Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins.

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Perforin-2, the product of the MPEG1 gene, limits the spread and dissemination of bacterial pathogens in vivo. It is highly expressed in murine and human phagocytes, and macrophages lacking Perforin-2 are compromised in their ability to kill phagocytosed bacteria. In this study we used Salmonella enterica serovar Typhimurium as a model intracellular pathogen to elucidate the mechanism of Perforin-2’s bactericidal activity. In vitro Perforin-2 was found to facilitate the degradation of antigens contained within the envelope of phagocytosed bacteria. In contrast, degradation of a representative surface antigen was found to be independent of Perforin-2. Consistent with our in vitro results a protease sensitive, periplasmic superoxide dismutase (SodCII) contributed to the virulence of S. Typhimurium in Perforin-2 knockout but not wild-type mice. In aggregate our studies indicate that Perforin-2 breaches the envelope of phagocytosed bacteria facilitating the delivery of proteases and other antimicrobial effectors to sites within the bacterial cell.

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Vibrio are widely distributed in aquatic environments (Thompson et al., 2006) and are commonly associated with marine living species and include many important pathogens for farmed animals and humans who consume contaminated seafood or polluted drinking water. Pathogenic mechanisms are only clearly understood for V. cholerae and V. parahaemolyticus, but not for the majority of other vibrios (Faruque et al., 1998;Nishibuchi and Kaper, 1995;Thompson et al., 2006). Many virulence genes have been elucidated, including the tdh gene (coding thermostable direct haemolysin) in V. parahemolyticus, ctxAB genes (coding the potent cholera toxin) in V. cholera and others (Faruque et al., 1998;Nishibuchi and Kaper, 1995).Zot (zonula occludens toxin) was first discovered in V. cholerae, and has the ability to increase the permeability of the small intestinal mucosa by affecting the structure of the intercellular tight junction, or zonula occludens (Fasano et al., 1991). The toxS gene was also first discovered in V. cholerae, and lies downstream from the toxR gene, which encodes the transcriptional activator for the cholera toxin (ctx ) operon in V. cholerae (Dirita and Mekalanos, 1991;Miller et al., 1989). toxS acts in conjunction with toxR to activate expression of the ctx operon (Miller et al., 1989). tcpA is an important virulence gene of V. cholerae, encoding the major subunit of the toxin-coregulation pilus (TCP), which exerts important roles in infection as the colonization factor (Faast et al., 1989). The importance of the flagellum in virulence has been demonstrated for several species, as either a motility organelle or an organelle that carries an adhesive component (Lee et al., 2006;Milton et al., 1996). V. cholerae contains five different structural genes of the flagellum: flgI, flgJ, flgM, flgL and flaA, and these genes encode the Pand L-ring proteins, the hook-associated proteins 1,3 and the flagellin core protein of the flagellum, respectively (Das et al., 1998).V. harveyi is a serious pathogen of marine animals . However, the virulence factors of V. harveyi are not completely understood. Previous studies have shown that the virulence factors, such as VHH haemolysin, protease, phospholipse, lipopolysaccharide and bacteriocin-like substances may contribute to the virulence of this species Zhang et al., 2001). It has been proposed that the virulence genes could be horizontally transferred among Vibrio spp. (Waldor and Mekalanos, 1996). Previously, we found that the vhh/tlh haemolysin gene and toxR gene exit in all V. harveyi strains detected (Pang et al

ABSTRACT. Lipid-associated membrane proteins (LAMPs) are important in the pathogenicity of the Mycoplasma genus of bacteria. We investigated whether Mycoplasma hyopneumoniae LAMPs have pathogenic potential by inducing apoptosis in a St. Jude porcine lung epithelial cell line (SJPL). LAMPs from a pathogenic strain of M. hyopneumoniae (strain 232) were used in the research. Our investigation made use of diamidino-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) analysis, B. Ni et al. 11430©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (3): 11429-11443 (2015) and Annexin-V-propidium iodide staining. After LAMP treatment for 24 h, typical changes were induced, chromosomes were concentrated, apoptotic bodies were observed, the 3'-OH groups of cleaved genomes were exposed, and the percentage of apoptotic cells reached 36.5 ± 11.66%. Caspase 3 and caspase 8 were activated and cytochrome c (cyt c) was released from the mitochondria into the cytoplasm; poly ADP ribose polymerase (PARP) was digested into two fragments; p38 mitogen-activated protein kinase (MAPK) was phosphorylated; and the expression of pro-apoptosis protein Bax increased while the antiapoptosis protein Bcl-2 decreased. LAMPs also stimulated SJPL cells to produce nitric oxide (NO) and superoxide. This study demonstrated that LAMPs from M. hyopneumoniae can induce apoptosis in SJPL cells through the activation of caspase 3, caspase 8, cyt c, Bax, and p38 MAPK, thereby contributing to our understanding of the pathogenesis of M. hyopneumoniae, which should improve the treatment of M. hyopneumoniae infections.