The characterization of leptospiral outer membrane proteins represents an important step toward the understanding of leptospirosis pathogenicity. To date, there has been little functional analysis of Leptospira
membrane proteins, in spite of their unquestionable relevance to host-pathogen interactions. Bioinformatic analysis of the genome sequences of L. interrogans
serovar Copenhageni revealed more than 200 predicted outer membrane proteins that merit further studies [7
]. In this report, we have characterized one of those hypothetical proteins, a surface leptospiral adhesin of 21 kDa, named Lsa21 that may play a role in pathogenesis.
The LIC10368 expression is mostly detected in low-passage virulent strains (Figs. , ). Culture attenuation has also been reported for the LigA and B proteins, leptospiral antigens recognized during the acute host infection [26
]. Although a larger sample set will be required for definite conclusion, our findings suggest a correlation between Lsa21 protein and virulence.
The LIC10368 gene is up regulated by osmolarity, another distinguishing feature shared with ligA
]. The addition of 120mM NaCl to the culture medium reproduces the host's serum osmolarity (~300 mosM), thus providing a more physiological environment for leptospiral growth. In fact, EMJH-salt supplementation enhanced Lsa21 expression (Fig. ). It is worth mentioning that several predicted outer membrane proteins currently under study in our laboratory are responsive to physiologic osmolarity (unpublished data). It is not surprising because 6% of the L. interrogans
genes are susceptible to osmoregulation [27
]. Osmotic control of gene expression has been reported as an environmental cue associated with virulence in a variety of pathogens [28
]. In V. cholerae
, optimum expression of cholera toxin and Tcp pilli
occurs within an osmolarity range that probably represents that of mucosal secretions [37
]. Transcription levels of invA
, an S. Typhimurium
invasion gene, have also been reported to be considerably higher on media with high osmolarity [38
]. Moreover, genes controlling the P. aeruginosa
capsule, an important Pseudomonas
virulence factor, are also subjected to osmoregulation [39
Temperature induction of LIC10368 expression was also observed (Fig. ). Maximal mRNA levels were detected at 37°C, the body core temperature of most mammalian species. Intriguingly, LIC10368 is not represented among the leptospiral genes found to be up-regulated in response to temperature or physiologic osmolarity, as assessed by whole-genome microarrays [27
]. This could be explained by the fact that LIC10368 expression rapidly decreases after three or four passages in culture medium (Fig. ). In addition, optimum expression might occur when both physiological parameters (mammalian body core temperature and host's serum osmolarity) are combined.
Lsa21 protein exhibits extracellular matrix-binding properties. It is thus possible that it may play a role in the attachment to host tissues. Several leptospiral adhesins have been described, including a 36-kDa fibronectin-binding protein [11
], a 24-kDa laminin-binding protein named Lsa24 [9
], LigA and LigB proteins [10
]. Recently, it has been demonstrated that L. interrogans
contain five additional paralogs of Lsa24 [9
], designated as Len-like proteins [13
]. The Len proteins were all found to bind laminin and in addition LenB, LenC, LenD, LenE and LenF have shown affinity to bind host fibronectin [13
Lsa21 protein exhibits a broader spectrum binding profile because it interacts with laminin, collagen IV and fibronectin. Similarly, other adhesins, namely the Lig proteins [10
], have been reported to bind to different ECM macromolecules. In fact, attachment of Leptospira
to several ECM macromolecules, including laminin, collagen I, collagen IV, cellular fibronectin, and plasma fibronectin was previously shown [9
]. Interestingly, Lsa21 shares neither sequence similarity nor a common domain with the Len-like and the Lig proteins. Emp, a cell surface protein of Staphylococcus aureus
, strongly interacts with fibronectin, fibrinogen, collagen, and vitronectin [40
]. The outer membrane protein YadA of Yersinia enterocolitica
has been shown to bind to laminin, fibronectin, and several types of collagens [41
]. Enterococcus faecalis
adhesin Ace mediates attachment to laminin, and to collagens I and IV [44
]. Finally, the Haemophilus influenzae
Hap autotransporter protein exhibits the same binding profile of Lsa21 protein: it interacts with laminin, fibronectin and collagen IV [45
]. Chemical disruption of laminin carbohydrate moieties by sodium metaperiodate caused significant reduction in the binding activity of Lsa21 protein, thus indicating the sugar moiety involvement in interactions between this recombinant protein and ECM macromolecules. Possibly Lsa21 protein interacts with a particular structural epitope shared by the ECM components mentioned above. A good candidate would be complex sugars of glycosylated proteins. During infection, injury of wall vessels exposes a repertoire of adhesive glycoproteins that constitute major targets for initial adherence of pathogens [46
]. The extracellular matrix-binding properties of Lsa21 together with the data of indirect immunofluorescence suggest that this protein is surface-exposed.
The presence of Lsa21 antigen as demonstrated by IHC in cells, chiefly macrophages, of the inflammatory infiltrate both in human liver and kidney is expected because leptospirosis is an acute septicemic disease involving several organs and blood vessels. Liver-plate disarray is a pathological finding described in human and experimental leptospirosis and in the human liver, its prominence is closely related to the interval between patient's death and the autopsy, thus indicating that post-mortem changes might accentuate the lesion. In any circumstance, liver-plate disarray supports speculation that cell membrane lesion might play an important role in leptospirosis pathogenesis [47
]. Although cross-reactivity between anti-Lsa21 serum and other leptospiral proteins is not excluded, immunohistochemical detection of leptospiral antigens on the sinusoidal side of human hepatocytes is an infrequent finding in human leptospirosis when standard whole bacterial polyclonal sera were used [47
]. The fact that Lsa21 anti-serum stained hepatocyte membranes more often might strengthen the possible role of cell membrane lesion in the pathogenesis of the disease.
Tubulo interstitial nephritis is the main manifestation of acute renal failure in leptospirosis and the renal injury is usually associated with polyuria. Hypokalemia appear frequently with an elevated urinary fractional excretion of potassium, possibly due to proximal tubular lesions that is expressed both in human and experimental leptospirosis [47
]. The IHC detection of the leptospiral antigenic protein Lsa21 on the epithelial membrane of the distal nephron, particularly distal tubules and collecting ducts, might be, up to a certain point, unexpected when a functional correlation is attempted. However, it is known that in a normal state, urine is concentrated because of the combined functions of Henle's loop and the collecting duct. Therefore, we might speculate that the Lsa21 deposits detected by IHC chiefly in collecting ducts might be interfering with the renal water absorption in leptospirosis, in spite of the absence of definite morphological alterations. Altogether these results strongly suggest a role of this protein in the pathogenesis of the disease because no reactivity was seen in non-leptospirosis human fatal cases.