Membrane Cofactor Protein (MCP), C4b binding protein (C4BP),
Factor I] and the common Terminal Pathway (e.g., CD59, vitronectin, clusterin). Some pathogens are able to evade the Complement System because they acquire Complement regulatory
proteins from the host.
As early as in the mid-1960s, a bactericidal activity of normal
human serum (NHS) against saprophytic but not pathogenic Leptospira strains was reported by Johnson and Muschel [2]. Since
then, it became clear that the Complement System had a role in
eliminating nonpathogenic Leptospira strains. More recently, a
number of studies aimed at unraveling the mechanisms by which
virulent Leptospira circumvent Complement activation have been
conducted (reviewed in [3]). Some of the general strategies used by
pathogenic Leptospira to counteract Complement include:
(1) binding of negative Complement regulators including FH,
FHL-1, C4BP, and vitronectin [4–7]; (2) cleavage of Complement
proteins of the Alternative, Lectin, and Classical Pathways by
secreted proteases [8, 9], and (3) the acquisition of host proteases
such as plasminogen, known to degrade Complement proteins
once converted to its active form, plasmin [10, 11].
In the following sections, we describe in detail some of the
most frequently used assays to evaluate Leptospira Complement
resistance:
1. Serum susceptibility assay—Leptospires susceptible to Complement attack fragment into fine particles, losing their typical
morphology. They acquire a finely granulated aspect, as
observed under the microscope. Serum susceptibility can be
assessed by incubating freshly harvested leptospires with normal human serum (NHS) followed by counting viable bacteria
under a dark-field microscope.
2. Serum adsorption assays using intact leptospires to evaluate binding of negative Complement regulators—Acquisition of negative
Complement regulators such as FH, FHL-1, C4BP, or vitronectin by intact leptospires can be assessed by incubating bacteria in NHS-EDTA. Surface-bound proteins are subjected to
Western blotting, and Complement regulators are detected by
specific antibodies.
3. Cleavage of Complement proteins by secreted proteases—
Pathogenic Leptospira such as L. interrogans serovar Pomona
strain Pomona, L. interrogans serovar Kennewicki strain
Fromm, L. interrogans serovar Copenhageni strain 10A,
L. interrogans serovar Icterohaemorrhagiae strain RGA,
L. interrogans serovar Pyrogenes strain Salinem, L. kirshneri
serovar Cynopteri strain 3522C, and L. noguchii serovar Panama strain CZ 214 secrete proteases that cleave several Complement proteins like C3 and Factor B (Alternative Pathway),
188
Lourdes Isaac and Angela Silva Barbosa
Factor I] and the common Terminal Pathway (e.g., CD59, vitronectin, clusterin). Some pathogens are able to evade the Complement System because they acquire Complement regulatory
proteins from the host.
As early as in the mid-1960s, a bactericidal activity of normal
human serum (NHS) against saprophytic but not pathogenic Leptospira strains was reported by Johnson and Muschel [2]. Since
then, it became clear that the Complement System had a role in
eliminating nonpathogenic Leptospira strains. More recently, a
number of studies aimed at unraveling the mechanisms by which
virulent Leptospira circumvent Complement activation have been
conducted (reviewed in [3]). Some of the general strategies used by
pathogenic Leptospira to counteract Complement include:
(1) binding of negative Complement regulators including FH,
FHL-1, C4BP, and vitronectin [4–7]; (2) cleavage of Complement
proteins of the Alternative, Lectin, and Classical Pathways by
secreted proteases [8, 9], and (3) the acquisition of host proteases
such as plasminogen, known to degrade Complement proteins
once converted to its active form, plasmin [10, 11].
In the following sections, we describe in detail some of the
most frequently used assays to evaluate Leptospira Complement
resistance:
1. Serum susceptibility assay—Leptospires susceptible to Complement attack fragment into fine particles, losing their typical
morphology. They acquire a finely granulated aspect, as
observed under the microscope. Serum susceptibility can be
assessed by incubating freshly harvested leptospires with normal human serum (NHS) followed by counting viable bacteria
under a dark-field microscope.
2. Serum adsorption assays using intact leptospires to evaluate binding of negative Complement regulators—Acquisition of negative
Complement regulators such as FH, FHL-1, C4BP, or vitronectin by intact leptospires can be assessed by incubating bacteria in NHS-EDTA. Surface-bound proteins are subjected to
Western blotting, and Complement regulators are detected by
specific antibodies.
3. Cleavage of Complement proteins by secreted proteases—
Pathogenic Leptospira such as L. interrogans serovar Pomona
strain Pomona, L. interrogans serovar Kennewicki strain
Fromm, L. interrogans serovar Copenhageni strain 10A,
L. interrogans serovar Icterohaemorrhagiae strain RGA,
L. interrogans serovar Pyrogenes strain Salinem, L. kirshneri
serovar Cynopteri strain 3522C, and L. noguchii serovar Panama strain CZ 214 secrete proteases that cleave several Complement proteins like C3 and Factor B (Alternative Pathway),
188
Lourdes Isaac and Angela Silva Barbosa