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This investigation establishes that there is a genetic difference in the enolase loci that correlates with both vegetative growth and virulence phenotypes. DNA sequence analysis of enolase loci from seven P. larvae strains reveals a single SNP that alters the protein sequence at position 331 (Table 3). These two enzyme isoforms segregate along subspecies classification and within ERIC genotypes, except for ERIC II. It is possible that one of the ERIC II strains was misclassified. Interpretation of ERIC classification can be problematic between laboratories (Tyler et al., 1997; Johnson and Clabots, 2000; Di Pinto et al., 2011). Strain SAG 10367, originally identified as ERIC III (Dingman, 2015), has a banding pattern that best matches the ERIC-PCR electrophoretic banding pattern presented by ERIC II and has been reclassified (Genersch et al., 2006). The ERIC II genotype is problematic and may contain isolates that are intermediates to Pll and Plp (Genersch et al., 2006). Further, differences could be due to use of strains grouped within the same genotype that most likely have been geographically segregated for an extensive time. Strain SAG 10367 was originally isolated in South America, while strain DSM 25430 was an isolate from Germany.
The single amino PPADS tetrasodium salt change at position 331 in the P. larvae enolase produced a different affinity and turnover rate for 2-PGA. The enolase found within strains of Plp demonstrated an affinity for substrate (i.e., Km) 2.8 times, and an enzymatic efficiency 1.6 times, that of enolase present in Pll (ERIC I) strains. The replacement of glycine by alanine within a α-helix structure of a protein is known to stabilize that structure (Scott et al., 2007) in that alanine provides less flexibility in the protein (Yan and Sun, 1997). Differences in rigidity can effect enzymatic activity by influencing conformational changes or producing structural stress at the reactive site (Yan and Sun, 1997). Although the change in P. larvae enolase occurred between a α-helix and β-sheet structure, it was within 4 amino-acids of one of the three conserved amino-acids defined as forming the active site of enolase (Feng et al., 2009). Lowered flexibility at this location, due to the presence of alanine, may explain the lower enzymatic affinity for substrate observed in the Pll strains. This “rigidity,” by stabilizing the reactive site, may also explain the increased turnover rate observed for the Pll strains. What advantage/disadvantage this amino-acid change in enolase might have provided for the Pll strains is unknown considering that all other paenibacilli examined (Table 3) contained glycine at position 331.
What advantage/disadvantage this amino-acid change in enolase might have provided for the Pll strains is unknown considering that all other paenibacilli examined (Table 3) contained glycine at position 331.
A change in enolase flexibility may also influence plasminogen binding properties or its interaction in and function of the RNA degradosome. The change in amino acid at position 331 is outside the presumed binding regions for plasminogen (nucleotides 250–255 and C terminus) and magnesium (enzyme cofactor; 241D, 285E, and 312D) (Feng et al., 2009). Future investigations are needed to determine any effect of protein sequence on plasminogen binding or RNA degradosome properties.
Introduction Visceral leishmaniosis (VL) is a disease caused by the parasitic protozoan Leishmania infantum in the Americas, of which dogs are considered domestic reservoirs of these parasites. To reduce the disease’s dissemination, strategies to control canine visceral leishmaniosis (CVL) are essential, since seroprevalence varies in endemic regions, ranging from 3.4% to 40% of the animals, and reveals the potential for surveillance of canine infection as a marker of transmission between humans and sand flies (Dantas-Torres et al., 2006, Prado et al., 2011).