Proteomic and bioinformatic analyses of putative Mannheimia haemolytica secretome by liquid chromatography and tandem mass spectrometry

Abstract

Mannheimia haemolytica is a major bacterial contributor to bovine respiratory disease complex that costs the livestock industry a billion dollars a year in USA. Commercial vaccines are only partially efficacious under field conditions. Earlier studies found that outer membrane protein preparations and culture supernatants can induce immune responses that enhance resistance to challenge by M. haemolytica strains. The objective of this study was to characterize secretome of two M. haemolytica stains grown under two different media. Bacteria-free concentrated supernatants from M. haemolytica culture was subjected to LC-MS/MS. The secretome of M. haemolytica from both strains yielded 923 proteins. Using bioinformatic tools, 283 were identified as secreted proteins. Further breakdown of 283 proteins showed that 114 (40.2%), 184 (65.0%), 138(48.7%), 151 (53.3%) and 172 (60.7%) were characterized as secreted proteins by SignalP 4.1, SecretomeP 2.0, LipoP, Phobius, and PRED-TAT, respectively. A total of 95 (33.56%) proteins were characterized as being secreted via non-classical pathway as opposed to the majority that were secreted in signal peptide dependent pathway. The demonstrated proteins include all previously immunologically characterized M. haemolytica proteins. The potential of using secretome analysis in the design and development of a multivalent vaccine is discussed.

Mannheimia haemolyticain bovine respiratory disease: immunogens, potential immunogens, and vaccines

“…In addition, Ayalew et al . (2017 b ) demonstrated by proteomic analyses a putative IgA protease in M. haemolytica culture supernatant. IgA proteases in other bacteria are components of autotransporter molecules, enhance bacterial invasion of mucosal surfaces, and assist in bacterial escape from host defenses (Mistry and Stockley, 2006).…”

“…Gs60 is a surface-exposed, 60 kDa outer membrane lipoprotein found in M. haemolytica culture supernatant (Moore et al ., 2011; Ayalew et al ., 2017 b ). A fragment of the gene was cloned, characterized, and expressed, and antibodies directed against the expressed protein fragment correlated with resistance against experimental challenge (Weldon et al ., 1994).…”

“…O-Sialoglycoprotease (also referred to as O-sialoglycoprotein endopeptidase and glycoprotease) is a 35.2 kDa endopeptidase that hydrolyzes peptide bonds within glycoproteins with a marked specificity for sialylated glycoproteins (Otulakowski et al ., 1983; Abdullah et al ., 1990, 1992; Lee et al ., 1994 a , 1994 b ; Mellors and Lo, 1995). Its enzyme activity was originally identified in M. haemolytica culture supernatants, the protein was demonstrated by proteomic analyses in supernatants, and the enzyme was isolated and activity characterized as a neutral metalloprotease (Otulakowski et al ., 1983; Abdullah et al ., 1992; Ayalew et al ., 2017 b ). Subsequently, Lo et al .…”

“…Lkt gene expression increased between 6 and 12 h after challenge, whereas gs60 gene expression decreased (Sathiamoorthy et al ., 2012). The Gs60 protein was identified as a component of the putative M. haemolytica secretome but was not identified in an outer membrane immunoproteomics study of one M. haemolytica strain (Lo and Mellors, 1996; Ayalew et al ., 2010, 2017 b ). Recombinant Gs60 as a potential vaccine component was studied by several approaches including expression in alfalfa as a potential component of an edible vaccine and incorporation of M. haemolytica culture supernatant into immunostimulatory complexes (ISCOMs) with recombinant bovine C3d (Lee et al ., 2008; Moore et al ., 2011).…”

“…The protein was named PlpE, because it was the fifth lipoprotein associated with the outer membrane of then P. haemolytica, with PlpA-C being three contiguous 28–30 kDa lipoproteins and PlpD being a 31-kDa lipoprotein (Cooney and Lo, 1993; Murphy and Whitworth, 1993; Dabo et al ., 1994; Murphy et al ., 1998; Nardini et al ., 1998). PlpE was identified by immunoproteomics of the outer membrane and is in culture supernatants of M. haemolytica propagated under various growth conditions (Ayalew et al ., 2010, 2017 b ). Vaccination of cattle with recombinant PlpE plus adjuvant resulted in a reduction in lesion scores of >40% compared with controls after Mannheimia haemolytica S1 or S6 challenge (Confer et al ., 2003, 2006).…”