Leptospirosis is a disease of global significance (
3). Chronically infected mammalian hosts harbor pathogenic
Leptospira species in renal tubules of the kidney, from which they are shed via urine into the environment and survive under suitable moist conditions. The rat has long been recognized as a carrier host for
Leptospira species (
8,
31), with recent studies confirming its role in disease outbreaks in urban settings (
22,
29,
32). Humans are infected via broken skin and mucosal surfaces during contact with contaminated environments (
9). Clinical manifestations of acute human leptospirosis range from mild to severe forms. More recently, a severe pulmonary form of leptospirosis (SPFL) has been recognized that results in rapid disease progression with high mortality among patients (
24-
26,
30,
34). While there are indications that such severe manifestations of disease are due to an autoimmune response (
12) and the emergence of a virulent clonal isolate of
Leptospira (
20,
21,
27), pathogenic mechanisms of the pulmonary form of leptospirosis remain unclear.
Isolates of
Leptospira interrogans were cultured from patients suffering from SPFL in 1998 at the National Leptospirosis Reference Laboratory at the Oswaldo Cruz Institute-FIOCRUZ, Brazil (
23). These isolates have been used to develop relevant animal models of disease, including an acute lethal infection with pulmonary hemorrhage in guinea pigs, marmosets, and mice (
12,
14,
19). To date, the guinea pig model of SPFL has provided two significant insights into pathogenic mechanisms of severe forms of leptospirosis: first, alveolar septal deposition of immunoglobulin and complement parallels pulmonary hemorrhage in the absence of detectable leptospiral antigen, suggesting that lung hemorrhage is mediated in part by autoimmune mechanisms (
12); and second, novel assays employed to extract intact motile leptospires from in vivo sources during acute disease has confirmed that these host-tissue-derived
Leptospira isolates differ dramatically in the expression of lipopolysaccharide and protein constituents from the same isolates cultured in vitro (
13,
17).
Specific serovars of
Leptospira species are associated with clinically asymptomatic chronic infection of specific mammalian host species (
28).
Rattus norvegicus is a reservoir host of those strains of
Leptospira associated with human SPFL. Such host adaptation was first recognized by Babudieri, who observed a “biological equilibrium” between rodent hosts and certain
Leptospira serovars (
2). Leptospiruria in maintenance hosts is of high intensity, constant, and of long duration compared to that in accidental hosts, where it is of low intensity, intermittent, and of short duration (
4). In contrast to previous studies which focused on characterization of the proteome of in vitro-cultivated
Leptospira (IVCL) (
5,
16), the present study has exploited leptospiruria during a chronic animal disease model to provide, for the first time, a proteomic analysis of leptospires as excreted in urine of chronically infected hosts. Results confirm that leptospires differentially express proteins in the host, which in turn likely facilitates persistence in the presence of a specific host antibody response. A clearer understanding of changes in protein expression by pathogenic species of
Leptospira during infection is likely to lead to elucidation of pathogenic mechanisms of leptospirosis.
DISCUSSION
Rattus norvegicus is a reservoir host for
L. interrogans serovar Copenhageni, a causative agent of SPFL, which is a severe, rapid disease process with mortality rates greater than 50% (
3). Since human patients become infected by direct contact with urine from chronically infected animals or other environmental sources contaminated with such urine, it can be assumed that leptospires derived from such sources represent an inherently virulent form responsible for causing acute disease processes. The proteome of such excreted organisms has not yet been examined.
To date, the majority of proteomic studies seek to elucidate pathogenic mechanisms of leptospirosis by identifying the proteome of leptospires that have been cultured in vitro (
16,
18). Results confirm the dynamic nature of the proteome, particularly in response to culture conditions designed to mimic host conditions (
11,
15). In the present study, the development of an animal model of leptospirosis reflecting chronic disease was exploited to look at the proteome of
Leptospira organisms as excreted during transmission in infected urine. Interestingly, early work on rodent carrier models of
Leptospira infection suggested fundamental antigenic differences between IVCL organisms and leptospires derived from infected renal tissue, since antibody from infected urine, kidney suspensions, or serum of infected rodents was active against IVCL but inactive against leptospires derived from renal tissues (
6,
7). Therefore, the present study sought to establish the nature of these antigenic differences using, for the first time,
Leptospira organisms as excreted from chronically infected hosts.
Experimental infection of
Rattus norvegicus results in an asymptomatic carrier state accompanied by a persistent chronic interstitial nephritis, aggregations of organisms within tubules, and chronic excretion of organisms in urine. Numbers of leptospires excreted in infected urine were sufficient for proteomic analysis, as confirmed by immunoblotting of samples with antiserum specific for OMV of
Leptospira species (Fig.
3B). In addition, both IVCL and RUIL samples expressed the previously characterized outer membrane proteins Qlp42, LipL41, LipL32, LipL21, and Loa22. However, differential antigenic expression was observed in RUIL compared to IVCL when samples were separated by 2-D gel electrophoresis for immunoblotting with anti-OMV (Fig.
6).
Chronic infection of rats with
Leptospira species exemplifies the equilibrium between the host immune response and persistent infection. Despite the fact that experimentally infected rats produce antibody specific for IVCL by 7 days postinfection (data not shown) and continue to produce specific antibody during chronic disease, this immune response does not stop the shedding of leptospires in urine. The detection of a lymphocyte-rich inflammatory infiltrate in association with leptospires in infected kidney sections was interesting; however, it remains unclear if such an immune response would ultimately be successful enough to remove organisms and eliminate renal excretion. Of equal interest was the observation of leptospiral organisms within tubules devoid of any immune response, suggesting a possibility that such organisms may be shielded from the host immune response (Fig.
2). Immunoblotting of IVCL with chronic rat serum detected many antigens. However, immunoblotting of RUIL with chronic rat serum reacted with relatively fewer antigens. This observation would explain previously described differences in activity of chronic rat serum against in vitro-derived
Leptospira compared to kidney-derived
Leptospira (
7). It further confirms the dynamic nature of the leptospiral proteome in adapting to specific niche conditions and in this case regulating protein expression to minimize interaction with the host antibody response. While the results of immunoblotting using chronic rat serum shown in Fig.
4 might suggest a diminution of antigen expression by leptospires isolated from urine compared to that for IVCL, immunoblotting with serum specific for OMV of
Leptospira species confirmed the presence of several antigens common to both samples. This in turn suggests that the expression of those antigens specifically reactive with chronic rat serum is downregulated during chronic disease (Fig.
4). However, downregulation of specific antigens may correlate with the release of leptospires from tissues into urine. This is a major possibility if some of these antigens are adhesins that mediate adherence of leptospires to host tissues. Finally, different environmental signals associated with kidney and urine, e.g., pH, could influence protein expression.
Results illustrated prolonged survival of leptospires in renal tubules despite antibody production by the rat. It is likely that leptospires are exposed to antibody during their replication in renal tubules, since rat IgG is found in normal as well as infected rat urine (
33); however, the specificity of urinary antibodies for
Leptospira species in infected rats remains to be determined. The outer membrane protein LipL32 (also known as Hap-1) was expressed by RUIL and was reactive with chronic rat serum. This suggests that the antibody in chronic rat serum that is specific for LipL32 may not be able to bind leptospires in situ in renal tissues or if so is not functionally active.
This study has confirmed the utility of a chronic animal model of disease for studying the proteome of Leptospira species directly during the transmission process. Results highlight the dynamic nature of the leptospiral proteome in response to host conditions during infection and confirm that colonization of renal tubules is facilitated by differential protein expression. Identification of proteins expressed during persistent infection will provide insights into pathogenic mechanisms of disease and potential mechanisms that distinguish acute versus chronic disease processes.