br Transparency document br Acknowledgments This

Transparency document
Acknowledgments This study was funded by ICMR, Govt. of India. Alisha Dhiman acknowledges UGC-DSKPDF, India for post-doctoral fellowship and Monisha Gopalani acknowledges CSIR, India for Senior Research Fellowship. AIRF, JNU is acknowledged for TEM, confocal microscopy and SPR facility, and Dr. Gajendra Saini, Mr. Ashok Kumar Sahu and Mr. Manu Vashistha/Ms. Anu Singh for their technical help in the same. BSL-3 at SBT, JNU funded by Department of Biotechnology, Govt. of India, was used for work with virulent Mtb H37Rv strain and is gratefully acknowledged.
Introduction Paenibacillus larvae, a gram-positive spore-forming bacterium, is the cause of American foulbrood (AFB) and powdery scale in honey bee larvae (Apis mellifera Linnaeus) (White, 1906; Katznelson, 1950). Phenotypic and molecular characteristics place P. larvae into two subspecies P. larvae subsp. larvae (Pll) and P. larvae subsp. pulvifaciens (Plp) (Heyndrickx et al., 1996), A more recent molecular characterization, using ERIC-PCR technology, has generated 4 genotypes (i.e., ERIC I, II, III, IV) (Genersch et al., 2006). ERIC-PCR and subspecies classification do correlate in that ERIC I includes Pll strains and ERICs III, and IV contain Plp strains. Genotype ERIC II, predominantly contains isolates identified as Plp (Genersch et al., 2006) and, for this investigation, Plp is considered as containing genotypes ERIC II, III, and IV. Hitchcock and associates (Hitchcock et al., 1979) demonstrated that disease progression was more rapid for Plp than disease progression by Pll. In addition, ERIC I genotypes take longer to kill 100% of infected host samples than isolates grouped into ERIC II, III, and IV genotypes (i.e., ~12days vs ~7days, respectively) (Ashiralieva and Genersch, 2006; Genersch et al., 2005). An explanation(s) for virulence differences between subspecies, or genotypes, is not known. Strain variations that affect basic metabolic processes are potential candidates and are now accessible through kinesin 5 sequences. Enolase is of particular interest since it may influence the pathogenic process by three different routes: as a cell surface protein, as a component of the RNA degradosome or as an enzyme. In gram-positive bacteria enolase is found on cell surfaces and binds to laminin, fibronectin, and collagens (aiding bacterial cell adhesion) and to plasminogen (promoting conversion to plasmin) aiding virulence (Antikainen et al., 2007). Plasmin, a serine protease involved in fibrinolysis and extracellular matrix degradation, can enhance tissue invasion (Saksela and Rifkin, 1998). In P. larvae secreted enolase was identified as a potential virulence factor (Antúnez et al., 2010; Antúnez et al., 2011). Whether a plasminogen-binding property is associated with P. larvae enolase, and how that property might be associated with virulence is unknown. However, strong evidence exists of a plasminogen-like protein in A. mellifera (Grossi et al., 2016). Enolase is also part of the RNA degradosome that indirectly influences both metabolic loci involved in growth (Morita et al., 2004) as well as bacterial virulence by controlling response to oxidative stresses (Weng et al., 2016). Enolase (EC 4.2.1.11) is a key enzyme in the glycolic pathway facilitating the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP), and is abundant in many organisms (Pancholi, 2001), Enolase activity may serve as a rate-limiting growth factor for P. larvae and growth rate might influence speeds of lethality. This investigation examines vegetative growth characteristics and enolase genotypes in different P. larvae virulence segregated subspecies. In addition, this research determines the kinetics of two enolase forms.
Materials and methods
Results
Discussion Genetic differences between P. larvae subspecies determine virulence phenotypes (fast or slow progression) (Hitchcock et al., 1979), as well as the disparity in cell density at the stationary stage of vegetative growth (Fig. 1). It is as yet unclear if the growth and virulence characters are influences by the same or overlapping loci. Three reasons support the contention that the enolase gene is a candidate locus that influences both virulence and metabolism. First, enolase is a key metabolic enzyme involved in the conversion of 2-PGA to PEP during cellular glycolysis, and as such is directly involved in vegetative cell growth. Second, enolase is part of the RNA degradosome that indirectly influences both metabolic loci involved in growth (Morita et al., 2004) as well as bacterial virulence by controlling response to oxidative stresses (Weng et al., 2016). Third, enolase has a direct role in pathogenicity by acting as a cell surface binding protein (Genersch et al., 2005; Grossi et al., 2016) and is reported to be a virulence factor in P. larvae (Antúnez et al., 2011).