not in sufficient amounts during chronic infection of rats to generate a detectable antibody response. LigA is reactive with CRS but only slightly reactive with anti-OMV. Similar amounts of LigA are present in each IVCL sample, suggesting that the greater reactivity with CRS is due to the expression of LigA during chronic infection of rats. As with CRS, anti-OMV generally reacts with a smaller set of antigens in the HTL sample but does react with LipL32, LipL21, and Loa22. Reactivity with the putative lipoprotein NT03LI0010 and the conserved hypothetical protein NT03LIA0039 is not detected in the HTL sample, confirming their diminished expression in HTL, as already demonstrated by a lack of reactivity with CRS.
While LipL32 is expressed in both IVCL and HTL, it was noted that several lower-molecular-mass antigens are specifically reactive with LipL32 monospecific antiserum, indicating that these fragments are likely derived from the mature LipL32. It is of interest that different lower-molecular-mass fragments of LipL32 are detected in IVCL and HTL samples. The meaning of this finding, observed with both CRS and anti-OMV antiserum, is unclear at this time. However, breakdown products of LipL32 have previously been noted during experimental preparations of outer membranes of IVCL (2
In this report, we have described the hydrophobic proteome of guinea pig liver-derived HTL recovered during the course of acute lethal infection. The relative amounts of Loa22 and LipL32 were enhanced in HTL compared to IVCL samples. There is also a striking reduction in the relative content of other hydrophobic protein antigens in HTL relative to their representation in IVCL. We have recently demonstrated that the lipopolysaccharide O-antigen content of HTL found in guinea pig liver is markedly reduced compared to that of IVCL. Taken together, these findings indicate that the surface antigen structure of HTL differs markedly from that of IVCL. The role of these compositional changes in pathogenesis remains to be determined.